Ph h tid A Pl tf f C ll l Si liPhosphopeptide Array Platform for Cellular SignalingPhosphopeptide Array Platform for Cellular SignalingPhosphopeptide Array Platform for Cellular Signaling N k P i P fili i B C C llNetwork Protein Profiling in Breast Cancer CellsNetwork Protein Profiling in Breast Cancer CellsNetwork Protein Profiling in Breast Cancer Cellsg

S i i K i h th 1 Aili H 2 Zh h Li 1 R ij Zh 1 Xi W 1 Y l Zh 1 Qi Zh 4Srinivasan Krishnamoorthy 1, Ailing Hong2, Zhonghua Liu1, Ruijuan Zhu1, Xin Wang1, Yulu Zhang1, Qi Zhu4,Srinivasan Krishnamoorthy , Ailing Hong , Zhonghua Liu , Ruijuan Zhu , Xin Wang , Yulu Zhang , Qi Zhu , 2 2 1Xiaochuan Zhou2 Tongbin Li2 Xiaolian Gao1 Xiaochuan Zhou , Tongbin Li , Xiaolian Gao

1Dept of Biology&Biochemistry University of Houston; Houston TX 77004; 2LC Sciences Houston TX 770541Dept. of Biology&Biochemistry, University of Houston; Houston, TX 77004; 2LC Sciences, Houston, TX 77054p gy y, y ; , ; , ,

Ph h tid hi (PPEP hi ) d b i itIntroduction Signature Peptides for GRB2 SH2 Domain

Phosphopeptide array chips (PPEP array chip) was made by in situ synthesis on microfluidic microchips More information can be found from Microarray based PPEP binding are real Introduction Signature Peptides for GRB2 SH2 DomainValidation assays for peptide microarray datasynthesis on microfluidic microchips. More information can be found from http://www lcsciences com/applications/proteomics/ Thousands of PPEPs are

y gprotein-protein interactions in cellsValidation assays for peptide microarray datahttp://www.lcsciences.com/applications/proteomics/. Thousands of PPEPs are

designed based the known phosphorylation or signaling pathway informationprotein-protein interactions in cells

Tyrosine phosphorylation is the hallmark of activation of Receptor Tyrosine Cell lysis Immunoprecipitation and Western analysisdesigned based the known phosphorylation or signaling pathway information to allow probing proteins which mediate signaling transduction pathways such

Kinase (RTK) pathway proteins which regulate various aspects of cellular Cell lysis,Immunoprecipitation and Western analysisCells were washed 4 times in cold PBS and lysed in 1 2 mL of lysis buffer

to allow probing proteins which mediate signaling transduction pathways, such as kinases related SRC PLCG2 PIK3R1 adaptors LCK CSK FYN GRB2

division, multiplication, differentiation and apoptosis (1-2). Temporal and Cells were washed 4 times in cold PBS and lysed in 1-2 mL of lysis buffercontaining 20mM Tris (pH 8) 1% TX-100 10% glycerol 137mM NaCl 5mM

as kinases related SRC, PLCG2, PIK3R1, adaptors LCK, CSK, FYN, GRB2, GRB10, transcription activity factors STATs and phosphoatases SHP1 and

spatial misregulation of RTKs leads to various cancers (3). Phospho-protein i h l d i h hi h h h h d h

containing 20mM Tris (pH 8), 1% TX-100, 10% glycerol, 137mM NaCl, 5mMsodium orthovanadate along with 1% phosphatase inhibitor cocktail and 1%

GRB10, transcription activity factors STATs and phosphoatases SHP1 and SHP2. Each pY peptide we synthesized in triplicate and a control sequence

enrichment coupled with high-throughput mass spectrometry methods have l d t t l i f th d f h t i h h difi ti

sodium orthovanadate along with 1% phosphatase inhibitor cocktail and 1%protesase inhibitor cocktail (Roche) for 1 hour at 4 degree C The cells are

S ac p pept de e sy t es ed t p cate a d a co t o seque cewhere the tyrosine phosphomotifs (pY) was replaced by an alanine (A). Thus

lead to cataloguing of thousands of such tyrosine phospho modifications on t i d i till di idl (4 6) B t d t di th f ti l

protesase inhibitor cocktail (Roche) for 1 hour at 4 degree C. The cells arecentrifuged at 14K RPM for 20 min to remove the DNA and cellular debris.

