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INTERACTIONS AMONG THE MITOGEN-ACTIVATED PROTEIN KINASE CASCADES AND THE IDENTIFICATION OF A NOVEL cdc2-

RELATED PROTEIN KINASE

Susan Randall

A thesis submitted in conformity with the requirements for the Degree of Master of Science, Graduate Department of Medical Biophysics, in the

University of Toronto

O Copyright by Susan Randall 1997

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I I L b C L Q L b I U 1 1 3 Q U I V l l E bIAC LIII bUfjCIA'CLbbI V 4 b C U P L U LCILL N A l Q 3 t Z LQ3LQUC3 4 L 1 U b l l C

identification of a novel cdc2-related protein kinase.

Susan Randall Master of Science, 1997

Department of Medical Biophysics University of Toronto

ABSTRACT

Signal transduction is the means by which cells recognize and respond

to changes in their extemal environment through the modification of

cytoplasmic and nuclear events. In several cases, transduction of the signal

to the nucleus of the ce11 occurs through cascades of protein kinases.

In mammalian cells, structurally homologous mitogen-activated protein

kinase (MAPK) cascades regulate diverse biological functions. This raises the

question of how specificity is achieved in signalling via MAPKs. With the

yeast two hybrid system, we reveal that the three known MARK pathways,

ERK, SAPK, and p38, do not require scaffolding proteins to maintain signalling

specificity and that crosstalk among them does not readily occur in vivo.

The cloning of a novel67 kD cytoplasmic serine/threonine kinase is also

described. This kinase, termed NKIATRE, is the third member of a unique

family of kinases that are highly related to the MAPKs and the cyclin-

dependent kinases.

n w a u - v T V -UUUATLUAW A u

The work accomplished in this thesis would not have been possible

without the support of many people. Firstly, 1 would like to thank the

members of my supervisory committee, Dr. Brent Zanke, Dr. J im Woodgett,

Dr. Dwayne Barber, and the late Dr. Ron Buick, for not only their support but

their guidance. I would also like to thank both Kim Boudreau and Phyllis

Billia for helping me to master many of the laboratory techniques 1 have

learned and for motivating me to attend many more exercise classes than 1

would have on my own. To the remainder of my friends, my parents, my

sister, and especially Gabe, thanks for believing in me and putting up with me!

Abstract

Acknowledgements

Table of Contents

List of Tables

List of Figures

List of Abbreviations

List of S p b o l s

Chapter 1: Literature Review

1.1 Introduction

1.2 Kinases 1.2.1 Conserved Features 1.2.2 Three Dimensional Structure

xii

1.3 Mitogen-Activated Protein Kinase Signalling 19 1.3.1 The FUS3/KSS1 and ERKUERKZ Signalling Pathways 20 1.3.2 The SAPK Pathway 25 1.3.3 The HOGI and p38 Pathways 28 1.3.4 The ERK5 Signalling Pathway 33 1.3.5 The SAPK3/ERK6 Signalling Pathway 34 1.3.6 Crosstalk 35

1.4 The Eukaryotic Ce11 Cycle 1.4.1 Ce11 Regulation by CDKs 1.4.2 Cyclins as Activators of CDKs 1.4.3 Cyclin-CDK Complexes Throughout the Ce11 Cycle 1.4.4 Modulation of Cyclin-CDK Activity

1.4.4.1 Activation by Phosphorylation 1.4.4.2 Inhibition by Phosphorylation 1.4.4.3 CDK Inhibitors

1.5 cdc2-Related Protein Kinases 1.5.1 KKIALRE and KKIAMRE

Chapter 2: Examination of In Vivo Protein Interactions Among the Three Known Mitogen-Activated Protein Kinase Pathways 51

2.1 Introduction 52

2.2 Materials and Methods 2.2.1 The Yeast Two Hybrid System 2.2.2 Plasmid DNAs 2.2.3 Transformation of Yeast 2.2.4 Liquid Culture B-galactosidase Assays 2.2.5 Filter Assays for B-galactosidase Activity

2.3 Results 2.3.1 f3-galactosidase Liquid Culture Assays 2.3.2 bgalactosidase Filter Assays

2.4 Discussion 65

Chapter 3: The Cloning of a Serine/Threonine Specific Kinase with Homology to the Cyclin-Dependent Kinases and the Mitogen-Activated Protein Kinases 70

3.1 Introduction 71

3.2 Materials and Methods 3.2.1 PCR Amplification and cDNA Cloning 3.2.2 DNA Sequence Analysis 3.2.3 Amplification of 5' cDNA End 3.2.4 Southem Blot Analysis 3.2.5 Northern Blot Analysis 3.2.6 Prokaryotic Expression of NKIATRE 3.2.7 Preparation for Eukaryotic Expression 3.2.8 In Vitro Transcription and Translation

Results 3.3.1 Isolation of a Rat cDNA 3.3.2 Amplification of the 5' cDNA End 3.3.3 Sequence Analysis 3.3.4 Southern Blot Analysis 3.3.5 Northern Blot Analysis 3.3.6 Prokaryotic Expression of NKIATRE 3.3.7 In Vitro Transcription and Translation

UiX U I U U U U U I U A A

3.5 Future Work

Chapter 4: References

Table 1: Primers

vii

Figure 1:

Figure 2:

Figure 3:

Figure 4:

Figure 5:

Figure 6:

Figure 7:

Figure 8:

Figure 9:

Figure 10:

Figure 11:

Figure 12:

Figure 13:

Figure 14:

Figure 15:

Figure 16:

Figure 17:

Figure 18:

Figure 19:

Figure 20:

Figure 21:

The FUS3/KSS1 and ERK signalling pathways.

The SAPK signalling pathway.

The HOG and p38 signalling pathways.

The mammalian ceIl cycle.

The basis of the Yeast Two Hybrid System.

Flow diagram of the Yeast Two Hybrid System.

Functional map of pAS1-CYH2.

Functional map of pACTII.

$-galactosidase liquid culture assays.

&galadosidase filter assays.

Design of the degenerate oligonucleotide primers.

Flow diagram of the 5' RACE procedure.

PCR amplification product. 88

Sequence alignment of the cDNA clones. 90

Sequence of NKIATRE. 91

Product of the 5' RACE procedure. 92

Analysis of NKIATRE as a serine/threonine specific kinase. 93

Multiple sequence alignment. 97

Dendogram. 100

Southern blot of rat genomic DNA for NKMTRE. 102

Cross species Southern Hot for NKIATRE. 103

B . .

Vll l

I A ~ U A G YY. U ~ ~ & ~ U U I V L A VA A ~ A U X A A Y Y AAA I U U UAUUUYY. AVI

Figure 23: Prokaryotic expression of GST-NKIATRE fusion protein. 106

Figure 24: In vitro transcription and translation of NKIATRE. 107

Ac ADP ATP ATF BMK CAMP CAK cdc CDK CD1 cDNA cGMP cm dCTP DEPC Drosophila Dictyostelium Dig DNA dNTP E. coli. EDTA EGF EGFR ERIC GO G i G2 GCK GDP GST GTP HA HOG HPK IL IPTG JNK LPS M phase MAP MAPK

acetate adenosine diphosphate adenosine triphosphate activating transcSption factor big mitogen-activated protein kinase cyclic adenosine monophosphate CDK activating kinase ce11 division cycle cyclin-dependent kinase cyclin-dependent kinase inhibitor complimentary DNA cyclic guanosine monophosphate cyclin-dependent kinase inhibitor deoxycytidine triphosphate diethyl pyrocarbonate Drosophila melanogaster Dictyostelium discoideurn down-regulator of invasive growth deoxyribonucleic acid deoxyribonucleoside triphosphates Escherichia coli ethylenediaminetetraacetate epidermal growth factor epidermal growth factor receptor extracellular signal-regulated kinase quiescent state first gap phase second gap phase germinal centre kinase guanosine diphosphate glutathione-S-transferase guanosine triphosphate hemagglutinin high osmolarity glycerol response hematopoietic progenitor kinase interleukin isopropylthio-$-D-galactoside cJun NH,-terminal kinase lipopolysaccharide mitosis microtubule-associated protein mitogen-activated protein kinase

MAPKKK MAPKAPK MAT MBP MEK MEKK MKH MKK MLK MUK mRNA OD ONPG PAGE PAK PBS2 PCR PEG pfu PKA-Ca RACE Rb RNA RPTK RSK Rst SAPK S. cerevisiae SD SDS SEK SH2 SH3 SI30 SLN SOS S phase S. pombe SSC SSK STE TdT TE

mitoien-activated protein kinase kinase kinase MAP kinase-activated protein kinase menage a trois myelin basic protein MAPK/ERK kinase MEK kinase MAP kinase homologue MAP kinase kinase mixed lineage kinase MAPK upstream kinase messenger RNA optical density O-nitrophenylgalactoside polyacrylamide gel electrophoresis p2 1 activated protein kinase polymyxin B sensitivity 2 polymerase chah reaction pol yethylene glycol plaque forming units type a CAMP-dependent protein kinase rapid amplification of cDNA ends retinoblastoma protein ribonucleic acid receptor protein tyrosine kinase . ribosomal protein S6 kinase regulator of sterile twelve stress-activated protein kinase Saccharomyces cerevisiae synthetic deficient sodium dodecyl sulphate SAPK/ERK kinase src homology 2 domain src homology 3 domain synthetic, high osmolarity sensitive synthetic lethal of N-end rule son of sevenless DNA synthesis phase Schizosaccharomyces pombe sodium sodium citrate suppressor of sensor kinase sterile terminal deoxynucleotidyl tram ferase tris-EDTA

TNF-a tRNA UV x-gal n?D

turnour necrosis factor-a transfer RNA ultraviolet 5-bromo-4-chloro-3-indolyl-~-galactop~de yeast extract peptone dextrose

xii

bp OC cPm kb kD M mg ml mm mM mol ng pmol

U

base pair degree celsius counts per minute kilobase kilodalton molar milligram millilitre millimetre millimolar mole nanogram picornole revolutions per minute units

Fig microgram Pl microlitre PM micromolar pmol micromole

CHAPTER 1

Literature Review

&.a

1.5 INTRODUCTION

As unicellular organisms evolved into more complex multicellular fonns,

the ability of the ce11 to respond to extracellular stimuli became increasingly

important to many biological processes. The ability to coordinate growth and

differentiation of single cells into multicellular organisms requires the input

of many growth factors, growth hormones, and growth inhibitors. In addition,

exposure of cells to alterations in their external environment requires the

ability to sense these changing conditions and respond to them appropriately.

Cells must therefore be capable of recognizing a multitude of stimuli and

translating these into coordinated signals that evoke appropriate changes in

cytoplasmic and nuclear events.

The mechanism by which cells translate external stimuli t o cytoplasmic

and nuclear changes is known as signal transduction. Many signal

transduction pathways are composed of kinase cascades which involve series

of interacting proteins regulated by phosphorylation events. Kinases transfer

the terminal phosphate group of ATP to serine, threonine, or tyrosine residues

within their substrates. This change in phosphorylation leads to a

corresponding change in the substrate's activation state. Therefore, within

these cascades, the protein catalyzing each step is activated or inhibited by the

product of the preceding step. Not only do kinase cascades serve to amplify

the original signal but they enable the effects of that signal to be rapidly

reversed by dephosphorylation through the action of phosphatases.

Many different farnilies of kinases exist but two that have been studied

in extensive detail are the MAPKs and the CDKs. In the literature review of

this thesis, these kinases will be discussed in terms of their stmctures and

functions. Further, the signalling pathways that they comprise, in addition to

their involvement in cellular processes, will be highlighted.

1.2 KINASES

1.2.1 Conserved Features

The catalytic domain of a kinase imparts its enzymatic activity and

functions to bind and orientate the phosphate donor (either GTP or ATP), to

bind and orientate the protein substrate, and to transfer the y-phosphate from

the ATP or GTP t o the acceptor hydroxyl of a serine, threonine, or tyrosine

residue on the protein substrate (reviewed in Hanks and Hunter, 1995). An

alignment of 65 protein kinases by Hanks et al. revealed that an overall

similarity exists among the catalytic domains (Hanks et al., 1988). The

catalytic domains are not conserved uniformly but consist of regions of high

conservation interspersed with regions of low conservation. Within these 250

to 300 amino acids units are 12 major conserved subdomains. These are

numbered sequentially from subdomain 1 through subdomain XI, with two

subdomains VIA and VIB. These subdomains contain characteristic patterns

of conserved residues and are defined as regions never interrupted by large

amino acid insertions.

Twelve residues within the kinase domain are either invariant or nearly

invariant. Using the PKA-Ca catalytic subunit as an example (since it was the

first kinase structure to be solved), these residues include Gly50 and Gly52 in

subdomain 1 (within the motif GXGXXGXV), Lys72 in subdomain II, Glu91 in

subdomain III, Aspl66 and Asnl71 in subdomain VIB, Aspl84 and Gly186 in

subdomain VI1 (within the DFG motif), Glu208 in subdomain VI11 (within the

APE motif), Asp220 and Gly225 in subdomain M, and Arg280 in subdomain

XI (Hanks et al., 1988; Hanks and Quinn, 1991). Because these residues are

highly conserved, they are believed to be involved in either the binding of ATP

or the phosphotransfer reaction (reviewed in Hanks and Hunter, 1995).

Within the kinase superfamily two main subdivisions exist. These

include the serine/threonine specific protein kinases and the tyrosine specific

protein kinases. The amino acid residues described above are conserved among

al1 kinases; however, other short sequences are found in either the

serinekhreonine specific kinases or the tyrosine specific kinases (EIanks et al.,

1988; Hanks and Quinn, 1991). The first of these sequences lies between the

two conserved Asp and Asn residues of subdomain VI. A strong indicator of

serinekhreonine specific kinase activity is the consensus sequence DLKPEN,

whereas DLRAAN or D W N indicates tyrosine specificity. A second

sequence that indicates the specificity of the kinase can be found in Subdomain

VIII. This region is found immediately amino terminal to the conserved APE

sequence and has the consensus PIVK/RWT/M for tyrosine specific kinases and

GT/SXXY/FX for serinehhreonine specific kinases.

1.2.2 Three Dimensional Structure

Because of the homology among the various kinase domains, it is

believed that they al1 fold into similar three-dimensional structures that

phosphorylate their substrates by a common mechanism. The first three-

dimensional structure of a kinase to be solved was that of mouse PKA-Ca with

a substrate analog inhibitor (Knighton et al., 1991a; Knighton et al., 1991b).

Later, the structures of PKA-Ca with the peptide inhibitor and either MgATP

or MnAMP-PNP (a MgATP analog) were solved (Zheng et al., 1993;

Bossemeyer et al., 1993). The structures of other kinases including ERKS and

CDK2 have been solved (Zhang F et al., 1994; De Bondt et al., 1993). As a

result of these studies, precise functional roles have been assigned to the

highly conserved kinase domain residues (reviewed in Hanks and Hunter,

1995). These studies also suggest a two-lobed organization of the kinase

domain with a deep cleft between the two lobes where catalysis occurs. Within

the smaller amino-terminal lobe are located subdomains 1-IV, which contain

most of the residues necessary for MgATP binding. Within the larger carboxy-

terminal lobe are subdomains VIA-XI that contain the residues associated with

peptide binding and catalysis. Subdomain V residues span both lobes.

A U

1.3 MITOGEN-ACTIVA'IXD PROTEIN KINASE SIGNALLING

When cells are exposed to environmental change, adaptive signalling

pathways may become stimulated. Often such signal transduction pathways

utilize the MAPK family of enzymes. These enzymes have been implicated in

regulating several important biological functions and several MAPK isofonns

have been assigned to specific signal transduction pathways. Currently four

pathways have been described in yeast that are involved in the regulation of

osmotic stability, mating, cell wall integrity, and sponilation. In mammalian

cells five distinct pathways have also been described that are involved in the

regulation of growth and differentiation, osmotic stability and the immune

response, and the response to "stress" stimuli. Clearly, the MAPKs represent

an evolutionarily conserved group of enzymes that play an essential role in

diverse intracellular signalling processes from yeast to vertebrates.

Three protein kinases are central to MAPK signalling and form a

sequentid protein kinase cascade. Although the specific kinases differ from

one MAPK pathway to the next, they each contain a MAPKKK, a MAPKK, and

a MAPK. The MAPKKKs are a group of serine/threonine specific protein

kinases that can phosphorylate and activate the MAPKKs upon appropriate

stimulation. The MAPKKs; however, are a unique class of dual specificity

kinases. They activate their substrates, the W K s , by phosphorylation o n

both threonine and tyrosine residues found within a characteristic T M - T y r

motif of subdomain VIII. The MAPKs, like the MAPKKKs, are

serinelthreonine specific protein kinases.

Due to the nature of the work presented within this thesis, the

remainder of the discussion of MAPK signalling will focus on the MAPK

homologues that have been assigned to specific cellular responses. While other

members of the MAPK family have been identified, including ERK3, p63WLPK,

and ~ 9 7 - ~ , their roles in ce11 signalling have yet to be identified (Boulton et

al., 1991; Gonzalez et al., 1992; Zhu et al., 1994). Based on the existence of

multiple MAPK homologues in both mammalian cells and lower eukaryotes,

there may be many more members of this family that are involved in the

regdation of other important biological functions.

1.3.1 The FUS3KSSl and ERKILERK2 Pathways

Mating within the yeast S. cerevisiae involves a pheromone-induced

MAPK signal transduction pathway (Figure 1A) (reviewed in Herskowitz et al.,

1995). Binding of the pheromone to a seven transmembrane receptor (STE2

or STE3) triggers the dissociation of a trimeric G protein into its Ga (Gpal)

and GB, (STE4 and STE18) subunits. Free Gg, activates cdc42 and STESO

which in turn activate the MAPKKK homologue STE11. STES, a Zn2+ finger

domain containing protein, functions to tether STE11, STE7 (a MAPKK

homologue), and FUS3/KSS1 (a MAPK homologue) into a multikinase cornplex,

allowing for the efficient and sequential activation of STE7 and FUSB/KSSl.

