THE ADAPTOR PROTEIN APPL1 REGULATES CELL MIGRATION AND

ADHESION DYNAMICS

By

Joshua Allen Broussard

Dissertation

Submitted to the Faculty of the

Graduate School of Vanderbilt University

in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

in

Biological Sciences

August, 2012

Nashville, Tennessee

Approved:

Katherine L. Friedman, Ph.D.

Donna J. Webb, Ph.D.

Lilianna Solnica-Krezel, Ph.D.

Christopher J. Janetopoulos, Ph.D.

James R. Goldenring, Ph.D.

Irina Kaverina, Ph.D.

ii

ACKNOWLEDGEMENTS

I would first like to thank the members of my committee, Dr. Katherine Friedman

(Chair), Dr. Lilianna Solnica-Krezel (previous Chair), Dr. Christopher Janetopoulos, Dr.

Irina Kaverina and Dr. James Goldenring for all of their guidance. I would also like to

thank my advisor, Dr. Donna J. Webb for all of the time, effort and hard work she has

continued to put into me. I would like to thank all of the members of the Webb lab, past

and present, Devi Majumdar, Corey Evans, Lan Hu, Leolene Jean, Josh Hurley,

Mingfang Ao, Mingjian Shi and Bridget Lindsay. I would like to thank Claire Brown and

Brady Eason for their assistance in learning FRET, and a special thanks to Claire for

opening up her home to me while I stayed in Montreal. I would especially like to thank

Wan-Hsin Lin for helping to keep me sane with her insanity.

I am grateful to Jeffery Field, Margaret Frame, Steve Hanks, Brian Hemmings,

and Michiyuki Matsuda for providing us with reagents. I would like to thank Lan Hu and

Vanessa Hobbs for their assistance in preparing cDNA constructs. This work was

supported by National Institutes of Health (NIH) grants GM092914 and MH071674 to

D.J.W. J.A.B. was supported by predoctoral training grant CA78136 from NIH. FRET

imaging was conducted at the McGill University Life Sciences Complex Imaging Facility

on equipment purchased with funding from the Ministère du Développement

économique, Innovation et Exportation - Québec (MDEIE).

iii

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ................................................................................................ ii

LIST OF TABLES ........................................................................................................... vi

LIST OF FIGURES ........................................................................................................ vii

LIST OF ABBREVIATIONS ............................................................................................ ix

INTRODUCTION ............................................................................................................ 1

Cell Migration ............................................................................................ 1

Cell Adhesion - formation and composition ............................................... 1

Adhesion Turnover ................................................................................... 5

Coupling of adhesions with the contractile actin cytoskeleton ................... 8

Adhesions in the cell rear .........................................................................12

Microtubule support of adhesion asymmetry ............................................13

Akt and cell migration ...............................................................................16

Src and migration .....................................................................................18

The adaptor protein APPL1......................................................................19

Hypothesis: ..............................................................................................21

Chapter II ......................................................................................................................22

IDENTIFICATION OF PHOSPHORYLATION SITES WITHIN THE SIGNALING

ADAPTOR APPL1 BY MASS SPECTROMETRY ..............................................22

Abstract ...................................................................................................23

Introduction ..............................................................................................23

Materials and Methods .............................................................................25

Reagents and Plasmids ..............................................................25

iv

Protein Expression and Proteolytic Digestion ..............................25

Western Blot Analysis .................................................................26

Linear Ion Trap and LTQ-Orbitrap MS ........................................27

Bioinformatic Analysis .................................................................27

Results and Discussion ............................................................................28

Comprehensive Phosphorylation Map of Human APPL1 ............28

Phosphorylation Sites within APPL1 Functional Domains ...........34

Putative Kinases for Identified APPL1 Phosphorylation Sites ......37

Advantages and Challenges to Contemporary Phosphoproteomic

Methodologies ............................................................................39

Conclusion ...............................................................................................39

Chapter III .....................................................................................................................43

THE ENDOSOMAL ADAPTOR PROTEIN APPL1 IMPAIRS THE TURNOVER OF

LEADING EDGE ADHESIONS TO REGULATE CELL MIGRATION ..................43

Abstract ...................................................................................................44

Introduction ..............................................................................................44

Materials and Methods .............................................................................47

Reagents ....................................................................................47

Plasmids .....................................................................................48

Cell Culture, Transfection, and Immunoprecipitation ...................49

Migration Assay and Microscopy.................................................50

Immunocytochemistry and Image Analysis .................................50

Sensitized Emission Image Processing.......................................51

Sensitized Emission Image Analysis ...........................................52

Adhesion Turnover Assay ...........................................................55

v

Results ....................................................................................................56

The signaling adaptor APPL1 inhibits cell migration ....................56

Endosomal localization of APPL1 is required for its effects on

migration .....................................................................................60

APPL1 regulates leading edge adhesion dynamics in migrating

cells ............................................................................................62

APPL1 and Akt regulate cell migration and adhesion dynamics ..65

APPL1 reduces the amount of active Akt in cells ........................72

APPL1 regulates the tyrosine phosphorylation of Akt by Src .......78

Src-mediated tyrosine phosphorylation of Akt is critical for its

activation and function ................................................................80

Discussion ...............................................................................................85

Chapter IV .....................................................................................................................91

CONCLUSIONS AND FUTURE DIRECTIONS ..............................................................91

REFERENCES ............................................................................................................ 100

vi

LIST OF TABLES

Page

Table 1. Phosphorylation Sites within APPL1 Identified by LTQMS ...............................31

Table 2. Phosphorylation Sites Identified within APPL1 by LTQ-Orbitrap-MS ................32

Table 3. Comparison of Peptide Sequence Surrounding Identified Phosphorylation Sites

in APPL1 ............................................................................................................33

Table 4. Putative Kinases for Identified APPL1 Phosphorylation Sites ..........................38

Table 5. Putative kinases determined by Scansite .........................................................40

vii

LIST OF FIGURES

Page

Figure 1. The migration cycle. ........................................................................................ 2

Figure 2. Schematic of focal adhesion architecture and composition. ............................. 4

Figure 3. Model of asymmetric adhesion dynamics. ....................................................... 6

Figure 4. Schematic of the molecular clutch. .................................................................10

Figure 5. APPL1 protein purification. .............................................................................29

Figure 6. Map of APPL1 phosphorylation sites. .............................................................35

Figure 7. Phosphorylation in the BAR and PH domains of APPL1. ................................36

Figure 8. Tandem MS/MS spectrum acquired using an LTQ-Orbitrap illustrates peak

validation for accurate SEQUEST assignments. ................................................41

Figure 9. Tandem MS/MS spectrum of a phosphorylation site incorrectly assigned by

SEQUEST..........................................................................................................42

Figure 10. Calculation of FRET/CFP ratio images. ........................................................53

Figure 11. APPL1 regulates cell migration. ...................................................................58

Figure 12. APPL1 regulates cell migration in MDA-MB-231 cells. ..................................59

Figure 13. APPL1 localizes to vesicular structures, which is critical in its regulation of cell

migration. ...........................................................................................................61

Figure 14. APPL1 impairs adhesion turnover at the leading edge of cells. .....................63

Figure 15. Deletion of the PTB domain of APPL1 abolishes its effect on cell migration. 66

Figure 16. Akt plays an important role in the APPL1-mediated regulation of cell migration.

..........................................................................................................................68

viii

Figure 17. Akt knockdown inhibits cell migration. ...........................................................71

Figure 18. APPL1 reduces the amount of active Akt in cells. .........................................73

Figure 19. Schematic diagram of the Akt activation CFP-Venus FRET probe (Akind). ...75

Figure 20. Comparison of the FRET ratio images for wild-type and mutant Akind probes.

..........................................................................................................................76

Figure 21. APPL1 decreases Akt activity in cells. .........................................................77

Figure 22. APPL1 reduces the amount of active Akt in adhesions. ................................79

Figure 23. Src mediates tyrosine phosphorylation of Akt. .............................................81

Figure 24. Tyrosine phosphorylation of Akt regulates its activation and function. ...........83

Figure 25. Akt1 is the major Akt isoform in HT1080 cells. ..............................................88

Figure 26. Proposed model of APPL1-mediated effects on cell migration and adhesion

turnover. ............................................................................................................95

Figure 27. APPL1 and Src colocalize in the same vesicles. ...........................................97

Figure 28. APPL1 reduces the amount of active Rac in cells. ........................................99

ix

LIST OF ABBREVIATIONS

A Alanine

Abl Abelson kinase

APPL1 Adaptor protein containing a PH domain, PTB domain and leucine zipper motif 1

APPL1-AAA APPL1 mislocalization mutant in which R146, K152, and K154 are mutated to alanine

APPL1-PTB APPL1 truncation mutant in which the PTB domain has been deleted

Arp Actin related protein

BAR Bin-Amphiphysin-Rvs

CA Constitutively active

Cas Crk-associated substrate

Cdc42 Cell division cycle 42

CFP Cyan fluorescent protein

CLSM Confocal laser scanning microscope

DCC Deleted in colorectal cancer

DMEM Dulbecco’s modified Eagle medium

DMSO Dimethyl sulfoxide

DN Dominant negative

DNA Deoxyribonucleic acid

x

DTT Dithiothreitol

ECM Extracellular matrix

ER Endoplasmic reticulum

ERK Extracellular signal-regulated kinase

F Phenylalanine

F-actin Filamentous actin

FA Focal adhesion

FAK Focal adhesion kinase

FBS Fetal bovine serum

FN Fibronectin

FRET Fluorescence resonance energy transfer

FSHR Follicle-stimulating hormone receptor

FSM Fluorescence speckle microscopy

GAP GTPase-activating protein

GEF Guanine nucleotide exchange factor

GFP Enhanced green fluorescent protein

GIT1 GPCR kinase interacting protein 1

GPCR G-protein coupled receptor

xi

GTP Guanosine triphosphate

h Hour

HEK Human embryonic kidney

HGF Hepatocyte growth factor

IB Immunoblot

IP Immunoprecipitate

LC Liquid chromatography

MEF Murine embryonic fibroblast

MLCK Myosin light chain kinase

MMP Matrix metalloproteinase

MS Mass spectrometry

MT1-MMP Transmembrane type I MMP

OCRL Oculocerebrorenal syndrome of Lowe

PAGE polyacrylamide gel electrophoresis

PAK p21-activated kinase

PBS Phosphate buffered saline

PDK1 Phosphoinositide-dependent kinase 1

PH Pleckstrin homology

xii

PI3K Phosphoinositide 3-kinase

PIP2 Phosphatidylinositol (4,5)-bisphosphate

PIP3 Phosphatidylinositol (3,4,5)-trisphosphate

PIPKIg661 Phosphatidylinositol phosphatase kinase isoform-g661

PKL Paxillin kinase linker

PTB Phospho-tyrosine binding

PTP1B Protein tyrosine phosphatase 1B

R Arginine

Rac Ras-related C3 botulinum toxin substrate

Rho Ras homolog

RNA Ribonucleic acid

RNAi RNA interference

ROCK Rho kinase

S Serine

Scr Scrambled

SDS Sodium dodecyl sulfate

SEM Standard error of the mean

SH2 Src homology 2

xiii

Shp2 Protein tyrosine phosphatase 2

siRNA Small interfering RNA

Src Sarcoma

T Threonine

T1/2 Half-life

TIRF Total internal reflection fluorescence

VASP Vasodilator-stimulated phosphoprotein

Wt Wild-type

Y Tyrosine

1

Chapter I

INTRODUCTION

Cell Migration: Cell migration is central to numerous biological processes

including embryogenesis, the inflammatory response, tissue repair and regeneration, as

well as pathological processes such as cancer, arthritis, and atherosclerosis

(Lauffenburger and Horwitz 1996). Nevertheless, despite its importance, the molecular

mechanisms that control cell migration remain poorly understood.

Cell migration may be viewed as a multi-step cycle (Figure 1), which begins with

extension of an actin rich protrusion (Wang 1985; Carson, Weber et al. 1986; Borisy and

Svitkina 2000). This protrusion is then stabilized by the formation of adhesions, which

are points of contact between the cell and the extra-cellular matrix (ECM), near the

leading edge of the protrusion. These adhesions transmit the necessary forces to serve

as traction points required to move the cell body forward (Lee, Leonard et al. 1994;

Galbraith and Sheetz 1997; Beningo, Dembo et al. 2001). In turn, the adhesions at the

leading edge of the cell must disassemble for migration to continue (Laukaitis, Webb et

al. 2001; Webb, Donais et al. 2004). The process of adhesion assembly and

disassembly at the leading edge is termed adhesion turnover. Release of adhesions and

retraction at the rear completes the migratory cycle (Lee, Ishihara et al. 1993; Sheetz

1994; Lauffenburger and Horwitz 1996). Since adhesion dynamics play such an

important role in regulating cell migration, I will focus mainly on this aspect of migration

in this dissertation.

Cell Adhesion - formation and composition: Small nascent adhesions, which

are less than the 0.25 m resolution of most light microscopy techniques, form

2

Figure 1. The migration cycle. (1) Cell migration begins with the extension of an actin rich protrusion. (2) This protrusion is then stabilized by the formation of cell-matrix adhesions. (3) The cell body then translocates in the direction of migration. (4) Finally, the adhesions at the cell rear detach and the cell retracts. Reprinted from Molecular Biology of The Cell, Alberts 4th Ed.

3

concurrently with lamellipodial protrusion (Choi, Vicente-Manzanares et al. 2008). Some

of these will further mature into focal complexes (0.5 m) and then to focal adhesions

(FAs) (1-5 m) (Choi, Vicente-Manzanares et al. 2008). This dissertation will focus

mainly on FAs. FAs are points of contact between a cell and the ECM (for example,

fibronectin (FN)), which link the extracellular environment with the actin cytoskeleton

(actin stress fibers terminate at adhesion sites). They are highly complex and are

composed of more than 180 different protein-protein interactions (Zaidel-Bar, Itzkovitz et

al. 2007; Zaidel-Bar and Geiger 2010). However, adhesion proteins fall into some

general classes (Figure 2).

The physical link between the ECM and a cell is mediated by the integrin family

of transmembrane receptors, which are composed of - and -integrin heterodimers

(Hynes 2002). The extracellular integrin domains bind directly to motifs within ECM

proteins, such as FN, while the short intracellular tail domains interact with various

adhesion proteins (Hynes 2002). One major category of adhesion molecules is adaptor

proteins, such as paxillin, talin, vinculin and zyxin (Brown and Turner 2004; Zaidel-Bar,

Itzkovitz et al. 2007; Kanchanawong, Shtengel et al. 2010). They act as signaling and

scaffolding proteins, linking integrins to the cytoskeleton and recruiting other molecules

to adhesions. For example, talin links the cytoplasmic tail of integrins to actin as well as

vinculin (Campbell and Ginsberg 2004). Vinculin binds actin and -actinin (an actin

binding/bundling protein) (Otey and Carpen 2004; Ziegler, Liddington et al. 2006). There

are also other actin regulatory proteins present, such as vasodilator-stimulated

phosphoprotein (VASP), which is involved in actin nucleation and binding (Harbeck,

Huttelmaier et al. 2000). Kinases, such as focal adhesion kinase (FAK), Abelson kinase

(Abl) and Src kinase, regulate adhesion signaling by phosphorylating many adhesion

components, such as paxillin and Cas. This localizes guanine nucleotide exchange

4

Figure 2. Schematic of focal adhesion architecture and composition. Integrin extracellular domains interact with the ECM, while their cytoplasmic tails interact with the integrin signaling layer (including FAK and paxillin). There is then a force transduction layer, including talin and vinculin. Finally, there is an actin regulatory layer, which

includes zyxin, VASP and -actinin which link the adhesion to an actin stress fiber. Figure reprinted from (Kanchanawong, Shtengel et al. 2010).

5

factors (GEFs) and GTPase-activating proteins (GAPs), which regulate Rho family

GTPases (for example, Rho, Rac and Cdc42), and, in turn, modulate adhesion assembly

and disassembly (Parsons 2003; Defilippi, Di Stefano et al. 2006; Mitra and Schlaepfer

2006; Peacock, Miller et al. 2007).

Adhesion Turnover: Polarized cell migration requires asymmetric dynamics of

cell-ECM adhesions (Figure 3) that are closely coupled to reorganization of the actin

cytoskeleton. Migrating cells continuously form and disassemble their adhesions at the

leading edge in a process termed adhesion turnover (Webb, Parsons et al. 2002).

Nascent adhesions at the cell front exert tractional forces on the substrate that lead to

cell relocation (Beningo, Dembo et al. 2001). While the majority of nascent adhesions

undergo rapid turnover such that their components can be incorporated into newly

formed adhesion sites, a few mature behind the leading edge in response to tensile

stress (reviewed in (Bershadsky, Balaban et al. 2003)). Mature adhesions are, in turn,

either disassembled underneath the approaching cell body or diminished to form fibrillar

adhesions, which play critical roles in extracellular matrix modifications (Zamir, Katz et al.

2000; Rid, Schiefermeier et al. 2005).

Once formed, nascent adhesions either turnover or undergo maturation. The

molecular basis of this choice has received a great deal of attention over the last several

years because of its central role in cell migration. Locally regulated FAK appears to be a

key trigger for adhesion turnover. Fibroblasts from FAK null mice have an increased

number of large, peripheral adhesions (Ilic, Furuta et al. 1995). Local rises in Ca2+

concentration can induce adhesion disassembly by increasing the residency of FAK at

these sites (Giannone, Ronde et al. 2004). In addition, FAK downstream pathways can

cause a decrease in local myosin contractility. For example, FAK stimulates adhesion

disassembly through a signaling pathway that includes extracellular signal-regulated

6

Figure 3. Model of asymmetric adhesion dynamics. Nascent adhesions formed at the front either undergo turnover, which is predominantly controlled by kinase signaling, or mature in response to contractile forces. Mature adhesions can be transformed into fibrillar adhesions. Trailing adhesions arise as a result of fusion of additional nascent adhesions and remaining fibrillar adhesions. Once formed, trailing adhesions slide because of tension from attached stress fibers and either eventually disassemble or detach in the form of membrane ‘footprints’. Reprinted from (Broussard, Webb et al. 2008).

Cell Front

7

kinase (ERK) and myosin light chain kinase (MLCK) (Webb, Donais et al. 2004). FAK

can also facilitate adhesion disassembly through phosphorylation of Src and

p190RhoGAP, which decreases the activity of Rho and Rho kinase (ROCK) and recruits

a complex containing a Rac and Cdc42 GEF, PIX, and an effector, p21-activated kinase

(PAK), to adhesions (Schober, Raghavan et al. 2007). Thus, FAK downregulates

myosin-generated tension at the leading edge either by influencing MLCK or by tilting

the Rho/Rac antagonistic interplay toward Rho inhibition and Rac activation. These

findings are in agreement with studies implicating myosin contractility in nascent

adhesion formation (Rottner, Hall et al. 1999; Gupton and Waterman-Storer 2006).

Mechanistically, the regulation of adhesion turnover modulates the relative rates

of association versus dissociation of molecules at adhesion sites. Downstream targets of

Src and/or FAK kinase activity often regulate the recruitment of protein complexes to

adhesions. The phosphorylation of paxillin kinase linker (PKL) promotes the binding of

PKL to the adaptor paxillin (Brown, Cary et al. 2005). Paxillin is present early in adhesion

formation and can stimulate the formation of adhesions. However, paxillin likely induces

adhesion turnover depending on its phosphorylation state (Nayal, Webb et al. 2006;

Zaidel-Bar, Milo et al. 2007). PKL and another family member, GIT1, are part of a larger

protein complex containing PIX and PAK that are targeted to forming adhesions through

their interaction with paxillin (Brown, Cary et al. 2005; Nayal, Webb et al. 2006).

Besides phosphorylation-dependent regulation, it is likely that proteolysis of

adhesion components can also contribute to the turnover process. Talin proteolysis by

calpain has been shown to stimulate the dissociation of several major adhesion

components, including paxillin, vinculin, and zyxin (Franco, Rodgers et al. 2004). In this

case, FAK signaling to talin is able to stimulate adhesion growth. FAK promotes talin

association with the type I phosphatidylinositol phosphate kinase isoform-g661

8

(PIPKIg661), which ultimately results in the local generation of PI(4,5)P2 leading to the

recruitment of additional proteins to adhesions (Ling, Doughman et al. 2002). Calpain

serves to resist this unusual FAK activity and stimulates adhesion turnover.

Tyrosine phosphatases also play a role in determining whether adhesions turn

over or mature. For example, protein tyrosine phosphatase 1B (PTP1B) is principally

associated with the cytosolic face of the endoplasmic reticulum (ER) and travels to sites

of newly forming adhesions through projections of the ER, which are dependent on

microtubules (Hernandez, Sala et al. 2006). The maturation of these adhesions is

inhibited in PTP1B deficient cells, probably because of a loss in the activation of PTP1B

substrates, such as Src (Hernandez, Sala et al. 2006).

Coupling of adhesions with the contractile actin cytoskeleton: Initiation of

adhesion formation is closely coupled with actin assembly in protruding regions of cells

and requires activation of the same Rac or Cdc42 regulatory pathways as Arp2/3-

dependent actin polymerization. The Arp2/3 complex is recruited to adhesion sites

during initial stages of assembly through a transient interaction with vinculin in a PI3K

and Rac1 dependent manner (DeMali, Barlow et al. 2002). Additionally, efficient

adhesion assembly requires the actin-binding protein, cortactin, which could affect

adhesion assembly by interacting with the Arp2/3 complex (Bryce, Clark et al. 2005).

Adhesions that do not turn over at the leading edge mature by increasing in size

and molecular composition. A major trigger of adhesion maturation is an increase in

tensile force through interactions with the actin cytoskeleton. Accordingly, proteins that

produce force by causing contraction, such as both myosin II isoforms (A and B), are

required for adhesion maturation (Vicente-Manzanares, Zareno et al. 2007). In

persistently migrating cells, many mature adhesions decrease in size or totally

9

disassemble in the perinuclear region (Rid, Schiefermeier et al. 2005). An important part

of adhesion disassembly is likely driven by reversing maturation signaling, such as a

reduction in tensile force because of myosin or Rho inhibition. The interplay between the

exchange of adhesion molecules and tensile force applied to adhesions is thought to

play a decisive role in regulating cell migration rates (Gupton and Waterman-Storer

2006).

A number of recent findings connect tension with the molecular regulation of

adhesion assembly. For example, recent work has shown that directional tension on

integrin cytoplasmic tails triggers the and subunits to separate, which could promote

their activation and recruit cytoplasmic proteins (Zhu, Luo et al. 2008). Tensile force

directly applied to p130Cas changes its conformation allowing phosphorylation by Src

family kinases (Sawada, Tamada et al. 2006), which is proposed to stimulate tension-

induced adhesion growth (Geiger 2006). In addition, force induced unfolding of talin

stimulates binding of vinculin (del Rio, Perez-Jimenez et al. 2009). Another likely

mechanism that stimulates adhesion maturation is driven by protein tyrosine

phosphatase 2 (Shp2), which inactivates FAK by dephosphorylation (von Wichert,

Haimovich et al. 2003). Dephosphorylation prevents FAK from phosphorylating -actinin.

Dephosphorylated -actinin, which is able to bind actin, accumulates at adhesion sites

and establishes a link between integrin-based adhesions and the actin network. This link

brings tensional force into adhesions, which allows them to mature (von Wichert,

Haimovich et al. 2003). The extent of coupling between an adhesion and actin transmits

tensile force to the adhesion and is described as a ‘molecular clutch’ (Figure 4). Such

coupling plays a critical role in adhesion dynamics at all stages (Hu, Ji et al. 2007).

Cell protrusion is mediated by the lamellipodium, which is rich in cross-linked,

dendritic F-actin arrays facilitated primarily by the Arp2/3 complex and cofilin

10

Figure 4. Schematic of the molecular clutch. (a) At the leading edge, actin monomers (blue spheres) are added to the barbed end of the actin filament (arrow). Dark lines are integrin transmembrane proteins. (b) If the clutch is disengaged, as actin is assembled (blue spheres), the filament moves away from the leading edge due to the force generated by the polymerizing actin along with the action of myosin motors (yellow oval). This leads to retrograde actin flow. (c) If the clutch is engaged, there is now an indirect interaction between the actin filament and the ECM through FA molecules (red and green). This restrains the filament allowing new actin polymerization to propel the protrusion of the leading edge and the myosin-mediated forces to be transmitted through the FA into traction on the ECM. Reprinted from (Gardel, Schneider et al. 2010).

11

(Small 1988; Bailly, Macaluso et al. 1999; Ichetovkin, Grant et al. 2002; Pollard and

Borisy 2003). Monomers of actin are added to the barbed ends of actin filaments in the

lamellipodium in a direction facing the leading edge (Okabe and Hirokawa 1991;

Symons and Mitchison 1991). This generates a pushing force that drives membrane

protrusion in the direction of migration, while also pushing the actin network away from

the cell edge, a process is called retrograde actin flow (Heath 1983; Forscher and Smith

1988; Holifield, Ishihara et al. 1990; Lin and Forscher 1995; Pollard and Borisy 2003;

Ponti, Machacek et al. 2004). This retrograde actin flow is coupled to the ECM through

adhesions, which act as a molecular clutch (Mitchison and Kirschner 1988). This allows

for either the filament polymerization to cause protrusion of the leading edge membrane

or traction against the ECM to pull the cell body forward via myosin-driven pulling forces

(Figure 4c) (Harris 1973; Suter and Forscher 2000; Jurado, Haserick et al. 2005).

Disengagement of the molecular clutch corresponds to increased actin retrograde flow,

low traction forces and no leading edge protrusion (Figure 4b). However, high levels of

engagement lead to diminished retrograde flow, high force generation and leading edge

protrusion (Figure 4c).

To date, the best molecular candidates for this clutch are talin and vinculin

(Calderwood and Ginsberg 2003; Jiang, Giannone et al. 2003; Critchley 2004). Vinculin

binds to talin and actin, while talin binds both to actin and the cytoplasmic tail of integrins

(Kaufmann, Piekenbrock et al. 1991; Gilmore and Burridge 1996; Calderwood, Zent et al.

1999). The interaction between talin and vinculin is tension dependent, which suggests

that the strength of the interaction between FAs and actin may intensify under increased

mechanical tension through a vinculin/talin/actin interaction (del Rio, Perez-Jimenez et al.

2009). In addition, when talin is depleted, cells exhibit impaired FA formation, an

12

increase in actin retrograde flow and impaired traction capability (Zhang, Jiang et al.

2008).

Adhesions in the cell rear: Much less is known about the regulation of trailing

adhesions as compared to those at the front. It is clear that in persistently moving cells a

relatively small number of adhesions originating at the leading edge survive disassembly

and reach the cell rear (Rid, Schiefermeier et al. 2005). There, they increase in size and

fuse with additional focal adhesions formed at small protrusions around the trailing edge

as well as with new adhesions formed beneath stress fiber termini (Rid, Schiefermeier et

al. 2005). Resulting trailing adhesions serve to resist tensile stress induced at the

leading lamella and support cell spreading. For cell translocation to occur, they must

move along with the cell body or be released. Adhesion sliding can be accomplished by

movement of whole adhesions relative to the substrate along with rapid molecule

exchange at adhesion sites. Fluorescence speckle microscopy (FSM) analysis of

multiple adhesion proteins suggests that some are coupled with actin movement along

stress fibers (Hu, Ji et al. 2007), while other components close to the membrane, such

as integrins, must be exchanged for the adhesion to slide (Ballestrem, Hinz et al. 2001;

Hu, Ji et al. 2007).

Adhesion sliding at the trailing edge, although a common phenomenon, is not

conducive to rapid migration. In migrating cells, trailing adhesions are periodically

completely detached from the substrate (reviewed in (Kirfel, Rigort et al. 2004)).

Detachment can be accomplished by actomyosin stress fiber contraction, which requires

Rho activity (Peacock, Miller et al. 2007) and is likely driven by the MIIA myosin isoform

(Vicente-Manzanares, Zareno et al. 2007). Before detachment, adhesions must be

destabilized because a simple increase in tensional force results in a traumatic

detachment of adhesions from the cell body and a loss of a significant part of the

13

adhesion (Laukaitis, Webb et al. 2001; Peacock, Miller et al. 2007). Destabilization is

most likely triggered by mechanisms similar to those driving adhesion turn over at the

cell front. Sometimes, a combination of adhesion disassembly with cell retraction may

lead to cell detachment even though integrins are still engaged with ECM. In this case,

patches of membrane are left behind forming ‘footprints’ that have been implicated in

intercellular guidance (Palecek, Schmidt et al. 1996; Mayer, Maaser et al. 2004).

Molecular bonds connecting integrins with adhesions are broken at the level of the

‘molecular clutch’ (Hu, Ji et al. 2007) such that integrins, but not paxillin or -actinin, are

detached (Laukaitis, Webb et al. 2001). However, many observations argue that

adhesion destabilization at the rear of motile cells can be significant enough for complete

detachment of the trailing edge (Kirfel, Rigort et al. 2004; Rid, Schiefermeier et al. 2005).

Microtubule support of adhesion asymmetry: It is clear that the coordinated

asymmetry of adhesion disassembly as well as assembly is required for directional cell

migration. How is the polarized balance of the diverse mechanisms of adhesion

disassembly and assembly maintained in a motile cell? Microtubules play a key role in

the polarized distribution of signals within a cell and not surprisingly, have been

implicated in the asymmetric regulation of adhesion dynamics (Kaverina, Krylyshkina et

al. 1999). Multiple microtubule targeting of adhesions behind the leading edge or at the

cell rear leads to their disassembly (Kaverina, Krylyshkina et al. 1999; Rid,

Schiefermeier et al. 2005). A number of mechanisms have been proposed to explain this

phenomenon. First, microtubules could facilitate adhesion disassembly by local release

of tension. Indeed, the tyrosine kinase Arg, which requires microtubules for proper

localization and activity, can inhibit Rho and p190RhoGAP. Depletion of Arg results in

the inhibition of adhesion turnover at the front as well as disassembly at the rear

(Peacock, Miller et al. 2007). Additionally, microtubules could release tensile forces by

14

the uncoupling of adhesion proteins through calpain-driven proteolysis (Chan, Bennin et

al. 2010). In addition to these processes potentially destabilizing the adhesion structure,

microtubules can disassemble adhesion components by dynamin-driven endocytosis

(Ezratty, Partridge et al. 2005). Interestingly, such disassembly requires FAK activity,

suggesting that dynamin can disintegrate adhesions only in interplay with FAK-induced

destabilization (Ezratty, Partridge et al. 2005).

Finally, the ripping off of adhesions by cell edge retraction at the rear may also

be microtubule dependent since it requires myosin contractility. Contractility is strongly

regulated by the microtubule-associated Rho GEFs, H1 and Lfc (Glaven, Whitehead et

al. 1999; Krendel, Zenke et al. 2002). These GEFs reside on the microtubule lattice in an

inactive form where they can be released and cause local Rho activity upon microtubule

depolymerization (Zenke, Krendel et al. 2004). Importantly, the microtubule-regulated Lfc

activity was shown to be essential for cell migration in culture and for morphogenetic cell

movement in the developing Xenopus embryo (Kwan and Kirschner 2005).

Though microtubules have been traditionally described in the context of adhesion

disassembly, they can probably also stimulate adhesion formation and growth. For

example, since microtubule polymerization activates Rac (Waterman-Storer, Worthylake

et al. 1999), the persistently growing ‘pioneer’ microtubules at the cell front could induce

formation of nascent Rac-dependent adhesions (Waterman-Storer and Salmon 1997).

This function could be accomplished by the activity of Rac and Cdc42 exchange factors,

ASEFs, which depend on the microtubule plus tip protein APC (Kawasaki, Senda et al.

2000; Kawasaki, Sagara et al. 2007). The well established microtubule role in the

delivery of vesicular carriers to the cell front can be implicated in the delivery of adhesion

components or in the exportation of ECM proteins, which prepares a proper adhesive

substrate for a moving cell.

15

The ability of microtubules to differentially regulate adhesion dynamics at the

front and the rear of a motile cell can be explained by the general functional and

structural asymmetry of the microtubule network. Although microtubules in an interphase

cell radiate from the cell center to the periphery, their distribution is far from a perfect

radial symmetry. Indeed, microtubule dynamics in the front half of the cell body are

characterized by long-lived detyrosinated and/or acetylated microtubule arrays

(Gundersen and Bulinski 1988), which could serve as preferential tracks for kinesin-

driven transport (Kreitzer, Liao et al. 1999). Diverse plus-end capturing molecular

clusters (reviewed in (Akhmanova and Hoogenraad 2005; Watanabe, Noritake et al.

2005)) are located behind the leading edge, in areas of adhesion turnover and

disassembly and serve as a basis for rapid microtubule rescues and extensive targeting

of adhesions by microtubules without long depolymerization phases (Rid, Schiefermeier

et al. 2005). Finally, the extending protrusions, where adhesions are formed, are

associated with Rac-dependent persistent microtubule growth because of local stathmin

inhibition (Wittmann, Bokoch et al. 2003; Wittmann, Bokoch et al. 2004).

In contrast to the front, microtubules at the rear are short-lived and undergo

frequent Rho-dependent catastrophes of an unclear mechanistic nature (Wadsworth

1999). Additionally, the front-oriented microtubule array is expanded by a non-

centrosomal population of Golgi-derived microtubules (Efimov, Kharitonov et al. 2007).

This population depends on a microtubule-stabilizing factor, CLASP, and may bear

specific properties and/ or functions (Efimov, Kharitonov et al. 2007). The asymmetric

network of described dynamic properties can serve in the initiation of adhesions in

protrusive regions of the cell, because of Rac activation and integrin delivery. This

network can also function in adhesion turnover as well as disassembly behind the

leading edge and in the cell center (e.g. because of Arg delivery and subsequent local

16

relaxation) and in adhesion destabilization and detachment at the rear by local Arg-

dependent relaxation succeeded by contraction because of GEF release from

depolymerizing dynamic microtubules (Figure 3).

Akt and cell migration: Akt is a serine/threonine kinase that was originally

identified as an oncogene within the mouse leukemia virus AKT8 (Bellacosa, Testa et al.

1991). Akt is activated downstream of PI3K signaling (Franke, Yang et al. 1995).

Activation of PI3K generates the second messenger phosphatidylinositol (3,4,5)-

trisphosphate (PIP3) from phosphatidylinositol (4,5)-bisphosphate (PIP2). Akt is

recruited to the membrane via an interaction between its PH domain and PIP3, and it is

then activated by phosphorylation by kinases such as phosphoinositide-dependent

kinase 1 (PKD1) (Kim, Kim et al. 2001). Upon activation, Akt phosphorylates proteins

and regulates various cellular processes, including cell growth, survival, and proliferation

(Toker and Yoeli-Lerner 2006).

Akt is well known for its role in modulating cell growth and survival, but more

recently, there has been a growing interest in the function of Akt in regulating cell

migration. Moreover, Akt mutation, usually leading to hyperactivation, is commonly

reported in human cancers including gastric, breast and pancreatic cancer (Suzuki,

Freije et al. 1998; Perez-Tenorio and Stal 2002; Schlieman, Fahy et al. 2003; Panigrahi,

Pinder et al. 2004; Hennessy, Smith et al. 2005). The three isoforms of Akt (Akt1, Akt2,

and Akt3) appear to play differing roles in regulating cell migration. Akt1 stimulates the

migration of epithelial cells, fibroblasts, and fibrosarcomas, and promotes the invasion of

breast carcinomas and fibrosarcomas (Kim, Kim et al. 2001; Irie, Pearline et al. 2005;

Zhou, Tucker et al. 2006; Ju, Katiyar et al. 2007). These and other properties of Akt1

point to a central role for this molecule in tumor cell progression and indeed, in mice,

loss of Akt1 delayed cancer development (Maroulakou, Oemler et al. 2007). In addition,

17

Akt1 is one of the most frequently activated kinases in a number of human cancers

(Vivanco and Sawyers 2002). The function of Akt2 in regulating cell migration is not as

clear. Akt2 suppresses the migration of fibroblasts, but promotes motility in a number of

breast and ovarian cancer cells (Arboleda, Lyons et al. 2003; Irie, Pearline et al. 2005;

Yoeli-Lerner, Yiu et al. 2005; Zhou, Tucker et al. 2006). The effects of Akt3 on cell

migration are presently not known.

Akt regulates the phosphorylation of PAK on serine 21 (Zhou, Zhuo et al. 2003).

PAK is recruited to adhesions in a complex containing a Rac and Cdc42 GEF, PIX

(Schober, Raghavan et al. 2007). This phosphorylation event modulates the binding of

the adaptor protein, Nck, to PAK, which regulates migration (Zhou, Zhuo et al. 2003).

Nck is important in recruiting paxillin kinase linker (PKL) to adhesions, where it regulates

cell spreading and motility (Brown, Cary et al. 2005). In addition, PAK induces

phosphorylation of serine 273 in paxillin, regulating adhesion turnover (Nayal, Webb et al.

2006). This phosphorylation event increases cell migration, protrusion, and adhesion

turnover by increasing paxillin-GIT1 binding and promoting the localization of the GIT1-

PIX-PAK complex to the leading edge (Nayal, Webb et al. 2006).

Akt also plays an important role in metastasis through its ability to regulate

secretion of matrix metalloproteinases (MMPs). MMPs are responsible for degrading the

ECM and allowing tumor cells to invade through tissue (Kim, Kim et al. 2001). Akt has

been shown to regulate the secretion of MMP-9, which promotes migration and invasion

(Kim, Kim et al. 2001).

Akt activation requires multiple phosphorylation events including serine 473 and

threonine 308 (Fayard, Tintignac et al. 2005). However, tyrosine phosphorylation of Akt

by the non-receptor tyrosine kinase Src has also recently been shown to be important in

18

both the activation of Akt as well as its biological function (Chen, Kim et al. 2001;

Choudhury, Mahimainathan et al. 2006; Bouchard, Demers et al. 2007).

