Author response: Time-resolved phosphoproteomics reveals scaffolding and catalysis-responsive patterns of SHP2-dependent signaling (2021)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract SHP2 is a protein tyrosine phosphatase that normally potentiates intracellular signaling by growth factors, antigen receptors, and some cytokines, yet is frequently mutated in human cancer. Here, we examine the role of SHP2 in the responses of breast cancer cells to EGF by monitoring phosphoproteome dynamics when SHP2 is allosterically inhibited by SHP099. The dynamics of phosphotyrosine abundance at more than 400 tyrosine residues reveal six distinct response signatures following SHP099 treatment and washout. Remarkably, in addition to newly identified substrate sites on proteins such as occludin, ARHGAP35, and PLCγ2, another class of sites shows reduced phosphotyrosine abundance upon SHP2 inhibition. Sites of decreased phospho-abundance are enriched on proteins with two nearby phosphotyrosine residues, which can be directly protected from dephosphorylation by the paired SH2 domains of SHP2 itself. These findings highlight the distinct roles of the scaffolding and catalytic activities of SHP2 in effecting a transmembrane signaling response. Introduction SHP2 is a non-receptor tyrosine phosphatase that is essential for mammalian development (Saxton et al., 1997). In humans, germline mutations of PTPN11 cause the developmental disorders Noonan and LEOPARD syndromes (Tartaglia and Gelb, 2005). Somatic PTPN11 activating mutations are also found frequently in juvenile myelomonocytic leukemia and, to a lesser extent, in solid tumors (Bentires-Alj et al., 2004). Numerous studies have shown that SHP2 acts as a positive effector of receptor tyrosine kinase (RTK) signaling (Bennett et al., 1994; Easton et al., 2006; Tang et al., 1995). SHP2 facilitates the full induction of Ras-dependent extracellular signal-regulated kinase (ERK) proteins following stimulation of cells with epidermal growth factor (EGF) or other receptor tyrosine kinase ligands. SHP2 also serves as a positive regulator of numerous other signaling systems, including cytokine (Xu and Qu, 2008), programmed cell death (Yokosuka et al., 2012), and immune checkpoint pathways (Gavrieli et al., 2003). The SHP2 protein consists of two SH2 domains (N-SH2, C-SH2), followed by a phosphatase (PTP) domain, with a natively disordered C-terminal tail that contains tyrosine residues known to become phosphorylated (Bennett et al., 1994; Keegan and Cooper, 1996). X-ray structures of the SHP2 protein core, encompassing the two SH2 domains and the PTP domain, show that the wild-type protein normally adopts an autoinhibited conformation in which the N-SH2 domain occludes the active site of the PTP domain (Hof et al., 1998). Activation of the enzyme requires disengagement of the N-SH2 domain from the PTP domain and subsequent recruitment of SHP2 to phosphotyrosine docking sites on substrate proteins. Recruitment can rely on the N-SH2 domain, C-SH2 domain, or both domains. Most oncogenic mutations of SHP2 lie at the interface between the N-SH2 and PTP domains, thereby increasing the propensity of the enzyme to adopt an open, active conformation (LaRochelle et al., 2018; LaRochelle et al., 2016). Numerous studies in a range of cell types have identified phosphotyrosine (pY) residues on adaptor proteins (e.g. GAB1 and GAB2 [Arnaud et al., 2004; Cunnick et al., 2001]) or on RTKs themselves (e.g. PDGFβR [Rönnstrand et al., 1999]) thought to recruit SHP2 to sites of active signaling following RTK activation (such sites include multiprotein complexes formed on RTK intracellular tails and IRS-1 adapters). Genetic and mutational studies have implicated specific pY residues on RTKs as putative SHP2 substrates (Agazie and Hayman, 2003; Bunda et al., 2015; Klinghoffer and Kazlauskas, 1995). The availability of a potent and highly selective allosteric inhibitor of SHP2 now allows us to capture the whole proteome dynamics of the SHP2 dependence of the EGF response. Thus, we use here the allosteric inhibitor SHP099 (Garcia Fortanet et al., 2016) to study the role of SHP2 on phosphoprotein dynamics at the whole proteome level following EGF stimulation of a breast cancer cell line carrying an EGFR amplification. Global analysis of time-resolved changes in pY abundance at over 400 tyrosine residues reveals several distinguishable