Time-resolved phosphoproteomics reveals scaffolding and catalysis-responsive patterns of SHP2-dependent signaling.

Vidyasiri Vemulapalli,Morvarid Mohseni,Kimberley Stegmaier,Kartik Subramanian,Jonathan Larochelle,Ruili Cao,Khal-Hentz Gabriel,Michael G Acker,Anamarija Pfeiffer,Peter K Sorger,Stephen C Blacklow,Matthew J Lamarche,Alison Erickson,Lily A Chylek,Steven P Gygi

doi:10.7554/elife.64251

Abstract

SHP2 is a protein tyrosine phosphatase that normally potentiates intracellular signaling by growth factors, antigen receptors, and some cytokines, yet is frequently mutated in human cancer. Here, we examine the role of SHP2 in the responses of breast cancer cells to EGF by monitoring phosphoproteome dynamics when SHP2 is allosterically inhibited by SHP099. The dynamics of phosphotyrosine abundance at more than 400 tyrosine residues reveal six distinct response signatures following SHP099 treatment and washout. Remarkably, in addition to newly identified substrate sites on proteins such as occludin, ARHGAP35, and PLCγ2, another class of sites shows reduced phosphotyrosine abundance upon SHP2 inhibition. Sites of decreased phospho-abundance are enriched on proteins with two nearby phosphotyrosine residues, which can be directly protected from dephosphorylation by the paired SH2 domains of SHP2 itself. These findings highlight the distinct roles of the scaffolding and catalytic activities of SHP2 in effecting a transmembrane signaling response.

Highlights

SHP2 is a non-receptor tyrosine phosphatase that is essential for mammalian development (Saxton et al, 1997)
We investigated the role of SHP2 in the responsiveness to epidermal growth factor (EGF) by using the EGFR
(condition iii) revealed p-ERK1/2 induction with a kinetic profile similar to that of EGF stimulation in the absence of drug, showing that SHP099 inhibition of SHP2 is rapidly reversible in MDA-MB-468 cells (Figure 1A, Figure 1-Figure supplement 1A)

Summary

Introduction

SHP2 is a non-receptor tyrosine phosphatase that is essential for mammalian development (Saxton et al, 1997). Germline mutations of PTPN11 cause the developmental disorders Noonan and LEOPARD syndromes (Tartaglia and Gelb, 2005). Somatic PTPN11 activating mutations are found frequently in Juvenile Myelomonocytic. Numerous studies have shown that SHP2 acts as a positive effector of receptor tyrosine kinase (RTK) signaling (Bennett et al, 1994; Easton et al, 2006; Tang et al, 1995). Proteins following stimulation of cells with epidermal growth factor (EGF) or other receptor tyrosine kinase ligands. SHP2 serves as a positive regulator of numerous other signaling systems, including cytokine (Xu and Qu, 2008), programmed cell death (PD-1) (Yokosuka et al., 2012), and immune checkpoint pathways (Gavrieli et al, 2003)

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Conclusion