Cellular compartmentalization in insulin action: Altered signaling by a lipid-modified IRS-1

While most receptor tyrosine kinases signal by recruiting SH2 proteins dire ctly to phosphorylation sites on their plasma membrane receptor, the insuli n receptor phosphorylates intermediary IRS proteins that are distributed be tween the cytoplasm and a state of loose association with intracellular mem branes. To determine the importance of this distribution to IRS-1-mediated signaling, we constructed a prenylated, constitutively membrane-bound IRS-1 by adding the COOH-terminal 9 amino acids from p21(ras), including the CAA X motif, to IRS-1 (IRS-CAAX) and analyzed its function in 32D cells express ing the insulin receptor. IRS-CAAX migrated more slowly on sodium dodecyl s ulfate-polyacrylamide gel electrophoresis than did IRS-1 and demonstrated i ncreased levels of serine/threonine phosphorylation, Insulin-stimulated tyr osyl phosphorylation of IRS-CAAX was slightly decreased, while IRS-CAAX-med iated phosphatidylinositol 3'-kinase (PI3'-kinase) binding and activation w ere decreased by approximately 75% compared to those for wild-type IRS-1. S imilarly, expression of IRS-CAAX desensitized insulin stimulated [H-3]thymi dine incorporation into DNA by about an order of magnitude compared to IRS- 1. By contrast, IRS-CAAX-expressing cells demonstrated increased signaling by mitogen-activated protein kinase, Akt, and p70(S6) kinase in response to insulin. Hence, tight association with the membrane increased IRS-1 serine phosphorylation and reduced coupling between the insulin receptor, PI3'-ki nase, and proliferative signaling while enhancing other signaling pathways. Thus, the correct distribution of IRS-1 between the cytoplasm and membrane compartments is critical to the normal balance in the network of insulin s ignaling.