SUPPLEMENTARY INFORMATION

Transcription

1 doi:.8/nature8 Zheng et al. Temporal regulation of EGF signaling networks by the scaffold protein Shc SUPPLEMENTARY TEXT EGF concentration selection and optimization EGF levels vary greatly in various bodily fluids, from low ( ng/ml) in plasma, serum, and saliva to medium ( ng/ml) in tears, follicular fluid, sperm, and seminal plasma, to high ( ng/ml) in bile, urine, milk, and prostate fluid -. To select an optimal EGF concentration for Shc signaling analysis by smrm, we stimulated Rat- fibroblasts with increasing amounts of EGF and quantified the levels of Shc phosphopeptides and Shc-interacting proteins by smrm. The results confirmed that the measured Shc-interaction network is EGF-dependent. In general, both Shc phosphorylation and levels of interacting proteins showed typical Michaelis Menten kinetics with a saturating concentration of ng/ml (Supplementary Fig. ), which is on the high end of physiological concentrations. Different concentrations of EGF can result in different kinetics of protein interactions and physiological responses. Indeed, stimulating cells with ng/ml or ng/ml EGF resulted in slower rates of protein-protein interactions and phosphorylation, yet the sequence of Shc phosphorylation and the nature and kinetics of Shc protein interactions were very similar to those observed upon stimulation with ng/ml (data not shown). However, stimulating cells with very low EGF doses only allowed us to detect a subset of Shc-interacting proteins, likely because the signal intensity for some binders was out of the sensitivity range of our smrm assay. Thus, to ensure reproducibility across different biological repeats as

2 RESEARCH well as the linearity of our quantification, we chose to use ng/ml EGF in all subsequent studies. Reference Westergaard, L. G. et al. Epidermal growth factor in small antral ovarian follicles of pregnant women. J Endocrinol, 6-6 (99). Richards, R. C. et al. Epidermal growth factor receptors on isolated human placental syncytiotrophoblast plasma membrane. Placenta, -8 (98). Grau, M. et al. Relationship between epidermal growth factor in mouse submandibular glands, plasma, and bile: effects of catecholamines and fasting. Endocrinology, 8-86 (99). Rationale for using Rat- fibroblasts as the model system Although Rat- fibroblasts are immortalized, they are still considered to be phenotypically normal, non-transformed cells for studying receptor tyrosine kinase signaling. They have previously been shown to respond to EGF simulation -8. We also chose Rat- fibroblasts for a number of technical reasons. Firstly, Rat- fibroblasts are highly transfectable and easy to handle in large-scale tissue culture. More importantly, Rat- fibroblasts provide much higher concentration of proteins compared to MEFs, allowing for a higher coverage of the Shc protein interaction network and better data quality for smrm quantifications. Finally, the smrm assay we have designed using Rat- fibroblasts can be easily applied to mouse cell lines

3 RESEARCH due to high sequence homology between rat and mouse, facilitating later studies in the various MEF cell lines that we have constructed. References Nicholson, P.R. et al. ShcA tyrosine phosphorylation sites can replace ShcA binding in signalling by middle T-antigen. EMBO J. :6-66 (). Wu, J. et al. Inhibition of the EGF-activated MAP kinase signaling pathway by adenosine ','-monophosphate. Science 6:6-69 (99). 6 Qiu, R.G.et al. An essential role for Rac in Ras transformation. Nature :- 9 (99). Haley, J.D. et al. Analysis of mammalian fibroblast transformation by normal and mutated human EGF receptors. Oncogene :-8 (989). 8 Kim, B.C. et al. Rac GTPase activity is essential for EGF-induced mitogenesis. Mol Cells 8:9-9 (998). Choosing Shc normalizing peptides for data normalization The abundance of proteins and phosphopeptides measured by smrm was normalized to the protein level of Shc using five Shc normalizing peptides as references. Ideally, peptides without any post-translational modifications should be used as references for normalization. However, due to limited availability of such peptides, we used Shc peptides with Met, Trp, or His residues, which may undergo oxidation (peptide,, and in Supplementary Fig. ). To increase the accuracy of smrm quantification, we measured the major oxidized forms, as well as unmodified forms of these peptides in all experiments. For data normalization, we then used the summed extracted ion chromatograms (XICs) for any given Shc peptide as the reference.

4 RESEARCH Phosphorylation on Shc Y9/ motif We only detected the Y9/ motif of Shc as a singly phosphorylated species in our survey scans due to the weak ion intensity of the corresponding peptide. In order to determine if this motif can be doubly phosphorylated, we specifically targeted the doubly-phosphorylated form by smrm using electronically predicted MS properties and confirmed the presence of both singly and doubly phosphoforms only in EGF-stimulated samples. This observation may represent different activation states, as indicated by differentially phosphorylated forms of Shc during signal transduction. The temporal profile of the doubly phosphorylated Y9/ site is similar to that of the singly phosphorylated form, although higher background interference was observed due to low signal to noise ratio. Therefore, the subsequent analyses were conducted on the singly phosphorylated Y9/ motif (termed py9/).

5 RESEARCH Time control of immunoprecipitation experiments Analysis of signaling complexes by mass spectrometry typically relies on immunoprecipitations, as is the case for our study. Given the dynamic nature of protein interactions, proteins often associate and disassociate from a complex during isolation in vitro, making the final composition of immunoprecipitates dependent on the amount of time the proteins in the complex had to exchange during cell lysate processing. As a result, there may be a subset of proteins that are lost or gained during our analysis. However, this issue should not affect our comparison between samples since each set of samples were processed at the same time under the same conditions. To further ensure consistency and minimize potential artifacts, we implemented several quality control steps in our experimental protocol including a defined 6-hour antibody incubation, timed buffer washes and a consistent amount of antibody. Technical and biological replicates For each MRM quantification experiment, we used - technical replicates, depending on the amount of available sample. We also performed a minimum of three biological replicates for each experiment. The error bars shown in the results were standard deviations from technical replicates within one biological experiment.

6 RESEARCH SUPPLEMENTARY TABLES Supplementary Table EGF-induced phosphorylation in Shc-mediated signaling networks. Phosphorylated residues are highlighted in red. Novel sites are marked by asterisks. Residue numbers are based on Rat sequence. Protein Residue Position Sequence Novel Binding partners Upstream kinase Shc Serine 9 HGSFVNKPTR Ptpn Shc Threonine LVTPHDR * Unknown Shc Tyrosine ELFDDPSYVNIQNLDK Grb Shc Tyrosine 9 / MAGFDGSAWDEEEEEPPDHQYYNDFPGK Grb Shc Serine QAGGGAGPPNPSLNGSAPR * Unknown Egfr Tyrosine RPAGSVQNPVYHNQPLHPAPGR Grb, Egfr Threonine 69 ELVEPLTPSGEAPNQAHLR Unknown Erk Egfr Tyrosine GSHQMSLDNPDYQQDFFPK Shc Src 6 Egfr Tyrosine 96 GPTAENAEYLR Shp Erbb Tyrosine DVFAFGGAVENPEYLVPR Crk 8 Src 6 Gab Serine VTSVSGESGLYNLPR Unknown Gab Serine 9 SYSHDVLPK Unknown 9 Gab Threonine SNTISTVDLNK Unknown 9 Gab Serine 6 SSSLEGFHNQLK Unknown Gab Tyrosine 6 QVEYLDLDLESGK Ptpn Gab Tyrosine 686 VDYVVVDQQK Ptpn Gab Serine 8 NVLAAGNVSGEELDENYVPMNPNSPPR Unknown 9 Erk 9 Gab Serine 8 VKPAPLEIKPLSEWEELQAPVRSPITR Unknown 9 Erk 9 Gab Serine SSPAEFSSSQHLLR Unknown Gab Tyrosine 6 KPSTSSVTSDEKVDYVQVDKEK Shp 9 Sos Serine 96 TSISDPPESPPLLPPREPVR Unknown Erk, Sos Serine 6 HLPSPPLTQEVDLHSIAGPPVPPR Unknown Erk, 6

