Insulin receptor-mediated p62dok tyrosine phosphorylation at residues 362 and 398 plays distinct roles for binding GTPase-activating protein and Nck and is essential for inhibiting insulin-stimulated activation of Ras and Akt.
A GTPase-activating protein (GAP)-associated 60-kDa protein has been found to undergo rapid tyrosine phosphorylation in response to insulin stimulation. However, whether this protein is a direct in vivo substrate for the insulin receptor (IR) tyrosine kinase and whether the tyrosine phosphorylation plays a role in insulin signaling remain to be established. Here we show that the insulin-stimulated tyrosine phosphorylation of the GAP-associated protein, now identified as p62(dok), is inhibited by Grb10, an adaptor protein that binds directly to the kinase domain of the IR, both in vitro and in cells. Replacing Tyr(362) and Tyr(398) with phenylalanine greatly decreased the IR-catalyzed p62(dok) tyrosine phosphorylation in vitro, suggesting that these two residues are the major IR-mediated phosphorylation sites. However, mutations at Tyr(362) and Tyr(398) only partially blocked insulin-stimulated p62(dok) tyrosine phosphorylation in cells, indicating that p62(dok) is also a target for other cellular tyrosine kinase(s) in addition to the IR. Replacing Tyr(362) with phenylalanine abolished the interaction between p62(dok) and Nck. Mutations at Tyr(362/398) of p62(dok) disrupted the interaction between p62(dok) and GAP and decreased the inhibitory effect of p62(dok) on the insulin-stimulated activation of Ras and Akt, but not mitogen-activated protein kinase. Furthermore, the inhibitory effect of p62(dok) on Akt phosphorylation could be blocked by coexpression of a constitutively active Ras. Taken together, our findings indicate that p62(dok) is a direct substrate for the IR tyrosine kinase and that phosphorylation at Tyr(362) and Tyr(398) plays an essential role for p62(dok) to interact with its effectors and negatively regulate the insulin signaling pathway.
Wick
M J
MJ
Department of Pharmacology and Biochemistry, the University of Texas Health Science Center, San Antonio, Texas 78229, USA.
Dong
L Q
LQ
Hu
D
D
Langlais
P
P
Liu
F
F
D048868:Adaptor Proteins, Signal Transducing; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D002478:Cells, Cultured; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D051383:GRB10 Adaptor Protein; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D020935:MAP Kinase Signaling System; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015513:Oncogene Proteins / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D016601:RNA-Binding Proteins; D015398:Signal Transduction; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry
D048868:Adaptor Proteins, Signal Transducing; D000596:Amino Acids; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D051383:GRB10 Adaptor Protein; D020558:GTP Phosphohydrolases; D020690:GTPase-Activating Proteins; D005982:Glutathione Transferase; D015513:Oncogene Proteins; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D011506:Proteins; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D016601:RNA-Binding Proteins; D014443:Tyrosine; C489560:DOK1 protein, human; C074561:GAP-associated protein p62; C078332:Nck protein; C494918:AKT1 protein, human