A hotspot phosphorylation site on SHP2 drives oncoprotein activation and drug resistance

Domain: chemistry
Published: 2026-03-03
Editorial outcome: major_revision

Abstract
SHP2 is a phosphatase and a critical mediator of receptor tyrosine kinase (RTK)-driven
RAS/mitogen-activated protein kinase (MAPK) signaling. Despite promising preclinical data,
SHP2 inhibitors have shown minimal clinical efficacy, with no defined clinical mechanisms of
primary resistance. Here, we elucidate phosphorylation of SHP2 at tyrosine 62 (pY62) as a
hotspot phosphorylation site in the proteome and RTK-driven tumor types in patients. We
demonstrate that SRC family kinases directly phosphorylate SHP2 at Y62, downstream of but not
directly phosphorylated by RTKs. Using biochemical and biophysical analyses, we show that
SHP2Y62D enforces an open, active conformation, resulting in constitutive phosphatase activation
that is sufficient to activate MAPK signaling and confer resistance to allosteric SHP2 inhibitors.
These findings establish that SHP2 pY62 is a phosphorylation hotspot phenocopying mutational
activation, a mechanism of primary resistance to SHP2 inhibitors, and a cancer drug target distinct
from wildtype SHP2.

Introduction
SHP2, encoded by the PTPN11 gene, is a non-receptor protein tyrosine phosphatase1-5 that
serves as a critical mediator of receptor tyrosine kinase (RTK) signaling to the RAS/MAPK
pathway6,7. In its basal state, SHP2 adopts an autoinhibited closed conformation, in which the N-
terminal Src homology 2 (SH2) domain occludes the protein tyrosine phosphatase (PTP) domain
active site8,9 (Fig. 1a). Upon activation of RTKs, SHP2 binds to phosphotyrosine-containing motifs
through its SH2 domains, relieving autoinhibition and enabling its catalytic activity. In addition to
its phosphatase function, SHP2 acts as a signaling scaffold, integrating RTK inputs and facilitating
the activation of downstream effectors such as RAS and ERK10-15.

Activating mutations in SHP2 have been well characterized as oncogenic drivers in hematologic
malignancies, including juvenile myelomonocytic leukemia (JMML) and subsets of acute myeloid
leukemia (AML), and as germline mutations which underlie Noonan syndrome (NS)16. More
recently, SHP2 has emerged as an essential signaling node in RTK-driven solid tumors, where
SHP2 is rarely mutated17-19. Functional genetic studies have demonstrated that SHP2 is required
for survival in RTK-driven cancer models, including those driven by oncogenic epidermal growth
factor receptor (EGFR), anaplastic lymphoma kinase (ALK), and fibroblast growth factor receptor
(FGFR) alterations20. These findings catalyzed the development of allosteric SHP2 inhibitors
(SHP2i) that function as molecular glues, stabilizing the autoinhibited conformation of the protein
(e.g., SHP099, RMC4550, TNO155)21. Although these agents have demonstrated potent
preclinical activity, clinical trials to date have reported negligible objective response rates (ORR =
~0%)22-24 with stable disease at best, and have been associated with dose-limiting toxicities,
including fatigue, rash, and cytopenias. These challenges highlight the need to investigate
mechanisms of primary resistance to SHP2i and identify biomarkers that modulate SHP2
dependence and therapeutic response.

Considerable preclinical effort has gone into uncovering genetic resistance mechanisms to SHP2i
including activating SHP2 mutations that lock the protein in an open, active state, making it
resistant to allosteric inhibitors designed to bind the inactive form. However, activating mutations
are rare in RTK-driven solid tumors. Additionally, much attention has focused on phosphorylation
of SHP2 at its canonical C-terminal tyrosines, Y542 and Y580, which are known to promote
scaffolding interactions and downstream signaling15,25-27, but their direct effect on SHP2’s catalytic
activity remains unclear28.

Despite the clinical momentum behind SHP2 inhibitors, emerging evidence points to alternative
phosphorylation sites, including SHP2 Y62 (pY62) as potential modulators of inhibitor sensitivity.
pY62 was initially discovered in cytokine-stimulated hematopoietic stem cells29. More recently,
pY62 was found to be enriched in SHP099 resistant FLT3-driven acute myeloid leukemia cells
and was associated with SHP099 resistance possibly through enforcing an open, active
conformation of SHP230. Whether SHP2 pY62 occurs broadly across cancer types—and in which
biological contexts this modification meaningfully alters signaling, protein biochemistry, and
structure—remains incomplete.

Our interest in SHP2 pY62 was driven by two independent observations that underscore its
potential significance. First, a comprehensive phosphoproteomic analysis identified SHP2 pY62
as one of the most recurrent phosphorylation events in the human proteome. Second, SHP2 pY62
is one of the most differentially enriched phosphopeptides across multiple RTK-driven tumor types
in patient samples. This set of observations in normal cells and tissues, cancer cells, and patient
samples prompted us to investigate the functional, mechanistic, and therapeutic consequences
of SHP2 Y62 phosphorylation.

Here we show that SHP2 pY62 is conserved across cell and cancer types and that SRC family
kinases directly phosphorylate SHP2 at Y62, downstream of RTKs. We also show that SHP2Y62D
enforces an open, active conformation, resulting in constitutive phosphatase activation that is
sufficient to activate MAPK signaling and confer resistance to allosteric SHP2 inhibitors.

Results
SHP2 pY62 is a hotspot phosphorylation in normal cells/tissues and cancer cells and is
enriched in diverse RTK-driven tumor types

Inspired by cancer genomics efforts to define frequent “mutational hotspots”31-33, we performed a
comprehensive analysis of the human phosphoproteome using PhosphositePlus34. We identified
239,530 human proteins phosphorylation sites (phosphosites) across normal cells and tissues,
and cancer cells (Fig. 1b) (Supplementary Data 1 ). We determined the reported frequency of
every phosphosite by computing the sum of MS_LIT, the number of published studies reporting
the phosphosite, and MS_CST, the number of unpublished mass spectrometry experiments
performed and curated by Cell Signaling Technology, on PhosphoSitePlus. We posited that the
most frequent phosphosites may be enriched for functionality, and focused analysis on the top
2400 (1%) of phosphosites. In line with this assertion, many of the most frequent phosphosites
were known kinase activation loop sites including GSK3α pY279, GSK3β pY216, and ERK2
pY187 (Supplementary Data 1). We examined proteins with multiple phosphorylation sites and
found that the top differentially phosphorylated protein was not a kinase, but the protein
phosphatase SHP2 (Fig. 1c). The known SHP2 C-terminal tyrosine phosphosites Y542 and Y580
(pY542, pY580) (Fig. 1a) were abundant, but surprisingly, the most abundant SHP2 phosphosite
was the tyrosine Y62 (pY62) (Fig. 1c).

We next determined the experimental characterization frequency by comparing LT_LIT, the
number of low-throughput (LT), experimentally validated studies in PhosphositePlus
(Supplementary Data 1), with MS_LIT + MS_CST. By this metric, SHP2 pY62 was reported far
less frequently than the canonical C-terminal phosphorylation sites pY542 and pY58034 (Fig. 1d).
Despite this underrepresentation, SHP2 Y62 is evolutionarily conserved across vertebrates, down
to Xenopus (Supplementary Fig. 1). Notably, SHP2 Y62 is not conserved in the closely related
human phosphatase SHP1, where it aligns with phenylalanine 60 (F60), a non-phosphorylatable
residue. In contrast, the adjacent SHP2 Y63 is conserved and aligns with SHP1 Y61, indicating
selective evolutionary retention of the neighboring tyrosine, rather than the Y62-equivalent site.
SHP2 Y62 is located in a structured core on a loop between the βD and βE strands of the SHP2
N-terminal SH2 (nSH2) domain8. Among the 110 SH2-domain containing proteins in humans,
Y62-homologous tyrosines are present in 12 proteins, some of which are frequently
phosphorylated (Supplementary Fig. 1)35.

Protein phosphorylation can differ between cancer cell lines and tumors36. To understand the role
of SHP2 pY62 in patients, we determined its abundance and enrichment across genomically
defined human tumor types based on publicly available data from the Clinical Proteomic Tumor
Analysis Consortium (CPTAC)37 and National Omics Data Encyclopedia (NODE)38. SHP2 pY62
has been reported as the single most differentially increased phosphosite in patients with EGFR-
mutated non-small cell lung cancer (NSCLC), compared to their wild-type (WT) counterparts39
(Fig. 1e). In fact, SHP2 pY62 was more increased than phosphorylated EGFR itself (Fig. 1e,
Supplementary Data 2). Additionally, SHP2 pY62 was the second most differentially increased
phosphosite in patients with FGFR2-altered cholangiocarcinoma40 (Fig. 1f), and differentially
increased in CPTAC datasets in patients with EGFR-amplified head and neck squamous cell
carcinoma41, EGFRvIII-positive glioblastoma42, and EGFR-altered high grade glioma43, compared
to WT counterparts. In contrast, SHP2 pY62 was one of the most significantly decreased
phosphosites in KRAS-mutant NSCLC relative to wild-type NSCLC39 (Fig. 1g, Supplementary
Data 2). Our identification of SHP2 pY62 as one of the most frequently detected phosphosites in
the human phosphoproteome, along with its evolutionary conservation and selective retention
across orthologs and SH2 domains, and its high frequency in RTK-driven patient tumor types
suggests that Y62 phosphorylation may exert distinct conformational or functional effects that
contribute to RTK-driven oncogenesis.

SHP2 pY62 is downstream of RTKs, but Y62 is not phosphorylated by RTKs

Given the high abundance of pY62 across RTK-driven tumors, we hypothesized that RTKs signal
to and directly phosphorylate SHP2 Y62 in cells (Fig. 2a). To test this hypothesis, we developed
a phosphospecific SHP2 pY62 antibody and validated it in SHP2 knock out (KO) cells
overexpressing SHP2 and the phosphodead mutant Y62F (Supplementary Fig. 2a, b). We serum-
starved U-2 OS (osteosarcoma) and BT20 (EGFR-amplified triple-negative breast cancer) cells
and exposed them to various RTK ligands (EGF, FGF1, PDGF). RTK stimulation led to RTK
phosphorylation at 5-10 minutes, with eventual detection of SHP2 pY62 much later, suggesting
SHP2 Y62 phosphorylation is downstream of RTKs (Fig. 2b, Supplementary Fig. 2c-e). However,
when we exposed RTK stimulated cells to RTK inhibitors against EGFR, FGFR, and PDGFR in
serum starved media, there was no decrease in SHP2 pY62 (Fig. 2c), showing that SHP2 Y62 is
not directly phosphorylated by RTKs.