7 RESEARCH References for Supplementary table. Faisal A, et al. Serine/threonine phosphorylation of ShcA. Regulation of protein-tyrosine phosphatase-pest binding and involvement in insulin signaling. J Biol Chem., - ().. van der Geer P, et al. The Shc adaptor protein is highly phosphorylated at conserved, twin tyrosine residues (Y9/) that mediate protein-protein interactions. Curr Biol. 6, - (996).. Okutani T, et al. Grb/Ash binds directly to tyrosines 68 and 86 and indirectly to tyrosine 8 of activated human epidermal growth factor receptors in intact cells. J Biol Chem. 69, - (99).. Xin Li, et al. ERK-dependent threonine phosphorylation of EGF receptor modulates receptor downregulation and signaling. Cell Signal., (9).. A G Batzer, et al. Hierarchy of binding sites for Grb and Shc on the epidermal growth factor receptor. Mol Cell Biol., 9 (99). 6. Lombardo CR, et al. In vitro phosphorylation of the epidermal growth factor receptor autophosphorylation domain by c-src: identification of phosphorylation sites and c-src SH domain binding sites. Biochem., (99).. Keilhack H, et al. Phosphotyrosine mediates binding of the protein-tyrosine phosphatase SHP- to the epidermal growth factor receptor and attenuation of receptor signaling. J Biol Chem., 89-6 (998). 8. Dankort D, et al. Multiple ErbB-/Neu Phosphorylation Sites Mediate Transformation through Distinct Effector Proteins. J Biol Chem. 6, 89-8 (). 9. Lehr S, et al. Identification of major ERK-related phosphorylation sites in Gab. Biochem., - ().. Cunnick JM, et al. Phosphotyrosines 6 and 69 of Gab constitute a bisphosphoryl tyrosine-based activation motif (BTAM) conferring binding and activation of SHP. J Biol Chem. 6, 8- ().. Corbalan-Garcia S, et al. Identification of the mitogen-activated protein kinase phosphorylation sites on human Sos that regulate interaction with Grb. Mol Cell Biol. 6, 6-8 (996).. Yuji Kamioka, et al. Multiple Decisive Phosphorylation Sites for the Negative Feedback Regulation of SOS via ERK. J Biol Chem. 8, 8 ().

8 RESEARCH Supplementary Table List of identified EGF-dependent Shc-interacting proteins targeted by smrm. Novel interactors are marked by asterisks. Protein Accession MW Function Novel Apa O99 96 Endocytosis Aps P68 8 Endocytosis Arhgef Q 68 Rho GEF * Asap Q9 9 Arf GAP * Cblb QVWQ8 6 E ubiquitin-protein ligase * Dabip O 6 Ras GAP * Egfr P Receptor tyrosine kinase Erbb Q8 666 Receptor tyrosine kinase Erbb Q9UQC 8 Receptor tyrosine kinase Errfi Q9H6 986 Negative regulator of Egfr family protein * Fam9a P699 6 Grb binding protein * Gab P66 9 Scaffold protein Gab P Scaffold protein Grb P6 8 Adaptor Lrrk P Serine/threonine kinase * Pikca O9 8 Lipid kinase Pikr P66 Lipid kinase subunit Pikr Q9 886 Lipid kinase subunit Pppca Q9UJF 88 Serine/threonine phosphatase * Pppcg Q86YV 9 Serine/threonine phosphatase * Ptpn Q9H9 96 Tyrosine phosphatase Ptpn P9 68 Protein tyrosine phosphatase Rasal QTFU9 9 Ras GAP * SgK Q8NEM 69 Rnd effector * SgK69 O 899 Atypical protein kinase * Shcbp Q6 686 SH domain binding Ship Q889 6 Lipid phosphatase Sos Q89 99 Ras GEF Sos Q8SD 9 Ras GEF 8

9 RESEARCH Supplementary Table smrm transitions used in this study. Q - mass-to-charge ratio (m/z) for the peptide ion selected in Q; Q - m/z for the fragment ion selected in Q; CE - collision energy; [Oxi] - oxidation; [Pho] - phosphorylation; [Dea] - deamination; y - y ion; b - b ion. Protein Peptide sequence Q Q CE RT Fragment Shc HLLLVDPEGVVR y Shc HLLLVDPEGVVR y6 Shc HLLLVDPEGVVR b6 Shc HLLLVDPEGVVR y6 Shc H[Oxi]LLLVDPEGVVR y Shc H[Oxi]LLLVDPEGVVR y6 Shc VMGPGVSYLVR y8 Shc VMGPGVSYLVR y9 Shc VM[Oxi]GPGVSYLVR y8 Shc VM[Oxi]GPGVSYLVR y9 Shc VEGGQLGGEEWTR y Shc VEGGQLGGEEWTR y8 Shc VEGGQLGGEEWTR y Shc VEGGQLGGEEWTR y6 Shc VEGGQLGGEEW[oxi]TR y Shc VEGGQLGGEEW[oxi]TR y8 Shc VEGGQLGGEEW[oxi]TR y Shc VEGGQLGGEEW[oxi]TR y8 Shc EAEALLQLNGDFLVR y Shc EAEALLQLNGDFLVR y Shc EAEALLQLNGDFLVR y9 Shc ALDFNTR y Shc ALDFNTR y Shc HGS[Pho]FVNKPTR y Shc HGS[Pho]FVNKPTR b6-98 Shc HGS[Pho]FVNKPTR b-98 Shc HGSFVNKPTR b6 Shc HGSFVNKPTR b Shc HGSFVNKPTR y8 Shc HGSFVNKPTR y9 Shc ELFDDPSY[Pho]VNIQNLDK y Shc ELFDDPSY[Pho]VNIQNLDK b8 Shc ELFDDPSY[Pho]VNIQNLDK y8 Shc ELFDDPSY[Pho]VNIQNLDK y Shc ELFDDPSY[Pho]VNIQNLDK y Shc ELFDDPSY[Pho]VNIQNLDK y8 Shc ELFDDPSYVNIQNLDK y Shc ELFDDPSYVNIQNLDK y Shc ELFDDPSYVNIQNLDK y8 Shc ELFDDPSYVNIQNLDK y Shc ELFDDPSYVNIQNLDK y8 Shc ELFDDPSYVNIQNLDK b8 9

