1. Biochemistry and Chemical Biology
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Time-resolved phosphoproteomics reveals scaffolding and catalysis-responsive patterns of SHP2-dependent signaling

  1. Vidyasiri Vemulapalli
  2. Lily A Chylek
  3. Alison Erickson
  4. Anamarija Pfeiffer
  5. Khal-Hentz Gabriel
  6. Jonathan LaRochelle
  7. Kartik Subramanian
  8. Ruili Cao
  9. Kimberley Stegmaier
  10. Morvarid Mohseni
  11. Matthew J LaMarche
  12. Michael G Acker
  13. Peter K Sorger
  14. Steven P Gygi
  15. Stephen C Blacklow  Is a corresponding author
  1. Department of Cancer Biology, Dana-Farber Cancer Institute Boston, United States
  2. Department of Biological Chemistry & Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, United States
  3. Laboratory of Systems Pharmacology, Harvard Medical School, United States
  4. Department of Cell Biology, Harvard Medical School, United States
  5. Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Denmark
  6. Department of Pediatric Oncology, Dana Farber Cancer Institute, United States
  7. Novartis Institutes for Biomedical Research, United States
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Cite this article as: eLife 2021;10:e64251 doi: 10.7554/eLife.64251

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
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

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
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
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
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

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
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
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.

https://cdn.elifesciences.org/articles/64251/elife-64251-fig3-data2-v2.xlsx

Allosteric inhibition of SHP2 results in reduced pY abundance at its interaction motifs

SHP099 allosterically stabilizes the autoinhibited conformation of SHP2 (Chen et al., 2016), thereby not only inhibiting the catalytic activity of the enzyme, but also suppressing binding of its two SH2 domains to pY-containing motifs. Because the third pattern of response shows accumulation of pY after SHP2 is released from inhibition, we performed a dynamic analysis of whether SHP2 directly protects sites in this class from dephosphorylation and whether this protection is lost upon SHP099 binding by Western blot. Y659 of GAB1 and Y643 of GAB2 both show dramatic reductions in pY abundance upon SHP099 treatment and recover their pY marks upon compound washout (Figure 4), as judged by the phosphoproteomic data and confirmed across an extended time course by Western blot. These data are fully consistent with prior work indicating that the SH2 domains of SHP2 can bind to the bisphosphotyrosine-containing motifs of GAB1 (pY627/pY659) and GAB2 (pY614/pY643) to relieve SHP2 autoinhibition (Arnaud et al., 2004; Cunnick et al., 2001).

Figure 4 with 1 supplement see all
SHP2 inhibition results in reduced pY abundance at its interaction motifs.

(A, B) TMT signal-to-noise intensities of GAB1 pY659 (A) and GAB2 pY643 (B) peptides showing dynamic changes in phosphorylation under DMSO- (solid line) and SHP099-treated (dotted line) 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 nM). Total GAB1, total GAB2, GAB1 pY659, and GAB2 pY643 were analyzed by Western blot with both anti-protein and phosphospecific antibodies as a function of time after EGF addition. Western blot results are representative of at least two independent biological replicates.

Figure 4—source data 1

Table showing TMT relative abundance values for Y659 of GAB1.

https://cdn.elifesciences.org/articles/64251/elife-64251-fig4-data1-v2.xlsx
Figure 4—source data 2

Table showing TMT relative abundance values for Y643 of GAB2.

https://cdn.elifesciences.org/articles/64251/elife-64251-fig4-data2-v2.xlsx

To determine whether pY sites of GAB1 and GAB2 exhibit the same pattern of response in other cellular contexts, we also treated other EGFR-driven cancer cell lines with SHP099. KYSE520, an EGFR-amplified esophageal cancer cell line, showed loss of GAB1 Y659 phosphorylation with SHP099 treatment under conditions of EGF stimulation (Figure 4—figure supplement 1A). H1975 cells, which carry an EGFR activating mutation, displayed high levels of constitutive GAB2-Y643 phosphorylation. SHP099 treatment eliminated phosphorylation of this site, and washout of the drug restores the mark within 5 min (Figure 4—figure supplement 1B). In addition, we tested whether deletion of the SHP2 gene recapitulated the effects of chemical inhibition on the phosphorylation of Y659 of GAB1. For these studies, we used a PTPN11 knockout U2OS cell line prepared by CRISPR/Cas9 mediated genome editing (LaRochelle et al., 2018). When stimulated with EGF, SHP2-null U2OS cells showed a reduction in GAB1 pY659 levels when compared to parental cells, and reintroduction of SHP2 fully rescued the level of accumulated pY659 (Figure 4—figure supplement 1C). Stimulation of U2OS cells with PDGFββ also showed an induction of the GAB1 pY659 mark that is lost upon treatment with SHP099 (Figure 4—figure supplement 1D). Similarly, stimulation of the T cell receptor on Jurkat cells with an anti-CD3 antibody induced GAB1 and GAB2 phosphorylation, and this induction was attenuated by SHP099 treatment (Figure 4—figure supplement 1E). These findings show that the effect of SHP2 on the abundance of GAB1 and GAB2 pY marks is broadly shared among a range of growth factor and antigen receptor signaling systems.

To determine how inhibition of SHP2 reduced pY abundance at these sites, we generated a set of site-specific SHP2 mutants and asked whether they could rescue GAB1-pY659 phosphorylation in U2OS PTPN11 null cells. We constructed point mutations that abolish autoinhibition (E76K), eliminate catalytic activity (C459E), or disable both autoinhibition and catalytic activity (E76K/C459E). We also created protein truncations that contain only the catalytic domain (PTP) or only the SH2 domains (SH2) (Figure 5A). When stimulated with EGF, C459E, E76K, and the E76K/C459E double mutant, all restored GAB1-pY659 phosphorylation to a level similar to that of cells rescued with wild-type SHP2 (Figure 5B). When the SH2 or PTP constructs were introduced into U2OS cells lacking endogenous SHP2, the PTP fragment failed to rescue, whereas the SH2 fragment was as active as wild-type SHP2 (Figure 5C).

Figure 5 with 3 supplements see all
The SH2 domains of SHP2, but not its PTP domain, protect specific GAB1 and GAB2 pY marks.

(A) Schematic representation of wild-type, point mutants, and deletion constructs of SHP2 tested. (B, C) Parental-, SHP2 knockout-, or SHP2 knockout U2OS cells stably expressing various SHP2 ‘rescue’ constructs were cultured with or without EGF stimulation (10 nM) for 10 min. Cells were lysed and subjected to Western blotting using anti-pY659-GAB1 and anti-GAB1 antibodies. (D) MDA-MB-468 cells stably expressing wild-type, PTP, or SH2 domains pre-treated with SHP099 were stimulated with EGF and immunoblotted after lysis using the specified antibodies. Western blot results are representative of at least n = 2 independent biological replicates.

Since MDA-MB-468 cells cannot survive without SHP2 (see also the gene essentiality data from avana_public_18Q2 library, Project Achilles; Meyers et al., 2017), we stably expressed the SH2 or PTP fragments in parental MDA-MB-468 cells and chemically inhibited endogenous SHP2. These cell lines were stimulated with EGF and probed for phospho-GAB1 and phospho-GAB2. As expected, SHP099 treatment significantly reduced the abundance of pY659 on GAB1, as well as that of pY643 on GAB2. GAB1 pY627, which has been reported to bind to the N-SH2 domain of SHP2 (Cunnick et al., 2001), also showed reduced phosphorylation. Consistent with the results from U2OS cells, the SH2 fragment, but not the PTP fragment was active in increasing pY abundance at these positions of GAB1 and GAB2 (Figure 5D). Indeed, the SH2 fragment stabilizes these GAB1 and GAB2 phospho marks even in the absence of EGF stimulation (Figure 5D), consistent with a dominant protective function that is independent of the catalytic activity of the enzyme. In addition, the SHP099-resistant variant of SHP2 (T253M/Q257L) rescues protection of the GAB1 and GAB2 pY sites (Figure 6A,B), and point mutation of the key arginine residue of each SH2 domain (R32M of the N-SH2 domain or R138M of the C-SH2 domain) suppresses phosphosite protection of these sites, as does the R32M/R138M double mutant (Figure 6C,D). Together, these findings show that the catalytic activity of SHP2 is not required for the protection of the pY659 and pY643 marks on GAB1 and GAB2, respectively, and argue that the presence of tandem SH2 domains in SHP2 is needed to bind and protect pYs on GAB1 and GAB2 from phosphatase-mediated dephosphorylation.