Among the MAPK cascades, the yeast mating pheromone pathway is the only

Pherornone 7 0

Growth Factor

hospholipase A2 \

Figure 1: The FUSS/KSSl and ERK signalling pathways. (A) Binding of pheromone to G protein coupled receptors on the surface of the yeast ce11 leads t o activation of the FUS3lKSS1 kinase cascade. (B) Activation of the ERK pathway by growth factor binding to RPTKs. Double lines represent plasma membranes and single lines represent nuclear membranes. Arrows indicate activation.

one described to date that utilises a binding protein, STE5, to ensure protein

kinase specificity in the presence of multiple MAPK pathways.

FUS3 and KSS1, which are functionally redundant MAP kinases,

activate many substrates involved in mating. For example, the protein Far1

which mediates pheromone induced G1 arrest is activated by FUSS/KSSl

(Elion et al., 1993; Peter et al., 1993). In addition, FUS3/KSSl activates the

transcription factor STE 12 by phosphorylating two recently identified proteins,

Rstl and Rst2 (also known as Digl and Dig2), that directly inhibit STEl2

(Tedford et al., 1997; Cook et al, 1996).

Interestingly, the mating pheromone pathway also mediates the invasive

growth response initiated when the same yeast are exposed to nutrient

limitation (reviewed in Herskowitz et al., 1995). Therefore, a single MAPK

pathway mediates two developmental programs. The invasive growth pathway

also depends upon many of the same components, including STE20, cdc42,

STE11, STE7, KSS1, Rstl, Rst2, and STE12 (Roberts and Fink, 1994; Tedf'ord

et al., 1997). Unlike the mating pathway, STE5 is not required for the

invasive growth response and it has therefore been suggested that an

analogous protein might be involved in mediating this signal o r that STE5

functions to effectively isolate the kinase components thereby preventing their

interactions with other pathways (Yashar et al., 1995).

In mammalian cells, a homologous pathway exists that is involved in the

regulation of ce11 growth and differentiation. The first members of this

pathway to be identified were the MAPKs, ERKl and ERK2 (also referred to

as RSK kinase, MAP-2 kinase, MBP kinase, and MAPK), based on their

capacity to activate RSK, a kinase initially thought to be responsible for the

phosphorylation of ribosomal protein S6 (Chung et al., 1991). Cloning of the

two ERKs reveals that they are most homologous to the yeast kinases FUS3

and KSS1, sharing 55% identity a t the amino acid level (Boulton et al., 1990;

Boulton et al., 1991). ERK1, which encodes a protein with a molecular size of

43 kD, is 90% identical t o ERK2 which encodes a protein with a molecular

weight of 41 kD. The ERKs are activated during meiotic maturation and upon

exposure to various stimuli including EGF, insulin, nerve growth factor,

platelet derived growth factor, insulin-like growth factor, and phorbol esters

(Sanghera et al., 1990; Ahn et al., 1990; Hoshi etal., 1988; C-mez et al., 1990;

Ray and Sturgill, 1987).

ERKl and ERK2 are activated by MEK (also referred to as MKK), a

unique kinase that possesses dual specificity for the regulatory threonine and

tyrosine residues found within their subdomain VI11 TEY motifs (Nakielny et

al., 1992). Cloning of the cDNAs for two distinct ME&, MEKl and MEK2,

reveals that they encode proteins with predicted molecular weights of 42 kD

and 44 kD respectively (Ashworth et al., 1992; Crews et al., 1992; Zheng and

Guan, 1993). MEKl and MEK2 are approximately 80% identical and are most

homologous to the S. cerevisiae MAPKK STE7. In addition, MEK is also

dependent on serinehhreonine phosphorylation for its activation (Gomez and

Cohen, 199 1).

The ERK pathway can become activated through receptors coupled to

heterotrimeric G proteins, through receptors linked to protein kinase C,

through receptors coupled to cytoplasmic tyrosine kinases, and through

receptor protein tyrosine kinases. Of these mechanisms, the most information

exists regarding ERK activation via receptor protein tyrosine kinases (reviewed

in Blenis et al., 1993).

One of the most extensively studied rnechanisms of RPTK coupling to

the ERK pathway involves the binding of an adaptor protein termed Grb2, via

its SH2 domain, to a phosphotyrosine on the receptor (Figure 1B) (reviewed in

Schlessinger and Bar-Sagi, 1994). Typically, ligand binding induces receptor

dimerization which leads to trans-phosphorylation of sites outside of the

catalytic domain. Grb2 binds to a phosphotyrosine on the receptor via its SH2

domain while its two SH3 domains interact with the proline-rich binding sites

on the guanine nucleotide exchange factor SOS (Lowenstein et al., 1992; Egan

et al., 1993). SOS catalyzes the dissociation of GDP from Ras thereby '

initiating the loading of GTP and the subsequent activation of Ras (Buday and

Downward, 1993). GTP-bound Ras binds to the amino-terminus of Raf thus

recruiting Raf to the membrane where it becomes activated (Vojtek et al., 1993;

Warne et al., 1993; Zhang et al., 1993). Raf can then activate MEKYMEK2 by

phosphorylation on two serine residues (Kyriakis et al., 1992; Yan and

Templeton, 1994; Alessi et al., 1994). In tum MEKVMEK2 activates

Ad

ERK1/ERK2 by phosphorylating the threonine and tyrosine residues within the

consensus TEY (Nakielny et al., 1992). Following activation, ERK1CERK2 can

phosphorylate cytoplasmic substrates such as cytosolic phospholipase A2 and

other protein serine/threonine kinases such as RSK which can then enter the

cell's nucleus and activate specific transcription factors (Lin et al., 1993;

Sturgill et al., 1988). In addition, activated ERKIlERK.2 can enter the nucleus

and phosphorylate transcription factors such as Elkl (Marais et al., 1993).

1.3.2 The SAPK Pathway

SAPK (also known as JNK) was first purified in active form from the

livers of cycloheximide treated rats as a 54 k D protein that could

phosphorylate MAP-2 on serinehhreonine residues (Kyriakis and Avruch,

1990). Subsequent cloning of the cDNA for SAPK revealed the existence of

three distinct genes that were subject to differential splicing and could

generate up to eight proteins of 46 k D and 54 kD (Kyriakis et al., 1994). The

three genes, termed cc, B, and y, share 85-92% identity with each other, and

approximately 43% identity with ERKl and the yeast MAPKs, FUS3 and

KSS 1.

Like the MAPKs, the SAP& are serine/threonine specific kinases that

are dependent on threonine and tyrosine phosphorylation for activation

(Kyriakis et al., 1991). In contrast to the ERKs which contain a TEY motif as

the site of activating phosphorylation, the SAPKs contain a TPY motif,

suggesting a distinct regulating kinase. In support of this notion, the SAPKs,

unlike the ER&, are poorly activated by mitogens and phorbol esters (Kyriakis

et al., 1994). Further, the SAP& appear to be involved with a stress-activated

signalling pathway since their activity is greatly enhanced in the presence of

agents like TNF-a, IL-1, anisomycin, cycloheximide, heat shock, UV

irradiation, sodium arsenite, muscarinic acetylcholine receptors, and ischemia-

reperfusion (Figure 2) (Kyriakis et al., 1994; Derijard et al., 1994; Bird et al.,

1994; Coso et al., 1995a; Pombo et al., 1994).

SEKl (also referred to as MKK4 and JNKK) is the physiologic activator

of SAPK (Sanchez et al., 1994). As expected, this kinase is most homologous

to the MAPKKs, with the highest homology of 48% to a yeast MAPKK, PBS2

(see section 1.3.3). In addition, SEK potently activates the SAPKs in vitro and

in vivo without interfering with the ERK pathway (Sanchez et al., 1994). Once

activated by SEK, SAPK can phosphorylate the transcription factors c-Jun and

ATF2 within their amino-terminal transactivation domains on two serine or

threonine residues respectively (Pulverer et al., 199 1; Gupta et al., 1995).

SEK is activated by the serinelthreonine kinase MEKK1, which was

originally thought to be the activator of MEK (Lange-Carter et al., 1993).

Expression of MEKKl at physiological levels is incapable of activating the ERK

pathway but can still stimulate the SAPKs, suggesting that it is a physiological

SEK kinase and not a physiological MEK kinase (Yan et al., 1994, Minden et

al., 1994). Other MAPKKK family members or STE 11 homologues,

TNFa, IL 1 , Anisomycin, W, Heat S hock, Sodium Arsenite Ischemia-Reperfusion, Cycloheximide

Figure 2: The SAPK signalling pathway. Through an unclear mechanism stressful agonists in the cell's external environment initiate the SAPK cascade. Dashed arrows represent an undetermined set of events and solid arrows represent direct activation. The double line indicates the plasma membrane and the single line represents the nuclear membrane.

including MUK, Tpl-2, and MLK3, activate the SAPK pathway (Hirai et al.,

1996; Salmeron et al., 1996; Tibbles et al., 1996). While it remains unclear

how MUK and Tpl-2 are regulated, two STEPO homologues, HPKl and GCK,

activate the SAPK pathway via MLK3 (Kiefer et al., 1996; Tibbles et al., 1996).

The PAK family of protein kinases are also STEPO homologues that are

activated by the srnall GTP-binding proteins Racl and cdc42 (Bagrodia et al.,

1995). Racl and cdc42 can regulate the activity of the SAPK pathway and

recently human PAKl has been suggested to act upstream of MEKK (Coso et

al., 1995; Brown et al., 1996). However, a direct interaction between PAK and

MEKK has yet to be been demonstrated.

1.3.3 The HOGl and p38 Pathways -

An osmosensing signal transduction pathway in yeast was first descnbed

when mutagenized yeast eells were screened for the failure to grow on high-

osrnolarity medium (Brewster et al, 1993). From these mutants, two different

yeast genes were isolated that are required for restoring the osmotic gradient

across the ce11 membrane in response to exposure to extemal osmolarity. The

product of the first of these genes, termed HOM, was demonstrated to become

rapidly tyrosine phosphorylated by the product of the second gene, PBS2, in

response to increases in extracellular osmolarity. Sequence cornparisons of

HOGl and PBS2 indicated that HOGl was most similar to the MAPK family

members and that PBS2 was most homologous to the MAPKKs.

Recent work on this yeast MAP kinase cascade has suggested its

regulation by a two-component osmosensor composed of a transrnembrane

sensor histidine kinase termed SLNl and a cytoplamic response regulator

termed SSKl (Figure 3A) (Maeda et al., 1994). Under normal salinity

conditions the SLNl histidine kinase represses the activation of the PBS2-

HOGl kinase cascade through phosphorylation of SSK1. In the presence of

high-osmolarity media however, the histidine kinase is inactive, allowing the

unphosphorylated SSKl to activate the PBS2-HOG1 kinase cascade. Further

work has identified two MAPKKKs, SSK2 and SSK22, that are under the

control of the SLN1-SSK1 two-component osmosensor and t,hat activate PBS2

(Maeda et al., 1995). PBS2 is also activated by the interaction of its proline-

rich amino terminal motif with the SH3 domain of a putative transmembrane

osmosensor termed SH01 (Maeda et al., 1995).

A mammalian homologue of HOGl was first identified from studies

involving LPS, an activator of cells of the immune and infiammatory systems

(Han et al., 1994). When mammalian prolymphocytes are exposed to LPS, a

protein kinase cascade is initiated resulting in the tyrosine phosphorylation of

a 38 kD protein, termed p38. Sequence analysis of this clone indicates that it

shares homology with HOGl(52% identity) and the other mammalian W K s

(46-49% identity). p38 also complements a yeast HOGl mutation and becomes

tyrosine phosphorylated upon exposure of cells t o increased osmolarity.

Like the other MAPKs, both HOGl and p38 possess threonine and

Hi& Osmolarity LPS, TNFa, IL 1, UV, H,O,, Arsenite, High ~smolari@

Figure 3: The HOG and p38 signalling pathways. (A) High osmolarity leads to activation of the HOG pathway in yeast through two possible osmosensors. (B) Through an unclear mechanism stressful agonists in the cell's external environment activate the p38 pathway. Double lines represent plasma membranes and single lines represent nuclear membranes. Dashed arrows represent an unclear series of events, solid arrows indicate direct activation.

tyrosine residues in subdomain VI11 which must be phosphorylated for

enzymatic activity (Raingeaud et al., 1995). p38 and HOGl have a TGY

sequence while the ERKs and the SAP& have TEY and TPY sequences

respectively. This suggests that p38 and HOGl form a distinct subset of the

MAPK family that is regulated by different upstream kinases and under

differing conditions.

As described, p38 was first identified in marnmalian cells from studies

involving LPS; however, further work has shown that p38 activation can be

observed under a multitude of stimuli including TNF-a, IL-1, high osmolarity,

W irradiation, heat shock, and arsenite (Rouse et al., 1994; Raingeaud et al.,

1995). While many of these agonists overlap with the corresponding SAPK

profile, it appears that p38 has a unique role in the idammatory response

based on its specific inhibition by a class of pyridinyl imidazoles that block

LPS-induced TNF and IL-1b production (Lee et al., 1994).

To further support the hypothesis that p38 is a member of a distinct

MAPK signalling pathway in marnmalian cells, several new MAPKKs have

been implicated in its activation (Figure 3B). For example, MKK3 and MKK4,

which activate p38 in vitro, were cloned as the human homologues of PBS2

(Derijard et al., 1995). Humaii MKK3b, a n alternatively spliced form of

MKK3, was cloned by complementation of a yeast PBS2 deletion mutant with

cDNA libraries in p38-dependent manner and shown to be activated by osmotic

shock (Moriguchi e t al., 1996a). In addition, another mammalian MAPKK

U A

termed MKK6, and MKK3, are p38 activators in vivo (Han et al., 1996;

Raingeaud et al., 1996). While little information currently exists regarding the

upstream activators of MKK3 or MKKG, in vitro and in vivo studies have

shown that the MAPKKK TAKl can work as a direct activator for both

(Moriguchi et al., 199613). Similarly, it has recently been demonstrated that

MLK3 can activate both MKK3 and MKK6 (Tibbles et al., 1996). Of the

multiple in vitro substrates of p38, only MAPKAPK2, MAPKAPK3, and the

transcription factors ATF2 and CHOP have been suggested to play a role in

vivo (Freshney et al., 1994; Rouse et al, 1994; McLaughlin et al., 1996;

Raingeaud et al., 1996; Wang and Ron, 1996). However, new evidence suggests

that ATF2 is not Iikely a physiologie p38 substrate (Cuenda et al., 1997).

A new member of the p38 family, termed p38$, was isolated (Jiang e t

al., 1996). p38B is slightly larger that p38, the product of a separate gene, and

is approximately 74% identical to p38. Like p38, p38p contains the TGY dual

phosphorylation site which is required for its kinase activity and is also

activated by pro-inflammatory cytokines and environmental stress. p38$ is

preferentially activated by MKK6 when CO-expressed in cultured cells whereas

p38 is activated nearly equally by MKK3, MKK4, and MKK6. Further, in vitro

and in vivo experiments show a much stronger substrate preference of p38$ for

ATF2 than seen with p38. Lastly, like p38, p38B is inhibited by the anti-

inflammatory pyridinyl imidazole drug implying that it too is involved in the

anti-inflammatory response.

1.3.4 The ERK5 Signalling Pathway

Two components of a new human protein kinase signal transduction

pathway, ERK5 (also known as BMK1) and MEKS, were recently identified

(Zhou et al., 1995; Lee JD et al., 1995; English et al., 1995). MEK5 which is

most closely related to MEKl(48% identical) has two alternative splice forms,

the larger of which, MEK5a, contains a n extra 23 amino acids thought to

associate with the actin cytoskeleton (English et al., 1995). In addition, an

amino terminal sequence, present in both isoforms, may be important in

coupling GTPase signalling molecules to this cascade (Zhou et al., 1995).

ERK5 and BMKI, which are 98% identical, are believed to be

differentially spliced products of the same gene (Lee JD at al., 1995). ERK5,

which is 50% identical to FUS3, encodes a n 815 amino acid protein with a

predicted molecular weight of 98kD. ERK5 is therefore approximately twice

the size of dl known MAP kinases, with an extra 400 amino acid carboxy-

terminal domain. Within this domain lie two proline-rich regions that are

believed to be involved in targeting the kinase to the cytoskeleton and

regulating its kinase activity (Zhou et al., 1995). Like ERK1, ERK5 also has

a TEY motif; however, the extra carboxy-terminal domain and the unique loop

between subdomain VI1 and VI11 suggest that it is involved i n a previously

unknown pathway in mammalian cells.

Consistent with the belief that these two kinases compose a distinct

signalling cascade are data that show the inability of MEKS to phosphorylate,

- * in vitro, any of the other known mammalian MAPKs (English et al., 1995).

Further, in vitro and in vivo binding assays demonstrate the ability of'ERK5

to bind MEK5 but not MEKl or MEK.2 (Zhou et al., 1995). Lastly, studies to

define ERK5 regdation have shown it to be stimulated upon exposure of cells

to sorbitol and H,O, but not to agonists that are strong activators of ERKY2,

raising the possibility that it is a redox-sensitive kinase (Abe et al., 1996).

1.3.5 The SAPK3/ERK6 Signalling Pathway

The most recent member of the MAPK family to be described was cloned

independently from both rat and human cDNA libraries and termed SAPK3

and ERK6 respectively (Mertens et al., 1996; Lechner et al., 1996). Sequence

cornparison reveals that this kinase shares approximately 60% identity with

p38, 45% identity with SAPK, and 40% identity with the ER&. Like p38,

ERK6 also contains a TGY motif in the activation domain and is activated by

many of the same stimuli that activate both p38 and SAPK (Cuenda et al.,

1997).