10 RESEARCH Shc LVT[Pho]PHDR y-98 Shc LVT[Pho]PHDR y6-98 Shc LVT[Pho]PHDR y Shc LVT[Pho]PHDR y6-98 Shc LVT[Pho]PHDR y Shc LVTPHDR y Shc LVTPHDR y Shc LVTPHDR y Shc QAGGGAGPPNPS[Pho]LN[Dea]GSAPR y-98 Shc QAGGGAGPPNPS[Pho]LN[Dea]GSAPR y9 Shc QAGGGAGPPNPS[Pho]LN[Dea]GSAPR y9-98 Shc [PGQ]QAGGGAGPPNPS[Pho]LN[Dea]GSAPR y-98 Shc [PGQ]QAGGGAGPPNPS[Pho]LN[Dea]GSAPR y9 Shc [PGQ]QAGGGAGPPNPS[Pho]LN[Dea]GSAPR y9-98 Shc QAGGGAGPPNPSLN[Dea]GSAPR y Shc QAGGGAGPPNPSLN[Dea]GSAPR y9 Shc QAGGGAGPPNPSLN[Dea]GSAPR y Shc QAGGGAGPPNPSLN[Dea]GSAPR y9 Shc [PGQ]QAGGGAGPPNPSLN[Dea]GSAPR y Shc [PGQ]QAGGGAGPPNPSLN[Dea]GSAPR y9 Shc MAGFDGSAWDEEEEEPPDHQY[Pho]Y[Pho]NDFPGK y Shc MAGFDGSAWDEEEEEPPDHQY[Pho]Y[Pho]NDFPGK y Shc MAGFDGSAWDEEEEEPPDHQYY[Pho]NDFPGK y(+) Shc MAGFDGSAWDEEEEEPPDHQYY[Pho]NDFPGK y Shc MAGFDGSAWDEEEEEPPDHQYY[Pho]NDFPGK y(+) Shc MAGFDGSAWDEEEEEPPDHQYY[Pho]NDFPGK y Shc MAGFDGSAWDEEEEEPPDHQYYNDFPGK y Shc MAGFDGSAWDEEEEEPPDHQYYNDFPGK y(+) Shc MAGFDGSAWDEEEEEPPDHQYYNDFPGK y Shc MAGFDGSAWDEEEEEPPDHQYYNDFPGK y(+) Shc ESTTTPGQYVLTGLQSGQPK y8 Shc ESTTTPGQYVLTGLQSGQPK y9 Shc ESTTTPGQYVLTGLQSGQPK y8 Shc ESTTTPGQYVLTGLQSGQPK y9 Sos DGNSVIFSAK y Sos DGNSVIFSAK y Sos TEEGNPEVLR y Sos TEEGNPEVLR y8 Sos EYIQPVQLR y Sos EYIQPVQLR y6 Sos TFLTTYR y Sos TFLTTYR y Sos FEIPEPEPTEADR y8 Sos FEIPEPEPTEADR y Sos TSISDPPES[Pho]PPLLPPREPVR y(+) Sos TSISDPPES[Pho]PPLLPPREPVR y-pho(+) Sos TSISDPPES[Pho]PPLLPPREPVR y(+) Sos HLPSPPLTQEVDLHSIAGPPVPPR y Sos HLPSPPLTQEVDLHSIAGPPVPPR y8 Sos HLPS[Pho]PPLTQEVDLHSIAGPPVPPR y Sos HLPS[Pho]PPLTQEVDLHSIAGPPVPPR y

11 RESEARCH Gab NVLAAGNVSGEELDENYVPMNPNSPPR y9 Gab NVLAAGNVSGEELDENYVPMNPNSPPR y9(+) Gab NVLAAGNVSGEELDENYVPMNPNSPPR y Gab NVLAAGNVSGEELDENYVPMNPNS[Pho]PPR y9 Gab NVLAAGNVSGEELDENYVPMNPNS[Pho]PPR y(+) Gab NVLAAGNVSGEELDENYVPMNPNS[Pho]PPR y Gab VKPAPLEIKPLSEWEELQAPVRS[Pho]PITR y8(+) Gab VKPAPLEIKPLSEWEELQAPVRS[Pho]PITR y9-pho(+) Gab SNT[Pho]ISTVDLNK y Gab SNT[Pho]ISTVDLNK y6 Gab SNTISTVDLNK y Gab SNTISTVDLNK y9 Gab SYS[Pho]HDVLPK b-pho Gab SYS[Pho]HDVLPK y9-pho(+) Gab SYS[Pho]HDVLPK y8-pho(+) Gab SYS[Pho]HDVLPK y-pho(+) Gab SYSHDVLPK y8(+) Gab SYSHDVLPK y(+) Gab VDY[Pho]VVVDQQK y Gab VDY[Pho]VVVDQQK b Gab VDYVVVDQQK y6 Gab VDYVVVDQQK y Gab QVEY[Pho]LDLDLESGK y8 Gab QVEY[Pho]LDLDLESGK y Gab QVEY[Pho]LDLDLESGK y9 Gab QVEYLDLDLESGK y8 Gab QVEYLDLDLESGK y Gab QVEYLDLDLESGK y9 Gab SSS[Pho]LEGFHNQLK y-pho(+) Gab SSS[Pho]LEGFHNQLK y9 Gab SSS[Pho]LEGFHNQLK y-pho(+) Gab SSS[Pho]LEGFHNQLK y Gab SSS[Pho]LEGFHNQLK y-pho(+) Gab SSSLEGFHNQLK y(+) Gab SSSLEGFHNQLK y Gab VTS[Pho]VSGESGLYNLPR y Gab VTS[Pho]VSGESGLYNLPR y8 Gab VTSVSGESGLYNLPR y Gab VTSVSGESGLYNLPR y8 Gab LTGDPDVLEYYK y8 Gab LTGDPDVLEYYK y Gab HVSISYDIPPTPGNTYQVPR y9 Gab HVSISYDIPPTPGNTYQVPR b8 Gab TFPEGTLGQSSK y Gab TFPEGTLGQSSK y8

12 RESEARCH Grb ESESAPGDFSLSVK y9 Grb ESESAPGDFSLSVK y Grb FNSLNELVDYHR y8 Grb FNSLNELVDYHR y Grb FNSLNELVDYHR y Grb FNSLNELVDYHR y Grb DIEQVPQQPTYVQALFDFDPQEDGELGFR y Grb DIEQVPQQPTYVQALFDFDPQEDGELGFR b Grb N[Dea]YVTPVNR y Grb N[Dea]YVTPVNR y6 Grb FGNDVQHFK y Grb FGNDVQHFK y8(+) Grb ATADDELSFK y8 Grb ATADDELSFK y Egfr GPTAENAEY[Pho]LR y Egfr GPTAENAEY[Pho]LR y6 Egfr GPTAENAEYLR y6 Egfr GPTAENAEYLR y Egfr RPAGSVQNPVY[Pho]HNQPLHPAPGR y Egfr RPAGSVQNPVY[Pho]HNQPLHPAPGR b8 Egfr RPAGSVQNPVYHNQPLHPAPGR y Egfr RPAGSVQNPVYHNQPLHPAPGR b8 Egfr ELVEPLT[Pho]PSGEAPNQAHLR y Egfr ELVEPLT[Pho]PSGEAPNQAHLR y Egfr ELVEPLTPSGEAPNQAHLR y Egfr ELVEPLTPSGEAPNQAHLR y Egfr GSHQMSLDNPDY[Pho]QQDFFPK b9 Egfr GSHQMSLDNPDY[Pho]QQDFFPK y8 Egfr GSHQM[Oxi]SLDNPDY[Pho]QQDFFPK b9 Egfr GSHQM[Oxi]SLDNPDY[Pho]QQDFFPK y8 Egfr GSHQMSLDNPDYQQDFFPK b9 Egfr GSHQMSLDNPDYQQDFFPK y8 Egfr YLVIQGDER y Egfr YLVIQGDER y6 Egfr NLQEILIGAVR y8 Egfr NLQEILIGAVR y9 Egfr ELILEFSK y Egfr ELILEFSK y6 Egfr VLGSGAFGTVYK y Egfr VLGSGAFGTVYK y8 Ptpn INAAEIESR y Ptpn INAAEIESR y Ptpn FDSLTDLVEHYK y8 Ptpn FDSLTDLVEHYK y6 Ptpn ESAAHDYTLR y Ptpn ESAAHDYTLR y8(+) Ptpn ESAAHDYTLR y Ptpn ESAAHDYTLR y6 Ptpn IQNTGDYYDLYGGEK y8 Ptpn IQNTGDYYDLYGGEK y Ptpn IQNTGDYYDLYGGEK y9