Effects of SHP099-resistance and pY binding-site mutations on pY abundance at protected sites on GAB1 and GAB2.

(A) Schematic illustrating wild-type and SHP099-resistant forms of SHP2. (B) Parental MDA-MB-468 cells or MDA-MB-468 cells stably expressing wild-type or T253M/Q257L SHP2 proteins were pre-treated with SHP099 (10 µM). The cells were then mock treated or stimulated with EGF (10 nM) for 10 min and immunoblotted after lysis using the specified antibodies. (C) Schematic illustrating wild-type and mutated forms of SH2-only tandem fragments of SHP2. (D) Parental MDA-MB-468 cells or MDA-MB-468 cells stably expressing wild-type or mutated tandem SH2 fragments of SHP2 (SH2) as indicated (R32M, R138M, and R32M/R138M, denoted DM here) were pre-treated with SHP099 (10 µM). The cells were then mock treated or stimulated with EGF (10 nM) for 10 min and immunoblotted after lysis using the specified antibodies. Western blot results are representative of at least n = 2 independent biological replicates.

To assess whether this protection mechanism applies to other SHP2 interacting proteins that show reduced pY abundance in the presence of SHP099, we studied MPZL1, a cell surface receptor involved in extracellular matrix-induced signaling (Beigbeder et al., 2017; Zhao et al., 2002). MPZL1 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) that, when doubly phosphorylated at Y241 and Y263, can bind the tandem SH2 domains of SHP2 (Zhao and Zhao, 2000). Our phosphoproteomic data show that basal pY abundance at these sites is greatly reduced by SHP099 treatment and that the dependence of pY modifications on SHP2 at these two sites occurs independent of EGF (Figure 5—figure supplement 1A,B). Using immunoblot assays, we found that the tandem SH2 domains alone are also sufficient to protect the phosphate groups on Y241 and Y263 of MPZL1 (Figure 5—figure supplement 1C). It is also notable that the two pY sites on the SHP2 C-terminal tail exhibit a protection-type pattern in the presence of SHP099. Because the allosteric inhibitor locks SHP2 in the closed conformation, we speculate that the inhibitor interferes with membrane recruitment of SHP2 by preventing the SH2 domains from binding to pY residues deposited at membrane proximal sites by activated kinases and that removal of inhibitor permits membrane recruitment to sites of EGFR activation, phosphorylation of the SHP2 C-terminal tail, and subsequent GRB2 recruitment.

Multiple studies have reported that GAB1, GAB2, and MPZL1 can be phosphorylated by Src family kinases (SFKs) (Chan et al., 2003; Kong et al., 2003; Kusano et al., 2008). Indeed, MDA-MB-468 cells treated with an SFK inhibitor (saracatinib/AZD0530) showed greatly reduced modification of GAB1-Y659 and GAB2-Y643 (Figure 5—figure supplement 2A), suggesting that SFK activity is required to phosphorylate the SHP2 binding sites on GAB1 and GAB2. SHP099, however, had no detectable effect on the abundance of pY at Y416 of the activation loop of SFKs (Figure 5—figure supplement 2B), or on the abundance of pY at Y527, which maintains SFKs in their autoinhibitory conformation (Roskoski, 2005). Although it is possible that SFK activity in the GAB complex is not reflected by assessment of bulk SFK activity in the whole-ell lysate, when combined with the phosphosite protection results from the studies with various forms of SHP2 above, these data suggest that SHP2 acts to increase the half-life of pY modifications at sites on GAB1 and GAB2 that are phosphorylated by SFKs and then bound by SHP2.

SHP2 is required for membrane localization of GAB1

To determine whether activation of SHP2 is required for recruitment of GAB1 to EGFR at the plasma membrane, we used a proximity ligation assay (PLA). MDA-MB-468 cells were mock treated or stimulated with EGF in the presence of DMSO or SHP099, fixed in paraformaldehyde, and probed for co-localization of GAB1 and EGFR. Under serum-starved (basal) conditions, the PLA signal was minimal and unaffected by SHP2 inhibition with SHP099. In contrast, within 2 min of EGF addition, cells treated with DMSO alone displayed a strong PLA signal indicative of GAB1 and EGFR co-localization, whereas SHP099 treatment reduced the number of binding events by 85% (p<0.05; Figure 5—figure supplement 3). These results suggest that SHP2 is required to assemble the EGFR-GAB1 signaling hub crucial for MAPK and PI3K signaling.

Discussion

Here, we performed quantitative phosphoproteomics to determine how the responses of cells to EGFR stimulation are modulated by inhibition of SHP2. We identified over 400 pY sites that exhibit increases or decreases in phosphorylation in response to SHP099 addition and washout. The dynamics of these changes in pY abundance reveal six distinct response signatures that provide a number of insights into how cancer cells depend on SHP2 in RTK signaling. Three of the six patterns identified by hierarchical clustering correspond to EGFR-dependent pY abundance changes that are also modulated by SHP2. These patterns include an early SHP2 substrate-like pattern (early responders that increase in abundance upon SHP2 inhibition), a late SHP2 substrate-like pattern (late responders also increased upon SH2 inhibition), and a pattern in which pY levels are unexpectedly decreased when SHP2 is inhibited (protected sites).

Among proteins that exhibit a substrate-like pattern, occludin, ARHGAP35, and PLCγ2 were also confirmed to have specific pYs that are SHP2 substrate sites, opening up new avenues for future study of the downstream effects of SHP2 on cell migration and adhesion. A number of other early responder sites lie on proteins that transmit information from activated RTKs to Ras/MAPK and PI3K signaling cascades. For example, the pY209 mark on the adaptor protein Grb2, which inhibits Sos binding and downstream Ras activation (Chardin et al., 1993; Li et al., 2001), rapidly accumulates upon SHP2 inhibition, suggesting that one important role of SHP2 in these cells is to remove an inhibitory mark and promote Grb2-dependent protein–protein interactions that drive immediate early signaling. Similarly, the adapter Crk also has two early responder pY sites at positions 108 and 136 that accumulate upon SHP2 inhibition; their removal by SHP2 may promote the ability of Crk to propagate the EGFR signal. Likewise, the PI3K regulatory subunits PI3KR1 and PI3KR2 accumulate pY marks in their inter-SH2 (iSH2) domains at analogous positions between residues 452–470 that may suppress effector function when SHP2 is inhibited, and PI3KR3 also has a pY residue in its iSH2 domain that also shows a substrate-like response at later timepoints. Not surprisingly, SHP2 inhibition also affects the pY abundance on numerous proteins involved in cell adhesion and migration, as well as on cytoskeletal components (Figure 2—figure supplement 2D).

Our data also reveal many proteins with SHP2-dependent changes in pY abundance that function in cellular processes previously unlinked to SHP2. These proteins include effectors of distal signaling responses such as phospholipase C enzymes (PLCγ1 and PLCγ2). Also noteworthy are the late-response, substrate-like patterns that predominate among proteins implicated in mRNA processing and endocytosis factors (hnRNPA2B1, hnRNPA3, EPN1/2, RAB10, FNBP1L, ITSN2, STX4) (Figure 2—figure supplement 2D). Dissecting the role of SHP2 in these events should be a fertile area of future investigation.