SRC family kinases (SFKs) phosphorylate SHP2 Y62, Y542, and Y580

To determine the direct kinase for SHP2 Y62, we leveraged The Kinase Library, a recently
developed global atlas of protein kinome in vitro substrate specificities44,45, based on the peptide
primary sequence surrounding the phosphosite (Supplementary Data 3). For SHP2 pY62, RTKs
were predicted as the top candidate kinases (Fig. 3a). JAK3, a non-RTK, was also highly
predicted. However, exposure of cells to the JAK3 inhibitor tofacitinib did not alter the level of
SHP2 pY62 (Supplementary Fig. 3a). Interestingly, non-RTKs, such as SRC family kinases
(SFKs) SRC, YES1, and FYN, were in the bottom quartile with lower confidence scores. Since
inhibiting RTKs did not alter the SHP2 Y62 phosphorylation, we wanted to test if SFKs can
phosphorylate SHP2 Y62.

To test this hypothesis, we first used pharmacologic methods (Fig. 3b). Dasatinib is a multi-kinase
inhibitor that targets not only SFKs but also ABL, PDGFR, and c-KIT46 (Fig. 3b). Treatment with
dasatinib readily decreased SHP2 pY62 levels in U-2 OS cells (Fig. 3c) and HCC827 cells (EGFR-
mutant NSCLC) (Fig. 3d). Dasatinib treatment also decreased SHP2 pY542 and pY580 levels in
U-2 OS cells (Fig. 3c) but only pY542 in HCC827 cells (Fig. 3d). Since dasatinib has a wide range
of targets including SFKs, ABL, and c-KIT, we next chose the selective SFK inhibitor SU665647
which inhibits SRC, YES1, FYN, and LYN; asciminib48 which binds to the myristoyl binding pocket
and inhibits ABL; and ripretinib49, which inhibits c-KIT, to narrow down candidate kinases (Fig.
3b). Treatment of U-2 OS cells with SU6656 decreased SHP2 pY62, pY542, and pY580 but there
was no change in SHP2 phosphorylation with SU6656 in HCC827 cells (Fig. 3d). Treatment with
asciminib or ripretinib did not change SHP2 phosphorylation levels (Supplementary Fig. 3b).

The SFKs comprises 8 human kinases50, three of which–-SRC, YES1, and FYN–-are ubiquitously
expressed in all tissues. In contrast, other SFK kinases (BLK, FGR, HCK, LCK, and LYN) are
expressed in specific tissue types51. To our knowledge, there are no SFK inhibitors selective to
individual SFKs. To better deconvolute the contributions of specific SFK kinases, we tested the
SFK inhibitors saracatinib52 (SRC and ABL inhibitor), CH695375553 (YES1 inhibitor), and PP254
(FYN and LCK inhibitor) (Fig. 3b) which are more selective than dasatinib or SU6656 but less
potent. In HCC827 cells, SHP2 Y62 phosphorylation was inhibited by saracatinib, CH6953755
and PP2, and Y542 phosphorylation was altered by PP2 and saracatinib at high concentrations,
but not Y580 phosphorylation (Fig. 3e). In U-2 OS cells, SHP2 Y62 phosphorylation was inhibited
by saracatinib only at high concentrations, while Y542 and Y580 phosphorylation were inhibited
by saracatinib even at 1 µM (Supplementary Fig. 3c), but not by CH6953755 (Supplementary Fig.
3d) or PP2 (Supplementary Fig. 3e).

We further investigated the cell-type specificity of SHP2 Y62 phosphorylation. Since 1) dasatinib
decreased SHP2 pY62 levels in HCC827 and U-2 OS cells, 2) inhibitors of the non-SFK kinase
targets of dasatinib did not decrease SHP2 phosphorylation, and 3) selective SFK inhibitors
inhibited SHP2 pY62 levels in HCC827 but not in U-2 OS cells, we hypothesized that SHP2 Y62
phosphorylation is maintained by distinct SFK family members in a context-dependent manner,
and that broad SFK inhibition is required to suppress SHP2 pY62 across diverse cell types.

To test this hypothesis, we tested double and triple combinations of the SFK selective inhibitors
saracatinib, CH6953755, and PP2 in HCC827 and U-2 OS cells. Combining two drugs reduced
SHP2 pY62, pY542, and pY580 levels more than single drugs, and combining all three drugs
reduced levels more than combining two drugs (Fig. 3f, Supplementary Fig. 3f).

We also used mouse embryonic fibroblasts (MEFs) from the SYF knock out mouse (SRC-/-, YES1
-/-, FYN -/-)55 as a genetic tool to test the hypothesis that SFKs phosphorylate SHP2 pY62. We
found that SHP2 pY62, pY542, and pY580 levels were inhibited compared to WT MEFs (Fig. 3g).
Thus, pharmacologic inhibition and genetic loss of the SFKs SRC, YES1, and FYN reduce SHP2
phosphorylation at Y62, Y542, and Y580.

Our initial growth factor experiments showed that SHP2 Y62 phosphorylation is downstream of
RTKs (Fig. 2b). We used our pharmacologic and genetic models to situate SFKs in RTK-SHP2
signaling. EGF-dependent SHP2 phosphorylation at Y62, Y542, and Y580 was abrogated when
U-2 OS cells were pretreated with dasatinib or SU6656 (Supplementary Fig. 3g). EGF-, FGF1-,
and PDGDF-dependent SHP2 phosphorylation at Y62 was abrogated in our SYF KO MEF models
compared to WT MEFs (Supplementary Fig. 3h-j). In contrast, SHP2 Y542 and Y580
phosphorylation were abrogated in SYF KO MEFs under FGF1 stimulation but not under EGF or
PDGF stimulation (Supplementary Fig. 3h-j).

Finally, to further validate that SFKs phosphorylate SHP2 Y62, we performed in vitro kinase
assays using bacterially purified recombinant SHP2. SRC, YES1, and FYN kinases
phosphorylated SHP2 WT at Y62 but not the Y62F phosphodead mutant (Fig. 3h). SRC, YES1,
and FYN also phosphorylated Y542 and Y580 in vitro (Fig. 3h). Together these data show that
SHP2 Y62 is a direct substrate of SFKs, and that SHP2 Y542 and Y580 can also be
phosphorylated by SFKs.

SHP2 Y62 phosphomimetic protein constitutively increases phosphatase activity

SHP2 Y62 phosphorylation has been proposed to disrupt its autoinhibited state through the
introduction of negative charge at the N-SH2/PTP interface, thereby enhancing phosphatase
activity, and this has been supported by biochemical and cellular experiments with the Y62
phosphomimetic mutants30,56,57. We corroborated these prior results by conducting Michaelis-
Menten enzyme kinetics with the Y62D mutant, which showed a 2.4-fold increase in k /K when
cat m
compared with wild-type SHP2 (Fig. 4a). Tyrosine to aspartate mutation is commonly used as a
phosphomimetic to model the effects of phosphorylation, though it may not fully recapitulate the
structural or binding consequences of a bona fide phosphotyrosine at this site. By contrast, the
Y542 and Y580 phosphorylation sites are thought to mediate intramolecular interactions with the
SHP2 SH2 domains or mediate intermolecular interactions with other SH2-containing proteins.
SH2-mediated interactions with tyrosine-phosphorylation proteins cannot be readily mimicked
using D/E substitutions, and indeed, the Y542D and Y580D mutations did not significantly impact
SHP2 activity (Fig. 4a). Furthermore, the combination of Y542D or Y580D with Y62D did not
appreciably impact the gain-of-function effect of Y62D (Fig. 4a).

When SHP2 auto-inhibition is disrupted by mutations, it becomes less responsive to activation by
phosphopeptides58. Indeed, unlike wild-type SHP2, Y62D-containing SHP2 variants could not be
activated by binding to an IRS-1-derived phosphopeptide (Fig. 4b). To further examine whether
these mutations disrupt auto-inhibition, we used differential scanning fluorimetry (DSF)
(Supplementary Fig. 4). Previous studies have shown that SHP2 variants with disrupted
autoinhibition tend to have lower melting temperatures59. Indeed, only SHP2 variants containing
the Y62D mutation, but not Y542D or Y580D, had decreased melting temperatures (Fig. 4c).
Collectively, these experiments show that the Y62D mutation disrupts SHP2 auto-inhibition and
corroborates the assertion that Y62 phosphorylation may disrupt auto-inhibition through
electrostatic effects.

SHP2 Y62D samples mixed open and closed conformations through increased exchange
in its buried nSH2 domain

Since the SHP2Y62D phosphomimetic protein exhibited increased enzymatic activity (Fig. 4a) and
decreased thermal instability (Fig. 4c), and since SHP2 oncogenic mutation propensity for an
open conformation correlates with increased catalysis and increased dynamic and solvent
exposure60,61, we hypothesized that SHP2 Y62 phosphorylation increases catalysis by enforcing
an open, dynamic conformation. We investigated this hypothesis using computational, structural,
and biophysical methods. We used AlphaFold2 to analyze conformational changes in SHP2Y62D
(Supplementary Fig. 5, Supplementary Data 4) and compared the predicted structure to crystal
structures of SHP2WT, which adopts a closed conformation (PDB: 2SHP)8, and SHP2E76K, the only
SHP2 mutant with a crystal structure resolved in an open conformation (PDB: 6CRF)58. While
AlphaFold2 is not specifically powered for optimal structural prediction of missense mutations,
nonetheless, SHP2 Y62D was predicted to have a 22 Å root mean square deviation (RMSD) when
aligned with the WT crystal structure, but a 1.1 Å RMSD when aligned with the SHP2E76K structure
oncogenic mutant crystal structure, suggesting an open confirmation (Fig. 5a). We crystallized
the SHP2Y62D mutant and solved its structure at 2.63 Å resolution (Fig. 5b, Supplementary Table
1). The Y62D crystal structure exhibited a closed conformation, with a 0.5 Å RMSD when aligned
with the SHP2WT crystal structure (Fig. 5b).

Given the discordance between our biochemical and computational data and compared to our
structural data, we hypothesized that SHP2 Y62 phosphorylation samples both the closed and
open state conformations. To investigate the conformational state of the SHP2Y62D mutant, we
performed hydrogen-deuterium exchange mass spectrometry (HDX-MS) with recently developed
peptide disambiguation and analysis tools62 comparing SHP2Y62D to SHP2WT and the SHP2
phosphomimetic mutant Y542D/Y580D. Compared to WT, SHP2Y62D exhibited increased
deuterium uptake across peptides spanning not only the nSH2 domain (where Y62 is located),
but also in the PTP domain mediated by amino acids that bind to and maintain nSH2 domain
autoinhibitory interactions (Fig. 5c, Supplementary Fig. 6). Notably this PTP domain peptide
region (496-510) also includes S502, G503, and Q506 which bind to the nSH2 domain, are known
cancer-associated mutations61, and activate SHP2 in deep mutational scanning studies63. This
pattern is consistent with increased destabilization of the closed, autoinhibited conformation and
adoption of an open conformation relative to SHP2WT. In contrast, the Y542D/Y580D mutant
showed minimal differences in hydrogen exchange relative to WT, indicating that these
phosphomimetic substitutions do not strongly alter the conformational ensemble (Supplementary
Fig. 6). Together, these data demonstrate that the Y62D mutation promotes an open, active-like
conformation of SHP2, distinct from WT and Y542D/Y580D.