13 RESEARCH Ptpn ESQSHPGDFVLSVR y Ptpn ESQSHPGDFVLSVR y Sgk69 LIVSNFSQAK y8 Sgk69 LIVSNFSQAK y Sgk69 FGVDSWSDFR y Sgk69 FGVDSWSDFR y6 Sgk69 EVPHLTVADFVR y Sgk69 EVPHLTVADFVR y Sgk69 ERPVPSTANSISSLATLSIK y9 Sgk69 ERPVPSTANSISSLATLSIK y6 Sgk69 LAPEIITATQYK y8 Sgk69 LAPEIITATQYK y Ptpn SFDGNTLLNR y Ptpn SFDGNTLLNR y8 Ptpn TLLLEFQNESR y8 Ptpn TLLLEFQNESR y Ptpn AIAQLFEK y6 Ptpn AIAQLFEK y Ptpn SSKPQELSSGDLK y Ptpn SSKPQELSSGDLK b Ptpn SSKPQELSSGDLK y6 Ptpn SSKPQELSSGDLK y Ptpn ESEGLITSGNDK y6 Ptpn ESEGLITSGNDK y Fam9a IEIPVHYAGQFK y Fam9a IEIPVHYAGQFK y6 Fam9a SDFLLDPSR y Fam9a SDFLLDPSR y6 Fam9a DELTQSFHR y6 Fam9a DELTQSFHR y Fam9a DELTQSFHR y Fam9a DELTQSFHR b6 Fam9a SPFGSPSAEALSSR y9 Fam9a SPFGSPSAEALSSR y Fam9a LLNAPPVPPR y6 Fam9a LLNAPPVPPR y8 Arhgef SFLILPFQR y Arhgef SFLILPFQR y Arhgef LLLQNILK y6 Arhgef LLLQNILK y Arhgef SGELTALEFSVSPGLK y9 Arhgef SGELTALEFSVSPGLK y Arhgef FLVFDHAPFSSIR y6 Arhgef FLVFDHAPFSSIR y(+) Arhgef VYLPYVTNQTYQER y8 Arhgef VYLPYVTNQTYQER y Arhgef VYLPYVTNQTYQER y8 Arhgef VYLPYVTNQTYQER y Errfi HLSYVVSP b Errfi HLSYVVSP b6

14 RESEARCH Errfi VVPDPNPPPPQSHR y6 Errfi VVPDPNPPPPQSHR y8 Errfi SHS[Pho]GPAGSFNKPAIR y Errfi SHS[Pho]GPAGSFNKPAIR y6 Errfi SHS[Pho]GPAGSFNKPAIR y Sgk LAPEIVSASQYR y(+) Sgk LAPEIVSASQYR y6 Sgk GSLNQDLHGSGEEPLVVQGLSSR y Sgk GSLNQDLHGSGEEPLVVQGLSSR y Sgk AEPEVYER y6 Sgk AEPEVYER y Sgk LGGLYTQSLAR y6 Sgk LGGLYTQSLAR y Sgk SASFAFEFPK y6 Sgk SASFAFEFPK y8 Pppcg AHQVVEDGYEFFAK y Pppcg AHQVVEDGYEFFAK b Pppcg AHQVVEDGYEFFAK b8 Pppcg AHQVVEDGYEFFAK y Pppcg AHQVVEDGYEFFAK y Pppcg AHQVVEDGYEFFAK y Pppcg EIFLSQPILLELEAPLK y Pppcg EIFLSQPILLELEAPLK y Pppcg EIFLSQPILLELEAPLK y Pppcg EIFLSQPILLELEAPLK y8 Pppca LNLDSIIGR y Pppca LNLDSIIGR y6 Pppca LNLDSIIGR y Pppca NVQLTENEIR y6 Pppca NVQLTENEIR y Pppca NVQLTENEIR y8 Pppca YPENFFLLR y6 Pppca YPENFFLLR y Asap GDDNTGENNIVQELTK y Asap GDDNTGENNIVQELTK y6 Asap GDDNTGENNIVQELTK y9 Asap ASIEIANESGETPLDIAK y6 Asap ASIEIANESGETPLDIAK y Asap ASIEIANESGETPLDIAK y Asap EIISEVQR y Asap EIISEVQR y6 Asap GVDLLQNLIK y6 Asap GVDLLQNLIK y8 Asap NTVAAIEEALDVDR y8 Asap NTVAAIEEALDVDR y Gab GSGESASWSAESPGK y Gab GSGESASWSAESPGK y9 Gab TQALQNTMQEWTDVR y9 Gab TQALQNTMQEWTDVR y Gab TQALQNTMQEWTDVR y

15 RESEARCH Gab TQALQNTM[Oxi]QEWTDVR y Gab TQALQNTM[Oxi]QEWTDVR y9 Gab TQALQNTM[Oxi]QEWTDVR y Gab SS[Pho]PAEFSSSQHLLR y Gab SS[Pho]PAEFSSSQHLLR y8 Gab GSEIQPPPVNR b Gab GSEIQPPPVNR y6 Gab GSEIQPPPVNR y8 Sos TVQDVEER y6 Sos TVQDVEER y Sos DENSVIFAAK y6 Sos DENSVIFAAK y Sos LPGYSSAEYR y6 Sos LPGYSSAEYR y8 Sos GEQPISADLK y Sos GEQPISADLK y Sos VLDDAVELSQDHFK y Sos VLDDAVELSQDHFK y8 Erbb DVFAFGGAVENPEYLVPR y9 Erbb DVFAFGGAVENPEYLVPR y Erbb DVFAFGGAVENPEYLVPR y Erbb DVFAFGGAVENPEY[Pho]LVPR y Erbb DVFAFGGAVENPEY[Pho]LVPR y Erbb DVFAFGGAVENPEY[Pho]LVPR y Erbb DVFAFGGAVENPEY[Pho]LVPR y8 Erbb DVFAFGGAVENPEY[Pho]LVPR b8 Erbb GLQSLSPHDLSPLQR y Erbb GLQSLSPHDLSPLQR y9 Erbb EILDEAYVM[Oxi]AGVGSPYVSR y Erbb EILDEAYVM[Oxi]AGVGSPYVSR y9 Erbb EILDEAYVMAGVGSPYVSR y Erbb EILDEAYVMAGVGSPYVSR y9 Erbb WM[Oxi]ALESILR y Erbb WM[Oxi]ALESILR y6 Erbb WMALESILR y Erbb WMALESILR y6 Erbb GMSYLEDVR y Erbb GMSYLEDVR y Ship TLSEVDYSPGPGR y8 Ship TLSEVDYSPGPGR y Ship VDQLNLER y6 Ship VDQLNLER y Ship VDQLNLER y Ship NQNYLDILR y6 Ship NQNYLDILR y Ship TGIANTLGNK y6 Ship TGIANTLGNK y Ship LDVTLGDLTK y Ship LDVTLGDLTK y8