Strikingly, the most frequent change in pY pattern following SHP2 inhibition is not substrate-like, but rather one in which pY levels fall. Others have also recently reported that binding of SHP2 can prevent dephosphorylation of certain pY sites in the response to stimulation of other RTKs (Batth et al., 2018). This pattern of response is seen at pY residues on proteins recruited to the EGFR signaling hub, such as GAB1, GAB2, as well as on EGFR and SHP2 itself. To better understand this mode of pY regulation, we studied SHP2-dependent protection of pY sites on GAB1 and GAB2, adapters that induce SHP2 binding and activation upon tyrosine phosphorylation (Arnaud et al., 2004; Crouin et al., 2001; Cunnick et al., 2001). Binding and mutagenesis studies clearly demonstrate that the tandem SH2 domains of SHP2 shield pY binding sites on GAB1 and GAB2 from dephosphorylation. Also intriguing is the SHP2-mediated protection of nearby pairs of pY residues on several proteins, including MPZL1-pY241/pY263, a fibronectin-activated adhesion protein that can assemble into complexes containing SHP2 and Grb2 in a pY-dependent manner (Figure 5—figure supplement 1 and Beigbeder et al., 2017). In fact, decreased phosphoabundace is observed on multiple proteins harboring nearby tyrosine residues (Figure 7—figure supplement 1), all of which may experience protection of their pY marks by the tandem SH2 domains of SHP2. The protection of pY sites by SHP2 highlights the scaffolding role of SHP2 in stimulating pY engagement by SH2 domains at sites of RTK activation (Figure 7). SHP2 scaffolding activity is particularly relevant to Noonan and LEOPARD syndromes, both of which result from missense SHP2 mutations. Though distinct, the two syndromes have overlapping clinical features. Mutations found in Noonan syndrome destabilize the autoinhibited form of the enzyme without substantially affecting the intrinsic catalytic activity of the phosphatase domain, leading to increased basal phosphatase activity, elevated scaffolding activity, and downstream induction of ERK (Araki et al., 2004; Niihori et al., 2005). LEOPARD syndrome mutations also destabilize the autoinhibited conformation of the enzyme, enhancing scaffolding activity, but in addition cripple the catalytic activity of the phosphatase domain. The increased preference for the open conformation in both Noonan and LEOPARD syndrome mutations promotes the protective ‘arm’ of SHP2 signaling, whereas only Noonan syndrome mutations retain the catalytic activity needed to drive the substrate-dependent features of signaling. The work reported here shows how central the scaffolding function is at the EGFR signaling hub and in the engagement of pY sites on GAB1 and GAB2 at the site of activation, with the phosphatase-dependent events, through adapter proteins like Grb2 and key signaling nodes like PI3K, required for full propagation of a growth factor induced signal to downstream effectors.

Figure 7 with 1 supplement see all
Model highlighting coordinated scaffolding and catalytic activities of SHP2.

(A) Schematic model showing pY mark protection by stable engagement of a partner protein (e.g. GAB1) with the tandem SH2 domains of SHP2. (B) In the presence of SHP099, pY marks on putative SHP2 interactors are vulnerable to dephosphorylation, and SHP099 is not recruited to sites of RTK signaling. It is also possible that deposition of certain phosphate marks is SHP2 dependent (lower panel).

Materials and methods

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Recombinant DNA reagentpCMV-SHP2-WTAddgeneRRID:Addgene_8381, plasmid # 8381
Recombinant DNA reagentmigR1-IRES-GFPAddgeneRRID:Addgene_27490, plasmid # 27490
Chemical compound, drugSHP2 inhibitor (SHP099)DC chemicalsCatalog # DC9737
Chemical compound, drugSrc inhibitor (AZD0530)Dr. Nathanael Gray (Dana-Farber Cancer Institute)
AntibodyMouse monoclonal CD3 (UCHT1)Thermo Fisher ScientificRRID:AB_468857; Catalog # 16-0038-8510 µg/ml for TCR stimulation of Jurkat cells
AntibodyRabbit monoclonal Phospho-Tyr-1000Cell Signaling TechnologyRRID:AB_2687925; Catalog # 8954Western blot (1:2000);
Immunoprecipitation (1:100)
AntibodyRabbit polyclonal Phospho-Thr202/Tyr204-Erk1/2Cell Signaling TechnologyRRID:AB_331646; Catalog # 9101Western blot (1:2000)
AntibodyRabbit polyclonal Erk1/2Cell Signaling TechnologyRRID:AB_330744; Catalog # 9102Western blot (1:2000)
AntibodyRabbit polyclonal GAB1-pY659Cell Signaling TechnologyRRID:AB_2798014; Catalog # 12745Western blot (1:1000)
AntibodyRabbit polyclonal GAB1-pY627Cell Signaling TechnologyRRID:AB_2305002; Catalog # 3231Western blot (1:500)
AntibodyRabbit polyclonal GAB2-pY643Thermo Fisher ScientificRRID:AB_2554386; Catalog # PA5-37778Western blot (1:1000)
AntibodyRabbit polyclonal GAB1Cell Signaling TechnologyRRID:AB_2304999; Catalog # 3232Western blot (1:2000)
AntibodyRabbit monoclonal GAB2Cell Signaling TechnologyRRID:AB_10698601; Catalog # 3239Western blot (1:2000)
AntibodyRabbit polyclonal pY263-MPZL1Cell Signaling TechnologyRRID:AB_10715080; Catalog # 5543Western blot (1:1000)
AntibodyRabbit monoclonal pY241-MPZL1Cell Signaling TechnologyRRID:AB_10891793; Catalog # 8131Western blot (1:1000)
AntibodyRabbit polyclonal MPZL1Cell Signaling TechnologyRRID:AB_1904189; Catalog # 4157Western blot (1:2000)
AntibodyRabbit polyclonal pY1105-ARHGAP35Thermo Fisher ScientificRRID:AB_2553676; Catalog # PA5-36713Western blot (1:1000)
AntibodyRabbit polyclonal ARHGAP35Cell Signaling TechnologyRRID:AB_2115740; Catalog # 2562Western blot (1:2000); Immunoprecipitation (1:100)
AntibodyRabbit monoclonal SHP2Cell Signaling TechnologyRRID:AB_2174959; Catalog # 3397Western blot (1:500)
AntibodyRabbit polyclonal SHP2BethylRRID:AB_1040019; Catalog # A301-544AWestern blot (1:1000)
AntibodyMouse monoclonal SHP2Santa CruzRRID:AB_628252; Catalog # sc-7384Immunoprecipitation (2 µg antibody per500 µg cell lysate)
AntibodyMouse monoclonal PLCG2Santa CruzRRID:AB_628120; Catalog # sc-5283Western blot (1:500); Immunoprecipitation (2 µg antibody per500 µg cell lysate)
AntibodyRabbit polyclonal OCLNBethylRRID:AB_2631690; Catalog # A305-297AWestern blot (1:1000); Immunoprecipitation (5 µg antibody per1 mg cell lysate)
AntibodyMouse monoclonal GRB2BD Transduction LaboratoriesRRID:AB_397518; Catalog # 610112Western blot (1:2000)
Cell line (H. sapiens)MDA-MB-468ATCCRRID:CVCL_0419; HTB-132
Cell line (H. sapiens)KYSE-520DSMZRRID:CVCL_1355; ACC 371
Cell line (H. sapiens)U2OSATCCRRID:CVCL_0042; HTB-96
Cell line (H. sapiens)JurkatATCCRRID:CVCL_0367; TIB-152
Cell line (H. sapiens)NCI-H1975ATCCRRID:CVCL_UE30; CRL-5908
Cell line (H. sapiens)SHP2 knockout U2OS cell lineLaRochelle et al., 2018
Peptide, recombinant proteinHuman EGFGibcoPHG0311
Peptide, recombinant proteinHuman PDGF-BBPeprotechCatalog # 10771–922
Peptide, recombinant proteinIRS1_pY1172–PEG–pY1222 peptideThis paperSLN{pY}IDLDLVK–dPEG8–LST{pY}ASINFQK; (custom synthesized by Genscript)
Peptide, recombinant proteinOCLN_pY443 peptideThis paperTGLQE{pY}KSLQS; (custom synthesized by Genscript)
Peptide, recombinant proteinPLCG2_pY818 peptideThis paperTRIQQ{pY}FPSNY
(custom synthesized by Genscript)
Peptide, recombinant proteinGRB2_pY209 peptideThis paperMFPRN{pY}VTPVN;
(custom synthesized by Genscript)
Peptide, recombinant proteinARHGAP35_pY1105 peptideThis paperEENI{pY}SVPHDS;
(custom synthesized by Genscript)
Peptide, recombinant proteinCSF-1R_pY708 peptideThis paperIHLEKK{pY}VRRDSGF; (custom synthesized by Genscript)
Peptide, recombinant proteinTMEM134_pY57 peptideThis paperKQSRLR{pY}QNLEND; (custom synthesized by Genscript)
Peptide, recombinant proteinLysyl Endopeptidase, Mass Spectrometry Grade (Lys-C)FUJIFILM Wako Pure Chemical CorporationCatalog # 125–05061Protein cleavage for mass spectrometry (10 µg per 1 mg total protein)
Peptide, recombinant proteinSequencing Grade Modified TrypsinPromegaCatalog # V511CProtein cleavage for mass spectrometry (10 µg per 1 mg total protein)
Peptide, recombinant proteinWild-type SHP2 purified proteinLaRochelle et al., 2016
Commercial assay or kitQuikChange II Site-Directed Mutagenesis kitAgilentCatalog # 200523
Commercial assay or kitBCA protein assayPierceCatalog # 23225
Commercial assay or kitMalachite Green assay kitSigma–AldrichCatalog # MAK307
Commercial assay or kitPLA kitSigma–AldrichCatalog # DUO92014
OtherNTA Magnetic Agarose beadsQiagenCatalog # 36113
OtherTMT 11-plex reagentThermo ScientificCatalog # A37725
Software, algorithmGraphPad Prism 9.0N/ARRID:SCR_002798; https://www.graphpad.com/scientific-software/prism/
Software, algorithmR 3.6.3N/ARRID:SCR_001905; https://www.r-project.org/
Software, algorithmCellProfilerN/ARRID:SCR_007358; https://cellprofiler.org/
Software, algorithmReactome databaseN/ARRID:SCR_003485; http://software.broadinstitute.org/gsea/msigdb/annotate.jsp
Software, algorithmGene set enrichment analysisMootha et al., 2003; Subramanian et al., 2005RRID:SCR_003199
Software, algorithmHierarchical clusteringMATLABRRID:SCR_001622; https://www.mathworks.com/products/matlab.html
Software, algorithmPrincipal component analysisPythonRRID:SCR_008394; https://www.python.org/
Software, algorithmDependence classificationThis paperSee ‘Dependence classification of phosphorylation site time courses’ in Methods
Software, algorithmLogo analysisCrooks, 2014RRID:SCR_010236