response signatures following SHP099 exposure and washout. Putative substrate sites fall into two classes, with increased pY abundance at early and/or late timepoints, respectively. Proteins in this category include previously unidentified substrate sites on proteins such as occludin, ARHGAP35, and PLCγ. Yet another class, which contains the largest number of dynamic pY sites, exhibits decreased pY abundance. These latter sites are enriched on proteins with two nearby pY residues, some of which are directly protected from dephosphorylation by the SH2 domains of SHP2. These data emphasize the two distinct and interrelated biochemical activities of SHP2 – dephosphorylation and phosphosite protection – and identify specific sites relevant to the activity of SHP099 and similar molecules such as TNO155 as therapeutic lead compounds (LaMarche et al., 2020). Results Dynamic regulation of the EGF-responsive phosphoproteome by SHP2 We investigated the role of SHP2 in the responsiveness to EGF by using the EGFR-amplified cell line MDA-MB-468, derived from a patient with triple-negative breast cancer. To identify timepoints for in-depth proteomic analysis, we monitored the influence of SHP2 on the response to EGF stimulation using ERK1/2 phosphorylation as a readout. Cell extracts were prepared at a series of timepoints from three treatment conditions: (1) following pretreatment with dimethyl sulfoxide (DMSO) and then stimulation with 10 nM EGF, (2) following pretreatment with the SHP2 inhibitor SHP099 for 2 hr and then stimulation with EGF, and (3) following pretreatment with SHP099 and then stimulation with EGF for 10 min, after which drug was washed out and medium containing EGF replenished. Because the response of the EGF receptor to EGF stimulation occurs in the 1–2 min time period (Jadwin et al., 2016), we focused attention on the 5–30 min time window to ensure the observation of SHP2-dependent events downstream of EGF receptor stimulation. Immunoblot analysis revealed the expected effect of EGF stimulation: a dramatic increase in phospho-ERK1/2 (p-ERK1/2) levels followed by a decline toward basal levels by 30 min. The induction of p-ERK1/2 was greatly attenuated by pretreatment of cells with 10 μM SHP099. SHP099 washout in the continued presence of EGF (condition 3) revealed p-ERK1/2 induction with a kinetic profile similar to that of EGF stimulation in the absence of drug (condition 1), showing that SHP099 inhibition of SHP2 is rapidly reversible in MDA-MB-468 cells (Figure 1A, Figure 1—figure supplement 1A). Figure 1 with 3 supplements see all Download asset Open asset Phosphoproteomic studies in MDA-MB-468 cells. (A) Left: Western blot showing the phosphoERK1/2 abundance after EGF stimulation alone, EGF stimulation in the presence of SHP099, and EGF stimulation in the presence of SHP099, followed by drug washout 10 min after EGF stimulation. Right: quantification of the Western blot data, calculated from the ratio of [pERK1/2]/[ERK1/2] band intensities. (B) Schematic illustration of treatment conditions and mass spectrometry workflow. Western blot and phosphoproteomic data are both representative of two independent biological replicates (n = 2). (C) Venn diagram showing the overlap of phosphopeptides identified in biological replicates 1 and 2. Figure 1—source data 1 Table showing Phospho-ERK1/2 levels normalized to total-ERK1/2 levels (as quantified by measuring band intensities using ImageJ) in MDA-MB-468 cells treated with SHP099 and EGF as indicated. https://cdn.elifesciences.org/articles/64251/elife-64251-fig1-data1-v2.xlsx Download elife-64251-fig1-data1-v2.xlsx To obtain an in-depth view of the effects of SHP2 inhibition on EGFR signaling, we performed quantitative phosphoproteomics, monitoring dynamic changes in pY abundance as a function of time under DMSO, SHP099, and washout conditions (Figure 1B). Tryptic peptides from DMSO, SHP099, and SHP099-washout groups were enriched for phosphopeptides using immobilized metal affinity chromatography (IMAC) prior to labeling with 11-plex isobaric tandem mass tags (TMT). Tyrosine phosphorylated (pY) peptides were then immunoprecitiated from the TMT-labeled, pooled samples using an anti-pY antibody, and the recovered pY-containing peptides were analyzed by LC-MS3 mass spectrometry (Supplementary files 1 and 2 and Figure 1—figure supplement 1B,C). Principal component analysis revealed a high degree of consistency between matched samples from the two biological replicates (Figure 1—figure supplement 2), which yielded relative quantification for several hundred pY-containing peptides, with good concordance between biological replicates (Figure 1C and Supplementary files 1 and 2). The Western blot (Figure 1A) and mass spectrometry data for the dynamics of pY204 of ERK1 (Figure 1—figure supplement 1B) and pY187 of ERK2 (Supplementary files 1 and 2) show the same pattern of response, confirming that key pY marks associated with EGF-induced signaling events were accurately determined in the TMT experiment. The phosphoproteomic data revealed a spectrum of dependencies on EGF and SHP2 (Figure 1—figure supplement 3A). These were classified on the basis of both direction of change (increase or decrease) and timing. EGF dependencies were established based on responses to EGF addition in the absence of drug and were classified into five categories: fast, medium, or slow increases, neutral (no significant change), or decrease. The dependence of each phosphosite on SHP2 activity was established by assessing how phosphorylation levels were altered when SHP2 was inhibited: negative (more phosphorylation with inhibition; Figure 1—figure supplement 3A, top two rows), positive (less phosphorylation with inhibition; Figure 1—figure supplement 3A, bottom two rows), or neutral (Figure 1—figure supplement 3A, middle row). Positive and negative dependencies were further subdivided based on whether the change was present before EGF stimulation (Figure 1—figure supplement 3A, first and fourth rows) or only after (Figure 1—figure supplement 3A, second and fifth rows). As anticipated, SHP2 inhibition resulted in increased abundance of pY marks at a number of sites (Figure 1—figure supplement 3A, top two rows), the expected effect of blocking a tyrosine phosphatase. Remarkably, however, we found an even larger family of pY sites that showed a decreased abundance when SHP2 is inhibited even at early time points (Figure 1—figure supplement 3A, bottom two rows), indicating that SHP2 inhibition had direct and/or indirect activities other than direct dephosphorylation of substrates. Additionally, SHP2 exhibited EGFR-independent regulation of a number of pY sites: a subset of sites was regulated (either positively or negatively) by SHP2 prior to EGF addition (Figure 1—figure supplement 3A, first and fourth rows), showing that SHP2 had a role in shaping the basal signaling state of these cells. Motif analysis of phosphosites associated with different classes of SHP2 responsiveness did not reveal enrichment for specific motifs (residues flanking the modified tyrosine residue; Figure 1—figure supplement 3B). Remarkably, sites from different regulatory classes could even occur together in the same protein (see below), highlighting the importance of considering specific sites of phosphorylation when evaluating the impact of a phosphatase on a protein substrate. Hierarchical clustering was performed to identify groups of pY sites with similar kinetics in DMSO, SHP099, and washout conditions. Among the six kinetic profiles that emerged (Figure 2—figure supplement 1), we highlight three clusters of sites implicated in EGFR signaling and that display distinct responses to the drug (Figure 2A). The first class of responses, which includes the regulatory subunits of PI3K, occludin, Syntaxin 4 (Figure 2B), the adapter proteins CRK and GRB2, and ARHGAP35 (also known as p190RhoGAP), among other proteins, shows quantitatively increased tyrosine phosphorylation in the presence of SHP099 that rapidly disappears upon drug washout (Figure 2A), as predicted for a SHP2 substrate. The second class of sites accumulates pY marks slowly under SHP099 inhibition (at 30 min after EGF treatment), and not when SHP099 is omitted or washed out. This pattern, observed for pY sites on CBL E3 ligase, RAB10, hnRNPs, PLCγ proteins, and CAPRIN1 (Figure 2C), also matches the response predicted for a SHP2 substrate, but with a time delay, suggesting that the action of SHP2 on these proteins might require an intervening event, such as relocalization (e.g. after endocytosis of active EGFR signaling complexes), or alternatively, that the effect is indirect. The large number of potential substrates identified in this work suggests that SHP2 may catalyze dephosphorylation of many different proteins on different