# Defining and targeting substrate specificity of protein tyrosine phosphatases

> **NIH NIH R01** · UNIVERSITY OF VIRGINIA · 2022 · $343,726

## Abstract

Cells possess enzymes and proteins that can write (kinase), erase (phosphatase), and read (phospho-specific
binding domains) phosphorylation modifications across the proteome to form a complex signaling system for
regulating proliferation, differentiation, and transcriptional activation. Aberrant phosphotyrosine (pTyr) signaling
underlie many human diseases and the identification of molecular components of pTyr processing has
uncovered fundamental principles of signal transduction and furnished new targets for therapy. A key example
is the oncogenic BCR-ABL fusion protein that exhibits constitutive tyrosine kinase activity resulting in excessive
pTyr modifications to transform cells in cancer. The development of small molecule (GleevecTM) and antibody
(HerceptinTM) therapeutics that target pTyr signaling continues to transform anti-cancer treatment options in the
clinic.
 Despite enormous clinical potential, a critical barrier that remains in the biomedical field is the ability to
assign site specific phosphorylation events to functional changes for therapeutic targeting in human disease.
Technologies capable of overcoming the low abundance and substoichiometric phosphorylation-site occupancy
of phosphoproteins are needed to address the challenge of functional phosphoproteomic profiling. This is
especially true for phosphorylation of Tyr. Compared with phospho-Ser and -Thr, pTyr modifications represent a
rare subset (~1%) of the human phosphoproteome.
 The objective of the proposed studies is to apply a site trapping by covalent probes (dubbed SiteTraP)
methodology to assign substrate specificity to the PTP oncogene, tyrosine-protein phosphatase non-receptor
type 11 (SHP2). The significance of the proposed studies is development of a chemical proteomics strategy to
assign substrate specificity – at the protein and pTyr site level – to individual PTPs directly on native proteins in
lysate and cellular studies.
 We will test 2 independent, yet related specific aims directed at benchmarking: (Aim 1) pTyr specificity of
SiteTraP in the presence of phospho-Ser and -Thr in complex proteomes, (Aim 1) Sensitivity of SiteTraP for
capturing SHP2-specific dephosphorylation of pTyr sites in complex proteomes, (Aim 2) Capability of SiteTraP
for capturing global pTyr activation in PTP-disrupted live cells, and (Aim 2) Sensitivity and specificity of SiteTraP
for assigning substrate specificity to SHP2 and determining how SHP2 inhibitors disrupt these networks in live
cells.
 Broadly, our proposed studies will be important for the biomedical community by 1) guiding PTP inhibitor
development for targeting specific pTyr modifications in human disease, 2) revealing differences in active site
and allosteric SHP2 inhibitor mode of action for basic and translational understanding of PTP pharmacology,
and 3) gain a deeper understanding of PTP-substrate networks and regulation in live cells.

## Key facts

- **NIH application ID:** 10341499
- **Project number:** 1R01GM144472-01
- **Recipient organization:** UNIVERSITY OF VIRGINIA
- **Principal Investigator:** Ku-Lung Hsu
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $343,726
- **Award type:** 1
- **Project period:** 2022-01-01 → 2025-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10341499

## Citation

> US National Institutes of Health, RePORTER application 10341499, Defining and targeting substrate specificity of protein tyrosine phosphatases (1R01GM144472-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10341499. Licensed CC0.

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