Experiments to examine the role of ERK6 in ce11 signalling reveal that

it is activated, both in vitro and in vivo, by MKK6, and not MKK1, MKK4, or

MKK3, (Cuenda et al., 1997). Additional experiments demonstrate that the

substrate specificity of ERK6, in vitro, is similar t o p38; however, significant

distinctions exist. For example, ERK6 is far less effective at activating the

MAPKAPKs and, unlike p38, is able to phosphorylate ATF2 on d l residues

U V

believed to be critical for its in vivo activation (Cuenda et al., 1997). In

addition, ERK6 is not inhibited by one of the p38 inhibiting drugs described

in section 1.3.3 (Cuenda et al., 1997). These findings suggest that ERK6 may

be responsible for mediating some of the functions previously attributed to

either SAPK or p38.

1.3.6 Crosstalk

Although the previously described pathways seem physiologically

distinct, a substantial amount of interaction, or crosstalk, occurs among thern.

In support of this belief, activation of the ERK, SAPK, and p38 pathways is not

mutually exclusive, since the stimuli of one these kinases can partially activate

the other two (Kyriakis et al., 1994; Raingeaud et al., 1994). However, when

a broad range of agonists are analyzed by comparing their efficiencies of

activation of the MAPKs, it is clear that there is discrimination between the

ERKs and SAPK (Kyriakis et al., 1994).

It has been demonstrated in vitro and with in vivo overexpression

studies that the MAPKKs from one pathway can activate the MAPKs of a

homologous pathway. For example, SEK can phosphorylate both p38 and

SAPK in vitro and CO-transfection assays show that both MKK3 and MKK6 can

phosphorylate p38 (Derijard et al., 1995; Han et al, 1996). As demonstrated

in the past, these experiments can lead to false associations between members

of different pathways. For example, MEKK was originally described as the

activator of MEK and not as the activator of SEK (Lange-Carter et al., 1993).

With the increasing numbers of kinases being described that are involved with

MAPK signalling, it will be important to determine how these pathways are

regulated such that only a specific subset of kinases become activated.

1.4 THE EUKARYOTIC CELL CYCLE

The eukaryotic ce11 cycle is the process by which a single ce11 gives rise

to two genetically identical daughter cells (reviewed in Norbury and Nurse,

1992). This cycle is composed of four main phases: G1, S, G2, and M (Figure

4). Proliferating cells must progress sequentially through al1 four stages of the

ce11 cycle from G1 to S to G2 and finally to M. DNA synthesis occurs during

S phase and mitosis occurs during M phase. The gaps between the M phase

and S phase (Gl) and the S phase and M phase (G2), enable the ce11 t o prepare

for DNA synthesis and ce11 division respectively. In addition to these four

stages, a non-proliferative, quiescent state also exists, terrned GO. While cells

in this phase are not actively dividing, they are not terminally differentiated

and therefore retain the ability to re-enter the proliferative ce11 cycle.

The initial progression of cells from GO or G1 into the S phase requires

the presence of growth factors specific for the ce11 type. However, once the

cells reach a restriction point in late Cl, they are committed to completing the

ce11 cycle, even in the absence of the growth factors that initiated the

progression (Pardee, 1974).

Figure 4: The mammalian cell cycle. A schematic of the stages of the ce11 cycle showing the points of action of the various cyclin-CDK complexes and the CKIs. R point = restriction point (serum independence).

For the accurate transmission of genetic information from rnother ce11

to daughter, the order and timing of events within the cycle are critical.

Failing to repair DNA damage, entering mitosis with unreplicated DNA, or

comrnencing anaphase before correct alignment of the chromosomes, leads to

dead, mutant, or aneuploid cells. Therefore, to ensure that the ce11 cycle is

properly maintained, biochemical pathways, known as checkpoints, exist that

prevent the initiation of new ce11 cycle events until the accurate completion of

others.

1.4.1 Cell Cycle Regulation by CDKs

Progression of the cell cycle is governed by a group of CDKs that

regulate ce11 cycle checkpoints in response to environmental or intracellular

conditions such as the presence of damaged or unreplicated DNA. In yeast, a

single CDK, either cdc2 in S. pombe or CDC28 in S. cerevisiae, is responsible

for the major transitions of the ce11 cycle. The genes encoding these kinases

were identified by temperature-sensitive mutated forms which could arrest

cells a t specific points within the ce11 cycle, namely a t the Gl/S and the G2/M

transitions (Nurse and Bissett, 198 1; Hartwell e t al., 1973; Reed, 1980; Piggott

et al., 1982). A human homologue to cdc2 was cloned by its ability to 4

complement an S. pombe temperature-sensitive CDC2' mutant strain (Lee and

Nurse, 1987). Unlike the yeast cdc2, mammalian cdc2 controls only the G2/M

transition since the G U S transition is regulated by different cdc2 homologues,

namely CDK2 and CDK4 (Fang and Newport, 1991). In mammalian cells,

multiple CDKs exist whose activities are regulated by their phosphorylation

and dephosphorylation, by the binding of cyclins, and by the activity of cyclin-

dependent kinase inhibitors.

1.4.2 Cyclins as Activators of CDKs

Cyclins are a family of structurally related proteins that function to bind

and activate CDK catalytic subunits in a regulatory fashion. Homology

between the different members of the cyclin family is not extensive and is

mainly confined to a stretch of 100 amino acids termed the cyclin box (Nugent

et al., 1991). The cyclin box lies in the middle of the 400 amino acid cyclin

sequence and is involved in CDK binding and activation (Kobayashi et al.,

1992).

Based on the crystal structures of both an inactive monomeric CDK and

an active cyclin-CDK complex, it is believed that cyclins activate CD& by

altering the conformation of the catalytic domain (De Bondt et al., 1993;

Jeffrey et al., 1995). Specifically, activation of the CDK through cyclin binding

is thought to result from two structural changes. First, binding of a cyclin

changes the conformation of the amino-terminal lobe of the CDK, thereby

reorienting the bound ATP and facilitating phosphotransfer to the CDK

substrate. Second, cyclin binding displaces a region in the middle of the CDK,

termed the T loop, such that it no longer obscures the substrate binding cIeft

of the enzyme.

To date, cyclins can be classified into two different families depending

on how they are degraded within the ce11 (reviewed in Pines, 1996). The G1

cyclins, D and E, are short lived proteins that have half lives of approximately

30 minutes. The instability of these cyclins is conferred by PEST amino acid

sequences that are carboxy-terminal to the cyclin box; however, the biochemical

basis of this proteolysis has not yet been elucidated. Because of their

instability, the levels of the G1 cyclins are determined by their rates of

transcription. In contrast, the mitotic cyclins or G2 cyclins, A and B, are

stable throughout the ce11 cycle except during mitosis when they are rapidly

degraded in a ubiquitin-dependent manner (Glotzer et al., 1991). This

instability is dependent on a region located amino-terminal to the cyclin box,

termed the destruction box (Glotzer et al., 1991). Because CD& are stable

throughout the ce11 cycle, inactivation of a cyclin-CDK complex occurs mainly

through the ce11 specific degradation of the cyclin partner.

Cyclins also play an important role in targeting their partner kinase to

specific parts of the cell. For example, cyclin A is a nuclear protein whereas

cyclin B is a cytoplasmic protein (Pines and Hunter, 1991). Because most

cyclin-CDK complexes recognize the same basic consensus sequence in vitro,

WR-Sm-P-X-WR, the targeting of these complexes to different locations in the

ce11 would likely restrict the substrates available to them (reviewed in Pines,

1995).

1.4.3 Cyclin-CDK Complexes Throughout the Cell Cycle

In mammalian cells CDKs complex with various cyclins throughout the

stages of the ce11 cycle (Figure 4). The first cyclin-CDK complex to form once

cells are released from quiescence is cyclin D with either CDK4 or CDK6,

depending on the ce11 type (Matsushime et al., 1992; Meyerson and Harlow,

1994). The formation of this complex is highly dependent on the presence of

growth factors since cyclin D levels drop immediately when growth factors are

removed (Matsushime et al., 199 1). Microinjection of anti-cyclin D 1 antibodies

into normal fibroblasts during G1 prevents cells from entering S phase,

indicating that this CDK-cyclin complex functions in middle to late G1 and is

important for the initiation of S phase (Baldin et al., 1993).

The second CDK-cyclin complex to become activated after release from

quiescence is the G1 cyclin, cyclin E, and CDK2 (Dulic et al.,1992; Koff et al,

1992). This complex plays a crucial role in the progression from G1 to S phase

and regulates different aspects of G1 than the cyclin D-CDK4/6 complex

(Reznitzky and Reed, 1995).

Following cyclin E degradation in S phase, CDK.2 associates with cyclin

A (Pagano et al., 1992). This complex is required for DNA replication since

cyclin A antibodies or antisense cyclin A cDNA will inhibit DNA synthesis

when micro-injected into cells (Pagano et al., 1992). Cyclin A and CD= also

localize to subnuclear replication foci, further suggesting a role of this cyclin-

CDK complex in DNA replication (Cardoso et al., 1993).

Entry into mitosis is triggered by formation of the Maturation Promoting

Factor which is composed of cdc2 and cyclin B (reviewed in King et al., 1994).

In addition, cyclin A also plays an essential role in the initiation of mitosis by

forming a complex with cdc2 (Pagano et al., 1992). Experiments with

Drosophila cells that are mutant in both cyclin A and cyclin B suggest that the

two cyclins act synergistically to initiate mitosis but that once in mitosis the

cyclin B-cdc2 complex plays a more central role in its progression (Knoblich

and Lehner, 1993). FoIlowing destruction of cyclin B at the metaphase-

anaphase transition, cdc2 becomes inactivated and the cells exit from mitosis

(reviewed in King et al., 1994).

While the complexes of cyclins A, B, D, and E with their respective

CD& clearly have a function in the regulation of the ce11 cycle, i t is believed

that other cyclin-CDK complexes exist that have other functions than

participating directly or exclusively in ce11 cycle control. For example, the

cyclin H-CDK7 complex forms part of the general transcription factor TFIIH

and is also part of C M (see section 1.4.4.1) (Shiekhattar et al., 1995). Another

complex, composed of CDK8 and cyclin C, is also involved in mammalian

transcription (Tassan et al., 1995b). In addition, two other cyclins have been

identified, cyclins F and G, but their cell cycle role, if any, remains to be

demonstrated (Bai et al., 1994; Okamoto and Beach, 1994 ).

1.4.4 Modulation of Cyclin-CDK Activity

1.4.4.1 Activation by Phosphorylation

Complete CDK activation requires not only cyclin binding but also its

phosphorylation at a conserved threonine residue within the T loop. In cdc2

this threonine corresponds to amino acid 161, whereas in CDK2 this

corresponds to amino acid 160 (Solomon et al., 1992; Gu et al., 1992). While

cyclin binding is thought to facilitate phosphorylation of this threonine residue,

its phosphorylation is also predicted to strengthen cyclin binding.

The enzyme responsible for phosphorylating ThrlGO in cdc2 and Thrl61

in CDK2 was originally named CAK, but it has since been realized that, like

its substrates, it is also a cyclin-CDK complex composed of cyclin H, CDK7,

and an assembly factor termed MAT1 (Fisher and Morgan, 1994; Fesquet et

al., 1993; Poon et al., 1993; Solomon et al., 1993; Tassan et al., 1995a). In

addition to activating both cdc2 and CDK2 complexes, CAK has also been

demonstrated to activate cyclin D-CDK4 (Matsuoka et al., 1994). This suggests

that a single CAK might be responsible for activating the major CD& involved

in cell cycle control.

Throughout the ce11 cycle, phosphorylation of CDK residue Thr1601161

parallels cyclin binding. Changes in CDK phosphorylation a t this site are

probably not due to changes in CAK activity but rather in the ability of cyclin

binding to stimulate CDK phosphorylation, since CAK activity does not change

in a cell cycle-dependent manner (Matsuoka et al., 1994).

1.4.4.2 Inhibition by Phosphorylation

In mammalian cells cyclin-CDK inactivation is regulated by cyclin

degradation, dephosphorylation of Thr160/161, and by phosphorylation of two

amino acid residues near the amino terminus of the CDK (Norbury et al., 1991;

Gu et al., 1992). These two residues, Thrl4 and Tyr15 in both cdc2 and CDK2,

are found within the ATP binding site buried beneath the T loop (De Bondt et

al., 1993). Phosphorylation of Tyr15 is dependent on cyclin binding and is

carried out by the Weel kinase (Parker et al., 1991; McGowan and Russell,

1993). In yeast, phosphorylation occurs only on Tyr15 and is camed out by the

redundant Weel and Mikl kinases (Lundgren et al., 1991). In animal cells,

Thrl4 is phosphorylated by an unidentified membrane-bound kinase activity

which is distinct from that of the Weel kinase (Kornbluth et al., 1994).

In yeast, dephosphorylation of Tyrl5, and hence activation of the cyclin-

CDK complex, is accomplished by the product of the cdc25 gene (Russell and

Nurse, 1986). In mammalian cells at least three members of this family exist

which appear to interact with different cyclin-CDK complexes. cdc25A is

phosphorylated and activated by cyclin E-CDK2 at the initiation of DNA

synthesis. Following its activation, cdc25A is thought to dephosphorylate and

activate one of the CDKs functioning early in the ce11 cycle, most likely CDK2

in complex with cyclin A (Hoffman et al., 1994; Jinno et al., 1994). At the

onset of mitosis cdc25C is activated and dephosphorylates Thrl4 and Tyr15 in

cdc2, leading to activation of the cyclin B-cdc2 complexes (Hoffman et al.,

1993). A positive feedback loop is initiated since cyclin B-cdc2 activates more

cdc25C such that the activation of cyclinB-cdc2 occurs rapidly and irreversibly

(Hoffmann e t al., 1993). The point of action of cdc25B in the ce11 cycle is

unclear .

1.4.4.3 CDK Inhibitors

Cyclin-CDK complexes are also regulated by a diverse family of proteins,

known as CKIs or CDIs, that bind to and inactivate the cyclin-CDK complexes.

Two families of CKIs have been isolated from animal cells. The first family is

composed of 161NK4", l ~ ~ ~ ~ ~ ~ , 181NK4c, and while the second family

is comprised of p21C'Pmm1, ~27~'''~ and ~ 5 7 ~ ~ . Together, these proteins play

a n important role in mediating extracellular negative signals that arrest the

ce11 at various points throughout its cycle (Figure 4).

The INK4 farnily members, p15, p16, p18, and p19, bind only to CDK4

and CDK6, apparently competing for cyclin D binding sites, thereby arresting

cells in G1 (Hannon and Beach, 1994; Guan et al., 1994; Hirai et al., 1995

Serrano et al., 1993). The extracellular signals regulating the activities of pl6,

p18, and p l9 have yet to be defined; however, it is known that inhibition by

p l6 and pl8 occurs in an Rb-dependent fashion (Serrano et al., 1993; Guan et

al., 1994). p l5 mediates ce11 cycle arrest induced by the negative growth factor

TGFB (Hannon and Beach, 1994).

The second family of CKIs have a preference for CDK2- and CDK4-cyclin

r u

complexes. The first member of this family to be described, p21, binds and

inhibits a vanety of cyclin-CDK complexes including cyclin D-CDK4 and CDK2

with either cyclin E or A (Harper e t al., 1993). p21 transcription is activated

by p53, suggesting that increased p21 synthesis occurs when DNA is damaged

in Gl (El Deiry et al., 1993). This would prevent the cells from entering S

phase by inhibiting both cyclin D-CDK4 and cyclin E-CDK2 complexes.

p27, a cyclin E-CDK2 inhibitor, blocks cells in late G1 in response to

TGFB or contact inhibition (Polyak et al., 1994). However, p27 is competitively

bound by cyclin D-CDK4 complexes suggesting that when p27 is sequestered

by cyclin D-CDK4 i t relieves its inhibition of cyclin E-CDK2 (Polyak e t al.,

1994). p27 also mediates CAMP induced G1 arrest by binding to cyclin D-

CDK4 complexes and preventing their activation by CAK (Kato et al., 1994).

p57 which potently inhibits cyclin E-CDK2, cyclin D-CDK4, and cyclin A-

CDK2, is thought to be involved in the decision to exit the ce11 cycle since most

cells expressing p57 are terminally differentiated (Lee MH et al., 1995;

Matsuoka e t al., 1995).

1.5 cdc2-RELATED PROTEIN KINASES

A series of cdc2-related protein kinases exist that have greater than 40%

amino acid identity with cdc2. Unlike the CDKs, these kinases have not been

demonstrated to associate with a cyclin or to posses activities that fluctuate in

a ce11 cycle-dependent fashion. Therefore, they have been named after their

- .

amino acid sequences corresponding to the characteristic PSTAIRE motif,

found in cdc2 and CDK2, which is involved in cyclin binding.

To date, seven cdc2-related kinases have been described whose roles, if

any, in the cell cycle have yet to be determined. Within this group are

PITALRE, PISSLRE, PITAIRE, PITSLRE, PCTAIRE, KKIALRE, and

KKIAMRE. The majority of these kinases were initially isolated by PCR based

techniques in a n atternpt to identify other kinases involved in the cell cycle

(Grana et al., 1994a; Grana et al., 199413; Lapidot-Lifson et al., 1992; Bunnell

et al., 1990; Okuda et al., 1992; Meyerson et al., 1992; Taglienti et al., 1996).

Because of the limited information that exists regarding these kinases and

their potential functions, the remainder of this discussion will be limited to

KKIALRE and KKIAMRE whose sequences most closely resemble each other

and a novel protein kinase, NKIATRE, which forms the subject of this thesis.

1.5.1 KKIALRE and KKIAMRE

KKIALRE and KKIAMRE were isolated by cDNA library screening

using degenerate oligonucleotides corresponding to conserved regions of cdc2

and the MAPKs respectively (Meyerson et al., 1992; Taglienti et al., 1996).

