new c-src targets revealed by chemical rescue and proteomics

New c-Src targets revealed by chemical rescue and proteomics

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Identification of Targets of c-Src Tyrosine Kinase by Chemical Complementation and

Phosphoproteomics

Isabel Martinez-Ferrando1, Raghothama Chaerkady2,4, Jun Zhong2, Henrik Molina2,5, Harrys

Kishore2,4, Katie Herbst-Robinson1,6, Beverley M. Dancy1, Vikram Katju2,4,7, Ron Bose1,8, Jin

Zhang1, Akhilesh Pandey2,3, and Philip A Cole1,*

1Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine.

Baltimore, MD, USA, 2McKusick-Nathans Institute of Genetic Medicine and Department of

Biological Chemistry, Johns Hopkins School of Medicine. Baltimore, MD, USA, 3Pathology and

Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA, 4Institute of

Bioinformatics, International Technology Park, Bangalore, India, 5Present address: Rockefeller

University Proteomics Research Center. The Rockefeller University, New York, NY, USA,

6Present address: Institute on Aging, University of Pennsylvania, Philadelphia, PA, USA,

7Present address: M.D. Anderson Cancer Center. The University of Texas, Houston, TX, USA,

8Present address: Division of Oncology, Department of Medicine. Washington University School

of Medicine. St Louis, MO, USA

*Corresponding author: Department of Pharmacology and Molecular Sciences, Johns Hopkins

School of Medicine, 725 North Wolfe Street, Baltimore, MD, 21205, USA. Tel.: +1 410 614

8849; Fax: +1 410 614 7717; E-mail: [email protected]

MCP Papers in Press. Published on April 12, 2012 as Manuscript M111.015750

Copyright 2012 by The American Society for Biochemistry and Molecular Biology, Inc.


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Abbreviations

FAK, focal adhesion kinase; PTM, post-translational modifications; LTQ- Orbitrap Velos, linear

ion trap orbitrap Velos mass spectrometer; RapGEF1 (C3G), Rap guanine exchange factor;

SILAC, stable isotope labeling of amino acids in cell culture; SYF/MEFs, Src-, Yes-, and Fyn-

knock out mouse embryonic fibroblasts.


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Summary

The cellular proto-oncogene c-Src is a non-receptor tyrosine kinase involved in cell growth and

cytoskeletal regulation. Despite being dysregulated in a variety of human cancers, its precise

functions are not fully understood. Identification of c-Src’s substrates remains a major challenge,

as there is no simple way to directly stimulate its activity. Here we combine the chemical rescue

of mutant c-Src and global quantitative phosphoproteomics to obtain the first high-resolution

snapshot of the range of tyrosine phosphorylation events that occur in the cell immediately after

specific c-Src stimulation. After enrichment by anti-phosphotyrosine antibodies, we identified 29

potential novel c-Src substrate proteins. Tyrosine phosphopeptide mapping allowed the

identification of 382 non-redundant tyrosine phosphopeptides on 213 phosphoproteins. SILAC-

based quantitation allowed the detection of 97 non-redundant tyrosine phosphopeptides whose

level of phosphorylation is increased by c-Src. A large number of previously uncharacterized c-

Src putative protein targets and phosphorylation sites are presented here, a majority of which

play key roles in signaling and cytoskeletal networks, particularly in cell adhesion. Integrin

signaling and focal adhesion kinase (FAK) signaling pathway are two of the most altered

pathways upon c-Src activation through chemical rescue. In this context, our study revealed the

temporal connection between c-Src activation and the GTPase Rap1, known to stimulate

integrin-dependent adhesion. Chemical rescue of c-Src provided a tool to dissect the

spatiotemporal mechanism of activation of the Rap1 guanine exchange factor, C3G, one of the

identified potential c-Src substrates that plays a role in focal adhesion signaling. In addition to

unveiling the role of c-Src in the cell and, specifically, in the Crk-C3G-Rap1 pathway, these


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results exemplify a strategy for obtaining a comprehensive understanding of the functions of

non-receptor tyrosine kinases with high specificity and kinetic resolution.


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INTRODUCTION

The discovery of c-Src (cellular, wild-type Src) as the proto-oncogene of v-Src (viral,

mutant Src) has led to persistent interest in this non-receptor protein tyrosine kinase in studies of

cell signaling. It is now known that c-Src is involved in regulating cellular growth, adhesion,

motility and invasion. c-Src is frequently overexpressed in human cancer, such as

gastrointestinal, breast, ovarian, and other cancers (1), and it is considered a drug target. In spite

of its linkage to disease and breadth of functions, c-Src’s specific roles in signaling are still not

fully understood.

A variety of biochemical and cellular approaches have been used to identify direct and

indirect tyrosine phosphorylated substrates of Src: many of these cellular substrate identification

studies have used the hyperactive, dysregulated form of Src, v-Src (2, 3), which lacks normal

downregulation by C-terminal phosphorylation on Tyr 527, or constitutively active Src mutants

(for example, Y527F) (4,5). However, v-Src forms are rarely found in human cells, even in

cancer (6). Instead, it would be informative to pursue these studies focusing on the cellular

proto-oncogene c-Src.

Analyzing cellular protein tyrosine phosphorylation targets of c-Src using a proteomics

strategy would require an approach that can directly and specifically monitor c-Src kinase action

rather than previously used indirect methods, such as growth factors activating growth factor

receptor tyrosine kinases that indirectly stimulate c-Src (7). Related work has been done in this

regard combining chemical genetics of kinases (8-10) and proteomics (11). For our goals, the

challenge was to achieve specific and rapid activation of c-Src in living cells that will allow


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identification of substrates temporarily close to c-Src activation. An attractive strategy to pursue

these objectives involves chemical rescue of mutant c-Src tyrosine kinase.

It has previously been shown that mutation of a highly conserved Arg (390 in c-Src) in

protein tyrosine kinases (PTKs) results in a dramatic reduction in catalytic activity (200-5000-

fold), presumably because of the loss of a key hydrogen bonding side chain responsible for

orienting the substrate tyrosine phenol for phosphoryl transfer (12-14). A variety of di- and

triamino compounds added to the enzyme reaction buffer have been shown to complement this

defective kinase activity, the most efficient being imidazole (12-14). Structural and pH studies

suggest that positively charged imidazolium occupies the unnatural cavity found in R/A mutant

PTKs and serves to rescue the catalytic function without significantly affecting c-Src substrate

selectivity (14) (Figure 1A). Indeed, in the case of the Src kinase domain, treatment of R390A v-

Src with imidazole stimulates catalytic activity by about 100-fold to within 40% of the wild-type

level and appears to have native substrate specificity and regulation in vitro (3). It was also

shown that imidazole, a relatively non-toxic small molecule, could rescue R390A v-Src in cell

culture (3).

By combining the specific and controlled activation of c-Src by chemical rescue with the

ability of quantitative SILAC phosphoproteomics to provide the phosphorylation fingerprint of

cells in different conditions, we have identified a number of potential novel c-Src targets and

discuss the potential ramifications of these phosphorylation events in cell signaling. Chemical

rescue has also served as a powerful tool to dissect the mechanism of activation of one of the

identified targets (C3G) in a spatiotemporal manner.


