Molecular mechanism for a role of SHP2 in epidermal growth factor receptor signaling.
Journal: 2003/December - Molecular and Cellular Biology
ISSN: 0270-7306
PUBMED: 14560030
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.
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Mol Cell Biol 23(21): 7875-7886

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

Department of Molecular Genetics and Microbiology, Health Sciences Center, Stony Brook University, Stony Brook, New York 11794-5222
Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Stony Brook, NY 11794-5222. Phone: (631) 632-8792. Fax: (631) 632-8891. E-mail: ude.bsynus.cc.sm@namyahm.
Received 2003 May 19; Revised 2003 Jun 16; Accepted 2003 Jul 29.

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.

Abstract

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, E. H. Fischer, J. Ihle, K. Imai, F. Jirik, B. Neel, T. Pawson, S. Shen, M. Thomas, A. Ullrich, and Z. Zhao, Letter, Cell 85:15, 1996). The binding of a cytokine to its cognate receptor also elicits similar signaling events. However, tyrosine phosphorylation of proteins, including the receptors themselves, is carried out by associated cytoplasmic tyrosine kinases, since these receptors lack intrinsic tyrosine kinase activity (7, 58, 59).

Recent advances in the area of phosphotyrosylphosphatases (PTPs) indicate that tyrosine dephosphorylation is as important as tyrosine phosphorylation in the transduction of signals elicited by growth factors and cytokines. Among the hundred or slightly more transmembrane and nontransmembrane phosphotyrosine (pY) phosphatases known to date, the ubiquitously expressed mammalian cytoplasmic phosphatase termed SH2 phosphatase 2 (SHP2) is a well-known positive effector of tyrosine kinase signaling (23, 32, 53, 54). Also, Corkskrew, the Drosophila melanogaster counterpart of SHP2, is an essential positive effector of the Torso RTK signaling (8). On the other hand, SHP1, which is expressed primarily in hematopoietic cells, is a negative regulator of tyrosine kinase signaling, although it has significant structural similarity to SHP2.

SHP2 possesses two tandemly arranged SH2 domains in its N-terminal region and a phosphatase domain in its C-terminal region (15, 16). It also possesses a stretch of proline-rich sequences and tyrosine phosphorylation sites in its extreme C-terminal region. Both the SH2 domains and the phosphatase domain have been shown to be absolutely essential for the biological activity of SHP2 (10). However, no biological role has been definitively ascribed to the tyrosine phosphorylation sites or the proline-rich region. Deletion of the N-SH2 or mutation of the conserved cysteine residue to serine in the active site of the phosphatase domain eliminates the biological activity of SHP2 (15, 16). Transgenic mice homozygous for the N-SH2 deletion mutation die in the uterus before day E10.5 from multiple defects in mesoderm development (4, 39, 40). Also, microinjection of the N-SH2 deletion or phosphatase-dead mutant mRNA into Xenopus laevis eggs causes abnormal embryonal development (48). These findings have established that SHP2 is a requisite for normal growth and development.

SHP2 interacts directly with autophosphorylated RTKs such as EGFR and PDGFR or indirectly via tyrosine-phosphorylated adaptor proteins through its SH2 domains (9, 18, 25, 26, 41, 52). These interactions have been shown to be essential for RTK signaling. However, how these interactions contribute positively to tyrosine kinase signaling is virtually unknown. Moreover, how pY dephosphorylation by SHP2 drives the signaling message forward and what substrates are acted upon by SHP2 are unknown. Recently, we developed an efficient substrate-trapping mutant of SHP2 and demonstrated that one of its physiological substrates is EGFR (2). In the present report, we describe the molecular mechanism of SHP2 in the mitogenic signaling pathway by using EGFR as a model. We find that SHP2 acts upstream of Ras in the EGFR pathway and functions by increasing the half-life of activated Ras (GTP-Ras) in the cell. It does so by interfering with the process of Ras inactivation (conversion to GDP-bound form) catalyzed by GTPase-activating protein (RasGAP) via inhibition of tyrosine phosphorylation-dependent translocation of RasGAP to the plasma membrane (PM). Furthermore, we demonstrate that Y992 of the EGFR is a negative-regulatory autophosphorylation site that acts as a binding site for RasGAP.

Acknowledgments

We thank D. Shalloway, D. Bar-Sagi, B. Myers, O. Petrenko, and J. Schlessinger for providing valuable reagents.

This work was supported by NIH Public Service grants CA28146 and CA42573 to M.J.H.

Acknowledgments

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