Molecular Mechanism for a Role of SHP2 in Epidermal Growth Factor Receptor Signaling

Abstract

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,

Autophosphorylation of the carboxyl‐terminal domain is not required for oncogenic transformation by lung‐cancer derived EGFR mutants

“…First, autophosphorylation of EGFR can have either a positive or a negative role in the regulation of EGFR activity depending upon the site and temporal–spatial balance. We hypothesized that absence of two major negative regulatory phosphorylation sites at Y1016 and Y1069, in the L858R/CYF10, Ex19Del/CYF10 or Ex20Ins/CYF10 EGFR mutants are sufficient to induce oncogenic signaling cascades through aberrant activation of the Ras‐ERK signaling and loss of EGFR ubiquitination and subsequent receptor degradation, respectively 31, 32. However, this is not the case in wild‐type EGFR/CYF10 mutants, suggesting that ligand induced multiple autophosphorylation is required for cellular transformation in wild‐type EGFR.…”

Inhibition of SHP2 leads to mesenchymal to epithelial transition in breast cancer cells

“…First, SHP2 is a positive effector of mitogenic and cell survival signaling. 9,12,30 Second, SHP2 promotes cell transformation induced by v-Src and the constitutively active form of FGFR3. [1][2][3] and associated leukemia.…”

“…7,8 In the epidermal growth factor receptor (EGFR) signaling pathway, SHP2 promotes Ras activation by blocking the Ras GTPase-activating protein (RasGAP)-induced downregulation of Ras, via dephosphorylation of a docking site on the EGFR, Tyr992. 9 Similarly, the Drosophila ortholog of SHP2, Corkscrew, transduces the Torso RTK signaling by blocking Ras inactivation. 10 Other reports also show that SHP2 dephosphorylates a RasGAPbinding site on the adapter protein Gab1, 11 leading to the same conclusion that SHP2 promotes Ras activation by blocking RasGAP.…”

Annexin II binds to SHP2 and this interaction is regulated by HSP70 levels

“…Horseradish peroxidase-conjugated secondary antibodies were purchased from Amersham. Construction of plasmids that express HSP70, wild-type SHP2 (WT-SHP2), D425A, C459S -SHP2 (DM-SHP2), or N-terminal FLAG-tagged WT/DM SHP2 proteins was described previously (FSHP2WT, FSHP2DM) [3,14]. C-terminal 3x FLAG tagging Annexin II was a gift from Dr. S. Tsirka (SUNY at Stony Brook, New York, USA).…”

“…SHP2 binds to several RTK [2,3], adaptor proteins [4][5][6][7][8][9][10][11][12], and SHPS-1 [13]. These interactions are essential for growth factor-or cytokine-induced activation of the multiple receptor-evoked functions.…”