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Experimental Procedures

Cell Culture and SILAC

Src-, Yes-, and Fyn- triple knockout mouse embryonic fibroblast cells (hereafter referred

to as SYF cells) (ATCC) stably transfected with either R390A c-Src or D388N c-Src constructs

as previously described (3) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)

supplemented with 10% (v/v) qualified fetal bovine serum (FBS) (Invitrogen), 1% (v/v) of

penicillin-streptomycin-glutamine (PSG) (Invitrogen) and 300 µg/ml of Hygromycin B (Roche)

and incubated at 37ºC in the presence of 5% CO2. R390A c-Src SYF MEF cells (hereafter

referred to as R390A/ SYF MEF cells) were adapted to the SILAC media. A detailed protocol

for SILAC media preparation can be obtained from http://www.silac.org. Arginine- and lysine-

free DMEM media were bought from Athena Enzyme Systems. c-Src R390A stably transfected

SYF cells were cultured in Arg-/ Lys- free DMEM media supplemented with light (12C6 Arg/

12C6 Lys) amino acids from Sigma or heavy (13C6 Arg/ 13C6 Lys) amino acids from Cambridge

Isotope Laboratories, Inc. Both media contained 10% qualified FBS (Invitrogen), 1% PSG

(Invitrogen) and 300 µg/ml of Hygromycin B (Roche). The cells were adapted to the SILAC

medium by growing them in these media for at least 5 replication cycles, as described earlier

(15). When starved, cells were cultured in the same medium lacking serum. The p-Tyr

proteomes were assessed from cells actively growing and motile at ~75% confluency.

Untransfected SYF cells and wt MEFs were grown as above without hygromycin

treatment.


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Cell treatment and peptide preparation

Thirty 150mm diameter plates of R390A/SYF cells (per condition) were serum-starved

for 16 hours. Cells grown in 13C6 Arg/ 13C6 Lys- containing media (heavy media) were treated

with 10 mM imidazole, pH 7.5 for 5 minutes, whereas cells grown in 12C6 Arg/ 12C6 Lys-

containing media (light media) were left unstimulated and used as a control. Both cells sets were

then washed once with ice cold PBS (Invitrogen) and immediately lysed in Urea lysis buffer (20

mM HEPES pH 8.0, 9 M urea, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 1

mM ß-glycerophosphate) by 3 bursts of ultrasonication (Duty cycle 40%, output control at 4, on

Sonifier 250, Branson) at 4 °C. After sonication, the lysate was cleared by centrifugation at

20,000 x g for 15 min and the supernatant collected. Three biological replicates were conducted.

Protein concentrations were calculated by Bradford (Supplementary Figure 1A). As a further

loading control equal amount of lysates (50 µg) were mixed with SDS loading buffer and boiled

at 95ºC for 5 minutes, analyzed by SDS-PAGE and stained with Colloidal Coomasie

(Supplementary Figure 1A).

Tyrosine phosphopeptide enrichment

Phosphotyrosine protein profile with and without imidazole treatment was analyzed by

immunoaffinity purification of phosphoproteins with 4G10 anti-phosphotyrosine antibody

followed by western blot using 4G10 anti-phosphotyrosine antibody (Supplementary Figure 1B).

“Heavy” and “Light” lysates were mixed in protein concentration ratios 1:1. As a result, a total


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amount of 35 mg (first biological replicate), 50 mg (second biological replicate) and 52 (third

biological replicate) were generated for phosphotyrosine peptide profiling. Two of the 3

biological replicates had two technical replicates with respect to LC-MS/MS. LC-MS analysis of

sample aliquots were performed to observe the equal mixing of heavy and light cell lysate

proteins using a Micromass quadrupole time-of-flight mass spectrometer (QTOF) Ultima API

(Waters) (Supplemental Figure 1C).

Proteins were reduced with DTT at a final concentration of 4.5 mM and alkylated with 10

mM iodoacetamide. Lysates were diluted in 20 mM HEPES pH 8.0 to a final concentration of 2

M urea. 1/100 vol/vol of 1mg/ml trypsin- TPCK (Worthington Biochemical Corporation) was

added to the diluted protein extracts and digestion was performed overnight at 25 ºC. The lysates

were analyzed before and after digestion as a control (Supplementary Figure 1D) using SDS-

PAGE and colloidal Coomassie staining.

The protein digests were acidified with trifluoroacetic acid (TFA) at a final concentration

of 1%. The acidified peptide solutions were centrifuged at 1,800 x g for 5 minutes to remove the

precipitate. The clear supernatants were loaded onto Sep-Pak C18 cartridges (Waters, cat no:

WAT051910) previously wetted with 100% acetonitrile and equilibrated with 0.1% TFA. The

cartridges were then washed with 0.1% TFA and acidified peptide samples were passed through

cartridge by gravity. The peptides were eluted by 40% acetonitrile (ACN) /0.1% TFA. The

eluted peptide solutions were lyophilized to remove residual TFA completely.

Immunoaffinity purification of tyrosine phosphopeptides


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Anti phosphotyrosine antibody based immunoaffinity purification (IAP) of

phosphopeptides was carried out according to manufacturer protocol (16). Lyophilized peptides

from each of the fractions (3 biological replicates, 2 technical replicates) were dissolved with 1.4

ml of IAP buffer (50 mM MOPS pH 7.2, 10 mM sodium phosphate, 50 mM NaCl) aided by

shaking and brief sonication. Solution was cleared by centrifugation for 5 minutes at 1,800 x g.

The phosphotyrosine antibody-linked beads (Cell Signaling Technology) (250 µg in 40 µl) were

washed with IAP buffer (1.4 ml IAP buffer, mix by invert 5 times) once and incubated with the

cleared peptide solution for 30 minutes at 4ºC with gentle shaking. The phosphopeptide bound

antibody beads were washed twice with IAP buffer and twice with water at 4ºC.

Phosphopeptides were eluted from the beads by incubation with 55 µl of 0.15% TFA at 20-25ºC

for 10 minutes followed by second extraction with 45 µl of 0.15% TFA.

The eluted phosphopeptides were desalted using Stage-Tip protocol described previously

(17). The peptide eluate was then dried at 25ºC in Speed-Vac for mass spectrometry analysis.

LC-MS/MS analysis of tyrosine phosphorylated peptides

LC-MS/MS analysis of phosphopeptides enriched by IAP was carried out using a

reversed-phase liquid chromatography system interfaced with Fourier transform LTQ-Orbitrap

Velos mass spectrometer (FT-MS) on a “high-high” mode (both MS and MS/MS analyses were

carried out in FT-MS mode) (Thermo Scientific). The reversed phase setup consisted of a trap

column (2 cm × 75 µm, Magic C18 AQ material 5µm, 100 Å) and an analytical column (10 cm ×

75 µm, Magic C18 AQ material 5 µm, 100 Å). After concentration and desalting on the pre-

column, peptides were eluted and separated on the analytical column using a gradient increasing


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from 100% solvent A/ 0% solvent B (Solvent A: 0.1% formic acid, solvent B: 100% acetonitrile,

0.1% formic acid) to 40% buffer A/60% solvent B in 135 min. Each survey scan involved

precursor scan from 350-1600 m/z at 60,000 resolution and data dependent higher collision

dissociation (HCD) MS/MS scans for 10 most intense precursor ions at 7,500 resolution.

Normalized collision energy was 35% and monoisotopic precursor selection enabled with

isolation width of 1.9 m/z.

Data analysis

The acquired mass spectra were processed and searched using MaxQuant (version 1.2)

(18), Mascot (19) and Sequest (20) since these algorithms are complementary which can broaden

protein and site identification (21). Searches using the latter two were submitted via Proteome

Discoverer (version 1.3, Thermo Scientific), which allowed merging search results and

quantitating the SILAC pairs. It should be noted that although there was a large difference between

Mascot and Proteome Discoverer for redundant peptides (about 10-fold), perhaps related to initial

curation stringency, this difference becomes minor when comparing non-redundant peptides (<2-

fold). We believe that the similarity in non-redundant peptides is the more important comparison.

All MS/MS spectra were searched against the RefSeq mouse protein database (v. 40, containing

31,183 entries). For all searches, two missed cleavages were allowed; carbamidomethylation of

cysteines were set as a fixed modification. N-terminal acetylation, deamidation of asparagine and

glutamine, oxidation at methionine, phosphorylation at serine, threonine and tyrosine together

with the ‘heavy’ lysine and arginine were used as variable modifications.