Both KKIALRE and KKIAMRE, over their total sequences, share

approximately 40% amino acid identity with cdc2 and approximately 34%

amino acid identity with the ERKs (Bestfit program, Wisconsin Genetics

Computer Group). In contrast, KKIALRE and KKIAMRE share 50% amino

acid identity with each other (Bestfit program, Wisconsin Genetics Computer

Group). Both KKIALRE and KKIAMRE contain the arnino acid residues

described in section 1.4.4 that are necessary for activation as CDKs. In

contrast, both KKIALRE and -RE contain the motif Thr-X-Tyr in

subdomain VI11 of their catalytic domains that is characteristic of the members

of the MAPK family (see section 1.3). Further, this sequence, Thr-Asp-Tyr, is

the same in both KKLALRE and KKIAMRE, and is unique to the MAPKs,

suggesting that as MAPKs, these may be part of novel signalhg pathways.

KKIALRE is expressed in human ovary and kidney, moderately in the

brain and lung, and at a low level in placenta, spleen, prostate, and duodenum

(Taglienti et al., 1996). KKIAMRE is expressed in human testis and kidney

with a lower level detected in brain and lung (Taglienti et al., 1996).

KKIAMRE and KKIALRE have a similar tissue distribution with the exception

that KKIAMRE is found in the testis while KKIALRE is expressed in the

ovary. The differential expression of these two kinases would suggest that

they rnay play a specialized role in meiosis or the production of germ cells.

c-Myc and the EGFR are in vitro substrates for both KKLAMRE and

KKIALRE (Taglienti et al., 1996). In addition, both KKIALRE and KKIAMRE

can become activated in COS cells stimulated with EGF but this activation

does not require phosphorylation of the Thr and Tyr residues in the Thr-Asp-

Tyr motif (Taglienti et al., 1996). MEKl is also able to phosphorylate

KKIAMRE in vitro but this is 50-fold weaker than the phosphorylation of

1"

ERKL! by MEKl (Taglienti et al., 1996). Together, these data indicate that

KKIAMRE and KKIALRE may not function as either MAPKs or CDKs and

therefore represent a new subfamily of proline-directed protein kinases.

1.6 SUMRlARY

The mechanism by which cells alter their cytoplasmic and nuclear

events, in response to extracelhlar signals, is a complex process that often

involves the input of many different signalling molecules such as protein

kinases. This introduction has reviewed two important classes of kinases that

are invo1ve.d in regulating diverse biological functions. The MAPKs enable

cells to grow and differentiate and adapt to multiple changes in their extemal

environment, whereas the CDKs regdate progression of the cell cycle. In

addition, two members of a subfamily of cdc2-related protein kinases,

KKIALRE and KKIAMRE, have been introduced. These two kinases' are

unique since they contain conserved features characteristic of both the MAPKs

and the CD&.

Chapter 2 of this thesis describes experiments using the yeast two

hybrid approach that demonstrates that the three known MAPK pathways,

ERK, SAPK, and p38, do not require scaffolding proteins to maintain sipalling

specificity and that crosstalk among them does not readily occur in vivo. In

chapter 3, the search and discovery of a novel serinekhreonine specific protein

kinase, NKIATRE, is described. This kinase appears t o be the third member

of a novel group of kinases that includes KKIALRE and KKIAMRE.

CHAPTER 2

Examination of In Vivo Protein Interactions Among the Three Known

Mitogen-Activated Protein Kinase Pathways

Parts of this chapter have been previously published in: "Mammalian Mitogen-activated Protein Kinase Pathways Are Regulated through Formation of Specific Kinase-Activator Complexes" Zanke BW, Rubie EA, Winnett E, Chan J , Randall S, Parsons M, Boudreau K, McInnis M, Yan M, Templeton DJ, and Woodgett JR The Journal of Biological Chemistry, 1996, Vol. 271 No. 12: 29876-29881

2.1 INTRODUCTION

Structurally similar MAPK pathways have been identified in both yeast

and mammalian cells. While these pathways regulate diverse biological

events, they each contain a conserved kinase cascade consisting of a MAP-,

a MAPKK, and a MAPK. Of the MAPK pathways in mammalian cells, the

most highly characterized involves the sequential kinase cascade of Raf (a

MAPKKK), MEK (a MAPKK), and ERK (a MAPK), upon stimulation of cells

with growth factors (see section 1.3.1). Two other MAPK pathways that have

also been studied in detail are the SAPK and p38 kinase cascades (see sections

1.3.2 and 1.3.3). Unlike the ERK pathway, when cells are exposed to stressful

agonists including W irradiation, heat shock, osmotic shock, TNFa, and IL-1,

the SAPK pathway becomes activated. This pathway is typically composed of

MEKK, SEK, and SAPK, although other MAPKKKs have been demonstrated

to activate SEK. Similarly, when cells are exposed to UV irradiation, TNFa,

IL-1, and osmotic shock, the p38 pathway becomes activated. Although not as

clearly defined as the ERK and SAPK pathways, p38 is activated by both

MKK3 and MKK6, which are both activated by TAKl and MLK3.

Although it may seem that these three pathways are physiologically

distinct, data obtained to date has been less than conclusive. In vitro

experiments and in vivo overexpression studies have lead to false conclusions

in the past regarding interactions between molecules. For example, MEKK

was originally described as the activator of MEK both in vitro, and when

expressed at high levels in COS-7 cells (Lange-Carter et al., 1993). However,

at physiologie levels MEKK specifically activates the SAPK pathway via

activation of SEK (Yan et al., 1994; Minden et al., 1994). It has been

demonstrated that activation of these three pathways is not mutually exclusive

since TNFa and heat shock can partially activate the ERK cascade and EGF

can partially activate the SAPK pathway (Kyriakis et al., 1994). Further, it

has been shown that SEK can phosphorylate both p38 and SAPK in vitro

(Derijard et al., 1995). Therefore, it would appear that a significant amount

of crosstalk could occur between the different MAPK pathways.

To maintain signalling specificity in the presence of multiple kinases

with high structural homology, mechanisms must exist that prevent

interactions between members of the different W K cascades. In yeast cells,

STE5 functions as a scaffolding protein that brings together KSSUFUS3,

STE20, and STE11, thereby preventing the activation of any other kinases

(Choi et al., 1994). Because no STE5 homologue has been identified in

mammalian cells, we decided to investigate how the specificity of MAPK

signalling is maintained. Further, we have chosen to examine the extent of

crosstalk that occurs between these three homologous signalling pathways.

With the yeast two hybrid system, a yeast based genetic assay that

detects protein-protein interactions in vivo, we have evaluated the physical

interactions between the MAPKs, ERK, SAPK, and p38, and the MAPKKs,

MEK, SEK, MKK3, and M W . Here we describe that the ERK, SAPK, and

-

p38 signalling pathways do not require specific scaffolding proteins to maintain

signalling specificity. Further, these experiments, in combination with other

data by Zanke et al., indicate that the mammalian MAPK pathways are

distinct signalling cascades and that crosstalk does not readily occur in vivo

(Zanke e t al., 1996).

2.2 MATERLAILS AND lMETHODS

2.2.1 The Yeast Two Hybrid System

The two hybrid system is a yeast based genetic assay that detects

protein-protein interactions in vivo. The assay works by reconstituting the

function of the transcription factor GAIA which can be can be separated into

a DNA-binding domain and a transcriptional activation domain. Nomally

both domains are located in the same protein; however, separated components

can be assembled in vivo to give a functional activator (Ma and Ptashne, 1988;

Brent and Ptashne, 1985). In the MatchmakerTM Two Hybrid System

(Clontech, Pa10 Alto CA, USA) the gene for one protein is cloned into the vector

pAS1-CYH2, enabling the generation of a hybrid with the GAL4 DNA-binding

domain. The gene for a putative interacting protein is cloned into the vector

pACTII, enabling the generation of a hybrid protein with the GAL4 activation

domain.

Following construction of the two plasmids, they are cotransformed into

the yeast strain Y190 which contains the lacZ and HIS3 reporter genes under

the control of upstream GALA binding sites. Because the vector pAS1-CYH2

carries the TRPl gene and the vector pACTII carries the LEU2 gene,

transformant yeast can be selected by growth in the absence of tryptophan and

leucine. If the two hybrid proteins interact with each other, the GAL4 DNA-

binding domain and the GAL4 activation domain will be brought into close

proximity, allowing reconstitution of the transcription factor function and

expression of the two reporter genes (Figure 5).

To identi@ transformant colonies positive for expression of the lacZ gene,

f3-galactosidase assays are performed (Figure 6). With this assay, the yeast

will develop a blue colour in the presence of X-gal due to expression of the

enzyme P-galactosidase from the lac2 gene. Similarly, expression of the HIS3

gene can be detected by the ability of transformant yeast to grow in the

absence of histidine.

2.2.2 Plasmid DNAs

The complete coding sequences of SAPKa, p38AGF (a kinase-inactive

version of p38 which has the two sites of activating phosphorylation mutated

from threonine and tyrosine to alanine and phenylalanine respectively), and

ERR1, were cloned into the multiple cloning site of vector pAS1-CYH2 such

that in-frame fusion proteins could be generated (Figure 7). SAPKa was

cloned into the NcoI site while p38AGF and ERKl were cloned into the NcoI

and BamHI sites. Because of difficulties in growing yeast cultures expressing

(B) 1 GALl UAS 1 Prornoter 1 lac2 or HIS3 reporter

(A)

Figure 5: The basis of the yeast two hybrid system. (A) The GAIA DNA- binding domain (BD) hybrid binds to the GALl upstream activating sequence (UAS) but cannot activate transcription. (B) The hybrid of protein Y and the GAL4 activation domain (AD) cannot localize to the UAS and activate transcription. (C) Interaction between the two proteins, X and Y, leads to reconstitution of the GAL4 transcription factor and expression of the reporter genes lac2 and HISS.

GAL 1 UAS

I (C)

Promoter lacZ or HIS3 reporter

lacZ or HIS3 reporter GAL 1 UAS Promoter

TRP 1 LEU 2

1 Cotransform into Y 190

Select transformed yeast in absence of Trp or Leu

I Assay for P- galactosidase activity

Figure 6: Flow Diagram of the yeast two hybrid system. The interaction between two known proteins, X and Y, is assayed using the yeast two hybrid System as described in the text. If the two proteins interact, al1 colonies are expected to tum blue in a P-galactosidase filter assay.

Nco 1 / Sfi 1

fl+ ori I

Figure 7: Functional map of pAS1-CYH2. The cloning vector provided in the yeast two hybrid kit for construction and expression of GAL4 DNA-binding domain (BD) fusion proteins. ERK, SAPK, and p38 were cloned into this vector as described in the text.

wild type p38, the kinase-inactive version was used in these experiments. The

coding sequences for SEK1, MEK, MKK3, and MKK6 were cloned into the

multicloning site of vector pACTII, also ensuring that in-frame fusion proteins

would be generated (Figure 8). SEKl was cloned into the NcoI and XhoI sites,

MER was cloned into the EcoRI and XhoI sites, MKK3 was cloned into the

NcoI and SmaI sites, and MKK6 was cloned into the EcoRI and the XhoI (with

a blunt end due to fill-in by Klenow) sites of pACTII. Plasmid DNA was

prepared from bacterial cells for use in the transformations using either the

Qiagen system (Qiagen, Studio City CA, USA) or by alkali lysis and

polyethylene glycol purification (Sarnbrook et al., 1989).

2.2.3 Transformation of Yeast

Competent yeast cells were prepared by inoculating 20 ml of YPD broth

with a single colony of the yeast strain Y190. The culture was grown overnight

a t 30°C with shaking. The following day, enough of the culture was added to

300 ml of YPD to give an OD,,, of approximately 1.0. This culture was grown

for three additional hours, as before. The cells were then spun a t 3000 rpm for

5 minutes, and washed in 50 ml of sterile water. Following, the cells were

resuspended in 1.5 ml of 1X LiAcITE (100 mM LiAc pH 7.5, 10 mM Tris pH

7.5, 1 mM EDTA) and left at room temperature for 10 minutes. For

cotransformations, 1-10 pg of a given MAPK-pAS1-CYH2 vector was combined

with 1-10 pg of a MAPKK-pACTII vector and 100 pg of salmon sperm carrier

2p ori P r COI ~1 O~ 1

Xhol (4991) P = promoter T = Transcription termination sequence

Figure 8: Functional map of pACTII. The cloning vector provided in the yeast two hybrid kit for construction and expression of GAL4 activation domain fusion (AD) proteins. MEK, SEK, MKK3, and MKK6 were cloned into this vector as described in the text.

DNA. 100 pl of competent cells was added to each sample, followed by 600 p l

of PEG/LiAc solution (50% PEG 4000, 10 mM Tris pH 7.5, 1 mM EDTA, 100

mM LiAc). The mixtures were then incubated at 30°C for 30 minutes with

shaking. Following, 70 pl of dimethyl sulfoxide was added to each

transformation reaction, and mixed gently. The cells were then heat shocked

for 15 minutes at 42OC and chilled on ice. The reactions were spun for 5

seconds at full speed in a microcentrifuge, and resuspended in 500 pl of TE (10

mM Tris pH 7.5, 1 mM EDTA). Transformed cells were spread on SD plates

lacking leucine and tryptophan, to select for the pACTII and pAS1-CYH2

vectors respectively, and grown at 30°C for 3-4 days.

2.2.4 Liquid Culture ~galactosidase Assays

Five ml of liquid media (SD lacking tryptophan and leucine) was

inoculated with an entire yeast colony, and the culture was incubated

oveniight at 30°C with shaking. The following morning 4 ml of YPD was

inoculated with 1 ml of overnight culture, and this was incubated as described

until the OD, reached 0.5-0.8. Each culture was vortexed to disperse any ce11

clumps, and the OD,,, was recorded. One and a half ml of culture was spun

at 14 000 rpm for 30 seconds, and the pellet was washed and resuspended in

300 pl of Z buffer (16.1 gA Na2HPO;7H,0, 5.5 g/l NaH,PO;H,O, 0.75 gA KCI,

0.246 gA MgS0;7H20, pH 7.0), thereby concentrating the cells 5-fold. One

hundred pl of cells was transferred to a fresh tube and placed in liquid

nitrogen for 1 minute. The cells were then thawed a t 37OC for 1 minute. To

each reaction tube, 700 pl of Z buffer with B-mercaptoethanol (100 ml of Z

buffer to 0.27 ml of P-mercaptoethanol) was added, in addition a blank was set

up with 100 pl of Z buffer. A timed incubation of each lysate and 160 pl of

ONPG (4 mg/ml ONPG in Z buffer) was performed. The tubes were incubated

at 30°C until a yellow colour developed. At this point, 400 pl of a 1 M Na,CO,

solution was added to the reaction. Once al1 reactions were complete, the

tubes were spun at 14 000 rpm to pellet ce11 debris, and the supernatant was

transferred to a cuvette. The degree of colour intensity was determined by

measuring the OD,,, of the samples. To compare different samples, f3-

galactosidase units (where 1 unit is the amount of enzyme which hydrolyzes

1 pmol of ONPG to o-nitrophenol and D-galactose) were calculated using the

following formula:

p-galactosidase units = 1000 x OD,, / (t x V x OD,,,)

where: t = elapsed time V = 0.1 ml x concentration factor

2.2.5 Filter Assays for P-galactosidase Activity

Once the transformant yeast colonies growing on the SD plates lacking

leucine and tryptophan were 1-2 mm in diameter, a nylon filter was placed

over top of the agar and slowly removed such that the majority of the yeast

colonies adhered. These filters were submerged in liquid nitrogen for 10

- -

seconds and then thawed a t room temperature. Once thawed, the nylon filters

were placed inside the lid of a petri dish on top of Whatman filter paper that

had been presoaked in 2 ml of Z buffer and X-gal solution ( 100 ml Z buffer, 270

$-mercaptoethanol, 1.67 ml of a 20 mdml X-gal stock in

dimethylformamide). The dishes were then covered and sealed with parafilm

so that the b d e r did not evaporate. Following, the filters were incubated at

30°C for up to 30 hours so that the blue colour could develop.

2.3 RlESULTS

2.3.1 p-galactosidase Liquid Culture Assays

To examine the protein-protein interactions occurring between members

of the ERK, SAPK, and p38 pathways, four separate trials of $-galactosidase

liquid culture assays were performed on yeast cotransformed with each

combination of a MAPK-pAS1-CYH2 vector and a MAPKK-pACTII vector

(Figure 9). Since an interaction was seen with the ERK hybrid and the

pACTII empty vector, we can not conclude that any of the interactions seen

with the ERK hybrid were not artifactual. However, no interactions were seen

with any of the remaining controls implying a tme interaction between SEK

and SAPK, SEK and p38, and MKK6 and p38. Further, no interactions were

detected between MEK and SAPK, MEK and p38, MKK3 and SAPK, or MKK3

and p38.

Figure 9:

SEK MEK MKK3 MKK6

p-galactosidase liquid culture assays. Each bar represents the interaction in &galactosidase k t s of a given MAPK and WKK. The combination of MAPK (SAPK, p38, or ERK) and MAPKK (SEK, MEK, MKK3, or MKK6) tested is indicated beneath each bar. Those bars marked with MAPK-C or MAPKK-C represent controis that assay for interactions between the given kinase and the opposite empty vector. The pAS1-CYH2 and pACTII bars are the results of control transformations with empty vector. For further description see text.

2.3.2 B-galactosidase Filter Assays

To examine the interactions between components of the p38 and SAPK

pathways further, yeast cotransformed with members of these pathways were

analyzed by the B-galactosidase filter assays. The experiment was repeated

five times, and the results indicate that SAPK interacts with SEK but not with

MKK3 or MKK6 and that p38 interacts with MKK3, MKK6, and SEK (Figure

10). No interactions were seen in the controls which were cotransformed with

either a MAPK-pAS1-Cm2 and empty pACTII or a MAPKK-pACTII and

empty pAS1-CYH2 (data not shown).

