The Src homology 2-containing phosphotyrosine phosphatase (SHP2) is primarily a positive effector of receptor tyrosine kinase signaling. However, the molecular mechanism by which SHP2 effects its biological function is unknown. In this report, we provide evidence that defines the molecular mechanism and site of action of SHP2 in the epidermal growth factor-induced mitogenic pathway. We demonstrate that SHP2 acts upstream of Ras and functions by increasing the half-life of activated Ras (GTP-Ras) in the cell by interfering with the process of Ras inactivation catalyzed by Ras GTPase-activating protein (RasGAP). It does so by inhibition of tyrosine phosphorylation-dependent translocation of RasGAP to the plasma membrane, to its substrate (GTP-Ras) microdomain. Inhibition is achieved through the dephosphorylation of RasGAP binding sites at the level of the plasma membrane. We have identified Tyr992 of the epidermal growth factor receptor (EGFR) to be one such site, since its mutation to Phe renders the EGFR refractory to the effect of dominant-negative SHP2. To our knowledge, this is the first report to outline the site and molecular mechanism of action of SHP2 in EGFR signaling, which may also serve as a model to describe its role in other receptor tyrosine kinase signaling pathways.The process of protein tyrosine phosphorylation and dephosphorylation is central to growth factor, cytokine, and integrin signal transduction. The enzymes that catalyze phosphorylation reactions are either the receptors themselves with intrinsic tyrosine kinase activity, known as receptor tyrosine kinases (RTKs), or cytoplasmic tyrosine kinases. The substrates for these kinases are the receptors themselves and/or downstream signaling proteins. Some of the most extensively studied RTKs include the epidermal growth factor receptor (EGFR), the platelet-derived growth factor receptor (PDGFR), and the fibroblast growth factor (FGF) receptor (13,21,22,27,29,37,42,49,55,57). Upon the binding of a cognate ligand, RTKs dimerize and autophosphorylate tyrosine residues in their cytoplasmic domain or tyrosylphosphorylate downstream substrates (5,6,9,11,17,20,25,30,34,41). More often than not, phosphorylated tyrosine residues serve as binding sites for Src homology 2 (SH2)-domain-containing signaling proteins (38). These interactions mediate the formation of multiprotein signaling complexes by which signals are transduced down the cascade. At least two known functions are effected by these interactions: recruitment of enzymes to substrate microdomains and/or induction of enzyme activity (3,16,18,26,30,41,43; M. Adachi,

The Src homology phosphotyrosyl phosphatase 2 (SHP2) plays a positive role in HER2-induced signaling and transformation, but its mechanism of action is poorly understood. Given the significance of HER2 in breast cancer, defining a mechanism for SHP2 in the HER2 signaling pathway is of paramount importance. In the current report we show that SHP2 positively modulates the Ras-extracellular signal-regulated kinase 1 and 2 and the phospoinositide-3-kinase-Akt pathways downstream of HER2 by increasing the half-life the activated form of Ras. This is accomplished by dephosphorylating an autophosphorylation site on HER2 that serves as a docking platform for the SH2 domains of the Ras GTPase-activating protein (RasGAP). The net effect is an increase in the intensity and duration of GTP-Ras levels with the overall impact of enhanced HER2 signaling and cell transformation. In conformity to these findings, the HER2 mutant that lacks the SHP2 target site exhibits an enhanced signaling and cell transformation potential. Therefore, SHP2 promotes HER2-induced signaling and transformation at least in part by dephosphorylating a negative regulatory autophosphorylation site. These results suggest that SHP2 might serve as a therapeutic target against breast cancer and other cancers characterized by HER2 overexpression.The Src homology phosphotyrosyl phosphatase 2 (SHP2) 2 functions as a positive effector of cell growth and survival (1-4), migration and invasion (5-8), and morphogenesis and transformation (9 -11). In receptor-tyrosine kinase signaling (12-14), SHP2 positively transduces the Ras-extracellular signal-regulated kinase 1 and 2 (ERK1/2) and the phosphoinositide-3-kinase-Akt (or protein kinase B) signaling pathways. SHP2 also promotes cell transformation induced by the constitutively active form of fibroblast growth factor receptor 3 and v-Src (9, 11). The discovery of germline-activating SHP2 mutations in Noonan and LEOPARD syndrome patients (15-18) and the subsequent experimental demonstration of these phenotypes in knockin and transgenic mice expressing these mutants (19,20) has led to the conclusion that disregulation of SHP2 is responsible for these disease states. Furthermore, somatic activating SHP2 mutations were discovered in juvenile myelomonocytic leukemia, acute myelogenous leukemia, and chronic myelomonocytic (18,21) and are suggested to play a causative role.SHP2 possesses two Src homology 2 (SH2) domains in the N-terminal region that allow the protein to localize to substrate microdomains after tyrosyl phosphorylation of interacting proteins. The phosphotyrosyl phosphatase (PTP) domain in the C-terminal region is responsible for dephosphorylation of target substrates (13,22). Mutation of the critical Cys residue in the active site of SHP2 abolishes its phosphatase activity, leading to the production of a dominant-negative protein (23). The activity of SHP2 is regulated by an intramolecular conformational switch. SHP2 assumes a "closed conformation" when inactive and an "open conformation" when active. In t...