y p p (p ) p y ( )the pY PPEP array synthesis is similar to conventional peptide synthesis using

proteins and is still expanding rapidly (4-6). But understanding the functional significance of these phospho motifs on proteins in modulating signals that

centrifuged at 14K RPM for 20 min to remove the DNA and cellular debris.The protein supernatant is collected and filtered through a 0.2um filter and

p y y p p y gBoc chemistry (9-11).

significance of these phospho-motifs on proteins in modulating signals that directs cells to attain the abnormal cancerous state and metastasize is still

p p gthe filtered protein lysate is collected and aliquoted in to 500uL volume andSurface treatments: the PPEP array chip surface undergo stringent GRB2 RTK pY proteome interaction networks indirects cells to attain the abnormal cancerous state and metastasize is still

an enigma

p y qstored at minus 80 degree deep freezer. The concentration is determinedpretreatment for uniform, selective cell lysate protein binding. GRB2-RTK pY proteome interaction networks in

breast cancer cellsan enigma. gusing DC assay kit (BioRad) based on Lowry method.pY peptide-GRB2 Binding assay and Data Processing: After equilibration

i h i bi di b ff (PBB) d l i h bi ibreast cancer cells

Comprehensive maps of protein networks regulated by such phospho-motifs About 500 g of total protein was incubated with the appropriatewith protein binding buffer (PBB), we used total either recombinant protein (200 / L) ll l t (1 / L) t t d f ll f i ht

MCF10A MDAMB231MCF7T47DComprehensive maps of protein networks regulated by such phospho motifs will lead to identification of nodal signaling protein motifs and open up phosphospecific antibody for one hour at 4 ºC. Protein SEPHAROSE A/G(200ng/mL) or cell lysate (1mg/mL) extracted from cancer cells for overnight

bi di t 40C Thi f ll d b i tib d i b ti (1 h fwill lead to identification of nodal signaling protein motifs and open up avenues for better therapeutic intervention strategies. Using a set of breast beads (Dyna beads from invitrogen) were used to collect the antigen

tib d l f b t h I i it t thbinding at 40C. This was followed by primary antibody incubation (1 hr for recombinant protein at RT; overnight incubation for cell lysate) and secondaryp g g

cancer cells, we have generated a detailed map of interaction between antibody complexes for about one hour. Immunoprecipitates were thenh d th ti ith ld l i b ff P t i i th IP l

recombinant protein at RT; overnight incubation for cell lysate) and secondary antibody binding (1hr at RT) with washing (1 hour with PBB) between each Signature GRB2~Phosphoprotein complexes from Breast cancer cells. The , g p

phospho-motifs representing various RTK pathway proteins and an RTK washed three times with cold lysis buffer. Proteins in the IP complex wereanalyzed by resolving in 8 12% PAGE gels and blotting with appropriate

antibody binding (1hr at RT) with washing (1 hour with PBB) between each step to remove unbound reagents The chip is mounted on a chip holder and

g p p pwestern blot analyses demonstrate the direct GRB2 mediated and the indirect g y

adaptor protein GRB2. We also describe a novel high throughput tyrosine analyzed by resolving in 8-12% PAGE gels and blotting with appropriateantibody Signals were detected using the infra red dye conjugated

step to remove unbound reagents. The chip is mounted on a chip holder and scanned using the microchip array scanner Anon GenePix 4400A (Molecular

(non-GRB2 mediated) interactions observed on the phosphopeptide microarray

GF RTK t d b SH2 P t i N t kphosphopetide microarray platform (µParaflo® PepArray Microchip antibody. Signals were detected using the infra-red dye conjugated

secondary antibodies and signals detected using Odyssey IR imagescanned using the microchip array scanner Anon GenePix 4400A (Molecular devices) using the Genepixpro7 software sand the binding image obtained is GF RTKs connected by SH2 Protein Networkssystem) and its potential applications in biomarker and drug discovery. We secondary antibodies and signals detected using Odyssey IR image

analyzer (LICOR Biosciences)devices) using the Genepixpro7 software sand the binding image obtained is saved as TIFF file. y

have uncovered several novel interactions of tyrosine phospho-motifs with GRB2 i b t ll t

analyzer (LICOR Biosciences).saved as TIFF file.Using another software program called Array-Pro Analyzer and the numerical

GRB2 in breast cancer cell systems.

