university of geneva master’s in proteomics and

36
University of Geneva Master’s in Proteomics and Bioinformatics 2009 Analysis of c-Src kinase mutants for understanding conformational plasticity Emilio E. Espínola Master’s thesis director: Prof. Leonardo Scapozza, Ralitza Boubeva LCT – Biochimie Pharmaceutique, Section des Sciences Pharmaceutiques

Upload: others

Post on 02-May-2022

1 views

Category:

Documents


0 download

TRANSCRIPT

Page 1: University of Geneva Master’s in Proteomics and

University of Geneva

Master’s in Proteomics and Bioinformatics

2009

Analysis of c-Src kinase mutants for understanding conformational plasticity

Emilio E. Espínola

Master’s thesis director: Prof. Leonardo Scapozza, Ralitza Boubeva

LCT – Biochimie Pharmaceutique, Section des Sciences Pharmaceutiques

Page 2: University of Geneva Master’s in Proteomics and

2

Table of contents

Abstract............................................................................................................................. 3

Introduction ...................................................................................................................... 4

Protein tyrosine kinases ............................................................................................... 4

Discovery and oncogenicity of c-Src ............................................................................ 6

Structural domains of c-Src.......................................................................................... 6

Conformational plasticity ............................................................................................. 8

Aim of the work........................................................................................................... 10

Materials and Methods ................................................................................................... 11

Plasmid ....................................................................................................................... 11

Design of mutations .................................................................................................... 11

Site directed mutagenesis ........................................................................................... 13

Transformation of Escherichia coli competent cells .................................................. 14

DNA sequencing and analysis .................................................................................... 14

Co-transformation in Escherichia coli BL21DE3 strain............................................ 15

Protein expression ......................................................................................................15

Protein purification .................................................................................................... 15

Verification of absence of post-translational modifications ...................................... 17

Western blot................................................................................................................ 17

Kinase assay and inhibition of protein activity by imatinib ....................................... 18

Results ............................................................................................................................ 20

Discussion....................................................................................................................... 29

Concluding remarks........................................................................................................ 33

References ...................................................................................................................... 34

Page 3: University of Geneva Master’s in Proteomics and

3

Abstract

Protein tyrosine kinases are enzymes that play critical roles in eukaryotic cells. They are

involved in the regulation of intracellular signal-transduction pathways that mediates

development and intracellular communication. They all catalyze the same reaction, the

transfer of γ-phosphate from ATP to a protein substrate. These enzymes can adopt two

extreme conformations: an inactive state, which has no enzymatic activity, and an active

state, which shows maximal activity. The structure of the inactive state is more

divergent than the active state. The aim of this work was to identify important amino

acids involved in the switch between the active and inactive conformations of c-Src.

Our strategy consisted in mutating some amino acids located at a hydrophobic interface

of c-Src kinase domain, and assessing how these mutations influence the binding of

imatinib, a drug developed to target the inactive conformation of c-Abl, a related

tyrosine kinase. Seven c-Src mutant proteins were expressed, purified, and analyzed.

We found that: 1) three out of five mutants (i.e. G318, L317I-V377L, and G318-

V378L) showed an increase in relative affinity towards imatinib at the micromolar

range, compared to the wild type; and 2) the c-Src mutants L322I and L317I-L322I

showed similar affinity values with respect to the wild type, showing that L322I can

reverts completely the influence of L317I, which is a reported c-Src mutant that

increases the affinity of imatinib to the nanomolar range. The identification of amino

acids that govern the kinase plasticity is an important step toward the understanding of

inhibitor specificity.

Page 4: University of Geneva Master’s in Proteomics and

4

Introduction

Protein tyrosine kinases

Protein tyrosine kinases (PTKs) are enzymes that play critical roles in eukaryotic cells.

They are involved in the regulation of intracellular signal-transduction pathways that

mediate development, immune system function, and intracellular communication [1].

These functions are achieved by phosphorylation of tyrosine residues of specific protein

substrates, using adenosine triphosphate (ATP) as phosphate donor [2]. In normal cells,

the activity of PTKs is tightly regulated. In some cases, however, their deregulation

results in uncontrolled phosphorylation activity. The consequences of the deregulation

of PTKs can be disordered cell growth, cell division, and apoptosis, which ultimately

can lead to cancer [3]. Therefore, PTKs are important candidates for the development of

new drugs [4].

Protein tyrosine kinases can be divided in two main groups, according to their

localization within the cell: the receptor tyrosine kinases (RTKs), and the non-receptor

tyrosine kinases (NRTKs) [1]. To date, there are 90 tyrosine kinases described for the

human genome. From these, 58 are RTKs which show a transmembrane domain, and 32

are NRTKs which are “free” within the cell [5]. The receptor and non-receptor tyrosine

kinases are further classified into 20 and 10 families, respectively, according to the

sequence similarity of the kinase domain.

Receptor tyrosine kinases are transmembrane proteins usually found as monomers,

which are structurally divided in: 1) an extracellular region carrying one or more ligand

binding domains; 2) a transmembrane helix, that cross the plasma membrane; and 3) an

intracellular region that posses tyrosine kinase activity (Figure 1 on next page). Upon

ligand binding at the extracellular region, a dimerization occurs, which triggers the

autophosphorylation of the kinase domain and allows the binding of other proteins

carrying phosphotyrosine binding domains.

Non-receptor tyrosine kinases are found in the cytoplasm and nucleus of cells, and some

of them can bind to cell membranes via an N-terminal lipid [6]. In general, they contain:

1) two regulatory domains, named SH2 and SH3; and 2) a tyrosine kinase domain,

which is also named SH1 (Figure 2 on next page). Among the 10 described NRTK

families, the first characterized and most studied was the Src family [6]. There are nine

Page 5: University of Geneva Master’s in Proteomics and

5

members of Src family, which are expressed in different tissues (Src, Yes, Fgr, Yrk, and

Fyn) or only in hematopoietic cells (Lyn, Hck, Lck, and Blk,) [7].

Figure 1. Example of RTKs. The extracellular region is shown at the top, and the intracellular region at

the bottom. The picture was obtained from Hubbard et al. [1].

Figure 2. Examples of NRTKs, showing different domain structures for the 10 families. On the right,

some members of each family are cited. The picture was obtained from Blume-Jensen and Hunter [3].

Page 6: University of Geneva Master’s in Proteomics and

6

Discovery and oncogenicity of c-Src

In 1911, Peyton Rous discovered a new virus as a cause of an avian cancer called

sarcoma [8]. This virus, called Rous sarcoma virus (RSV) in honor to its discoverer,

was the first oncovirus to be described. The RSV is a member of the Retroviridae

family, and has a genome composed by a single strand RNA that codifies four genes. In

1970, the gene responsible for the transforming properties of RSV was found. It was

called v-src (or viral-src) [9, 10], and was the first oncogene to be described [11]. In

1976, Bishop and Varmus discovered that a homologous gene to v-src was present in

the genomic DNA of chicken cells and this cellular precursor or proto-oncogene was

called c-src (or cellular-src) [12]. The idea that we all have proto-oncogenes that can

lead (if altered) to human cancers was a great discovery [11].

c-Src is ubiquitously expressed in tissues and is highly conserved through evolution. It

is an enzyme that interacts with numerous substrates involved in the regulation of the

three main cellular functions such as adhesion, invasion and motility [13]. Therefore,

alterations in c-Src activity might also contribute to tumor progression and metastasis.

c-Src is the member of Src kinase family the most often implicated in cancer growth.

The oncogenic effect of c-Src has an impact on several cell signaling pathways and

results in the increase of motility and invasiveness of cancer cells [7]. As an example of

its function, c-Src promotes the loss of cell-cell adhesion by disrupting the adherens

junctions. Adherens junctions are protein complexes composed of cadherins and

catenins that occur at junctions in epithelial tissues. The activation of c-Src affects both

protein families that build up the adherens junctions. It stimulates the E-cadherin

inhibition on adjacent cells through ubiquitylation, and phosphorylates β-cathenin

which prevents its binding to E-cadherin [13].

Structural domains of c-Src

All the members of Src kinase family share a common structure. In order from the N- to

C-terminus, they contain: 1) a short membrane anchor or SH4 domain; 2) a poorly-

conserved “unique” region; 3) an SH3 domain of 50 residues, which can bind to specific

proline-rich sequences; 4) an SH2 domain of 100 residues, which can bind to specific

sites of tyrosine phosphorylation; 5) a catalytic (tyrosine kinase) domain of 250

residues, also named SH1 domain; and 6) a short C-terminal tail containing a conserved

tyrosine residue (Tyr527) [14, 6] (Figure 3 on next page).