Monoisotopic peptide tolerance was set to 10 p.p.m. for precursors and 0.05 mDa for

MS/MS fragment ions. Peptides fulfilling a FDR of 1% were reported. Peptides with inconsistent


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ratios or unusual high ratios altered were manually checked. We established a threshold H/L

ratio value of 1.2. Phosphopeptides showing an average (from the three biological replicates)

SILAC ratio of 1.2 or above from Proteome Discoverer, MaxQuant, or both were considered

upregulated upon c-Src rescue. Four filtering criteria were used to obtain a high-confidence data

set of quantified phosphotyrosine peptidesIf manual validation was satisfactory and showed a

rounded SILAC ratio of 1.2 or above, the peptides were included on the list. A detailed flow-

chart of the process is included in Supplementary Figure 2.

Tyrosine phosphoprotein enrichment and quantitative analysis

SYF/MEF cells stably transfected with R390A c-Src were cultured, isotopically labeled and

treated as described above. Immunoaffinity purification of tyrosine-phosphorylated proteins was

carried out as described previously (3). The enriched tyrosine-phosphorylated proteins were

subjected to in-gel digestion as described previously (3). The resulting tryptic peptides were

analyzed by reversed phase LC-MS/MS using a quadrupole-time-of-flight mass spectrometer

(QSTAR Pulsar, MDS Sciex, Concord, Ontario, Canada) coupled to an 1100 series CapLC

(Agilent, Santa Clara, CA). Acquired data were queried against the RefSeq mouse protein

database (v.26, containing 20311 entries) using MASCOT v.2.2.2 (Matrixscience, London,

England) and quantitated using MSQuant v1.4.3a39 (http://msquant.sourceforge.net). Mass

tolerance of 1.1 Dalton was used for precursors and 0.1 Da for fragment ions. Fixed modification

with carbamidomethylation of cysteines was set while N-terminal acetylation, deamidation of

asparagine and glutamine, oxidation at methionine, phosphorylation at serine, threonine and


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tyrosine together with ‘heavy’ lysine and arginine was set as variable modifications. Quantitation

was performed using MSQuant as described on http//msquant.sourceforge.net.

Network Modeling and Literature Curation

Phosphosite (http://phosphosite.org), Human Protein Research Database (HPRD) (22)

(http://hprd.org), and literature curation were used to differentiate previously unidentified and

known substrates from the list of proteins showing increased phosphorylation upon c-Src rescue.

Phosphosite was used to establish the protein type classification of the detected proteins. The

Ingenuity pathway analysis software (Ingenuity ® Systems, www.ingenuity.com) and literature

curation were used to investigate previously observed connections between c-Src and the protein

of study.

The Ingenuity pathway analysis software was used to perform ontology and canonical pathway

enrichment analysis of the proteins associated with the found tyrosine phosphopeptides

experiencing an increase in their tyrosine phosphorylation level upon c-Src rescue. The online

web tool DAVID Bioinformatics Resources 6.7 (http://david.abcc.ncifcrf.gov. (23) and the

Ingenuity pathway analysis software were used to obtain a representation of the role of c-Src on

focal adhesion pathways. The list of proteins corresponding to the peptides identified as

upregulated upon c-Src rescue was uploaded in the DAVID tool, which incorporated the newly

discovered proteins to the to the pathway. The pathway was completed using Ingenuity Systems.

Both the known and the newly discovered potential c-Src phosphorylation sites were

incorporated to the graph manually and validated with Phosphosite and HPRD.


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Immunobloting and Immunoprecipitations

After imidazole treatment cells were washed once with PBS and lysed for 30 min at 4ºC

in RIPA buffer (Tris-HCl 50 mM pH 7.4, Na-deoxycholate 0.25%, NaCl 150 mM, EDTA 1mM,

1% NP-40, Na3VO4 1 mM, NaF 1 mM, PMSF 1 mM), and centrifuged at 13,000 x g at 4ºC. The

supernatants were collected for immunobloting and immunoprecipitation.

For immunobloting the supernatants were mixed with SDS loading buffer and boiled at

95ºC for 5 minutes, analyzed by SDS-PAGE. The proteins were electroblotted into PVDF

membrane (Invitrogen). The membrane was probed with appropriate antibody, followed by

incubation with the correspondent HRP- conjugated secondary antibody. Antibodies were used at

the recommended concentration in the manufacturer´s instructions. Bands were visualized using

SuperSignal West Pico chemiluminescent substrate (Pierce).

For immunoprecipitation, the supernatants were pre-cleared with protein A or G agarose

and subsequently incubated overnight with the antibody at 4ºC. Antibodies were used at the

recommended concentration in the manufacturer’s instructions. Mixtures were treated with

protein A or G agarose and then incubated at 4ºC overnight. The agarose beads were

subsequently washed three times by RIPA buffer, resuspended in SDS loading buffer with β-

Mercaptoethanol and boiled. The eluates were analyzed by SDS-PAGE and Western Blot

analysis.

Antibodies were obtained as follows: PHOSPHOTYROSINE: (4G10) from Upstate

Biotechnology. FAK: 06-543 (Fak IP and WB) from Upstate Biotechnology. PAXILLIN: 05-417

(Paxillin IP and WB) from Millipore. VIMENTIN: ab8978 (Vimentin IP), ab58462 (Vimentin


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WB) from Abcam, Inc. C3G: sc869 (C3G IP and WB) from Santa Cruz Biotechnology. TENSIN:

sc28542 (Tensin IP and WB) from Santa Cruz Biotechnology. MATRIN: sc55724 (Matrin IP and

WB) from Santa Cruz Biotechnology. NUCLEOPHOSMIN: ab10530 (Nucleophosmin IP and WB)

from Abcam. GSK3β: CST9315 (GSK3β IP) from Cell Signaling Technology, sc81462 (GSK3β

WB) from Santa Cruz Biotechnology, CST9315 (GSK3β WB) from Cell Signaling Technology.

C3G sc-15359 (C3G WB) from Santa Cruz Biotechnology, pTyr514-C3G sc-32612 (pTyr514-

C3G WB) from Santa Cruz Biotechnology. C3G IP/ CRK WB: sc-15359 (C3G IP) from Santa

Cruz Biotechnology, 610035BD (Crk WB) from BD Transduction Laboratories. CRK (PTYR221)

WB: ab76227 (WB) from Abcam, Inc. All Western blots were performed at least twice and

results shown are representative. It should be noted that baseline values between rescuable and

non-rescuable cells may not be the same due to subtle alterations in the cell passage number,

growth conditions, or western blot conditions (such as exposure time).

Immunofluorescence and Confocal Microscopy

Cells were seeded and grown in 8-well glass microscopy slides (Lab-Tek). Cells were

processed for immunofluorescence staining in the following way: After imidazole treatment (10

mM, 5 minutes), cells were washed once with ice cold PBS. Cells were fixed with 4%

formaldehyde (Tousimis Reserch Corporation) for 1 h at 4°C followed by washing with 10mM

Glycine in PBS. Permeabilization was performed with 0.2% (v/v) of Triton X100 in PBS for 5

min at room temperature. After washing with PBS, cells were incubated in blocking solution

(10mg/ml BSA in PBS).