SHP2 Y62 phosphomimetic mutation activates MAPK signaling and causes resistance to
SHP2 inhibitors

Since SHP2 pY62 is enriched in RTK-driven tumors, we tested the role of SHP2 Y62
phosphorylation in cancer cells. We knocked out endogenous PTPN11/SHP2 using CRISPR-
Cas9 in BT20 and U-2 OS cells, which are frequently used to model SHP2 alterations30,58
(Supplementary Fig. 2a), and complemented this with lentiviral overexpression of SHP2WT,
aspartate and phenylalanine mutants at Y62 and Y542/Y580. Complementation with empty vector
control was not tolerated by cells, consistent with PTPN11/SHP2 loss being synthetic lethal in
RTK-driven cancer cells20. SHP2Y62D, but not SHP2Y62F, sustained maximal MAPK signaling as
measured by phosphorylated MEK (pMEK) and phosphorylated ERK (pERK) in SHP2 KO BT20
cells, suggesting that SHP2 pY62 is necessary for maximal MAPK signaling (Fig. 6a). To
delineate how upstream SFKs modulate MAPK signaling, we used our SYF KO MEF models.
SYF KO showed decreased pMEK and pERK as compared to WT MEFs (Fig. 3g).

Given the abundance of SHP2 pY62 across cancer cells and tumors, and the minimal response
rates conferred by SHP2i in the clinic, we hypothesized that SHP2 pY62 confers resistance to
SHP2i. We performed biochemical inhibition assays using purified recombinant SHP2WT and
SHP2Y62D proteins. SHP099 IC values were 0.2319 μM for WT and 1.356 μM for Y62D
50
(Supplementary Fig. 7), a statistically significant shift. These data show that the Y62D variant has
reduced sensitivity to allosteric SHP2 inhibition. We tested varied allosteric SHP2 inhibitors
including SHP099, RMC4550, and TNO155, which are based on similar scaffolds that stabilize
the SHP2 autoinhibited conformation20,64,65. SHP2 Y62 phosphorylation as modeled by SHP2Y62D
resulted in resistance to SHP099, RMC4550, and TNO155, as measured by sustained pERK
signaling, as well increased GAB1 phosphorylation which is a direct readout of SHP2 scaffolding
function66 (Fig. 6b-d).

Methods
Data reproducibility statement
To ensure the rigor and reproducibility of our findings, all experimental procedures were
conducted following standardized protocols with appropriate controls. Detailed methods, including
reagent sources, catalog numbers, and RRIDs, are provided to enable exact replication. All
experiments were conducted with at least three biological replicates, and statistical analyses were
performed in accordance with established guidelines.

In silico analysis of pY62 abundance
Phosphorylation_site_dataset.gz was obtained from PhosphoSitePlus34. MS_LIT (the number of
published studies reporting the phosphosite) was added to MS_CST (the number of unpublished
mass spectrometry experiments performed by Cell Signaling Technology reporting the
phosphosite) to obtain the number of observations in Fig. 1b and c. LT_LIT (the number of low
throughput, experimentally validated publications supporting the site) was divided by the total
number of observations [(LT_LIT+1)/(MS_CST+MS_LIT)] to obtain the ratio of literature support
and abundance of phosphopeptide observation in Fig. 1d.

The phosphosite abundance in KRAS mutations vs WT tumors and EGFR mutations vs WT
tumors in lung adenocarcinoma was obtained from Gillette et al (2020) – Supplementary table
S4 and S4M39. The phosphosite abundance in FGFR altered vs non-altered tumors in
intrahepatic cholangiocarcinoma was obtained from Dong et al (2022)40. We analyzed relative log
fold-changes within each dataset, without normalization to total protein levels and plotted with
GraphPad (Prism, RRID:SCR_002798). We used published phosphoproteomic summary
statistics and focused on relative ranking of phosphosite enrichment within each dataset;
therefore, no new hypothesis testing, confidence intervals, or multiple-comparison adjustments
were performed.

Cell lines used in this study
HEK293T LentiX (ATCC - CRL-3216, RRID:CVCL_0063), U-2 OS (ATCC - HTB-96™,
RRID:CVCL_0042), BT20 (ATCC - HTB-19™, RRID:CVCL_0178), HCC827 (ATCC - CRL 2868,
RRID:CVCL_2063), SYF KO (ATCC - CRL-2459, RRID:CVCL_6461), and SV40 immortalized
Mouse embryonic fibroblast – MEFs (Gift from Assistant Prof. Iok In Christine Chio, Columbia
University) were used in this study and frequently checked for mycoplasma contamination. U-2
OS, SYF KO MEFs, and WT MEFs were maintained in DMEM media (Gibco, Cat# 11965-092)
with 10% FBS (Sigma, Cat# F0926) and 1% Pen Strep (Gibco, Cat# 15070-063). BT20 was
maintained in DMEM/F12 media (Gibco, Cat# 11330-032) with 10% FBS and 1%
Penicillin/Streptomycin. HCC827 was maintained in RPMI media 1640 (Gibco, Cat# 11875-093)
with 10% FBS and 1% Pen Strep. TrypLE™ Express (Gibco, Cat# 12604-021) was used to detach
the cells from the plates.

RTK stimulation
The stock solution (100 mg/mL) of Human EGF (Sigma-Aldrich, Cat# E9644), FGF1
(SignalChem, Cat# F819-40N-50), and PDGF-ββ (Sigma-Aldrich, Cat# SRP3138) were made
with ddH O and stored at -20 oC. The cells were serum starved for 24 hours prior to the RTK
2
stimulation. The ligands were diluted in appropriate media and spiked in the cells. The stimulation
was stopped at different time points by washing with ice cold PBS wash.

Drug treatment
All drugs were diluted with DMSO to acquire 10 mM stock concentration and were diluted with
appropriate media prior to the drug treatments. Erlotinib (Cat# S7786), PDGFR inhibitor 1 (Cat#
S8721), Tofacitinib (Cat# S2789), dasatinib (Cat# S1021), saracatinib (Cat# S1006), SHP099
(Cat# S6388), TN0155 (Cat# S8987), and RMC4550 (Cat# S8718) were purchased from
Selleckchem, Houston, TX, USA. Infigratinib (Cat# HY-13311), asciminib (Cat# HY-104010),
ripretinib (Cat# HY-112306), SU6656 (Cat# HY-B0789), CH6953755 (Cat# HY-135299), and PP2
(Cat# HY-13805) were purchased from MedChemExpress, NJ, USA. For drug treatment assays
(RTK inhibitors and SFK inhibitors), the cells were treated with final concentrations of the desired
drugs in the normal media. For RTK stimulation in the presence of inhibitors, the cells were serum
starved for 24 hours, followed by an hour drug treatment and RTK stimulation at timepoints. For
SHP2 inhibitors treatment with EGF stimulation, the cells were serum starved for 24 hours and
treated with allosteric SHP2 inhibitors for an hour and followed by 10 minutes EGF stimulation
(final concentration 100 ng/mL). The reactions were stopped by washing with ice cold PBS wash.

Western blotting analysis
Cells were lysed in Pierce™ RIPA lysis buffer (Thermo Scientific, Cat# PI87788) containing
protease (Selleckchem, Cat# B14001) and phosphatase inhibitors (Selleckchem, Cat# B15001).
BCA assay (Thermo Scientific, Cat# 23225) was performed to determine the protein
concentration. Samples were run on PAGE gels (ThermoFisher) and transferred to Trans-Blot
transfer membranes (Bio-Rad, Cat# 1620115) using the Trans-Blot® Turbo™ Transfer System.
The membranes were blocked with 5% milk in 1x TBST (10x TBS - Bio-Rad, Cat# 1706435;
Tween® 20- Fisher, Cat# 240186) and probed with the following primary antibodies, pEGFR-
Y1068 (1:1000, Cat# 3777S, RRID:AB_2096270), EGFR (1:1000, Cat# 4267S,
RRID:AB_2864406), pFGFR-Y653/654 (1:1000, Cat# 3471S, RRID:AB_331072), pPDGFR-Y751
(1:1000, Cat# 3161S, RRID:AB_331053), pGab1-Y627 (1:1000, Cat# 3233S), pSRC-Y416
(1:1000, Cat# 2101S, RRID:AB_331697), SRC (1:1000, Cat# 2110T, RRID:AB_10691385),
pSHP2-Y62 (1:500, This study), pSHP2-Y542 (1:1000, Cat# 3751S, RRID:AB_330825), pSHP2-
Y580 (1:1000, Cat# 3703S), SHP2 (1:1000, Cat# 3397S, RRID:AB_2174959), pMEK-S217/S221
(1:1000, Cat# 9154S), MEK (1:1000, Cat# 8727S), pERK-T202/Y204 (1:1000, Cat# 4370S), ERK
(1:1000, Cat# 4696S), Vinculin (1:1000, Cat# 13901S, RRID:AB_2728768), -actin (1:5000, Cat#
8457T, RRID:AB_10950489) and V5 (1:1000, Cat# 13202S, RRID: AB_2687461) from Cell
Singaling Technologies and secondary antibodies (anti-Mouse IgG (1:4000, Cat# 7076S, RRID:
AB_330924), and anti-Rabbit IgG (1:4000, Cat# 7074S, RRID: AB_2099233) from Cell Singaling
Technologies before developing with SuperSignal West Atto Ultra-Sensitivity Chemiluminescent
Substrate (ThermoFisher: A38556). Blots were detected using the ChemiDoc MP Imaging System
(Bio-Rad).

Protein expression and purification
cDNA of SHP2 (UNIPROT ID Q06124) was ordered as a Genestring and cloned into a pGEX-5X-
1 vector (Addgene, Cat# 27-4584-01) with a N-terminal GST tag and thrombin cleavage site for
in vitro experiments. For all other in vitro, the cDNA was cloned into a pHAT vector (RRID:
Addgene_112582) with a non-removable N-terminal his-tag. For lentiviral overexpression, the
cDNA was cloned into pLX304 (RRID: Addgene_25890) with a C-terminal v5 tag. pGEX-5X-
1_SHP2C495S, pGEX-5X-1_SHP2Y62F, pHAT_SHP2Y62D, pHAT_SHP2Y542D, pHAT_SHP2Y580D,
pHAT_SHP2Y62D_Y542D, pHAT_SHP2Y62D_Y580D, pHAT_SHP2Y542D_Y580D,
pHAT_SHP2Y62D_Y542D_Y580D, pLX304_SHP2Y62F, pLX304_SHP2Y62D, pLX304_SHP2Y542F_Y580F,
pLX304_SHP2Y542D_Y580D were prepared through site directed mutagenesis, designed with
NEBaseChanger.

pGEX-5X-1_SHP2 or pHAT_SHP2 constructs were transformed into electrocompetent E. coli
BL21(DE3) (NEB, Cat# C2527H). All constructs were expressed in 0.5-1L LB with 100 µg/mL
carbenicillin/ampicillin, induced at OD600 0.4-0.6 with 0.1 mM IPTG and grown overnight at 20
°C. Cell pellets were washed with binding buffer (20 mM Tris, 500 mM NaCl, 5 mM Imidazole, pH
8.0) and spun down.

