Roles of the Complex Formation of SHPS-1 with SHP-2 in Insulin-stimulated Mitogen-activated Protein Kinase Activation

doi:10.1074/jbc.273.15.9234

Roles of the Complex Formation of SHPS-1 with SHP-2 in Insulin-stimulated Mitogen-activated Protein Kinase Activation*

From the Second Department of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650 and^§Pharmacia Biotech, Nishinakajima, Yodogawa-ku, Osaka, Japan

Abstract

SHPS-1 is a receptor-like protein that undergoes tyrosine phosphorylation and binds SHP-2, an SH2 domain-containing protein tyrosine phosphatase, in response to insulin and other mitogens. The overexpression of wild-type SHPS-1, but not of a mutant SHPS-1 in which all four tyrosine residues in its cytoplasmic region were mutated to phenylalanine, markedly enhanced insulin-induced activation of mitogen-activated protein kinase in Chinese hamster ovary cells that overexpress the human insulin receptor. Mutation of each tyrosine residue individually revealed that the major sites of tyrosine phosphorylation of SHPS-1 in response to insulin are Tyr⁴⁴⁹ and Tyr⁴⁷³. In addition, mutation of either Tyr⁴⁴⁹ or Tyr⁴⁷³ abolished the insulin-induced tyrosine phosphorylation of SHPS-1 and its association with SHP-2. Surface plasmon resonance analysis showed that glutathioneS-transferase fusion proteins containing the NH₂-terminal or COOH-terminal SH2 domains of SHP-2 bound preferentially to phosphotyrosyl peptides corresponding to the sequences surrounding Tyr⁴⁴⁹ or Tyr⁴⁷³, respectively, of SHPS-1. Furthermore, phosphotyrosyl peptides containing Tyr⁴⁴⁹ or Tyr⁴⁷³ were effective substrates for the phosphatase activity of recombinant SHP-2 in vitro. Together, these results suggest that insulin may induce phosphorylation of SHPS-1 at Tyr⁴⁴⁹ and Tyr⁴⁷³, to which SHP-2 then binds through its NH₂-terminal and COOH-terminal SH2 domains, respectively. SHPS-1 may play a crucial role both in the recruitment of SHP-2 from the cytosol to a site near the plasma membrane and in increasing its catalytic activity, thereby positively regulating the RAS-mitogen-activated protein kinase signaling cascade in response to insulin.

Footnotes

↵* This work was supported by a grant-in-aid for cancer research and a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan, Grant 96-22809 from the Princess Takamatsu Cancer Research Fund, a grant from the Yamanouchi Foundation for Research on Metabolic Disorders, a grant from the Ciba-Geigy Foundation (Japan) for the Promotion of Science, and a grant from Kirin Brewery Co.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ To whom correspondence should be addressed: Second Department of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650, Japan. Tel.: 81-78-341-7451 (ext. 5522); Fax: 81-78-382-2080; E-mail: matozaki{at}med.kobe-u.ac.jp.
↵1 The abbreviations used are: PTPase, protein tyrosine phosphatase; SH2, SRC homology 2; PDGF, platelet-derived growth factor; EGF, epidermal growth factor; MAP, mitogen-activated protein; IR, insulin receptor; IRS-1, IR substrate-1; SHPS-1, SHP substrate-1; CHO, Chinese hamster ovary; FBS, fetal bovine serum; mAb, monoclonal antibody; GST, glutathione S-transferase; HRP, horseradish peroxidase; PBS, phosphate-buffered saline; SPR, surface plasmon resonance; TCR, T cell receptor; BCR, B cell antigen receptor; pY, phosphotyrosine.
- Received October 16, 1997.
- Revision received January 13, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.