Intracellular location and regulation of the IRS-1/P1 3-kinase complex in adipocytes: Potential involvement of a membrane cytoskeleton

Abstract

Numerous growth factors, cytokines and hormones appear to regulate mammalianncell growth, cell fate and cell function via an overlapping repertoire of intracellularnsignalling pathways. One signalling molecule capable of mediating several diversenbiological responses such as thrombin-induced platelet activation and growth factorregulatednapoptosis is a phospholipid kinase referred to as phosphatidylinositide (PI) 3-kinase. Establishing those factors that enable PI 3-kinase to mediate unique biologicalneffects in response to different stimuli, remains an important objective in studies of signalntransduction. The studies described herein focus on the role of PI 3-kinase in mediatingninsulin-regulated glucose transport in 3T3-L1 adipocytes. Insulin regulates glucosenuptake in these cells by promoting the movement of GLUT4 glucose transporters from annintracellular site to the cell surface, and PI 3-kinase is necessary for this process. Thisncomprises a suitable paradigm for studies of biological specificity because while othernfactors such as platelet-derived growth factor (PDGF), are equally effective in stimulatingnPI 3-kinase in adipocytes, they do not mimic the effect of insulin in regulating GLUT4ntranslocation in these cells.A feature that distinguishes insulin signal transduction is the utilisation of ansubstrate of the insulin receptor (IR) tyrosine kinase, referred to as insulin receptornsubstrate-1 (IRS-1), to dock and activate PI 3-kinase. In the present studies, subcellularnfractionation analysis of adipocytes showed that the majority of IRS-1 is located in annintracellular membrane fraction, referred to as the high speed pellet (HSP). The HSP alsonconstitutes the major insulin-regulatable pool of IRS-1 and PI 3-kinase. In contrast,nPDGF treatment of adipocytes causes the recruitment and activation of PI 3-kinase in anfraction that is distinct from the HSP. Thus the targeting of IRS-1 and PI 3-kinase to andistinct intracellular location may be central to the specificity with which insulin promotesnGLUT4 translocation.To dissect the basis upon which IRS-1 and PI 3-kinase associate with thenadipocyte HSP, this fraction was further analysed using a variety of biochemicalntechniques. Treatment of the HSP with non-ionic detergent, liberated all membranenconstituents, whereas IRS-1 and PI 3-kinase remained insoluble. Conversely, at highnionic strength, membranes remained intact, whereas IRS-1 and PI 3-kinase became freelynsoluble. Sucrose density sedimentation analysis of the HSP resulted in the segregation ofntwo sub-fractions: one enriched in IRS-1, tyrosyl-phosphorylated IRS-1 and PI 3-kinase,nas well as cytoskeletal elements, and another enriched in membranes, includingnintracellular GLUT4 vesicles. The predicted molecular weight of the IRS-l/PI 3-kinasencomplex is greater than 660 kDa. These properties of the IRS-l/PI 3-kinase complex arenindicative of an association with a large proteinaceous matrix, possibly the cytoskeleton.In response to insulin or PDGF, PI 3-kinase catalyses the 3' phosphorylation ofnmembrane-bound phosphatidylinositides, to form specific docking sites for thenrecruitment and activation of downstream signalling molecules, including protein kinasenB (PKB). Microinjection of a PKB substrate peptide or an antibody to PKB into 3T3-L1nadipocytes inhibited insulin-stimulated GLUT4 translocation to the plasma membrane byn66% and 56%, respectively, suggesting PKB performs an essential role in this process.nAntisera raised against different PKB isoforms detect the preferential expression ofnPKBb in rat and 3T3-L1 adipocytes. In the latter cells, insulin caused the robustnphosphorylation of PKBb, whereas PDGF had no significant effect. Furthermore, innadipocytes, insulin preferentially stimulated the translocation of PKBb to membranes,nincluding the plasma membrane.n n n n n n n n