2.4 DISCUSSION

Within a ce11 multiple signalling pathways exist that regulate diverse

physiological functions. Because these cascades utilize many kinases with

seemingly similar sequences and structures, mechanisms must exist which

allow the components of a distinct cascade t o phosphorylate and activate very

specific targets. In the yeast mating pathway, the protein STE5 functions to

bind together and effectively isolates the three kinase components KSSUFUS3,

STEBO, and STEll from al1 other kinases that might interfere with this

signalling pathway. While STE5 appears to be effective at compartmentalising

these kinases, to date no homologous scaffolding proteins have been found in

mammalian cells. This would indicate that another mechanism might be

responsible for rnaintaining the specificity required for signalling within higher

MKK3 MKK6 SEK

SAPK

SAPK

Figure 10: fbgalactosidase filter assays. The association of the MAPKs, p38 and SAPK with the MAPKKs, MKK3, MKK6, and SEK is presented. (A) The yeast were streaked uniformly on SD agar plates, lacking tryptophan and leucine, and were grown for approximately 48 hours at 30°C. (B) A filter lift of the yeast colonies was assayed for B-galactosidase activity as described in section 2.2.5. Those yeast which tumed blue demonstrated an interaction between the respective W K and MAPKK.

v I

eukaryotic cells. Because it is unlikely that a yeast ce11 would contain the

appropriate scaffolding proteins that could effectively bind the kinase

components of a signalling cascade from a higher eukaryote, the yeast two

hybrid system was exploited to examine how members of the mammalian

MAPK pathways might maintain specificity. The results of these experiments

suggest that scaffolding proteins are not involved in maintaining the specificity

of a given pathway, but that another mechanism, possibly protein-protein

interactions, may be largely responsible.

Not only has this yeast work been integral in examining how the

different MAPK cascades maintain their functional distinctiveness, but it has

helped to further clarify the degree of crosstalk that occurs between the

different pathways. Previous work has shown -that activation of the ERK

pathway, the SAPK pathway, and the p38 pathway are not mutually exclusive

and that a significant overlap in the agonist profiles of SAPK and p38 exists

(see section 1.3.6). Crosstalk among the different pathways has also been

noted when in vitro kinase assays or transient transfection over-expression

studies are performed (see section 1.3.6).

The results of these yeast two hybrid experiments combined with other

work from Our group demonstrates that crosstalk does not readily occur in vivo

under physiologie conditions (Zanke et al., 1996). For example, we have shown

that SAPR and p38 are differentially activated by a variety of stress stimuli

and that these results are not only agonist- but ce11 type-dependent. Further,

we have demonstrated MEKK over-expression does not result in p38 activation

and that SEK coprecipitates from cells with SAPK but not with p38. These

data support the hypothesis that. SEK does not activate p38. 0the;

coimmunoprecipitation experiments have shown that MEK will coprecipitate

with ERK but not with SAPK or p38. A dominant-negative SEK mutant also

impairs SAPK activation but not p38 activation when transfected into cells

pnor to stimulation with anisomycin. Similarly a dominant-negative MKK3

impairs the activation of p38 but not SAPK when transfected into cells prior

to stimulation with either sorbitol or anisomycin. Lastly, a dominant-negative

version of MKK6 prevents p38 activation in response to hyperosmolar stress

in COS cells, but has no effect on the activation of SAPK.

While the results of the yeast two hybrid -work is generally consistent

with the aforementioned data, a few minor discrepancies exist. Firstly, p38

was shown to interact with both SEK and MKK6 by the liquid culture assays

and SEK, MKKG, and MKK3 by the filter assays. In contrast, the results of

Our group demonstrates that p38 interacts with both MKK3 and MKK6 but not

SEK (Zanke et al., 1996). Because X-gal is 105 fold more sensitive than ONPG,

a transient o r weak interaction would not be quantifiable by the liquid culture

assay. Therefore, by combining al1 of this information one could conclude that

SEK and SAPK are components of one distinct signalling pathway, MEK and

ERK are members of a second, and that p38 with either MKK3 or MKK6 are

members of a third. Clearly, more work d l be critical to further elucidating

the role of MKK3 andlor MKK6 in p38 signalling.

CHAPTER 3

The Cloning of a Serine/threonine Specific Kinase with Homology to the

Cyclin-Dependent Kinases and the Mitogen-Activated Protein Kinases

3.1 INTRODUCTION

The regulation of cellular activities following exposure to differing

environmental stimuli is a complex process that includes the phosphorylation

and dephosphorylation of many members of multiple signalling pathways. One

such signalling pathway that has been studied extensively is the MAPK

cascade (see section 1.3). Homologues of this pathway exist in both lower and

higher eukaryotes that regulate physiologically distinct processes. For

example, in yeast, the FUS3lKSSl pathway is involved in mating, the HOGl

pathway is involved in osmoregulation, the SMKl pathway is involved in

sporulation, and the MPKl pathway is involved in ce11 wall biosynthesis (see

sections 1.3.1 and 1.3.3) (Krisak et al., 1994; Mazzoni et al., 1993). In

mammalian cells other homologues of the MAPK-family exist. The ERKs are

involved in growth and differentiation and the SAP& and the p38 family are

involved in the stress response (see sections 1.3.1, 1.3.2, and 1.3.3).

Just as changes in the cell's phenotype resulting from exposure to

mitogenic and stressful stimuli are regulated by phosphorylation and

dephosphorylation events, ce11 division is also regulated by similar mechanisms

(see section 1.4). The ce11 cycle is regulated, in part, by a family of protein

kinases termed cyclin dependent kinases, of which eight have been identified

to date. cdc2 plays a critical role in the transition from G2 t o mitosis while

CDK2, CDK3, CDK4, and CDK6 are believed to be involved in the progression

from G l to S phase (see section 1.4.3). CDK5 has been implicated in neuronal

development and CDK7 and CDK8 appear t o be involved in ce11 transcription

(see section 1.4.3). In addition, a series of cdc2-related protein kinases exists.

This subgroup includes PITSLRE, PCTAIRE, PITAIRE, PITALRE, PISSLRE,

KKIAMRE, and KKIALRE (see section 1.5). The association of these kinases

with any cyclins or their involvement with ceIl cycle progression has yet to be

determined.

With the goal of isolating a novel MAPK member, we performed PCR on

cDNAs from a rat jejunum library with degenerate oligonucleotide primers

designed to regions specific to known MAPK family members. A product of

this reaction was used to screen a rat jejunum cDNA library and a rat brain

library, and in combination with the RACE technique, we isolated a cDNA for

a novel protein kinase. This cDNA encodes a rat serine/threonine specific

kinase that is related to the MAPK famiiy, but most closely resembles

KKIAMRE and -RE, members of the cdc2-related protein kinase family.

This kinase has been named NKIATRE based o n its sequence corresponding

to the PSTAIRE motif characteristic of CD&.

3.2 MATERIALS AND METHODS

3.2.1 PCR Amplification and Library Screening

To clone a novel MAPK, an alignment of 20 sequences of MAPK

homologues from various species was created. Two regions that were highly

conserved among these kinases were selected and compared to the

corresponding sequences found within the other families of kinases as aligned

by Hanks and Quinn (Hanks and Quinn, 1991). Because of their restriction

to the members of the MAPK family, the degenerate oligonucleotide primers

SRSP and SRAP were designed to the sequences TLREIK in subdomain III and

YVA/VTRW in subdomain VI11 respectively (Figure 11, Table 1). Although

both of these primers were designed to be used for the PCR amplification,

NKIATRE was isolated by use of SRAP and the M13/pUC Forward

amplification primer (Gibco BRL, Gaithersburg MD, USA), a specific primer

that recognizes a sequence flanking the cDNA insert site within the phage.

To obtain a satisfactory product by PCR, it is necessary to optirnize the

reaction conditions for any particular combination of template and primers.

Primer concentrations are important since limiting concentrations will r e s a

in low yields and excessive amounts will result in contamination due to

binding at non-target sequences. Because the levels of dNTPs and

oligonucleotides are the major contributors of phosphate groups to the reaction,

any change in their concentration will affect the levels of available Mg?

Therefore, it is important to optimize not only the primer concentration but the

Mg+ concentration as well.

NKIATRE was isolated from the pooled products of four slightly

different PCR amplifications. Each of these reactions contained 1 ug of cDNA

isolated from a rat jejunum cDNA library (Stratagene, La Jolla CA, USA) as

described by Sambrook et al. (Sambrook et al., 1989). Conditions for the first

SUBDOMAIN III SUBDOMAIN VI11

MPK1 MPK1 HOGl HOGl KSSl FUS3 ERK2 ERK2 ERK2 ERK2 SPK1 SAPKa SAPKb SAPKc SAPKd ERK1 ERK1

ERKl (cer)

... CKRSLRE LKLLR ............ LTEYVATRWYRA ...

... CKRALRE LKLLQ ............ MTEYVATRWY RA ...

... AKRTRYELRLLK ............ MTGYVATRWYRA ...

... AKRTYRELKLLK ............ MTGYVSTRYYRA ... VTRT 1 RE IKLLR MTEYVATRWYRA ... ............ ...

... ALRTLRE IKI LK ............ MTEYVATRWYRA ...

... CQRTLRE IKI LL ............ LTEYVATRWYRA ...

... CQRTLRE IKI LL ............ LTEYVATRWYRA ... CQRTLRE IKI LL LTEYVATRWYRA ... ............ ...

... CQRTLRE IKI LL ............ LTEYVATRWYRA ... ............ ... CLRTLKE 1 KLLR MTEYVATRWYRA ...

... AKRAYRELVLLK ............ MT PYVVTRY YRA ...

... AKRAY RE LVLMK ............ MT PYVVTRY Y RA ...

... AKRAY RE LVLMK ............ MT PY VVTRY Y RA ...

... MTPYVVTRYYRA ............ MTPYVVTRY YRA ...

... TKRTLRE IHLLR ............ MTEYVATRWYRA ... CQRTLRE IQI LL LTEYVATRWYRA ... ............ ...

... CQRTLRELQI LL ............ LTEYVATRWYRA ...

... CLRTLRELKLLK ............ MTEYVATRWYRA ...

TLREIK YVATRW v

Figure 11: Design of the degenerate oligonucleotide primers. To create the degenerate primers SRAP and SRSP, two regions conserved within the mammalian MAPK family members were selected. Primer SRSP was designed corresponding to t he amino acid sequence TLREIK within subdomain III of the catalytic domain and primer SRAP was designed corresponding to the sequence YVNVTRW in subdomain VIII. Nucleotide sequence of the prirners can be seen in Table 1. Abbreviations are a s follows: cer = S. cereuisiae, alb = Candida albicans, hum = human, mou = mouse, bov = bovine, and dct = Dictyostelium .

I SEQUENCE 5'-3' II Forward, M 13LacZ

1 ATC AAG CCT CCT CTC ACT CT II

GCC AGG GTT TTC CCA GTC ACG A

Reverse M13/LacZ

1 CCA CCG AGA TAT AAA GCC TG

GAG CGG ATAA CAA TT' CAC ACA GG

1 GGT GGT TCT TCC TCA AGT TC

l

MKH-4

MKH-5

1 CAG GCT TTA TAT CTC GGT GG

TGT CTT C ï T CAG GTC TGG GA

GGACTAGTCTTAATGGCCACTATCCGC

MKH-6

MKK-7

- -

CTT TCC TGT CGC ATT TGG

TCC CAG ACC TGA AGA AGA CA

MKH-9

MKH- 10

1 TCC CTD AAA GAA CAC AGA GG

ACC ATG AGG TGT CTG TM' CC

GACGCACCTCTTCACAAACT

M m - 1 1 GAG ATG AAA GGG ATG GAG GT II

II SRSP

M 131pUC Forward

S W

1 ATG GAA TTC ACC/A CTG C/AGI/C GAGIA ATCIT AAGIA II

CCC AGT CAC GAC GTI' GTA AAA CG

ATG GGA TCC CCA IICCGPT GIAGT GIAGIAC CPTIAAC GIATA

1 ATG CTC GAG ATG GAG ATG TAT GAA ACC

1 ATG AAG CTT TGC A m CTG ACC TGT TCG

and sequencing NKIATRE.

50 pl reaction was 10 pmol primer SRAP, 20 pmol M13/pUC Fonvard

amplification primer, 2 mM MgCl,, 0.2 mM dNTP, 50 mM KC1, 10 mM Tris-

HCl (pH 9.0), 0.1% Triton X-100, and 0.1 U/pl Taq DNA polymerase (Promega,

Madison WI, USA). The other three reactions differed by having 10 pmol of

each primer and MgCl, concentrations of either 1.5 mM, 2.0 mM, or 2.5 mM.

Before the addition of Taq DNA polymerase each reaction was heated to 94OC

for 3 minutes. For 30 cycles the reaction mixtures were denatured at 94OC for

1 minute, allowed to anneal at 57OC for 1 minute and 30 seconds, and extended

at 72OC for 2 minutes. Before a 4OC soak, an additional cycle was performed

as above with a 5 minute primer extension at 72OC.

After al1 cycles were complete, the four reactions were pooled and 4 U

of DNA polymerase I Klenow fragment (Amersham, Amersham UK) were

added to create blunt termini by incubation at 37OC for 30 minutes. A 0.5 kb

product was purified from an agarose gel using the Geneclean II kit (Bio 101,

La Jolla CA, USA) and cloned into the SmaI site of the pUC18 plasmid.

Several products of the PCR amplification were analyzed by DNA

sequencing, as described in section 3.2.2. These sequences were compared to

those found in the GenBank database using the Blast program (Wisconsin

Genetics Cornputer Group). One unique product was selected to screen the

original rat jejunum cDNA library and a second, rat brain cDNA library

(Stratagene, La Jolla CA, USA).

For library screening, plating cultures were prepared by inoculating LB

broth, supplemented with 0.2% maltose and 10 mM MgSO,, with a single

colony of the bactenal strain XL1 Blue. Cultures were grown ovemight a t

30°C to ensure that the cells would not overgrow. The following day the cells

were spun down for 10 minutes at 2000 rpm and resuspended in 10 mM

MgSO, so that the OD, = 0.5. To 6 ml of the cells, 5x10~ pfu of the

appropriate library were added and the solution was gently mixed and

incubated at 37OC for 15 minutes. To each of 10, 150 mm, NZY plates, 600 pl

of the phage and bacteria mixture with 8 ml of top agar was poured. The

plates were incubated at 37OC until the bacteria cells were completely lysed

(approximately 12 hours).

The phage DNA from the lysed bacteria cells was transferred to Hybond-

N nylon membranes (Amersham, Amersham UK) for 2 minutes. Duplicate

filters were allowed to transfer for 5 minutes. M e r transfer, the filters were

denatured by submersion for 2 minutes in a solution of 1.5 M NaCl and 0.5 M

NaOH, and neutralized by submersion for 5 minutes in a solgtion of 1.5 M

NaCl and 0.5 M Tris-HCl (pH 8). The filters were then briefly rinsed in 2x

SSC and allowed to dry at room temperature. DNA was UV crosslinked to the

filters using the W Stratalinker (Stratagene, La Jolla CA, USA) and the agar

plates were stored at 4OC.

For screening, the filters were pre-hybridized in a solution of 50%

formamide, 5x SSC, 5x Denhardt's reagent, 20 mM Na2P0,, and 0.2 m g h l

salmon sperm DNA for two hours at 42OC. Hybridization was carried out

overnight a t 4 2 ' ~ in a solution of 50% formamide, 5x SSC, 5x Denhardt's

reagent, 20 mM Na2P0,, 10% dextran sulphate, 0.2 mg/ml salmon sperm DNA,

and lx 106 cpm/ml of probe. To make the probe, 25 ng of the PCR product was

radiolabelled with 50 pCi of ~ a - ~ v l d C ~ P using the Multiprime DNA labelling

system (Amersham, Amersham UK) and unincorporated label was removed by

gel filtration through 7 cm of Sephadex G-50. Following hybridization, the

filters were washed at room temperature in 2x SSC, and 0.1% SDS until the

wash b d e r radioactivity could no longer be detected by a Geiger counter. This

was followed by two 20 minute washes a t 55OC in 0 . h SSC and 0.1% SDS.

Filters were exposed to film overnight a t -70°C with an intensifying screen.

Purification of the hybridizing plaques was performed by secondary,

tertiary, and quaternary screens, as described above. Excision of the cDNA

inserts from the purified phage followed the manufacturer's lambda ZAP II

automatic excision protocol (Stratagene, La Jolla CA, USA).

3.2.2 DNA Sequence Analysis

Plasmid DNA was purified for sequencing with the Wizard Minipreps

DNA Purification System (Promega, Madison WI, USA) and approximately 0.2

ug was sequenced with either [a-35S]dATP and the Amplicycle Sequencing Kit

(Perkin Elmer, Branchburg NJ, USA) or [~x-~~P]dclNTPs and the Thermo

Sequenase radiolabeled terrninator cycle sequencing kit (Amersham,

Amersham UK). DNA sequence was read manually following exposure of the

. - gels to film a t room temperature for 24 - 48 hours.

Sequencing primers used included the Forward M13(lacZ) Primer and

the Reverse MlS(LacZ) Primer (Perkin Elmer, Branchburg NJ, USA) (Table 1).

In addition, twelve primers, MKH1-12, were constructed to allow overlapping

sequence determination from the 5' and 3' ends of the clones (Table 1).

3.2.3 Amplification of 5' cDNA End

Anplification of the 5' end of the cDNA was accomplished with the 5'

RACE System (Gibco BRL, Gaithersburg MD, USA). This technique allows for

the amplification of nucleic acid sequences from an mRNA template between

a defined interna1 site and unknown sequences at the 5' end (Figure 12). The

general strategy of this protocol is as follows: first strand cDNA synthesis is

primed using a gene-specific oligonucleotide which penaits conversion of the

specific RNA and mavimizes the potential for complete extension to the 5' end.

