Roles of the complex formation of SHPS-1 with SHP-2 in insulin-stimulated mitogen-activated protein kinase activation.
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 glutathione S-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.
Takada
T
T
Second Department of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650, Japan.
Matozaki
T
T
Takeda
H
H
Fukunaga
K
K
Noguchi
T
T
Fujioka
Y
Y
Okazaki
I
I
Tsuda
M
M
Yamao
T
T
Ochi
F
F
Kasuga
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000943:Antigens, Differentiation; D001665:Binding Sites; D016466:CHO Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D006224:Cricetinae; D017931:DNA Primers; D004789:Enzyme Activation; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D039842:Neural Cell Adhesion Molecule L1; D019006:Neural Cell Adhesion Molecules / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D016133:Polymerase Chain Reaction; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011972:Receptor, Insulin / Q000096:biosynthesis / Q000502:physiology*; D011971:Receptors, Immunologic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D014162:Transfection; D018909:src Homology Domains
D000943:Antigens, Differentiation; D017931:DNA Primers; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D039842:Neural Cell Adhesion Molecule L1; D019006:Neural Cell Adhesion Molecules; D010446:Peptide Fragments; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; C501920:SIRPA protein, human; D011972:Receptor, Insulin; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C516724:PTPN11 protein, human; C509249:PTPN6 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases