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Protein kinase C modulation of insulin receptor substrate-1 tyrosine phosphorylation requires serine 612
1Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305, USA.
Abstract:
Activation of the endogenous protein kinase Cs in human kidney fibroblast (293) cells was found in the present study to inhibit the subsequent ability of insulin to stimulate the tyrosine phosphorylation of an expressed insulin receptor substrate-1. This inhibition was also observed in an in vitro phosphorylation reaction if the insulin receptor and its substrate were both isolated from cells in which the protein kinase C had been activated. To test whether serine phosphorylation of the insulin receptor substrate-1 was contributing to this process, serine 612 of this molecule was changed to an alanine. The insulin-stimulated tyrosine phosphorylation and the associated phosphatidylinositol 3-kinase activity of the expressed mutant were found to be comparable to those of the expressed wild-type substrate. However, unlike the wild-type protein, activation of protein kinase C did not inhibit the insulin-stimulated tyrosine phosphorylation of the S612A mutant nor its subsequent association with phosphatidylinositol 3-kinase. Tryptic peptide mapping of in vivo labeled IRS-1 and the S612A mutant revealed that PMA stimulates the phosphorylation of a peptide from wild-type IRS-1 that is absent from the tryptic peptide maps of the S612A mutant. Moreover, a synthetic peptide containing this phosphoserine and its nearby tyrosine was found to be phosphorylated by the insulin receptor to a much lower extent than the same peptide without the phosphoserine. Activation of protein kinase C was found to stimulate by 10-fold the ability of a cytosolic kinase to phosphorylate this synthetic peptide as well as the intact insulin receptor substrate-1. Finally, cytosolic extracts from the livers of ob/ob mice showed an 8-fold increase in a kinase activity capable of phosphorylating this synthetic peptide, compared to extracts of livers from lean litter mates. These results indicate that activation of protein kinase C stimulates a kinase which can phosphorylate insulin receptor substrate-1 at serine 612, resulting in an inhibition of insulin signaling in the cell, posing a potential mechanism for insulin resistance in some models of obesity.
Insights
Activation of protein kinase C inhibits insulin signaling by phosphorylating insulin receptor substrate-1 at serine 612. This finding reveals a potential mechanism for insulin resistance in obesity.
Area of Science:
- Biochemistry
- Cell Biology
- Metabolic Research
Background:
- Insulin signaling is crucial for glucose homeostasis.
- Insulin resistance is a hallmark of type 2 diabetes and obesity.
- Protein kinase C (PKC) activation is implicated in various cellular processes.
Purpose of the Study:
- To investigate the inhibitory effect of protein kinase C activation on insulin signaling.
- To identify the specific mechanism by which PKC interferes with insulin receptor substrate-1 (IRS-1) function.
- To explore the role of serine phosphorylation of IRS-1 in insulin resistance.
Main Methods:
- Utilized human kidney fibroblast (293) cells expressing insulin receptor and IRS-1.
- Performed in vitro phosphorylation assays with isolated insulin receptors and IRS-1.
- Generated a serine 612 to alanine mutant (S612A) of IRS-1 to assess phosphorylation site importance.
- Analyzed tryptic peptide mapping of wild-type and mutant IRS-1.
- Measured phosphatidylinositol 3-kinase (PI3K) activity.
- Assessed kinase activity in cytosolic extracts from lean and obese (ob/ob) mice.
Main Results:
- PKC activation inhibited insulin-stimulated tyrosine phosphorylation of IRS-1 and subsequent PI3K activity.
- The S612A IRS-1 mutant showed resistance to PKC-mediated inhibition of insulin signaling.
- PKC activation stimulated a cytosolic kinase to phosphorylate IRS-1 at serine 612.
- This phosphorylation at serine 612 reduced the ability of the insulin receptor to phosphorylate nearby tyrosine residues.
- Obese (ob/ob) mouse liver extracts exhibited increased activity of the kinase phosphorylating a synthetic peptide mimicking IRS-1 serine 612.
Conclusions:
- PKC activation phosphorylates IRS-1 at serine 612, inhibiting insulin receptor binding and downstream signaling.
- Serine 612 phosphorylation of IRS-1 is a key mechanism contributing to insulin resistance.
- This pathway represents a potential therapeutic target for obesity-related insulin resistance.