16 RESEARCH Dabip LGSFSTAAEELAR y9 Dabip LGSFSTAAEELAR y8 Dabip NSYLGLVSLPAASVAGR y Dabip NSYLGLVSLPAASVAGR y8 Dabip LISASLFLR y8 Dabip LISASLFLR y Shcbp GAGIEIYPGSK y Shcbp GAGIEIYPGSK y9 Shcbp LSTFTLANVK y8 Shcbp LSTFTLANVK y Shcbp TSAELFM[Oxi]K y6 Shcbp TSAELFM[Oxi]K y Shcbp TSAELFMK y6 Shcbp TSAELFMK y Shcbp TPFPRIIQTNELLFYER y Shcbp TPFPRIIQTNELLFYER y6 Shcbp LIENPLLR y Shcbp LIENPLLR y Apa GLAVFISDIR y Apa GLAVFISDIR y6 Apa VVHLLNDQHLGVVTAATSLITTLAQK y6 Apa VVHLLNDQHLGVVTAATSLITTLAQK y Apa NADVELQQR y Apa NADVELQQR y Apa FVNLFPEVK y Apa FVNLFPEVK y Aps FILIQNR y Aps FILIQNR y Aps LIEEVHAVVTVR y6 Aps LIEEVHAVVTVR y Pikr GFLLALPAAVVTPEAASEAYR y9 Pikr GFLLALPAAVVTPEAASEAYR y Pikr GFLLALPAAVVTPEAASEAYR y Pikr VALQALGVADGGER y8 Pikr VALQALGVADGGER y Pikr DTPDGTFLVR y6 Pikr DTPDGTFLVR y8 Pikr ALSEIFSHVLFR y6 Pikr ALSEIFSHVLFR y Pikr ISEIIDSR y Pikr ISEIIDSR y6 Pikr NESLAQYNPK y8 Pikr NESLAQYNPK y6 Pikr TQSSSNPAELR y9 Pikr TQSSSNPAELR y Pikr TQSSSNPAELR y8 Pikca LINLTDILK y Pikca LINLTDILK y6 Pikca EAGFSYSHTGLSNR y6 Pikca EAGFSYSHTGLSNR y8 Pikca EAGFSYSHTGLSNR y 6

17 RESEARCH Erbb ELANEFTR y Erbb ELANEFTR y6 Erbb SPSQVQVADFGVADLLPPDDK b9 Erbb SPSQVQVADFGVADLLPPDDK y6 Erbb GESIEPLDPSEK y Erbb GESIEPLDPSEK y8 Erbb GESIEPLDPSEK y Erbb LAEIPDLLEK y6 Erbb LAEIPDLLEK y8 Cblb ALEIAQNNLEVAR y9 Cblb ALEIAQNNLEVAR y8 Cblb LAQLSENEYFK y Cblb LAQLSENEYFK y8 Cblb EGFYLYPDGR y Cblb EGFYLYPDGR y6 Lrrk GIRPVLGQPEEVQFHR b Lrrk GIRPVLGQPEEVQFHR y Lrrk LLVLTHGADPENYAVR y Lrrk LLVLTHGADPENYAVR y Lrrk STLLEILQTGK y Lrrk STLLEILQTGK y8

18 RESEARCH Supplementary Table smrm transitions from αcasein spiked in as the internal control. Protein Peptide sequence Q Q CE RT Fragment acasein_s ALNEINQFYQK y6 acasein_s ALNEINQFYQK y acasein_s VPQLEIVPNS[Pho]AEER y-98 acasein_s VPQLEIVPNS[Pho]AEER y Supplementary Table Summary of Ser/Thr phosphorylation sites in Shc signaling network. Data extracted from the phosphositeplus.org database at Cell Signaling Technology. All proteins are human. Protein Accession Evidence count for ps/t Biological insight APA O99 S/T sites detected < times No follow up APS P68 S/T sites detected < times No follow up ARHGEF Q S; S6 detected > times No follow up CBLB Q9 S6, S, S, S9, S6, S886 > times No follow up DABIP QVWQ8 S, S, S9////8, S99, S, S8 > times S8 regulates molecular association ASAP O S, S, S8, S8 > times No follow up EGFR P T68, T69, S69, S99, S99, S6, S, S, S8, S66 > times T68, T69, S69, S99, S, S, S9 have various functions GAB Q8 S66, S6, T6, S9, T68, S68, T68 > times No follow up GAB Q9UQC S, S, S6, S, T9, S, S, S, S, S, S6, S6 > times S9, S, T9, S6 regulate molecular interactions - FAM9A Q9H6 S, S6, S6 > times No follow up GRB P699 > times No follow up ERBB P66 T, S, S > times T686 is implicated in receptor internalization - ERBB P86 S686 > times No follow up PIKCA P6 S > times No follow up PIKR P986 T6, S68 > times S68 involved in "activation 8 PIKR O9 T68 > times No follow up PPPCA P66 T > times T: Inhibitory site 9 PPPCC P68 T No follow up PTPN Q9 S9, S, S, S9, T9, T9, T69, S, S6, S66, S68, S6 > times S9 implicated in inhibition RASAL Q9UJF S, S6, T6, S6, S86, T9 > times No follow up SGK Q86YV S9, S69, S, S8 > times No follow up SGK69 Q9H9 S8, S68, S, T6, T6, S, S > times No follow up SHC P9 S9 > times S9 implicated in molecular association SHCBP Q8NEM S/T sites detected < times No follow up SHIP O S, S98, S, S, T, S, S6 > times T98 involved in "phosphorylation" PTPN Q6 S, T, T, T68, S9 > times No follow up SOS Q889 S8, S, S, S6 > times Regulation of molecular association by S, S6, S8, S9, S9 SOS Q89 S/T sites detected < times No follow up LRRK Q8SD S6, T8 detected > times No follow up ERRIF Q9UJM S9,,, 6 No follow up 8

19 RESEARCH References for Supplementary Table. Zhang H, et al. RIP-mediated AIP phosphorylation at a ---binding site is critical for tumor necrosis factor-induced ASK-JNK/p8 activation. J Biol Chem 8, ().. Brummer T, et al. Phosphorylation-dependent binding of -- terminates signaling by the Gab docking protein. EMBO J, -6 (8).. Lynch DK, Daly RJ PKB-mediated negative feedback tightly regulates mitogenic signaling via Gab. EMBO J, -8 ().. Aud M, et al. Phosphorylation of Grb-associated binder on serine 6 by ERK MAPK regulates its association with the phosphatase SHP- and decreases STAT activation. J Immunol, 96- ().. Monje PV, et al. Protein kinase A-mediated gating of neuregulin-dependent ErbB-ErbB activation underlies the synergistic action of camp on Schwann cell proliferation. J Biol Chem 8, 8- (8). 6. Ouyang X, et al. The duration of phorbol-inducible ErbB tyrosine dephosphorylation parallels that of receptor endocytosis rather than threonine-686 phosphorylation: implications for the physiological role of protein kinase C in growth factor receptor signaling. Carcinogenesis 9, -9 (998).. Ouyang X, et al. Human cancer cells exhibit protein kinase C-dependent c-erbb- transmodulation that correlates with phosphatase sensitivity and kinase activity. J Biol Chem, 86-9 (996). 8. Foukas LC, et al. Regulation of phosphoinositide -kinase by its intrinsic serine kinase activity in vivo. Mol Cell Biol, 966-(). 9. Guo CY, et al. Ionizing radiation activates nuclear protein phosphatase- by ATM-dependent dephosphorylation. J Biol Chem, 6-6().. Garton AJ, Tonks NK PTP-PEST: a protein tyrosine phosphatase regulated by serine phosphorylation. EMBO J, 6-(99).. Faisal A, et al. Serine/threonine phosphorylation of ShcA. Regulation of protein-tyrosine phosphatase-pest binding and involvement in insulin signaling. J Biol Chem., - ().. Artemenko Y, et al. Regulation of PDGF-stimulated SHIP tyrosine phosphorylation and association with Shc in T-L preadipocytes. J Cell Physiol, 98-6 ().. Corbalan-Garcia S, et al. Identification of the mitogen-activated protein kinase phosphorylation sites on human Sos that regulate interaction with Grb. Mol Cell Biol 6, 6-8 (996). 9