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The primary antibodies used were rabbit polyclonal antibody against C3G (SC-13359

from Santa Cruz Biotechnology) and rabbit polyclonal antibody against pTyr514-C3G (SC-

12926 R from Santa Cruz). The secondary antibodies used were Alexa-Fluor 555 goat anti-rabbit

(A-21430 from Invitrogen) and Alexa-Fluor 488 chicken anti-rabbit (A-21441 from Invitrogen)

respectively. The slides were mounted with ProLong® Gold Antifade Reagent (With DAPI)

from Invitrogen. Samples were acquired with the Zeiss Meta Confocal Laser Scanning

Microscope System, utilizing AIM software version 4.0. An Argon laser exciting at 488nm and

a red HeNe at 561 nm wavelengths were used to obtain optical sections. Narrow band emission

filters (nm) were utilized to eliminate channel cross-talk and a 1.0 µm confocal aperture was

used to obtain z-plane sections. Slides were imaged with 40x and 100x oil immersion Plan

Apochromat objective lens (N.A. 1.4) through a Zeiss Axiovert inverted microscope. For

quantification, at least 40 cells were counted for each condition and scored according to whether

or not they had peripheral staining. Percent of cells with a peripheral staining was calculated for

each 40x window. Averages and standard errors are shown and statistical analysis was done

using the Student’s t test.

FRET Microscopy

R390A and D388N c-Src SYF MEF cells were plated to glass bottom dishes (MatTech

Corp.) and transiently transfected with 2µg Raichu-Rap1 (24) reporter using lipofectamine

(Invitrogen). The cells were starved for 16 hours. After 15 h cells were washed once and media

was replaced with Hanks Balanced Salt Solution (HBSS). The dish was loaded onto the stage of

an inverted Zeiss Axiovert 200M microscope controlled by METAFLUOR software (Universal


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Imaging, Downingtown, PA). Dual emission ratio imaging was performed using a 420DF20

excitation filter and a 450DRLP dichroic mirror and appropriate emission filters, 475DF40 for

CFP and 535DF25 for YFP. Images were captured with a MicroMAX BFT512 CCD camera

(Roper Scientific, Trenton, NJ). Baseline values were captured every 30 seconds for 2 minutes.

Cells were treated with 10mM imidazole at time zero and imaged for an additional 15-20

minutes. Images were quantified using the Metafluor software. Regions of interest were selected

and average intensities of the pixels in each region for each channel were quantified. FRET ratios

were calculated by dividing the yellow emission over the cyan emission at each time point.


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RESULTS

c-Src chemical rescue

The chemical rescue of mutant v-Src provides precise temporal control of its intracellular

kinase activity (3). In order to test whether we could observe similar results with cellular c-Src,

we used SYF (Src-, Yes-, and Fyn-triple knockout) MEF cells stably transfected with R390A c-

Src (R390A/SYF cells). The triple knockout reduces the background from endogenous c-Src, as

well as two other members of the Src kinase family, Yes and Fyn (25). We observed that

treatment of SYF MEF cells stably transfected with R390A c-Src using a range of 2.5-5 minutes

and 2-10 mM imidazole induced a time-dependent, dose-dependent increase in range and

intensity of tyrosine-phosphorylated protein bands as visualized by western blot analysis of

whole cell lysate with 4G10 anti-phosphotyrosine antibody (Supplementary Figure 3 and Figure

1). The imidazole-insensitive inactive c-Src mutant D388N/SYF cells were used as a negative

control since they express an inactive Src kinase at similar levels (3) and should be most similar

to the untreated R390A/SYF cells. In prior studies (3), it was shown that D388N/SYF cells are

unresponsive to imidazole. We confirmed here that D388N/SYF cells showed no such change in

protein tyrosine phosphorylation with 10 mM imidazole and 5 minute treatment, compared with

the imidazole-sensitive R390A/SYF c-Src mutant (Figure 1B), suggesting that the imidazole

effect was specific. The modest baseline western blot differences (minus imidazole) between

D388N/SYF and R390A/SYF cells are not consistent (see ref. 3) and likely represent subtle

differences in passage numbers and clonal populations. To further ensure that imidazole had no


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major effects on cellular tyrosine phosphorylation outside of chemical rescue under these

conditions, we investigated its effect on SYF and WT MEF cells. As shown in Supplementary

Figure 4, there were no appreciable changes in anti-pTyr western blots of cell lysates treated with

or without imidazole.

After a series of optimization experiments, we selected 10 mM imidazole and 5 minutes

exposure as the optimal conditions for c-Src rescue in our R390A/SYF cells. Since our goal was

to identify the c-Src-induced phosphoproteome, we combined c-Src chemical rescue with mass

spectrometry and stable isotope labeling with amino acids in cell culture (SILAC) (Figure 2)

(26). R390A c-Src-expressing SYF cells were labeled with either 13C6-Arg/ 13C6-Lys (heavy) or

12C6-Arg/ 12C6-Lys (light). “Heavy” cells were treated with 10 mM imidazole for 5 minutes and

“light” cells were left untreated (control) to distinguish the c-Src induced tyrosine

phosphorylation events from the background of steady-state tyrosine phosphorylated proteins in

the cell.

Phosphoprotein identification and quantitation

As an initial approach to test the combined advantages of chemical rescue and a

proteomics analysis, protein enrichment was performed by immunoprecipitation with anti-

phosphotyrosine antibody (Figure 2A). The phosphotyrosine proteins were analyzed by tandem

mass spectrometry. The complete list of identified proteins is shown in Supplementary Table 1,

and a summary of fold change in phosphorylation upon c-Src chemical rescue for all proteins is

included in Figure 3A. It can be surmised that the 35 proteins showing SILAC ratios greater

than or equal to 1.2 are targets of c-Src-mediated tyrosine phosphorylation and in fact, at least six


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of these are previously documented c-Src substrates. While it is tempting to infer that larger

SILAC ratios suggest greater confidence in undergoing c-Src-mediated phosphorylation, this is

not necessarily the case. For example, the well-established c-Src substrate focal adhesion kinase

(FAK) showed a modest ratio of 1.2. The proteins with a SILAC ratio of 1.2 or above were

classified into five protein type classes (Figure 3B) according to the Phosphosite bioinformatics

resource (www.phosphosite.org) (27). Interestingly, the most represented cellular functions were

signaling pathways (37%) – with proteins such as nucleophosmin, G3BP-2, Map kinase, C3G,

and cullin 5 – and adhesion and cytoskeleton (26%) – vimentin, tensin 1, matrin 3, plectin 1,

fibronectin 1, and myosin X. A subset of the 29 potentially novel c-Src targets have been

functionally linked to c-Src signaling pathways such as vimentin (28) and nucleophosmin (29),

although the experiments here show unique evidence that they are closely temporally linked to c-

Src kinase action.

Confirmation of c-Src targets by immunoprecipitation-western blot analysis

We thus selected a set of identified proteins to confirm chemical rescue-induced tyrosine

phosphorylation using an immunoprecipitation-western blot approach in c-Src R390A/SYF cells.

The D388N/SYF cells serve as negative controls. As described previously, paxillin and Fak are

well-established c-Src substrates and therefore served as positive controls, which showed

imidazole-enhanced signal intensity when blotting with 4G10 anti-phosphotyrosine antibody

after immunoprecipitation (Supplementary Figure 5).

We showed that C3G, vimentin, tensin 1, matrin 3, nucleophosmin, and Gsk-3ß could be

efficiently immunoprecipitated from SYF cells (Figure 4). Analysis of these immunoprecipitated


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proteins with anti-phosphotyrosine antibody showed an increase in the phosphorylation of each

of these proteins from R390A/SYF but not D388N/SYF cells when treated with 10 mM

imidazole for 5 minutes. The loading controls were performed by immunoprecipitation with a

specific protein antibody followed by blotting with an antibody against the same protein. These

data suggest that tyrosine phosphorylation of each of these proteins depends on c-Src kinase

activity, with a close temporal connection to c-Src rescue.