Src and migration: Src was the first identified oncogene and tyrosine kinase

(Martin 2001; Courtneidge 2002). Src regulates many normal cellular processes

including: cell proliferation and survival, control of cell shape, cytoskeletal regulation,

adhesions dynamics and cell migration (Thomas and Brugge 1997; Yeatman 2004). Src

has many important functions in regulating adhesion dynamics and cell migration. The

cytoplasmic tail of some integrins can interact with Src and activate it, which leads

ultimately to the activation of Rac and local actin-based protrusion (Huveneers and

Danen 2009). In addition, FAK is able to recruit Src and Src substrates to sites of

integrin engagement, and the FAK-Src complex coordinates the assembly and

disassembly of leading edge adhesions as well as disruption of adhesions in the cell rear

(Parsons 2003; McLean, Carragher et al. 2005; Berrier and Yamada 2007). Moreover,

paxillin in adhesions binds to FAK and is subsequently phosphorylated by Src, and

paxillin can recruit the PAK-PIX-GIT/PKL complex to adhesions, which is important for

activating Rac and PAK as well as promoting paxillin dissociation, which promotes their

turnover (Zhao, Manser et al. 2000; Parsons 2003; Hoefen and Berk 2006). Src mainly

behaves as a negative regulator of adhesions, as cells deficient in Src show strong

adhesions, reduced adhesion turnover and impaired migration (Horwitz and Parsons

1999; Yeatman 2004).

Moreover, Src deregulation (usually overexpression or hyperactivation) is one of

the major oncogenic signatures in a large number of human cancers including: breast,

colorectal, pancreas, prostate, lung, head and neck, melanoma, glioma and different

types of sarcomas (Summy and Gallick 2003; Bild, Yao et al. 2006; Shor, Keschman et

al. 2007). Tumor cells must be able to degrade ECM and migrate through tissues to be

19

invasive, and this function is promoted by invadopodia, which are actin-rich membrane

protrusions with components similar to FAs (Gimona, Buccione et al. 2008; Buccione,

Caldieri et al. 2009). Invadopodia accomplish this matrix degradation through the

recruitment of transmembrane type I MMP (MT1-MMP), which induces localized

activation of secreted MMP-2 and MMP-9 (Seiki 2003; Steffen, Le Dez et al. 2008). Src

is a potent inducer of invadopodia formation, as well as a key regulator of invadopodia

function (Myoui, Nishimura et al. 2003; Artym, Zhang et al. 2006; Rucci, Recchia et al.

2006; Oser, Yamaguchi et al. 2009; Hu, Mukhopadhyay et al. 2011). Src is also known

to promote the expression of MMPs as well as inhibit their endocytosis (Hsia, Mitra et al.

2003; Rivat, Le Floch et al. 2003; Wu, Gan et al. 2005; Meng, Jin et al. 2009; Van

Slambrouck, Jenkins et al. 2009; Hu, Mukhopadhyay et al. 2011). Interestingly, recent

work has shown a novel function for FAs, in tumor cells, in degrading ECM, which is

dependent on Src, FAK, p130-Cas and MT1-MMP (Wang and McNiven 2012). This

study further blurs the line between FAs and invadopodia.

The adaptor protein APPL1: Even though kinases and phosphatases have

received much attention, adaptor molecules are emerging as important regulators of cell

migration. These molecules function as integrators of signaling pathways by bringing

together and targeting protein-binding partners to specific locations within cells. The

adaptor protein containing a PH domain, PTB domain, and leucine zipper motif 1

(APPL1) was originally identified through its association with Akt (Mitsuuchi, Johnson et

al. 1999) and was subsequently shown to interact with transmembrane receptors of

different classes, including the tumor suppressor DCC (deleted in colorectal cancer), and

to regulate DCC-mediated signals impacting cell survival (Liu, Yao et al. 2002). In

addition, APPL1 also interacts with the small GTPases Rab5 and Rab21 and with the

Rab effector Oculocerebrorenal Syndrome of Lowe (OCRL), which is an inositol 5-

20

phosphatase linked to Lowe syndrome (Miaczynska, Christoforidis et al. 2004; Erdmann,

Mao et al. 2007; Zhu, Chen et al. 2007). Rab5 has previously been shown to be

important in regulating actin dynamics, in some cases including the formation

lamellipodia (Spaargaren and Bos 1999). Recent work has suggested that this effect on

actin can be mediated through the regulation of the small GTPase Rac, where endocytic

trafficking of Rac is necessary for the spatial restriction of Rac signaling (Palamidessi,

Frittoli et al. 2008). This is accomplished by the RacGEF, Tiam1, which is recruited to

Rab5 early endosomes (Palamidessi, Frittoli et al. 2008). Finally, APPL1 has recently

been shown to decrease the activation of Akt on phagosomes in a process that is

dependent on OCRL (Bohdanowicz, Balkin et al. 2012).

The ability of APPL1 to interact with various signaling and trafficking proteins

puts it in an ideal position to modulate signaling pathways that regulate cellular behavior.

Indeed, APPL1 has been linked to cell survival and proliferation (Miaczynska,

Christoforidis et al. 2004; Schenck, Goto-Silva et al. 2008). In zebrafish, APPL1 has

been shown to be required for cell survival during normal development, as knockdown of

APPL1 using morpholinos leads to widespread apoptosis in a process dependent on Akt

(Schenck, Goto-Silva et al. 2008). Conversely, knockout of APPL1 in mouse showed no

gross effects on embryonic development or on Akt signaling in T-cells (Tan, You et al.

2010; Tan, You et al. 2010). Interestingly, thus far, the only observed phenotype in the

APPL1 knockout mice is an effect on growth-factor induced survival and migration of

MEFs, which is dependent on APPL1 regulation of Akt signaling (Tan, You et al. 2010).

However, presently, the function of APPL1 in cell migration is poorly understood.

APPL1 mediates its function through a series of domains, including an N-terminal

Bin-Amphiphysin-Rvs (BAR), a central Pleckstrin homology (PH), and a C-terminal

phospho-tyrosine binding domain (PTB) (Mitsuuchi, Johnson et al. 1999; Miaczynska,

Christoforidis et al. 2004). Both the BAR and PH domains are involved in binding to cell

21

membranes. The BAR domain is a dimerization motif associated with the sensing and/or

induction of membrane curvature while the PH domain binds to phosphoinositol lipids

(Varnai, Lin et al. 2002; Habermann 2004). In APPL1, the BAR and PH domains are

thought to act together as a functional unit forming an integrated, crescent-shaped,

symmetrical dimer that mediates membrane interactions (Li, Mao et al. 2007; Zhu, Chen

et al. 2007). The C-terminal PTB domain of APPL1 has been shown to be critical in

APPL1’s ability to bind Akt (Mitsuuchi, Johnson et al. 1999; Schenck, Goto-Silva et al.

2008).

Hypothesis: Cell migration is a highly complex process, which consists of

membrane protrusion, adhesion, cell body translocation and rear retraction. Adaptor

proteins are emerging as critical regulators of multiple aspects of the migration process.

The adaptor protein APPL1 has been shown to be an important regulator of the

serine/threonine kinase Akt, being able to regulate both its activation and function. Akt is

a known regulator of cell migration, and we therefore hypothesized that APPL1 can

regulate cell migration in a process involving modulation of Akt activity and function.

22

Chapter II

IDENTIFICATION OF PHOSPHORYLATION SITES WITHIN THE SIGNALING ADAPTOR APPL1 BY MASS SPECTROMETRY

Joshua A. Broussard,*2,3 Randi L. Gant-Branum,*1,3,4 Ablatt Mahsut,1,3,4 Donna J. Webb,2,3 and John A. McLean1,3,4

1Department of Chemistry, 2Department of Biological Sciences, 3Vanderbilt Institute for Chemical Biology (VICB), 4Vanderbilt Institute for Integrative Biosystems Research and Education (VIBRE), Vanderbilt University, Nashville TN 37215 *These authors have contributed to an equal extent. J.A.B. prepared all biological samples and contributed to Figures 5-6 and Tables 3-5. A.M. generated Figure 7. R.L.G-B. conducted the M.S. experiments and contributed to Figures 6, 8 and 9 as well as Tables 1 and 2. Both J.A.B and R.L.G-B. contributed equally to the preparation of the manuscript. This article has been published under the same title in Journal of Proteome Research. 2010 March 5; 9(3): 1541–1548.

23

Abstract

APPL1 is a membrane-associated adaptor protein implicated in various cellular

processes, including apoptosis, proliferation, and survival. Although there is increasing

interest in the biological roles as well as the protein and membrane interactions of

APPL1, a comprehensive phosphorylation profile has not been generated. In this study,

we use mass spectrometry (MS) to identify 13 phosphorylated residues within APPL1.

By using multiple proteases (trypsin, chymotrypsin, and Glu C) and replicate

experiments of linear ion trap (LTQ) MS and LTQ-Orbitrap-MS, a combined sequence

coverage of 99.6% is achieved. Four of the identified sites are located in important

functional domains, suggesting a potential role in regulating APPL1. One of these sites

is within the BAR domain, two cluster near the edge of the PH domain, and one is

located within the PTB domain. These phosphorylation sites may control APPL1 function

by regulating the ability of APPL1 domains to interact with other proteins and

membranes.

Introduction

Adaptor protein containing a PH domain, PTB domain and Leucine zipper motif 1

(APPL1) is a 709 amino acid membrane associated protein that has been reported to

play a key role in the regulation of apoptosis, cell proliferation, cell survival, and vesicular

trafficking (Mitsuuchi, Johnson et al. 1999; Deepa and Dong 2009). APPL1 is widely

expressed and found in high levels in the heart, brain, ovary, pancreas, and skeletal

muscle (Mitsuuchi, Johnson et al. 1999). Although a significant amount of interest has

been generated in the interactions and function of APPL1, the complete phosphorylation

profile of this protein has not been described. To date, phosphorylation of three residues,

threonine 399, and serines 401 and 691, which were identified from global profiling

studies (Ballif, Villen et al. 2004; Beausoleil, Jedrychowski et al. 2004; Matsuoka, Ballif

24

et al. 2007; Sugiyama, Masuda et al. 2007; Dephoure, Zhou et al. 2008) are reported in

protein databases, including Phosphosite, Proteinpedia/Human Protein Reference

Database, and Expasy-SwissProt.

APPL1 mediates its function through a series of domains, including an N-terminal

Bin–Amphiphysin–Rvs (BAR), a central Pleckstrin homology (PH), and a C-terminal

phospho-tyrosine binding domain (PTB) (Mitsuuchi, Johnson et al. 1999; Miaczynska,

Christoforidis et al. 2004). Both the BAR and PH domains are involved in binding to cell

membranes. The BAR domain is a dimerization motif associated with the sensing and/or

induction of membrane curvature while the PH domain binds to phosphoinositol lipids

(Varnai, Lin et al. 2002; Habermann 2004). The BAR domain has also been shown to be

critical in the ability of APPL1 to localize to endosomal structures (Schenck, Goto-Silva

et al. 2008). In APPL1, the BAR and PH domains are thought to act together as a

functional unit forming an integrated, crescent-shaped, symmetrical dimer that mediates

membrane interactions (Li, Mao et al. 2007; Zhu, Chen et al. 2007). Moreover, the BAR

and PH domains function together to create the binding sites for Rab5, which is a small

GTPase involved in endosomal trafficking (Zerial and McBride 2001; Zhu, Chen et al.

2007). The C-terminal PTB domain of APPL1 has been shown to be critical in the ability

of APPL1 to bind to several signaling molecules, including the serine/threonine kinase

Akt, the neurotrophin receptor TrkA, the adiponectin receptors AdipoR1 and AdipoR2,

Human Follicle-Stimulating Hormone (FSHR), and the tumor suppressor DCC (deleted

in colorectal cancer) (Mitsuuchi, Johnson et al. 1999; Liu, Yao et al. 2002; Nechamen,

Thomas et al. 2004; Lin, Quevedo et al. 2006; Mao, Kikani et al. 2006).

In this study, phosphorylation sites were identified on APPL1 using contemporary

MS-based methods, namely by liquid chromatography (LC)-coupled to data-dependent

tandem MS on both an LTQMS and LTQ-Orbitrap-MS. The bioinformatics algorithm

SEQUEST was used to process the MS/MS data obtained in these phosphorylation

25

mapping experiments. However, spectral assignments required manual validation of all

identified phosphorylation site spectra. To obtain near-complete coverage of APPL1,

multiple proteases were used in parallel phosphorylation site mapping experiments.

Proteolysis digestion with Glu C, trypsin, and chymotrypsin yielded sequence coverages

of 44.6%, 88.3%, 81.1%, respectively, with a combined sequence coverage of APPL1 of

greater than 99%. A total of 13 phosphorylation sites were detected and four of these

sites were found within APPL1 interacting domains, suggesting a potential regulatory

role in APPL1 function.

Materials and Methods

Reagents and Plasmids: FLAG M2-agarose affinity gel, FLAG peptide

(DYKDDDDK), and mouse IgG agarose were purchased from Sigma (St. Louis, MO).

Calyculin A was purchased from Calbiochem (San Diego, CA). Sodium vanadate was

obtained from Fischer Scientific (Fairlawn, NJ). Peroxovanadate was prepared as

previously described (Posner, Faure et al. 1994). FLAG-GFP plasmid was prepared by

inserting the FLAG epitope sequence into pcDNA3 (Invitrogen, Carlsbad, CA) and

cloning EGFP C1 (Clonetech) into the vector at KpnI and BamHI sites. Human APPL1

(accession number GI:124494248) was then cloned into the FLAG-GFP plasmid at

EcoRI and the insertion as well as orientation of APPL1 was confirmed by sequencing.

Protein Expression and Proteolytic Digestion: Human embryonic kidney 293

(HEK-293) cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM)

(Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Hyclone) and

penicillin/streptomycin (Invitrogen). HEK-293 cells were transfected with FLAG-GFP-

APPL1 (12 µg per 150 mm dish) using Lipofectamine 2000 (Invitrogen). After 36 h, cells

were incubated with 1 mM peroxovanadate and 50 nM calyculin A in DMEM with 10%

FBS for 30 minutes and extracted with 25 mM Tris, 100 mM NaCl, 0.1% NP-40 (pH 7.4).

26

The lysates were precleared twice with mouse IgG-agarose for 1 h at 4oC, and

immunoprecipitated with FLAG-agarose (Sigma, St. Louis, MO) for 2 h at 4oC. Samples

were washed three times with 25 mM Tris, 100 mM NaCl, pH 7.4 and FLAG tagged

APPL1 was eluted by incubation of the beads with 0.2 mg/ml FLAG peptide in 25 mM

Tris, pH 7.4 for 1 h at 4oC. Purified APPL1 protein was subjected to sodium dodecyl

sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie blue

staining. The concentration of APPL1 was quantified with a LI-COR Biosciences

ODYSSEY Infrared Imaging System using bovine serum albumin (BSA) as a standard.

For MS analyses, APPL1 was separated into three equal aliquots and

proteolytically digested by trypsin, chymotrypsin, and Glu C proteases, respectively.

Briefly, proteolysis was performed by taking 2.6 μg of APPL1 (20 μl) and diluting to 25 μl

with 25 mM ammonium bicarbonate. Cysteine sulfhydryl groups were reduced by the

addition of 1.5 μl of 45 mM dithiothreitol (DTT) for 30 min at 55°C followed by alkylation

with 2.5 μl of 100 mM iodoacetamide for 30 min at room temperature in the dark.

Digestion was performed using 100 ng (1:40 enzyme: substrate, wt:wt) of trypsin gold

(Promega, Madison, WI), chymotrypsin (Princeton Separations, Freehold, NJ), or

endoproteinase Glu C (Calbiochem EMD Biosciences, Gibbstown, NJ) at 37°C for 16, 4,

or 6 h respectively. Proteolysis was quenched by adding 1 µl of 88% formic acid.

Subsequently, the digest was lyophilized and then reconstituted in 25 µl of 0.1% formic

acid.

Western Blot Analysis: Purified APPL1 protein was subjected to SDS-PAGE,

and then transferred to a nitrocellulose membrane. The membrane was incubated with

primary antibody against GFP (Invitrogen) or 4G10 (a kind gift from Steve Hanks,

Vanderbilt University) at a dilution of 1 µg/ml. The membrane was then incubated with

IRDye 800 Conjugated Affinity Purified anti-Rabbit IgG or anti-Mouse IgG (Rockland) at

27

a dilution of 0.1 µg/ml, and visualized using a LI-COR Biosciences ODYSSEY Infrared

Imaging System.

Linear Ion Trap and LTQ-Orbitrap MS: LC-MS/MS analyses of APPL1 digests

were performed using a linear ion trap mass spectrometer (LTQ, Thermo Electron, San

Jose, CA) equipped with an autosampler (MicroAS, Thermo) and an HPLC pump

(Surveyor, Thermo), and Xcalibur 2.0 SR2 instrument control. Ionization was performed

by using nanospray in the positive ion mode. Spectra were obtained by using data-

dependent scanning tandem mass spectrometry in which one full MS scan, using a

mass range of 400–2000 amu, was followed by up to 5 MS/MS scans of the most

intense peaks at each time point in the HPLC separation. Incorporated into the method

was data-dependent scanning for the neutral loss of phosphoric acid or phosphate (−98

m/z, −80 m/z), for which MS3 was performed. Dynamic exclusion was enabled to

minimize redundant spectral acquisitions. High resolution data was collected using a

similar strategy on a LTQ-Orbitrap mass spectrometer with the exception that the full

MS3 scan was performed in the Orbitrap at 30,000 resolution, rather than at unit mass

resolution on the LTQMS. Further instrumental details are available in the Electronic

Supplementary Material.

Bioinformatic Analysis: Tandem MS/MS spectra acquired in LTQMS and LTQ-

Orbitrap-MS experiments were identified using SEQUEST (University of Washington).

MS/MS spectra were extracted from the raw data files into .dta format with spectra

containing fewer than 25 peaks being excluded. Files labeled as singly charged were

created if 90% of the total ion current occurred below the precursor ion, and all other

spectra were processed as both doubly- and triply-charged ions. Proteins were identified

using the TurboSEQUEST version 27 (rev. 12) algorithm (Thermo Electron) and the IPI

Human database version 3.33 (67837) sequences. Search parameters are outlined in

28

the Electronic Supporting Information. Manual verification was performed on all

phosphorylation assignments having an Xcorr value above 1, 2, and 2.5 for charges +1,

+2, and +3, respectively. Validation was performed as previously described.(Nichols and

White 2009) All spectra are included in the Electronic Supporting Information according

to MIAPE standards (Taylor, Paton et al. 2007).