Page 7: University of Geneva Master’s in Proteomics and

7

(a) (b)

Figure 3. (a) Representation of the primary amino acid sequence of c-Src. The numeration of amino acids

can change with other members of the Src family. (b) Three dimensional structure of c-Src, obtained from

Xu et al. [15].

The N-terminal membrane anchor or SH4 domain is myristylated in order to be attached

to the membrane. The “unique” region has no particular similarity to other Src family

members, and appears to be important for mediating interactions with receptors or

proteins which are specific to each member [14].

The SH2 and SH3 domains are highly conserved. They are required for the regulation of

the kinase activity, and mediate protein-protein interactions in cellular signaling

cascades. Both domains are connected to the kinase domain by a linker of about 14

amino acids. Interestingly, SH2 and SH3 domains are found also in many proteins

outside the Src family [16].

The kinase domain is divided in two lobes: a small N-terminal lobe, and a large C-

terminal lobe. They are connected by a flexible “hinge” region. The structural

determinants of the N-terminal lobe are: 1) a five stranded antiparallel β-sheet; 2) a

nucleotide binding loop (P-loop or G-rich loop), where the phosphotransfer reaction

takes place through a conserved glycine rich sequence that allows the interaction with

the γ-phosphate of an ATP molecule; and 3) one α-helix (αC helix) [14]. The C-terminal

lobe is mostly α-helical, and includes: 1) the catalytic loop; and 2) the activation loop,

or A-loop. The A-loop is an important segment in the kinase domain, which can

interferes with the binding of either ATP or substrate or both. The regulatory tyrosine

residue located on the A-loop (Tyr416 in chicken Src, or Try419 in human Src) is

important for the stabilization of its conformation. Upstream of the A-loop is located the

highly conserved DFG motif (Aspartate-Phenylalanine-Glycine) [17].

Page 8: University of Geneva Master’s in Proteomics and

8

Conformational plasticity

As member of the PTK family, c-Src adopts at least two extreme conformations: an

“on” state that is maximally active, and an “off” state that has no enzymatic activity

(Figure 4). PTKs catalyze the same reaction: the transfer of the γ-phosphate of ATP to

the hydroxyl group of tyrosine. Thus, upon activation c-Src adopts a catalytically active

“on” conformation that is structurally similar to the active state through all tyrosine

kinases. The inactive “off” state, however, is not subject to the chemical constraints that

the active state must satisfy, and so we have distinct inactive conformations for PTKs in

general [18].

Figure 4. Representation of the two extreme conformations that c-Src adopts, detailing the arrangements

of the activation loop (showed with arrows) and the helix αC (in purple). The picture was obtained from

Yaffe [2]. On the right, a summary of the main structural arrangements between the active and inactive

conformations that determine the kinase plasticity.

The transition between the active and inactive states in c-Src is highly regulated at the

intramolecular level, and implies the precise arrangements of several catalytic residues

and domains [18]. To achieve the active state, the following is necessary: 1) the

dephosphorylation of Tyr527 at the C-terminal “tail” [19], with the release of the SH2

domain that was bound to the phosphorylated Tyr527; 2) the autophosphorylation of

Tyr416 on the activation loop, which stabilizes it in an “open” conformation and

facilitates the substrate binding [19]; 3) the orientation of helix αC towards the active

site; this helix contains a conserved Glutamate residue (Glu310) which forms a salt

bridge with another conserved Lysine (Lys295). This salt bridge serves to position the

Lys295 side chain in order to coordinate the α- and β-phosphate of ATP molecule; 4)

the correct position of the DFG motif (which is situated at the beginning of the

Structural element Inactive Active

DFG motif out in

Glu310 out in

A-loop closed openP-loop closed open

Page 9: University of Geneva Master’s in Proteomics and

9

activation loop), specially the position of the Aspartate towards the active site in order

to coordinate the interaction between a Magnesium cation and the phosphate groups of

ATP; at the same time, the Phenylalanine should point out of the active site; and 5) the

positioning of the P-loop in an open conformation, which facilitates the ATP binding

[18].

In the inactive state, the Tyr416 is not phosphorylated and Tyr527 is phosphorylated by

a specific c-Src kinase (Csk). This phosphorylation inhibits c-Src activity by engaging

SH2 in an intramolecular interaction with the phosphotyrosine, and at the same time

inducing an interaction between the SH3 domain and the SH3-kinase linker [2] (Figure

4). The inactive conformation differs also from the active conformation by the

orientation of the helix αC, which is displaced following the interaction between the

SH3 domain and the SH3-kinase linker. This displacement moves out the Glu310,

breaking the salt bridge between G310 and Lys295 and inhibiting the ATP binding [19].

The phosphotransfer to protein substrates is also inhibited by the closed conformation of

the P-loop and the “out” conformation of the DFG motif. Strikingly, c-Src adopts an

inactive conformation similar to the one of Cdk, a serine/threonine kinase but differs

from the one of related tyrosine kinases like c-Kit or Abl [20]. In summary, a wealth of

information is available about the structure and function of c-Src. Nevertheless, one

issue remains to be solved: it is still unknown whether there are amino acids dictating

the transition between the active and inactive states at the kinase domain.

Page 10: University of Geneva Master’s in Proteomics and

10

Aim of the work

The aim of this work was to identify important amino acids involved in the switch

between the active and inactive conformations of c-Src. Four observations have guided

our study:

1) There is more difference in structure of tyrosine kinases in their inactive state.

2) A new drug called imatinib (Gleevec®, Novartis) was developed to target

specifically the inactive conformation of the fusion protein BCR-Abl, c-Abl, c-Kit, and

the platelet-derived growth factor receptor (PDGFR) with high affinity.

3) There is a resistance of c-Src toward imatinib, even though: a) the residues

interacting with imatinib in c-Abl and c-Kit are conserved in c-Src; and b) the crystal

structure of c-Src in complex with imatinib closely resembles the one of Abl-imatinb,

but differs significantly from the inactive apo-form of Src family kinases [21].

4) Amino acids located at a hydrophobic interface between the N- and C-lobes (H1 for

the hydrophobic region at N-lobe, and H2 for the one at C-lobe) were described as

having a concerted movement between the active and inactive conformations (more

than the rest of the protein), indicating a possible role in the transition between both

states [22].

Our strategy to achieve the aim consisted in using mutagenesis to mutate some amino

acids located at the H1-H2 hydrophobic interface of c-Src, and to assess their

importance in conformational plasticity via the evaluation of the affinity of the mutants

towards imatinib.

Page 11: University of Geneva Master’s in Proteomics and

11

Materials and Methods

Plasmid

The plasmid pET-28a(+) containing the chicken c-Src kinase domain was used (a gift

from Markus Seeliger, University of California [23]). It was constructed using the NdeI

and XhoI restriction sites, and subcloned into a pET-28a(+) vector (Novagen) in order

to have the kinase domain (amino acids 251 to 533), according to the sequence of the

complete c-src gene from Gallus gallus (GenBank accession number V00402). The

pET-28a(+) vector carries an N-terminal hexahistidine tag, and was further modified to

carry the amino acid sequence ENLYFQG, that can be cleaved by the tobacco etch virus

(TEV) protease between the amino acids Q and G.

Design of mutations

The mutations were designed in order to swap some amino acids of c-Src located at the

hydrophobic interface between the N- and C-lobes to the corresponding amino acids in

c-Abl, c-Kit, or PDGFR. Briefly, the amino acid sequences of chicken c-Src, human c-

Abl, human c-Kit, human PDGFR, and human INSR were retrieved from UniProtKB

database, release 14.8 [24], under the following accession numbers: P00523, P00519,

P10721, P09619, and P06213. A multiple sequence alignment was carried out using

CLUSTAL W [25]. Then, the fourteen amino acids located at the hydrophobic interface

of INSR were identified in the remaining kinases, and the numbering was changed to

the one of chicken c-Src (Figure 5 on next page). The seven mutations designed for this

study were as follow: 1) an insertion of Glycine at position 318 (i.e. mutant G318),

which was based on the Glycine observed in the sequence of c-Kit and PDGFR; 2) the

mutation L322I, which was based on an Isoleucine found in c-Kit; 3) the mutation

V377L, which was based on a Leucine found in c-Abl; 4) the double mutant L317I-

L322I, which was based on an Isoleucine found in c-Abl and c-Kit, respectively; 5) the

double mutant L317I-V377L, which was based on an Isoleucine and a Leucine found

both in c-Abl; 6) the double mutant G318-V378L, which was based on an insertion of

Glycine and a swapping to Leucine (found in c-Kit-PDGFR and c-Abl, respectively). It

should be noted that the insertion causes a shift in the numbering upstream this

mutation; and 7) the triple mutant G318-L323I-V378L, which was based on an insertion

Page 12: University of Geneva Master’s in Proteomics and

12

of Glycine, and swapping to Isoleucine and Leucine, which are found in c-Kit-PDGFR,

c-Kit, and c-Abl, respectively.