Phosphosite mapping by chemical rescue of mutant c-Src

Given the success in our preliminary attempt to combine c-Src chemical rescue and

phosphoproteomics, we decided to perform an extensive phosphopeptide mapping and

quantitation of phosphorylation changes in the cell upon c-Src rescue. The protocol described

above for quantitative analysis of protein tyrosine phosphorylation was modified such that

trypsin proteolysis of extracted proteins was performed prior to affinity based enrichment with

anti-phosphotyrosine antibody (Figure 2B). In addition, three biological replicates and two

technical replicates were performed (Supplementary Figure 2) and analyzed using high

resolution, high accuracy mass spectrometry (30). Mascot and Sequest led us to the

identification of an initial list of redundant tyrosine phosphopeptides (Supplementary Table A).

That translated into a list of 335 non-redundant peptide sequences (corresponding to 205

proteins). In parallel, the quantitative proteomics software MaxQuant led us to the identification

of an initial list of redundant tyrosine phosphopeptide spectrum matches (Supplementary Table

B) that translated into a list of 216 non-redundant tyrosine phosphopeptides (corresponding to


22

147 proteins). We assembled a proteome comprising 382 non-redundant peptide sequences,

corresponding to 334 tyrosine phosphorylation sites on 213 proteins (Supplementary Table 2).

We set a threshold value of 1.2-fold for SILAC ratio (heavy vs. light ≥ 1.2) to filter for

tyrosine-containing peptides with increased phosphorylation level upon c-Src rescue. A high-

confidence data set of quantified phosphotyrosine peptides was obtained through a four filtering

criteria described in the Experimental Procedures section. After manual validation we obtained a

list of 97 non-redundant peptide sequences, corresponding to 85 tyrosine phosphorylation sites

(corresponding to 52 proteins), as upregulated in response to chemical rescue of c-Src,

suggesting that they may be c-Src targets. A complete list of the mapped proteins with sites

showing an increased phosphorylation level upon c-Src chemical rescue is provided in

(Supplementary Table 3).

Several of these sites were identified in proteins that also showed an increased

phosphorylation level at the protein level (such as C3G, tensin 1, Pard3 and Lpp), adding further

confidence to their significance. The proteins containing the tyrosine residues which showed an

increased phosphorylation level upon c-Src rescue can be classified into several categories

(Figure 5) including actin modeling, adaptor/scaffold, adhesion, guanine exchange factors/

GTPase-activating proteins, non-receptor protein kinases, phosphatases, cell polarity, and

receptor tyrosine kinases and a group of proteins with diverse functionalities.

We analyzed the c-Src upregulated phosphoproteome with the Ingenuity Pathways

Knowledge Software (Ingenuity ® Systems, http://www.ingenuity.com) and looked for

enrichment of both ontology (Figure 6A) and canonical pathways (Figure 6B). Not surprisingly

two of the most enriched pathways are the integrin and the focal adhesion signaling pathways.

The cellular movement category appears to be the functionality that is especially impacted upon


23

c-Src activation. Both integrin-mediated focal adhesion formation, and adherens junction

formation and focal adhesion disassembly (turnover) mediate cellular motility and are involved

in cancer invasiveness and progression (1). A large number of the identified sites are involved in

cell adhesion, particularly in focal adhesion regulation, where c-Src plays key role (31, 32). In

this context, several phosphosites have been found in potential novel c-Src substrates. Also,

novel sites in well-established c-Src substrates have shown an increased tyrosine

phosphorylation level (Figure 7).

Although the tyrosine phosphoproteome provided here is not likely to include the entire

c-Src phosphoproteome, it is the first snapshot of the tyrosine phosphorylation events that take

place immediately (5 minutes) after c-Src specific activation and the first one that has been

obtained through high-resolution, high accuracy mass spectrometry.

The role of c-Src in C3G-mediated mechanism of Rap1 activation

We next explored the potential of chemical rescue as a tool to explore the biological

significance of the phosphorylation of one of the detected proteins, C3G, in response to c-Src

chemical rescue. C3G was identified in both proteomic approaches (phosphoprotein enrichment

and phosphopeptide enrichment) and was validated through immunoprecipitation and western

blot as mentioned above. Furthermore, C3G has shown to play a role in focal adhesion signaling

(33) as can be shown in Figure 7. C3G is a guanine nucleotide exchange factor for the small

Ras-related G-proteins Rap1, Rap2, and R-Ras (34-37). It is known that C3G is phosphorylated

on Tyr514 and this PTM has been shown to be critical for the activation of C3G (38). Previous


24

studies have suggested possible roles of Hck, Jak2, and c-Src kinases in Tyr514 phosphorylation

(38-40). In this study, we have observed that imidazole treatment of R390A/SYF cells but not

D388N/SYF cells led to an increase in Tyr514 phosphorylation of C3G as observed by western

blot with an anti-pTyr514 antibody (Figure 8A). This increase was observed within 1 minute of

imidazole exposure supporting the possibility that C3G is a direct substrate of c-Src. Additional

tyrosine phosphorylation sites mat account for the large overall increase in C3G overall tyrosine

phosphorylation level (Figure 4), such as the newly identified site, Tyr 571, which shows an

approximate 3-fold for SILAC ratio upon c-Src rescue.

In addition to C3G phosphorylation, it has been shown that C3G activation occurs

through membrane recruitment (39, 42-44). Redistribution of C3G within the cell, probably to

sites where Rap1 is located, occurs through binding of C3G proline-rich regions to the SH3

domain of the adaptor protein Crk, whose SH2 domains bind to phosphorylated sites resulting

from activated receptors (40, 41, 45). In order to further dissect this signaling process, we

explored the potential changes in protein-protein interactions between C3G and Crk as a result of

c-Src chemical rescue. After imidazole treatment of R390A/SYF cells, C3G protein was pulled

down and co-immunoprecipitated Crk was detected by Western blot (Figure 8B). We observed

that increased Crk was pulled down by C3G immunoprecipitation only 1 minute after rescue of

R390A/SYF but not D388N/SYF cells. The increased binding of C3G and Crk was maintained

until 5 minutes, when it experienced a decrease. We could therefore conclude that c-Src

activation leads to an immediate increase in binding between C3G and Crk.

In order to gain a better understanding of the dynamics of the Crk-C3G interaction, we

explored the possible cause of the observed decrease in binding after 5 minutes upon c-Src

rescue. Crk is negatively regulated when it is phosphorylated on Tyr221, which occurs as a


25

result of various types of stimulation (46-48). We examined the phosphorylation level of Tyr221

site with a phosphospecific antibody after chemical rescue at the same time points used to

observe C3G-Crk interaction (Figure 8C). Indeed, phosphorylation of Crk on Tyr221 reaches a

peak at 5 minutes, the same time as when C3G-Crk dissociation is first observed.

To further examine the connections between c-Src and C3G, we explored the subcellular

localization of C3G using immunofluorescence and confocal microscopy after chemical rescue.

As expected, C3G was mainly cytoplasmic at baseline. However, upon imidazole treatment, a

greater concentration was observed at the cell periphery in R390A c-Src expressing SYF cells

(Figure 8D) but this was not observed in D388N c-Src expressing SYF cells (Supplementary

Figure 6). This was detected 1 minute after imidazole treatment and persisted until 5 minutes,

after which it showed a decrease (Figure 8D, Supplementary Figure 6). Cells with peripheral

staining showed both membrane and subcortical localization. Moreover, localization of

pTyr514-C3G before and after c-Src rescue showed a similar trend as C3G, with a peak in

peripheral localization of pTyr514-C3G at 2.5 minutes (Figure 8D, Supplementary Figure 6).

Rap1 is a small G-protein of the Ras family that antagonizes Ras by sequestering their

common target, Raf1, in its inactive form (49-51). It has also been suggested to play an

important role in integrin-mediated cell-adhesion (52). Thus we wanted to examine the effects

of c-Src-mediated activation of C3G on Rap1. We employed the cell-based reporter Raichu-

Rap1 (24), which is designed to undergo a FRET change upon Rap1 activation. As shown in

Figure 8E, imidazole treatment of R390A/SYF cells but not D388N/SYF cells indeed stimulates

Rap1 within 5 minutes primarily at the cell periphery. C3G activation of Rap1 appears therefore

to be controlled by c-Src activation.