20 RESEARCH SUPPLEMENTARY FIGURES a KDa - +EGF IB dt-shc p66 p endogenous Shc p6 Lysates b dt-shc complex purification Initial discovery smrm quantification EGF smrm method α-flag Cell lines LC/MS/MS Method validation & optimization dt-shc On-bead tryptic digestion smrm experiments Supplementary Figure. Workflow for smrm analysis. a, Expression level of dt-shc in a stable Rat- fibroblast cell line. Lysates prepared from Rat- fibroblasts stably expressing dt-shc were immunoblotted with anti-shc antibodies. b, Workflow for smrm analysis. dt-shc was affinity purified from EGF-stimulated cells and digested with trypsin. The peptides were analyzed by LC/MS in discovery mode to identify a Shc- based interaction network. This information was used to build an smrm method, which was employed to quantify dynamic changes in the Shc network.

21 RESEARCH a. Egfr Relative abundance EGF concentration (ng/ml) Grb Ptpn Sgk69 Apa Ptpn Fam9a Gab Arhgef Asap b Relative abundance of dt-shc phosphorylation EGF concentration (ng/ml) c Relative abundance EGF concentration (ng/ml) Supplementary Figure. EGF dose optimization. a-b, Cells were stimulated with increasing concentrations of EGF for min. Interacting proteins and Shc phosphopeptides in purified dt-shc complexes were quantified by smrm. c, Normalized Shc levels. Error bars are +/- s.d. (n=).

22 RESEARCH a Peptides: PTB SH Shc No. Sequence m/z VEGGQLGGEEWTR 9. VMGPGVSYLVR 89. ALDFNTR 8. ESTTTPGQYVLTGLQSGQPK 6. HLLLVDPEGVVR 6.9. b Relative abundance Peptide Peptide Peptide Peptide Peptide Peptide Peptide Peptide Peptide Injected sample volume (μl) Peptide Supplementary Figure. Normalization peptides and linearity of smrm assay for Shc complexes. a, Relative positions and sequences of dt-shc peptides used for normalization. b, Linearity plot of dt-shc normalizing peptides. Tryptic digested dt-shc immunoprecipitates were prepared as described in Fig. b and the indicated volumes were injected. Relative abundance of dt-shc normalizing peptides were measured by smrm. dt-shc peptides are numbered as in a and the transitions for each peptide are graphed.

23 RESEARCH a 8 Ion intensity (cpm) H O (%) ACN (%) Retention time (minutes) b Log (injection B, ) Log (injection A, ) Supplementary Figure. Reproducibility of smrm technical repeats. a, The overlay of three LC-sMRM chromatographs from three consecutive injections of the same biological sample (pink: injection a; blue: injection b; orange: injection c). Each chromatograph consists of the whole set of smrm-targeted proteins for the Shc interactome. Each peak represents a detected peptide fragment (transition) from the Shc interactome. The peak areas for major peaks are labeled. The LC gradient for peptide elution is also shown by dotted lines (Blue: H O %; Red: ACN %). cpm=count per minute. b, Verification of the linear regression of smrm data in a. The Log values of all data points (circles) from the first two injections (injection a and injection b) are plotted against each other. Each data point represents the ion intensity of a given peptide transition (peak area) quantified by the smrm assay.

24 RESEARCH a Relative abundance Y py9/ Relative abundance Y py9/ Time after EGF addition (minutes) (real time scale) Time after EGF addition (minutes) (quasi-logarithm time scale ) b Relative abundance S9 T S Y c Time after EGF addition (minutes) (real time scale) Relative abundance Time after EGF addition (minutes) Supplementary Figure. Temporal profiles of dt-shc phosphotyrosine sites after EGF stimulation. a, dt-shc was affinity purified from EGF-stimulated fibroblasts. Dynamic phosphorylation on tyrosine (Y) and tyrosine 9/ (py9/, as singly phosphorylated form; see supplementary text for details) was measured by smrm (left panel). Phosphorylation kinetics were plotted using a quasi-logarithm time scale to expand the early time points (right panel). b, Dynamics of all Shc phosphorylation sites plotted on a real time scale. c, Normalized dt-shc levels. Results are representative of three independent biological repeats. Error bars are +/- s.d. from all transitions for each phosphopeptide from all technical repeats.

25 RESEARCH a b Supplementary Figure 6. Principle component analysis (PCA) of the dynamic profiles of phosphorylation sites from the Shc-signaling network shown in Fig. b. a, Distribution of eigenvalues among PCA components, showing components and (in blue) with the highest contributions among all variances. b, PCA of analyzed phosphorylation sites in the Shc-signaling network. The centre of the open circles marks the mean PCA values for each phosphorylation site. Open circles: red, tyrosine sites; blue, serine/threonine sites.

26 RESEARCH - + EGF Inhibitor: - - EGFR Mek PIK IB: pakt S Akt Lysates perk Erk dt-shc Supplementary Figure. Monitoring kinase inhibitor activity by immunoblotting. The inhibition efficiencies of indicated kinase inhibitors were monitored by immunoblotting for Erk and Akt (S) phosphorylation. Inhibitor used for EGFR is AG8; Mek is PD989; PIK is LY9. Results are representative of three independent biological repeats. 6

27 RESEARCH a Relative abundance of S9.. inhibitors - - PIKpan (GDC) Pα (PIK9) Pβ (TGX) EGF Lysates GDC PIK9 TGX LY IB pakt S pakt T8 pakt T ps6k T89 ps6k T/ perk - + EGF p-dt-shc Y dt-shc b Relative abundance S9 T Y inhibitors - - Akt Mek EGFR inhibitors EGF Lysates - - EGFR Mek Akt IB pakt S Akt - + EGF Supplementary Figure 8. EGF-induced phosphorylation of Shc Ser9 is sensitive to Akt inhibition and requires PIK pα. a, Effects of indicated inhibitors on dt-shc S9 phosphorylation. Fibroblasts were pretreated for min with isoform-specific PIK inhibitors. Phosphorylation on dt-shc S9 was quantified by smrm (left panel). The efficiency of PIK inhibition was monitored by measuring the phosphorylation on Akt and S6k using immunoblotting (right panel). b, Effects of an Akt-specific inhibitor (Akt inhibitor IV) on dt-shc phosphorylation compared to Mek and EGFR inhibitors (left panel) as monitored by smrm. The inhibition efficiency was monitored by immunoblotting for phospho-akt (right panel). GDC: pan-pik inhibitor; PIK9: PIK pα isoform-specific inhibitor; TGX: PIK pβ isoform-specific inhibitor, LY: pan-pik inhibitor (LY9). To inhibit Akt, we used Akt inhibitor IV; Mek, PD989; EGFR, AG8. Results are representative of three independent biological repeats. Error bars are s.d. from all transitions for each phosphopeptide from all technical repeats. Data are the representative of three biological replicates.