time scales. Figure 2 with 2 supplements see all Download asset Open asset Quantitative phosphoproteomics reveals distinct dynamic responses to SHP2 inhibition. (A) Heatmap showing three classes of dynamic response in which inhibition of SHP2 modulates the effect of EGF stimulation on pY abundance. Specific examples from each cluster are indicated to the left of the heatmap. (B) Plot of pY abundance as a function of treatment condition for Y251 of STX4, an example of an early substrate-like response pattern to SHP2 inhibition. (C) Plot of pY abundance as a function of treatment condition for Y818 of PLCG2, an example of a late substrate-like response pattern to SHP2 inhibition. (D) Plot of pY abundance as a function of treatment condition for Y1100 of ARHGEF5, an example of a site where the abundance of the mark decreases when SHP2 is inhibited, and rebounds after compound washout. Figure 2—source data 1 Table showing TMT relative abundance values for Y251 of STX4. https://cdn.elifesciences.org/articles/64251/elife-64251-fig2-data1-v2.xlsx Download elife-64251-fig2-data1-v2.xlsx Figure 2—source data 2 Table showing TMT relative abundance values for Y78 of CAPRIN1. https://cdn.elifesciences.org/articles/64251/elife-64251-fig2-data2-v2.xlsx Download elife-64251-fig2-data2-v2.xlsx Figure 2—source data 3 Table showing TMT relative abundance values for Y1100 of ARHGEF5. https://cdn.elifesciences.org/articles/64251/elife-64251-fig2-data3-v2.xlsx Download elife-64251-fig2-data3-v2.xlsx The third pattern of response observed is one in which accumulation of pY in response to EGF depends on the release of SHP2 from inhibition by SHP099. Examples of sites that fall into this category include Y1100 of ARHGEF5 (Figure 2D), Y659 of GAB1, Y643 of GAB2, as well as Y546 and Y584 of SHP2 itself. Functional classification of EGF-responsive pY sites (by GSEA and Reactome) differentially regulated by SHP099 (Figure 2—figure supplement 2A–C) reveals enrichment of six major cellular processes (Figure 2—figure supplement 2D). As expected, proteins implicated in MAPK and PI3K signaling, including EGFR and SHP2 itself, contain numerous EGF-responsive phosphosites differentially regulated by SHP099 treatment. The largest functional group includes proteins implicated in adhesion and migration, including tight junction proteins, catenins, Rho-GEF, and Rho-GAP proteins. These findings, along with data on the abundance of cytoskeletal proteins differentially affected by SHP099 (Figure 2—figure supplement 2D), are consistent with earlier results, suggesting that SHP2 promotes migration in MDA-MB-468 cells by regulating EGF-induced lamellipodia persistence (Hartman et al., 2013). Proteins implicated in transcription comprise another functional class with a number of differentially regulated phosphosites (Figure 2—figure supplement 2D). Two additional functional categories enriched in the analysis comprise endocytosis and mRNA processing, neither of which have previously been linked to SHP2 activity. Most of the sites in these two categories display a substrate-like response with SHP099 treatment. In addition, SHP099 also affected phosphorylation of CBL and CBLB, E3 ligases that ubiquitinate EGFR and facilitate its recruitment to clathrin-coated pits for endocytosis. Together, these results highlight the diversity of influence of SHP2, both in the function of proteins that it affects and in the manner in which it affects their phosphorylation patterns. Identification of new substrates of SHP2 Among proteins that show a substrate-like pattern, only one of the phosphosites observed has previously been reported to be dephosphorylated by SHP2 (GRB2 pY209; ARHGAP35 has also been reported to be a substrate, but its pY site has not been reported) (Bregeon et al., 2009; Chardin et al., 1993). Therefore, we carried out follow-up studies to determine whether any of the proteins with sites newly identified in this proteome-wide analysis are indeed directly dephosphorylated by SHP2. Of the potential substrates identified from the mass spectrometry data, we identified 20 proteins with sites that (1) showed an increase upon EGF stimulation, (2) showed an increase of greater than twofold in the presence of SHP099 when compared to the DMSO control for at least one timepoint, (3) were not previously reported as SHP2 substrates, and (4) had commercial antibodies available for immunoprecipitation