The cDNAs are then tailed with TdT which adds homopolymeric tails to the 3'

ends. Following, the target cDNA is amplified by PCR using a nested gene

specific primer which anneals 5' to the first gene-specific primer and an anchor

primer that permits amplification from the homopolymeric tail.

To ampli6 the 5' end of NKIATRE, rat poly(A)+RNA (see section 3.2.5

for preparation of poly(A)+RNA) was used as a template and sequencing primer

MKH4 was used to prime first strand cDNA synthesis (Table 1). PCR

amplification of the NKIATRE cDNA utilised MKH8 (Table 1) as the nested

5' (A) n-3'

4 4 , . !.-.. .:

(A) n-3' - - * I L '

Anchor Primer

Figure 12: Flow diagram of the 5' RACE procedure. (A) Gene specific primer (GSP) 1 is annealed to mRNA. (B) First strand cDNA synthesis is performed with reverse transcriptase. ( C ) mRNA template is degraded with RNase H. (D) cDNA is purified and tailed with dCTP by terminal transferase. (E) The cDNA is PCR amplified with GSP2, a second primer that recognizes a sequence 5' t o GSP1, and an anchor primer that recognizes the tailed region. To further increase specificity a second PCR amplification can be performed with the anchor primer and GSP3, a third primer that recognizes a sequence 5' to GSP2.

gene specific primer in a 50 pl reaction volume as described in the

manufacturer's manual. For 35 cycles the reaction mixture was denatured a t

94OC for 1 minute, allowed to anneal at 60°C for 30 seconds, and extended a t

72OC for 2 minutes. One final cycle was performed as above with a 10 minute

extension at 72OC.

To further enrich the PCR product for the specific cDNA, 1 pl of the PCR

reaction was re-amplified under the outlined conditions with primer MKH5 as

a second nested gene specific primer (Table 1). Following amplification, 1 U

of DNA polymerase 1 Klenow fragment (Amersham, Amersham UK) was added

to the reaction to create blunt termini by incubation a t 37OC for 30 minutes.

Products were cloned into the SmaI site of pBluescript KS and analyzed by

sequencing with the Forward M13/lacZ primer and the Reverse M13/LacZ

primer (Perkin Elmer, Branchburg NJ, USA) as previously described.

3.2.4 Southern Blot Analysis

For Southern blot analysis, 10 pg of rat genomic DNA was digested

overnight at 37OC with either EcoRI, BamHI or HindIII. For the cross species

Southem blot, 10 pg of either rat, pig, mouse, human or Drosophila genomic

DNA was digested overnight at 37OC with EcoRI. Each of these digests were

fractionated overnight by electrophoresis on a 1.0% agarose gel. Following

electrophoresis, the gel was incubated with shaking for 15 min a t room

temperature in 0.1 M HC1, followed by 30 min in a solution of 0.5 M NaOH,

1.5 M NaCl, and then for 45 min in a solution of 1 M Tris pH 7.0, 1.5 M NaCl.

The genomic DNA was transferred to nylon membrane by capillary transfer

(Sambrook et al., 1989). For al1 blots, the 1.3 kb EcoRf fragment (nucleotides

1-1372) of the rat jejunum cDNA was radiolabelled with [ ~ L - ~ ~ P I ~ C T P , as

described in section 3.2.1, and used as the probe. Prehybridization,

hybridization, and wash conditions were perfonned as previously described for

the library screens (section 3.2.1). Filters were visualized by Phosphor Imager

(Molecular Dynamics) using the Image Quant software (Molecular Dynamics)

3.2.5 Northern Blot Analysis

Total cellular RNA was prepared using the TRIzol Reagent (Gibco BRL,

Gaithersburg MD, USA) and from this poly(A)+RNA was prepared using Oligo

(dT) cellulose (Boehringer Mannheim Biochemica, Laval PQ, Canada) as

follows. 1 g of Oligo (dT) cellulose was suspended in 20 ml of loading b f i e r

(400 mM NaCl, 20 mM Tris pH 7.4, 10 mM EDTA, 0.2% SDS) and mixed for

20 minutes at room temperature. Oligo (dT) cellulose was added to each

sample of total RNA (1 g of cellulose binds 10 mg of total RNA), and each

mixture was gently rotated at room temperature overnight. Al1 samples were

then spun for 5 minutes a t 2000 rpm, washed 2-3 times with 20 ml of loading

buffer, and then 2-3 times with 20 ml of wash bufYer (100 mM NaCl, 10 mM

Tris pH 7.4, 1 mM EDTA, 0.2% SDS). Cellulose samples were then

resuspended in 10 ml of wash buffer and poured into columns (Bio Rad,

Hercules California, USA). Columns were packed with 10 ml of wash bufTer

and then poly(A)+RNA was eluted with Gve 1 ml volumes of elution buffer pre-

warmed to 37OC (1 mM Tris pH 7.4, 1 mM EDTA, 0.2% SDS). Poly(A)'RNA

was precipitated overnight at -20°C by the addition of 30 pg tRNA, 0.6 ml 3 M

sodium acetate, and 12 ml 99% ethanol. Pellets were air dried and

resuspended in DEPC-treated water.

Ten pg of poly(A)+RNA from intestine, brain, muscle, lung, spleen, heart,

liver, and thymus were used to generate a rat tissue blot. RNA was

fractionated overnight by electrophoresis on a 1.2% formaldehyde agarose gel

with buffer recirculation. The gel was then washed thoroughly in DEPC

treated water and the RNA was transferred to nylon membrane by capillary

transfer (Sambrook et al., 1989). Like the Southern blots, the Northern blot

was probed with the 1.3 kb EcoRI fragment .of the rat jejunum cDNA,

radiolabelled with [a-32P]dCTP, as described in section 3.2.1. To control for the

level of RNA in each lane, a rat P-actin probe was also used. Prehybridization,

hybridization, and wash conditions were carried out as described in section

3.2.1. Exposure of the filter and visualization of the image was as described

in section 3.2.4.

3.2.6 Prokaryotic Expression of NKIATRE

To express and purify NKIATRE from prokaryotic cells, the vector

pGEX- 1hT was utilised (Pharmacia, Picataway NJ, USA). This vector contains

the sequence for GST just upstream of the multiple cloning site, allowing for

u;E

the generation of a GST fusion protein. To create an in-frame GST-NKIATRE

fusion, the rat jejunum cDNA was digested with the restriction endonuclease

EcoRI and the 1.38 kb fragment containing the 5' end of the cDNA was

purified and cloned into the EcoRI site of the prokaryotic expression vector

pGEX-lhT.

Fusion proteins were expressed in the E. Coli. strain BL21(DES)pLysS,

which carries the plasmid pLysS, that codes for T7 lysozyme, an enzyme that

lowers the basal expression of target genes (Novagen, Madison WI, USA).

Induction of protein expression in large bacterial cultures was accomplished

with the addition of 1 mM IPTG. Bacteria were then lysed by gentle

sonication in 50 mM Tris pH 8.0, 10 mM EDTA, 2.5% Nonidet P40, 50 mM

NaCl, and fusion proteins were affinity purified with glutathione-Sepharose.

Following, proteins were eluted with 3 mg/ml glutathione in 50 mM Tris pH

8.0 and dialysed against phosphate buffered saline (140 mM NaCI, 2.7 mM

KC1, 1.5 mM KH,P04, 8 mM Na2HP04) at 4OC. Protein integrity was

codrmed by 0.25% Coomassie Brilliant Blue staining of SDS-PAGE products.

3.2.7 Preparation for Eukaryotic Expression

To obtain a complete cDNA for NKIATRE, the rat intestine clone and

the rat brain clone #2 were digested with the enzyme BglI and the 1.3 kb and

2.0 kb bands were purified from an agarose gel utilizing the Geneclean II kit

(Bio 101, La Jolla CA, USA). Following ligation of the two products, PCR was

used to generate the complete cDNA with approximately 100 ng of ligated DNA

and 2 pl each of the 20 PM primers 5C1 and 3C1 (Table 1). Conditions for a

total reaction volume of 50 pl were 2 mM MgCl,, 0.2 mM dNTP, 50 mM KCl,

10 mM Tris-WC1 (pH 9.0), 0.1% Triton X-100, and 0.1 U/pl Taq DNA

polymerase (Promega, Madison WI, USA). Before addition of the Taq DNA

polynerase, the reaction was heated to 94% for 3 minutes. For 30 cycles the

reaction mixture was denatured at 94OC for 1 minute, allowed to anneal at

57OC for 1 minute and 30 seconds, and extended at 72OC for 2 minutes. Before

a 4% soak, an additional cycle was performed as above with a 5 minute primer

extension at 72OC. After the reaction was complete, 4 U of DNA polymerase

1 Klenow fragment (Amersharn, Amersham UK) were added to create blunt

termini by incubation at 37OC for 30 minutes. Following, the reaction was

purified from an agarose gel by the Geneclean II kit (Bio 101, La Jolla CA,

USA), and cloned into the SmaI site of pBluescript KS.

To allow for immunoprecipitation from eukaryotic cells, NKIATRE was

tagged with three HA. repeats and ten histidine residues by subcloning into the

vector pBluescript-3HA-10His. pBluescript-3H.A-1OHis is a pBlues~ript

derivative with the tags at the 3' end of the multiple cloning site. The

complete NKIATRE cDNA was cut out of pBluescript KS with XhoI and

HindIII (sites that were engineered into the PCR primers) and cloned into

pBluescript-3HA-lOHis digested with the same enzymes, creating NKIATRE-

pBluescript-3HA-10His.

To subclone the complete, tagged, cDNA into the eukaryotic expression

vector, pcDNA3 (Invitrogen, Carlsbad CA, USA), NKIATRE-pBluescript-3HA-

lOHis was cut with XhoI and XbaI. Following, the fragment containing the

cDNA and tags was ligated to pcDNA3 digested with the same enzymes and

the integrity of the construct was validated by diagnostic restriction

endonuclease digestions.

3.2.8 In Vitro Transcription and Translation

To linearize the vectors for in vitro transcription and translation,

NKIATRE-pcDNA3 and control pcDNA3 were digested with the enzyme XbaI

which cuts to the 3' side of NKIATRE. The DNA was purified by

phenoYchloroform extraction and ethanol precipitation, and was resuspended

in DEPC treated water. Transcription and translation were coupled using the

TNT T7 Coupled Wheat Germ Extract System (Promega, Madison WI, USA),

and 40 pCi of 35S-methionine. Following transcription and translation, the

products were separated by SDS-PAGE and analyzed using a Phosphor Imager

(Molecular Dynamics) and the Image Quant SoRware (Molecular Dynamics).

3.3 RESULTS

3.3.1 Isolation of a Rat cDNA

To isolate a novel MAPK, degenerate oligonucleotide primers were

designed to two regions specific for sequences within the kinase catalytic

domain of the MAPK family mernbers. PCR amplification using the antisense

degenerate primer, SRAP, and the M13/puc18 Forward primer generated a

product of approximately 500 bp from a template of r a t jejunum cDNAs (Figure

13). Sequence analysis of 44 of these products led to the discovery a single

clone that was a putative M W kinase.

To obtain the complete cDNA, the rat jejunum cDNA library and a rat

brain cDNA library were screened with the PCR product. Three clones were

isolated from the jejunum l ib rav which upon sequence analysis were shown

to be identical. From the brain library two additional positive phage were

isolated, each representing independent clones. Criteria for a full length cDNA

included the presence of a consensus translation initiation sequence of

GCCGCCA/GCCAUGG a t a potential initiator methionine codon, and the

presence of an upstream stop codon in the same reading frame as the initiation

codon (Kozak, 1987; Kozak, 1989). Sequencing of al1 three clones demonstrated

that none were complete by these criteria. The jejunum clone was truncated

with a premature stop codon afier amino acid 457 and the putative start codon

was within 15 nucleotides from the 5' end of the clone. Although both brain

clones contained the 3' end of the coding region, they were both missing the 5'

end. Clone #2 commenced at amino acid 95 and clone #6 commenced a t amino

acid 62 (Figure 14). Of note, clone #2 contained a sequence of unknown origin,

that had no homology t o any known kinase, immediately upstream of the codon

Figure 13: PCR amplification. The products of PCR amplification from rat jejunum cDNAs with combinations of primers SRAP (A), SRSP (S), M13/puc18 Reverse (R), and M13/puc18 Forward (F) are shown above. M g * concentrations are indicated above each set of reactions. The first three sets of reactions had lOpmol of each primer indicated. The fourth set of reactions, labelled 20:10, had a Mg2' concentration of 2mM and contained 2Opmol of primer R or F and lOpmol of primer A or S. A positive control (+) reaction had lOpmol each of the primers R and F. A negative control (-) reaction had lOpmol each of the primers R and F but no template DNA. See text for more detailed description of conditions. Size markers indicated are in kb.

for amino acid 95. By combining al1 of this data, it is apparent that the

deduced amino acid sequence of the complete clone encodes a protein of 505

amino acids (Figure 15), with a predicted molecular weight of 67 kD.

3.3.2 Amplification of the 5' cDNA End

To determine if the putative initiation codon was the true start of

translation, based on the previously outlined criteria, a 5' RACE was

performed on rat brain poly{A)+RNA. The initial PCR amplification with the

anchor primer and primer MKH8 generated a product of approximately 700 bp.

To increase specificity, re-amplification of a small sample of this product with

the internal primer MKH5 and the anchor primer, resulted in a product of

approximately 400 bp. As expected from the distance between the two internal

primer sites on the clones, the size of the product was approximately 300 bp

shorter (Figure 16).

Sequence analysis of the cloned products revealed a n upstream stop

codon forty-two nucleotides from the putative initiation methionine.

Cornparison of the sequence surrounding this methionine codon to the Kozak

consensus sequence, demonstrates that seven of the thirteen nucleotides are

conserved a t this site, including the purine at position -3 and the guanine at

position +4 (Figure 17). Of the thirteen nucleotides, the purine at position -3

and the guanine at position +4 have been determined to be the most highly

consemed nucleotides in all eukaryotic mRNAs (Kozak, 1986). Further, by

considering only these two positions one can predict the relative strength of the

brain2 brain6

j e j unum

brai112 brain6

je junum

brain2 bxain6

je junum

brain2 brain6

j e junum

brain2 brain6

j e j unum

brain2 brain6

j e j unum

brain2 brai116

j e j unum

brain2 brain6

j e j unum

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MEMYETLGKV GEGSYGTVMK CKHKDTGRIV AIKIFYEKPE KSVNKIATRE IKFLKQFRHE

61 120 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HGLESK RLRKYLFQIL RAIEYLHNNN .LVNLIEVFR QKKKIHLVFE FIDHTVLDEL QHYCHGLESK RLRKYLFQIL RAIEYLHNNN NLVNLIEVFR QKKKIHLVFE FIDHTVLDEL QHYCHGLESK RLRKYLFQIL RAIEYLHNNN

? 2 1 180 IIHRDIKPEN ILVSQSGITK LCDFGFARTL AAPGDVYTDY VATRWYRAPE LVLKDTTYGK IIHRDIKPEN ILVSQSGITK LCDFGFARTL AAPGDVYTDY VATRWYRAPE LVLKDTTYGK IIHRDIKPEN ILVSQSGITK LCDFGFARTL AAPGDVYTDY VATRWYRAPE LVLKDTTYGK

1 8 1 240 PVDIWALGCM IIEMATGNPY LPSSSDLDLL HKIVLKVGNL TPHLHNIFSK SPIFAGVVLP PVDIWALGCM IIEMATGNPY LPSSSDLDLL HKIVLKVGNL TPHLHNIFSK SPIFAGVVLP PVDIWALGCM IIEMATGNPY LPSSSDLDLL HKIVLKVGNL TPHLHNIFSK SPIFAGVVLP

241 300 QVQHPKNARK KYPKLNGLLA DIVHACLQID PAERISSTDL LHHDYFTRDG FIEKFIPELR QVQHPKNARK KYPKLNGLLA DIVHACLQID PAERISSTDL LHHDYFTRDG FIEKFIPELR QVQHPKNARK KYPKLNGLLA DIVHACLQID PAERISSTDL LHHDYFTRDG FIEKFIPELR

301 360 AKLLQEAKVN SFIKPKENFK ENEPVRDEKK PVFTNPLLYG NPTLYGKEVD RDKRAKELICV AKLLQEAKVN SFIKPKENFK ENEPVRDEKK PVFTNPLLYG NPTLYGKEVD RDKRAKELKV WLLQEAKVN SFIKPKENFK ENEPVRDEKK PVFTNPLLYG NPTLYGKEVD RDKRAKELKV

361 420 RVIKAKGGKG DVPDLKKTES EGEHRQQGTA EDTHPTSLDR KPSVSELTNP VHPSANSDTV RVIKAKGGKG DVPDLKKTES EGEHRQQGTA EDTHPTSLDR KPSVSELTNP VHPSANSDTV RVIKAKGGKG DVPDLKKTES EGEHRQQGTA EDTHPTSLDR KPSVSELTNP VHPSANSDTV

4 2 1 480 KEDPHSGGCM IMPPINLTSS NLLAANPSSN LSHPNSRLTE RTKKRRTSSQ TIGQTLSNSR KEDPHSGGCM IMPPINLTSS NLLAANPSSN LSHPNSRLTE RTKKRRTSSQ TIGQTLSNSR KEDPHSGGCM IMPPINLTSS NLLAANPSSN LSHPNSR*.. . . . . . . . . . . . . . . . . . . . .

4 8 1 506 QEDTGPTQVQ TEKGAFNERT GQNAK* QEDTGPTQVQ TEKGAFNERT GQNAK*

Figure 14: Sequence alignment of the cDNA clones. The amino acid sequences for the three cDNA clones are presented. Astensks represent in frame stop codons.