(a) G318 [H1] (b) L322I [H1] 310 321 310 320 |....|.....|....| |....|..-..|....| Chicken c-Src EAQVMKKL-RHEKLVQL Chicken c-Src EAQVMKKL-RHEKLVQL Human c-Kit ELKVLSYLGNHMNIVNL Human c-Kit ELKVLSYLGNHMNIVNL Human PDGFRb ELKIMSHLGPHLNVVNL

(c) V377L [H2] 370 380 |....|....|....| P00523-Chicken c-Src IASGMAYVERMNYVHR P00519-Human c-Abl ISSAMEYLEKKNFIHR

(d) L317I-L322I [H1] 310 320 310 320 |....|....|....| |....|..-..|....| Chicken c-Src EAQVMKKLRHEKLVQL Chicken c-Src EAQVMKKL-RHEKLVQL Human c-Abl EAAVMKEIKHPNLVQL Human c-Kit ELKVLSYLGNHMNIVNL

(e) L317I-V377L [H1-H2] 310 320 370 380 |....|....|....| |....|....|....| Chicken c-Src EAQVMKKLRHEKLVQL IASGMAYVERMNYVHR Human c-Abl EAAVMKEIKHPNLVQL ISSAMEYLEKKNFIHR

(f) G318-V378L [H1-H2] 310 321 371 381 |....|.....|....| |....|....|....| Chicken c-Src EAQVMKKL-RHEKLVQL Chicken c-Src IASGMAYVERMNYVHR Human c-Kit ELKVLSYLGNHMNIVNL Human c-Abl ISSAMEYLEKKNFIHR Human PDGFRb ELKIMSHLGPHLNVVNL

(g) G318-L323I-V378L [H1-H1-H2] 310 321 371 381 |....|.....|....| |....|....|....| Chicken c-Src EAQVMKKL-RHEKLVQL Chicken c-Src IASGMAYVERMNYVHR Human c-Kit ELKVLSYLGNHMNIVNL Human c-Abl ISSAMEYLEKKNFIHR Human PDGFRb ELKIMSHLGPHLNVVNL

Figure 5. Multiple sequence alignments of amino acid sequences of chicken c-Src, human c-Abl, human

c-Kit, and human PDGFR. (a-g) Insertion and swapping of amino acids on c-Src to the corresponding

ones in c-Abl, c-Kit, or PDGFR are depicted in gray bars for each c-Src mutant. The localization of each

mutation at the H1 or H2 interface or both is shown between brackets.

Page 13: University of Geneva Master’s in Proteomics and

13

Site directed mutagenesis

Mutants of c-Src protein were created by in vitro site directed mutagenesis. The reaction

was performed in 50 µl reaction mixture, and contained 5 ng of plasmid pET-28a(+) c-

src (wild type), 2.5U of Pfu DNA polymerase, Pfu Buffer 10X, 50 pmol of each primer

(forward and reverse; see Table 1 for details), and 0.25 mM of Deoxynucleotide

triphosphates (dNTPs) mix. Two negative controls were included, one without plasmid

and the other without DNA polymerase. Three more conditions were tested, with 5%

Dimethyl Sulfoxide (DMSO), 5% Dimethylformamide (DMF), and 5% Glycerol,

respectively. The oligonucleotides were designed using the online softwares PrimerX

[26] and Oligocalc [27] (Table 1).

Table 1. Primer sequences for the construction of c-src mutants by site directed mutagenesis. Codons that

were mutated are underlined. The first three mutants were constructed directly in one PCR reaction.

Among the mutants market by an asterisk (*), double mutants were constructed in two PCR steps: first,

the single mutation was introduced, and afterwards this was used as template for the second mutation.

The triple mutant was constructed using the plasmid coding for c-Src V377L as template.

The criteria to design the primers using PrimerX were as follow: 1) the mutation should

be in the middle of the primer sequence; 2) terminate in Guanine (G) or Cytosine (C); 3)

of approximately 25 to 45 base pairs; 4) with a melting temperature between 65°C and

75°C; and 5) a GC content between 40% and 60%. The theoretical formation of

secondary structures and dimers was verified with Oligocalc, and avoided when

possible.

Mutation Sense Primer sequence (5' → 3')

G318 Forward CCAAGTGATGAAGAAGCTCGGACGGCATGAGAAGCTGG

Reverse CCAGCTTCTCATGCCGTCCGAGCTTCTTCATCACTTGG

L322I Forward CCGGCATGAGAAGATTGTTCAGCTGTACGC

Reverse GCGTACAGCTGAACAATCTTCTCATGCCGG

V377L Forward CGGCATGGCCTATTTGGAGAGGATGAACTACG

Reverse CGTAGTTCATCCTCTCCAAATAGGCCATGCCG

L317I-L322I*

L317I-V377L*

G318-V378L*

G318-L323I-V378L* Forward GCTCGGACGGCATGAGAAGATTGTTCAGCTGTACGCAGTGG

Reverse CCACTGCGTACAGCTGAACAATCTTCTCATGCCGTCCGAGC

Page 14: University of Geneva Master’s in Proteomics and

14

The Polymerase Chain Reaction (PCR) was carried out in a thermocycler (model

Techgene TC-312, Techne, UK). The initial denaturation at 94°C for 2 minutes was

followed by 18 PCR cycles each consisting of denaturation at 94°C for 30 seconds,

annealing at 52°C for 1 minute, and extension at 68°C for 14 minutes. The final

extension was at 68°C for 8 minutes. The parental plasmid was digested with 1 µl of

DpnI (applied directly to PCR reactions) at 37°C for 1.5 hours. The mutated plasmids

were purified using the GenEluteTM PCR Clean-Up Kit (SIGMA, USA), and eluted in

10 µl of 10% elution buffer (diluted with H20).

Transformation of Escherichia coli competent cells

The entire volume of purified plasmid (10 µl), usually corresponding to 100 ng of DNA,

was used to transform Escherichia coli (E. coli) competent cells, NovaBlue strain

(Novagen). Competent cells were prepared using Calcium Chloride, as described

elsewhere [28]. Transformation was carried out using an in-house protocol. Briefly, 10

µl of plasmid containing the mutated c-src gene was added to 60 µl of E.coli competent

cells, let on ice for 20 minutes, and incubated at 42°C for 30 seconds. Then, 200 µl of

Super Optimal broth with Catabolite repression (SOC) medium was added and

incubated at 37°C, at 200 rpm for 1 hour. Transformed cells were plated on Lysogeny

Broth (LB) agar plates containing Kanamycin at 50 µg/ml, and incubated overnight at

37°C.

DNA sequencing and analysis

To know whether the mutation was inserted successfully, the mutated c-src gene

inserted in the plasmid has to be sequenced at the DNA level. Briefly, obtained colonies

were selected and inoculated in 5 ml of LB medium containing Kanamycin at 50 µg/ml,

and incubated overnight at 37°C. The plasmid was purified using the GenEluteTM

Plasmid Miniprep Kit (SIGMA, USA), and eluted in 50 µl of elution buffer. The

purified plasmid was diluted up to 0.8 µg in 10 µl of water, and sent for sequencing at

Microsynth (Balgach, CH) by the Sanger method [29], using the T7 promoter primer.

Obtained electropherograms were verified using Chromas 2.31 (Technelysium Pty Ltd.,

Helensville, Queensland, Australia), and edited using BioEdit v7.0.0 [30]. The

comparison between electropherograms was carried out with CodonCode Aligner 2.0.6

[31]. Nucleotide and deduced amino acid sequences were aligned with CLUSTAL W

[25].