26

In this way we were able to further elucidate the mechanism of C3G activation following

c-Src chemical rescue in a precise spatiotemporal manner that could have been very difficult to

obtain with traditionally used methods for the study of signal transduction.

Discussion

Previous studies have focused on proteomic analysis on oncogenic viral v-Src (3) or

constitutively active Src mutants (4, 5). In addition, some of these studies have examined

tyrosine phosphorylation patterns several hours or days after Src is expressed, allowing chronic

changes in phosphoproteins to be ascertained. Such analyses are well-suited to identification of

possible biomarkers in v-Src expressing tumors, which appear to be very rare, but likely reflect

many secondary changes in cellular protein levels relating to Src’s well established effects on

growth and gene expression. A recent study focused on cellular c-Src’s kinase action in response

to treatment with SU6656 inhibitors and posterior stimulation with platelet-derived growth factor

(PDGF) (7). Proteomic analysis after the acute activation of tyrosine kinases by ligands such as

growth factors have been particularly informative because they have revealed how a cascade of

signaling is initiated in a temporal fashion, but give rise to indirect information about non-

receptor kinases.

In order to identify temporally direct substrates of the proto-oncogene c-Src, it would be

ideal to use a method to rapidly and specifically switch on c-Src activity in the cell. In contrast

to receptor tyrosine kinases, most non-receptor tyrosine kinases cannot readily controlled in this

fashion. However, the chemical rescue of the R390A c-Src mutant provides a strategy that

perfectly fits this purpose since it provides a rapid, specific and reversible mechanism of c-Src


27

activation by the small molecule imidazole, allowing the detection of the immediate effects of

the restored Src activity in cells (3). Prior in vitro studies on the tyrosine kinases Csk, Src, and

Abl have indicated that the imidazole concentration used here (<30 mM) has no effect (<10%)

on wt kinase activity (3, 12,14). Furthermore, previous experiments on chemical rescue of R/A

Csk and Src with imidazole in cell culture have revealed that this chemical rescue approach

recapitulates, without apparent toxic effects, expected tyrosine kinase activities involved in stress

fiber formation, vascular tube formation, cell migration and transformation, and guanylyl cyclase

modulation (3, 12-14, 53, 54). Extensive western blot analysis from an earlier report (3) and

contained in the current study show no evidence of cellular tyrosine phosphorylation mediated

by imidazole in control cell lines. Although we cannot completely rule out that at the level of

mass spectrometric analysis some imidazole effects may be non-specific, we believe that the

accumulated evidence suggests that the chemical rescue approach is a robust technique for

phosphoproteomics.

Combining the power of chemical rescue with proteomics led us to the identification of

28 potential new c-Src protein substrates and 85 tyrosine phosphorylation sites (corresponding to

52 proteins), which showed an increased phosphorylation level upon c-Src rescue. It is

reassuring that several of the putative protein substrates of c-Src that we have uncovered in this

work have been linked to Src in prior studies. We note that 16 of the proteins found here using

c-Src rescue were found among the 140 putative Src kinase targets identified in a combined list

(Supplementary Table 4) of the four prior mass spectrometry phosphoproteomics studies (3-5,7)

reported previously. The lack of greater overlap is likely due in part to the divergent

experimental approaches. It must be cautioned that even chemical rescue cannot unequivocally

assign a protein as a direct kinase substrate of c-Src since the changes in phosphorylation we see


28

can result from secondary effects, which can happen within the time frame used in our

observations (5 minutes). Indeed, the class of proteins that show a decrease in tyrosine

phosphorylation likely reflects examples of a secondary phenomenon where a protein tyrosine

phosphatase is activated by phosphorylation. For example, protein tyrosine phosphatases SHP1

and SHP2 are known to be activated by tyrosine phosphorylation and such a mechanism could

contribute to specific dephosphorylation events (55, 56). Nevertheless, in order to define the

characteristics of signaling networks, it is helpful to elucidate the temporal connection among

phosphoprotein changes, even if the protein interactions are indirect.

Activation of c-Src by growth factors or activating mutations leads to an increased

cellular proliferation, invasion and motility and a decreased cell-cell (adherens junctions) and

cell-matrix adhesion (focal adhesions) (1). A significant number of the phosphopeptides

detected in our chemical rescue analysis are involved in cell adhesion and, in particular, in focal

adhesion pathways (Figure 7). c-Src is known to play a role in the regulation and disassembly of

focal adhesions, which link the extracellular matrix to the actin cytoskeleton (57) and are also

involved in signaling pathways that regulate proliferation and gene transcription (58). Our work

here provides a number of possible novel connections in this regard. A network map that

includes the role of c-Src in focal adhesions and highlights the novel proteins and

phosphopeptides identified in our study is shown in Figure 7.

Focal adhesions form when integrin connects the ECM to a complex of proteins that acts

as a link between integrins and the actin cytoskeleton. Several tyrosine sites from cytoskeletal

proteins that are part of this supramolecular structure and have not previously been described as

c-Src substrates experienced an increase in their phosphorylation level include talin (Tyr436),

filamin B (Tyr2502), tensin 1 (Tyr1477, Tyr1480, Tyr1558), and tensin2 (Tyr483, Tyr705,


29

Tyr770, Tyr770). Tensin 3 is a recently discovered c-Src target (59) but the site found in this

study, Tyr584, has never shown to be phosphorylated by c-Src.

The activation of RhoA is one of the key events of focal-adhesion assembly (60). c-Src

blocks downstream signalling by RhoA through activation of p190 RhoGAP, leading to focal-

adhesion disruption and increased motility (61). Two other RhoGAPs are known to be

phosphorylated by c-Src: RhoGAP3 (Tyr21-known (60)), and RhoGAP5 (63) (Tyr342-known,

749-known). Interestingly, we detected two other RhoGAPs that have never been described as c-

Src substrates: RhoGAP42 (Tyr342-unknown; Tyr759-unknown) and RhoGAP32 (Tyr2025-

unknown).

It is known that c-Src phosphorylates and activates Fak at sites Tyr614 and Tyr615

promoting maximal Fak catalytic activity (64). Interestingly we detected a new Fak

phosphorylation site, Tyr5, which underwent a c-Src mediated increase in phosphorylation and

has never been identified as a c-Src substrate. Similarly, p130Cas is a well-known substrate of

active Fak-Src. Several known sites and a new phosphorylation site, Tyr331, showed an

increased phosphorylation level upon c-Src rescue.

The Rap1 guanine exchange factor (C3G) was also detected in our proteomic studies and

plays a role in focal adhesion signaling as indicated in Figure 7. The temporal connection

between c-Src and C3G identified here allow us to propose a molecular mechanistic pathway for

Rap1 control. It has been shown that Crk is constitutively associated with C3G (65). We show

that rapid activation of c-Src enhances interaction between C3G and Crk by 1 minute. Such

regulation of this complex may account for reported responses after the stimulation of cells by

insulin, integrin engagement, and other natural triggers (66-68). Presumably, the adaptor protein

Crk drives C3G to the cell periphery, where we detected it within minutes after c-Src rescue.


30

Increased phosphorylation of C3G on Tyr514 is known to facilitate C3G’s activity to catalyze

GTP exchange on Rap1 (38), consistent with our live cell reporter assay, which showed a

maximum level of activity at 5 minutes. Interestingly, the binding between C3G and Crk shows

a decrease at this time, possibly initiating retrograde signaling in this pathway (66).