Results and Discussion

Comprehensive Phosphorylation Map of Human APPL1: In this study, a

comprehensive phosphorylation profile of APPL1 is described for the first time. To

accomplish this, FLAG-GFP-APPL1 was expressed in HEK-293 cells and subsequently

immunoprecipitated for MS analysis according to the purification scheme outlined in

Figure 5A. A major band corresponding to the molecular mass of APPL1 was observed

when the immunoprecipitate was subjected to SDS-PAGE and stained with Coomassie

blue (Figure 5B). The band was confirmed to be APPL1 by Western blot analysis

(Figure 5C). Before subjecting APPL1 to MS analysis, we examined the phosphorylation

state of this protein using 4G10 phospho-tyrosine antibody. APPL1 was phosphorylated

on tyrosine residues as determined by Western blot analysis with 4G10 (Figure 5C).

Several other minor bands were detected in the immunoprecipated samples, which

could correspond to endogenous APPL1 or APPL1 binding proteins. However,

insufficient peptide signal from MS analyses precluded positive protein identification of

these additional minor bands.

At least 13 (as discussed below) phosphorylation sites with 99.6% total amino

acid sequence coverage were identified using multiple proteases, including trypsin,

chymotrypsin, and Glu C, followed by LC-MS analyses using both an LTQMS instrument

and an LTQ-Orbitrap instrument. Of these reported phosphorylation sites, three could

29

Figure 5. APPL1 protein purification. (A) Schematic showing the generalized protocol used for purifying FLAG-tagged proteins. (B) SDS-PAGE gel of immunoprecipiated FLAG-GFP-APPL1 stained with Coomassie blue. Arrow points to purified FLAG-GFP-APPL1. (C) Western blot with GFP-specific antibody (left panel) or phospho-tyrosine antibody (right panel). Left panel shows the purified protein is FLAG-GFP-APPL1 (IB: GFP) and right panel shows that APPL1 is phosphorylated on tyrosine residues (IB: 4G10).

30

not be located to a single amino acid (i.e. phosphorylation was determined to exist within

a range of potential sites within a peptide).

Table 1 shows each confirmed phosphorylation site assignment by sequence

position using the LTQMS instrument. In total, ten phosphorylation sites were identified

by combining the data obtained for trypsin, chymotrypsin, and Glu C digests to obtain a

sequence coverage of 95.3%. Of these ten sites, only two could not be located to a

specific residue, i.e. phosphorylation was confirmed to exist between amino acids 97-98

(SS) and 401-403 (SPS). Table 2 shows the confirmed phosphorylation sites using the

LTQ-Orbitrap instrument. By combining the data obtained for Glu C, trypsin, and

chymotrypsin digests, nine phosphorylation sites were identified with a sequence

coverage of 99.6%. Several of these phosphorylation sites were detected in multiple

peptides derived from proteolytic miscleavages corresponding to the same site of

phosphorylation. Of these nine sites, two could not be located to a specific residue, but

were confirmed to exist between amino acids 401-403 (SPS) and 689-691 (SSS). A

comparison of the phosphorylation sites identified using the LTQMS and LTQ-Orbitrap

yielded four unique sites by the former and three unique sites by the later. We detected

five phosphorylation sites, including serines 401/403, 459, 691, 693, and 696 by both

methods. Interestingly, most of the phosphorylation sites we detected in human APPL1

are conserved in rat and mouse APPL1 (Table 3), raising the possibility that these sites

serve a functional role.

Two of the previously identified phosphorylation sites in APPL1, 401S and 691S,

were detected in our analysis while one additional site, 399T, was not definitively

31

Table 1. Phosphorylation Sites within APPL1 Identified by LTQMS

Peptidesa

Sequence

Positionb

Proteasec

[M+H]+

(m/z)

92 VIDELSSCHAVLSTQLADAMMFPITQFK

119 ‡ Trp 3175.53

376 QIpYLSENPEETAAR

389 378Y Trp 1700.75

390 VNQSALEAVTPSPSFQQR

407 ‡ Trp 2038.99

456 DIIpSPVC*EDQPGQAKAF

472 459S Chymo 1954.93

479 TNPFGESGGSTKpSETEDSILHQLFIVR

505 491S Trp 3029.46

595 SESNLSSVCpYIFESNNEGEK

614 604Y Trp 2315.94

669 LIAASSRPNQASSEGQFVVLpSSSQSEESDLGEGGK

703 689S Trp 3631.71

683 GQFVVLSSpSQSEESDLGEGGKKRE

706 691S Glu C 2633.24

683 GQFVVLSSSQpSEEpSDLGEGGKKRE

706

693S,

696S Glu C 2713.24

a. "p" denotes phosphorylation, asterisk, “*” denotes carboxyamidomethylation. b. “‡” denotes sequence regions where single residue is known to be phosphorylated between the residues underlined. Phosphorylation on specific residue on those regions cannot be confirmed. c. represents digestion by multiple proteases. Trp, Chymo and Glu C correspond to the proteases, trypsin, chymotrypsin, and Glu C, respectively.

32

Table 2. Phosphorylation Sites Identified within APPL1 by LTQ-Orbitrap-MS

Peptidea

Seq

uen

ce

Po

sit

ion

b

Pro

tea

se

c

[M+

H]+

(m/z

)

Mass

err

or

(pp

m)

367 IC*TINNIpSKQIYLSENPEETAARVNQSAL

395 374S Chymo 3356.66 3.30

390 VNQSALEAVTPSPSFQQR

407 ‡ Trp 2038.96 -2.45

415 AGQSRPPTARTSpSSGSLGSESTNL

438 427S Chymo 2428.11 -0.62

418 SRPPTARTSpSSGpSLGSESTNL

438

427S,

430S Chymo 2251.96 0.93

418 SRPPTARTSpSSGSLGSESTNL

438 427S Chymo 2171.99 1.10

451 TPIQFDIIpSPVC*EDQPGQAKAF

472 459S Chymo 2541.17 0.08

456 DIIpSPVC*EDQPGQAKAF

472 459S Chymo 1954.91 -1.64

457 IIpSPVC*EDQPGQAKAF

472 459S Chymo 1954.86 0.33

669 LIAASSRPNQASSEGQFVVLSSSQSEESDLGEGGK

703 ‡ Trp 3631.68 -3.71

683 GQFVVLSSpSQSEESDLGEGGKKRE

706 691S Glu C 2633.21 -0.46

683 GQFVVLSSSQpSEESDLGEGGKKRESE

708 693S Glu C 2849.28 5.58

683 GQFVVLSSSQpSEESDLGEGGKKRE

706 693S Glu C 2633.21 -0.57

686 VVLSSpSQSEESDLGEGGKKRE

706 691S Glu C 2301.06 0.13

686 VVLSSSQpSEEpSDLGEGGKKRE

706

693S,

696S Glu C 2381.03 -1.89

a."p" denotes phosphorylation, asterisk, “*” denotes carboxyamidomethylation. b. “‡” denotes sequence regions where single residue is known to be phosphorylated between the residues underlined. Phosphorylation on specific residue cannot be confirmed. c. represents digestion by multiple proteases. Trp, Chymo and Glu C correspond to the proteases, trypsin, chymotrypsin, and Glu C, respectively.

33

Table 3. Comparison of Peptide Sequence Surrounding Identified Phosphorylation Sites in APPL1

Site Position Homologues Peptide Sequence

‡ 97/98S 92-119 Human VIDELSSCHAVLSTQLADAMMFPITQFK

Rat VIDELSSCHAVLSTQLADAMMFPISQFK

Mouse ----------------------------

374S, 378Y 367-395 Human ICTINNISKQIYLSENPEETAARVNQSAL

Rat ICTINNISKQIYLSENPEETAARVNQSAL

Mouse ICTINNISKQIYLSENPEETAARVNQSAL

‡ 401/403S 390-407 Human VNQSALEAVTPSPSFQQR

Rat VNQSALEAVTPSPSFQQR

Mouse VNQSALEAVTPSPSFQQR

427S, 430S 418-438 Human SRPPTARTSSSGSLGSESTNL

Rat SRPPTARTSSSGSLGSESTNL

Mouse SRPPTARTSSSGSLGSESTNL

459S 451-472 Human TPIQFDIISPVCEDQPGQAKAF

Rat TPIQFDIISPVCEDQPGQAKAF

Mouse TPIQFDIISPVCEDQPGQAKAF

491S 479-505 Human TNPFGESGGSTKSETEDSILHQLFIVR

Rat TNPFGESGGSTKSETEDSILHQLFIVR

Mouse TNPFGESGGSTKSETEDSILHQLFIVR

604Y 595-614 Human SESNLSSVCYIFESNNEGEK

Rat SESNLSSVCYIFESNNEGEK

Mouse SESNLSSVCYIFESNNEGEK

689S, 691S, 693S, 696S

669-703 Human LIAASSRPNQASSEGQFVVLSSSQSEESDLGEGGK

Rat LIAASSRPSQSGSEGQ-LVLSSSQSEESDLGEEGK

Mouse LIAASSRPNQAGSEGQ-LVLSSSQSEESDLGEEGK

‡ 689/690/691S

669-703 Human LIAASSRPNQASSEGQFVVLSSSQSEESDLGEGGK

Rat LIAASSRPSQSGSEGQ-LVLSSSQSEESDLGEEGK

Mouse LIAASSRPNQAGSEGQ-LVLSSSQSEESDLGEEGK

“‡” denotes sequence regions where single residue is known to be phosphorylated between the residues underlined. Phosphorylation on specific residue on those regions cannot be confirmed.

34

assigned. Phosphorylation of 401S was initially identified in epithelial carcinoma (HeLa)

cells as part of a large-scale characterization of nuclear phosphoproteins and in an

analysis of protein phosphorylation in developing mice brains (Ballif, Villen et al. 2004;

Beausoleil, Jedrychowski et al. 2004). This site was subsequently shown to be

phosphorylated in HeLa cells in two additional studies (Sugiyama, Masuda et al. 2007;

Dephoure, Zhou et al. 2008). Phosphorylation of 691S was detected in HEK-293 cells in

response to DNA damage using ionizing radiation.(Matsuoka, Ballif et al. 2007) We also

identified phosphorylation of this site in HEK 293 cells under physiological conditions.

Phosphorylation at 399T was identified in a global profiling study, but a positive

identification could not be definitively made in our experiments. Our spectra potentially

suggested phosphorylation at 399T, but in these spectra, this site was not the highest

confidence assignment. Furthermore, the previous study examined protein

phosphorylation during mitosis using HeLa cells arrested in the mitotic phase of the cell

cycle while our analysis was performed in HEK-293 cells under conditions in which they

were progressing through the cell cycle. Thus, it is possible that phosphorylation of this

site is transient if it is regulated by cell cycle progression and difficult to detect.

Phosphorylation Sites within APPL1 Functional Domains: The confirmed

phosphorylation sites obtained on both instruments are shown in Figure 6. Of the

confirmed sites, four were found in APPL1 interacting domains. Namely serines 97/98

were located in the BAR domain, raising the possibility that phosphorylation at these

sites could disrupt APPL1 dimerization as well as endosomal localization. Interestingly,

as shown in the crystal structure of the BAR and PH domains, serines 97/98 are located

on the concave surface of the BAR domain, which is thought to interact with membranes

(Figure 7) (Tanabe, Hosaka et al. ; Li, Mao et al. 2007). Therefore, phosphorylation at

this site could potentially regulate membrane interactions. Serine 374 and tyrosine 378

35

Figure 6. Map of APPL1 phosphorylation sites. (A) Phosphorylation sites identified in APPL1, using LTQMS and LTQ-Orbitrap MS. Underlined sites indicate that one phosphorylation is known to exist within the region. (B) A schematic of APPL1 is shown with identified phosphorylation sites relative to the position of APPL1 domains. Interacting regions within APPL1 for several proteins and receptors are also indicated.

36

Figure 7. Phosphorylation in the BAR and PH domains of APPL1. Crystal structure of the BAR and PH domains of APPL1 is shown. Phosphorylation site serine 97/98 and serine 374 are highlighted in red and their positions are indicated with arrows. Note that although tyrosine 378 neighbors the PH domain, it was not included in the crystal structure from the Protein Data Bank (2ELB).

37

are clustered near the edge of the PH domain (Figure 7), suggesting a potential link to

APPL1 localization. Collectively, these sites in the BAR and PH domains, may contribute

to altered APPL1 binding to Rab5, since together these domains are important for this

interaction. Finally, tyrosine 604 was found in the PTB domain, which is typically

involved in protein-protein interactions, and phosphorylation in this domain may regulate

the ability of APPL1 to bind to its interacting protein partners. Interestingly, a significant

number of identified phosphorylation sites are found outside of known domains. Even

though these sites are outside described domains, it does not imply a lack of functional

significance. These sites may have importance in regulating the structure and molecular

interactions of APPL1.

Putative Kinases for Identified APPL1 Phosphorylation Sites: Table 4 shows

putative kinases for eight of the identified APPL1 phosphorylation sites obtained using

NetPhosK 1.0 and Scansite (medium stringency) (Obenauer, Cantley et al. 2003; Blom,

Sicheritz-Ponten et al. 2004). These potential kinases include the serine/threonine

kinases casein kinase 1 and casein kinase 2, which are involved in Wnt signaling

(Davidson, Wu et al. 2005; Gao and Wang 2006). Casein kinase 1 is predicted to

phosphorylate serines 430, 693 and 696, while casein kinase 2 is predicted to

phosphorylate serines 491, 691, and 696. The serine/threonine kinase cell division cycle

2 (cdc2) is an important regulator of cell cycle progression, and is predicted to

phosphorylate serines 401, 689, and 691 (Aleem, Kiyokawa et al. 2005). The

serine/threonine kinase Akt, also known as protein kinase B, binds to APPL1 via the

PTB domain, and is predicted to phosphorylate serine 427 in the area between the PH

and PTB domain (Mitsuuchi, Johnson et al. 1999). Moreover, the serine/threonine kinase

glycogen synthase kinase 3 (GSK3), a downstream effector of Akt, is predicted to

phosphorylate APPL1 at serine 401 (Cross, Alessi et al. 1995). Putative kinases

38

Table 4. Putative Kinases for Identified APPL1 Phosphorylation Sites

Sequence Position

MS-based method Putative Kinasesa

401 S LTQMS/ LTQ-Orbitrap cell division cycle 2

glycogen synthase kinase 3 cyclin dependent kinase 5

427 S LTQ-Orbitrap

ribosomal s6 kinase Akt

protein kinase A

430 S LTQ-Orbitrap protein kinase C casein kinase 1

491 S LTQMS casein kinase 2

689 S LTQ-Orbitrap cell division cycle 2

691 S LTQMS/ LTQ-Orbitrap casein kinase 2

DNA protein kinase cell division cycle 2

693 S

LTQMS/ LTQ-Orbitrap casein kinase 1

696 S

LTQMS/ LTQ-Orbitrap casein kinase 2 casein kinase 1

a. Kinases predicted to phosphorylate the indicated sites using NetPhosK 1.0 (0.5 threshold) and Scansite (Medium Stringency).

39

obtained from Scansite (low, medium, and high stringency) are shown in Table 5.

Advantages and Challenges to Contemporary Phosphoproteomic

Methodologies: Figures 8 and 9 demonstrate the necessity of manual verification of

bioinformatics analyses. For example, the peptide in Figure 8,

GQFVVLSSSQpSEESDLGEGGKKRE, was identified correctly, but because the

incorrect peak was used as the monoisotopic peak, the mass error of the precursor ion (-

381 ppm) was outside of the acceptable range (-5 to 5 ppm). Conversely, an example of

an erroneous SEQUEST assignment is shown in Figure 9. Although b and y ion

coverage bracketing the phosphorylation site is sufficient for a high X-corr value and

high sequence coverage confidence, high abundance peaks do not correspond to b and

y ions or their respective neutral losses. Collectively, these examples and ambiguity

arising from gas-phase rearrangement illustrate the continuing need to validate

sequencing data in phosphorylation site mapping experiments (Palumbo and Reid 2008;

Palumbo, Tepe et al. 2008).

Conclusion

Emerging data indicate an important role for APPL1 in regulating various cellular

processes, such as cell proliferation, apoptosis, and survival, which points to a need to

gain insight in to the regulation of this protein (Mitsuuchi, Johnson et al. 1999; Deepa

and Dong 2009). Since phosphorylation is an important regulatory mechanism, we

generated a comprehensive map of phosphorylation sites within APPL1. We detected 13

phosphorylation sites within APPL1, with four of these being identified in functional

domains. These sites have potential implications in regulating APPL1 function and

interactions, which represents an important avenue for future study.

40

Table 5. Putative kinases determined by Scansite

Sequence

Position Putative Kinases Score Percentile Stringency

427S Akt 0.5939 0.962% medium and low

430S casein kinase 1 0.3641 0.132% high, medium and low

protein kinase C delta 0.4422 0.634% low

protein kinase C epsilon 0.4934 2.673% low

491S casein kinase 2 0.5636 2.386% low

689S glycogen synthase kinase 3 0.6119 2.146% low

691S ataxia telangiectasia mutated kinase 0.5612 1.541% low

DNA protein kinase 0.6199 3.346% low

693S casein kinase 2 0.6084 4.799% low

DNA protein kinase 0.5419 1.027% low

696S casein kinase 2 0.4160 0.214% medium and low

a. The score values start at 0.000 if the sequence optimally and they increase for sequences as they diverge from this motif. b. The percentile tells how good a score is for a given motif, the lower the percentile the better the score. For example, the cut off value for high stringency is 0.2%.

41

Figure 8. Tandem MS/MS spectrum acquired using an LTQ-Orbitrap illustrates peak validation for accurate SEQUEST assignments. Inset illustrates a situation whereby the instrument selected the peak at 878.7426 as the monoisotopic peak resulting in erroneous mass accuracy (-381 ppm). Manual validation of the data correctly assigns the accurate monoisotopic peak at 878.4084 resulting in a mass accuracy for the parent species of 0.56 ppm.

42

Figure 9. Tandem MS/MS spectrum of a phosphorylation site incorrectly assigned by SEQUEST. SEQUEST assignments report 15% b-ion sequence coverage and 55% y-ion coverage from the y5 ion to the y16 ion. Of the eight most abundant peaks in the spectrum, six ions, indicated by an asterisk, correspond to neither b nor y ions, or to characteristic neutral losses. Manual verification was performed to detect such errors in the bioinformatic assignments. Additionally, the b and y ion coverage fails to bracket the suggested sites of phosphorylation, namely tyrosine 378 and serine 380.

43

Chapter III

THE ENDOSOMAL ADAPTOR PROTEIN APPL1 IMPAIRS THE TURNOVER OF LEADING EDGE ADHESIONS TO REGULATE CELL MIGRATION

Joshua A. Broussard1, Wan-hsin Lin1, Devi Majumdar1, Bridget Anderson1, Brady Eason3, Claire M. Brown3, and Donna J. Webb1,2

1Department of Biological Sciences and Vanderbilt Kennedy Center for Research on Human Development, and 2Department of Cancer Biology, Vanderbilt University, Nashville, Tennessee 37235. 3Life Sciences Complex Imaging Facility, McGill University, Montreal, Canada, H3G 0B1. This article has been published under the same title in Molecular Biology of the Cell. 2012 Apr;23(8):1486-99. Epub 2012 Feb 29.