Page 15: University of Geneva Master’s in Proteomics and

15

Co-transformation in Escherichia coli BL21DE3 strain

Purified pET-28a(+) plasmids containing the designed mutations on c-src were

transformed into E.coli BL21DE3 competent cells (Novagen). The E.coli BL21DE3

cells have been previously transformed with a pCDFDuet-1 plasmid (Novagen) [23],

modified to express the full-length YopH phosphatase from the bacteria Yersinia pestis

[32]. The co-expression of a tyrosine phosphatase has been shown to rescue the lethality

of unregulated c-Src expression in yeast [33]. Therefore, the c-Src co-expression

together with a phosphatase was adopted to maintain the kinase in the unphosphorylated

state, to improve the yield of soluble protein, and to produce a preparation of protein

which will be conformationally homogeneous.

Co-transformed cells were selected on LB-agar plates containing Kanamycin (50 µg/ml)

and Streptomycin (50 µg/ml), and incubated overnight at 37°C.

Protein expression

Co-transformed E.coli BL21DE3 cells from LB-agar plates were resuspended in 10 ml

of LB media, containing Kanamycin (50 µg/ml) and Streptomycin (50 µg/ml). The

culture was incubated overnight at 37°C, with agitation at 200 rpm. The next day, 1 liter

of selective Terrific Broth (TB) medium was inoculated with the 10 ml culture, let at

37°C for 16 hours with shaking, and then cooled for 3 hours at 18°C prior to induction

with 0.2 mM of IPTG, at 18°C for 16 hours. Cell culture was splitted in six bottles of

250 ml capacity, and centrifuged in a pre-cooled Sorvall centrifuge at 4°C, at 5’000 rpm

for 10 minutes. The pellet was recovered and stored at -20°C.

Protein purification

Cell pellets were diluted up to 30 ml in Lysis buffer, composed of Buffer A with 1 mM

Ethylene Diamine Tetraacetic Acid (EDTA) and 20 mg of DNase (Deoxyribonuclease I

from bovine pancreas, SIGMA, USA). Cell lysates were obtained using a mechanical

French Press, by three cycles of homogenization at 15’000 psi of pressure, at 4°C. Cell

debris and insoluble protein was sedimented by centrifugation at 9’500 rpm, at 4°C for

40 minutes.

The protein was purified by three consecutive types of chromatography on an

ÄKTA TMFPLCTM (GE Healthcare, UK): 1) immobilized Nickel (Ni)-affinity

chromatography, to isolate the histidine tagged recombinant c-Src; 2) ion exchange

chromatography, to eliminate residual amounts of YopH phosphatase, which tends to

Page 16: University of Geneva Master’s in Proteomics and

16

bind to Ni-affinity resin despite the lack of a histidine tag; and 3) size exclusion

chromatography, to recover the homogenous monomeric protein. Thus, the supernatant

was filtered through a 0.45 µm filter and loaded onto a Ni affinity column (HisTrap HP,

GE Healthcare, UK), equilibrated with Buffer A (50 mM Tris pH 8.0, 500 mM NaCl,

5% Glycerol, 25 mM Imidazole). The column was washed with five column volumes of

Buffer A, and the protein was eluted with a linear gradient of 0-50% of Buffer B (i.e.

Buffer A with 0.5 M Imidazole). The fractions of protein were put on ice.

To verify the presence of the purified recombinant protein in the fractions, a 12%

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) was

performed, followed by Coomassie Blue staining, as described elsewhere [34]. The

histidine tag was not cleaved for the kinase inhibition analysis, since its presence did not

show any interference with the activity of c-Src (data not shown).

Peak fractions containing c-Src mutants were assembled and dialyzed through a 8 kDa

Molecular Weight Cut Off (MWCO) membrane in Dialysis buffer (20 mM Tris pH 8.0,

100 mM Sodium Chloride [NaCl], 5% Glycerol, and 1 mM Dithiothreitol [DTT]),

overnight at 4°C.

For the second step of purification (anion exchange chromatography), the dialyzed

protein was diluted to 20 ml in Buffer QA (20 mM Tris pH 8.0, 5% Glycerol, and 1 mM

DTT), filtered through a 0.45 µm filter and loaded onto the ion exchange column

(HiTrap HP, GE Healthcare, UK), equilibrated with Buffer QA. The column was

washed with five column volumes of Buffer QA, and the protein was eluted with a

linear gradient of 0-35% of Buffer QB (i.e. Buffer QA with 1 M NaCl). The fractions of

protein were put on ice. The presence of our protein in the fractions was verified by a

12% SDS-PAGE (as shown above).

The fractions containing the c-Src protein were concentrated to 1 ml, using the Amicon

Ultra 4 falcon tube 10 kDa MWCO (Millipore, USA), upon centrifugation at 3’500 rpm,

at 4°C for 20 minutes. Then, the concentrated protein was loaded onto a size-exclusion

column (S75, GE Healthcare, UK), and equilibrated with Buffer S75 (50 mM Tris pH

8.0, 100 mM NaCl, and 5% Glycerol). The presence of recombinant protein in the

fractions was verified by a 12% SDS-PAGE (as shown above). Purified c-Src protein

was stored at -80°C in aliquots of 100 µl, until needed.

Page 17: University of Geneva Master’s in Proteomics and

17

Verification of absence of post-translational modifications

ESI-time-of-flight mass spectrometry (TOF MS/MS) analyses were performed using a

QSTAR XL quadrupole TOF mass spectrometer (AB/MSD Sciex, Toronto, Canada), by

the Laboratory of Life Sciences Mass Spectrometry (Section of Pharmaceutical

Sciences, University of Geneva). This analysis was carried our in order to verify the

absence of phosphorylation or any post-translational modifications on the c-Src kinase

domain that may have occurred during the expression or purifications of the proteins.

Calculated masses were obtained from the deduced amino acid sequences of each

mutant, using ProtParam [35].

Western blot

Autophosphorylation of c-Src wild type and mutants was analyzed by Western blot. We

performed this analysis for the following reasons: 1) to know the phosphorylation state

of purified mutants, which should be unphosphorylated; 2) to test whether the purified

mutants were enzymatically active; and 3) to compare the autophosphorylation rate of

each purified mutant against the wild type. In this assay, we used 1 µg of purified

protein, 5 mM Magnesium Chloride (MgCl2), 20 µM ATP, and Buffer S75 up to 20µl

reaction. The mix was incubated at 30°C without ATP, and the reaction was started by

adding ATP with different times of incubation (0 second, 15 seconds, 1 minute, 10

minutes, and 30 minutes), and stopped by denaturating the protein with Sample Buffer

3X (0.24 M Tris-Cl pH 6.8, 6% SDS, 30% Glycerol, 16% B-mercaptoethanol, and 8.9

mM Bromophenol blue). A negative control was included (without MgCl2 and ATP), as

well as a positive phosphorylation control (EGF-Stimulated A431 cell lysate, Millipore,

USA). All the samples were boiled at 95°C for 5 minutes, and separated by 12 % SDS-

PAGE (see above). The nitrocellulose membrane and two Watman® papers (with the

obtained gel) were equilibrated in water and Transfer buffer (Tris 25 mM pH 8.5, 0.2 M

Glycine, 20% Methanol, and 0.05% SDS), respectively, for 30 minutes. The protein

transfer was carried out at 19 V, 400 mA, for 2.5 hours, in a Trans-Blot Semi-Dry

Transfer Cell (Bio-Rad, UK). Then, the nitrocellulose membrane was blocked with

Blocking buffer (1X Tris-buffered saline [TBS], and 3% nonfat dry milk) for 1 hour at

room temperature with constant agitation. After that, it was incubated with 0.5 µg/ml of

Monoclonal Anti-Phosphotyrosine Antibody, clone 4G10® (Millipore, USA), diluted in

freshly prepared Blocking buffer, overnight with agitation at 4°C. Then, the

nitrocellulose was washed twice with water, and incubated with the secondary antibody

Page 18: University of Geneva Master’s in Proteomics and

18

(Goat Anti-Mouse Horseradish Peroxidase Conjugate, Bio-Rad, UK) diluted 1:3’000

times in Blocking buffer for 1.5 hours at room temperature with constant agitation. The

nitrocellulose was washed twice with water, then once with TBS-0.05% Tween® 20 for

5 minutes, and five times with water. Bound antibodies were detected using the ECL

Plus Western Blotting Detection System (GE Healthcare, UK), after 5 minutes of

incubation at room temperature. A FUGI Medical X-Ray film (FUJIFILM Corporation,

Tokyo) was used to image the nitrocellulose membrane after 5 seconds of exposure.

