INSULIN-RECEPTOR SUBSTRATE-1 IS REQUIRED FOR INSULIN-MEDIATED MITOGENIC SIGNAL-TRANSDUCTION

Insulin treatment of mammalian cells immediately stimulates the tyrosi ne phosphorylation of a cellular protein of 185 kDa referred to as pp1 85 or IRS-1 (insulin receptor substrate 1). The potential role of the IRS-1 protein in insulin signaling has been examined by microinjecting affinity-purified antibodies into living cells. Stably transfected Ra t-1 fibroblasts, which overexpress the human insulin receptor, were mi croinjected and subsequently stimulated with insulin or other growth f actors. Progression through the cell cycle was monitored by using a si ngle-cell assay, which employs bromodeoxyuridine labeling of DNA and a nalysis with immunofluorescence microscopy. Microinjection of anti-IRS -1 antibody completely inhibited incorporation of bromodeoxyuridine in to the nuclei of cells stimulated with insulin or insulin-like growth factor I but did not affect cells stimulated with serum or a variety o f purified growth factors. These studies indicate that IRS-1 is a crit ical component of the insulin and insulin-like growth factor I signali ng pathways, which lead to DNA synthesis and cell growth.