Plasmids, compounds, and ligands

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Constructs used to rescue SHP2 knockout U2OS cells were obtained by cloning the SHP2 cDNA from pCMV-SHP2-WT (RRID: Addgene_8381) into the migR1-IRES-GFP vector (RRID:Addgene_27490). Point mutants (E76K, C459E, and DM) were generated from the parental SHP2-migR1 construct using site-directed mutagenesis (QuikChange II, Agilent, Catalog # 200523). SH2-only and phosphatase-only mutants were generated by cloning the PTP (220–525 aa) and N-SH2-C-SH2 (1–219 aa) fragments, respectively, into the migR1-IRES-GFP vector. SHP099 was obtained commercially from DC chemicals (Catalog # DC9737). AZD0530 (Saracatinib) was a gift from Dr. Nathanael Gray (Dana-Farber Cancer Institute). The CD3 monoclonal antibody (UCHT1) was purchased from Thermo Fisher Scientific (RRID:AB_468857). Recombinant human EGF and PDGF-BB were purchased from Gibco (Catalog # PHG0311) and Peprotech (Catalog # 10771–922), respectively.

Antibodies

Antibodies used in this study were obtained commercially from the following sources: Phospho-Tyr-1000 (RRID:AB_2687925), Phospho-Thr202/Tyr204-Erk1/2 (RRID:AB_331646), Erk1/2 (RRID:AB_330744), GAB1-pY659 (RRID:AB_2798014), GAB1-pY627 (RRID:AB_2305002), GAB2-pY643 (RRID:AB_2554386), GAB1 (RRID:AB_2304999), GAB2 (RRID:AB_10698601), pY263-MPZL1 (RRID:AB_10715080), pY241-MPZL1 (RRID:AB_10891793), MPZL1 (RRID:AB_1904189), pY1105-ARHGAP35 (RRID:AB_2553676), ARHGAP35 (RRID:AB_2115740), SHP2 (RRID:AB_2174959, RRID:AB_1040019, RRID:AB_628252), PLCG2 (RRID:AB_628120), OCLN (RRID:AB_2631690), and GRB2 (RRID:AB_397518).

Cell culture and generation of stable cell lines

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All the parental cell lines used in this study (MDA-MB-468 [RRID:CVCL_0419], KYSE520 [RRID:CVCL_1355], U2OS [RRID:CVCL_0042], Jurkat [RRID:CVCL_0367], and NCI-H1975 [RRID:CVCL_UE30]) were purchased from ATCC. The SHP2 knockout U2OS cell line was created previously (LaRochelle et al., 2018). To generate rescued SHP2-null U2OS lines, knockout cells were infected with retrovirus harboring the wild-type or mutant SHP2 cDNAs and FACS sorted for GFP-positive cells. MDA-MB-468 parental cells stably expressing full-length GAB1-GFP, GAB2-GFP, SHP2, PTP, and N-SH2-C-SH2 cDNAs were also generated similarly. U2OS cell lines were grown in McCoy’s 5A media with 10% fetal bovine serum (FBS). MDA-MB-468 cells were cultured in Leibovitz’s L-15 media with 10% FBS without CO2. All other cell lines (KYSE520, Jurkat, and NCI-H1975) were maintained in RPMI-1640 supplemented with 10% FBS at 5% CO2. Cell lines were acquired from sources provided in the key resources table. All cell lines used in this study tested negative for mycoplasma contamination.

Ligand stimulation

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For ligand stimulation experiments analyzed by Western blotting, cells were seeded at 70% confluence in 10 cm petri dishes (Nunc, Thermo Fisher Scientific), serum starved for 24 hr, and treated with DMSO carrier or SHP099 (10 µM) for 2 hr before stimulation with ligands (EGF [10 nM], PDGF-BB [5 nM], or anti-CD3 [10 µg/ml]) for the indicated time periods. Cells were then quickly washed with ice-cold phosphate-buffered saline (PBS) twice, lysed in Ripa buffer (25 mM Tris–HCl [pH 7.6], 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 2 mM EDTA) with protease and phosphatase inhibitors (cOmplete Mini and PhosSTOP, Roche) and analyzed by Western blotting. For all Western blot assays, the figures shown are representative results from at least two independent experiments.

Sample preparation for phosphoproteomics

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MDA-MB-468 cells were seeded in 15 cm cell culture dishes (Corning) in full growth media (Leibovitz’s L-15 with 10% FBS and 1% penicillin/streptomycin). At 80% confluence, cells were washed twice with warm HBSS buffer and incubated in serum-free media for 24 hr. On the following day, cells were pre-treated with DMSO or SHP099 (10 µM) for 2 hr prior to ligand stimulation. As outlined in Figure 1A, DMSO and SHP099 groups were left untreated (0 min) or stimulated with EGF (10 nM) for 5, 10, or 30 min. The SHP099 washout group was stimulated with EGF (10 nM) for 10 min, washed three times with warm HBSS, and stimulated with EGF again for 5, 10, or 30 min. To terminate stimulation, cells were immediately washed with ice-cold PBS, harvested, centrifuged, flash frozen in liquid N2 and stored at −80°C until all 11 samples were prepared. Cell pellets were lysed in lysis buffer (2% SDS, 150 mM NaCl, 50 mM Tris [pH 8.5–8.8]) containing protease and phosphatase inhibitors (cOmplete Mini, PhosSTOP, Roche; 2 mM Sodium Orthovanadate, NEB), sonicated to shear chromatin, and centrifuged to remove cellular debris. A bicinchoninic acid (Pierce, Catalog # 23225) assay was performed according to manufacturer’s instructions and lysates were normalized to a protein concentration of 1 mg/ml, reduced with DTT (5 mM), alkylated with iodoacetamide (14 mM), and quenched with further addition of DTT (5 mM). Total protein (1 mg) was precipitated with methanol-chloroform, reconstituted in 8 M urea at pH 8.5, sonicated, diluted to 4 M urea, and digested with Lys-C (FUJIFILM Wako, Catalog # 125–05061) overnight and Trypsin (Promega, Catalog # V511C) for 6 hr at an enzyme-to-protein ratio of 1:100. The percentage of missed cleavages was monitored using aliquots from a few representative samples (total 3 µg) that were desalted and analyzed by mass spectrometry. Digests were acidified with 2% formic acid and desalted using C18 Sep-Pak cartridges (WAT054960, Waters). Samples were dried by vacuum centrifugation, resuspended in 80% acetonitrile/0.15% trifluoroacetic acid (TFA), and normalized to 1 mg/ml for enrichment of phosphopeptides.

Phosphopeptide enrichment

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Phosphopeptides were enriched using IMAC with NTA Magnetic Agarose beads (Qiagen, Catalog # 36113) stripped of Ni2+ and reloaded with Fe(III). Five hundred microliters of beads were incubated in 1 ml EDTA (40 mM) for 30 min at room temperature to remove bound Ni2+, followed by three washes with 1 ml of HPLC-grade water. The beads were then charged with iron using FeCl3 (100 mM) for 30 min at room temperature. Excess iron was removed by washing the beads three times with water followed by acidification with 80% acetonitrile/0.15% TFA. Peptides (1 mg per sample) were incubated with the IMAC beads for 30 min at room temperature. Phosphopeptide-bound beads were washed with 1 ml of 80% acetonitrile/0.15% TFA to remove non-specifically bound peptides. Phosphopeptides were eluted with 300 µl of 50% acetonitrile/0.7% NH4OH, acidified with 4% formic acid, immediately vacuum centrifuged to dryness, and subjected to desalting using SOLA HRP 10 mg Sep-Pak cartridges (Thermo Fisher).