A picture of each western blot film was taken using the software Quantity One 4.5.1

(Bio-Rad, UK). A densitometric analysis of each gel was carried out, in order to know

whether we can correlate the autophosphorylation rate of each mutant with its capacity

to stabilize the inactive conformation. Briefly, the intensities of each band were

calculated in duplicates and averaged, using the Scion Image software (Scion

Corporation, USA). The highest calculated autophosphorylation score (after 30 minutes

of incubation, at 30°C with ATP/MgCl2) was scaled to 100%. Each value was corrected

for background by subtracting the value from a negative control (protein incubated for 0

second at 30°C, without ATP/MgCl2).

Kinase assay and inhibition of protein activity by imatinib

The kinase activity of c-Src in vitro was monitored using a continuous

spectrophotometric assay, as described elsewhere [36]. Briefly, the consumption of ATP

is coupled via the Pyruvate kinase (PK)/Lactate dehydrogenase (LDH) enzyme pair to

the oxidation of Nicotinamide adenine dinucleotide (NADH) to NAD+. Since NADH

absorbs Ultraviolet (UV) light at 340 nm, and the reaction is equimolar, the decrease in

absorption of NADH is directly linked to the kinase activity (Figure 6).

Figure 6. Representation of c-Src kinase assay in vitro.

Page 19: University of Geneva Master’s in Proteomics and

19

Reaction mixtures contained 100 mM Tris (pH 8.0), 10 mM MgCl2, 275 µM ATP, 1

mM Phosphoenolpyruvate (PEP), 180 µM NADH, 1 mM DTT, 75 U/ml Pyruvate

kinase, 105 U/ml Lactate dehydrogenase, and 0.75 mM of peptide as substrate for c-Src

(sequence: EAIYAAPFAKKK, molecular weight of 1336.6 Da). The reaction mix was

diluted in Buffer S75, to a final volume of 75 µl, and pre-incubated for 5 minutes at

30°C. Protein inhibition was measured at different concentrations of imatinib (from 1

nM to 500 µM). Imatinib was dissolved in DMSO, which was maintained constant at

5% for each measurement. The assay was initiated with the addition of peptide

(incubated at 30°C) at a final concentration of 120 nM. The reactions were carried out at

30°C, and the decrease in absorbance at 340 nm was monitored over 30 minutes in a

Varian Cary 50 UV-Vis spectrophotometer (Varian, Inc., USA). The background

activity of the proteins in 5% DMSO was calculated without the substrate peptide and

imatinib, and subtracted from the kinase assay. The slope value of the obtained curve at

1 nM of imatinib was used as measure for the highest protein activity, because this

concentration showed no interference with protein activity. In total, three series of

measurements were carried out for each c-Src mutant. The half maximal inhibitory

constant (IC50), or the concentration of inhibitor which causes 50 percent inhibition of

an enzymatic reaction , was calculated for each c-Src mutant by using GraphPad Prism

4.0 (GraphPad Software, Inc., USA) using the non-linear four parameter logistic

equation, as shown below:

Y = Bottom + (Top – Bottom) / (1+10^ ( (LogIC50 – X) * Hill Slope) )

where X is the logarithm of concentration, and Y is the response.

Inhibitory constants (Ki) were calculated from each IC50 values, according the following

relationship [37, 38]:

50

[ ]1

i

ICK

ATP

Km

=+

where [ATP] is the concentration of ATP in the assay (275 µM), and Km is the

Michaelis-Menten constant for ATP, which was determined for c-Src to be 70 µM [21].

Page 20: University of Geneva Master’s in Proteomics and

20

Results

In this work, seven mutants of c-Src protein were constructed. They were all located at a

hydrophobic interface of the kinase domain, between the N- and C-lobes, called the H1-

H2 interface. All the mutants were analyzed and contained the desired mutations

(Figure 7 on next page). The group of mutants was formed as follow: an insertion of a

Glycine at position 318 (i.e. G318), two single mutants (L322I and V377L), three

double mutants (L317I-L322I, L317I-V377L, and G318-V378L), and one triple mutant

(G318-L323I-V378L).

All the c-Src mutants were purified and showed the same “profile” of purification. As

an example, we show the results of purification of the mutant L317I-L322I (Figure 8 on

page 22). Chromatograms from Ni-affinity chromatography showed two peaks (Figure

8a). The first peak, formed by fractions 5 to 8, corresponded to a contamination by

YopH phosphatase which was co-expressed to prevent c-Src phosphorylation. The

second peak, formed by fractions 13 to 17, corresponded to the c-Src mutant. This was

verified by a 12% SDS-PAGE gel, in which the phosphatase was seen at the expected

Molecular Weight (MW) of 50.9 kDa. The c-Src mutants were observed in fractions of

the second peak, at the expected MW (~35 kDa). These fractions containing c-Src

mutant were used for the second purification, the anion exchange chromatography

(Figure 8b). Here, the chromatogram showed only one peak (fractions 5 to 8)

corresponding to a pure c-Src mutant. This was verified by a second SDS-PAGE gel.

Finally, the third purification (size-exclusion chromatography) was carried out using

these fractions (Figure 8c). The chromatogram showed one peak corresponding to pure

and monomeric c-Src mutant (fractions 10 to 12), which was verified subsequently by a

third SDS-PAGE gel. All the purifications of c-Src mutants were carried out following

the same steps. The yield of purified protein ranged from 0.3 mg to 5.8 mg per liter of

TB culture, which was sufficient for the subsequent analyses.

Page 21: University of Geneva Master’s in Proteomics and

21

(a) (b) (c) (d)

(e) (f)

(g)

Figure 7. Extract of electropherograms after DNA sequencing of c-src mutants. Each mutant is compared

with c-src wild type (wild type sequence is shown over the mutant sequence). Mutated codons are shown

in a frame. (a) G318 insertion. (b) L322I. (c) V377L. (d) L317I-L322I. (e) L317I-V377L. (f) G318-

V378L. (g) G318-L323I-V378L.

Page 22: University of Geneva Master’s in Proteomics and

22

(a)

(b)

(c)

Figure 8. Protein purification of mutant L317I-L322I. On the left are shown the chromatograms depicting

the UV absorbance of purified proteins (in blue). The green line shows the gradient of the buffer

containing imidazole. On the right are presented 12% SDS-PAGE gels stained by Coomassie Blue, after

each purification. (a) Ni-affinity chromatography. (b) Anion exchange chromatography. (c) Size-

exclusion chromatography. Purified c-Src mutants appears at ~35 kDa.

104.4 → 97.3 → 50.4 → 37.2 → 29.2 → 20.2 →

kDa M FP P AF FT F13 F14 F15 F17

104.4 → 97.3 → 50.4 → 37.2 → 29.2 → 20.2 →

kDa M D AF FT F5 F6 F7 F8

104.4 → 97.3 → 50.4 → 37.2 → 29.2 → 20.2 →

kDa M FT C F11 F12 F13

M : marker FP: after French Press P: pellet AF: after filtration FT: flow-through F13-F17: purified protein fractions

M : marker D: after dialysis AF: after filtration FT: flow-through F5-F8: purified protein fractions

M : marker FT: flow-through C: after concentration F11-F13: purified protein fractions

Page 23: University of Geneva Master’s in Proteomics and

23

A western blot analysis was performed for all recombinant c-Src mutants to verify

whether the mutants were purified in their unphosphorylated state, and whether they

were active (Figure 9).

(a) G318 (b) L322I (c) L317I-L322I

(d) L317I-V377L (e) G318-V378L (f) V377L

(g) G318-L323I-V378L

Figure 9. Western blots showing the autophosphorylation of c-Src kinase mutants. Proteins were detected

by immunostaining with an Anti-Phosphotyrosine Antibody, after incubation with ATP and MgCl2 at

30°C. On each blot we have, from left to right: a negative control (protein incubated without

ATP/MgCl2), and purified proteins collected after different times of incubation (0 second, 15 seconds, 1

minute, 10 minutes, 30 minutes) with 20 µM ATP, and 5 mM MgCl2. (g) Comparison of the

autophosphorylation rate between c-Src wild type and the triple mutant, after 30 minutes of incubation in

presence of ATP and MgCl2. The arrow shows a low level of autophosphorylation for the triple mutant.

Six out of seven c-Src mutants were purified in their unphosphorylated state, which can

be verified by the lack of detected phosphorylation at 0 second of incubation with ATP

and MgCl2. One mutant, V377L, was purified in its phosphorylated state (Figure 9f).