For pHAT_SHP2 constructs, the washed cells were resuspended with 10-15 mL lysis buffer
(binding buffer, 1x Bugbuster (Millipore, Cat# 7092 1), 1 tablet protease inhibitor/50 mL (Pierce,
Cat# A32963), 1 mg/mL Lysozyme from chicken egg white (Sigma, Cat# L8676), 0.2 µL/mL
nuclease (Pierce, Cat# 88701) and lysed for 30 minutes at room temperature. The supernatant
was separated (15000g, 30 minutes) and added to 1.5 mL CV of nickel resin (His-select nickel
affinity gel, Merck, P6611 on a gravity-based column. Nickel resin was washed with 5 CV binding
buffer with 2 mM TCEP before being eluted with 2.5 mL elution buffer (20 mM Tris, 500 mM NaCl,
500 mM Imidazole, 2 mM TCEP pH 8.0). The eluted protein was dialyzed with PD10 columns
(Cytiva, Cat# 17085101) to SHP2 buffer (25 mM Tris-HCl, 150 mM NaCl, 2 mM DTT, pH 8.0).
The dialyzed protein was size excluded on a Superdex SD75 column (Cytiva, Cat# 10/300 GL) and
concentrated through Amicon centrifugal filters (Millipore, Cat# UFC803024, 4 mL, 30K). All
proteins were aliquoted for single-use, flash-frozen, and stored at -80 °C.

For pGEX-5X-1_SHP2 constructs, the washed cells were resuspended with 10-15 mL lysis buffer
(PBS (Gibco, Cat# 20012-027), 1x Bugbuster, 1 tablet protease inhibitor/50 mL (Pierce,
Cat# A32963), 1 mg/mL Lysozyme from chicken egg white (Sigma, Cat# L8676), 0.2 µL/mL nuclease
(Pierce, Cat# 88701) and lysed for 30 minutes at room temperature. The supernatant was
separated (15000g, 30 min) and combined with 2 mL CV glutathione resin (Pierce Glutathione
Agarose, Cat# 16101) washed with equilibration buffer (50 mM Tris, 150 mM NaCl, pH 8.0)
rotating overnight at 4 °C. The resin-supernatant mixture was added to a gravity column and the
flow through was discarded. The resin was washed by resuspending the resin with 5 mL
equilibration buffer. Thrombin (Sigma-Aldrich, Cat# T4648-1KU) resuspended in 1 mL cold PBS
(1U/μL), was diluted 10x in equilibration buffer, and 100 units of thrombin (1 mL) were added to
the column, cleaving overnight at 4 °C. One mL of protease inhibitor solution (Pierce, Cat#
A32963, one tablet/50 mL) was added to the column, collecting the eluted fraction. The eluted
fraction was dialyzed to RTK buffer (20 mM Tris, 300 mM NaCl, 1 mM TCEP, 5 mM MgCl2, 20%
glycerol, pH 8.0) after which ~5 v/v% of concentrated protease inhibitor was added (1 tablet
protease inhibitor/mL, Pierce, Cat# A32963). All proteins were aliquoted for single-use, flash-
frozen and stored at -80 °C.

In vitro kinase assay
SRC (Cat# S19-18G), YES1 (Cat# Y01-10G), and FYN (Cat# F15-10G) kinase proteins were
purchased from SinoBiological. Reaction mixture was prepared with the 10x Kinase buffer (Cell
Signaling Technology, Cat# 9802S), ATP (Cell Signaling Technology, Cat# 9804S, final
concentration 1 mM), SHP2 recombinant protein (1 µg), the kinase of interest (0.2 µg) and ddH2O
to make the final volume of 50 µL. The reaction mixture was incubated at 30C in Thermocyler for
1 hour and stopped by adding 4x sample buffer (Life Technologies, Cat# NP0008) in the reaction
mixture and incubation at 95C for 5 mins.

Michaelis-Menten kinetics
Phosphatase activity was measured using the EnzChek® Phosphatase Assay kit for SHP2
activity (Invitrogen, Cat# E12020) according to the manufacturer’s protocol. Briefly, the reactions
were initiated by the addition of 0-2000 µM 6,8-Difluoro-4-Methylumbelliferyl Phosphate
(DiFMUP) to the reaction mixture containing purified SHP2 variants in 1x reaction buffer (100 mM
sodium acetate (pH 5.0). The fluorescence was measured using excitation at 360 nm and
emission was recorded at 455 nm with a Tecan Infinite M200 Pro Plate Reader. Initial reaction
rates were determined and divided by the final enzyme concentration to determine specific
reaction rates. The specific reaction rates for each SHP2 variant were plotted against substrate
concentration and the curves were fitted to the Michealis-Menten equation (1) using GraphPad
(Prism, RRID:SCR_002798) to establish turnover number k (2) and Michaelis constant K
cat m
values.

(1) Y= V *X / (K + X)
max m
Where X is substrate concentration, Y is enzyme velocity, V is maximum reaction rate and K
max m
is Michaelis constant

(2) k = V / [E]
cat max
Where k is turnover number and [E] is total enzyme concentration
cat

p-IRS stimulation
p-IRS (Y1172) (GLN{pTyr}IDLDLVKD, GenScript, Lot# U644VGB170-1/PE4713) was
resuspended in DMSO to a concentration of 3 mg/mL. SHP2 variants (0.15 μM final, in SHP2
buffer) were combined with a linear titration of p-IRS in HEPES buffer (10 μM- 0.01 μM final) and
incubated for 30 minutes at room temperature. DiFMUP (100 μM final) was added to start the
reaction, and fluorescence was measured at t = 30 min by the Tecan Infinite M200 Pro Plate
Reader (RRID:SCR_019033).

T measurements
m
SHP2 variants were diluted to 2 mg/mL in SHP2 buffer (25 mM Tris-HCl, 150 mM NaCl, 2 mM
DTT, pH 8.0). SYPRO orange (5000x, Thermo Fisher) was diluted to a 25x solution in SHP2
buffer. In a 20 µL reaction, 5 µL SHP2 (0.5 mg/mL final), 4 µL SYPRO orange (5x final) was
combined in SHP2 buffer in triplicate. The sample was measured on an Applied Biosystems 7500
Fast Real-Time PCR (RRID:SCR_018051), using ROX as a reporter with a 0.1 °C/sec slope from
25 to 99°C. Melting temperature (Tₘ) was recorded at the inflection point of fluorescence vs.
temperature curve.

AlphaFold2 alignments
The AlphaFold model of SHP2WT (entry: Q06124) was obtained from AlphaFold website (version:
AF-Q06124-F1-v4, accessed on March 9, 2023). SHP2Y62D mutant primary sequence was
generated by modifying the wildtype SHP2 (UNIPROT ID Q06124) where tyrosine residue (Y) at
62 was replaced with aspartate (D). SHP2Y62D mutant structure models were predicted using
AlphaFold267. AlphaFold2-predicted structures of SHP2Y62D mutant and SHP2WT were
superimposed onto crystal structures of SHP2E76K (PDB: 6CRF) and SHP2WT (PDB: 2SHP).

Crystallography
• Protein Production
The SHP2Y62D construct used for crystallization comprises residues 1-528 of SHP2, the mutation
Y62D and a non-cleavable C-terminal hexahistidine tag and was based on a previously described
construct68. The crystallization construct was produced in E. coli BL21 (DE3) cells. The cells were
lysed and the protein was purified by Ni2+-affinity chromatography, ion-exchange chromatography
and size-exclusion chromatography. The purified protein was concentrated to 13.7 mg/mL in 50
mM NaCl, 20 mM TRIS-HCl pH 7.8, 1 mM EDTA and 1 mM DTT, cooled in liquid nitrogen and
stored at -80°C. The identity of the protein was confirmed by intact mass spectrometry and peptide
mass fingerprinting.

• Crystallization
SHP2Y62D was crystallized by hanging-drop vapor-diffusion at 20°C. 1.0 µL of SHP2Y62D (13.7
mg/mL in 50 mM NaCl, 20 mM TRIS-HCl pH 7.8, 1 mM EDTA and 2 mM DTT) was mixed with
1.0 µL of crystallization solution (0.2 M BICINE pH 8.5 and 17% w/v PEG 4000) and equilibrated
against a reservoir containing crystallization solution. The crystals were mounted after three days.
Crystals were cryo-protected in crystallization solution supplemented with 20% v/v Glycerol (final
concentration) and cooled in liquid nitrogen. Data were collected at beamline BL18U1 of the
Shanghai Synchrotron Radiation Facility (Shanghai, China).

• Structure determination
Diffraction data were integrated, analyzed and scaled with XDS69, POINTLESS, AIMLESS and
STARANISO70 in AUTOPROC71. The structures were determined by molecular replacement with
PHASER72 using a previously determined model of SHP2 (without any ligands) based on PDB ID
2SHP [https://doi.org/10.2210/pdb2SHP/pdb] as a starting model. The model was improved
through manual rebuilding of the model in COOT73 and restrained refinement with BUSTER74.
Atomic displacement factors were modelled with a single TLS group per chain and a single
isotropic B-factor per atom. The backbone geometry was analyzed with MOLPROBITY75.

HDX/MS Protocol
H O buffer (25 mM Tris-HCl, 150 mM NaCl, 2 mM DTT) was prepared in deionized water and
2
lyophilized using Labconco FreeZone 4.5 lyophilizer in 5 ml aliquots for at least 24 hours, then
resuspended in D O to produce deuterated buffer. HX liquid handling was performed using the
2
LEAP system (Trajan) controlled by Chronos 5.8.3 software (Trajan). Protein stock integrity was
verified by MALDI-MS (Bruker Autoflex Speed TOF/TOF). The protein stock solution was
manually pipetted into reaction vials (to avoid syringe clogging), diluted tenfold in deuterated
buffer (7.5 µl protein stock in 75 µl buffer), and exchanged at 15 ºC for the specified time, then
diluted twofold (75 µl exchange reaction) in a quench solution (75 µl, 3% acetonitrile, 1% formic
acid in H O, all MS-grade, Fisher Scientific) at 2 ºC. 140 μL of the quenched reaction was injected
2
into the protease column, which was chilled to 7 °C. Solvent A (3% acetonitrile, 0.15% formic acid
in H O) was pumped (UltiMate 3000, ThermoFisher Scientific) at 150 μL/min through the protease
2
column. Peptides eluted from the protease column were trapped on a 1x10 mm C18 column
(Hypersil GOLD, 3 μM poresize, ThermoFisher Scientific). For Rep1 and Rep2, we used an
immobilized protease type XIII/pepsin column (FP, 1:1 w/w, 2.1 × 30 mm, NovaBioAssays), for
Rep3 we used an alanyl aminopeptidase/pepsin column (AP/pepsin 2.1 x 20 mm, Affipro)
(Supplementary Table 2).

Fully deuterated sample buffer was prepared by adding 3 M GuHCl to deuterated buffer. Full-D
control samples were prepared by manually diluting protein stock 1:10 in Full-D buffer and
exchanging at 37 ºC for at least 24 hours. Exchanged Full-D reaction was pipetted into reaction
vials (82.5µl per vial) and subsequently quenched and injected exactly as for other samples.