SHP2 is a widely overexpressed signalling protein in IDC breast tumours. Given SHP2's positive role in cell growth, transformation and stem cell survival, the positive relationship of its overexpression to lymph node metastasis, nuclear accumulation of hormone receptors and higher tumour grade suggests that SHP2 promotes breast oncogenesis.

Vascular smooth muscle cells respond to the purinergic agonist ATP by increasing intracellular calcium concentration and increasing the rate of cell proliferation. In many cells the extracellular signal-regulated kinase (ERK) cascade plays an important role in cellular proliferation. We have studied the effect of extracellular ATP on ERK activation and cell proliferation. ATP binding to a UTP-sensitive P2Y nucleotide receptor activates ERK1/ERK2 in a time- and dose-dependent manner in coronary artery smooth muscle cells (CASMC). ATP-induced activation of ERK1/ERK2 is dependent on the dual-specificity kinase mitogen-activated protein kinase/ERK kinase (i.e., MEK) but independent of phosphatidylinositol 3-kinase (PI3K) activity. We provide evidence that both ERK1/ERK2 and PI3K activities are required for CASMC proliferation. Thus ATP-stimulation of CASMC proliferation requires independent activation of both the ERK and PI3K signaling pathways.

Receptor tyrosine kinases (RTKs) such as the fibroblast growth factor receptor (FGFR) and the epidermal growth factor receptor are overexpressed in a variety of cancers. In addition to overexpression, the FGFRs are found mutated in some cancers. The Src homology 2 domaincontaining phosphotyrosine phosphatase (SHP2) is a critical mediator of RTK signaling, but its role in oncogenic RTK-induced cell transformation and cancer development is largely unknown. In the current report, we demonstrate that constitutively activated FGFR3 (K/E-FR3) transforms NIH-3T3 cells, and that SHP2 is a critical mediator of this transformation. Infection of K/E-FR3-transformed 3T3 cells with a retrovirus carrying a dominant-negative mutant of SHP2 (C/S-SHP2) retarded cell growth, reversed the transformation phenotype and inhibited focus-forming ability. Furthermore, treatment of K/E-FR3-transformed NIH-3T3 cells with PD98059 or LY294002, specific inhibitors of MEK and PI3K, respectively, inhibited focus formation. Biochemical analysis showed that K/E-FR3 activates the Ras-ERK and the PI3K signaling pathways, and that the C/S SHP2 mutant suppressed this effect via competitive displacement of interaction of the endogenous SHP2 with FRS2. However, the C/S SHP2 protein did not show any effect on receptor autophosphorylation, FRS2 tyrosine phosphorylation or interaction of Grb2 with K/E-FR3 or FRS2. Together, the results show that K/E-FR3 is transforming and that the Ras-ERK and the PI3K-Akt signaling pathways, which are positively regulated by SHP2, are important for K/E-FR3-induced transformation.