TMT labeling

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Prior to isobaric labeling, phosphopeptides were reconstituted in 200 mM EPPS (pH 8.3) and anhydrous acetonitrile to 30% (v/v). Each sample was labeled with 5 µl of a TMT 11-plex reagent (20 µg/µl; A37725, Thermo Scientific) for 90 min at room temperature. The reactions were quenched with 5 µl of 5% hydroxylamine for 15 min, combined, acidified with TFA to 1.0% (v/v), desalted using 10 mg SOLA Sep-Pak cartridges (Thermo Fisher), and lyophilized for 2 days to remove residual TFA.

Phosphotyrosine immunoaffinity purification

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The pY antibody (p-Tyr-1000, Cell Signaling Technology) was coupled with agarose beads one day prior to the immunoaffinity purification (IAP) as follows. A Protein A agarose bead slurry (Sigma–Roche) (60 µl) was washed four times with 1 ml of cold PBS. The p-Tyr-1000 antibody (Cell Signaling Technology) (30 µl) was gently mixed with 1.5 ml PBS and added onto the washed agarose beads and incubated on a rotator overnight at 4°C. The beads were washed four times in 1 ml of cold PBS to remove excess uncoupled antibody. Lyophilized peptides were reconstituted with 500 µl of IAP buffer (50 mM MOPS/NaOH pH 7.2, 10 mM Na2HPO4, 50 mM NaCl) and incubated with antibody conjugated beads for 2 hr at 4°C with gentle rotation. The non-specific binding phosphopeptide (phospho-serine and -threonine peptides) flow through was collected after centrifugation and stored at −80°C. The pY-bound peptides were washed once with 1 ml of IAP buffer and transferred into a 0.2 µM filter spin column followed by two 400 µl washes with cold HPLC-grade water. pY peptides were eluted twice with 75 µl of 100 mM formic acid. The pY eluate was desalted using a C18 StageTip, dried by vacuum centrifugation, and reconstituted in 3% acetonitrile/0.5% formic acid for MS analysis.

Mass spectrometry

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Phosphoproteomic mass spectrometric data were acquired on an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) equipped with a Proxeon EASY-nLC 1000 liquid chromatography system (Thermo Fisher Scientific). Phosphopeptides were separated on a 75 µm inner diameter microcapillary column packed with ~35 cm Sepax GP-C18 resin (1.8 µm, 150 A, Thermo Fisher Scientific). Phosphopeptides (~2 µg) were separated using a 2 hr gradient of acetonitrile in 0.125% formic acid.

The phosphoproteome MS analysis scan sequence began with the collection of an FTMS1 spectrum (120,000 resolution with mass range 400–1400 Th). The top 10 most intense ions were selected for MS/MS and fragmented via collision-induced dissociation (CID, CE = 35%) with a maximum injection time of 200 ms and an isolation window of 0.5 Da. FTMS3 precursors were fragmented by high-energy collision-induced dissociation (HCD, CE = 55%) and analyzed at 50,000 resolution (200 Th) with a maximum ion injection time of 300 ms and an isolation window of 1.2. The MultiNotch MS3-based TMT method was applied as described in McAlister et al., 2014.

Data processing

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Mass spectra were processed using a SEQUEST-based software pipeline. A modified version of ReAdW.exe was used to convert spectra (.raw) to mzXML. All spectra were searched against a database containing the human proteome downloaded from Uniprot (February 4, 2014) and common contaminating protein sequences. Database searches were performed using a peptide mass tolerance of 50 ppm and a fragment ion tolerance of 0.9 Da. TMT on lysine residues and peptide N termini (+229.163 Da) and carbamidomethylation of cysteine residues (+57.021 Da) were fixed modifications, while oxidation of methionine residues (+15.995 Da) and phosphorylation of serine, threonine, and tyrosine residues were set as a variable modification (+79.966 Da).

Peptide-spectrum matches were filtered by linear discriminant analysis to a false discovery rate (FDR) of 2% at the peptide level based on matches to reversed sequences. Linear discriminant analysis considered the following parameters: XCorr, ΔCn, missed cleavages, adjusted PPM, peptide length, fraction of ions matched, charge state, and precursor mass accuracy. Filtered peptides were collapsed further to a final protein-level FDR of 2%. Phosphopeptides were quantified from MS3 scans after filtering with a total TMT reporter signal-to-noise ratio > 200 and isolation specificity at 0.5. Localization of phosphorylation sites was determined using AScore (Huttlin et al., 2010), and sites with an AScore > 13 were selected for further analysis.

Dependence classification of phosphorylation site time courses

Each phosphorylation time course was classified according to its response to (1) EGF stimulation and (2) SHP2 inhibition. This analysis was performed using the union of the two biological replicates, averaging measurements for sites that were found in the intersection of replicates. EGF classes were defined based on phosphorylation dynamics in control cells only. Significant increases and decreases were defined as changes of at least 1.5-fold relative to time 0 (unstimulated cells). The classes comprised the following: (1) fast increase (increased phosphorylation within 5 min of EGF stimulation), (2) medium increase (increased phosphrylation within 10 min), (3) slow increase (increased phosphorylation within 30 min), (4) neutral (no change at any timepoint), and (5) decrease (decreased phosphorylation by any point in the time course).

EGF classes

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NameEarliest timepoint where
change is detected
Fast increase5 min
Medium increase10 min
Slow increase30 min
Neutraln/a
DecreaseAny

Classes of SHP2 responses were defined based on comparison between control cells and cells treated with SHP099 for two hours prior to EGF stimulation. The classes comprised the following: (1) pre-stimulation negative (increased phosphorlyation in SHP099-treated cells compared to control cells, prior to EGF addition), (2) post-stimulation negative (increased phosphorylation in SHP099-treated cells at any point after EGF addition), (3) neutral (no difference between treatment and control at any timepoint), (4) pre-stimulation positive (decreased phosphorylation in SHP099-treated cells compared to control cells, prior to EGF addition), and (5) post-stimulation positive (decreased phosphorylation in SHP099-treated cells at any point after EGF stimulation).

SHP2 classes

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NameChange with
SHP099 treatment
Timing of change
Pre-stimulation negativeIncreaseBefore EGF addition
Post-stimulation negativeIncreaseAny timepoint after EGF addition, but not before
NeutralNonen/a
Pre-stimulation positiveDecreaseBefore EGF addition
Post-stimulation positiveDecreaseAny timepoint after EGF addition, but not before

Bioinformatic analysis

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Volcano plots were generated for all sites that were detected in either of the biological replicates. For each site at each timepoint (5 min, 10 min, and 30 min), the mean of [SHP099/DMSO] and [SHP099/WO] fold-changes was plotted against –log10(p-value). Significantly altered phosphorylation sites were visualized based on their membership in the Reactome database (http://software.broadinstitute.org/gsea/msigdb/annotate.jsp, RRID:SCR_003485). Gene set analysis was performed using publicly available GSEA tools (RRID:SCR_003199) (Mootha et al., 2003; Subramanian et al., 2005), which were used to compute the overlap between detected proteins and the REACTOME pathway database. Hierarchical clustering was performed using the clustergram function of MATLAB (RRID:SCR_001622), with a Euclidean distance metric.

Immunoprecipitation assays

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MDA-MB-468 cells were seeded at 80% confluence in 10 cm dishes (Nunc, Thermo Fisher Scientific), serum starved for 24 hr, and treated with DMSO carrier or SHP099 (10 µM) for 2 hr before stimulation with EGF (10 nM) for 5 min, 10 min, or 30 min. Cells were then quickly washed with ice-cold PBS, lysed in 1 ml of RIPA buffer (25 mM Tris–HCl (pH 7.6), 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 2 mM ethylenediaminetetraacetic acid [EDTA]) containing protease and phosphatase inhibitors (cOmplete Mini and PhosSTOP, Roche) on ice for 5 min and sonicated briefly to dissolve the pellet. After centrifugation at 16,000 g for 20 min, the lysates were pre-cleared with protein A/G agarose (20421, Pierce) for 1 hr at 4°C. Pre-cleared lysates were incubated with the specified antibodies overnight at 4°C. Immunoprecipitates were then pulled down by incubating lysates with protein A/G agarose for 1 hr at 4°C, washed four times with ice-cold RIPA buffer, eluted in protein loading buffer, and analyzed by Western blotting. For all immunoprecipitation – Western blotting assays, the figures shown are representative results from at least two independent experiments.