An auxiliary pump (MX-Class, Teledyne) flowed additional Solvent A over the trap C18 at 150
μL/min. After digesting and desalting (3 min for rep1, 200 s for rep2), the system valve was
switched so that the binary pump (Infinity II, Agilent) was in-line with the trap C18, flowing in the
opposite direction. The binary pump used a gradient of Solvent A and Solvent B (96.85%
acetonitrile, 0.15% formic acid) to elute peptides from a 50 x 1 mm C18 analytical column (Hypersil
GOLD, 1.9 μM pore size, ThermoFisher Scientific). The elution method consisted of isocratic flow
at 10% B for 1 min, a linear gradient to 40% B from 1 to 15 min, a linear gradient to 95% B from
15 to 19 min, isocratic flow at 100% B from 19 to 24 min, a linear gradient to 10% B from 24 to 30
min, and then isocratic flow at 10% B from 30 to 45 min, at a flow rate of 0.04 mL/min. The
protease column was washed between sample injections with injections of quench solution.

Eluted peptides entered the mass spectrometer (Bruker Maxis II ETD) via electro-spray ionization,
where full-scan mass spectra were collected in positive ion mode from 100-2250 m/z with a
spectra rate of 1.0 Hz, 500 V end plate offset, 4200 V capillary, 1.7 bar nebulizer, 8 L/min dry gas,
and 180 °C dry temp. We also ran tandem mass spectrometry (MS/MS) experiments for each
sample with the same full MS settings as described above with the exception of the spectra rate
of 1.20 Hz and the 200 °C dry temp. We used Bruker Compass Hystar 5.1 software to acquire
the data.

HDX/MS data analysis
We used Compass DataAnalysis 5.1 (Bruker) to identify and deconvolute peptides. For peptide
matching and disambiguation, we used PIGEON, using 30 ppm initial cutoff, 7ppm narrow cutoff,
0.020 Da error for MSMS, score cutoff 0.05, peptide length 3-15, charge cutoff 3, 0.1 D/e and 0.5
min degeneracy cutoffs, using the WT MSMS data. The resultant peptide pool was imported to
HDExaminer 3.3.0 (Sierra Analytics, Inc. 2020) and all spectra were fit for each replicate, all
peptide assignments and fits were manually checked for quality. Peptide pool results and spectra
were exported from HDExaminer and used for peptide-level analysis of both replicates in
FEATHER, with envelope centroids from HDExaminer used to produce uptake plots. Deuterated
envelope centroids were compared to un-deuterated and to the number of exchangeable residues
in each peptide to determine deuteration levels as % of maximum, determined by averaging full-
D centroid values across all replicates and mutants for each peptide, or using the theoretical
maximum if no full-D spectra were obtained. Deuteration levels of all peptides covering a residue
were averaged and subtracted between WT and both mutants and averaged across all timepoints
to calculate pseudo-residue level 𝑑𝐷̅. Fully deuterated controls were used for peptide-level back-
exchange correction; although percent-exchange comparisons across different peptides are
feasible in principle, all analyses here are confined to comparisons of identical peptide sequences
across matched protein variants.

Generation of SHP2 mutant cell lines
The endogenous SHP2 protein was knocked out using CRISPR/Cas9 gRNA obtained from
GeneScript (Supplementary Table 3). The three sgRNAs were cloned into pLENTICRISPR V2
backbone (RRID: Addgene_140206).

• Cloning the sgRNA plasmids
The cloned plasmids control, guide 1,2,3 was transformed into competent E. coli DH5⍺ cells
(NEB, Cat# C2987H), using the heat shock method. 1 μL of DNA was added to 50 μL cell aliquot
and equilibrated for 5 min. the cells were heat shocked by putting the tube in a water bath set at
42 °C for 40 s. Then, it was placed on ice for 2 min. 500 μL of SOC media was added and
incubated on the shaker at 37 °C for an hour. After an hour, the tubes were spun down and 400
μL of supernatant was removed. The bacterial pellet was resuspended, and the suspension was
added to the antibiotic agar plate. The colonies were picked next day and bacterial DNA miniprep
(Macherey-Nagel, Cat# 740727) was setup.

• Generating lentivirus
Day 0: 293T LentiX cells was seeded in 10 cm plate (5-8 million cells/plate) in 10 mL DMEM.
Day 1: The amount of plasmid DNA was calculated, JetprimeⓇ transfection media (Polyplus),
and packaging plasmids (psPAX2, RRID: Addgene_12260 and pMD2.G, RRID: Addgene_12259)
required for transfection (the amount was calculated as per the protocol given by Polyplus).
The manufacturer’s protocol was followed. Refresh media after 4 hours.
Day 3: The lentivirus was harvested. The media of HEK293T LentiX cell plates was aspirated with
a 10 mL syringe, and the 0.45 µM filter was attached to the syringe, and the virus was filtered out
into the falcon tube. (If not using the virus immediately for infection, make aliquots in cryovials
and store them in a -80 °C freezer).

• Infecting the target cells to knock out SHP2
The target cells were seeded (U-2 OS and BT20) in 6 well plates (3 wells for virus + 1 well for no
virus control).
Next day, lentivirus infection was performed. The virus was thawed, and 1.5 mL of the respective
virus was added to each well and 1.5mL media to no virus well. 8 µg/mL of polybrene was added
to each well to increase infection efficiency. The volume was made up to 2 mL, and the plates
were incubated overnight. The next day, the media was refreshed to remove the virus.
On the following day, the cells were trypsinized if required, and antibiotic selection (blasticidin)
was started. The antibiotic selection was performed until all the cells in the no-virus well were
dead.