We showed that the level of phosphorylation of Crk on Tyr221 increases upon c-Src

activation. Interestingly the observed decrease in Crk-C3G binding occurs when

phosphorylation of Crk on Tyr221 reaches a maximum level. Subsequently, Rap1 activation

appears to be indirectly modulated downward in a fashion that is correlated with Crk

phosphorylation on residue Tyr221. Phosphorylation of Crk on Tyr221 and the consequent

dissociation of the C3G-Crk complex at 5 minutes might contribute to the decrease in Rap1

activity that follows. Abl kinase has been identified as Crk upstream kinase on Tyr 221 (47). In

this regard, our phosphopeptide mapping revealed that sites Tyr439 of Abl/Arg, which has

additive effects on Abl activation (69) and has not been described previously as a c-Src substrate,

increased phosphorylation level significantly 5 minutes after c-Src chemical rescue. It is also

noteworthy that rescue induced an ~3-fold increase in phosphorylation of a new site, Tyr571

(Supplementary Figure 7), on C3G that may also modulate C3G’s ability to activate Rap1.

However, we caution that the exact timing of each event: C3G phosphorylation, C3G-Crk

binding, C3G translocation to the membrane, and Rap1 activation cannot be revealed with

certainty because of the varying techniques and limited temporal resolution associated with these

methods. This may account for the slight delay in Rap1 activity decrease relative to Crk-C3G

binding disruption and C3G phosphorylation.

In summary, the chemical rescue approach has allowed a kinetic description of c-Src

signaling events that would otherwise have been difficult to obtain. In principle, this technique


31

can be applied to other non-receptor PTKs to provide a more complete portrait of tyrosine

phosphorylation networks in cell signaling.

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43

Acknowledgments

We thank the NIH for financial support. Isabel Martinez- Ferrando is a recipient of a Fulbright

Graduate Scholarship from the International Institute of Education- Fulbright Spain, Government

of Spain. We thank Dr. Michiyuki Matsuda for providing us with the Raichu-Rap1 FRET

Reporter. We thank Barbara Smith and Michael Delannoy from the Microscope Facility at the

Johns Hopkins School of Medicine for their assistance. We thank Bob Cotter and Bob O’Meally

from the Mass Spectrometry and Proteomics Facility for help.

Footnotes

The raw data associated with this manuscript may be downloaded from ProteomeCommons.org

Tranche, http://proteomecommons.org/tranche/, using the following hash:

TzGvo1FbhJkLaM/FDLKTewGrBYDqWRPO/Oe9KHmXiohZonXBuLlolUe2NuY4G/UIubBh

NQjySdOasclB3Os1vYmje8gAAAAAAAAJtw==

This article contains the following supplementary data: Supplementary Table 1, a complete list

of tyrosine phosphorylated proteins quantitated by SILAC. Supplementary Table 2, a complete

list of tyrosine phosphorylated peptides quantitated by SILAC. Supplementary Table 3, a list of

c-Src induced,upregulated phosphorylated peptides, Supplementary Table 4, a list of putative Src

substrates from references 3-5,7. Supplementary Table A, a list of phosphoPSM identified by

Mascot and SEQUEST. Supplementary Table B, a list of phosphoPSM identified by MaxQuant.


44

Supplementary Figure 1, Quality control studies of proteomics samples used for

phosphotyrosine-site mapping. Supplementary Figure 2, Flow-chart representing the complete

data analysis process corresponding to the phosphopeptide enrichment by SILAC and LC-

MS/MS. Supplementary Figure 3, Optimization of c-Src rescue conditions. Supplementary

Figure 4, Anti-pTyr western blot with imidazole treatment of SYF and WT MEF cells.

Supplementary Figure 5, Validation of protein phosphorylation of known Src substrates Fak and

paxillin. Supplementary Figure 6, C3G and pTyr514-C3G increased localization at the cell

periphery after c-Src rescue. Supplementary Figure 7, Mass spectrum (MS) and tandem mass

spectrum (MS/ MS) of phosphopeptide from Rap Guanine Exchange Factor 1 (C3G).

Conflict of Interest: The authors declare no conflict of interest.

Figure Legends

Figure 1. c-Src chemical rescue by imidazole. (A) Proposed R390A c-Src chemical rescue

mechanism by imidazole indicating interactions of imidazole with the catalytic base Asp388 and

Tyr from the substrate (Based on PDB 3GEQ). (B) Comparison of the phosphotyrosine profile

of imidazole-treated cells compared to that of untreated cells. Tyrosine phosphorylation induced

by imidazole treatment (10 mM, 5 minutes) of SYF cells stably transfected with c-Src R390A

and D388N SYF (negative control). Cells were subjected to lysis and whole lysates were

subjected to Western blotting with 4G10 anti- phosphotyrosine antibody.

Figure 2. Scheme of the experimental design of SILAC-based quantitative

phosphoproteomic approach. SYF MEF cells stably transfected with R390A c-Src were


45

divided into two populations that were grown in ‘light’ or ‘heavy’ medium. The population of

cells cultured in ‘heavy’ medium was treated with 10 mM imidazole for 5 minutes and the one

cultured in ‘light’ media was left untreated. Cell lysates were combined in equal proportions. (A)

The protein mixture was incubated with anti-phosphotyrosine antibodies for enrichment of

tyrosine- phosphorylated proteins. The enriched fraction was subjected to in-gel trypsinization.

(B) The protein mixture was first digested with trypsin and then incubated with anti-

phosphotyrosine antibodies for enrichment of tyrosine- phosphorylated peptides. In both cases,

the enriched samples were analyzed by LC- MS/MS.

Figure 3. Distribution of identified and quantified phosphoproteins. (A) Summary of fold

change in phosphorylation upon c-Src chemical rescue for all proteins in R390A c-Src SYF MEF

cells. Known Src protein substrates are indicated. Proteins with a heavy to light (H/L) ratio > 1.2

(red) increased their tyrosine phosphorylation level, and those with H/L ratio < 0.8 (green)

decreased their tyrosine phosphorylation level. Proteins that showed no significant change in

their tyrosine phosphorylation level (0.8 < H/L ratio < 1.2) are indicated in gray. The pie chart

indicates the distribution of proteins with either up regulated (H/L >1.2), constant (0.8 < H/L <

1.2) or down-regulated (H/L <0.8) tyrosine phosphorylation levels. (B) Functional distribution

of identified proteins with increased tyrosine phosphorylation level upon c-Src rescue.

Figure 4. Validation of protein phosphorylation by immunoprecipitation- immunoblot

analysis of R390A and D388N c-Src SYF cells after treatment with 10 mM for 5 min.

Immunoprecipitation was carried out with commercially available antibodies corresponding to

the protein of interest and immunoblotting was carried out with the 4G10 pTyr- antibody as well


46

as protein-specific antibodies. Vimentin, C3G, tensin, matrin, nucleophosmin and GSK-3β are

some novel potential direct c-Src substrates, which were validated by immunoprecipitation-

immunoblot analysis.

Figure 5. Overview of identified phosphopeptides with upregulated tyrosine

phosphorylation upon c-Src rescue in R390A c-Src SYF MEF cells. All the detected

upregulated phosphopeptides are shown whether they are novel (red) or known (red and black

dot) c-Src substrate sites. As shown, the corresponding proteins span a wide variety of cellular

functions, such as polarity, actin remodeling, adaptors, kinases, phosphatases, GEF/GAP proteins

and are modulated by tyrosine phosphorylation following c-Src activation.

Figure 6. Ingenuity Pathway analysis of the c-Src upregulated phosphoproteome. (A)

Enrichment plots of ontology (x-axis) built on the list of 52 c-Src upregulated phosphoproteins.