R ltg p g y y

pixel density values are obtained as text file (output data). The pixel data is

We find that several GRB2 phosphpoprotein interactions from our peptide Resultsp y ( p ) pmerged with layout file using an In-house micro-array analysis program

We find that several GRB2-phosphpoprotein interactions from our peptide array have been validated by previous studies We have many novel

Resultsg y g y y g(macros in excel) where the data is background subtracted. Based on several

R bi t SH2 t i bi diarray have been validated by previous studies. We have many novel interactions involving phospho motifs not reported so far that are worth

other parameters the significant data values are reported as detectables. Recombinant SH2 protein binding assayinteractions involving phospho-motifs not reported so far that are worth

further follow up studies The interaction dynamics as measured by theThe data is further analyzed for the biology of each protein that represent the p g yfurther follow up studies. The interaction dynamics as measured by the

amount of GRB2 bound to the phospho-motif was sufficient to distinguishphosphomotifs (pathway analysis, GO term analysis etc).

amount of GRB2 bound to the phospho motif was sufficient to distinguish the cellular signature of a non-tumorogenic mammary epithelial cell AA

SH2 domain Proteins (PPBD) pY bindingthe cellular signature of a non tumorogenic mammary epithelial cell (MCF10A) from breast cancer cells (MCF7, T47D and MDAMB231). These A B CA B CSH2 domain Proteins (PPBD) – pY binding(MCF10A) from breast cancer cells (MCF7, T47D and MDAMB231). These interaction signatures are diagnostic for each cancer type we analyzed. We g g yp yobserve a generic downregulation of phospho-proteome-GRB2 interaction g g p p pnetwork in the metastatic tumor cell MDA-MB-231 compared to tumorogenic S H 2

# A A M W F u n c t io n M o t if a

MCF7 and T47D cells. p ro te in s# A A M W F u n c t io n M o t if

Protein carton drawings with purple ellipse are SH2 proteinsZ A P -7 0 6 1 9 7 0 K D K in a s e

We have validated some of these interactions obtained from phospho-id i hi d i I ffi i i i i d blpeptide microchip data using Immuno-affinity precipitation and western blot l i O lt d t t th t i h h tif i l

G rb 2 2 1 7 2 5 K D A d a p to rD 45 ×10KED 45 ×10KEanalysis. Our results demonstrate that various phosphomotifs on a single

t i diff ti ll l t d d l th t ti l f t ti i lD

40

45pY peptidesA peptides

ED40

45pY peptidesA peptides

E Table 1. Examples of phosphomotif-Grb2 interactions observed on PPEP array protein are differentially regulated and reveal the potential of targeting single phospho motif for therapeutic intervention

B T K 6 5 9 7 6 K D K in a s e30

35

y

A peptides

30

35

y

A peptides p p p yare validated by what were reported in previous studies phospho-motif for therapeutic intervention.

20

25

tens

ity

20

25

tens

ity

y p pS rc 5 3 6 6 0 K D K in a s e

10

15

0

In

10

15

0

In

Ph h tif R t d P t i

Technology and Methods 5

10

5

10 Phosphomotifs interacting with GRB2 Sequence (N' to C') Swiss prot ID Reported Protein

interactions Reference

Technology and Methods Cancer cells for Protein Profiling 0

EKLSVQVPANIR

VNIVKSEGVSEVDGNHIM

VMTMFAEGAAMVEKLNLH

YNANIMVNASKGAENIH

L

0

EKLSVQVPANIR

VNIVKSEGVSEVDGNHIM

VMTMFAEGAAMVEKLNLH

YNANIMVNASKGAENIH

L CSF3R_787 yENLWF Q99062 SYK,SH3BP2 Hunter MG, et al. (2004), de Koning JP, et al. (1996)

®

gy Cancer cells for Protein Profiling

YCNEYLN

SVYENV

YGNIYKNIYENSEYVNVYANVDYMNHYHNVYHNMYENEGYDNAMYCNEYGNL

YINA

YQNMVYLN

ASYSVGA

YKN

P tid

YCNEYLN

SVYENV

YGNIYKNIYENSEYVNVYANVDYMNHYHNVYHNMYENEGYDNAMYCNEYGNL

YINA

YQNMVYLN

ASYSVGA

YKN

P tid

_DYST_959 yQNVLT Q03001 GRB2 not reportedEPOR 489 yENSLI P19235 LYN SH3BP2 Sulahian R Cleaver O Huang LJ (2009) Protein carton drawings with purple ellipse are SH2 proteins