44

Abstract

Cell migration is a complex process that requires the integration of signaling

events that occur in distinct locations within the cell. Adaptor proteins, which can

localize to different subcellular compartments, where they bring together key signaling

proteins, are emerging as attractive candidates for controlling spatially coordinated

processes. However, their function in regulating cell migration is not well understood. In

this study, we demonstrate a novel role for the adaptor protein containing a pleckstrin

homology (PH), phosphotyrosine binding (PTB), and leucine zipper motif 1 (APPL1) in

regulating cell migration. APPL1 impairs migration by hindering the turnover of

adhesions at the leading edge of cells. The mechanism by which APPL1 regulates

migration and adhesion dynamics is by inhibiting the activity of the serine/threonine

kinase Akt at the cell edge and within adhesions. In addition, APPL1 significantly

decreases the tyrosine phosphorylation of Akt, by the non-receptor tyrosine kinase Src,

which is critical for Akt-mediated cell migration. Thus, our results demonstrate an

important new function for APPL1 in regulating cell migration and adhesion turnover

through a mechanism that is dependent on Src and Akt. Moreover, our data further

underscore the importance of adaptor proteins in modulating the flow of information

through signaling pathways.

Introduction

Adaptor proteins are emerging as important regulators of key signaling events

that control cellular behaviors underlying many biological and pathological processes

(Flynn 2001). They can accomplish this, through their multiple functional domains, by

bringing together and targeting protein binding partners to specific locations within cells

(Pawson 2007). This capability places adaptor proteins in an ideal position to integrate

and direct signals that control highly complex, spatiotemporally regulated processes

45

such as cell migration. Indeed, recent work has pointed to a role for these integrators in

the regulation of cell migration (Nayal, Webb et al. 2006; Yu, Deakin et al. 2009;

Meenderink, Ryzhova et al. 2010); however, their function in modulating this process is

not well understood.

APPL1 is a 709 amino acid endosomal protein which was first identified through

its association with Akt (also known as protein kinase B) in a yeast two-hybrid screen

(Mitsuuchi, Johnson et al. 1999). APPL1 contains an N-terminal Bin-Amphiphysin-Rvs

(BAR) domain, a central PH domain, as well as a C-terminal PTB domain (Mitsuuchi,

Johnson et al. 1999; Miaczynska, Christoforidis et al. 2004). The BAR domain is a

dimerization motif associated with sensing and/or induction of membrane curvature

(Habermann 2004; Peter, Kent et al. 2004; Chial, Lenart et al. 2010). Similarly, the PH

and PTB domains of APPL1 have been reported to bind to phosphoinositol lipids (Li,

Mao et al. 2007; Chial, Wu et al. 2008). The BAR and PH domains of APPL1 cooperate

to form a functionally unique BAR-PH domain that differentiates it from other members of

the BAR domain-containing protein family (Li, Mao et al. 2007; Zhu, Chen et al. 2007).

APPL1 interacts with the early endosomal protein Rab5 via the BAR-PH domain

(Miaczynska, Christoforidis et al. 2004; Zhu, Chen et al. 2007). Moreover, the PTB

domain is the critical region of APPL1 that is responsible for binding Akt (Mitsuuchi,

Johnson et al. 1999).

Akt is a serine/threonine kinase that is activated downstream of

phosphatidylinositol 3-kinase (PI3K) (Franke, Yang et al. 1995). PI3K signaling recruits

Akt to the plasma membrane where it becomes activated following phosphorylation on

two conserved residues, threonine 308 (T308) and serine 473 (S473) (Alessi,

Andjelkovic et al. 1996). Interestingly, Akt activation also occurs on signaling

endosomes, whereby PI3K is recruited to endosomal membranes and promotes the

46

activation of Akt (Wang, Pennock et al. 2002). Active Akt phosphorylates its

downstream effectors to regulate several cellular processes including cell growth,

survival, and proliferation (Manning and Cantley 2007). Moreover, there has recently

been a growing interest in the function of Akt in the regulation of cell migration. Akt has

been shown to stimulate the migration of epithelial cells, fibroblasts, and fibrosarcomas,

and to promote the invasion of breast carcinomas and fibrosarcomas (Kim, Kim et al.

2001; Irie, Pearline et al. 2005; Zhou, Tucker et al. 2006; Ju, Katiyar et al. 2007).

In addition to the regulatory phosphorylation at T308 and S473, recent work has

shown that Akt also undergoes tyrosine phosphorylation (Chen, Kim et al. 2001). Akt

tyrosine phosphorylation is mediated by the non-receptor tyrosine kinase Src (Chen, Kim

et al. 2001). Src-mediated tyrosine phosphorylation of Akt is reported to be important in

both the activation and function of Akt (Chen, Kim et al. 2001; Choudhury,

Mahimainathan et al. 2006; Bouchard, Demers et al. 2007). However, nothing is

currently known about the role of Akt tyrosine phosphorylation in the regulation of cell

migration.

Cell migration is initiated in response to an external stimulus and begins with the

extension of an actin-rich protrusion, which is stabilized by the formation of nascent

adhesions at the leading edge (Carson, Weber et al. 1986; Zaidel-Bar, Ballestrem et al.

2003; Ponti, Machacek et al. 2004; Nayal, Webb et al. 2006). These adhesions can then

mature into large, stable adhesions through subsequent recruitment of signaling, adaptor,

and cytoskeleton-related proteins, or they can disassemble (Miyamoto, Teramoto et al.

1995; Zaidel-Bar, Milo et al. 2007; Choi, Vicente-Manzanares et al. 2008; Dubash,

Menold et al. 2009; Kanchanawong, Shtengel et al. 2010). For migration to proceed in

an efficient manner, adhesions at the leading edge of the cell must continually form

47

(assemble) and disassemble in a process termed adhesion turnover (Laukaitis, Webb et

al. 2001; Webb, Donais et al. 2004; Dubash, Menold et al. 2009).

Here we show that the adaptor protein APPL1 is an important regulator of cell

migration and adhesion dynamics. APPL1 modulates these processes in a manner that

is dependent on its ability to regulate Akt activity and function. Moreover, APPL1 inhibits

the ability of Akt to promote migration by impairing Src-mediated tyrosine

phosphorylation of Akt.

Materials and Methods

Reagents: An APPL1 rabbit polyclonal antibody was made using the peptides

CSQSEESDLGEEGKKRESEA and CSQSEESDLGEGGKKRESEA (21st Century

Biochemicals, Marlboro, MA). Primary antibodies used for this study include:

phosphorylated Akt (Thr-308) polyclonal antibody (Santa Cruz Biotechnology, Santa

Cruz, CA), pan-Akt C67E7, Akt1 C73H10, Akt2 D6G4, and Akt3 62A8 monoclonal

antibodies (Cell Signaling, Beverly, MA), paxillin monoclonal antibody (BD Bioscience

Pharmingen, San Diego, CA), phosphotyrosine clone 4G10 monoclonal antibody

(Millipore, Billerica, MA), β-actin clone AC-15 monoclonal antibody, and FLAG M2

monoclonal antibody (Sigma, St Louis, MO). Secondary antibodies used for

immunocytochemistry were Alexa Fluor 488 and 555 anti-rabbit as well as Alexa Fluor

488 and 555 anti-mouse (Molecular Probes, Eugene, OR). Secondary antibodies for

Western blot analysis included IRDye 800 anti-mouse and 800 anti-rabbit (Rockland

Immunochemicals, Gilbertsville, PA). Fibronectin was purchased from Sigma (St Louis,

MO). Anti-FLAG M2 agarose, mouse IgG-agarose, and PP2 were purchased from

Sigma (St Louis, MO).

48

Plasmids: Full-length human APPL1 cDNA was produced via reverse

transcription of HEK293 cell RNA with subsequent amplification with the SuperScript

One-Step RT-PCR kit (Invitrogen, Carlsbad, CA) using the following primers: 5’-

ATGCCGGGGATCGACAAGCTGCCC-3’ (forward) and 5’-TGCTTCTGATTCTCTC-

TTCTTTCC-3’ (reverse). Then, the APPL1 cDNA was sequenced and cloned into

pEGFP-C3 vector (Clontech Laboratories). Small interfering RNA (siRNA) constructs

were prepared as previously described (Zhang and Macara 2008). Briefly, sense and

antisense 64-mer oligonucleotides, containing the 19-nucleotide targeting sequence,

were ligated into pSUPER vector. The siRNA oligos targeting sequences included:

APPL1 siRNA 1, 5’-CACACCTGACCTCAAAACT-3’; APPL1 siRNA 2, 5’-AAGAGTGGA-

TCTGTACAAT-3’; Akt siRNA 1, 5’-GGAGGGTTGGCTGCACAAA-3’; Akt siRNA 2, 5’-

CTACAACCAGGACCATGAG-3’. APPL1 siRNA 1 and both Akt target sequences have

been previously described (Miaczynska, Christoforidis et al. 2004; Pore, Jiang et al.

2006; Degtyarev, De Maziere et al. 2008; Bristow, Sellers et al. 2009). mCherry-paxillin

was kindly provided by Steve Hanks (Vanderbilt University, Nashville, TN). DN-Akt1

(K179A/T308A/S473A) and CA-Akt1 (T308D/S473D) were generously provided by Brian

Hemmings (Friedrich Miescher Institute, Basel, Switzerland) and Jeffrey Field (University

of Pennsylvania, Philadelphia, PA). The Akind FRET probe was kindly provided by

Michiyuki Matsuda (Kyoto University, Kyoto, Japan). GFP-Src-Y527F (CA-Src) was a

generous gift from Margaret Frame (University of Edinburgh, Edinburgh, UK). The cDNA

for GFP-APPL1-∆PTB (amino acids 1-466) was generated from full length GFP-APPL1

using PCR with the following primers: 5’-TCGAATTCGGCTTATGCCGG-3’ and 5’-

TCGGTACCTGGCTACTTCCAAATCCTC-3’. The PCR product was then cloned into the

pEGFP-C3 vector (Clontech Laboratories) at EcoRI and KpnI. GFP-APPL1-AAA was

prepared by site-directed mutagenesis of full length GFP-APPL1 using a Quikchange II

kit (Agilent Technologies, Santa Clara, CA). For GFP-APPL1-AAA, the following primers

49

were used to mutate amino acids R146, K152, K154 to alanines: 5’-

CGATTAATGCATATAGCCGTTTATCAGCAAAAGCAGAAAATGACAAGG-3’ and 5’-

GCGATTAATGCATATAGCCGTTTATCAGCAAAAAGAG-3’. HA-FLAG-Akt1 was

purchased from Addgene (Cambridge, MA). Akt-Y315F/Y326F and Akt-

T308D/S473D/Y315F/Y326F were generated by site-directed mutagenesis of HA-FLAG-

Akt1 using a Quikchange II kit. For Akt-T308D/S473D/Y315F/Y326F, the following

primers were used: T308D - 5’-GGTGCCACCATGAAGGACTTTTGCGGCACA-3’;

Y315F - 5’-GGCACACCTGAGTTCCTGGCCCCCGAGGTG-3’; Y326F - 5’-CAC-

TGCACGGCCGAAGTCATTGTCCTCCAG-3’; and S473D - 5’-TTCCCCCAGTTCGAC-

TACTCGGCCAGCGGC-3’.

Cell Culture, Transfection, and Immunoprecipitation: HT1080 cells were

maintained in Dulbeco’s Modified Eagles Medium (DMEM) (Invitrogen) with 10% fetal

bovine serum (FBS) (HyClone, Logan, UT) and 1% penicillin/streptomycin (Invitrogen).

Cells were transiently transfected with Lipofectamine 2000 (Invitrogen) according to the

manufacturer’s instructions.

For immunoprecipitation of Akt, HT1080 cells were co-transfected with 0.5 g of

FLAG-Akt cDNA and various other cDNA(s) using Lipofectamine 2000. After 24 h, cells

were incubated with 1 mM sodium vanadate and 2 mM H2O2 (peroxovanadate) in

DMEM for 15 min and then lysed with 1% NP-40 in 25 mM Tris, 100 mM NaCl, with

protease inhibitors, pH 7.4. In some experiments, cells were incubated with PP2 (1 –

7.5 M) for 1.5 h prior to peroxovanadate treatment. Cell lysates were pre-cleared by

incubation with non-immune mouse-IgG agarose in 25 mM Tris, 100 mM NaCl, pH 7.4

overnight at 4oC. FLAG-Akt was immunoprecipitated with anti-FLAG M2 agarose in 25

mM Tris, 100 mM NaCl, pH 7.4 for 2 h at 4oC followed by elution with 0.2 mg/ml FLAG

peptide (Sigma, St Louis, MO) in 25 mM Tris, pH 7.4.

50

Migration Assay and Microscopy: Cells were plated on tissue culture dishes,

which were coated with 2.5 µg/mL fibronectin for 1 h at 37oC, and permitted to adhere

for 1 h at 37oC in culture media. Then, cells were imaged using phase contrast

microscopy on an inverted Olympus IX71 microscope (Melville, NY) with a 10X objective

(NA 0.3) and a Retiga EXi CCD camera (QImaging, Surrey, BC). Cells were kept at

37oC in CCM1 (HyClone) supplemented with 2% FBS (HyClone) at pH 7.4 during

imaging. Images were acquired at 5 min intervals for at least 6 h, using MetaMorph

software (Molecular Devices, Sunnyvale, CA). Cell movement was tracked from the

time-lapse images using MetaMorph, and migration speed was calculated by dividing the

total distance moved in microns by the time. Wind-Rose plots were generated by

transposing X-Y coordinates of cell tracks to a common origin.

Immunocytochemistry and Image Analysis: Cells were incubated on glass

coverslips, which were coated with 2.5 µg/mL fibronectin, for 1 h at 37oC and

subsequently fixed in either 4% paraformaldehyde with 4% glucose in PBS for 15 min at

room temperature or methanol for 5 min on ice. After fixation, cells were permeabilized

by incubation with 0.2% (v/v) Triton X-100 for 3 min, and then blocked with 20% goat

serum in PBS. Following blocking, appropriate primary and second antibodies, diluted in

5% goat serum with 0.2% Triton-X-100 in PBS, were added to the coverslips. After each

step, coverslips were rinsed three times with PBS. Coverslips were then mounted using

Aqua Poly/Mount (Poly-sciences, Warrington, PA). Images were acquired using

MetaMorph software and an Olympus PlanApo 60X OTIRFM objective (NA 1.45)

(Melville, NY). TIRF images were acquired by exciting with either a 488 or 543 nm laser

line from a HeNe laser (Prairie Technologies, Middleton, WI). For GFP and Alexa Fluor

488, an Endow GFP Bandpass filter cube (excitation HQ470/40, emission HQ525/50,

Q495LP dichroic mirror) (Chroma, Brattleboro, VT) was used. A TRITC/Cy3 cube

51

(excitation HQ545/30, emission HQ610/75, Q570LP dichroic mirror) was used for

mCherry and Alexa Fluor 555. An ET-CFP filter cube (excitation ET436/20, emission

ET480/40, T455LP dichroic mirror) was used for CFP. For TIRF imaging a z488/543 rpc

filter was used.

For quantification of phosphorylated Akt (Thr308), the background subtracted,

integrated fluorescence intensity from individual cells was measured and normalized to

the unit area using MetaMorph software. Phosphorylated Akt (Thr308) was quantified in

adhesions by thresholding paxillin fluorescent staining, and creating an image mask of

adhesions using the Integrated Morphometry Analysis package of MetaMorph. These

masks were then applied to background subtracted, TIRF images of phosphorylated Akt,

and the average level of active Akt in adhesions was quantified using the Integrated

Morphometry Analysis package. For this analysis, objects with an area less than 0.2

m2 were excluded, because of the difficulty in distinguishing them from background

puncta.

Sensitized Emission Image Processing: In order to calculate the CFP/FRET

ratio image, it is necessary to go through a number of image processing steps. Typically,

all of the images must first be corrected for any field non-uniformity in the sample

excitation and for any background intensity within the images(Lacoste, Young et al.

2012). In this case, the illumination field was very uniform because we used a confocal

laser scanning microscope (CLSM) with a x63/1.4 NA Planapo oil immersion lens, and

an image zoom factor of 2. Therefore, no correction was necessary. The background

correction was done by measuring the average intensity of an ROI in the image where

there are no cells and subtracting this intensity from each pixel grey value within the

image. Correction factors for the CFP emission cross-talk and the Venus direct

52

excitation cross-talk were also calculated. Following this, the corrected FRET image

(FRETCorr) can be calculated based on Equation 1 (Figure 10A).

Equation 1:

Where FRETRaw is the original FRET image collected on the microscope (corrected for

background and field non-uniformity (if necessary)). A is the percentage of Venus

excitation cross-talk in the FRET image calculated from the control Venus sample.

Venus is the corrected image of Venus expression in the sample following direct

excitation by the 514 nm laser. B is the percentage of CFP emission cross-talk in the

FRET channel calculated from the control CFP sample. CFP is the corrected image of

the sample collected in the CFP channel. The ratio image is then calculated from

Equation 2 (Figure 10B and C).

Equation 2:

( )

( )

⁄ * 1000

The numerator is essentially the normalized FRETCorr image and the denominator is the

normalized CFP image. IMax is the maximum grey value in the FRETCorr (numerator) or

CFP image (denominator). Images are based on integer values so the factor of 1000 is

included to avoid fractional pixel intensities in the resultant ratio image.

Sensitized Emission Image Analysis: HT1080 cells were plated on fibronectin-

coated glass coverslips for 1 h at 37oC and then fixed by incubation in 4%

paraformaldehyde with 4% glucose in PBS for 15 min at room temperature. Images were

acquired with a Zeiss 710 CLSM attached to an Axiobserver inverted. The emission

53

Figure 10: Calculation of FRET/CFP ratio images. (A) Background subtracted CFP, Venus and FRETRaw images (first 3 panels) are used with Equation 1 (where Venus is the Acceptor and CFP is the Donor) to create a FRETCorr image (far right panel). (B) Upper panels, the newly created FRETCorr image and the CFP image from panel a are shown with a rainbow or pseudo-color coding look up table to emphasize subtle changes in intensity across the cells. These images are then normalized using the indicated equations to produce images with similar maximum intensity values (lower panels). (C)

54

The normalized images from panel b are then used to create a FRET/CFP ratio image by dividing the FRETCorr image by the CFP image and multiplying the result by 1000 (left panel). A median filter was used to reduce the image noise (right panel). Images are shown in pseudo-color coding. The scale bars are 10 μm.

55

settings on the Zeiss 710 microscope were set to collect the following wavelengths: CFP:

454–568, Venus: 516–621 and RawFRET: 516-621. For CFP and RawFRET, a 405 nm

dichroic was used, and for Venus, a 458/514 nm dichroic was used. Background

subtracted, FRET/CFP ratio images were generated using MetaMorph software. The

equation used to calculate the FRET image for our experimental conditions was: FRET =

RawFRET - 0.042 X Venus - 0.184 X CFP, where CFP is the image of CFP excited by

the 405 nm laser, and Venus is the image of Venus excited directly by the 514 nm laser.