In vitro phosphate release assay

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Phosphate released from synthetic phosphopeptide substrates by SHP2 was measured using Malachite Green assay (MAK307, Sigma–Aldrich). The dephosphorylation reaction contained 50 nM wild-type SHP2(1–525), 6 µM bisphosphorylated IRS1 ligand for activating SHP2, and varying concentrations of phosphopeptide substrates (0–0.5 mM) in a final volume of 40 µl buffer (60 mM HEPES [pH 7.2], 75 mM KCl, 75 mM NaCl, 1 mM EDTA, 0.05% Tween 20, and 2 mM DTT). The reactions were incubated at room temperature for 5 min in a 384-well plate in triplicate and terminated by the addition of 10 µl malachite green solution (Sigma–Aldrich, Catalog # MAK307) to each well. After further incubation for 30 min at room temperature for color development, absorbance was measured at 620 nm on a plate reader. The amount of phosphate released was determined by comparison with a malachite green standard curve. The reaction rate, defined as micromoles of phosphate released per minute, reflects the dephosphorylation activity of SHP2 toward its phosphopeptide substrates. The sequences of the peptides used in the assay were:

  • IRS1_pY1172–PEG–pY1222 – SLN{pY}IDLDLVK–dPEG8–LST{pY}ASINFQK

  • OCLN_pY443 – TGLQE{pY}KSLQS

  • PLCG2_pY818 – TRIQQ{pY}FPSNY

  • GRB2_pY209 – MFPRN{pY}VTPVN

  • ARHGAP35_pY1105 – EENI{pY}SVPHDS

  • CSF-1R_pY708 – IHLEKK{pY}VRRDSGF

  • TMEM134_pY57 – KQSRLR{pY}QNLEND

Proximity ligation assay

Request a detailed protocol

MDA-MB-468 cells were grown to 70% confluence on eight-chambered glass slides (Nunc Lab-Tek II, Thermo Scientific), starved in serum-free media for 24 hr, and treated with DMSO carrier or SHP099 (10 µM) for 2 hr prior to stimulation with 1 nM EGF for 2 min. Cells were fixed with ice-cold 4% (v/v) paraformaldehyde for 15 min and permeabilized with 0.125% Tween-20 in PBS for 5 min prior to incubation in 1× blocking solution (1× PBS, 10% BSA, 10% donkey serum, 10% goat serum) for 1 hr at room temperature. Cells were then incubated with mouse anti-EGFR (1:1000, clone 13G8, Millipore) and rabbit anti-GAB1 (1:100, Catalog # SAB4501060, Sigma) overnight at 4°C in a humidified chamber. The proximity ligation assay was conducted using a DuoLink In Situ Mouse/Rabbit PLA kit (Sigma–Aldrich, Catalog # DUO92014) according to the manufacturer’s instructions. All images were collected on a confocal laser scanning microscope (Zeiss LSM 880 with Airyscan) equipped with a 63× oil immersion objective lens (Plan Apo, NA = 1.4) and analyzed using CellProfiler software (RRID:SCR_007358). Three independent replicates (n = 3) were performed.

References

    1. Keegan K
    2. Cooper JA
    (1996)
    Use of the two hybrid system to detect the association of the protein-tyrosine-phosphatase, SHPTP2, with another SH2-containing protein, Grb7
    Oncogene 12:1537–1544.

Decision letter

  1. Jonathan A Cooper
    Senior and Reviewing Editor; Fred Hutchinson Cancer Research Center, United States
  2. Bruce J Mayer
    Reviewer; University of Connecticut School of Medicine, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

The authors used phosphotyrosine proteomics to uncover novel aspects of signaling by PTPN11/SHP2, a phosphotyrosine phosphatase that contains two SH2 domains and is involved in Noonan's Syndrome and cancer. By creative use of knockouts, mutants, and a chemical inhibitor that locks SHP2 in a state where its SH2 domains are unavailable, the authors provide evidence for two classes of phosphoproteins, those that are SHP2 substrates and those that appear to be protected from other PTPs by the SHP2 SH2 domains. The results help explain how SHP2 has both positive and negative effects on signaling.

Decision letter after peer review:

Thank you for submitting your article "Time resolved phosphoproteomics reveals scaffolding and catalysis-responsive patterns of SHP2-dependent signaling" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Bruce J Mayer (Reviewer #1).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

This manuscript addresses the importance of the Shp2 tyrosine phosphatase in shaping the response of cells to EGF stimulation. The profile of EGF-induced phosphotyrosine (pTyr)-containing peptide sites was measured with and without SHP099, a Shp2 allosteric inhibitor. As expected, the abundance of a number of phosphopeptides was increased by inhibitor treatment, consistent with being Shp2 substrates. In contrast, the phosphorylation of a number of peptides, such as those on Gab1/2, was decreased upon Shp2 inhibition. The authors go on to present evidence that this is likely due to phosphosite protection by the Shp2 SH2 domains.

This is a technically outstanding piece of work that makes extensive use of quantitative mass spectrometry. The concepts of SH2 protection and of Shp2 scaffold function are not novel, and conclusions regarding potential new Shp2 substrates are over-interpreted, but the results will be of general interest to the field due to the importance of Shp2 in stimulating RTK-mediated MAPK and PI3K pathways. The implication of Shp2 in important and cancer-relevant signaling pathways increases potential impact. That said, the value of this study is more in developingig new testable hypotheses rather than in providing new insights into the actual signaling mechanisms. The review consensus is that paper would be acceptable with only minor experimental work and rewriting, but would be much more interesting with deeper investigation of the proposed new substrates or the scaffolding functions.

Essential revisions:

1. Figure 4F: While we commend the authors for showing that Shp2 can dephosphorylate occludin, ARHGAP35 and PLCg2 peptides, additional work would be needed to conclusively demonstrate that these are indeed natural substrates. Does Shp2 interact with any of these proteins in cells and in cell-free systems? Does the enzymatic dead mutation affect the interaction? In the phosphatase enzymatic assay, it is nice to include CSF-1R substrate as a negative control. How about a type III peptide? Would the enzymatic dead mutant of SHP2 have any impact on phosphate release? What were the 20 candidate proteins initially screened? Were any of these 20 candidates established Shp2 substrates? Overall, the conclusions relating to potential Shp2 substrates are over interpreted. Adding more experimental information would be desirable, but, failing that, the authors should temper their conclusions.

2. As interesting as the SH2 phosphosite protection may be, the actual role of this phenomenon in Shp2 signaling is not really clarified. In other words, is protection merely a consequence of Shp2 recruitment, or does it have functional consequences beyond bringing the catalytic activity of Shp2 to membrane signaling complexes? The authors discuss potential scaffolding functions, but it is not clear what they mean-do they propose other signaling proteins are bound to other domains/sites of Shp2 that are critical for downstream signaling? The Shp2 mutants and knockout cell lines used here would be ideal to test a few simple ideas. For example, to what extent can the EK/CE (open/dead) mutant rescue EGF-dependent downstream signaling compared to the EK (open/active) mutant? Similarly, does the Shp2 SH2-only construct have any biological activity when compared to EK/CE? The impact of the manuscript would be much greater if some insight into the role of SH2 protection in Shp2 signaling were gained.

3. Regarding potential scaffold function: phosphorylation of Shp2 itself is increased when the Shp2 inhibitor is removed, falling into the same category as GAB Y659. Given the potential role of Shp2 phosphorylation in recruiting Grb2, the authors should discuss the possible scaffolding role of the Shp2 pY sites and their interpretation of why phosphorylation of those sites increases when Shp2 inhibitor is removed.