Paraflo® PPEP Array SC ll li l t ER PR P53 C ll t C lt M diPeptidesPeptides EPOR_489 yENSLI P19235 LYN,SH3BP2, Sulahian R, Cleaver O, Huang LJ (2009)

ETV6_314 yMNHIM P41212 GRB2 Million, RP et al 2004FAK1 925 ENVTG Q05397 SRC STAT3 SHC1 GRBMit SK t l (2006) K d T t l (2008)Paraflo PPEP Array SummaryCell line gene cluster ER PR P53 Cell type Culture Medium

C WT /

FAK1_925 yENVTG Q05397 SRC,STAT3,SHC1,GRBMitra SK, et al. (2006) , Kaneda T, et al. (2008)FLT1_1213 yVNAFK P17948 PTPN11,GRB2,NCK1,P Ito N et al (1998, 2001) y

S f f fMCF10A basal B - - +/-WT Normal mammary epitheliumDMEM/F12 FLT3_768 yENQKR P36888 GRB2 Masson K, et al. (2009)

FLT3 955 yQNVDG P36888 GRB2 Masson K, et al. (2009)

μParaflo® Biochip Technology • Streamlined procedures are established for reproducible profiling of l t t i f ll lt ti l W d t t th t

MCF7 luminal + + +/-WT invasion ductal carcinoma DMEM, 10%FBS3_955 yQ G P36888 GRB2 , ( )

FLT3_969 yQNRRP P36888 GRB2 Masson K, et al. (2009) FRS2 196 yVNTTG Q8WU20 GRB2 Hadari YR et al (1998)μParaflo Biochip Technology lysate proteins from cell culture, tissue samples. We demonstrate that

d G b2 i ll t t f t i t iMDA-MB231 basal B - - ++WT adenocarcinoma DMEM, 10%FBSFRS2_196 yVNTTG Q8WU20 GRB2 Hadari YR et al (1998) GSTP1_199 yVNLPI P09211 GRB2 Okamura T, et al. (2009)

endogenous Grb2 is a an excellent reporter for protein or protein complexes binding to PPEP probes Differential profiling patterns were

,T47D luminal + + ++WT invasion ductal carcinoma RPMI 10%FBS

IRS1_47 yENEKK P35568 GRB2,SH3BP2 not reportedLAT 200 yVNVPE O43561 GRAP2,GRAP,VAV1,PLCZhang W, et al. (1998, 2000), Malbec O, et al. (2004) complexes binding to PPEP probes. Differential profiling patterns were

obtained for more than ten cancer cell linesT47D luminal + + ++ invasion ductal carcinoma RPMI, 10%FBS _ y , , , g ( ) ( )

LAT_220 yVNVSQ O43561 VAV1,GRB2,PLCG1,GR Malbec O, et al. (2004) LAX1 268 yVNMTG Q8IWV1 GRB2 PIK3R1 not reported obtained for more than ten cancer cell lines . LAX1_268 yVNMTG Q8IWV1 GRB2,PIK3R1 not reportedLAX1_294 yENVPA Q8IWV1 GRB2 not reported

Q8IWV1 GRB2 ( )

PGA chemistry and • By combining in silico analysis (PepArray pro) and binding experimentsPPEP Array binding assayLAX1_373 yENVLT Q8IWV1 GRB2 Mayya V.et al. (2009) NFAM1_220 yTALQR Q8NET5 SYK,ZAP70 Yang J.et.al (2003). Ohtsuka M et al. (2004)y

high-fidelity synthesis• By combining in silico analysis (PepArray pro) and binding experiments,

we systematically screened signature peptides/panels by using Grb2PPEP Array binding assay NTAL_136 yQNFSK Q9GZY6 GRB2 Iwaki S, et al. (2008)

NTAL 95 yENVLI Q9GZY6 GRB2 Iwaki S, et al. (2008) we systematically screened signature peptides/panels by using Grb2, BTK, Src and ZAP70 as PPBDs. Calibration with signature peptides of

NTAL_95 yENVLI Q9GZY6 GRB2 Iwaki S, et al. (2008)PDGFRB_716 ySNALP P09619 GRB2,GRB7 Arvidsson AK, et al. (1994) PILRA 246 ENIRN Q9UKJ1 PTPN11 PTPN6 t t d BTK, Src and ZAP70 as PPBDs. Calibration with signature peptides of