The CFP and Venus correction factors (0.184 and 0.042, respectively) were calculated

from cells expressing CFP or Venus fluorescent protein alone and imaged in the FRET

channel under the same conditions as the RawFRET images (excited by the 405 nm

laser). The total FRET/CFP ratio was normalized to the unit area, and the average

FRET/CFP ratio per cell was calculated. Line scan analysis was performed using

MetaMorph software with a line length of 5 µm and width of 1.32 µm, and the average

FRET/CFP ratio was calculated as a function of distance from the cell edge. FRET/CFP

images shown were processed with a 3x3 median filter using MetaMorph software to

reduce noise.

Adhesion Turnover Assay: HT1080 cells were co-transfected with 1 µg

mCherry-paxillin cDNA and 3 µg cDNA of the indicated constructs, plated on fibronectin-

coated microscopy dishes, and imaged as previously described (Bristow, Sellers et al.

2009). Briefly, fluorescent time-lapse images were obtained at 15 sec intervals, and the

t1/2 values for adhesion assembly and disassembly were quantified (Webb, Donais et al.

2004; Bristow, Sellers et al. 2009).

56

Results

The signaling adaptor APPL1 inhibits cell migration: The multi-domain

adaptor protein APPL1 (Figure 11A) has been shown to interact with various signaling

and trafficking proteins, putting it in an ideal position to spatiotemporally coordinate

signaling pathways that underlie processes such as cell migration. This led us to

hypothesize that APPL1 is an important regulator of migration. To begin to test our

hypothesis, we expressed GFP and GFP-APPL1 in HT1080 cells, plated them on

fibronectin, and assessed their migration using live-cell imaging. The migration of

individual cells was tracked using MetaMorph software, and Rose plots were generated

from these data (Figure 11B). The migration paths for GFP-APPL1 expressing cells

were significantly shorter than those of control cells expressing GFP, suggesting that

APPL1 decreased the rate of migration in HT1080 cells (Figure 11B). Indeed,

quantification of the migration speed revealed a 1.7-fold decrease in GFP-APPL1

expressing cells compared with control cells expressing GFP (Figure 11B). To further

show a function for APPL1 in migration, we expressed GFP-APPL1 in MDA-MB-231

cells, which have similar endogenous levels of APPL1 as HT1080 cells (Figure 12A). As

with HT1080 cells, expression of GFP-APPL1 significantly reduced the migration speed

of MDA-MB-231 cells (Figure 12B). Collectively, these results point to a role for APPL1

in the regulation of cell migration.

We continued to probe the function of APPL1 in modulating migration by

generating two small interfering RNA (siRNA) constructs to knock down endogenous

expression of this protein. Although APPL1 siRNA 1 had been previously reported to be

very effective (Miaczynska et al., 2004), we confirmed its ability to knock down

expression of APPL1. When wild-type HT1080 cells were transfected with APPL1

siRNA 1, endogenous expression of APPL1 was decreased by over 80% compared to

57

58

Figure 11. APPL1 regulates cell migration. (A) A schematic with the domain structure of APPL1 is shown. (B) HT1080 cells were transfected with either GFP or GFP-APPL1, plated on fibronectin, and subjected to time-lapse microscopy. Migration was quantified for individual cells, and Rose plots of migration tracks are shown (left panels). Quantification of the migration speed for GFP and GFP-APPL1 expressing cells is shown (right panel). Error bars represent the S.E.M. for 49-64 cells from at least three separate experiments (*p<0.0001). (C) HT1080 cells were transfected with either empty pSUPER vector, a scrambled siRNA (Src siRNA) or APPL1 siRNA. Cell lysates were subjected to immunoblot analysis to determine the levels of endogenous APPL1 and β-actin (as a loading control) (left panel). Quantification of the endogenous levels of APPL1 in cells transfected with the indicated constructs is shown (right panel). Error bars represent the S.E.M. from four separate experiments (*p<0.0001, **p=0.0002). (D) Cells were transfected with empty pSUPER vector, a scrambled siRNA (Src siRNA) or APPL1 siRNA and used in migration assays three days later. Rose plots with migration tracks for cells transfected with the indicated constructs are shown (left panels). Quantification of the migration speed of cells transfected with the indicated constructs is shown (right panel). Error bars represent the S.E.M. for 46-64 cells from three individual experiments (*p<0.0009). For panels C and D, asterisks indicate a statistically significant difference compared with pSUPER transfected cells.

59

Figure 12. APPL1 regulates cell migration in MDA-MB-231 cells. (A) HT1080 and MDA-MB-231 cell lysates were subjected to immunoblot analysis to determine the levels of endogenous APPL1 and β-actin (as a loading control). (B) MDA-MB-231 cells were transfected with either GFP or GFP-APPL1, plated on fibronectin, and subjected to time-lapse microscopy. Quantification of the migration speed for GFP and GFP-APPL1 expressing cells is shown. Error bars represent the S.E.M. for 33-57 cells from three separate experiments (*p=0.0032).

60

either empty pSUPER vector or a scrambled siRNA, as determined by western blot

analysis (Figure 11C). APPL1 siRNA 2 similarly decreased endogenous levels of

APPL1 by approximately 65% compared to empty pSUPER vector or a scrambled

siRNA (Figure 11C), indicating the APPL1 siRNAs were effective in knocking down

expression of APPL1. Transfection of HT1080 cells with APPL1 siRNA 1 and APPL1

siRNA 2 led to a 1.4-fold and a 1.3-fold increase in migration speed, respectively,

compared to pSUPER or scrambled siRNA transfected cells (Figure 11D). These results

indicate that decreased expression of APPL1 enhances cell migration, thus implicating

APPL1 as an important regulator of this process.

Endosomal localization of APPL1 is required for its effects on migration:

Since APPL1 localizes to early endosomes and signaling events that take place on

endosomes are increasingly thought to play important roles in modeling cellular behavior

(Miaczynska et al., 2004; von Zastrow and Sorkin, 2007), we hypothesized the APPL1

localization to endosomes is critical for its ability to regulate cell migration. To determine

if APPL1 endosomal localization was necessary for its effects on migration, we mutated

three basic residues (R147A, K153A and K155A) within the BAR domain of APPL1,

which had previously been shown to be sufficient to disrupt its endosomal localization

(Schenck et al., 2008). GFP-APPL1, like endogenous APPL1, localized to vesicular

structures; however, GFP-APPL1 that contained the point mutations (GFP-APPL1-AAA)

no longer localized to endosomes when expressed in HT1080 cells (Figure 13, A and B).

The migration speed of cells expressing GFP-APPL1-AAA was not significantly different

from control GFP expressing cells (Figure 13, C and D). These results suggest that the

localization of APPL1 to endosomal membranes is critical for its ability to regulate cell

migration.

61

Figure 13. APPL1 localizes to vesicular structures, which is critical in its regulation of cell migration. (A) Wild-type HT1080 cells were immunostained for endogenous APPL1 (left panel) or transfected with either GFP-APPL1 (middle panel) or GFP-APPL1 in which three basic residues (147, 153, and 155) within the BAR domain were mutated to alanines (GFP-APPL1-AAA) (right panel). Unlike GFP-APPL1 or the endogenous protein, GFP-APPL1-AAA did not localize to endosomal structures. Scale bar = 15 µm. (B) High magnification images of the boxed regions from panel A are

shown. Scale bar = 15 m. (C) GFP and GFP-APPL1-AAA transfected cells were imaged using time-lapse microscopy and migration was analyzed. Rose plots with migration tracks for these cells are shown. (D) Quantification of the migration speed of GFP and GFP-APPL1-AAA expressing cells is shown. Error bars represent the S.E.M. for 52-66 cells from at least three separate experiments.

62

APPL1 regulates leading edge adhesion dynamics in migrating cells:

Adhesion assembly and disassembly at the leading edge of cells, termed adhesion

turnover, is required for efficient migration to occur (Webb et al., 2004; Nayal et al.,

2006). This led us to hypothesize that APPL1 affects migration through its ability to

regulate adhesion turnover. To determine if APPL1 affects the number and/or size of

adhesions, we expressed GFP and GFP-APPL1 in wild-type HT1080 cells and

immunostained for endogenous paxillin, which is a well characterized adhesion marker.

Cells expressing GFP-APPL1 exhibited a greater number of larger central adhesions

and fewer nascent peripheral adhesions compared to control cells expressing GFP

(Figure 14A). In GFP-APPL1 expressing cells, the larger central adhesions could arise

from their inability to efficiently turn over. We examined this possibility by quantitatively

measuring adhesion turnover using an assay that we previously developed (Webb et al.,

2004). GFP and GFP-APPL1 expressing cells that were transfected with mCherry-

paxillin were subjected to time-lapse fluorescence microscopy, and the t1/2s for adhesion

assembly and disassembly were assessed (Webb et al., 2004). Cells expressing GFP-

APPL1 exhibited a 1.8-fold increase in the apparent t1/2 for adhesion assembly as

compared to GFP controls (Figure 14, B and C), indicating that adhesions are forming

considerably slower in the GFP-APPL1 expressing cells. Moreover, GFP-APPL1

expression led to a 1.4-fold increase in the t1/2 for adhesion disassembly (Figure 14, B

and C).

In addition, we used the adhesion turnover assay to examine the effects of GFP-

APPL1-AAA on adhesion dynamics. Cells expressing this mislocalization mutant had

assembly and disassembly t1/2s of 2.1 ± 0.3 and 3.0 ± 0.3 min, respectively, which are

not significantly different from those observed in GFP controls (Figure 14C). Taken

63

Figure 14. APPL1 impairs adhesion turnover at the leading edge of cells. (A) Cells were transfected with either GFP or GFP-APPL1 and immunostained for the adhesion marker paxillin. Scale bar = 10 µm. (B) HT1080 cells were co-transfected with mCherry-paxillin and either GFP or GFP-APPL1 and imaged using time-lapse microscopy. In GFP-APPL1 expressing cells, the leading edge adhesions assemble and disassemble more slowly than those in control cells expressing GFP (arrows). Scale bar = 5 µm. (C) Quantification of the apparent t1/2 for adhesion assembly and the t1/2 for adhesion

64

disassembly for cells expressing the indicated constructs is shown. Error bars represent the S.E.M. for 26-30 adhesions from 4-6 cells from at least three independent experiments (*p<0.0001, **p≤0.007, ***p<0.013). Asterisks indicate a statistically significant difference compared with GFP cells.

65

together, these results demonstrate that APPL1 significantly slows the rate of adhesion

assembly and disassembly in cells, in a manner dependent on its endosomal localization.

We further corroborated a role for APPL1 in modulating adhesion turnover by

knocking down expression of the endogenous protein. Expression of APPL1 siRNA 1

and APPL1 siRNA 2 decreased the apparent t1/2 of adhesion assembly by 1.4-fold and

1.5-fold, respectively, compared to both scrambled siRNA and GFP controls (Figure

14C). In addition, APPL1 siRNA 1 and APPL1 siRNA 2 decreased the t1/2 of adhesion

disassembly by 1.7-fold and 1.8-fold, respectively, as compared to controls (Figure 14C).

These results reveal that cells turn over their adhesions much faster when endogenous

APPL1 expression is decreased, indicating an inhibitory role for APPL1 in the regulation

of leading edge adhesion dynamics.

APPL1 and Akt regulate cell migration and adhesion dynamics: Because Akt

has been previously shown to interact with APPL1, and Akt has recently been implicated

as a regulator of cell migration (Mitsuuchi et al., 1999; Kim et al., 2001; Arboleda et al.,

2003; Irie et al., 2005; Zhou et al., 2006; Ju et al., 2007; Schenck et al., 2008), APPL1

may affect migration via a mechanism involving Akt. Since the PTB domain of APPL1

mediates its interaction with Akt (Mitsuuchi et al., 1999; Schenck et al., 2008), we

expressed a GFP-APPL1 truncation mutant, which lacked the PTB domain (GFP-

APPL1-∆PTB), and assessed migration using time-lapse microscopy. Expression of

GFP-APPL1 significantly decreased the rate of migration compared with control GFP

expressing cells (Figure 15, A and B). However, the APPL1-induced decrease in

migration was abolished in GFP-APPL1-∆PTB expressing cells, whose migration speed

was similar to that observed in GFP control cells (Figure 15, A and B). This suggests

that Akt contributes to the effect of APPL1 on cell migration.

66

Figure 15. Deletion of the PTB domain of APPL1 abolishes its effect on cell migration. (A) Cells transfected with GFP, GFP-APPL1, or GFP-APPL1-∆PTB were imaged using time-lapse microscopy and migration was analyzed. Rose plots of individual cell tracks are shown for each of the indicated constructs. (B) Quantification of the migration speed of cells transfected with the indicated constructs is shown. Error bars represent the S.E.M. of 45-49 cells from at least three individual experiments (*p<0.0001). Asterisk indicates a statistically significant difference compared with GFP transfected cells.

67

We further investigated the relationship between APPL1 and Akt in the regulation

of cell migration by using a mutant based approach. We expressed either a dominant-

negative (DN-Akt) or a constitutively-active Akt1 mutant (CA-Akt) in wild-type HT1080

cells and analyzed migration using time-lapse microscopy. Cells expressing DN-Akt

showed a 1.7-fold decrease in their speed of migration, as compared to control cells

(Figure 16, A and B). In contrast, cells expressing CA-Akt exhibited a 1.3-fold increase in

migration, as compared to controls (Figure 16, A and B). Interestingly, the migration

speed of cells co-expressing either GFP-APPL1 and DN-Akt or GFP-APPL1 and CA-Akt

did not significantly differ from cells expressing GFP-APPL1 alone (Figure 16, A and B).

These results indicate GFP-APPL1 expression can suppress the CA-Akt-induced

increase in migration, while it does not provide an additive effect on migration when co-

expressed with DN-Akt.

To further investigate the ability of APPL1 to suppress Akt-induced migration, we

generated stable HT1080 cells expressing either GFP or GFP-APPL1. In the stable

GFP-APPL1 cells, the level of APPL1 expression was 1.5-fold over the endogenous

protein (Figure 16C). This expression level was comparable to that obtained with our

transient transfections in which GFP-APPL1 was expressed at 1.9-fold over endogenous

(Figure 16C). The GFP-APPL1 stable cells were then transfected with CA-Akt. As with

the transient transfections, expression of CA-Akt (3.1-fold over endogenous Akt, Figure

16D) did not significantly affect the migration of GFP-APPL1 stable cells (Figure 16E).

However, when the expression level of CA-Akt was increased to 5.3-fold over

endogenous Akt (2X CA-Akt, Figure 16D), the migration speed of the GFP-APPL1 stable

cells was increased (Figure 16E). These results indicate that while

68

Figure 16. Akt plays an important role in the APPL1-mediated regulation of cell migration. (A) HT1080 cells were co-transfected with GFP or GFP-APPL1 and either empty vector, constitutively active Akt (CA-Akt), or dominant negative Akt (DN-Akt) and used in migration assays. Rose plots with individual migration tracks for cells transfected with the indicated constructs are shown. (B) Quantification of the migration speed of cells transfected with the indicated constructs is shown. Error bars represent the S.E.M. of 35-65 cells from at least three individual experiments (*p<0.0001). (C)

69

Lysates from HT1080 cells transiently transfected with GFP-APPL1 (Transients) and HT1080 cells stably expressing GFP-APPL1 (Stables) were subjected to immunoblot analysis to determine the levels of total APPL1 (endogenous and exogenously expressed GFP-APPL1) (upper panels). Quantification of the relative amounts of GFP-APPL1 compared to endogenous APPL1 is shown (lower panel). Error bars represent the S.E.M. from at least three separate experiments (*p<0.05). Asterisks indicate a statistically significant difference compared with endogenous APPL1. (D) Stable HT1080 cells expressing GFP were transfected with empty vector (GFP). Stable HT1080 cells expressing GFP-APPL1 were transfected with empty vector (GFP-APPL1), 1.5 µg of CA-Akt cDNA (GFP-APPL1 + CA-Akt), or 3 µg of CA-Akt cDNA (GFP-APPL1 + 2X CA-Akt). Cell lysates were subjected to immunoblot analysis to determine the levels of total Akt (endogenous and exogenously expressed CA-Akt) and β-actin (as a loading control) (left panel). Quantification of the relative amount of Akt expression compared to that observed in control GFP cells is shown (right panel). Error bars represent the S.E.M. from three separate experiments (*p≤0.03). Asterisks indicate a statistically significant difference compared with control GFP cells. (E) Stable HT1080 GFP or GFP-APPL1 cells were transfected as described in panel D and used in migration assays. Quantification of the migration speed of transfected cells is shown. Error bars represent the S.E.M. of 80-91 cells from three individual experiments (*p<0.0001). Asterisks indicate a statistically significant difference compared with GFP cells.

70

GFP-APPL1 expression can inhibit low levels of CA-Akt from promoting migration,

higher expression of CA-Akt can overcome this inhibition.

We next generated two siRNA constructs to knockdown endogenous Akt.

Although we had previously used these two siRNA sequences to effectively knockdown

endogenous Akt (Bristow et al., 2009), we confirmed their efficacy by transfecting them

into HT1080 cells. Here, we obtained similar results, where Akt siRNA 1 knocked down

endogenous Akt to 61.8 ± 9.4% of control levels, while Akt siRNA 2 had an efficacy of

51.9 ± 4.7% (n= 4 separate experiments for each siRNA). Migration was then analyzed

to determine the effect of these constructs on this process. Cells transfected with Akt

siRNA 1 exhibited a 1.5-fold decrease in migration speed compared to either empty

pSUPER vector or scrambled siRNA expressing cells (Figure 17, A and B). Similarly,

Akt siRNA 2 transfected cells showed a 1.6-fold decrease in migration speed compared

with controls (Figure 17, A and B). Moreover, expression of GFP-APPL1 along with Akt

knockdown showed no further effect on migration (Figure 17, A and B), which is

consistent with the results obtained when GFP-APPL1 was co-expressed with DN-Akt

(Figure 17, A and B). Taken together, these results suggest that APPL1 is regulating

cell migration by inhibiting Akt function.

Since our results indicated that the APPL1-Akt association is critical in the

regulation of cell migration, we assessed the effect of APPL1 and Akt on adhesion

turnover. In cells expressing GFP-APPL1-∆PTB, the apparent t1/2 for adhesion

assembly and the t1/2 for adhesion disassembly were similar to those obtained for GFP

control cells, indicating that deletion of the PTB domain of APPL1 abolished its effect on

adhesion turnover (Figure 14C). We further probed the role of APPL1 and Akt in

modulating adhesion dynamics by co-expressing Akt mutants with GFP or GFP-APPL1.

Expression of CA-Akt decreased the t1/2 of adhesion assembly and disassembly, as

71

Figure 17. Akt knockdown inhibits cell migration. (A) HT1080 cells were co-transfected with empty pSUPER vector, a scrambled siRNA (Scr siRNA), or Akt siRNA and either GFP or GFP-APPL1, and imaged using time-lapse microscopy three days later. Rose plots are shown for the migration tracks of cells expressing the indicated constructs. (B) Quantification of the migration speed of cells transfected with the indicated constructs is shown. Error bars represent the S.E.M. of 56-76 cells from three separate experiments (*p<0.0001).