4. The EGF stimulation time course included only 5- 10- and 30-min time points. This misses a whole class of interesting/important phosphorylation events that occur very rapidly after EGF stimulation. While MAPK may take ~5 minutes to be fully activated, many pTyr sites are maximally phosphorylated within one or two minutes of EGF treatment, and in particular Shp2 SH2 binding sites have been reported to peak within a minute or two and then return close to baseline by 5 minutes (e.g. Jadwin et al., eLife 2016;5:e11835). It is not realistic to expect the authors to repeat the MS experiments with earlier timepoints, but it would be relatively trivial to provide overall anti-pTyr blots for the timepoints used for MS (to gauge the extent of overall pTyr changes with and without EGF, Shp2 inhibitor in these cells), and more importantly to provide more detailed timecourses for selected "SH2 protected" substrates such as GAB1 or EGFR with and without Shp2 inhibition. In addition, for the MAPK analysis, the time course should be extended to 2-4 hrs to include the decay period. The experiments should be quantitated (pERK/ERK).

5. The interpretation that MPZL and the GAB sites were being reduced in their level of phosphorylation as a result of SH2 domain protection is misleading and in its present form still open to the possibility raised by the authors that this is a Src-mediated phenomenon. There was no change in overall Src activity assayed using the pY416 antibody in total cell lysates but relevant changes in Src activity may be restricted to the precise protein complex in which it is modulated. One would need to determine the status of Src activation not in a whole cell lysate but rather the status of Src pY416 in the GAB complex. This is a well-established behavior of the SFKs, in general. Similar experiments should be performed with PZR in complex with Src as similar mechanisms are likely operative where Src can phosphorylate PZR, which has been shown, thereby creating binding sites for SH2 domains of Shp2. Deletion of the SH2 domains of Shp2, which binds Src, would fail to recruit Src to PZR resulting in reduced phosphorylation. These possibilities are all arguable and not in of themselves as presented "air-tight".

https://doi.org/10.7554/eLife.64251.sa1

Author response

Essential revisions:

1. Figure 4F: While we commend the authors for showing that Shp2 can dephosphorylate occludin, ARHGAP35 and PLCg2 peptides, additional work would be needed to conclusively demonstrate that these are indeed natural substrates. Does Shp2 interact with any of these proteins in cells and in cell-free systems? Does the enzymatic dead mutation affect the interaction? In the phosphatase enzymatic assay, it is nice to include CSF-1R substrate as a negative control. How about a type III peptide? Would the enzymatic dead mutant of SHP2 have any impact on phosphate release? What were the 20 candidate proteins initially screened? Were any of these 20 candidates established Shp2 substrates? Overall, the conclusions relating to potential Shp2 substrates are over interpreted. Adding more experimental information would be desirable, but, failing that, the authors should temper their conclusions.

We focused attention on potential substrates identified from the mass spectrometry data. We identified pY sites on 20 proteins (now included in the manuscript as Supplementary file 3) that met the following criteria:

1. The identified site was not previously reported as a SHP2 substrate

2. The pY abundance of the site was increased more than two-fold in the presence of SHP099 when compared to DMSO control (i.e. [SHP099/DMSO] fold-change > 2)

3. The pY abundance of the site is increased in response to EGF stimulation

4. Availability of commercial antibodies to the identified protein

We also used Y209 of GRB2 as a positive control for these studies because it is a well-established SHP2 substrate that met the other criteria (2-4) above. For 13 proteins, antibody sensitivity was insufficient to determine whether they are SHP2 substrates. For four proteins, Western blotting of the immunoprecipitates did not show increased pY abundance upon SHP099 treatment. The three proteins that did show increased pY abundance in response to SHP099 were occludin, ARHGAP35, and PLCγ2. The text has been modified on page 9 to include this information.

The conclusion that occludin, ARHGAP35, and PLCγ2 are substrates for SHP2 include both immunoprecipitation data showing that they have an increased amount of pY in the presence of SHP099 and in vitro confirmation that a pY-containing peptide spanning the site identified by MS is active as a substrate for the purified enzyme (Figure 3F). In contrast and as expected, a purified catalytically inactive mutant form of the enzyme (C459A), does not dephosphorylate these substrate phosphopeptides (wt and mutant data are plotted in Author response image 1).

Author response image 1
Dephosphorylation activity of wild-type or C459A SHP2, activated with 6 μM bisphosphorylated IRS-1 peptide [SLNY(p)IDLDLVKdPEG8-LSTY(p)ASINFQK], towards synthetic phosphopeptide substrates (OCLN_pY443, PLCG2_pY818, GRB2_pY209, ARHGAP35_1105, TMEM134_pY57, CSF-1R_pY708).

The C459A mutant is inactive in the dephosphorylation assay, as expected.

We did test whether SHP2 can be co-immunoprecipitated with occludin, ARHGAP35, or PLCγ2 in MDA-MB-468 cells, but did not see changes in bulk immunoprecipitation of any of these proteins with SHP2 after EGF stimulation. We note, however, that bulk recovery of a protein by co-IP is not a prerequisite for a bona-fide protein substrate.

Lastly, we are not certain what the reviewer meant by the term “Type III” peptide, but we interpreted the meaning as equivalent to what we defined in the manuscript as “Class III” peptides. The ability of SHP2 to dephosphorylate pY57 of TMEM134, a “Class III” peptide, is greatly reduced by comparison to the putative substrate peptides. These data are now included in the results reported in Figure 3F, and the text on pages 9-10 has been modified to include these new data. Lastly, as recommended, we have softened the concluding sentence of the paragraph to state that our results “suggest,” rather than “support the conclusion,” that ARHGAP35, PLCγ2, and occludin are newly identified substrates for SHP2.

2. As interesting as the SH2 phosphosite protection may be, the actual role of this phenomenon in Shp2 signaling is not really clarified. In other words, is protection merely a consequence of Shp2 recruitment, or does it have functional consequences beyond bringing the catalytic activity of Shp2 to membrane signaling complexes? The authors discuss potential scaffolding functions, but it is not clear what they mean-do they propose other signaling proteins are bound to other domains/sites of Shp2 that are critical for downstream signaling? The Shp2 mutants and knockout cell lines used here would be ideal to test a few simple ideas. For example, to what extent can the EK/CE (open/dead) mutant rescue EGF-dependent downstream signaling compared to the EK (open/active) mutant? Similarly, does the Shp2 SH2-only construct have any biological activity when compared to EK/CE? The impact of the manuscript would be much greater if some insight into the role of SH2 protection in Shp2 signaling were gained.

We agree with the reviewer that elucidating the functional role of phosphosite protection by the SH2 domains of SHP2 is an interesting future direction. While it is beyond the scope of the current work to investigate the functional implications of this phenomenon in detail, there is some evidence that the scaffolding/protective activity of the SH2 domains has a functional effect from the results of experiments performed with the SH2 domain-only SHP2 fragment (Figure 5D). The data show accumulation of protected pY marks on both GAB and GAB2 even in the absence of EGF stimulation, consistent with a dominant effect of the tandem SH2 domain fragment that is independent of the catalytic domain of the enzyme. This observation is now noted on Page 11 of the manuscript.

3. Regarding potential scaffold function: phosphorylation of Shp2 itself is increased when the Shp2 inhibitor is removed, falling into the same category as GAB Y659. Given the potential role of Shp2 phosphorylation in recruiting Grb2, the authors should discuss the possible scaffolding role of the Shp2 pY sites and their interpretation of why phosphorylation of those sites increases when Shp2 inhibitor is removed.

The presence of the allosteric inhibitor locks SHP2 in the closed conformation. Thus, the inhibitor interferes with membrane recruitment of SHP2 because its SH2 domains are not available to bind to pY residues deposited at membrane proximal sites by activated kinases. It is thus reasonable to postulate that removal of inhibitor permits membrane recruitment to sites of EGFR activation, phosphorylation of the SHP2 C-terminal tail, and subsequent GRB2 recruitment. We have added text discussing this issue to the manuscript on pages 12-13.

4. The EGF stimulation time course included only 5- 10- and 30-min time points. This misses a whole class of interesting/important phosphorylation events that occur very rapidly after EGF stimulation. While MAPK may take ~5 minutes to be fully activated, many pTyr sites are maximally phosphorylated within one or two minutes of EGF treatment, and in particular Shp2 SH2 binding sites have been reported to peak within a minute or two and then return close to baseline by 5 minutes (e.g. Jadwin et al., eLife 2016;5:e11835). It is not realistic to expect the authors to repeat the MS experiments with earlier timepoints, but it would be relatively trivial to provide overall anti-pTyr blots for the timepoints used for MS (to gauge the extent of overall pTyr changes with and without EGF, Shp2 inhibitor in these cells), and more importantly to provide more detailed timecourses for selected "SH2 protected" substrates such as GAB1 or EGFR with and without Shp2 inhibition. In addition, for the MAPK analysis, the time course should be extended to 2-4 hrs to include the decay period. The experiments should be quantitated (pERK/ERK).

As noted by the reviewer (see both Jadwin et al., eLife 2016;5:e11835 and Reddy et al., PNAS 2016;113:3114), we do appreciate that the kinetics of EGF stimulated tyrosine phosphorylation rapidly builds to a peak within a minute or two. Because the arrival of pY binding proteins at the membrane, such as GRB2 (and presumably other pY binders like SHP2; Jadwin et al., 2016), is delayed relative to the deposition of the pY mark, reaching a peak after ~5-10 min, we therefore chose 5, 10, and 30 minute timepoints for the mass spec measurements to maximize readout of the role of SHP2 downstream of EGF stimulation. This point is now noted on page 5 of the manuscript.

As requested by the reviewers, we now include quantification of the data in Figure 1A (right panel). We also provide more extended timecourse data that includes earlier timepoints (2 min EGF) and additional timepoints through 4 hours in the absence and presence of SHP099 for ERK1/2 (T202/Y204 of ERK1, T185/Y187 of ERK2), GAB1 (pY627 and pY659), GAB2 (pY614 and pY643), and phospho-tyrosine (P-Tyr-1000, CST). The ERK1/2 and pTyr-1000 data are reported in Figure 1—figure supplement 1A, and the GAB1 and GAB2 are presented in Figure 4C (replacing the prior figure panel, which did not show timecourse data), along with changes in the text on page 10.

5. The interpretation that MPZL and the GAB sites were being reduced in their level of phosphorylation as a result of SH2 domain protection is misleading and in its present form still open to the possibility raised by the authors that this is a Src-mediated phenomenon. There was no change in overall Src activity assayed using the pY416 antibody in total cell lysates but relevant changes in Src activity may be restricted to the precise protein complex in which it is modulated. One would need to determine the status of Src activation not in a whole cell lysate but rather the status of Src pY416 in the GAB complex. This is a well-established behavior of the SFKs, in general. Similar experiments should be performed with PZR in complex with Src as similar mechanisms are likely operative where Src can phosphorylate PZR, which has been shown, thereby creating binding sites for SH2 domains of Shp2. Deletion of the SH2 domains of Shp2, which binds Src, would fail to recruit Src to PZR resulting in reduced phosphorylation. These possibilities are all arguable and not in of themselves as presented "air-tight".

Point appreciated. It is certainly possible that the SFK activity in the GAB complex is not reflected by assessment of bulk SFK activity in the whole cell lysate. We have now noted this caveat in the manuscript on page 13, and tempered our conclusions as recommended by the reviewers. Further studies investigating the effect of SHP2 inhibition on SFK would lie beyond the scope of this manuscript.

https://doi.org/10.7554/eLife.64251.sa2

Article and author information

Author details

  1. Vidyasiri Vemulapalli

    1. Department of Cancer Biology, Dana-Farber Cancer Institute Boston, Boston, United States
    2. Department of Biological Chemistry & Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, United States
    Contribution
    Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3368-4766
  2. Lily A Chylek

    Laboratory of Systems Pharmacology, Harvard Medical School, Boston, United States
    Contribution
    Formal analysis, Methodology, Writing - review and editing, Visualization
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1216-1250
  3. Alison Erickson

    Department of Cell Biology, Harvard Medical School, Boston, United States
    Contribution
    Investigation, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
  4. Anamarija Pfeiffer

    Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark
    Contribution
    Validation, Investigation
    Competing interests
    No competing interests declared
  5. Khal-Hentz Gabriel

    1. Department of Cancer Biology, Dana-Farber Cancer Institute Boston, Boston, United States
    2. Department of Biological Chemistry & Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, United States
    Contribution
    Validation, Investigation
    Competing interests
    No competing interests declared
  6. Jonathan LaRochelle

    1. Department of Cancer Biology, Dana-Farber Cancer Institute Boston, Boston, United States
    2. Department of Biological Chemistry & Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, United States
    Contribution
    Conceptualization, Investigation, Methodology
    Competing interests
    No competing interests declared
  7. Kartik Subramanian

    Laboratory of Systems Pharmacology, Harvard Medical School, Boston, United States
    Contribution
    Conceptualization
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6900-8882
  8. Ruili Cao

    Department of Cancer Biology, Dana-Farber Cancer Institute Boston, Boston, United States
    Contribution
    Resources, Supervision, Expressed and purified SHP2 protein for enzymatic assays
    Competing interests
    No competing interests declared
  9. Kimberley Stegmaier

    Department of Pediatric Oncology, Dana Farber Cancer Institute, Boston, United States
    Contribution
    Supervision
    Competing interests
    No competing interests declared
  10. Morvarid Mohseni

    Novartis Institutes for Biomedical Research, Cambridge, United States
    Contribution
    Conceptualization, Supervision
    Competing interests
    Novartis employee while this work was performed.
  11. Matthew J LaMarche

    Novartis Institutes for Biomedical Research, Cambridge, United States
    Contribution
    Conceptualization, Supervision
    Competing interests
    Novartis employee while this work was performed.
  12. Michael G Acker

    Novartis Institutes for Biomedical Research, Cambridge, United States
    Contribution
    Conceptualization, Supervision, Methodology, Writing - review and editing
    Competing interests
    Novartis employee while this work was performed.
  13. Peter K Sorger

    Laboratory of Systems Pharmacology, Harvard Medical School, Boston, United States
    Contribution
    Conceptualization, Resources, Supervision, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3364-1838
  14. Steven P Gygi

    Department of Cell Biology, Harvard Medical School, Boston, United States
    Contribution
    Conceptualization, Resources, Supervision, Funding acquisition, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing
    Competing interests
    No competing interests declared
  15. Stephen C Blacklow

    1. Department of Cancer Biology, Dana-Farber Cancer Institute Boston, Boston, United States
    2. Department of Biological Chemistry & Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, United States
    Contribution
    Conceptualization, Resources, Supervision, Funding acquisition, Visualization, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    stephen_blacklow@hms.harvard.edu
    Competing interests
    SCB receives research funding for this project from Novartis, is a member of the SAB of Erasca, Inc, is an advisor to MPM Capital, and is a consultant on unrelated projects for IFM, Scorpion Therapeutics, and Ayala Therapeutics.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6904-1981

Funding

Novartis

  • Vidyasiri Vemulapalli
  • Khal-Hentz Gabriel
  • Jonathan LaRochelle
  • Kimberley Stegmaier
  • Stephen C Blacklow

National Cancer Institute (R35 CA220340)

  • Stephen C Blacklow

National Institutes of Health (U54-HL127365)

  • Lily A Chylek
  • Peter K Sorger

Novo Nordisk Foundation (NNF16CC0020906)

  • Anamarija Pfeiffer

National Cancer Institute (U54-CA225088)

  • Lily A Chylek

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Jon Aster, Huaixiang Hao, Aram Ghalali, Abhinav Dubey, and members of the Blacklow laboratory for helpful discussions. Funding: This work was supported by NIH grants R35 CA220340 (SCB), U54-CA255088 (LAC and PKS), and from the Dana Farber-Novartis Translational Drug Development Program. AP was supported by the Novo Nordisk Foundation Copenhagen Bioscience PhD Programme grant (grant number NNF16CC0020906). Competing Interests: SCB receives research funding for this project from Novartis, is a member of the SAB of Erasca, Inc, is an advisor to MPM Capital, and is a consultant on unrelated projects for IFM, Scorpion Therapeutics, and Ayala Therapeutics. PKS is a member of the SAB or Board of Directors of Merrimack Pharmaceutical, Glencoe Software, Applied Biomath, and RareCyte Inc and has equity in these companies. Sorger declares that none of these relationships are directly or indirectly related to the content of this manuscript. MGA, ML, and MM are former employees of Novartis. Data and materials availability: Quantitative proteomics data has been deposited in the mass spectrometry interactive virtual environment (MassIVE) database at: https://doi.org/doi:10.25345/C5HS6C

Senior and Reviewing Editor

  1. Jonathan A Cooper, Fred Hutchinson Cancer Research Center, United States

Reviewer

  1. Bruce J Mayer, University of Connecticut School of Medicine, United States

Publication history

  1. Received: October 23, 2020
  2. Accepted: March 21, 2021
  3. Accepted Manuscript published: March 23, 2021 (version 1)
  4. Version of Record published: April 6, 2021 (version 2)

Copyright

© 2021, Vemulapalli et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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