Grb2, we could detect Grb2 in cell lysates with sensitivity of low nM level. PPEPs are effectivePILRA_246 yENIRN Q9UKJ1 PTPN11, PTPN6 not reportedPTPN11_279 yKNILP Q06124 GRB2 Mitra S, et al (2008) , y yPPEPs are effective

probes for SH2PTPN11_546 yTNIKY Q06124 PTPN11,GRB2,FYN,SO Bennett AM, et al. (1994)PTPN11 584 yENVGL Q06124 PTPN11,GRB2,FYN Araki T et al (2003)

• Our binding data are consistent with what reported in the literature (Table probes for SH2 containing proteins

_ y Q06 ,G , ( )PTPRA_798 yANFK P18433 SRC,GRB2 Hao Q et al (2006); Chen M et al (2006)SHC1 427 yVNIQN P29353 GRB2 Ursini Siegel J et al (2008); Patrussi L et al (2005)

g p (1), thus PPEP profiling of proteins clearly demonstrates that these

His-

containing proteins SHC1_427 yVNIQN P29353 GRB2 Ursini-Siegel J, et al. (2008); Patrussi L, et al. (2005)SHIP1_556 yMNILR Q92835 GRB2,INPP5D CST curated data

Assay PPEPs are sensible and informative probes for cellular proteins, P P P

His-SH2

SOS1_974 yQNQPY Q07889 GRB2 not reprotedSPTAN1 2430 yQNLTR Q13813 GRB2 Pighi C, et al. (2011), Rikova K, et al. (2007)

µParaFlo™ microfluidic y

especially protein signaling complexes and indicate that these probes P P_

TNFL6_258 yVNVSE P48023 GRB2 not reportedXPO7 669 yTALGR Q9UIA9 PTPN11 PTPN6 not reportedµ

array chip to produce are active phosphomotifs in vivo and mediate protein interactions d t t d b GRB2 bi diPeptide

XPO7_669 yTALGR Q9UIA9 PTPN11,PTPN6 not reported

thousands peptides detected by GRB2 binding. Data indicate Direct and Indirect Grb2 mediated Peptide on chip Dye-labeled His-SH2 or dye-conjugated

The PPEP arra th s establishes a probe target platform for anal sis ofbinding of PPEPson chip y

antibodyy j g

secondary Ab• The PPEP array thus establishes a probe-target platform for analysis of

proteins in cellbinding of PPEPs

Digital Photolithography

Digital Photolithography

Digital proteins in cell. PhotolithographyPhotolithographyphotolithography

• Does the strength of binding affinity (quantitative intensity of GRB2GRB2 h h t i ll • Does the strength of binding affinity (quantitative intensity of GRB2 binding as measured by fluorescence) indicates the strength ofGRB2-phosphoproteome in cancer cells binding as measured by fluorescence) indicates the strength of phosphorylation of proteins represented by PPEPs

p p pphosphorylation of proteins represented by PPEPs .

• Working hypothesis to explain the peptide microarray data (GRB2 2

g yp p p p y (protein binding) of Breast cancer cells: High concentration of PPEPs Thousands of different peptides can be synthesized in situ on 1 cm2

i fl idi hi i t d d t B t ti i id dp g) g(probes) compete with low concentration motifs on phosphoproteins microfluidic chip using standard t-Boc protecting amino acids and a

photogenerated acid as deprotection reagent for the light gated parallel which holding the protein complex in vivo. Further proof for this photogenerated acid as deprotection reagent for the light-gated parallel synthesis (1 2)

assumption to be obtained from peptide based pull down assays from synthesis (1,2).

cell lysates.OO h Y P Y Q E

• The future direction is to identify all the proteins that bind to the h h tid b th hi Bi ti j t d tid b iBinding patterns and consensus sequences of BTK Grb2N CHC OR

OO

(CH3)3CN CHC OR

OO

(CH3)3C Solid Phase P tid S th i

h CD

YM

WD

NV

NT

phosphopeptide probes on the chip. Biotin conjugated peptides are being d i d t d ll d f ll l t d ID ll th t i