studies. Of these twenty proteins screened (Supplementary file 3), three that were reliably immunoprecipiated at endogenous abundance showed phosphosite enrichment after EGF stimulation in the presence of SHP099 as judged by Western blot with an anti-pY antibody. These three proteins are occludin, PLCγ2, and ARHGAGP35 (Figure 3A–C). Immunoprecipitation of pY-modified proteins with an anti-pY antibody in EGF-stimulated cells treated with SHP099 likewise showed that PLCγ2, occludin, and GRB2 accumulate pY in the presence of SHP099, as judged by Western blot (Figure 3—figure supplement 1). Because it was not possible to immunoprecipitate ARHGAP35 with either the anti-pY antibody or an antibody that specifically reognizes the pY1105 phosphosite, we immunoblotted anti-ARHGAP35 immunoprecipitates with the anti-pY1105 antibody to confirm that accumulation of this specific mark occurs when SHP2 is inhibited by SHP099 (Figure 3D,E). In addition, enzyme assays using purified SHP2 and the pY-containing peptides that contain the SHP099-enriched phsophosites identified in the proteomic studies confirm that SHP2 can robustly remove the phosphate mark from pY209 of GRB2, pY443 of occludin, pY1105 of ARHGAP35, and pY818 of PLCγ2, whereas neither a control peptide containing pY708 from CSF-1R nor a Class III peptide from TMEM134 that shows a protection-type pattern are robust SHP2 substrates (Figure 3F). Together, these data suggest that ARHGAP35, PLCγ2, and occludin are newly identified substrates for SHP2 in EGF-stimulated MDA-MB-468 cells. Figure 3 with 1 supplement see all Download asset Open asset Evaluation of occludin, ARHGAP35, and PLCγ2 as SHP2 substrates. MDA-MB-468 cells pre-treated with DMSO carrier or SHP099 (10 µM) for 2 hr were mock treated or stimulated with EGF (10 nM) for 10 min (A) or 30 min (B, C, and E). Total cell lysates were immunoprecipitated with occludin (A), PLCγ2 (B), or ARHGAP35 (C, E) antibodies and the eluted samples were subjected to Western blotting with the phosphotyrosine antibody pY1000 (A–C), pY1105-ARHGAP35 (E), and anti-protein antibodies as indicated (A–C, E). Input samples represent 1% of total cell lysate. Non-specific IgG was used as a negative control. (D) Plot of pY abundance as a function of treatment condition for Y1105 of ARHGAP35. (F) Dephosphorylation activity of full-length wild-type SHP2, activated with 6 µM bisphosphorylated IRS-1 peptide [SLNY(p)IDLDLVKdPEG8-LSTY(p)ASINFQK], toward synthetic phosphopeptides (OCLN_pY443, PLCG2_pY818, GRB2_pY209, ARHGAP35_1105, TMEM134_pY57, CSF-1R_pY708). Immunoprecipitation – Western blot assays are representative of at least two independent biological replicates. Figure 3—source data 1 Table showing TMT relative abundance values for Y1105 of ARHGAP35. https://cdn.elifesciences.org/articles/64251/elife-64251-fig3-data1-v2.xlsx Download elife-64251-fig3-data1-v2.xlsx Figure 3—source data 2 Table showing micromoles of phosphate released per minute for all phosphopeptides tested for dephosphorylation by IRS1-activated wild-type SHP2. Download inhibition of SHP2 results in reduced pY abundance at its motifs SHP099 allosterically the autoinhibited conformation of SHP2 et al., 2016), thereby not only the catalytic activity of the but also of its two SH2 domains to pY-containing Because the third pattern of response shows accumulation of pY after SHP2 is released from we performed a dynamic analysis of whether SHP2 directly sites in this class from dephosphorylation and whether this protection is upon SHP099 by Western Y659 of GAB1 and Y643 of GAB2 both show dramatic in pY abundance upon SHP099 treatment and their pY marks upon compound washout (Figure as judged by the phosphoproteomic data and an time by Western These data are consistent with prior work indicating that the SH2 domains of SHP2 can to the motifs of GAB1 and GAB2 to SHP2 et al., 2004; Cunnick et al., Figure 4 with 1 supplement see all Download asset Open asset SHP2 inhibition results in reduced pY abundance at its TMT intensities of GAB1 (A) and GAB2 (B) peptides showing dynamic changes in phosphorylation under and conditions. (C) MDA-MB-468 cells pre-treated with DMSO carrier or SHP099 (10 µM) for 2 hr were mock treated or stimulated with EGF (10 Total GAB1, total GAB2, GAB1 and GAB2 were analyzed by Western blot with both anti-protein and antibodies as a function of time after EGF Western blot results