This was verified by the presence of a band under the control conditions (incubation of

c-Src mutant without ATP and MgCl2, and incubation at 0 second with ATP and

MgCl2). This mutant, V377L, was not re-purified again due to lack of time. From the

six remaining mutant proteins, the triple mutant G18-L323I-V378L did not showed a

significant level of autophosphorylation (Figure 9g). This was verified comparing the

Page 24: University of Geneva Master’s in Proteomics and

24

level of autophosphorylation between c-Src wild type and the triple mutant, after 30

minutes of autophosphorylation in presence of ATP and MgCl2. Thus, c-Src mutants

V377L and G318-L323I-V378L obtained in this study were not taken into consideration

for the kinetic assays.

The next step of analysis consisted in verifying the absence of post-translational

modifications on the c-Src kinase domain. The analysis was carried out by Electrospray

ionization-time-of-flight mass spectrometry. Six out of seven c-Src mutants were

analyzed (except V377L) and showed no evidence for phosphorylation or other post-

translational modification on the kinase domain (Figure 10, and Table 2 on next page),

indicating that those mutants were purified in their inactive conformation.

Figure 10. Example of a mass spectrum (Electrospray ionization-time-of-flight) for c-Src mutant L317I-

L322I preparation. The calculated mass from the deduced amino acid sequence is 35,344.40 Da, and the

experimental mass is 35,344.80 Da (standard deviation 1 Da), indication that the c-Src kinase domain is

not post-translationally modified (see also Table 2 on next page).

Inte

nsi

ty

m/z

Page 25: University of Geneva Master’s in Proteomics and

25

Table 2. Results from Electrospray ionization-time-of-flight mass spectrometry analyses of six c-Src

mutants. The calculated masses agree with the experimental masses.

c-Src Mutation Calculated mass (Da) Experimental mass (Da)

G318 35,401.50 35,401.57

L322I 35,344.40 35,343.60

L317I-L322I 35,344.40 35,344.80

L317I-V377L 35,358.40 35,358.50

G318-V378L 35,415.50 35,415.70

G318-L323I-V378L 35,415.50 35,416.00

To see whether we can correlate the autophosphorylation rate of each c-Src mutant with

its capacity to stabilize the inactive conformation, a semi-quantitative analysis by

densitometry on western blots was carried out for each mutant through the time (Figure

11).

-20.00

0.00

20.00

40.00

60.00

80.00

100.00

120.00

0sec 15sec 30sec 1min 10min 30min

ATP/Mg2+ incubation at 30°C

Aut

opho

spho

ryla

tion

[%]

c-Src G318

c-Src L322I

c-Src L317I-L322I

c-Src L317I-V377L

c-Src G318-V378L

c-Src WT

Figure 11. Comparison of in vitro autophosphorylation rate over time between c-Src mutants and wild

type. The values were obtained from a densitometric analysis using western blots of each mutant at

different times of incubation with ATP and MgCl2.

We found that four out of five mutants (i.e. G318, L317I-L322I, L317I-V377L, and

G318-V378L) showed a lower rate of autophosphorylation over time compared to the

wild type (which reached almost complete autophosphorylation after 15 seconds of

Page 26: University of Geneva Master’s in Proteomics and

26

incubation in presence of ATP and MgCl2). However, the mutant L322I showed a

similar profile of autophosphorylation as the wild type, with more than 80% of

phosphorylation after 15 seconds of incubation in presence of ATP and MgCl2) (Figure

11). To be sure that the difference in autophosphorylation over time was due to intrinsic

properties of mutants (and no to an unequal volume of loaded proteins into gels), a

control 12% SDS-PAGE gel was stained by Coomassie Blue and showed that an equal

amount of proteins was loaded for each mutant (data not showed).

Before performing the test of inhibition of c-Src kinase activity, we verified that the

concentrations of c-Src (120 nM) and ATP (275 µM) that were going to be used in the

inhibition test did not trigger an autophosphorylation activity, since it may interfere with

the IC50 measurements as the inhibitor targets the inactive, unphosphorylated c-Src. For

this analysis, a western blot was made using c-Src wild type at 120 nM and 1 µM

(positive control), both with 275 µM ATP and 5 mM MgCl2 at different times of

incubation (see Materials and Methods). We showed that 120 nM of c-Src did not

showed any autophosphorylation activity after more than 1 hour, and that at a

concentration of 1 µM c-Src starts being autophosphorylated almost immediately

(Figure 12).

(a) (b)

Figure 12. Western blots showing the autophosphorylation rate of c-Src wild type over time, using 275

µM ATP, 5 mM MgCl2, and c-Src protein at (a) 120 nM, and (b) 1 µM. The mix was incubated at 30°C

through increasing times, and the reaction was started by the addition of ATP.

The test of inhibition of c-Src kinase activity by imatinib was performed for five out of

seven c-Src mutants (except the mutants V377L and G318-L323I-V378L, the first

purified in its active conformation and the second in a catalytically inactive state)

(Figure 13 on next page).

No ATP/ MW MgCl2 0 ' 2' 10' 30' 60' 120'

37 29

37 29

No ATP/ MW MgCl2 0 ' 2' 10' 30' 60' 120'

Page 27: University of Geneva Master’s in Proteomics and

27

(a) (b) (c)

(d) (e)

Figure 13. Inhibition of the enzymatic activity for five c-Src mutants by imatinib. The fitting was

calculated from mean values of triplicate measurements. The standard deviations are depicted as error

bars. The activity of the protein decreases with increasing concentration of imatinib (expressed at the x-

axis as the logarithm of imatinib concentrations in nanomoles).

All the mutants showed an IC50 value at the micromolar (µM) range, with inhibitory

constants (Ki) ranging from 14.6 to 50.7 µM (in the same order of magnitude as the wild

type, which shows an IC50 = 221 µM and Ki = 31.1 µM) (Table 3). It is interesting to

note that three out of five mutants (i.e. L317I-V377L, G318, and G318-V378L) showed

an increase in affinity compared to the wild type.

Table 3. Obtained IC50 values and inhibitory constants (Ki) of c-Src mutants towards imatinib. The

values for c-Src wild type (*) were obtained from Bendhiaf [39].

c-Src protein IC50 (µM) K i (µM)

Wild type* 221 31.1

G318 131 26.6

L322I 250 50.7

L317I-L322I 249 50.5

L317I-V377L 72 14.6

G318-V378L 149 30.2

Page 28: University of Geneva Master’s in Proteomics and

28

Thus, the mutant L317I-V377L showed a ~3 fold increase in affinity towards imatinib

(K i = 14.6 µM). Another finding was that two mutants (i.e. L322I, and L317I-L322I)

showed a similar IC50 value with respect to the wild type. If we compare our results,

however, to a recent finding that shows that the c-Src mutant L317I enhances the

affinity towards imatinib at the nanomolar range, this indicates that we did not reach the

same degree of improvement (see discussion).

Page 29: University of Geneva Master’s in Proteomics and

29

Discussion

The seven c-Src mutants constructed in this study were located at a hydrophobic

interface between the two lobes of the kinase domain, called H1-H2 interface (H1

corresponding to the N-lobe, and H2 corresponding to the C-lobe). This interface was

initially described for the insulin receptor (INSR) tyrosine kinase by molecular dynamic

simulations, and contains fourteen amino acids in total (six amino acids at the N-lobe,

and eight amino acids at the C-lobe) [22]. The interactions between amino acids at this

interface are mainly of the type van der Waals, and one polar interaction. It was

observed that the amino acids located at this interface were moving in a concerted way

during the transition from active to inactive conformations and vice versa, showing a

greater movement than the rest of the kinase domain [22]. Thus, the movements of N-

and C-lobe, described as rotations and torsions, apparently have their focus at this

interface, with changes of hydrophobic contacts and the conservation of the polar

interaction. It is interesting to note that other studies have described other hydrophobic

cores, but their formations are restricted to the active state [40, 41]. Thus, the idea of

this study was to address the importance of some of the amino acids located at the H1-

H2 hydrophobic interface of c-Src using site directed mutagenesis, to see whether we

can find the amino acids determining the equilibrium of the conformational switch

(from “on” to “off” states, and vice versa).