- -22 --- -2-8

1 3 atg ggc tga ggc ggc ggc gat t c r cag gac gag aag cca ggc t t g aaa ATG GAG ATG TAT 1 t M E M Y

76 GAA ACC CTT GGA AAA GTA GGA GAG GGA AGT TAT GGA ACG GTC ATG AAA TGC AAG CAT AAG 5 E T L G K V G E G S Y G T V M K C K H K

1 3 6 GAT ACT GGG CGG ATA GTG GCC ATT AAG ATA T T C TAT GAG AAA CCA GAA AAA T C T GTC AAC 2 5 D T G R I V A I K I F Y E K P E K S V N

1 9 6 AAA ATT GCA ACG AGA GAA ATA AAG T T T CTA AAG CAA T T T CGT CAT GAA AAC CTG GTC AAT 4 5 K I A T R E I K F L K Q F R H E N L V N

2 5 6 CTG ATT GAA G T T TTT AGA CAA AAA AAG AAA ATC CAT TTG GTA TTT GAG TTT A T T GAC CAC 6 5 L I E V F R Q K K K I H L V F E F I D H

3 1 6 ACG GTC TTA GAT GAG TTA CAG CAT TAT TGT CAC GGA TTA GAG AGT AAG CGA CTA AGA AAG 8 5 T V L D E L Q H Y C H G L E S K R L R K

3 7 6 TAC CTG T T C CAG ATC CTC CGA GCG ATC GAG TAT CTG CAC AAC AAC AAC ATT ATC CAC CGA 1 0 5 Y L F Q I L R A I E Y L H N N N I I H R

4 3 6 GAT ATA AAG CCT GAG AAT ATT TTA GTC TCC CAG TCA GGA ATT ACA AAG CTC TGC GAT TTT 1 2 5 D I K P E N I L V S Q S G I T K L C D F

4 9 6 GGG T T T GCG CGG ACA CTA GCA GCT CCT GGA GAC GTT TAC ACA GAC TAC GTG GCC ACC CGC 1 4 5 G F A R T L A A P G D V Y T D Y V A T R

5 5 6 TGG TAC AGA GCT CCA GAG CTG GTG TTG AAA GAC ACC ACC TAT GGA AAA CCA GTG GAT ATC 1 6 5 W R A P E L V L K D T T Y G K P V D I

6 1 6 TGG GCT TTG GGC TGT ATG ATC ATT GAA ATG GCC ACT GGC AAT CCC TAC CTT CCT AGC AGT 1 8 5 W A L G C M I I E M A T G N P Y L P S S

6 7 6 TCC GAT TTG GAT TTG CTC CAC AAG ATT GTT T T A AAA GTA GGC AAC CTG ACC CCG CAC CTG 2 0 5 S D L D L L H K f V L K V G N L T P H L

7 3 6 CAC AAT A T T T T T TCC AAG AGT CCC ATC TTT GCT GGG GTG GTT CTT CCT CAA G T T CAA CAT 2 2 5 H N f F S K S P I F A G V V L P Q V Q H

796 CCC AAA AAC GCA AGA AAG AAA TAC CCA AAG CTC AAC GGA TTG CTG GCA GAT ATA GTT CAT 2 4 5 P K N A R K K Y P K L N G L L A D I V H

856 GCT TGT TTA CAA ATT GAT CCT GCT GAG AGG ATA TCA TCC ACC GAT CTT TTG CAT CAC GAT 2 6 5 A C L Q I D P A E R I S , S T D L L H W D

9 1 6 TAC TTT ACT AGA GAT GGA T T T ATT GAG AAA T T T ATA CCA GAG CTG AGA GCT AAG TTA TTA 2 8 5 Y F T R D G F I E K F I P E L R A K L L

9 7 6 CAG GAA GCA AAG GTT AAT TCA TTT ATA AAG CCA AAA GAG AAT TTT AAG GAA AAT GAA CCT 3 0 5 Q E A K V N S F I K P K E N F K E N E P

1 0 3 6 GTG AGA GAT GAG AAA AAA CCA GTT T T T ACC AAC CCT CTG CTC TAT GGA AAC CCA ACA CTT 3 2 5 V R D E K K P V F T N P L L Y G N P T L

1 0 9 6 TAT GGA AAG GAA GTG GAC AGA GAC AAA AGG GCC AAG GAG CTC AAA GTC AGA GTC ATT AAG 3 4 5 Y G K E V D R D K R A K E L K V R V I K

1 1 5 6 GCC AAA GGG GGA AAA GGA GAC GTC CCA GAC CTG AAG AAG ACA GAG AGT GAA GGT GAA CAC 3 6 5 A K G G K G D V P D K K T E S E G E H

1 2 1 6 CGC CAG CAG GGC ACA GCT GAG GAC ACA CAC CCC ACA TCA CTG GAC AGG AAG C C T TCT GTC 3 8 5 R Q Q G T A E D T H P T S L D R K P S V

1 2 7 6 TCG GAA CTA ACA AAC CCT GTC CAT CCC AGT GCG AAT TCT GAC ACT GTC AAA GAA GAC CCA 4 0 5 S E L T N P V H P S A N S D T V K E D P

1 3 3 6 CAC TCT GGG GGC TGT ATG ATA ATG CCA CCT ATC AAC CTG ACA AGC AGT AAT TTG TTG GCC 4 2 5 H S G G C M I M P P I N L T S S N L L A

1 3 9 6 GCA AAT CCC AGT TCA AAC CTC TCC CAC CCC AAT TCA CGG TTA ACT GAA AGA ACA AAA AAG 4 4 5 A N P S S N L S H P N S R L T E R T K K

1 4 5 6 AGA CGC ACC T C T TCA CAA ACT ATT GGA CAA ACT TTG TCT AAT AGC AGA CAA GAG GAC ACA 4 6 5 R R T S S Q T I G Q T L S N S R Q E D T

1 5 1 6 GGT CCC ACA C A . GTC CAA ACA GAG AAA GGT GCA T T T AAT GAG CGA ACA GGT CAG AAT GCA 4 8 5 G P T Q V Q T E K G A F N E R T G Q N A

1 5 7 6 AAA TAG caagtggaaacaaaagaaaactgaatttttccaaatgcgacaggaaagaattccatttccctgagctgccgt 5 0 5 K *

1 6 5 4 tcacaatacaggcgaaggagatgaaagggatggaggttaaacagataaaagtgctgaagagagaatcaaagaaaacggat 1 7 3 4 tcacc

Figure 15: Sequence of NKIATRE. The nucleotide and deduced protein sequence of NKIATRE is presented.

Figure 16: Products of the 5' RACE procedure. (A) First round PCR amplification. Lane 1: 5' end of NKIATRE amplified with primer MKH8 and the anchor primer. Lane 2: Control PCR using only primer MKH8. Lane 3: Control PCR using only the anchor primer. Lane 4: Control PCR with primer MKH8 and the anchor primer using untailed template. (B) Second round PCR amplification. Lane 1: 5' end of NKIATRE amplified with primer MKH5 and the anchor primer. Lane 2: Control PCR with no template. For more detailed descriptions of PCR conditions see text. Size markers indicated are in bp.

1 cgg cgg gac tac agg

1 6 a tg ggc tga ggc ggc ggc gat t c t cag gac gag aag cca 1

I

76 GAA ACC CTT GGA AAA GTA GGA GAG GGA AGT TAT GGA ACG 5 E T L G K V & E & S - Y G T

II 136 GAT ACT GGG CGG ATA GTG GCC ATT AAG ATA TTC TAT GAG

2 5 D T G R 1 V A f & I F Y E III

196 AAA A T T GCA ACG AGA GAA ATA AAG TTT CTA AAG CAA TTT 4 5 K 1 A T R & I K F L K Q F

IV 2 5 6 CTG A T T GAA GTT T T T AGA CA74 AAA AAG AAA ATC CAT TTG

6 5 L I E V F R Q K K K I H L v

3 1 6 ACG GTC TTA GAT GAG TTA CAG CAT TAT TGT CAC GGA TTA 8 5 T V L D E L Q H Y C H G L

V I A 3 7 6 TAC CTG TTC CAG ATC CTC CGA GCG ATC GAG TAT CTG CAC

1 0 5 Y : F Q I L R A I E Y L H VIE

4 3 6 GAT ATA AAG CCT GAG AAT ATT TTA GTC TCC CAG TCA GGA 1 2 5 D I K P E N I L V S Q S G

4 9 6 GGG T T T GCG CGG ACA CTA GCA GCT CCT GGA GAC GTT TAC 1 4 5 & F A R T L A A P G D V - Y

v r n 5 5 6 TGG TAC AGA GCT CCA GAG CTG GTG TTG AAA GAC ACC ACC

1 6 5 W Y R A P _ k L V K D T T IX

6 1 6 TGG GCT TTG GGC TGT ATG ATC ATT GAA ATG GCC ACT GGC 1 8 5 W A L & C M I I E M A T G

6 7 6 TCC GAT TTG GAT TTG CTC CAC AAG ATT GTT TTA AAA GTA 2 0 5 S D L D L L H K I V L K V

7 3 6 CAC AAT ATT T T T TCC AAG AGT CCC ATC TTT GCT GGG GTG 2 2 5 H N I F S K S P I F A G V

7 9 6 CCC AAA AAC GCA AGA AAG AAA TAC CCA AAG CTC AAC GGA 2 4 5 P K N A R K K Y P K L N G

XI 8 5 6 GCT T C T TTA CAA ATT GAT CCT GCT GAG AGG ATA TCA TCC

2 6 5 A C L Q 1 D P A E A 1 S S 916 TAC T T T ACT AGA GAT GGA TTT ATT GAG AAA TTT ATA CCA

2 8 5 Y F T R D G F I E K F I P 976 CAG GAA GCA AAG GTT AAT TCA TTT ATA AAG CCA AAA GAG

3 0 5 Q E A K V N S F I K P K E 1036 GTG AGA GAT GAG AAA AAA CCA GTT TTT ACC AAC CCT CTG

3 2 5 V R D E K K P V F T N P L 1 0 9 6 TAT GGA AAG GAA GTG GAC AGA GAC AAA AGG GCC AAG GAG

3 4 5 Y G K E V D R D K R A K E 1 1 5 6 GCC AAA GGG GGA AAA GGA GAC GTC CCA GAC CTG AAG AAG

gcc gcc acc ATG G suc ttu aaa_ATC, aAG ATG TAT

M E M Y

GTC ATG AAA T G C AAG CAT AAG V M K C K H K

AAA CCA GAA AAA TCT GTC AAC K P E K S V N

CGT CAT GAA AAC CTG GTC AAT R H E N L V N

GTA T T T GAG T T T ATT GAC CAC V F E F f D H

GAG AGT AAG C G A CTA AGA AAG E S K R L R K

AAC AAC AAC A T T ATC CAC CGA N N N I I H R

VII ATT ACA AAG C T C TGC GAT TTT

r T K L C & F ACA GAC TAC G T G GCC ACC CGC

T D Y V A T R

TAT GGA M A C C A GTG GAT ATC Y G K P V & I

AAT CCC TAC C T T N P Y L X

GGC AAC CTG ACC G N L T

GTT CTT CCT CAA V L P Q

TTG CTG GCA GAT L L A D

CCT AGC AGT P S s

CCG CAC CTG P H L

GTT CAA CAT V Q H

ATA GTT CAT I V H

ACC GAT T D

GAG CTG E L

AAT TTT N F

CTC TAT L Y

CTC AAA L K

ACA GAG

CTT TTG CAT CAC GAT L L H H O

AGA GCT AAG TTA TTA R A K L L

AAG G A . AAT GAA CCT K E N E P

GGA AAC CCA ACA CTT G N P T L

GTC AGA GTC ATT AAG V R V I K

AGT GAA GGT GAA CAC

1216 CGC CAG CAG GGC A C . GCT GAG GAC ACA CAC CCC ACA TCA CTG GAC AGG AAG CCT TCT GTC 3 8 5 R Q Q G T A E D T H P T S L D R K P S V

1 2 7 6 TCG GAA CTA A C , AAC CCT GTC CAT CCC AGT GCG AAT TCT GAC ACT GTC AAA GAA GAC CCA 4 0 5 S E L T N P V H P S A N S D T V K E D P

1336 CAC T C T GGG GGC TGT ATG ATA ATG CCA CCT ATC AAC CTG ACA AGC AGT AAT TTG TTG GCC 4 2 5 H S G G C M I M P P I N L T S S N L L A

1396 GCA AAT CCC AGT TCA AAC CTC TCC CAC CCC AAT TCA CGG TTA ACT GAA AGA A C . AAA AAG 4 4 5 A N P S S N L S H P N S R L T E R T K K

1456 AGA CGC ACC TCT TCA CAA ACT ATT GGA CAA ACT TTG TCT AAT AGC AGA CAA GAG GAC ACA 4 6 5 R R T S S Q T I G Q T L S N S R Q E D T

1516 GGT CCC ACA CAA GTC CAA ACA GAG AAA GGT GCA TTT AAT GAG CGA ACA GGT CAG AAT GCA 4 8 5 G F T Q V Q T E K G A F N E R T G Q N A

1 5 7 6 AAA TAG caagtggaaacaaaagaaaactgaatttttccaaatgcgacaggaaagaattccatttccc~gagctgccgt 5 0 5 K *

1654 tcacaa~acaggcgaaggagatgaaaagggatggaggttaaacagataaaagtgctgaagagagaatcaaagaaaacggat 1734 tcacc

Figure 17: Analysis of NKIATRE as a serinehhreonine specific kinase. The twelve conserved amino acid residues characteristic of the kinase catalytic domain and the two sequences that denote serinelthreonine specificity are presented in bold font and are underlined with double lines. The twelve kinase domains are also indicated with italic bold font above the corresponding regions. The Kozak consensus sequence for the start of translation is highlighted and presented above the corresponding sequence of NKIATRE that is in bold font and underlined with a single line. In-frame stop codons are denoted by asterisks.

initiator codon (Kozak, 1986).

3.3.3 Sequence Analysis

Protein kinases share a core catalytic domain with various stmctural

motifs being conserved (see section 1.2.1). These features can be used not only

to help identie novel kinases, but to further classify them as serinekhreonine

specific or tyrosine specific. Analysis of the sequence of NKIATRE reveals that

it contains al1 twelve of the invariant or nearly invariant residues that are

characteristic of the kinase catalytic domain, and that it most closely resembles

a serinehhreonine specific kinase.

The 12 residues that are conserved within the kinase catalytic domain

and their positions within NKIATRE are as follows: Glyll and Gly13 within

subdomain 1 (GXGXXGXV motif), Lys33 in subdomain II, Glu50 in subdomain

III, Aspl25 and Asnl3O in subdomain VIB, Asp143 and Gly145 in subdomain

VI1 (DFG motif), Glu170 in subdomain VI11 (APE motif), Asp183 and Gly188

in subdomain IX, and Arg274 in subdomain XI (Figure 17).

Strong indicators of the specificity of the kinase can be found within two

short motifs in subdomain VI and subdomain VI11 (Figure 17). In subdomain

VI, the consensus DLKPEN represents serinehhreonine specificity whereas

DLRAAN or DLAARN represents tyrosine specificity. NKIATRE contains the

sequence DIKPEN (residues 125-130), which conforms most closely t o the

serinehhreonine specific kinase consensus sequence. In subdomain VIII, the

sequence GT/SXXY/FX is found to be conserved in serinehhreonine specific

kinases, whereas PI/VK/RWT/M is characteristic of the tyrosine specific

kinases. NKIATRE contains the sequence ATRWYR (residues 162-167) which

again is most closely related to the serinekhreonine specific kinases.

Cornparison of NKIATRE with al1 protein kinases reveals that it is most

homologous to the cdc2-related protein kinases KKIAMRE and -RE.

Within the kinase domain, at the amino acid level, NKIATRE shares 60%

identity to KKIAMRE, 50% identity to KKLALRE, 33% identity to rat ERK1,

32% identity to rat ERK2, 34% identity to mouse p38, and 33% identity to rat

SAPKa (Figures 18 and 19).

NKIATRE has conserved residues charactenstic of both the MAPKs and

the CD&. In subdomain VI11 are located Tb158 and Tyr161 which

correspond to the sites of activating phosphorylation for the MAPKs. Thr158

also corresponds to a critical site of activating phosphorylation for the CD&.

In subdomain 1 of the CD& are also located Thrl4 and Tyr 15 which are

involved in regulating CDK activity. While Tyr15 is consenred in NKIATRE,

the residue Thrl4 is analogous to a serine. In addition, a motif search

revealed no protein-protein interaction domains within NKIATRE.