Binding patterns and consensus sequences of BTK, Grb2, Src and ZAP70

NH

CHCCH2

ORO NH

CHCCH2

ORO Peptide SynthesisCyclePGA-P

EG

KNE

RG

YTR

VYL designed to do pull down assay from cell lysates and ID all the proteins

by mass spectrometrySrc and ZAP70.C 2

N

C 2

N

CyclePGA P K E S R L

Light activated by mass spectrometry. A. Binding profiles of 4 SH2 proteins on the same region of N

NHN

NH

Light activated acid deprotection

A k l d tg p p g

SH2-PPEP microarray. B. Heatmap of binding intensities of 4 NHNH

Peptides on a C S f

Standard

AcknowledgementsSH2 PPEP microarray. B. Heatmap of binding intensities of 4 SH2 proteins to related PPEPs. The binding signals were

Chip Surfacet-Boc AA gThi h i t d b t f NIH/NCI th Cli i l

SH2 proteins to related PPEPs. The binding signals were normalized into Z values The PPEPs with Z values ranked fromDigital light pattern This research is supported by grants from NIH/NCI, the Clinical

P i T h l f C (R33 CA126209) W h k D

normalized into Z values. The PPEPs with Z values ranked from 1 0-3 0 were selected to plot the heat map C Consensus

Digital light pattern

Proteomics Technology for Cancer (R33 CA126209). We thank Dr. 1.0 3.0 were selected to plot the heat map. C. Consensus sequences of binding sequences of 4 SH2 proteins The Heat map of log2 transformed binding

Xiaolin Zhang (Atactic Technologies) for synthesis instrument support.sequences of binding sequences of 4 SH2 proteins. The consensus sequences were generated by using Weblogoh h h h

V- Phosphopeptide binding of protein complexes in cells. Total eat ap o og t a s o ed b d g

Signals) from breast cells showR f

consensus sequences were generated by using Weblogo (http://weblogo berkeley edu/logo cgi )id l bilid l bil

V

PGA P/h H GH GGHHS Y Y SYYS S

Gp p p g p p

protein is isolated from cultured cells and is passed over theSignals) from breast cells show increasing signals for detected proteins References(http://weblogo.berkeley.edu/logo.cgi ). H+ H+acid labile

protecting group H+ H+acid labile protecting group

G

PGA-P/h S Y Y SYYSVI VIG G

SII

protein is isolated from cultured cells and is passed over the chip through microfluidics based circulation at 4 degree C for

increasing signals for detected proteins in T47D and MCF7 tumor cellsH H H H

PGA PG- IV GV V VG I

SH2 domain protein Our study Previous studies Refchip through microfluidics based circulation at 4 degree C for overnight Antibody based detection is used to identify the

in T47D and MCF7 tumor cellsNH NH NH NH NH NH NH NHNH NH NH NH NH NH NH NH NH NH NH NHNH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NHPGA-P … SH2 domain protein Our study Previous studies Ref

GRB2 Y E/V/Q/K N V/I/L Y E/Q N A/V/I/L/M/F/Y/W K l t l 2002overnight. Antibody based detection is used to identify the

t i f i t t th l B d i i1. Hunter T. Tyrosine phosphorylation: thirty years and counting. Curr Opin Cell Biol. 2009

GRB2 pY-E/V/Q/K-N-V/I/L pY-E/Q-N-A/V/I/L/M/F/Y/W Kessels et al 2002SRC Y E/S/A/T E/T/L/S L/I/V D/V F/L Y E/D/T E/N/Y L/I H t l 2008

protein of interest on these complexes. Based on in vivo SHC1 pY motif signal transductiony p p y y y g p

Apr;21(2):140-6. Epub 2009 Mar 9. Review.

(a)(a) (b) (c) SRC pY-E/S/A/T-E/T/L/S-L/I/V-D/V-F/L pY-E/D/T-E/N/Y-L/I Huang et al 2008/ /S / / / / / / / / / / / / / / / / / / /

substrate affinity of a specific phosphoprotein with its binding p g

in breast tumor cells 2. Pawson T. Specificity in signal transduction: from phosphotyrosine-SH2 domain interactions to complex cellular systems Cell 2004;116:191 203

Mi hi d i Ph h id i hiBTK pY-A/E/S-D/M/L/I-N/E/V/D-L/V/P/I/M pY-E/D/M/L/I-E/D/I-I/P/M/L Tzeng et al 2000protein, in vivo protein complexes bound to phospho-peptides

in breast tumor cells complex cellular systems. Cell 2004;116:191–203.