Our mutagenesis strategy consisted on swapping amino acids located at the H1-H2

interface from c-Src to the corresponding amino acids from c-Abl, c-Kit, and PDGFR,

because it is known that these later kinases can be inhibited by the drug imatinib with

high affinity [42]. The seven mutants constructed in this study were designed following

this rational. The first systematic attempt to discover important amino acids on c-Src

was carried out within other regions of the c-Src kinase domain: the activation loop,

beta3-αC loop, and the P-loop [21]. Kuriyan and co-workers studied 23 mutants of c-Src

by extracting the inhibition constant towards imatinib, but did not found any significant

increase in affinity. The second report came from a mutagenesis study of c-Src at the

activation loop and H1-H2 interface, which was carried out in our lab [39]. In this

study, an interesting mutation was done at the H1-H2 interface, which consisted in a

swap of Leucine from c-Src to the corresponding Isoleucine in c-Abl (L317I). This c-

Page 30: University of Geneva Master’s in Proteomics and

30

Src mutant showed an increase in relative affinity towards imatinib at the nanomolar

range (IC50 = 37nM, Ki = 9 nM), depicting for the first time an important amino acid

that could be involved in the conformational switch of c-Src. This prompted us to

further study this hydrophobic interface in order to address the question whether there

are other amino acids within this interface which are playing an important role in

shifting the conformational equilibrium.

The expression system used in this study consisted of a bacterial system, expressing

both the c-Src kinase domain and a bacterial phosphatase (YopH) at the same time [21].

This co-expression was designed to increase the amount of soluble protein to miligrams,

because it was reported that the yield of the tyrosine kinase in bacteria is very low [43,

44]. Other expression systems for c-Src exist, for instance insect cells [45, 46] or yeast

[33]. The disadvantages of these systems are that they are more demanding in cost and

time. The bacterial system used in this study, however, is not perfect. We still found

more than 90% of protein in the insoluble fraction, in accordance with published data

[23]. A possible explanation to this could be that c-Src proteins co-aggregate with

bacterial membranes, and thus are deposited in the pellet. Another possible explanation

is that c-Src is toxic for the bacteria, and thus it is confined in inclusion bodies.

Nevertheless, the obtained yield of protein (0.3 mg to 5.8 mg per liter of culture) was

sufficient for subsequent analyses.

During the purification of c-Src mutant proteins by Ni-affinity chromatography, it was

observed that YopH phosphatase was co-eluting with c-Src protein. Surprisingly, YopH

phosphatase was binding to the resin, even though it lacks the histidine tag. This

contamination, already seen before [23], could be due to a certain affinity of the

phosphatase for the resin. This problem was overcome by using another step of

purification, the anion exchange chromatography. A final size exclusion

chromatography was used in order to have pure and monomeric c-Src proteins.

In the process of activation of c-Src, it can autophosphorylate on Tyr416 at the

activation loop [47]. This phosphorylation constitutes a positive regulatory mechanism,

which stabilizes the activation loop in an open conformation, and allows the binding of

ATP and peptide for the phosphotransfer [18]. Another site of phosphorylation for c-

Src is on Tyr527, which is phosphorylated in vivo by the tyrosine kinase Csk and

Page 31: University of Geneva Master’s in Proteomics and

31

negatively regulate its activity [48]. In this study, we carried out a western blot analysis

for each c-Src mutant (at different times of incubation with ATP and MgCl2) and

observed that: 1) six out of seven c-Src mutants were purified in their unphosphorylated

state similar to the wild type, as expected; and 2) they could be activated, showing an

increasing autophosphorylation rate over time. Two exceptions to this were found. The

mutant V377L was purified in its phosphorylated state, possibly due to: 1) a lost of the

plasmid containing the phosphatase during the expression of c-Src proteins; or 2) a shift

towards a more active conformation, overcoming the regulation by the phosphatase.

This mutant was not further characterized due to lack of time. The other exception was

the triple mutant G318-L323I-V378L, which showed no significant level of

autophosphorylation over time. One possible hypothesis could be that this mutation can

stabilize the inactive conformation of c-Src more than the other mutations, taking more

time to autophosphorylate. This mutant seems to be properly folded. Two facts points

towards this direction: 1) the triple mutant was purified from a soluble fraction, which

usually requires a globular conformation; and 2) it showed a very low level of

autophosphorylation, which can only be achieved by adopting an active conformation.

Based on these experimental evidences, other experiments will be performed to

definitely answer this folding issue. The correct folding of the triple mutant can be

verified by some techniques, for instance by using Circular Dichroism spectroscopy.

Another point that arises here is whether the phosphorylation observed in western blot is

due to Tyr416 autophosphorylation or Tyr572 (which is usually phosphorylated by

Csk). Tyr527 is indeed also present on our constructs. A possible assay to address this

issue could be the use of a specific anti-phosphotyrosine antibody directed towards

phosphotyrosine-416 which does not cross react with phosphotyrosine-527, for instance

the Phospho-Src Family (Tyr416) antibody (Cell Signaling Technology, USA).

Can we correlate the autophosphorylation rate of c-Src mutant with its capacity to

stabilize the inactive conformation? In other words, a lower rate of autophosphorylation

is directly proportional to a higher stabilization of the inactive conformation?

Apparently no. A semi-quantitative analysis by densitometry on western blots showed

that even though this pattern can be observed for most of the mutants analyzed, the

mutant L322I showed a similar rate of autophosphorylation as the wild type.

Furthermore, there is no direct correlation when considering the kinetic analyses.

Page 32: University of Geneva Master’s in Proteomics and

32

The inhibition test was performed using concentrations of c-Src (120 nM) and ATP

(275 µM) that did not show any autophosphorylation activity, as observed in western

blot analyses. This was important since an autophosphorylation activity of c-Src may

interfere with the IC50 measurements because the inhibitor targets the inactive,

unphosphorylated c-Src.

The drug imatinib was used as a marker for the inactive conformation of c-Src. Other

tyrosine kinases (c-Kit and PDGFR) show a high affinity towards imatinib at the

nanomolar range [42]. Even though c-Src shows structural similarities with these

tyrosine kinases, its affinity towards imatinib remains at the micromolar range. This fact

was explained by the observation that the residue Phenylalanine from DFG motif was

flipped 180° in c-Src with respect to c-Abl, meaning that more energy is needed to

break this steric hindrance [21].

In this study, three out of five mutants (i.e. G318, L317I-V377L, and G318-V378L)

showed an increase in affinity towards imatinib at the micromolar range, compared to

the wild type. It was reported, however, that the mutation L317I located at the same

hydrophobic interface enhances the affinity of c-Src into the nanomolar range [39], a

finding that was not improved despite important efforts of the scientific community

[21]. Another important finding was that mutations L322I and L317I-L322I showed

similar IC50 values with respect to the wild type, showing that L322I can reverts

completely the influence of L317I. The importance of the mutation L322I to revert the

action of better mutations can be tested by constructing other c-Src mutants, for instance

G318-L322I, expecting to find again an affinity toward imatinib similar to the wild

type. At the same time, the c-Src mutant L317I-L322I is an interesting candidate to be

crystallized for future X-ray diffractions, to understand the structural basis of this

reversion.

Page 33: University of Geneva Master’s in Proteomics and

33

Concluding remarks

In this work, we have extended the search of amino acids that could govern the

plasticity of c-Src kinase domain. The strategy was based on swapping amino acids of

c-Src located at the hydrophobic interface between the N- and C-lobes to the

corresponding ones in c-Abl, c-Kit, and PDGFR, and to assess their importance in

conformational plasticity via the evaluation of the affinity of the mutants towards

imatinib. The affinity for imatinib was used as an indirect marker of the stabilization of

the inactive conformation of c-Src kinase domain. Seven c-Src mutants were

constructed following this rational. Five mutants were further analyzed by kinetic

assays, and three of them (i.e. G318, L317I-V377L, and G318-V378L) showed an

increase in affinity towards imatinib at the micromolar range, compared to the wild

type. The remaining two mutants (i.e. L322I, and L317I-L322I) showed similar affinity

values as the wild type. Interestingly, the mutation L322I reverted completely the action

of L317I (which drops the affinity at the nanomolar range). Future efforts can be

focused on: 1) assessing whether the mutation V377L is purified always in a more

active state; 2) analyzing whether the triple mutant adopts a correct folding; 3) testing

the importance of the mutation L322I to revert the action of better mutations by

constructing other c-Src mutants, like G318-L322I, in which we expect to find a similar

affinity towards imatinib as the wild type; 4) crystallizing the mutant L317I-L322I to

understand the structural basis of this reversion; 5) analyzing the importance of the rest

of amino acids localized at the H1-H2 interface; 6) extending the analysis to other

hydrophobic interfaces; and 7) extending this work on other tyrosine kinases, including

RTKs and NRTKs.

Page 34: University of Geneva Master’s in Proteomics and

34

References

1. Hubbard SR, Till JH (2000) Protein tyrosine kinase structure and function.

Annu. Rev. Biochem. 69: 373-398 2. Yaffe MB (2002) Phosphotyrosine-binding domains in signal transduction. Nat.

