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Modulation of insulin receptor substrate-1 tyrosine phosphorylation and function by mitogen-activated protein kinase
1Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305, USA.
Abstract:
Increased serine phosphorylation of insulin receptor substrate-1 (IRS-1) has been observed in several systems to correlate with a decreased ability of the insulin receptor to tyrosine-phosphorylate this endogenous substrate and to inhibit its subsequent association with phosphatidylinositol 3-kinase. In the present studies we have examined the potential role of the mitogen-activated protein (MAP) kinase in the increased serine phosphorylation of IRS-1 observed in human embryonic kidney cells treated with an activator of protein kinase C, phorbol 12-myristate 13-acetate. First, recombinantly produced kinase was shown to phosphorylate intact IRS-1 in a way that decreased the ability of isolated insulin receptor to phosphorylate the tyrosines recognized by the SH2 domains of the phosphatidylinositol 3-kinase. Second, an inhibitor of MAP kinase activation, PD98059, blocked the phorbol 12-myristate 13-acetate-induced inhibition of the insulin-stimulated increase in IRS-1 associated phosphatidylinositol 3-kinase. Third, activation of MAP kinase in intact cells via a regulatable upstream kinase, a RAF:estrogen receptor construct, could also inhibit the insulin-stimulated increase in IRS-1-associated phosphatidylinositol 3-kinase. Fourth, an in gel kinase assay showed that MAP kinase was the primary renaturable kinase in cell extracts capable of phosphorylating an IRS-1 fusion protein. Finally, IRS-1 was found to associate in coprecipitation studies with endogenous MAP kinase. These studies implicate MAP kinase as one of the kinases capable of phosphorylating and regulating IRS-1 tyrosine phosphorylation.
Insights
Mitogen-activated protein kinase (MAPK) directly phosphorylates insulin receptor substrate-1 (IRS-1), inhibiting insulin signaling. This finding reveals MAPK as a key regulator of IRS-1 activity and insulin receptor function.
Area of Science:
- Cellular signaling pathways
- Molecular endocrinology
- Signal transduction
Background:
- Increased serine phosphorylation of insulin receptor substrate-1 (IRS-1) impairs insulin receptor function.
- This impaired function is linked to reduced tyrosine phosphorylation of IRS-1 and diminished association with phosphatidylinositol 3-kinase (PI3K).
- The specific kinases responsible for IRS-1 serine phosphorylation in response to certain stimuli remain largely uncharacterized.
Purpose of the Study:
- To investigate the role of mitogen-activated protein kinase (MAPK) in the serine phosphorylation of IRS-1.
- To determine if MAPK activation mediates the inhibitory effects of phorbol 12-myristate 13-acetate (PMA) on insulin signaling.
- To elucidate the functional consequences of MAPK-mediated IRS-1 phosphorylation on insulin receptor activity and PI3K association.
Main Methods:
- In vitro kinase assays using recombinant MAPK and purified IRS-1.
- Cellular studies using human embryonic kidney cells treated with PMA and PD98059 (a MAPK inhibitor).
- Experiments involving a regulatable MAPK activation system (RAF:estrogen receptor construct).
- In-gel kinase assays and co-precipitation studies to identify and characterize interacting kinases and proteins.
Main Results:
- Recombinant MAPK phosphorylated IRS-1, reducing insulin receptor tyrosine phosphorylation.
- PD98059 blocked PMA-induced inhibition of insulin-stimulated IRS-1-associated PI3K.
- Activated MAPK in intact cells inhibited insulin-stimulated IRS-1-associated PI3K.
- MAPK was identified as the primary kinase phosphorylating an IRS-1 fusion protein in cell extracts.
- IRS-1 was found to associate with endogenous MAPK.
Conclusions:
- Mitogen-activated protein kinase (MAPK) directly phosphorylates insulin receptor substrate-1 (IRS-1).
- MAPK-mediated phosphorylation of IRS-1 inhibits insulin receptor tyrosine phosphorylation and downstream signaling.
- MAPK is implicated as a key kinase regulating IRS-1 function and insulin signal transduction.