Generating isogenic SHP2 mutant cell lines
We performed Gibson cloning to incorporate gRNAs of different SHP2 mutants into the pLX304
vector backbone (Supplementary protocol). The SHP2 mutant cell line generation was done using
the same protocol as above, using the JetprimeⓇ transfection and antibiotic selection
(puromycin).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Karunaraj, P., Scheele, R., et al. characterized biochemical and structural properties of the SHP2 Y62 phosphorylation using\nthe Y62D phosphomimetic mutant. The authors’ bioinformatics study attempted to substantiate the importance of the Y62\nphosphorylation. Using various kinase inhibitors, the authors identified SRC and other SFKs as the kinases that directly\nphosphorylate SHP2 at Y62.\nI appreciate that the authors employed multidisciplinary approaches to convey their point. However, there is insufficient\nevidence for SHP2 Y62 phosphorylation by SRC and other SFKs, as the authors claim. This is because the inhibitor studies\npresented (Fig. 2, 3, and Supplemental Fig. 3) lack essential data; none of these blots display the amount of the inhibitor-\ntargeted kinases. For example, the authors should include a blot showing depletion of active pY1068 EGFR with erlotinib\ntreatment, alongside pY62 SHP2 (Fig. 2c). All the authors’ inhibitor studies suffer from the same problem. The authors must\nshow that the added inhibitors had the intended effect on targeted kinases (decrease/disappearance of the active\nRTKs/SFKs) along with the level of SHP2 phosphorylation.\nAlso, where is the experimental evidence for pY62 being a drug-resistant mutation? Was there a previous study determining\nIC50 for the pY62 mutant against SHP2 inhibitors? If not, the authors should determine IC50 against one representative\nSHP2 inhibitor to make the title of this manuscript valid.\nThe authors presented Fig.6 as evidence for pY62 drug resistance. This suffers from the same issue as Figs. 2 and 3 as\nstated above. Fig.6B-D does not show phospho-EGFR level with EGF and the level of SHP2 after the transfection. What\nhappened to the level of SHP2 with each inhibitor?\nOther points are the following:\nFigure 1B-1G: The content of these figures is not clear at all.\nFig.1B and 1C: What does the Y-axis of the graph correspond to? I understand that the authors identified a total of 239,531\nphosphosites in all human proteins combined, but why are there multiple hits at y-axis (hits) = 1? Do these correspond to\nproteins with 1 phospho-sites? But, that doesn’t make sense since I see a lot of points above 100 hits. In summary, what\ndoes the “hit” correspond to? The authors must describe this point clearly in the manuscript.\nFig.1C and 1D:\nLine 107-110: These statements appear to be contradictory. According to the authors, SHP2 pY62 is the most abundant in\nthe database search, but pY62 is less reported than the canonical pY542 and pY580. Please provide a proper explanation\non this point.\nFig.1E-1G: The authors found pY62 SHP2 as the most abundant phosphorylation in different cancer cell lines except for\nKRAS-mutant NSCLC. Considering tyrosine phosphorylation comprises only ~ < 1-2 % of the entire proteome at most, the\nauthors’ finding is pretty shocking. Also, the phosphorylation abundance of different studies cannot be compared since\ndifferent sample preparation methods, LC, as well as mass spectrometry settings, are variable and influence the determined\nabundance (or amount) of phosphopeptides.\nIf the authors wish to highlight the significance of pY62, a previous study (ref 30) seems to be the most suitable for\nelaboration, instead of the Figure 1 results.\nAs to Reference 30, this study found that FLT3 phosphorylates SHP2 at Y62, as opposed to the authors’ finding. Please\nelaborate on this point.\nFigures 2 and 3: As stated on the first page, the level of inhibitor-targeted kinases should be shown on blots. Also, Fig.3a is\ndifficult to see.\nFigure 4B: This is probably the most interesting result in this manuscript. The Y62D mutant becomes insensitive to the\npresence of an allosteric peptide activator, while the peptide can activate the other mutants. Where is the peptide binding\nsite?\nFigure 5A: Structural alignment between an AlphaFold predicted model and a crystal structure of SHIP2 in a closed\nconformation (top right).\n• The figure does not appear to make sense. The PTP domain appears very similar in both structures, with the only\ndifference being the orientation of the SH2 domain. Did AlphaFold provide alternative structures that closely resemble the\nclosed conformation?\nFigure 5C: The percent exchange structure map\nThe preparation of the fully-deuterated standard poses an issue. The point of doing a fully-deuterated experiment is to obtain\nthe maximum deuterium uptake of each peptide under the same labeling strength (pH, temperature, and deuterium dilution\nfactor) as that of the labeling experiment. This allows us to normalize the deuterium uptake of each peptide and\nexpress them as the percent exchange. This is important since the structural flexibility of different regions in the same state can be\ncompared. For example, the structural flexibility of region A and region B in the same protein (state) can be compared since\na fully-deuterated experiment corrects for the back-exchange difference between the two regions (corrects for back-\nexchange dependence on amino acid composition/sequence of peptides). It also enables the comparison of the percent\nexchange between different proteins, provided that the fully-deuterated experiments are appropriately performed.\nThe labeling strength is highly dependent on labeling temperature. So, the authors’ result vastly underestimates structural\nflexibility due to the higher temperature employed for the full-deuterated experiment. For example, consider the following\nscenario where you are analyzing a 10-residue peptide. The maximum possible uptake is 9 (no proline), and you want to\ndetermine the fully-denatured/deuterated uptake of this peptide. The labeling strength is lower at 15 °C than at 37 °C. So, the\nexperimentally determined maximum uptake of all peptides will be higher at 37 °C (Say Duptake = 7 for this peptide) than at\n15 °C (Duptake = 5). If you then determine the same peptide uptake to be 3, the calculated percent exchange at 37 °C and\n15 °C is 43% and 60%, respectively. A viable remedy for this issue is to correct the deuterium uptake data using the different\ntemperature factor (or repeat the fully-deuterated experiments).\nAlternatively, since Fig.5C uptake plots (in Da, the same peptide uptake comparisons) are appropriate, I suggest that the\nauthors keep these exchange uptake data. It will be helpful if the authors indicate/highlight peptide regions in the SHP2\nstructure more clearly.\nAlso, based on the HDX-MS results, can the authors speculate how the pY62 mutant (Y62D) confers drug resistance? Is the\nY62D mutant drug resistant like the pY62 mutant?\nIn summary, the title of this manuscript does not align with the content. The manuscript requires major revision, with a\nsubstantial number of additional experiments needed to show a connection between pY62 activation and drug resistance.", "reviewer_2": "This paper reports a thorough exploration of phosphorylation of SHP2 on Y62 as a molecular mechanism of sustaining\nSHP2 in an active form and as a mechanism of resistance to SHP2 inhibition. The data generally support the conclusions\ndrawn by the authors. Overall, the work will be a meaningful contribution to the field, and I am supportive of publication after\nthe refinement of the x-ray structure is completed (see below) and the authors make the requisite textual edits to give the\nprior work proper recognition.\nMajor points:\n1) The authors unnecessarily downplay the prior identification of pY62 that was reported in two other manuscripts. They do\ncite both papers, but their text in the introduction and discussion minimizes the value of the earlier work and makes it appear\nas if the authors are making a breakthrough discovery of Y62 phosphorylation. They should be more forthright in\nacknowledging the prior work documenting Y62 phosphorylation as a resistance mechanism. What makes this work stand\nout is that it more fully explores the generality of Y62 phosphorylation across cancer cell types and the scope of contexts in\nwhich the modification may impact signaling, reports a pY62 phosphoselective Ab to probe direct versus indirect induction of\nthe pY mark, thereby assigning phosphorylation to SFKs, rather than the RTKs, and uses Y62D as a phosphomimetic for\nstructural analysis, HX-MS, and biochemical studies to complement the cellular work.\n2) Review of the x-ray structure table, text, pdb validation report, model, and map shows that the claim in the table of 2.63 Å\nresolution is not congruent with the text, which states a resolution of 2.9 Å, and that the data set is not complete, particularly\nin the outermost shell. The Rwork and Rfree values are unexpectedly high, even for the claimed 2.9 Å resolution, and the\nquality of the map and model fit could be substantially improved. Attention to the refinement might also clarify whether there\ncould be a bridging H2O molecule between D61 and D62.\nOne other minor point - the enzymatic data to measure catalytic parameters appear to substantially overestimate the activity\nof the WT (unmutated) protein compared to values published elsewhere. The basal activity of wt SHP2 is difficult to measure\naccurately, but it is not clear why that would be true.", "reviewer_3": "SHP2 is a phosphatase and a critical mediator of receptor tyrosine kinase (RTK)-driven RAS/mitogen-activated protein\nkinase (MAPK) signaling. Despite promising preclinical data, SHP2 inhibitors have shown minimal clinical efficacy, with no\ndefined clinical mechanisms of primary resistance. Therefore, it is important to elucidate the underlying mechanism on the\nlack of clinical response of SHP2 inhibitors in human cancer and to develop new therapeutic strategies to target SHP2.\nIn this study, the authors elucidated phosphorylation of SHP2 at tyrosine 62 (pY62) as a hotspot phosphorylation site in the\nproteome and RTK-driven tumor types in patients. They demonstrated that SRC family kinases directly phosphorylate SHP2\nat Y62, downstream of but not directly phosphorylated by RTKs. Using biochemical and biophysical analyses, the authors\nshowed that SHP2 Y62D (a phosphor tyrosine mimic) enforces an open, active conformation, resulting in constitutive\nphosphatase activation that is sufficient to activate MAPK signaling and confer resistance to allosteric SHP2 inhibitors. Their\nfindings establish that SHP2 pY62 is a phosphorylation hotspot phenocopying mutational activation, a mechanism of\nprimary resistance to SHP2 inhibitors, and a cancer drug target distinct from wildtype SHP2.\nThis study employed a number of complementary approaches and the experiments were well done. The conclusion was\nsolid and well justified. The manuscript was well written and easy to follow. This study answered an urgent and important\nquestion for the SHP2 filed and provided novel therapeutic approaches to target active SHP2, instead of inactive SHP2. It is\nrecommended that the study be published in Nature Communications."}, "author_response": "We thank the reviewer for acknowledging our multidisciplinary approach.\nHowever, there is insufficient evidence for SHP2 Y62 phosphorylation by SRC and other SFKs,\nas the authors claim. This is because the inhibitor studies presented (Fig. 2, 3, and\nSupplemental Fig. 3) lack essential data; none of these blots display the amount of the inhibitor-\ntargeted kinases. For example, the authors should include a blot showing depletion of active\npY1068 EGFR with erlotinib treatment, alongside pY62 SHP2 (Fig. 2c). All the authors’ inhibitor\nstudies suffer from the same problem. The authors must show that the added inhibitors had the\nintended effect on targeted kinases (decrease/disappearance of the active RTKs/SFKs) along\nwith the level of SHP2 phosphorylation.\nWe thank the reviewer for this comment. To address the need for on-target validation, we\nperformed immunoblots for active RTKs and SFKs under the same treatment conditions used in\nall inhibitor experiments. pEGFR, pFGFR, pPDGFR, and pSRC (Y416) blots now accompany\neach assay in the revised Figures 2 and 3 and Supplemental Figure 3, confirming effective\nsuppression of the targeted kinases. We note that the polyclonal pSRC (Y416) antibody (CST\n#2101) cross-reacts with pYES and pFYN.\nAs representative examples, erlotinib treatment markedly reduces pEGFR (Y1068) in the same\nsamples used for pY62 SHP2 analysis (revised Fig. 2C), and dasatinib reduces pSRC (Y416)\nunder conditions used for SFK inhibition (revised Fig. 3C).\nRevised Figure 2c Revised Figure 3c\n• Also, where is the experimental evidence for pY62 being a drug-resistant mutation? Was\nthere a previous study determining IC50 for the pY62 mutant against SHP2 inhibitors? If not,\nthe authors should determine IC50 against one representative SHP2 inhibitor to make the\ntitle of this manuscript valid.\nWe thank the reviewer for this question. We are not aware of any prior studies reporting SHP2\ninhibitor IC values for pY62 or its phosphomimetic Y62D. Therefore, we performed\n50\nbiochemical inhibition assays using purified recombinant SHP2 WT and Y62D proteins. SHP099\nIC values were 0.2319 μM for WT and 1.356 μM for Y62D (Supplementary Fig. 7), a\n50\nstatistically significant shift. These data show that the Y62D variant has reduced sensitivity to\nallosteric SHP2 inhibition.\nSupplementary Figure 7\n• The authors presented Fig.6 as evidence for pY62 drug resistance. This suffers from the\nsame issue as Figs. 2 and 3 as stated above. Fig.6B-D does not show phospho-EGFR level\nwith EGF and the level of SHP2 after the transfection. What happened to the level of SHP2\nwith each inhibitor?\nWe thank the reviewer for this comment. We have added blots for pEGFR (Y1068) and total\nSHP2 to the revised Figure 6B-D. As shown in the revised panels, pEGFR increases\nappropriately with EGF stimulation, and total SHP2 levels remain unchanged across the\nisogenic lines, indicating that the observed differences are not attributable to altered SHP2\nexpression. We have shown Revised Fig. 6B as a representative example here:\nRevised Figure 6b\nOther points are the following:\nFigure 1B-1G: The content of these figures is not clear at all.\n• Fig.1B and 1C: What does the Y-axis of the graph correspond to? I understand that the\nauthors identified a total of 239,531 phosphosites in all human proteins combined, but why\nare there multiple hits at y-axis (hits) = 1? Do these correspond to proteins with 1 phospho-\nsites? But, that doesn’t make sense since I see a lot of points above 100 hits. In summary,\nwhat does the “hit” correspond to? The authors must describe this point clearly in the\nmanuscript.\nWe thank the reviewer for pointing out this lack of clarity. In Figures 1B and 1C, the Y-axis\n(“hits”) represents the sum of two PhosphoSitePlus annotations: MS_LIT, the number of\npublished studies reporting a given phosphosite, and MS_CST, the number of unpublished\nmass-spectrometry experiments curated by Cell Signaling Technology. Most sites have only a\nsingle hit, typically from MS_CST, whereas higher values reflect sites detected repeatedly\nacross many studies or datasets.\nWe have removed the y-axis label “hits” and have relabeled the y-axis to “MS_LIT + MS_CST”\nin Figure 1B and 1C. The definitions covering MS_LIT and MS_CST have now been added to\nthe Results and Methods sections to ensure this point is clear in the manuscript.\n• Fig.1C and 1D:\nLine 107-110: These statements appear to be contradictory. According to the authors, SHP2\npY62 is the most abundant in the database search, but pY62 is less reported than the\ncanonical pY542 and pY580. Please provide a proper explanation on this point.\nWe thank the reviewer for highlighting this point. In Figure 1D, we quantified the characterization\nfrequency in the literature, using (LT_LIT + 1) / (MS_LIT + MS_CST). LT_LIT, another metric\nreported on PhosphoSitePlus, counts only “low-throughput” (LT), experimentally validated\nstudies and represents a small subset of MS_LIT. In the numerator, we added 1 so that\nphosphosites without LT_LIT entries (which are the majority of phosphorylation sites in\nPhosphoSitePlus) would still appear on the plot rather than being assigned a value of zero.\nUnder this metric, SHP2 pY62 has limited experimental characterization (LT_LIT = 1) despite\nfrequent detection in large data sets, whereas pY542 and pY580 have far more LT-validated\nreports (LT_LIT = 46 and 20, respectively). Above, we discussed how MS_LIT + MS_CST, the\ndenominator here, is higher for pY62 and lower for pY542 and pY580. Together, this explains\nthe lower value for (LT_LIT + 1) / (MS_LIT + MS_CST) for pY62 in Figure 1D and reflects its\nlimited targeted experimental follow-up, rather than a contradiction with Figure 1C. The\ndefinition covering LT_LIT has now been added to the Results and Methods sections to ensure\nthis point is clear in the manuscript.\n• Fig.1E-1G: The authors found pY62 SHP2 as the most abundant phosphorylation in\ndifferent cancer cell lines except for KRAS-mutant NSCLC. Considering tyrosine\nphosphorylation comprises only ~ < 1-2 % of the entire proteome at most, the authors’\nfinding is pretty shocking. Also, the phosphorylation abundance of different studies cannot\nbe compared since different sample preparation methods, LC, as well as mass spectrometry\nsettings, are variable and influence the determined abundance (or amount) of\nphosphopeptides. If the authors wish to highlight the significance of pY62, a previous study\n(ref 30) seems to be the most suitable for elaboration, instead of the Figure 1 results.\nWe thank the reviewer for this thoughtful comment. We agree that global phosphotyrosine\nabundance is low (<1-2% of all phosphorylation) and that absolute comparisons of phosphosite\nabundance across studies are not possible due to differences in sample preparation,\nenrichment, chromatography, and MS settings. In Figures 1E-G, our analysis relies only on\nwithin-dataset relative abundance (mutant versus WT tumors), using CPTAC-provided\nphosphosite intensity values that are not normalized to total protein levels. Our conclusions\ntherefore reflect relative differences within each dataset rather than absolute phosphosite\nstoichiometry.\nWe also agree that enrichment methods influence detectability. CPTAC uses pY-specific\nantibody enrichment for tyrosine sites and IMAC/TiO for serine/threonine sites, so pY and\n2\npS/pT values are not directly comparable. However, this methodological distinction primarily\naffects which peptides enter the dataset, not the rank-ordering of relative abundance within a\ngiven enrichment workflow. Importantly, because our analysis relies only on within-dataset\nrelative abundances, these methodological differences do not affect the conclusions drawn.\nNotably, in KRAS-mutant NSCLC—the one context where pY62 is not top-ranking—the most\nabundant phosphopeptide is a serine/threonine site (KRT8 pS475/pS478), indicating that the\nobserved pattern is not simply an artifact of pY enrichment.\nFinally, we appreciate the suggestion to reference the earlier mechanistic study (ref. 30), which\nwe have described more extensively in our Introduction and Discussion. That work identified\npY62 in the context of SHP099 resistant FLT3-driven AML cells. Our analysis is complementary:\nit arises from an unbiased, multi-dataset, multi-cancer interrogation of cell and tumor\nphosphoproteomic data, revealing pY62 as a recurrent and relatively enriched pY site across\ndiverse tumor contexts. Thus, Figure 1 provides independent large-scale support for the\nbiological relevance of pY62, which aligns with but does not duplicate the findings of ref. 30.\n• As to Reference 30, this study found that FLT3 phosphorylates SHP2 at Y62, as opposed to\nthe authors’ finding. Please elaborate on this point.\nWe thank the reviewer for raising this point. We respectfully note that the data presented in\nReference 30 do not demonstrate direct phosphorylation of SHP2 Y62 by FLT3, as suggested in\nits Abstract. In that study, FLT3-ITD autophosphorylation and SHP2 pY62 both increase in\nFLT3-ITD AML models of acquired SHP099 resistance, but the experiments do not test whether\nFLT3 directly phosphorylates Y62. Specifically, SHP2 pY62 levels were not assessed following\nFLT3 inhibition, and no in vitro kinase assays were performed to demonstrate FLT3-dependent\nphosphorylation of SHP2.\nWhat Reference 30 does show is a correlation between FLT3 activation and elevated SHP2\npY62. This observation is fully consistent with our model: we likewise find that SHP2 pY62\nincreases with RTK activation, but our biochemical, kinase, and inhibitor data indicate that the\nphosphorylation event is mediated by SFKs rather than by the RTKs themselves.\n• Figures 2 and 3: As stated on the first page, the level of inhibitor-targeted kinases should be\nshown on blots. Also, Fig.3a is difficult to see.\nWe have now included the levels of all inhibitor-targeted kinases as measured by decreased\nautophosphorylation in the revised blots for Figures 2 and 3, as requested in the first page of the\nreview. In addition, Figure 3A has been enlarged and reformatted to improve clarity and to\nprovide a more easily interpretable visualization of the predicted kinases within the kinome tree.\n• Figure 4B: This is probably the most interesting result in this manuscript. The Y62D mutant\nbecomes insensitive to the presence of an allosteric peptide activator, while the peptide can\nactivate the other mutants. Where is the peptide binding site?\nWe thank the reviewer for this insightful comment. SHP2-activating phosphopeptides bind the\ncanonical pY-binding cleft of the nSH2 (high-affinity) and cSH2 (low-affinity) domains (Eck,\nNature 1996). We are not aware of any crystal structures of human SHP2 nSH2 bound to pIRS1\n(Y1172), which was the peptide used in our experiments (characterized in Fujimoto, JBC 1994).\nStructures of Syp (mouse SHP2) nSH2 bound to pIRS1 (Y895) and pPDGFR (Y1009) as well\nas structures of human SHP2 nSH2 bound to pPDGFR (Y1009) have demonstrated that the\nphosphopeptides engage SHP2 nSH2 residues including R32, S33-N36, T42, T52-I56, L65-\nG68, Y81, L88-K91, and I96 (Lee, Structure 1994; Hof, Cell 1998; Eck, Nature 1996). These\ninteractions stabilize the open, active conformation by relieving nSH2-mediated autoinhibition.\nY62, however, is not part of the peptide-binding surface. It lies instead at the nSH2-PTP\ninterface, where phosphorylation—or the Y62D phosphomimetic—destabilizes autoinhibition\nand constitutively favors the open state. As a result, Y62D is already “activated” and cannot be\nfurther stimulated by the pIRS1 peptide, whereas other mutants retain an autoinhibited ground\nstate that the peptide can still unlock.\nThus, the peptide binds the established nSH2 interface, while Y62D activates SHP2 through a\ndistinct structural mechanism that renders it insensitive to additional allosteric activation.\n• Figure 5A: Structural alignment between an AlphaFold predicted model and a crystal\nstructure of SHIP2 in a closed conformation (top right). The figure does not appear to make\nsense. The PTP domain appears very similar in both structures, with the only difference\nbeing the orientation of the SH2 domain. Did AlphaFold provide alternative structures that\nclosely resemble the closed conformation?\nWe thank the reviewer for this insightful question. In Figure 5A, the structures were\nsuperimposed on the PTP domain because the defining conformational difference between\nopen and closed SHP2 states is the position of the SH2 domains; the PTP domain itself remains\nlargely unchanged. Because Y62D introduces a single substitution within the nSH2 domain,\nclose alignment of the PTP domains between the AlphaFold-predicted and experimentally\nsolved structures is expected and consistent with known SHP2 conformational behavior.\nRegarding alternative conformations, AlphaFold2 did generate multiple models for SHP2 Y62D.\nAs shown in the revised Supplementary Figure 5, the top 4 ranked models adopt open\nconformations, whereas the 5th ranked model more closely resembles the closed state. These\noutputs are in agreement with our HDX-MS and crystallographic data demonstrating that SHP2\nY62D can sample both conformations but preferentially occupies the open conformation. All\nrelevant AlphaFold models have now been included in Supplementary Figure 5.\nSupplementary Figure 5\n• Figure 5C: The percent exchange structure map\nThe preparation of the fully-deuterated standard poses an issue. The point of doing a fully-\ndeuterated experiment is to obtain the maximum deuterium uptake of each peptide under\nthe same labeling strength (pH, temperature, and deuterium dilution factor) as that of the\nlabeling experiment. This allows us to normalize the deuterium uptake of each peptide and\nexpress them as the percent exchange. This is important since the structural flexibility of\ndifferent regions in the same state can be compared. For example, the structural flexibility of\nregion A and region B in the same protein (state) can be compared since a fully-deuterated\nexperiment corrects for the back-exchange difference between the two regions (corrects for\nback-exchange dependence on amino acid composition/sequence of peptides). It also\nenables the comparison of the percent exchange between different proteins, provided that\nthe fully-deuterated experiments are appropriately performed.\nIn our HDX/MS workflow, which follows standard practice in the field, the fully deuterated control\nis used to determine the maximum possible deuterium uptake for each peptide. To obtain this,\nwe fully unfold the protein, deuterate it, and measure the resulting mass of each peptide. These\nvalues allow us to correct all forward-exchange data for D-to-H back-exchange, which occurs\nduring chromatographic processing. In our hands, back-exchange is typically 10-40%, and we\nexclude peptides with excessive back-exchange (>50%) to maintain high confidence.\nAfter this correction, deuterium uptake can be compared within the same peptide across\nconditions (e.g., WT vs mutant). However, as the reviewer notes, back-exchange correction\nalone does not make peptides with different amino-acid compositions directly comparable,\nbecause each sequence has its own intrinsic exchange rates that cannot be precisely\nnormalized on a residue-by-residue basis. For this reason, percent exchange should not be\ncompared across peptides of different sequences, but comparisons among identical peptides\n(e.g., WT vs point mutants) are valid and appropriate.\n• The labeling strength is highly dependent on labeling temperature. So, the authors’ result\nvastly underestimates structural flexibility due to the higher temperature employed for the\nfull-deuterated experiment. For example, consider the following scenario where you are\nanalyzing a 10-residue peptide. The maximum possible uptake is 9 (no proline), and you\nwant to determine the fully-denatured/deuterated uptake of this peptide. The labeling\nstrength is lower at 15 °C than at 37 °C. So, the experimentally determined maximum\nuptake of all peptides will be higher at 37 °C (Say Duptake = 7 for this peptide) than at 15 °C\n(Duptake = 5). If you then determine the same peptide uptake to be 3, the calculated\npercent exchange at 37 °C and 15 °C is 43% and 60%, respectively. A viable remedy for\nthis issue is to correct the deuterium uptake data using the different temperature factor (or\nrepeat the fully-deuterated experiments).\nWe thank the reviewer for raising this point and appreciate the opportunity to clarify the purpose\nof the full-D control in our HDX-MS experiments. We appreciate the reviewer’s broader point\nthat labeling conditions influence absolute percent-exchange values. The full-D control serves a\ndistinct purpose from the forward-exchange reaction and is not intended to replicate its\nconditions. Its sole purpose is to provide an accurate estimate of back-exchange so that\ndeuterium-uptake curves for each peptide can be properly corrected.