72

compared to GFP control cells, while DN-Akt expression led to a significant increase in

the t1/2 values (Figure 14C). When GFP-APPL1 was co-expressed with the Akt mutants,

the t1/2s were not significantly different from those observed in cells expressing GFP-

APPL1 alone (Figure 14C). Thus, as with migration, APPL1 inhibits the function of CA-

Akt in regulating adhesion turnover, while providing no additional effect on adhesion

dynamics when co-expressed with DN-Akt.

APPL1 reduces the amount of active Akt in cells: To begin to elucidate the

mechanism by which the APPL1-Akt association regulates migration and adhesion

dynamics, we examined the effect of APPL1 on the level of active Akt. Canonically, Akt

is activated through phosphorylation on two amino acids, Thr308 and Ser473 (Alessi et

al., 1996), and thus, phosphorylation-specific antibodies against these residues can be

used to detect active Akt. Cells expressing GFP and GFP-APPL1 were immunostained

with phospho-Thr308-Akt antibody and imaged using fluorescence microscopy. The

fluorescent intensity of active Akt was then quantified for individual cells using

MetaMorph software. Expression of GFP-APPL1 reduced the level of active Akt by

approximately 2-fold as compared to control cells expressing GFP (Figure 18A).

Knockdown of endogenous APPL1 using APPL1 siRNA 1 and APPL1 siRNA 2

increased the amount of active Akt by almost 1.5-fold compared to empty pSUPER

vector, while scrambled siRNA had no significant effect on the level of active Akt (Figure

18B). Interestingly, the GFP-APPL1-∆PTB mutant did not significantly affect the amount

of active Akt in HT1080 cells (101.7 ± 3.3% of control, n=85 cells), suggesting that an

association between APPL1 and Akt is necessary for the APPL1 effect on active Akt.

Moreover, the level of active Akt in GFP-APPL1-AAA expressing cells was similar to that

observed in GFP control cells (95.2 ± 3.4% of control, n=145 cells), indicating APPL1

regulates the amount of active Akt in cells in a manner dependent on its

73

Figure 18. APPL1 reduces the amount of active Akt in cells. (A) HT1080 cells were transfected with either GFP or GFP-APPL1, fixed, and immunostained for active Akt using a phospho-Thr308 specific antibody. Quantification of the fluorescent intensity in transfected cells is shown. The background subtracted, integrated fluorescent intensity was normalized to cell area (average intensity). Error bars represent the S.E.M. from greater than 150 cells from at least six individual experiments (*p<0.0001). (B) HT1080 cells were transfected with empty pSUPER vector, scrambled siRNA (Scr siRNA), APPL1 siRNA 1, or APPL1 siRNA 2 and immunostained for active Akt three days later. Quantification of the fluorescent intensity in transfected cells is shown. Error bars represent the S.E.M. of 78-121 cells from three separate experiments (*p<0.0001). Asterisks denote a statistically significant difference compared with pSUPER transfected cells.

74

endosomal localization. Collectively, these results indicate that APPL1 regulates the

amount of active Akt in cells and point to an important role for this function of APPL1 in

modulating cell migration.

We used a previously described Akind fluorescence resonance energy transfer

(FRET) probe (Yoshizaki et al., 2007) to further investigate the role of APPL1 in

regulating Akt activity. Akind is composed of the Akt PH domain (amino acids 1-144),

the fluorescent protein Venus, the Akt catalytic and regulatory domains (amino acids

133-480), and cyan fluorescent protein (CFP) (Yoshizaki et al., 2007). Upon activation,

Akind undergoes a conformational change that brings Venus and CFP into close enough

proximity to undergo FRET (Figure 19). In order to determine if Akind was being

properly activated in HT1080 cells, we compared Akt activity of the wild-type Akind

probe with a non-activatable Akind mutant in which 3 key residues (K179, T308, and

S473) have been mutated to alanine (3A-Akind). Results from using the Wt-Akind probe

show a significantly higher intensity in the FRET/CFP ratio image compared to the

dominant negative 3A-Akind probe (Figure 20A). Quantification of the results shows that

there is an approximately 3-fold increase in the FRET/CFP ratio for Akind relative to the

dominant negative 3A-Akind (Figure 20B). Therefore, Akind is being properly activated in

HT1080 cells.

Next, we assessed the effects of APPL1 on Akt activation by expressing the

Akind FRET probe with either mCherry-APPL1 or mCherry (as a control). Cells

expressing mCherry-APPL1 exhibited a 1.8-fold decrease in the average Akind

FRET/CFP ratio, when compared to mCherry expressing control cells (Figure 21A).

75

Figure 19: Schematic diagram of the Akt activation CFP-Venus FRET probe (Akind). This figure was designed based on Figure 1 from Yoshizaki et al. Molecular Biology of the Cell 18:119-128 (2007). Akind consists of an Akt pleckstrin (PH) homology domain, followed by Venus fluorescent protein, an Akt catalytic domain (CD) and finally CFP. a) In its basal state, there is minimal FRET, as the CFP and Venus fluorophores are not within 10 nm of one another. Thus, if CFP is excited, cyan fluorescence will be emitted. b) When Akind is recruited to the plasma membrane via an interaction between its PH domain and PIP3, it is activated by two phosphorylation events (P). These phosphorylation events cause a conformational change in the Akind probe, bringing the CFP and Venus fluorophores within close enough proximity to allow FRET to occur.

76

Figure 20: Comparison of the FRET ratio images for wild-type and mutant Akind

probes. (A) Representative FRET/CFP ratio images collected with a x63/1.4 NA

objective lens for HT1080 cells expressing either wild-type (Wt) Akind or a non-

activatable Akind mutant in which 3 key residues have been mutated to alanine (3A-

Akind) are shown. Scale bar is 10 µm. (B) Quantification of the average FRET/CFP ratio

in HT-1080 cells expressing either Wt-Akind or 3A-Akind is shown. Error bars represent

the S.E.M for at least 7 cells (p= 0.035). The asterisk indicates a statistically significant

difference compared with Wt-Akind.

77

Figure 21. APPL1 decreases Akt activity in cells. (A) HT1080 cells were co-transfected with either mCherry or mCherry-APPL1 and the Akt activity FRET probe Akind. Ratio images of FRET/CFP are shown in pseudo-color coding (left panels). Scale bar = 10 µm. Quantification of the average FRET/CFP ratio for cells co-transfected with either mCherry or mCherry-APPL1 and Akind is shown (right panel). Error bars represent the S.E.M. from greater than 34 cells from at least three individual experiments (*p<0.0001). (B) HT1080 cells were co-transfected with either mCherry or mCherry-APPL1 and Akind. Ratio images of FRET/CFP at the cell edge are shown in pseudo-color coding (left panels). Scale bar = 5 µm. A line scan analysis was performed on the boxed region, which represents an area 5 µm long and 1.3 µm wide. Quantification of the line scan analysis is shown (right panel). Error bars represent the S.E.M. of 52-59 total line scans from 20 cells from three separate experiments.

78

When we quantified Akt activity as a function of distance from the edge of cells, the

FRET/CFP ratio in control cells was high at the cell edge (Figure 21B), indicating active

Akt was localized to this region. In mCherry-APPL1 expressing cells, the FRET/CFP

ratio was decreased 2.9-fold at the cell edge compared with controls (Figure 21B). Akt

activity was also decreased 2.2-fold at a distance of 5 m behind the cell edge in

mCherry-APPL1 expressing cells (Figure 21B). Taken together, these results indicate

that APPL1 decreases the amount of active Akt in cells, and a significant reduction of

Akt activity is seen at the cell edge.

Since APPL1 affected the level of active Akt at the cell edge, and APPL1 and Akt

modulated the turnover of adhesions at the leading edge, we hypothesized that APPL1

regulates the amount of active Akt in adhesions. We addressed this by co-

immunostaining control and APPL1 expressing cells for active Akt, using the phospho-

Thr308-Akt antibody, and paxillin. Individual paxillin-containing adhesions were

visualized using total internal reflection fluorescence (TIRF) microscopy, and the levels

of active Akt were quantified in these adhesions. The amount of active Akt in adhesions

in APPL1 expressing cells was decreased 1.7-fold as compared to that observed in

control cells (Figure 22, A and B). This result suggests that APPL1 regulates cell

migration and adhesion turnover by reducing the amount of active Akt in adhesions.

APPL1 regulates the tyrosine phosphorylation of Akt by Src: Since tyrosine

phosphorylation of Akt by Src has recently been shown to be important in both the

activation of Akt as well as its biological function (Chen et al., 2001; Choudhury et al.,

2006; Bouchard et al., 2007), we hypothesized that Src-mediated tyrosine

phosphorylation of Akt was critical for its effects on migration. We began to test this

hypothesis by assessing tyrosine phosphorylation of Akt by Src in HT1080 cells. Wild-

type HT1080 cells were transfected with FLAG-Akt and subsequently treated with

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Figure 22. APPL1 reduces the amount of active Akt in adhesions. (A) HT1080 cells were transfected with CFP and either empty vector (Control) or FLAG-APPL1 (APPL1), fixed, and immunostained for active Akt, using a phospho-Thr308-specific antibody (P-Akt), and paxillin. TIRF images of paxillin (left panels) and P-Akt (middle panels) are shown. P-Akt images are shown in pseudo-color coding that indicates the range of fluorescent intensities to the assigned color (P-Akt, right panels). Overlays of paxillin and P-Akt are shown (far right panels). Scale bar = 2 µm. (B) Quantification of phospho-Thr308 Akt levels in adhesions in cells transfected with the constructs from panel A is shown. Error bars represent the S.E.M. of greater than 4782 adhesions from 58-63 cells from three separate experiments (*p=0.0251).

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various concentrations of the Src family kinase inhibitor PP2. Treatment with 1 M PP2

decreased Akt tyrosine phosphorylation by 1.8-fold compared to DMSO controls, while

7.5 M PP2 decreased the levels of tyrosine phosphorylation by 4.6 fold (Figure 23A).

To further support a role for Src in Akt tyrosine phosphorylation, we transfected HT1080

cells with constitutively-active Src (CA-Src). Expression of CA-Src resulted in a 10-fold

increase in the amount of Akt tyrosine phosphorylation compared to controls (Figure

23B), suggesting a critical role for Src in mediating Akt tyrosine phosphorylation.

We next assessed the ability of APPL1 to regulate Akt tyrosine phosphorylation.

When APPL1 was co-expressed with FLAG-Akt in HT1080 cells, tyrosine

phosphorylation of Akt was decreased 1.9-fold compared to control cells (Figure 23C).

Moreover, expression of APPL1 with CA-Src reduced Akt tyrosine phosphorylation by

2.4-fold (Figure 23D). Collectively, these data point to an important new function for

APPL1 in regulating the Src-mediated tyrosine phosphorylation of Akt.

Src-mediated tyrosine phosphorylation of Akt is critical for its activation

and function: Since our data indicated that APPL1 regulates the amount of active Akt in

cells, we thought it may be through a mechanism that involves Src and the tyrosine

phosphorylation of Akt. In initial experiments, we assessed the ability of APPL1 and Src

to regulate Akt T308 phosphorylation. Expression of APPL1 led to a 1.5-fold reduction in

Akt T308 phosphorylation as compared to control cells, which confirmed our previous

experiments showing that APPL1 decreases the amount of active Akt (Figure 24A). We

next examined the effects of Src activity on Akt T308 phosphorylation. Expression of CA-

Src resulted in a 4-fold increase in Akt T308 phosphorylation (Figure 24A). However,

when APPL1 was co-expressed with CA-Src in HT1080 cells, Akt T308 phosphorylation

was decreased significantly compared to that observed in cells expressing CA-Src

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Figure 23. Src mediates tyrosine phosphorylation of Akt. (A) FLAG-Akt transfected HT1080 cells were incubated with the indicated concentrations of PP2 for 1.5 h. FLAG-Akt protein was immunoprecipitated from cell lysates, and FLAG-Akt samples were subjected to immunoblot analysis to determine the levels of total FLAG-Akt, using FLAG M2 antibody (FLAG), and tyrosine phosphorylated Akt with 4G10 monoclonal antibody (4G10) (left panels). Quantification of the amount of Akt tyrosine phosphorylation is

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shown relative to the Control (0 µM PP2) (right panel). Error bars represent the S.E.M. from three separate experiments (*p≤0.0031). (B) HT1080 cells were co-transfected with FLAG-Akt and either GFP (Control) or GFP-CA-Src. Immunoprecipitated FLAG-Akt protein samples were immunoblotted for total FLAG-Akt (FLAG) and tyrosine phosphorylated Akt (4G10) (left panels). Quantification of the relative amount of Akt tyrosine phosphorylation compared to Control is shown (right panel). Error bars represent the S.E.M. from three separate experiments (*p=0.025). (C) FLAG-Akt was immunoprecipitated from lysates of cells expressing FLAG-Akt and either GFP (Control) or GFP-APPL1 (APPL1). Samples were subjected to immunoblot analysis to determine the levels of total FLAG-Akt (FLAG) and tyrosine phosphorylated Akt (4G10) (left panels). Quantification of the relative amount of Akt tyrosine phosphorylation compared to Control is shown (right panel). Error bars represent the S.E.M. from three separate experiments (*p≤0.0001). (D) HT1080 cells were co-transfected with FLAG-Akt and either mCherry + GFP-CA-Src (CA-Src) or mCherry-APPL1 + GFP-CA-Src (CA-Src + APPL1). Immunoprecipitated FLAG-Akt protein samples were subjected to immunoblot analysis to determine the levels of total FLAG-Akt (FLAG) and tyrosine phosphorylated Akt (4G10) (left panels). Quantification of the relative amount of Akt tyrosine phosphorylation compared that observed in Control cells from panel B is shown (right panel). Error bars represent the S.E.M. from three separate experiments (*p=0.048). Asterisk indicates a statistically significant difference compared with CA-Src transfected cells.

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Figure 24. Tyrosine phosphorylation of Akt regulates its activation and function. (A) HT1080 cells were co-transfected with FLAG-Akt and either mCherry + GFP

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(Control), mCherry-APPL1 + GFP (APPL1), mCherry + GFP-CA-Src (CA-Src), or mCherry-APPL1 + GFP-CA-Src (CA-Src + APPL1). After 24 h, FLAG-Akt was immunoprecipitated from cell lysates and subjected to immunoblot analysis to determine the levels of total FLAG-Akt (FLAG) and T308 phosphorylated Akt (T308) (left panels). Quantification of the relative amount of T308 phosphorylated Akt compared to Control is shown (right panel). Error bars represent the S.E.M. from at least ten separate experiments (*p<0.0001, **p≤0.002, ***p=0.031). (B) HT1080 cells were transfected with FLAG-Akt (Wt-Akt) or FLAG-Akt-Y315F/Y326F (Akt-Y315F/Y326F). Immunoprecipitated FLAG-Akt protein was subjected to immunoblot analysis to determine the levels of total FLAG-Akt (FLAG) and tyrosine phosphorylated Akt (4G10) (upper panels). Quantification of the relative amount of Akt tyrosine phosphorylation compared to Wt-Akt is shown (lower panel). Error bars represent the S.E.M. from four separate experiments (*p<0.0001). (C) HT1080 cells were transfected with GFP-CA-Src (CA-Src) and either FLAG-Akt (Wt-Akt) or FLAG-Akt-Y315F/Y326F (Akt-Y315F/Y326F). After 24 h, FLAG-Akt protein was immunoprecipitated from cell lysates, and samples were subjected to immunoblot analysis to determine the levels of total FLAG-Akt (FLAG) and tyrosine phosphorylated Akt (4G10) (upper panels). Quantification of the relative amount of Akt tyrosine phosphorylation compared to that observed in cells transfected with Wt-Akt + CA-Src is shown (lower panel). Error bars represent the S.E.M. from three separate experiments (*p<0.0001). (D) HT1080 cells were co-transfected with GFP and either empty vector (Control), constitutively active Akt (CA-Akt), or CA-Akt-Y315F/Y326F and used in migration assays. Rose plots with migration tracks for these cells are shown (left panels). Quantification of the migration speed for cells transfected with the indicated constructs is shown (right panel). Error bars represent the S.E.M. for at least 56 cells from at least three separate experiments (*p<0.0001, **p=0.0019).

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(Figure 24A). Thus, these results suggest APPL1 reduces the amount of active Akt in

cells by inhibiting tyrosine phosphorylation of Akt by Src. Since previous work had shown

that the major Src phosphorylation sites in Akt, which are important in regulating its

activity and function, are tyrosines 315 and 326 (Chen et al., 2001), we mutated these

tyrosine residues to phenylalanines. In cells expressing the Akt tyrosine mutant (Akt-

Y315F/Y326F), a 1.6-fold decrease in tyrosine phosphorylation was observed compared

to that seen in wild-type Akt (Wt-Akt) expressing cells (Figure 24B). In addition, the CA-

Src-mediated increase in Akt tyrosine phosphorylation was reduced by 1.7-fold in cells

expressing Akt-Y315F/Y326F compared to Wt-Akt expressing cells (Figure 24C). These

results suggest that residues 315 and 326 are major targets of phosphorylation by Src.

Next, we assessed the importance of phosphorylation at tyrosines 315 and 326

in regulating Akt-mediated migration. Consistent with our previous data, expression of

CA-Akt in HT1080 cells promoted a 1.2-fold increase in the migration speed compared

to controls (Figure 24D). In contrast, mutation of tyrosines 315 and 326 in CA-Akt

significantly reduced the migration of HT1080 cells. The migration speed of cells

expressing CA-Akt-Y315F/Y326F was decreased 1.5-fold compared to that observed in

control cells (Figure 24D). Taken together, these results indicate that tyrosine

phosphorylation by Src is a critical regulator of Akt-mediated cell migration, and APPL1

inhibits migration by decreasing this tyrosine phosphorylation.

Discussion

While the signaling adaptor APPL1 has been implicated in the modulation of

various cellular processes, such as proliferation and survival (Liu et al., 2002; Schenck

et al., 2008), its role in controlling cell migration is not well understood. Here we show

that APPL1 impairs the migration of HT1080 cells by regulating the assembly and

disassembly of leading edge adhesions. APPL1 modulates migration and adhesion

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dynamics through a molecular mechanism that is dependent on the Src-mediated

tyrosine phosphorylation of Akt. Interestingly, APPL1 was recently shown to affect the

ability of murine embryonic fibroblasts (MEFs) to migrate in response to HGF (Tan et al.,

2010), which is consistent with our data indicating that it is an important modulator of this

process. Intriguingly, this study found that APPL1 was dispensable for the survival of

MEFs, at least under normal culture conditions (Tan et al., 2010).

Our results indicate that APPL1 regulates cell migration through its multi-

functional domains, which mediate its interaction with other proteins as well as lipids.

When the PTB domain of APPL1 is deleted, it is unable to inhibit migration in HT1080

cells. This region of APPL1 has been previously shown to be important in its binding to

Akt (Mitsuuchi et al., 1999), suggesting APPL1 modulates migration through Akt.