3.3.4 Southern Blot Analysis

To examine if NKIATRE was a component of the rat genome, the 1.3 kb

NKIATRE KKIALRE KKIAMRE

CDC2 CDK2 ERKl ERKS SAPK P38

CONSENSUS

NKIATRE KKIALRE XKIAMRE

CDC2 CDKS ERKl ERK2 SAPK P3 8

CONSENSUS

NKIATRE KKIALRE KKIAMRE

CDC2 CDKS ERKl ERKS SAPK P38

CONS ENSUS

NKIATRE KKIALRE KKIAMRE

CDC2 CDK2 ERKl ERK2 SAPK P38

CONSENSUS

1 60 --M-ET-GK----Ç--T-M-CrnKD--R-- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . M - - K - E K I G K - - - - S - - V - - - C m - - Q - - - - K - E N - G L - - - - S - - M - M - C m - - R - - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . --D-TKIEK----T--V---GRRKT--Q-- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . --N-QmK----T--v---- m - - E - - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

EPRGTAGWPWPGEWWKGQ . . . . PFDVGPR-TQ-QY----A--M-SS-DmKTR- . . . . . . . MAAAAAAGPEMVRGQ .... VFDVGPR-TN-SY----A-AM--S-YDNLNKVR-

MSDSKSDGQFYSVQVADSTFTV-KR-QQ-KP--S-AQ-I--A-FDTVL-IN- . . . . . . . . . . . . . . . . . . MSQERPTFYRQELNKTIWEVP-R-QN-SP--S-A--S--A-FDTK--HR- - - - - - - - - - - - - - - - - - - - - - - - - - - - - --ME-Y--L--1GEG-YG-VCKA----TG-IV

61 120 - - - 1-YE.KPEKS-N-1-T---K---Q-----L-N-1---RQKI . . H----- I- - - - - -LESE-DPV-K-I-L---R--- Q-K-P-L-N-----RRKRR- . . . . . . H----- C- - - - - -LESD-DKM-K-I-M---K---Q-----L-N----CKKKKRW.... - - - - - - V-

rM--IRLES-EEG-PST-I---S---E---P--- S-Q- --MQDSR- ...... - - - - - - - s . -L--IRXDT-TEG-~ST-I---S---E-N-~---K---- IHTENK-..... - - - - - --H

- - - - IS.PF-HQTYCQRTL---QI-LG------ -GIR---.RAPT-EAMRDV-1-Q---- - - - - IS.PF-HQTYCQRTL---KI-LR------- GIN--1.RAPTIEQMKDV-1-Q---- - - - - - SRPFQNQTHA-R-Y--LV---CVN-K---S---DV---M---- - - - - -SRPFQSIIHA-RTY--LR---H-K-----G---- -TPARS-EEFNDV---TH--G AIKKF- - - - E---V-K-A-REI-LLK-LRHENIV-LLDVF-----L------YLVFE-m

121 180 HTVLDE-QHYCHG..-ESKRLRK------RA-E-L-NN------ 1--E-1--SQSGIT-- HTVLHE-DRYQRG..VPEHL-KSIT--T-QA-N-C-M-C----V--E-I--TKHSVI-- HTILDD-ELFPNG..-DYQV-QK-----IN--G-C--H------I--E-I--SQSGW-- M--KKY-DsIPPGQY-DsSL-KS------Q--V-C--RR-L------Q---- DDKGTI - - Q--KKF-DASALTG.IPLPL-KS----L-Q-LA-C--HR-L------Q----NTEGAI-- T--~KL-K....SQQ-S~H-C~------R-LK-I--A--L------~---- NTTCDL- 1 T--Y=-K .... TQH-SmH-CY------R-LK-1--A--L------ S---LNTTCDL-1 AN-CQVIH . . . . . ME-DHERMSY----M-C--KHL--AG--------S-IV-KSDCTL-1 A--NNIVK . . . . CQK-TDDH-QF-1----R-LK-1-----------S--A-mDCEL-1 -DL---L-------- L----V--YLYQIL-GI---Hs-N-IHRDLKP-NLL------- Ki,

181 240 - - - - - - -T-AA...-GDV------------ --LV-KDTT-GKP----AL--MII--ATGN - - - - - - -L-TG...-SDY-----------S--L-V-DTQ-Gpp----A--------- SGV - - - - - - -T-$,A., . -GEV---- - - - - - - - - - - L-V-DVK-GKA----A---LVT----MGE A----- -A-GI...-IRV--HE-V-L---S------ SAR-STp-------T--- - - ATKK A------ A-GV...-VRT --HE-V-L---------- CKY-STA-----L-------VTRR - - - - - - - IADpEHDHTGFL----------------NSKG-TKS------------- - SNR -------VADpDHDHTGFL----------------NSKG-TKS-------------- S m L - - - - - -TACTNF....MM-P--V--------- I--.MG-KEN------------- VLHK L------ HTDDE . . . . . . M - G - - - - - - - - - - - - - - ~ ~ N Q T - - - - - - - - - - - - - - TGR CDFGLAR-L----- P---YTDYVATRWYRAPEILLG---Y---VDTWSVGCIFAEML---

NKIATRE KKIALRE KKIAMRE

CDC2 CDK2 ERKl ERKZ SAPK P38

CONSENSUS

NKIATRE KKIALRE KKIAMRE

CDC2 CDK2 ERKl ERKZ SAPK P3 8

CONSENSUS

NKIATRE KKIALRE KKIAMRE

CDC2 CDK2 ERKl ERK2 SAPK P38

CONSENSUS

301 360 . . . . . . . . . . . . . . NGLm-IVHAC-QI--AE--SSTDL-H-D--TR DGFIEKFIPE-RA

. . . . . . . . . . SYP-LG--KGC-HM--TE-LTCEQL-H----ENSR-IE-MEHDKPTRK

. . . . . . . . . . SEWI--AK-C-HI--D--PFCAEL-H-D-- ....Q MDGFAERFSQE-QL . . . . . . . DENGL---S---1---A--- SGW- -N- - - - ND LDNQIKKM.. . . . . . . . . . . . . . . . . . . . . . . . . . . . DEDGRS--SQ--H---N---Sm--A----QD WKPVPH-RL

......... sDSK-L---DR--T-N-N---TVEE--A----EQn-PT-*EPv~EP-TF ADSK-L---D---T-N-H----EVEQ--A----EQm-PS-.EPIm-rn .........

PSESERDKIKTSQ-R---S---VI--D---SVDE--R---I~-PA-~APPPQI-DA . . . . . . . . . ANPL-V---E---V--SD---TAAQ--A-A--AQm-PD-.EPVm.P-DQ -------------A-DLL-KML--Dp-KRIII--Afi-Hpy~----D--~-------- k

361 420 K-LQEAKVNSFI-PKENFKENEPVRDEKKPVFTNPLLYGNPTLYGKEVDRDKRAKELKVR T-RKSRKHHCFTETSK- ........Q YLPQLTGSSIL ... P m D N m W C D T M m F P KVQKDARNVSLS-KSQN . . . . . . . . RKKEKEKDDSLVEERKTLWQDTNADPKIKDYKLF

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-ELDDLPKERL-ELI-QETARFQPGAPEAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-ELDDLPKEKL-ELI-EETARFQPGYRS . . .S . . . . . . . . . . . . . . . . . - - . . . . . . . . Q-EEREHAIEEW-ELI-KEVMDWEERSKNGVKDQPSDAAVSSKATPSQSSSINDISSMST S-ESRDLLIDEW-SLT-DEVISFVPPPLDQEEMES . . . . . . . . . . . . . . . . . . . . . . . . .

421 480 NKIATRE VIKAKGGKGDVPDLKKTESEGEHRQQGTAEDTHPTSLDRKPSVSELT . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . KKIALRE NI KKIAMRE KIKGSKIDGEKAEKGNRASNASCLHDSRTSHNKIVPSTSLKDCSWSVaHTRNPSVAIPP

CDC2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CDK2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ERKl

ERK2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SAPK EHTLASDTDSSLDASTGPLEGCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

481 540

NKIATRE . . . . . NPVHPSANSDTVKEDPHSGGCMIMPPINLTSSNLLPSSNLSHPNSRLTERTK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . KKIALRE

KKIAMRE LTHNLSAVAPSINSGMGTETIPIQGYR~EKTKKCSIPFVKPNRHSPSGIYNINVTTLVS CDC2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - . - . . . - . . . . . . . . . CDK2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ERKl ERKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SAPK .............................................................

NKIATRE KKIALRE KKIAMRE

CDC2 CDK2 ERKl ERK2 SAPK P3 8

CONSENSUS

541 582 KRRTSSQTIGQTLSNSRQEDTGPTQVQTEKGAFNERTGQNAK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GPPLSDDSGADLPQMEHQH . . . . . . . . . . . . . . . . . . . . . . .

Figure 18: Multiple Sequence Alignment. The deduced sequence of NKIATRE is compared with KKIALRE, -RE, cdc2, CDK2, ERK1, ERK2, SAPK, and p38 using the pileup program (Wisconsin Genetics Cornputer Group). Dashes indicate residues identical to the consensus and periods represent gaps introduced into the sequences to optimize the alignment.

n k i a t r e

LVU

% Identity Total Kinase

k k i a l r e

Figure 19: Dendogram. NKIATRE is compared to other serine/threonine kinases using the pileup program (Wisconsin Genetics Computer Group). The identity of the kinases with NKIATRE was calculated using the bestfit program (Wisconsin Genetics Computer Group). Total refers to the percent identity over the entire sequence of the kinase whereas kinase refers to the percent identity within the catalytic domain.

fragment of the rat jejunum cDNA clone was used to screen rat genomic DNA

digested with three separate restriction endonucleases (Figure 20). Results of

Southern blotting revealed that three bands hybridized to EcoRI digested DNA

with approximate sizes of 7.5 kb, 5.6 kb, and 4.7 kb. Two bands of

approximately 5.4 kb and 4.0 kb hybridized in BamHI digests, whereas only

two bands of 9.4 kb, and 4.7 kb were seen in the HindIII digested DNA. This

would suggest that NKIATRE is a single copy gene.

Results of cross species Southern blot analysis with the above described

probe demonstrate that NKIATRE hybridizes to EcoRI digested genomic DNA

from rat, pig, mouse, human, and hamster (data not shown) (Figure 21). No

hybridizing bands were seen in EcoRI digested DNA from either Drosophila,

Dictyostelium (data not shown) or S. cerevisiae (data not shown).

3.3.5 Northern Blot Analysis

To determine the tissue distribution of NKIATRE, a Northem blot of

poly(A)+mRNA from rat intestine, brain, muscle, lung, spleen, heart, liver, and

thymus was probed with the 1.3 kb EcoRl fragment of the rat jejunum cDNA

(Figure 22). Northern blotting revealed bands of approximately 7 kb in brain,

muscle, and heart, bands of approximately 4.4 kb in intestine, brain, muscle,

lung, and heart, bands of approximately 2.5 kb in brain, muscle and heart, and

a band of approximately 2.2 kb in muscle tissue. No expression of NKIATRE

was seen in rat spleen, liver, or thymus (not shown) tissue. Quality of

Figure 20: Southern blot of rat genomic DNA for NKIATRE. Rat genomic DNA was digested overnight with either EcoRI, BamHI, or HindIII and was probed with the 1.3 kb EcoRI fragment of the rat jejunum cDNA.

Figure 21: Cross species Southern blot for NKIATRE. Rat, pig, mouse, human, and Drosophila genomic DNAs were digested with EcoRI and probed with the 1.3 kb EcoRI fragment of the rat jejunum cDNA.

Figure 22: Expression of NKLATRE in rat tissues. (A) Expression of NKIATRE was examined by Northern blot analysis of poly(A)'mRNA from rat tissues with the 1.3 kb EcoRI rat jejunum fragment as a probe. (B) The sarne blot probed for B-actin.

poly(A)+mRNA was determined by probing for rat B-actin.

3.3.6 Prokaryotic Expression of NKIATRIZ

Expression of the GST-NKIATRE fusion protein from five independent

E. coli colonies yielded three clones with the NKIATRE cDNA in the correct

orientation. As seen in figure 23, a fusion protein of approximately 85 kD was

isolated whereas only GST was isolated from the other two clones.

3.3.7 In Vitro Transcription and Translation

Translation of full length NKIATRE is predicted to yield a protein

product of 67 kD. As seen i n figure 24, a product of 67 kD is. generated

following in vitro transcription and translation of the NKIATRE cDNA. In

addition, a srnaller product of approximately 50 kD is present which is likely

the result of degradation or interna1 initiatiodtermination. No protein was

seen in the negative control reaction which utilised pcDNA3 DNA as a

template.

3.4 DISCUSSION

In a n attempt to isolate a novel MAPK we have cloned a cDNA that is

most homologous to KKIALRE and KKIAMRE, members of the cdc2-related

protein kinase family. Before cloning the full cDNA for NKIATRE, we

confirmed by Southern blotting that the product of the initial PCR

NKIATRE

GST

Figure 23: Prokaryotic expression of GST-NKIATRE fusion protein. The 1.38kb EcoRl fragment from the rat jejunum cDNA library was expressed as a GST fusion protein in bacterial cells upon induction by IPTG. Fusion proteins were purified with glutathione-Sepharose and analyzed by SDS-PAGE. Lanes 1-2: Fusion protein resulting from NKIATRE cloned in the 3'-5' direction. Lanes 3-5: GST-NKIATRE fusion protein. Lane C: GST.

- - - - - - - - - - - - -

Figure 24: In vitro transcriptionand trandationofNHIATRE. Coupledin vitro transcription and translation of NKZATRE was accomplished with a T7 wheat germ extract system. Labelled protein products were visualized by Phosphor Imager following SDS-PAGE, as desciibed in the text.

amplification was a component of the rat genome and not an artifact of the

reaction. Further, by cross species Southeni blots we investigated whether

NKIATRE homologues exist in other mammals and lower eukaryotes. From

these results it does not appear that an NKIATRE homologue exists in lower

eukaryotes, although it remains possible that evolutionary changes have

resulted in sequence divergence that can not be readily detected by high

strlngency screening.

The tissue distribution of NKIATRE, as shown by the Northern blot,

suggests that the expression of NKIATRE is restricted to "non-proliferative"

tissues. This would iniply a discrete role in their biology, perhaps by playing

a role in suppressing the ce11 cycle and therefore proliferation. Further, the

multiple bands seen in several of the lanes on the Northern blot suggest the

existence of differentially spliced products .

While little is known about the function of NKJATRE, we have

determined from sequence analysis and searches of the Genbank database that

NKIATRE likely represents a single domain intracellular serine/threonine

specific kinase. Motif searches have identified possible phosphorylation sites

for other kinases but most of these sites are very short amino acid stretches

that probably play no role in the biological function of this kinase. The role of

NKLATRE in ce11 signalling will require further study.

Sequence cornparison of NKIATRE to the MAPK family members

indicates that it is most homologous to ERK2. However, if NKIATRE is a

MAPK, it will define a new subgroup of this family based on its sequence

within the site of activating phosphorylation. As described in chapter 1, the

MAPKs are unique in their ability to become activated by dual p,hosphorylation

on both a threonine and tyrosine residue. To date the members of this farnily

have been subdivided based on the amino acid separating these two sites of

phosphorylation. The ERKs contain the sequence TEY, p38 has TGY, and the

SAP% have TPY. NKIATRE contains the sequence TDY. Because these

sequences suggest different upstream activators, NKIATRE may be part of a

novel MAPK signalling cascade. However, t o be considered a member of the

MAPK family NKIATRE would have to become activated by phosphorylation

on both threonine 158 and tyrosine 160 in the presence of the appropriate

agonist and mutation of these sites must render-the protein kinase inactive.

NKIATRE is more likely a member of the cdc2-related protein kinase

family and appears t o be a third member of a new subgroup that includes

KKIAMRE and KKIALRE. Because these kinases are highly homologous and

share unique sequences that are conserved in both CDKs and MAPKs, it is

possible that they are involved in the regdation of similar cellular events. In

support of this, cornparison of the expression pattern of NKIATRE to that of

-RE and KKIAMRE reveals that they are highly similar. Over the

tissues examined, NKIATRE is the only one that is expressed in either heart

or muscle tissue. While the level of expression is only moderate in heart

tissue, a relatively high level is seen in muscle. Therefore, it is possible that

NKIATRE may have an additional role, especially in muscle cells, perhaps by

regulating their differentiation. As described in section 1.5.1, it has been

hypothesized that KKIALRE and KKIAMRE are involved in the production of

germ cells based upon their differential expression in ovary and testis. To

investigate whether NKIATRE may be involved in sirnilar cellular processes,

it will be important to examine its expression in testis and ovaries and to

compare these levels to that seen in brain and muscle.

One important question that remains to be answered is how these

kinases are regulated. As discussed in section 1.5.1, both KKIAMRE and

KKIALRE can be stimulated by EGF; however, this activation does not result

in phosphorylation of the TDY motif. While it seems that this family is not

regulated like the MAPKs upon exposure to EGF, it is possible that other

stimuli will phosphorylate them within the TDY motif or that they are

regulated by phosphorylation at other sites. The high sequence conservation

of this family of enzymes to the MAPKs and the CDKs suggests the existence

of an evolutionary relationship; however, it remains to be determined just how

related their functions really are.

3.5 WORK

As with KKIALRE and KKTAMRE, it will be necessary to resolve

whether NKIATRE is a CDK or a MAPK. To determine if NKIATRE functions

as a novel MAPK, external stimuli that elicit an increase in its kinase activity

will be sought. It will be critical to show that activation of the kinase upon

such stimulation occurs through dual phosphorylation within the TDY motif.

As mentioned above, both KKIALRE and K U R E also contain the TDY

motif; however, stimulation of the enzymatic activity of these two kinases upon

exposure to EGF does not require dual phosphorylation as would be expected

of a MAPK (Taglienti e t al., 1996).

To examine the physiological significance of NKIATRE it will be

important to identify upstream activators as well as downstream targets. The

interaction of NKIATRE with any of the known MAPKKs could be assessed

through in vitro kinase assays, CO-immunoprecipitation experiments, or with

the yeast two hybrid system. Based on the fact that NKIATRE contains an

aspartic acid residue between the threonine and tyrosine, which differs from

any other MAPK and suggests the existence of a novel upstream activator, i t

would be surprising if NKIATRE is phosphorylated well by a characterized

MAPKK. Lastly, as with any other W H sijpalling pathway, it will be

important to investigate what kinases or transcription factors lie downstream

of NKIATRE. This can be accomplished through in vitro kinase assays with

known MAPK substrates, CO-immunoprecipitation experiments or with the

yeast two hybrid system. If none of these experiments provide a clue as to the

physiological significance of NKIATRE, the yeast two hybrid system could be

used to screen a cDNA library for any proteins that will interact with it in

vivo.

cdc2-related kinases rnay be reclassified as CDKs once it has been

demonstrated that their kinase activities fluctuate in a ce11 cycle-dependent

manner, or that they are capable of binding a cyclin. NKIATRE will be

examined for these properties. However, neither KKIALRE nor KKIAMRE has

been demonstrated to bind any known cyclin or to have kinase activity that

fluctuates in a ce11 cycle-dependent manner. Although it is possible that the

cyclin partners of KKIALRE and KKIAMRE have yet to be identified, by

cornparison, it is unlikely that NKIATRE functions as a CDK. Clearly, much

work remains to elucidate the role of NKIATRE in either intracellular

signalling or ce11 cycle control.

CHAPTER 4

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