\nTo achieve complete deuteration, the protein must be fully unfolded, which we accomplish by\nincreasing the temperature before quenching. This ensures that all exchangeable amides are\nmaximally exposed and allows us to determine the true upper limit of deuterium incorporation.\nBecause this step is designed to measure back-exchange after quenching, rather than any\nproperty of the forward-exchange reaction, the full-D sample does not need to match the\ntemperature or folding state of the time-point samples prior to the quench.\nIf the full-D control were performed under the same folded-protein conditions as the time-point\nsamples, many residues would remain incompletely exchanged even after 48 hours, leading to\nan inaccurate estimate of back-exchange. For this reason, it is standard practice to use\nunfolded conditions for the full-D control to ensure complete deuteration and accurate peptide-\nlevel correction.\n• Alternatively, since Fig.5C uptake plots (in Da, the same peptide uptake comparisons) are\nappropriate, I suggest that the authors keep these exchange uptake data. It will be helpful if\nthe authors indicate/highlight peptide regions in the SHP2 structure more clearly.\nWe agree that the uptake plots in Fig. 5C are appropriate for comparing identical peptides\nacross matched proteins, and we have retained these data in the revised manuscript. To\nimprove clarity, as suggested, we have now indicated the N- and C-terminal positions of the four\npeptides directly on the SHP2 structural model and have revised Figure 5C accordingly. All\npeptides analyzed are clearly annotated in the Supplemental Information.\nRevised Figure 5c\n• Also, based on the HDX-MS results, can the authors speculate how the pY62 mutant\n(Y62D) confers drug resistance? Is the Y62D mutant drug-resistant like the pY62 mutant?\nSHP099 and related allosteric inhibitors act as molecular glues that bind at the nSH2-PTP\ninterface and stabilize the autoinhibited, closed conformation of SHP2. Our HDX-MS data show\nthat the Y62D phosphomimetic shifts the conformational equilibrium toward a more open state,\nreducing the stability of this autoinhibited interface. This provides a mechanistic basis for\nreduced inhibitor sensitivity: because the closed conformation is less populated, the SHP099\nbinding interface is correspondingly less accessible.\nConsistent with this model, the biochemical inhibition assays in Supplementary Figure 7\ndemonstrate that SHP2 Y62D exhibits decreased sensitivity to SHP099 compared with wild-type\nSHP2, indicating that the phosphomimetic is drug-resistant at the protein level.\nIn summary, the title of this manuscript does not align with the content. The manuscript requires\nmajor revision, with a substantial number of additional experiments needed to show a\nconnection between pY62 activation and drug resistance.\nWe have now performed the additional experiments requested throughout the review—including\ndirect biochemical measurements of inhibitor sensitivity for SHP2 Y62D, on-target validation of\nkinase-inhibitor treatments, and expanded structural and HDX-MS analyses. Together, these\nnew experiments strengthen the mechanistic linkage between pY62 activation and reduced\nsensitivity to allosteric SHP2 inhibition and bring the title and conclusions of the manuscript into\nfull alignment with the expanded experimental evidence.\nWe appreciate the Reviewer for these favorable comments.\nMajor points:\n• The authors unnecessarily downplay the prior identification of pY62 that was reported in two\nother manuscripts. They do cite both papers, but their text in the introduction and discussion\nminimizes the value of the earlier work and makes it appear as if the authors are making a\nbreakthrough discovery of Y62 phosphorylation. They should be more forthright in\nacknowledging the prior work documenting Y62 phosphorylation as a resistance\nmechanism. What makes this work stand out is that it more fully explores the generality of\nY62 phosphorylation across cancer cell types and the scope of contexts in which the\nmodification may impact signaling, reports a pY62 phosphoselective Ab to probe direct\nversus indirect induction of the pY mark, thereby assigning phosphorylation to SFKs, rather\nthan the RTKs, and uses Y62D as a phosphomimetic for structural analysis, HX-MS, and\nbiochemical studies to complement the cellular work.\nWe appreciate the reviewer’s point and agree that prior studies made important contributions by\nidentifying SHP2 pY62 in cytokine-stimulated hematopoietic cells and in FLT3-driven AML\nmodels with acquired SHP099 resistance. Our intent was not to minimize these findings, and we\nhave revised the Introduction and Discussion to acknowledge this work more directly and\nprominently.\nSpecifically, we now state in the Introduction that pY62 was first observed in cytokine-stimulated\nhematopoietic stem cells (Louria-Hayon, PNAS 2013) and that pY62 was later found to be\nenriched in SHP099 resistant FLT3-driven acute myeloid leukemia cells and was associated\nwith SHP-099 resistance possibly through enforcing an open, active conformation of SHP2\n(Pfeiffer, Cancer Res 2022). In the Discussion, we explicitly note that pY62 has been linked to\nSHP2 inhibitor resistance in leukemia and place our work in this context—emphasizing that our\nstudy extends these observations by defining the broader prevalence of pY62 across cancer\ntypes, demonstrating SFK-mediated phosphorylation using a phosphospecific antibody, and\nintegrating structural, biochemical, and HDX-MS analyses using the Y62D phosphomimetic.\n• Review of the x-ray structure table, text, pdb validation report, model, and map shows that\nthe claim in the table of 2.63 Å resolution is not congruent with the text, which states a\nresolution of 2.9 Å.., \nWe apologize for this inconsistency. We have corrected the reported resolution in the text from\n2.9 Å to 2.63 Å, which now matches the data processing and refinement statistics presented in\nthe table.\n• and that the data set is not complete, particularly in the outermost shell.\nWe agree that the dataset shows reduced completeness in the highest-resolution shell, and this\nis a direct consequence of the anisotropic diffraction that we observed across all SHP2 crystals\ncollected for this project. We attempted to improve the quality of the crystals through\noptimization of both the crystallization and cryo-protection conditions, but the quality of the\ncrystals did not improve significantly.\nTo ensure that all useful reflections were used during refinement the data were processed with\nSTARANISO (Global Phasing). STARANISO applies an anisotropic cutoff limit to include\nreflections from strongly diffracting regions of reciprocal space and exclude reflections from\nweakly diffracting regions of reciprocal space. The inclusion of the strong reflections during\nrefinement improves the accuracy of the model and the interpretability of the electron density\nmaps, while the exclusion of the weak reflections reduces the amount of noise in the electron\ndensity maps. While the use of an anisotropic cutoff limit results in an apparently reduced data\ncompleteness, this is only through the exclusion of reflections that would not improve the\naccuracy of the model or the quality of the electron density maps.\n• The Rwork and Rfree values are unexpectedly high, even for the claimed 2.9 Å resolution,\nand the quality of the map and model fit could be substantially improved.\nWe agree that the Rwork and Rfree values are higher than typically expected at this resolution,\nwhich is a recognized consequence of refining against an anisotropic dataset. Nevertheless, the\nelectron-density maps are sufficiently interpretable to support the key conclusion we draw from\nthe structure—namely, that SHP2 does not adopt an open conformation in this crystal form.\nBecause our structural interpretation relies on the absence of features consistent with the open\nstate rather than on modeling fine details of an activated conformation, the quality of the maps is\nadequate for this purpose.\n• Attention to the refinement might also clarify whether there could be a bridging H2O\nmolecule between D61 and D62.\nThere are two waters in the close vicinity of Asp61 and Asp62 (in chain A, W/117 and W/144).\nWe have included a figure here where the electron density around those two residues is\ndepicted alongside the final coordinates (rmsd 1.0 sigma, the depicted chain is chain A).\nFrom the attached image it becomes clear that the two waters and the carboxy groups of Asp61\nand Asp62 form a trapezoid. However, the distances between the waters and the carboxy\ngroups are too high to build a water network by themselves. There are no indications in the\nelectron density that further water molecules are bound close to Asp61 or Asp62.\nFigure: Electron density of Asp61 and Asp62 (in purple) including surrounding (5 Å) residues.\nProtein in green, the two water molecules are depicted as red spheres and the distance to\nAsp61/Asp62 is shown.\n• One other minor point - the enzymatic data to measure catalytic parameters appear to\nsubstantially overestimate the activity of the WT (unmutated) protein compared to values\npublished elsewhere. The basal activity of wt SHP2 is difficult to measure accurately, but it is\nnot clear why that would be true.\nIn revisiting our analysis, we realized that the initial activity values had not been normalized\nusing a standard calibration curve, which can lead to overestimation of absolute catalytic rates.\nWe have now reprocessed the data using an appropriate standard curve, and the revised\nvalues—shown in Figure 4A—are consistent with published basal activities reported for WT\nSHP2. We apologize for this error and thank the reviewer for drawing attention to this point.\nWe agree that accurately determining the basal activity SHP2, which is an intrinsically\nautoinhibited phosphatase, is inherently challenging particularly at very low substrate turnover.\nAlthough additional measurements at lower DiFMUP concentrations could further refine these\nestimates, the revised kinetic parameters now fall within the expected range and align with\npreviously reported values.\nWe appreciate the reviewer’s comments about our findings."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I thank the authors for taking considerable time to address my concerns. I would also like to commend them for performing through experiments and for providing a clear and thoughtful rebuttal to my comments.\nI would like to offer one additional point regarding HDX-MS analysis. Comparison of percent deuterium exchange across different peptides is appropriate provided that fully deuterated control experiments are performed. In response, the authors state that\n“back-exchange correction alone does not make peptides with different amino-acid compositions directly comparable,because each sequence has its own intrinsic exchange rates that cannot be precisely normalized on a residue-by-residue basis.”\nHowever, the purpose of back-exchange correction is precisely to normalize for sequence-dependent effect, including intrinsic exchange arising from amino acid composition. Indeed, examination of back-exchange values across peptides often reveal clear sequenced dependence – for example, histidine-containing peptides typically show higher back exchange – demonstrating that such correction explicitly account for intrinsic sequence effects. Once normalized using fully deuterated controls, percent deuterium exchange therefore allows meaningful comparison between different peptides.\nIf percent exchange across peptides were fundamentally invalid, the utility of back-exchange correction would be unclear. In that case, analysis would be restricted to absolute deuterium uptake (Da), limiting comparisons to identical peptides across conditions and substantially reducing the interpretive power of HDX-MS.\nFurthermore, full deuteration can be reliably achieved for most soluble proteins within 1-3 minutes at 25 C using high denaturant concentration (e.g., 6 M guanidine-DCl, or 8 M urea-d4), provided that the labeling strength is sufficient (~20-fold deuterium dilution). I recommend the authors to visit: https://hx2.med.upenn.edu/download.html, which calculates theoretical intrinsic exchange as well as exchange rates in various conditions (pH, temp). Although the site only calculates these rates for given peptide sequences, it may be useful to estimate full exchange times.", "reviewer_2": "The authors adequately addressed my comments about the referencing of prior work, their X-ray data, and the catalytic activity of the autoinhibited WT enzyme. I defer to the first reviewer regarding comments about SFKs as the kinases responsible for Y62 phosphoryation, as well as their other concerns."}, "author_response": "We thank the reviewer for this additional clarification and agree with the general principle that,\nwhen fully deuterated controls are appropriately performed, percent deuterium exchange can be\nused to compare structural flexibility across different peptides.\nIn our HDX-MS workflow, fully deuterated controls were performed for all peptides and used to\ncorrect for D-to-H back-exchange, with peptides exhibiting excessive back-exchange excluded.\nThis enables robust peptide-level normalization and supports valid comparison of deuterium\nuptake under matched conditions.\nIn the context of this study, we intentionally restrict interpretation to comparisons of identical\npeptide sequences across matched protein variants (WT versus point mutants), providing a\nconservative and direct assessment of mutation-induced conformational changes.\nTo make this analytical choice explicit, we have added the following sentence to the Methods\nsection: “Fully deuterated controls were used for peptide-level back-exchange correction;\nalthough percent-exchange comparisons across different peptides are feasible in principle, all\nanalyses here are confined to comparisons of identical peptide sequences across matched\nprotein variants.”\nWe believe this clarification addresses the reviewer’s point while accurately reflecting both\nstandard HDX-MS practice and the analytical framework used in this work.\nWe appreciate the reviewer’s comments."}]

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