Nevertheless, we cannot rule out contributions from other APPL1 interacting proteins,

since the tumor suppressor DCC, human follicle-stimulating hormone receptor, the

neurotrophin receptor TrkA, and the TrkA-interacting protein GIPC1 have also been

shown to bind to this region of APPL1 (Liu et al., 2002; Nechamen et al., 2004; Lin et al.,

2006). However, we provide additional results that strongly demonstrate APPL1

regulates migration by modulating Akt activity and function.

We show that Akt is a positive regulator of migration in HT1080 cells, where CA-

Akt increases migration speed, while DN-Akt and knockdown of endogenous Akt both

decrease migration. When APPL1 is exogenously expressed with CA-Akt (3-fold over

endogenous), it abolishes the CA-Akt-promoted increase in migration, indicating that

APPL1 inhibits Akt function. In contrast, increasing the amount of CA-Akt (5-fold over

endogenous) negates this effect of APPL1, demonstrating higher expression of CA-Akt

can overcome this inhibition by APPL1. When APPL1 is co-expressed with either DN-

Akt or in Akt knockdown cells, no further decrease in migration is observed, suggesting

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APPL1 and Akt are in the same signaling pathway that regulates migration. This role of

Akt in promoting cell migration is consistent with previous studies (Kim et al., 2001; Irie

et al., 2005; Ju et al., 2007; Bristow et al., 2009). Interestingly, some previous studies

looking at the relationship between APPL1 and Akt have shown APPL1 to be a positive

regulator of Akt activation (Nechamen et al., 2004; Lin et al., 2006; Saito et al., 2007;

Cheng et al., 2009; Wen et al., 2010), whereas our results indicate APPL1 decreases

the amount of active Akt. This discrepancy may be, at least in part, due to the isoform of

Akt being observed. The major isoform of Akt in HT1080 cells is Akt1 (Figure 25), while

most of the previous work was focused on Insulin/Akt2 signaling or on signaling in the

nervous system where Akt3 is the major isoform. Indeed, recent work has shown that

APPL1 inhibits Akt1 activity (Bohdanowicz et al., 2011; Tu et al., 2011).

Several residues (147, 153 and 155) within the BAR domain of APPL1 are

essential for its ability to regulate cell migration. The BAR domain of APPL1 is

structurally unique in that it interacts with the PH domain to form a functional unit (Li et

al., 2007; Zhu et al., 2007). This integrated functional dimer interacts with the

endosomal protein Rab5 and is responsible for APPL1’s endosomal localization

(Miaczynska et al., 2004; Li et al., 2007; Zhu et al., 2007). The endosomal localization is

important for APPL1 to regulate Akt substrate specificity (Schenck et al., 2008),

suggesting that APPL1 signaling on endosomes is critical to its function. Indeed, our

results indicate that APPL1 localization to endosomal membranes is critical for its ability

to regulate cell migration through Src and Akt. Akt activation, which is typically thought

to occur at the plasma membrane, has also been shown to take place on signaling

endosomes (Wang et al., 2002). In this context, APPL1 may function as a scaffold for

bringing signaling proteins to endosomal structures, which can be targeted to specific

regions within the cell in a spatiotemporal manner.

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Figure 25. Akt1 is the major Akt isoform in HT1080 cells. Lysates from HT1080 cells were subjected to immunoblot analysis to determine the expression levels of the indicated Akt isoforms and β-actin (as a loading control).

89

While several adaptor proteins have recently been reported to regulate

processes underlying migration, namely adhesion dynamics (Nayal et al., 2006; Zaidel-

Bar et al., 2007; Kanchanawong et al., 2010), the importance of APPL1 in contributing to

this process is unknown. We show that APPL1 is a negative regulator of adhesion

turnover where exogenous expression of APPL1 increases the apparent t1/2 for adhesion

assembly as well as the t1/2 for adhesion disassembly. Knockdown of endogenous

APPL1 has the opposite effect on adhesion turnover. This phenotype is dependent on

the PTB domain of APPL1, as expression of the APPL1-∆PTB mutant has no effect on

adhesion turnover. The dependence on the PTB domain suggests that Akt contributes to

the APPL1-mediated regulation of adhesion turnover. Indeed, we have previously

demonstrated a potential role for Akt in regulating adhesion dynamics (Bristow et al.,

2009) and show here that expression of CA-Akt stimulates more rapid adhesion turnover,

while DN-Akt induces slower turnover. Co-expression of exogenous APPL1 with CA-Akt

negates the CA-Akt-promoted increase in adhesion turnover, while co-expression with

DN-Akt has no additional effect. Moreover, expression of APPL1 causes a decrease in

the amount of active Akt at the cell edge as well as in adhesions. Thus, APPL1 may

regulate the assembly and disassembly of adhesions at the leading edge by inhibiting

Akt function. This would lead to impaired turnover of leading edge adhesions, which

could significantly slow cell migration.

Phosphorylation at threonine 308 and serine 473 has classically been thought to

activate Akt (Alessi et al., 1996). However, more recent work indicates that Akt activity

is also regulated by tyrosine phosphorylation, which is carried out by Src (Chen et al.,

2001). In our study, inhibition of Src with PP2 led to a decrease in the tyrosine

phosphorylation of Akt, while promotion of Src activity, through expression of CA-Src,

increased the level of tyrosine phosphorylated Akt, indicating that Src can tyrosine

90

phosphorylate Akt. In addition, APPL1 decreased tyrosine phosphorylation of Akt and

inhibited the CA-Src-promoted increase in Akt tyrosine phosphorylation. These

alterations in tyrosine phosphorylation are accompanied by corresponding changes in

T308 phosphorylation of Akt, which has not been previously shown. Moreover, mutation

of two previously described Src phosphorylation targets (tyrosines 315 and 326) (Chen

et al., 2001) to phenylalanines in CA-Akt reduced migration similarly to that observed

with co-expression of APPL1 with CA-Akt. Thus, APPL1 can inhibit Akt function by

reducing the tyrosine phosphorylation of Akt by Src, which hinders cell migration.

Our results support a working model in which the adaptor protein APPL1 inhibits

cell migration and adhesion dynamics through a mechanism involving the Src-mediated

tyrosine phosphorylation of Akt. Tyrosine phosphorylation of Akt by Src enhances the

activity of Akt. APPL1, in turn, decreases the amount of active Akt in adhesions and at

the cell edge by reducing Akt tyrosine phosphorylation. This leads to an inhibition of Akt

function particularly within regions of cells where Akt activity is high, such as the cell

edge and adhesions. As a result, the ability of cells to turn over their adhesions is

diminished, which leads to an impairment of cell migration.

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Chapter IV

CONCLUSIONS AND FUTURE DIRECTIONS

Cell migration is a complex process that requires highly coordinated,

spatiotemporally regulated signaling networks. Many of the major signaling molecules

(for example, kinases and phosphatases) are ubiquitously expressed, and yet they can

have cell-type and tissue-specific effects. One of the prevailing ideas as to how this

occurs is that other molecules in the cell’s protein milieu are able to differentially regulate

these signaling hubs. For example, adaptor proteins are able to localize signaling to

specific regions of cells, through the use of their multiple domains. They are able to bring

signaling components together as well as localize them to subcellular domains (for

example, the leading edge of a migrating cell) through interactions with other proteins as

well as lipids. In this way, they are strong candidates for modulating highly complex,

spatiotemporally regulated processes, such as cell migration.

Here, for the first time, we have generated a comprehensive phosphorylation

map of the adaptor protein APPL1. We identified a total of 13 phosphorylated residues

within APPL1 using mass spectrometry. Interestingly, four of these 13 phosphorylated

residues are located within functional domains of APPL1, one within the BAR domain,

two near the edge of the PH domain, and one within the PTB domain (Tables 1-2 and

Figures 6-7). These represent key potential targets for regulation of APPL1 function. We

expect that mutation of these phosphorylated residues, using site directed mutagenesis,

would lead to alterations in APPL1 function. For example, one of these potential sites,

serine 97/98, lies on the concave surface of the BAR domain (Figure 7). This domain is

essential in APPL1 dimerization and localization (Zhu, Chen et al. 2007; Chial, Lenart et

al. 2010). We will use site directed mutagenesis to determine the effects of both a non-

92

phosphorylatable (serinealanine) and a phosphomimetic (serineaspartic acid)

mutation on the localization of a GFP-tagged APPL1. We expect that phosphorylation

would lead to a dissociation of APPL1 from the membrane, as it is placing a bulky

charged group between the APPL1-BAR domain’s concave surface and the lipid

membrane. Therefore, we would expect the phosphomimetic mutant to no longer

properly localize to membrane structure, while the non-phosphorylatable APPL1 would

have no change or exhibit an increase in membrane localization. Moreover, serine 374

lies within the PH domain and is in an area important for APPL1 interaction with the

small GTPase Rab5 (Zhu, Chen et al. 2007). Therefore, phosphorylation at this site may

regulate the interaction of APPL1 and Rab5, and represents an interesting area for

future studies. Finally, we have identified serine 427 as a putative substrate of Akt

(Tables 4 and 5). This site is located near the APPL1 PTB domain (Figure 6), which is

the domain responsible for APPL1 and Akt interaction (Mitsuuchi, Johnson et al. 1999).

Therefore, Akt may regulate APPL1 by binding and phosphorylating APPL1 at this site.

Future studies, using site directed mutagenesis of this site, may lead to important

insights as to how APPL1 and Akt interact to regulate cell migration and adhesion

turnover.

Here, we show a novel role for the adaptor protein APPL1 as a regulator of cell

migration and adhesion turnover through its ability to modulate Akt activation and

function. Expression of APPL1 in HT1080 cells decreases the average speed of cell

migration. The ability of cells to both assemble and disassemble their adhesions

(adhesion turnover) at the leading edge is critical for efficient migration to occur (Webb,

Parsons et al. 2002). Interestingly, APPL1 expression leads to an increase in the t1/2

values of adhesion assembly and disassembly, indicating APPL1 functions to slow

adhesion turnover. It will be interesting to determine if other important aspects of cell

migration are also altered by APPL1, for example alterations of leading edge protrusion

93

and actin dynamics, or changes in cell rear detachment. These all represent potential

areas of future study. For instance, we will look at leading edge protrusion dynamics

using live cell imaging coupled with kymography to observe extension and retraction

rates of GFP-APPL1 expressing and control cells.

APPL1 was first identified as an Akt binding partner. Akt has previously been

shown to regulate cell migration. Therefore, we hypothesized APPL1 may regulate

migration through Akt. Expression of CA-Akt leads to an increase in the average speed

of HT1080 cells, while expression of DN-Akt has the opposite effect. Interestingly,

APPL1 is able to suppress CA-Akt-promoted migration, while it has no additive effect

with DN-Akt, suggesting these two proteins are in the same pathway that regulates

migration. Moreover, analysis of adhesion turnover was used here to show CA-Akt leads

to more rapid adhesion turnover, and APPL1 can abolish this effect. Also, DN-Akt leads

to less rapid adhesion turnover, and APPL1 has no additive effects on DN-Akt

suppressed adhesion turnover. These results are consistent with APPL1 reducing cell

migration and adhesion turnover by inhibiting Akt function.

The effects of APPL1 on Akt2 signaling, especially in the context of insulin

signaling, is relatively well characterized compared to the effects of APPL1 on the other

isoforms of Akt (Akt1 and Akt3). It is well established that APPL1 functions to increase

Akt2 activation (Cheng, Iglesias et al. 2009; Deepa and Dong 2009). Therefore, when

we first discovered that APPL1 reduced the amount of active Akt in HT1080 cells, we

were slightly puzzled. However, we soon discovered that the major isoform of Akt

present in these cells is Akt1, and there is little to no Akt2 or Akt3 (Figure 25). In addition,

others have recently shown that indeed there is an Akt isoform specific effect of APPL1,

where APPL1 reduces active Akt1 (Tan, You et al. 2010; Bohdanowicz, Balkin et al.

2012). This is quite exciting, as the role of Akt in migration seems to have some isoform

specificity (Stambolic and Woodgett 2006). It would be interesting to determine if these

94

disparities could be explained by differential regulation of Akt1 and Akt2 by APPL1, and

provides a very attractive avenue of future study, as there are also distinct effects of

Akt1 and Akt2 on tumor invasion and metastasis (Irie, Pearline et al. 2005; Maroulakou,

Oemler et al. 2007). Moreover, there is little known about the effects of Akt isoform

specificity on adhesion turnover.

In addition to phosphorylation of threonine 308 and serine 473, tyrosine

phosphorylation has also been shown to regulate Akt activation and function (Chen, Kim

et al. 2001; Choudhury, Mahimainathan et al. 2006). This phosphorylation is mediated

by Src. Here, we show that APPL1 can inhibit Src-mediated tyrosine phosphorylation of

Akt. Moreover, we have identified a novel role of Akt tyrosine phosphorylation in

mediating Akt-promoted cell migration. We propose a model in which APPL1 regulates

cell migration and adhesion turnover my modulating Src-mediated phosphorylation of

Akt (Figure 26).

We used an APPL1 mislocalization mutant (APPL1-AAA) to show that the

localization of APPL1 to endosomal membranes is critical to its function in modulating

the amount of active Akt in cells as well as regulating cell migration and adhesion

turnover. A recent study has shown that APPL1 reduces the amount of active Akt at

phagosomes by recruiting OCRL (an inositol 5-phosphatase) to these structures

(Bohdanowicz, Balkin et al. 2012). There, OCRL reduces the available PIPs needed to

activate Akt. It would be interesting to determine if APPL1, in HT1080 cells, reduces the

amount of active Akt on endosomes. We will use the Akt activation FRET probe, Akind,

to observe any changes in the activation of Akt on endosomal structures in APPL1

expressing and control cells. We expect to see a reduction in Akt activation in the

endosomal compartments when we express APPL1. This may explain why the APPL1

mislocalization mutant had no effect, as it may no longer reduce this localized population

of active Akt at endosomes.

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Figure 26. Proposed model of APPL1-mediated effects on cell migration and adhesion turnover. Akt functions to promote cell migration and adhesion turnover once fully activated (phosphorylated). Src phosphorylates (P) Akt at tyrosines 315 and 326 (Y315 and Y326). This promotes Akt activation and Akt is subsequently phosphorylated on threonine 308 and serine 473 (T308 and S473). Upon full activation, Akt then promotes an increase in migration speed and rate of adhesion turnover. APPL1 inhibits Src-mediated tyrosine phosphorylation of Akt and thereby inhibits Akt-promoted migration and adhesion turnover.

96

We discovered active Akt (as determined by threonine 308 phosphorylation state)

is present in adhesions, which, as far as we know, has not been previously described.

APPL1 expression reduces active Akt in adhesions, and we hypothesize this has an

effect on adhesion turnover. It would be exciting to determine if APPL1 also affects the

amount of tyrosine phosphorylated Akt in adhesions. We can determine this by immuno-

staining APPL1 expressing and control cells with an antibody that is specific to Akt

phosphorylated at tyrosines 315 and 326. Tyrosine phosphorylation of Akt regulates its

effects on migration. Therefore, we would expect that this would also be true for

adhesion turnover. We can also use CA-Akt in which we mutate the important tyrosine

phosphorylation sites (tyrosines 315 and 326), such that they can no longer be

phosphorylated (tyrosine phenylalanine), and ask what effect this has on Akt-

promoted adhesion turnover.

Tyrosine phosphorylation of Akt is mediated by the non-receptor tyrosine kinase

Src (Chen, Kim et al. 2001; Choudhury, Mahimainathan et al. 2006). Here, we show that

APPL1 can inhibit Src-mediated tyrosine phosphorylation of Akt. However, we do not

understand the mechanism by which APPL1 inhibits Src function. Interestingly, when

GFP-Src is co-expressed with mCherry-APPL1, we see these two proteins localized

together in a subpopulation of vesicles (Figure 27). We would like to determine if APPL1

is reducing Src-mediated tyrosine phosphorylation of Akt by altering Src localization,

perhaps retaining Src in an endosomal compartment in which it can no longer act on Akt.

We can use various fluorescently-tagged Src constructs to monitor the effects of APPL1

on Src localization. In this context, it would also be interesting to look at the effects of the

APPL1 mislocalization mutant (APPL1-AAA) on Src localization. We would expect that

APPL1 localization to endosomal membranes would be required to alter Src localization

in these structures.

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Figure 27. APPL1 and Src colocalize in the same vesicles. When GFP-Src and mCherry-APPL1 are co-expressed in HT1080 cells, we see a subpopulation of APPL1 vesicles that also contain Src (arrows).

98

Src typically exists in an auto-inhibited confirmation where its SH2 domain binds

to a phosphorylation site within its C-terminus (Guarino 2010). It becomes activated

when this phosphate is removed. Src then adopts an open, activated conformation, and

a phosphorylation event in the catalytic domain promotes full activation (Guarino 2010).

Therefore, APPL1 may regulate Src function by modulating the activation state of Src.

We can use phospho-specific antibodies against the key regulatory phosphorylation

sites in Src to observe any effects APPL1 has on Src activation using Western blot

analysis. Moreover, we can use these same antibodies, in conjunction with immuno-

fluorescent microscopy, to determine if APPL1 affects localized changes in active Src

within individual cells. This may lead to a better understanding of how APPL1 modulates

both Src and Akt function to regulate cell migration and adhesion turnover.

Finally, we have preliminary results suggesting that APPL1 expression leads to a

decrease in the active form of the Rho GTPase family member Rac. This was observed

in an active GTPase pulldown assay (Figure 28), see Bristow et al 2009 for methods. In

addition, use of an active Rac FRET probe, similar to the Akind FRET probe, showed

that APPL1 expressing cells have a reduced amount of active Rac (Figure 28). Further

studies are needed to determine if this reduction in active Rac is also part of the

APPL1/Src/Akt pathway that regulates cell migration and adhesion turnover.

Interestingly, previous studies have shown that Akt can be upstream of Rac activation

(Kobayashi, Hino et al. 2006). Moreover, Akt has been shown to regulate PAK and

GSK3, both of which have been shown to regulate Rac activation, and both are

involved in processes regulating cell migration, such as adhesion turnover (Zhou, Zhuo

et al. 2003; Irie, Pearline et al. 2005; Kobayashi, Hino et al. 2006; Zhou, Tucker et al.

2006). Therefore, these molecules are potential members of the APPL1 pathway that

regulates cell migration and adhesion turnover, and represent potentially fruitful avenues

for future study.

99

Figure 28. APPL1 reduces the amount of active Rac in cells. (Left, upper) Western blot showing APPL1 expression leads to a decrease in the amount of active Rac pulled down in an active GTPase assay compared to control cells. (Left, lower) Quantification of the relative amount of active Rac compared to Control is shown. Error bars represent the S.E.M. from four separate experiments (*p<0.0001). (Right, upper) HT1080 cells were co-transfected with either mCherry (Control) or mCherry-APPL1 (APPL1) and the Rac activity FRET probe. Ratio images of FRET/CFP are shown in pseudo-color (warm colors correspond to higher levels of active Rac compared to cooler colors). (Right, lower) Quantification of the average FRET/CFP ratio for cells co-transfected with either mCherry or mCherry-APPL1 and the Rac FRET probe is shown. Error bars represent the S.E.M. from greater than 16 cells (*p=0.0008).

100

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