Rev. Mol. Cell Biol. 3: 177-186 3. Blume-Jensen P, Hunter T (2001) Oncogenic kinase signalling. Nature 411:

355-365 4. Levitzki A (1999) Protein tyrosine kinase inhibitors as novel therapeutic agents.

Pharmacol. Ther. 82: 231-239 5. Robinson DR, Wu YM, Lin SF (2000) The protein tyrosine kinase family of the

human genome. Oncogene 19: 5548-5557 6. Brown MT, Cooper JA (1996) Regulation, substrates and functions of src.

Biochim. Biophys. Acta 1287: 121-149 7. Summy JM, Gallick GE (2003) Src family kinases in tumor progression and

metastasis. Cancer Metastasis Rev. 22: 337-358 8. Rous P (1966) The challenge to man of the neoplastic cell. Nobel Lecture,

Physiology or Medicine. http://nobelprize.org/ 9. Czernilofsky AP, Levinson AD, Varmus HE, Bishop JM, Tischer E, Goodman

HM (1980) Nucleotide sequence of an avian sarcoma virus oncogene (src) and proposed amino acid sequence for gene product. Nature 287: 198-203

10. Duesberg PH, Vogt PK (1970) Differences between the ribonucleic acids of transforming and nontransforming avian tumor viruses. Proc. Natl. Acad. Sci. U.S.A. 67: 1673-1680

11. Martin GS (2001) The hunting of the Src. Nat. Rev. Mol. Cell Biol. 2: 467-475 12. Stehelin D, Varmus HE, Bishop JM, Vogt PK (1976) DNA related to the

transforming gene(s) of avian sarcoma viruses is present in normal avian DNA. Nature 260: 170-173

13. Yeatman TJ (2004) A renaissance for SRC. Nat. Rev. Cancer 4: 470-480 14. Xu W, Harrison SC, Eck MJ (1997) Three-dimensional structure of the tyrosine

kinase c-Src. Nature 385: 595-602 15. Xu W, Doshi A, Lei M, Eck MJ, Harrison SC (1999) Crystal structures of c-Src

reveal features of its autoinhibitory mechanism. Mol. Cell 3: 629-638 16. Pawson T (1995) Protein modules and signalling networks. Nature 373: 573-580 17. Nolen B, Taylor S, Ghosh G (2004) Regulation of protein kinases; controlling

activity through activation segment conformation. Mol. Cell 15: 661-675 18. Huse M, Kuriyan J (2002) The conformational plasticity of protein kinases. Cell

109: 275-282 19. Cowan-Jacob SW, Fendrich G, Manley PW, Jahnke W, Fabbro D, Liebetanz J,

Meyer T (2005) The crystal structure of a c-Src complex in an active conformation suggests possible steps in c-Src activation. Structure 13: 861-871

20. Mol CD, Dougan DR, Schneider TR, Skene RJ, Kraus ML, Scheibe DN, Snell GP, Zou H, Sang BC, Wilson KP (2004) Structural basis for the autoinhibition and STI-571 inhibition of c-Kit tyrosine kinase. J. Biol. Chem. 279: 31655-31663

21. Seeliger MA, Nagar B, Frank F, Cao X, Henderson MN, Kuriyan J (2007) c-Src binds to the cancer drug imatinib with an inactive Abl/c-Kit conformation and a distributed thermodynamic penalty. Structure 15: 299-311

Page 35: University of Geneva Master’s in Proteomics and

35

22. Moretti L (2007) Exploring structure and plasticity of tyrosine kinase domains for drug discovery. Thesis N° 3904. University of Geneva, Switzerland

23. Seeliger MA, Young M, Henderson MN, Pellicena P, King DS, Falick AM, Kuriyan J (2005) High yield bacterial expression of active c-Abl and c-Src tyrosine kinases. Protein Sci. 14: 3135-3139

24. UniProtKB release 14.8. http://www.uniprot.org 25. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the

sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673-4680

26. Lapid C, Gao Y (2003) PrimerX: automated design of mutagenic primers for site-directed mutagenesis. http://www.bioinformatics.org/primerx/index.htm

27. Kibbe WA (2007) OligoCalc: an online oligonucleotide properties calculator. Nucleic Acids Res. 35: W43-46

28. Sambrook J, Russell D (2001) Molecular Cloning: A Laboratory Manual, Third edn. Cold Spring Harbor Laboratory Press, New York

29. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74: 5463-5467

30. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids. Symp. Ser. 41: 95-98

31. CodonCode Aligner 2.0.6. http://www.codoncode.com/aligner/ 32. Bliska JB, Guan KL, Dixon JE, Falkow S (1991) Tyrosine phosphate hydrolysis

of host proteins by an essential Yersinia virulence determinant. Proc. Natl. Acad. Sci. U.S.A. 88: 1187-1191

33. Weijland A, Neubauer G, Courtneidge SA, Mann M, Wierenga RK, Superti-Furga G (1996) The purification and characterization of the catalytic domain of Src expressed in Schizosaccharomyces pombe. Comparison of unphosphorylated and tyrosine phosphorylated species. Eur. J. Biochem. 240: 756-764

34. Smith BJ (1994) SDS polyacrylamide gel electrophoresis of proteins, in: Methods in Molecular Biology, Vol. 32: Basic Protein and Peptide Protocols (Walker JM, ed), Springer-Verlag New York, pp 23-34

35. ProtParam. http://www.expasy.ch/tools/protparam.html 36. Barker SC, Kassel DB, Weigl D, Huang X, Luther MA, Knight WB (1995)

Characterization of pp60c-src tyrosine kinase activities using a continuous assay: autoactivation of the enzyme is an intermolecular autophosphorylation process. Biochemistry 34: 14843-14851

37. Cheng HC (2001) The power issue: determination of KB or Ki from IC50. A closer look at the Cheng-Prusoff equation, the Schild plot and related power equations. J. Pharmacol. Toxicol. Methods 46: 61-71

38. Cheng Y, Prusoff WH (1973) Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22: 3099-3108

39. Bendhiaf SM (2008) Expression, purification and analysis of Src kinase domain mutants for understanding inhibitors's selectivity. Diploma Thesis. University of Geneva, Switzerland

40. Kornev AP, Haste NM, Taylor SS, Eyck LF (2006) Surface comparison of active and inactive protein kinases identifies a conserved activation mechanism. Proc. Natl. Acad. Sci. U.S.A. 103: 17783-17788

Page 36: University of Geneva Master’s in Proteomics and

36

41. Ten Eyck LF, Taylor SS, Kornev AP (2008) Conserved spatial patterns across the protein kinase family. Biochim. Biophys. Acta 1784: 238-243

42. Buchdunger E, Cioffi CL, Law N, Stover D, Ohno-Jones S, Druker BJ, Lydon NB (2000) Abl protein-tyrosine kinase inhibitor STI571 inhibits in vitro signal transduction mediated by c-kit and platelet-derived growth factor receptors. J. Pharmacol. Exp. Ther. 295: 139-145

43. Garcia P, Shoelson SE, George ST, Hinds DA, Goldberg AR, Miller WT (1993) Phosphorylation of synthetic peptides containing Tyr-Met-X-Met motifs by nonreceptor tyrosine kinases in vitro. J. Biol. Chem. 268: 25146-25151

44. Williams DM, Wang D, Cole PA (2000) Chemical rescue of a mutant protein-tyrosine kinase. J. Biol. Chem. 275: 38127-38130

45. Nagar B, Hantschel O, Young MA, Scheffzek K, Veach D, Bornmann W, Clarkson B, Superti-Furga G, Kuriyan J (2003) Structural basis for the autoinhibition of c-Abl tyrosine kinase. Cell 112: 859-871

46. Sicheri F, Moarefi I, Kuriyan J (1997) Crystal structure of the Src family tyrosine kinase Hck. Nature 385: 602-609

47. Smart JE, Oppermann H, Czernilofsky AP, Purchio AF, Erikson RL, Bishop JM (1981) Characterization of sites for tyrosine phosphorylation in the transforming protein of Rous sarcoma virus (pp60v-src) and its normal cellular homologue (pp60c-src). Proc. Natl. Acad. Sci. U.S.A. 78: 6013-6017

48. Thomas JE, Soriano P, Brugge JS (1991) Phosphorylation of c-Src on tyrosine 527 by another protein tyrosine kinase. Science 254: 568-571