Identification of enhanced serine kinase activity in insulin resistance.
Insulin receptor substrate (IRS) proteins play a crucial role as signaling molecules in insulin action. Serine phosphorylation of IRS proteins has been hypothesized as a cause of attenuating insulin signaling. The current study investigated serine kinase activity toward IRS-1 in several models of insulin resistance. An in vitro kinase assay was developed that used partially purified cell lysates as a kinase and glutathione S-transferase fusion proteins that contained various of IRS-1 fragments as substrates. Elevated serine kinase activity was detected in Chinese hamster ovary/insulin receptor (IR)/IRS-1 cells and 3T3-L1 adipocytes chronically treated with insulin, and in liver and muscle of obese JCR:LA-cp rats. It phosphorylated the 526-859 amino acid region of IRS-1, whereas phosphorylation of the 2-516 and 900-1235 amino acid regions was not altered. Phosphopeptide mapping of the 526-859 region of IRS-1 showed three major phosphopeptides (P1, P2, and P3) with different patterns of phosphorylation depending on the source of serine kinase activity. P1 and P2 were strongly phosphorylated when the kinase activity was prepared from insulin-resistant Chinese hamster ovary/IR/IRS-1 cells, weakly phosphorylated by the kinase activity from insulin-resistant 3T3-L1 adipocytes, and barely phosphorylated when the extract was derived from insulin-resistant liver. In contrast, P3 was phosphorylated by the serine kinase activity prepared from all insulin-resistant cells and tissues of animals. P1 and P2 phosphorylation can be explained by mitogen-activated protein kinase activity based on the phosphopeptide map generated by recombinant ERK2. In contrast, mitogen-activated protein kinase failed to phosphorylate the P3 peptide, suggesting that another serine kinase regulates this modification of IRS-1 in insulin-resistant state.
Qiao
L Y
LY
Endocrinology Division, University of Vermont College of Medicine, Burlington, Vermont 05405, USA.
Goldberg
J L
JL
Russell
J C
JC
Sun
X J
XJ
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D006224:Cricetinae; D066298:In Vitro Techniques; D055504:Insulin Receptor Substrate Proteins; D007333:Insulin Resistance; D008099:Liver / Q000201:enzymology; D009132:Muscles / Q000201:enzymology; D009765:Obesity / Q000201:enzymology; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats
D055504:Insulin Receptor Substrate Proteins; C526296:Irs1 protein, rat; D010748:Phosphopeptides; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases