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

Michael J. Wick, Lily Q. Dong, Derong Hu, Paul R Langlais, Feng Liu

Research output: Contribution to journalArticle

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Abstract

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 p62dok, is inhibited by Grb10, an adaptor protein that binds directly to the kinase domain of the IR, both in vitro and in cells. Replacing Tyr362 and Tyr398 with phenylalanine greatly decreased the IR-catalyzed p62dok tyrosine phosphorylation in vitro, suggesting that these two residues are the major IR-mediated phosphorylation sites. However, mutations at Tyr362 and Tyr398 only partially blocked insulin-stimulated p62dok tyrosine phosphorylation in cells, indicating that p62dok is also a target for other cellular tyrosine kinase(s) in addition to the IR. Replacing Tyr362 with phenylalanine abolished the interaction between p62dok and Nck. Mutations at Tyr362/398 of p62dok disrupted the interaction between p62dok and GAP and decreased the inhibitory effect of p62dok on the insulin-stimulated activation of Ras and Akt, but not mitogen-activated protein kinase. Furthermore, the inhibitory effect of p62dok 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 Tyr362 and Tyr398 plays an essential role for p62dok to interact with its effectors and negatively regulate the insulin signaling pathway.

Original languageEnglish (US)
Pages (from-to)42843-42850
Number of pages8
JournalJournal of Biological Chemistry
Volume276
Issue number46
DOIs
StatePublished - Nov 16 2001
Externally publishedYes

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GTPase-Activating Proteins
Phosphorylation
Insulin Receptor
Tyrosine
Carrier Proteins
Chemical activation
Insulin
Phenylalanine
Proteins
Mutation
Substrates
Mitogen-Activated Protein Kinases
Protein-Tyrosine Kinases
Phosphotransferases

ASJC Scopus subject areas

  • Biochemistry

Cite this

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. / Wick, Michael J.; Dong, Lily Q.; Hu, Derong; Langlais, Paul R; Liu, Feng.

In: Journal of Biological Chemistry, Vol. 276, No. 46, 16.11.2001, p. 42843-42850.

Research output: Contribution to journalArticle

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abstract = "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 p62dok, is inhibited by Grb10, an adaptor protein that binds directly to the kinase domain of the IR, both in vitro and in cells. Replacing Tyr362 and Tyr398 with phenylalanine greatly decreased the IR-catalyzed p62dok tyrosine phosphorylation in vitro, suggesting that these two residues are the major IR-mediated phosphorylation sites. However, mutations at Tyr362 and Tyr398 only partially blocked insulin-stimulated p62dok tyrosine phosphorylation in cells, indicating that p62dok is also a target for other cellular tyrosine kinase(s) in addition to the IR. Replacing Tyr362 with phenylalanine abolished the interaction between p62dok and Nck. Mutations at Tyr362/398 of p62dok disrupted the interaction between p62dok and GAP and decreased the inhibitory effect of p62dok on the insulin-stimulated activation of Ras and Akt, but not mitogen-activated protein kinase. Furthermore, the inhibitory effect of p62dok 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 Tyr362 and Tyr398 plays an essential role for p62dok to interact with its effectors and negatively regulate the insulin signaling pathway.",
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T1 - 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

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