28 RESEARCH a EGF (min) IB pakt S Lysates perk dt-shc b Relative intensity pakt pt Relative intensity perk ps Time after EGF addition (minutes) Time after EGF addition (minutes) Supplementary Figure 9. Temporal profiles of Akt and Erk phosphorylation upon EGF stimulation. a, EGF-induced Akt and Erk phosphorylation kinetics were quantified by quantitative immunoblotting (Odyssey infrared imaging system, LI-COR Biosciences). b, Overlay of temporal phosphorylation profiles, showing the difference in kinetics of Akt activation (pakt) versus Shc T phosphorylation (pt), and between Erk activation (perk) and Shc S9 phosphorylation (ps9) (compare with Fig. e). 8

29 . 6 RESEARCH Quantification of Grb in Shc complex Ion intensity Raw quantification data (Extracted ion chromatograph) Relative abundance. Kinetic curve for Shc binding (D view) Pseudo-D plot. 6 Time after EGF addition (minutes) Supplementary Figure. Conversion of smrm quantification data into pseudo-d plots to generate temporal profiles for Shc binding proteins using Grb as an example. The abundance (ion intensity) of each Grb transition (peptide fragment) was first converted into relative abundance using the highest value of that transition across all time points as.. The mean transitions from Grb were then plotted as a D kinetic curve, which was then converted into a pseudo-d view. The sizes of the dots are proportional to the relative abundance of Shc-associated Grb at the indicated time points. 9

30 RESEARCH Shc Erbb Egfr Erbb Grb Gab Sos Sos Supplementary Figure. Dynamic profiles of Cluster a Shc-interacting proteins. The individual temporal profiles of selected Shc-interacting proteins are shown (see Figb). Y-axis: relative abundance; X-axis: time after EGF addition (minutes). Results are representative of three biological replicates. Error bars are s.d. from all transitions for each protein from all technical repeats.

31 RESEARCH Pikr Fam9a Pikr Arhgef Pikca Lrrk Supplementary Figure. Dynamic profiles of Cluster b Shc-interacting proteins. The individual temporal profiles of Shc-interacting proteins from Cluster b are shown (see Figb). Y-axis: relative abundance; X-axis: time after EGF addition (minutes). Results are representative of three biological replicates. Error bars are s.d. from all transitions for each protein from all technical repeats.

32 RESEARCH Ptpn Aps Ship Apa Supplementary Figure. Dynamic profiles of Cluster Shc-interacting proteins. The individual temporal profiles of Shc-interacting proteins from Cluster are shown (see Figb). Y-axis: relative abundance; X-axis: time after EGF addition (minutes). Results are representative of three biological replicates. Error bars are s.d. from all transitions for each protein from all technical repeats.

33 RESEARCH SgK Sgk Asap Rasal Pppca Dabip Pppcc Shcbp Supplementary Figure. Dynamic profiles of Shcbp and Cluster Shc interacting proteins. The individual temporal profiles of Shc-interacting proteins are shown (see Figb). Y-axis: relative abundance; X-axis: time after EGF addition (minutes). Results are representative of three biological replicates. Error bars are s.d. from all transitions for each protein from all technical repeats.

34 RESEARCH a Shc KI phospho-kinetics dt-shc phospho-kinetics Relative abundance. Relative abundance. py ps9 pt ps..6 6 Time after EGF addition (minutes) Time after EGF addition (minutes) b py py9/ Shc KI phospho-kinetics dt-shc phospho-kinetics ps9 pt ps Supplementary Figure. Comparison of EGF-induced phosphorylation kinetics of endogenously expressed Shc vs. ectopically expressed dt-shc. a, Primary MEFs prepared from FLAG-tagged Shc knock-in (Shc KI) mice were stimulated with EGF for the indicated times. Phosphorylation kinetics of Shc KI were quantified by smrm and compared with those from dt-shc purified from stably expressing Rat- cells. Error bars are s.d. from all transitions for each protein from all technical repeats. Results are representative of three biological replicates. b, Overlay of phospho-kinetic curves of individual Shc sites.

35 RESEARCH a Shc KI dt-shc b Shc KI dt-shc Supplementary Figure 6. Comparison of the temporal profiles of EGF-induced phosphorylation of the endogenous Shc complex as compared with the dt-shc complex. a, Primary MEFs prepared from FLAG-Shc knock-in (Shc KI) mice were stimulated with EGF for the indicated times. Dynamic phosphorylation of the Shc complex purified from Shc KI cells was quantified by smrm and compared with that from dt-shc- expressing cells. b, Temporal profiles of Shc-associated proteins were compared from FLAG-Shc KI MEFs and dt-shc Rat- cells stimulated with EGF.

36 RESEARCH a Red: Grb vs. Grey: cluster a Red: Grb vs. Grey: cluster b b c Red: Grb vs. Green: cluster Red: Grb vs. Blue: cluster X-axis: time after EGF addition (minutes) as described in Fig. a. Y-axis: relative abundance. Supplementary Figure. Temporal profile comparisons. a, Shc-associated Grb vs. Cluster a and b proteins. b, Shc-associated Grb vs. Cluster proteins. c, Shc-associated Grb vs. cluster proteins. 6

37 RESEARCH a N-SH SH C-SH Grb wt Ex Ex Ex Ex Ex b Grb flx geo Ex sa-ires- geo-pa FLPe Grb flx loxp Ex loxp +Cre Excised Grb exon allele loxp c EGF (min) Grb WT Grb KO 6 6 IB Grb p66 p p6 dt-shc Endogenous Shc α-tubulin Lysates Supplementary Figure 8. Targeting of the mouse Grb gene with a conditional floxed allele. a, Diagrammatic representations of the wild-type mouse Grb gene. b, Cre-mediated deletion of exon introduces a frameshift mutation. c, Grb levels after -OH-tamoxifeninduced excision in MEFs homozygous for the Grbflox/flox allele and ectopically expressing dt-shc (WT wild-type MEFs, KO Grb-excised MEFs).

38 RESEARCH Rasal Shcbp Egfr Erbb Sos Gab Gab Asap Erbb Sos Ptpn Pppcc Pppca Shc py Grb Errfi Cblb SgK69 Sgk Dabip Ship Ap Ptpn Fam9a Arhgef Lrrk Pikr Pikc Cluster a Cluster b Cluster Cluster Shcbp Supplementary Figure 9. EGF-induced Shc-mediated signaling network consists of Grb-dependent and -independent sub-networks. Experiments were performed as described in Fig. b. WT or Grb KO cells were stimulated with EGF for minutes. Shc complexes were analyzed by smrm. A summary view of the data is shown in which proteins are separated based on their requirement for or independence of Grb for Shc binding. 8

39 RESEARCH a b EGF (min) Grb WT Grb KO 6 6 IB: pegfr Y68 Lysates pegfr Y pegfr Y8 EGFR Supplementary Figure. Increased tyrosine phosphorylation on Shc and EGFR in Grb-deficient cells. a, Grb-deficient cells expressing dt-shc were stimulated with EGF for the indicated times. The phosphorylation status of dt-shc was analyzed by smrm. b, Increased and sustained phosphorylation on various EGFR tyrosine sites in Grb-deficient MEFs was analyzed by immunoblotting using phospho-specific antibodies. 9

40 RESEARCH a Cluster a Cluster b Cluster Cluster b Time after EGF addition (minutes) Relative abundance SgK Sgk Dabip Relative abundance Pppcc Pppca Asap Time after EGF addition (minutes) Time after EGF addition (minutes) Time after EGF addition (minutes) WT F A Supplementary Figure. Grb-independent, serine/threonine-dependent Shc protein interactions. a, smrm analysis of Shc-interacting proteins in Shc-deficient fibroblasts stably expressing wild-type dt-shc (WT) or its mutants. b, Cluster proteins were also plotted with dynamic curves for ease of comparison. F: Y9//F; A: S9//TA. Error bars are s.d. from all transitions for each protein from all technical repeats. Results are representative of two biological replicates.