3 Choudhary C Olsen JV Brandts C et al (2009) Mislocalized activation of oncogenic RTKsMicroarray chip design: Phospho-peptide microarray chips ZAP70 pY-S/A/T-S/A/F/T-L pY-E-N-V/L-D Huang et al 2008p , p p p p p p(pY) on the chip from the pool of non-denatured total proteins

3. Choudhary C, Olsen JV, Brandts C, et al. (2009) Mislocalized activation of oncogenic RTKs switches downstream signaling outcomes. Mol Cell. 2009 Oct 23;36(2):326-39.

were designed using Peparray pro B d th l i f 100 PPEP ith G b2 SH2

(pY) on the chip from the pool of non denatured total proteins isolated from cells could be detected

g g ; ( )

4. Pawson T. (1995) Protein modules and signalling networks. Nature. 373(6515):573-80. g g p y p(http://www.pepcyber.org/PepArray/) program hosted by our Based on the analysis of over 100 PPEPs with Grb2 SH2

hi h bi di i l f d h f GRB2 SH2 d iisolated from cells could be detected. ( ) g g ( )

Review(http://www.pepcyber.org/PepArray/) program hosted by our lab (7) The SH2 protein array used a total of 1226 tyrosine high binding signals, we found that for GRB2 SH2 domain 5. Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, et al. (2006) Global, In Vivo, and Site-

S ifi Ph h l ti D i i Si li N t k C ll 127 635 648lab (7). The SH2 protein array used a total of 1226 tyrosine phosphomotifs (PPEPs) representing phosphomotifs from 423 I t t d l tf f h h tid i

the binding motif the consensus of phosphopeptides is pY- Specific Phosphorylation Dynamics in Signaling Networks. Cell 127: 635-648.

6 R h J M it A L KA G A G VL t l (2005) I ffi it fili f t iphosphomotifs (PPEPs) representing phosphomotifs from 423 t i di ti 2455 i t ti f P C b P PEP (8)

Integrated platform of phosphopeptide microarrayg p p p p p

E/V/Q/K-N-V/I/L. As per previous studies (12), this binding6. Rush J, Moritz A, Lee KA, Guo A, Goss VL, et al. (2005) Immunoaffinity profiling of tyrosine

phosphorylation in cancer cells Nature Biotechnol 23(1): 94-101proteins mediating 2455 interaction from PepCyber:P~PEP (8). E/V/Q/K N V/I/L. As per previous studies (12), this binding motif is similar with the reported (pY-E/Q-N-ψ (ψ represents

phosphorylation in cancer cells. Nature Biotechnol 23(1): 94 101.

7 Li T Zuo Z Zhu Q Hong A Zhou X Gao X (2009) Web-based design of peptideThe RTK pathway array peptides were collected by mining motif is similar with the reported (pY E/Q N ψ (ψ represents a hydrophobic residues) Based on our results and those

7. Li, T., Zuo, Z., Zhu, Q., Hong, A., Zhou, X., Gao, X. (2009) Web based design of peptide microarrays using mPepArray Pro. Invited review in “Peptide Microarrays” in Methods and

phosphoprotein data from various protein databases a hydrophobic residues). Based on our results and those f lit t th i i id t P+2 i ti l

Protocols. 570, 391-402. Ed. Cretich, M.p p p p(PepCyber, Phopho.ELM, PhosphoSite etc.) containing a total from literatures, the asparigine residue at P+2 is essential

f G S8. Gong, W., Zhou, D., Ren, Y., Wang, Y., Zuo, Z., Shen, Y., Xiao, F., Zhu, Q., Hong, A., Zhou, Z.,

Gao X and Li T (2008) PepCyber:P~PEP : A database of human protein-protein(PepCyber, Phopho.ELM, PhosphoSite etc.) containing a total number of 191 proteins with 644 tyrosine phosphor-motifs for Grb2 SH2 binding, whereas that selectivity at P+1 and Gao, X., and Li, T. (2008) PepCyber:P~PEP : A database of human protein-protein

interactions mediated by phosphoprotein binding domains. Nucleic Acids Res. 36, D679-D683.number of 191 proteins with 644 tyrosine phosphor-motifs. These include 52 RTKs 28 cytoplasmic TKs 32 signaling P+3 is apparent, but less stringent. These three amino acid