The y-axis represents the significance of the enrichment, which was calculated using the

Benjiamini-Hochberg multiple testing correction, as –log10 of P-values. (B) Enrichment plots of

canonical pathways (x-axis) built on the list of 59 c-Src upregulated phosphoproteins. The right

y-axis (line) represents the percentage enrichment (ratio between the number of c-Src

upregulated proteins and the total number of proteins included in the pathway). The left y-axis

(bars) represents the significance of the enrichment, which was calculated using the Benjiamini-

Hochberg multiple testing correction, as –log10 of P-values. From the complete list of pathways

obtained from the list of the c-Src upregulated phosphoproteins by Ingenuity Systems, the ones

with a value of -log (BH-P) ≥ 6 are represented.


47

Figure 7. Role of c-Src in Focal Adhesion Signaling. The signaling pathways involved in

focal adhesion activation by integrins and the resulting downstream signaling events are

represented. The biological functions resulting from focal adhesions are also summarized. For

each of the proteins involved the sites that are known substrates of c-Src are indicated. The

modulation of phosphotyrosine sites that were identified in our experiment is indicated as

significantly increased (red), not modulated (gray), or decreased (blue). Among the identified

sites some are known c-Src substrates (black dot).

Figure 8. Spatio-temporal dissection of the activation mechanism for the identified c-Src

substrate, C3G. (A) Immunoprecipitation with anti-C3G antibody and western blot using anti

pTyr514-C3G antibody showed an increase in the phosphorylation level of tyrosine 514 in C3G

after 2.5 min that was sustained until 7.5 minutes upon c-Src rescue in R390A c-Src SYF cells

but not in D388N c-Src SYF cells. (B) Co-Immunoprecipitation using C3G

immunoprecipitation and Crk blotting showed an increase in C3G-Crk binding after 1 minute

upon imidazole treatment which was sustained until 2.5 minutes and decreased at minute 5 in

R390A c-Src SYF cells but not in D388N c-Src SYF cells. (C) Western blot using anti-pTyr221

in Crk showed a gradual increase in phosphorylation upon c-Src rescue reaching the highest level

of phosphorylation at minute 5 in R390A c-Src SYF cells but not in D388N c-Src SYF cells. The

maximum level of phosphorylation was reached at the same point at which C3G and Crk showed

a decreased binding. (D) R390A c-Src/SYF cells were treated with 10 mM imidazole and fixed

at various time points. Localization of both C3G and pTyr514-C3G was detected by staining

with C3G (Rhodamine) and pTyr514-C3G (FITC) and nuclei stained by DAPI. Images were

acquired by confocal microscopy. Representative images at 100x magnification are shown. Cells


48

(n>40) were categorized according to phenotype (cytosolic vs peripheral) and quantified.

Quantitative analysis of C3G and pTyr514-C3G cellular localization was measured as a function

of imidazole treatment time. Images were captured by confocal microscopy at 40x magnification

as the ones shown in Supplementary Figure 6. Six representative images (at least 40 cells) were

quantified for each condition. Each cell was scored for whether it had peripheral staining. The

fraction of cells with peripheral staining was calculated for each image and averages were

calculated by combining the six images for each condition. Each time point was normalized to

time zero and standard errors are shown. Statistical significance (p<0.05) compared with time

zero was calculated using a Student’s T test and is indicated with an asterisk (*). (E) Rap1

activation upon c-Src rescue in R390A c-Src SYF cells (red) and D388N c-Src SYF cells (blue).

The FRET-based GTPase sensor, Raichu-Rap1 showed a 10% increase in FRET (Rap1

activation indicator) along the cell membrane after 8 min of treatment with imidazole to rescue

c-Src activity.

Figure 2 R390A c-Src SYF cells

12C-Arg, 12C-Lys media 13C-Arg, 13C-Lys media

IMIDAZOLE TREATMENT

“Light” Lysate “Heavy” Lysate

Combine lysates

Immunoafinity purification of pTyr-proteins

SDS- PAGE, in-gel trypsin digestion

Tyrosine phosphorylated PROTEIN identification

Tyrosine phosphorylated PEPTIDE identification

CONTROL (No treatment)

LC-MS/MS analysis

A B

Lysate trypsin digestion

Immunoaffinity purification of pTyr-peptides

0

0.5

1

1.5

2

2.5

3

3.5

Protein

Dok1

p130Cas

FAK

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Arhgef7 Src

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d C

hang

e

M

easu

red

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io [H

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B

32 % > 1.2

0.8 - 1.2

< 0.8

60 %

8 %

9 35

67

AFigure 3

H/L > 1.2 1.2 > H/L > 0.8 H/L < 0.8

1

2

3

4

5

Signaling Adhesion/ cytoskeleton

Metabolic/ Biosynthetic enzymes

RNA synthesis/ processing

Protein synthesis/ processing

13

9 3

6

4 37 %

26 %

9 %

17 % 11 %

Figure 4

Vimentin R390A D388N

imidazole - + - + IP: Vim WB: p-Tyr IP: Vim WB: Vim

C3G (Rap GEF1) R390A D388N

imidazole - + - + IP: C3G WB: p-Tyr IP: C3G WB: C3G

Tensin R390A D388N

imidazole - + - + IP: Tensin WB: p-Tyr IP: Tensin WB: Tensin

Nucleophosmin R390A D388N

imidazole - + - + IP: NPM WB: p-Tyr

IP: NPM WB: NPM

Matrin R390A D388N

imidazole - + - + IP: Matrin WB: p-Tyr IP: Matrin WB: Matrin

GSK3β R390A D388N

imidazole - + - + IP: GSK3β WB: p-Tyr IP: GSK3β WB: GSK3β

Figure 6

B

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Src Y343 (m) Y424 (m) Y444 (m) Y535 (m)

PI3K

Paxillin Y31 (m) Y40 (m) Y88 (m)

Y118 (m)

p130Cas Y132 (m) Y169 (m) Y183 (m) Y196 (m) Y238 (m) Y253 (m) Y271 (m) Y291 (m) Y310 (m) Y331 (m) Y391 (m) Y414 (m) Y668 (m)

Crk

p190RhoGAP 1087 (m) 1105 (m)

ArhGAP3 Y21 (m)

ArhGAP5 Y1089 (m) Y1107 (m) ArhGAP32 Y2025 (m) ArhGAP42 Y342 (m) Y759 (m)

C3G Y514(m) Y571(m)

Rap1

DOCK1

Rac

B-Raf

MEK1

JNK

Shc1 Y349(m) Y350(m) Y423(m)

Grb2 Y160(m) Sos Ha-Ras

Raf-1 Y340(m) Y341(m)

ERK1 Y205 (m)

ERK2 Y185 (m)

ELK1

PTEN

PIP2 PIP3

Vav1 Y174 (m)

Vav3 Y173 (m)

PAK

Talin Y436 (m)

Filamin B Y2502(m)

Actinin 1 Y193 (m)

Vinculin Y100 (m) Y692 (m) Y822 (m)

Y1065 (m)

α Actin

F Actin

Tensin 1 Y1477(m) Y1480(m) Y1558(m)

PDK1 Y9 (m)

Y373(m) Y376 (m)

Akt/PKB Y315 (m) Y326 (m)

GSK-3β Y216 (m)

β-Catenin Y86 (m)

Y489 (m) Y654 (m)

c-Jun

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Caveolin Fyn

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Wnt signaling pathway

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RhoGEF10L Y152 (m)

RhoA ROCK2 Y722 (m)

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MLC

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Tensin 2 Y483 (m) Y705 (m) Y770 (m) Y791 (m)

Tensin 3 Y584 (m)

Y1168 (m) Y1201 (m) Y1251 (m)

FAK Y5 (m))

Y428 (m) Y438 (m) Y614 (m) Y615 (m) Y899 (m) Y963 (m)

Integrin

Up (H/L > 1.2) Down (H/L < 0.8) None (0.8 < H/L < 1.2) Known c-Src phospho-site

pY modulation of identified sites Interactions Activation Inhibition Phosphorylation upon c-Src activation (5min)

Figure 7

new c-src targets revealed by chemical rescue and proteomics

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