41 RESEARCH Temporal clusters Cluster a Cluster b Cluster Cluster Supplementary Figure. Effect of alanine substitution of Shc Ser9 on EGFinduced Shc signaling complex assembly. smrm analysis of Shc-interacting proteins in Shc-deficient fibroblasts stably expressing wild-type dt-shc (WT) or S9A mutant. Note: For each protein, the highest value for association with WT Shc is set as %.

42 RESEARCH Temporal clusters Cluster a Cluster b Cluster Cluster Supplementary Figure. Effect of alanine substitution of Shc Ser on EGFinduced Shc signaling complex assembly. smrm analysis of Shc-interacting proteins in Shc-deficient fibroblasts stably expressing wild-type dt-shc (WT) or SA mutant. Note: For each protein, the highest value for association with WT Shc is set as %.

43 RESEARCH Temporal clusters Cluster a Cluster b Cluster Cluster Supplementary Figure. Effect of alanine substitution of Shc Thr on EGFinduced Shc signaling complex assembly. smrm analysis of Shc-interacting proteins in Shc-deficient fibroblasts stably expressing wild-type dt-shc (WT) or TA mutant. Note: For each protein, the highest value for association with WT Shc is set as %.

44 RESEARCH 9 a 8 SgK69 Pppr 6 Spectral counts Pppca Pppcb Pppcc GFP Bait proteins b Spectral counts PPPCA PPPCC PPPR SGK69 SGK GFP Bait proteins Supplementary Figure. SgK69 specifically associates with Pppca and Pppcc. a, Normalized spectral counts (across all replicates and conditions) for the interactions of SgK69 and Pppr with each of the three catalytic subunits of PP and with the negative control GFP. FLAG-tagged Pppca, Pppcb, Pppcc and GFP were purified from stablytransfected HEK9 cells and then analyzed by LC/MS. SgK69 is highly enriched with Pppca and Pppcc, but not detected as an interaction partner for Pppcb. By contrast, Pppr (also known as PNUTS) interacts with all three of the catalytic subunits (n ). b, SgK69, but not SgK, associates with Pppca and Pppcc. FLAG-tagged SgK69, SgK and GFP were overexpressed and purified from EGF-stimulated HEK9 cells, then analyzed by LC/MS. Normalized spectral counts of Pppca, Pppcc and Pppr are shown. n=. Error bars indicate s.d. from all transitions of each protein from all technical repeats.

45 RESEARCH SH (Grb) SH WW PTB (Shc) SgK69 6 aa Y6DN PPAPFPPP8 KRWISF98 PPLP6 NPTY88 Pseudokinase? GFG Pppca/cc Mg + Supplementary Figure 6. SgK69 contains multiple motifs involved in proteinprotein interactions. Potential motifs are predicted based on experimental data, literature searches and bioinformatics analyses. SgK69 lacks the conserved Asp residue within the conserved DFG motif, critical for binding Mg+ ions in protein kinases. This is replace by a Gly residue (highlighted in red). Phosphorylated tyrosine sites are highlighted in red.

46 RESEARCH. Relative abundance.8.6. Control shrna SgK69 shrna SgK69 shrna. Egfr Grb Sos Pppca Pppcc SgK69 Shc interacting proteins Supplementary Figure. Lentiviral-mediated knock-down of SgK69 effectively decreases EGF-dependent binding of Pppca and Pppcc to Shc in HeLa cells. Cells infected with SgK69 shrna (black bars), SgK69 shrna (blue bars), or luciferase shrna (open bars) were stimulated with EGF. Endogenous Shc complexes were purified by immunoprecipitation using an anti-shc antibody. The relative abundances of Shc- interacting proteins were quantified by smrm. Error bars are s.d. from all transitions for each protein from all technical repeats. Results are representative of three biological replicates. 6

47 RESEARCH. SgK69 shrna Ratio over wild type.. Dabip shrna Egfr Grb Sos Pppca Pppcc SgK SgK69 Asap Dabip Shc interacting proteins Supplementary Figure 8. Lentiviral-mediated knockdown of Dabip protein expression does not affect EGF-dependent association of SgK69-Pppca/cc complex to the Shc complex. HeLa cells infected with lentiviruses expressing shrnas for luciferase, SgK69, or Dabip were stimulated with EGF. The Shc signaling complex was affinity purified with anti-shc antibody. The indicated Shc-interacting proteins were quantified by smrm. Striped bars identify proteins that are the targets of shrna knockdown. Ratio over wild type=relative abundance of Shc binding partners in SgK69 or DabIP knockdown cells/relative abundance of Shc binding partners in control knockdown cells. Error bars are s.d. from all transitions for each protein from all technical repeats. Results are representative of two biological replicates.

48 RESEARCH a EGF (min) Vector SgK69 IB Shc EGFR SgK69 (HA) IP: Shc IP: Control IgG b EGF (min) SgK69 Non-specific band Vector SgK69 IB ptyr SgK69 (HA) Endogenous Shc IP: SgK69 (αha) c Control SgK69 SgK69 Y88F EGF (min) IP: Shc IB Shc SgK69 (HA) IP: SgK69 (αha) SgK69 (HA) Shc Supplementary Figure 9. SgK69 is tyrosine phosphorylated upon EGF stimulation. a, Shc interacts with SgK69 with delayed kinetics following EGF stimulation. EGF-stimulated MCFA cells stably expressing myc-sgk69-ha (SgK69) or empty vector were immunoprecipitated with anti-shc antibodies and immunoblotted as indicated. b, MCFA cells stably expressing myc-sgk69-ha (SgK69) or empty vector were serum-starved and stimulated with EGF for the indicated times. HA-tagged SgK69 was affinity purified. Tyrosine phosphorylation of SgK69 was examined using ptyr-specific antibodies (top panel), and associated Shc identified by blotting with anti-shc antibodies (lower panel). c, EGF-induced association of the SgK69 Y88F mutant with Shc was examined using immunoprecipitation and immunoblotting. 8

49 RESEARCH Supplementary Figure. Morphology of SgK69-induced MCFA acini. MCFA cells stably expressing empty vector (pretrox) or SgK69 were grown in Matrigel for days and photographed. Overexpression of WT SgK69 within this model system generates acini with a two-fold increase in diameter over those of control acini. Furthermore, these enlarged acini present with a multi-lobular morphology and non-cleared lumens, distinct from those of the rounded, hollow control acini. Both of these phenotypes induced by over-expression of WT SgK69 are rescued by mutation of Y88 to phenylalanine. This tyrosine residue sits within a canonical NPXY motif, responsible for the interaction between SgK69 and Shc. 9