U l ti f SHP 2 Y tif i t ti ith GRB29. Gao, X., Pellois, J. P., Na, Y., Kim, Y., Gulari, E., Zhou, X. (2004) High density peptide These include 52 RTKs, 28 cytoplasmic TKs, 32 signaling

d t t i d 82 th d t i li t i

pp , gpositions at the C-terminal of pY are sufficient for optimal Upregulation of SHP-2 pY motif interaction with GRB2

i T4 D d MCF llmicroarrays. In situ synthesis and applications. Mol Divers 8, 177-87.

adaptor proteins and 82 other downstream signaling proteins. positions at the C terminal of pY are sufficient for optimal Grb2 SH2 binding with high affinity and specificity The inT47D and MCF7 cells 10. Pellois, J. P., Zhou, X., Srivannavit, O., Zhou, T., Gulari, E., Gao, X. (2002 Individually

addressable parallel peptide synthesis on microchips Nat Biotechnol 20 922 6Grb2 SH2 binding with high affinity and specificity. The peptide signature for other proteins (SRC BTK and ZAP70)

addressable parallel peptide synthesis on microchips. Nat Biotechnol 20, 922-6.

11 Zhu Q Hong A Sheng N Zhang X Jun K Y Srivannavit O Gulari E Gao X and• A SH2 domain interacting

peptide signature for other proteins (SRC, BTK and ZAP70) l f lt f i t di (13) U i

11. Zhu, Q., Hong, A., Sheng, N., Zhang, X., Jun, K.-Y., Srivannavit, O., Gulari, E., Gao, X., and Zhou, X. (2007) “Microfluidic biochip for nucleic acid and protein analysis” in Methods Mol. • A SH2 domain interacting

phosphopeptide (SH2-PPEP)also conforms results from previous studies (13). Using , ( ) p p y

Biol. 382, 287-312, Microarrays. Vol. I. Ed. Rampal, J. B.phosphopeptide (SH2-PPEP) microarray was designed µParaflo® PepArray Microchip system, we not only 12. Kessels HW, Ward AC, Schumacher TN (2002) Specificity and affinity motifs for Grb2 SH2-

li d i t ti PNAS 99(13) 8524 9microarray was designed. detected experimentally proven interactions but also ligand interactions. PNAS 99(13):8524-9.

13 H H Li L W C S hibli D C l ill K M S Li C R P H K S Z P T• The PPEPs were from PepCyber p y p

validated many predicted interactions13. Huang H, Li L, Wu C, Schibli D, Colwill K, Ma S, Li C, Roy P, Ho K, Songyang Z, Pawson T,

Gao Y, Li SS. (2008) Defining the specificity space of the human SRC homology 2 domain.p y~PPEP (www.pepcyber.org).

validated many predicted interactions Gao Y, Li SS. (2008) Defining the specificity space of the human SRC homology 2 domain. Mol Cell Proteomics. 7(4):768-84.( p p y g)

Th PPEP i th i • By using four recombinant SH2 proteins specific and 14. Mitra SK, et al. (2006) Intrinsic FAK activity and Y925 phosphorylation facilitate an angiogenic • The PPEPs in the microarray

k b t t f SH2• By using four recombinant SH2 proteins, specific and

distinct binding profiles were obtainedswitch in tumors. Oncogene 25, 5969-84.

are known substrates of SH2 distinct binding profiles were obtained.Th i t t ith th

15. Ursini-Siegel J, et al. (2008) ShcA signalling is essential for tumor progression in mouse models of human breast cancer EMBO J 27 910-20domains • The consensus sequences are consistent with those models of human breast cancer. EMBO J 27, 910-20

Contact Information• Statistics of SH2-PPEP reported in literatures. Contact InformationXi li G U i it f H t @ h d

• Statistics of SH2-PPEP interactions • Recombinant binding assays can be used as

Xiaolian Gao, University of Houston, xgao@uh.edu; Xi h Zh LC S i h @l i

interactions g y“reference” to calibrate the cell lysate binding Interaction of GRB2 with all three pY motifs are upregulated in tumor cells (MCF7 Xiaochuan Zhou, LC Sciences, xzhou@lcscienes.com; reference to calibrate the cell lysate binding. Interaction of GRB2 with all three pY motifs are upregulated in tumor cells (MCF7

and T47D) but significantly down-regulated in MCF10A and MDA-MB231 cells.Srini Krishnamoorthy, University of Houston, ksriniva@central.uh.edu;

) g y g