are representative of at least two independent biological replicates. Figure data 1 Table showing TMT relative abundance values for Y659 of Download Figure data 2 Table showing TMT relative abundance values for Y643 of Download To determine whether pY sites of GAB1 and GAB2 the same pattern of response in other cellular we also treated other cancer cell with SHP099. an EGFR-amplified cancer cell showed of GAB1 Y659 phosphorylation with SHP099 treatment under conditions of EGF stimulation (Figure supplement 1A). which an EGFR activating high levels of SHP099 treatment phosphorylation of this and washout of the drug the mark min (Figure supplement 1B). In addition, we tested whether of the SHP2 the effects of inhibition on the phosphorylation of Y659 of these we used a PTPN11 cell line prepared by (LaRochelle et al., stimulated with EGF, cells showed a in GAB1 levels when compared to and of SHP2 the level of (Figure supplement of cells with also showed an induction of the GAB1 mark that is upon treatment with SHP099 (Figure supplement stimulation of the cell receptor on cells with an antibody GAB1 and GAB2 and this induction was attenuated by SHP099 treatment (Figure supplement These findings show that the effect of SHP2 on the abundance of GAB1 and GAB2 pY marks is among a range of growth factor and antigen receptor signaling To determine how inhibition of SHP2 reduced pY abundance at these sites, we a of SHP2 and whether could phosphorylation in PTPN11 cells. We mutations that catalytic activity or both and catalytic activity We also protein that contain only the catalytic domain (PTP) or only the SH2 domains (Figure stimulated with EGF, and the all phosphorylation to a level similar to that of cells with wild-type SHP2 (Figure the SH2 or PTP were into cells endogenous SHP2, the PTP to whereas the SH2 was as active as wild-type SHP2 (Figure Figure with 3 supplements see all Download asset Open asset The SH2 domains of SHP2, but not its PTP domain, specific GAB1 and GAB2 pY (A) Schematic of and of SHP2 (B, SHP2 or SHP2 cells SHP2 were with or EGF stimulation (10 nM) for 10 min. were and subjected to Western blotting using and (D) MDA-MB-468 cells or SH2 domains pre-treated with SHP099 were stimulated with EGF and immunoblotted after using the Western blot results are representative of at least = 2 independent biological replicates. MDA-MB-468 cells SHP2 (see also the data from et al., we the SH2 or PTP in MDA-MB-468 cells and inhibited endogenous SHP2. These cell were stimulated with EGF and for and As expected, SHP099 treatment reduced the abundance of on GAB1, as well as that of on GAB1 which has been reported to to the N-SH2 domain of SHP2 et al., also showed reduced with the results from the SH2 but not the PTP was active in increasing pY abundance at these of GAB1 and GAB2 (Figure the SH2 these GAB1 and GAB2 marks even in the absence of EGF stimulation (Figure consistent with a function that is independent of the catalytic activity of the In addition, the of SHP2 protection of the GAB1 and GAB2 pY sites (Figure and of the key of each SH2 domain of the N-SH2 domain or of the C-SH2 phosphosite protection of these sites, as the (Figure Together, these findings show that the catalytic activity of SHP2 is not for the protection of the and marks on GAB1 and GAB2, and that the presence of tandem SH2 domains in SHP2 is to and on GAB1 and GAB2 from Figure 6 Download asset Open asset of and pY mutations on pY abundance at protected sites on GAB1 and (A) Schematic wild-type and of SHP2. (B) MDA-MB-468 cells or MDA-MB-468 cells wild-type or SHP2 proteins were pre-treated with SHP099 (10 The cells were then mock treated or stimulated with EGF (10 nM) for 10 min and immunoblotted after using the (C) Schematic wild-type and mutated of tandem of SHP2. (D) MDA-MB-468 cells or MDA-MB-468 cells wild-type or mutated tandem SH2 of SHP2 as indicated and were pre-treated with SHP099 (10 The cells were then mock treated or stimulated with EGF (10 nM) for 10 min and immunoblotted after using the Western blot results are representative of at least = 2 independent biological replicates. To whether this protection to other SHP2 proteins that show reduced pY abundance in the presence of SHP099, we a cell receptor in extracellular signaling et al., et al., contains an when phosphorylated at and can the tandem SH2 domains of SHP2 and phosphoproteomic data show that