Intracellular Localization of Phosphatidylinositide 3-kinase and Insulin Receptor Substrate-1 in Adipocytes: Potential Involvement of a Membrane Skeleton

Abstract

Phosphatidylinositide (PI) 3-kinase binds to tyrosyl-phosphorylated insulin receptor substrate-1 (IRS-1) in insulin-treated adipocytes, and this step plays a central role in the regulated movement of the glucose transporter, GLUT4, from intracellular vesicles to the cell surface. PDGF, which also activates PI 3-kinase in adipocytes, has no significant effect on GLUT4 trafficking in these cells. We propose that this specificity may be mediated by differential localization of PI 3-kinase in response to insulin versus PDGF activation. Using subcellular fractionation in 3T3-L1 adipocytes, we show that insulin- and PDGF-stimulated PI 3-kinase activities are located in an intracellular high speed pellet (HSP) and in the plasma membrane (PM), respectively. The HSP is also enriched in IRS-1, insulin-stimulated tyrosyl-phosphorylated IRS-1 and intracellular GLUT4-containing vesicles. Using sucrose density gradient sedimentation, we have been able to segregate the HSP into two separate subfractions: one enriched in IRS-1, tyrosyl-phosphorylated IRS-1, PI 3-kinase as well as cytoskeletal elements, and another enriched in membranes, including intracellular GLUT4 vesicles. Treatment of the HSP with nonionic detergent, liberates all membrane constituents, whereas IRS-1 and PI 3-kinase remain insoluble. Conversely, at high ionic strength, membranes remain intact, whereas IRS-1 and PI 3-kinase become freely soluble. We further show that this IRS-1–PI 3-kinase complex exists in CHO cells overexpressing IRS-1 and, in these cells, the cytosolic pool of IRS-1 and PI 3-kinase is released subsequent to permeabilization with Streptolysin-O, whereas the particulate fraction of these proteins is retained. These data suggest that IRS-1, PI 3-kinase, as well as other signaling intermediates, may form preassembled complexes that may be associated with the actin cytoskeleton. This complex must be in close apposition to the cell surface, enabling access to the insulin receptor and presumably other signaling molecules that somehow confer the absolute specificity of insulin signaling in these cells.

Actin Filaments Facilitate Insulin Activation of the Src and Collagen Homologous/Mitogen-activated Protein Kinase Pathway Leading to DNA Synthesis and c-fos Expression

“…We attempted to investigate the effects of insulin and cytochalasin D on the subcellular distribution of Shc but were unable to detect any significant alterations in the distribution of this adaptor by these treatments (results not shown). Similar findings were also observed recently in 3T3-L1 adipocytes (58). It should be noted that Grb2 was localized to the cytosolic fractions of myotubes, and we were also unable to detect translocation to the actin-rich fractions.…”

Identification of a Novel Domain in Two Mammalian Inositol-polyphosphate 5-Phosphatases That Mediates Membrane Ruffle Localization

“…Subcellular Fractionation-Human glioblastoma U87 cells were homogenized with 15 strokes of a motor-driven Teflon pestle, and the subcellular fractions were obtained by sequential differential centrifugation as described previously (24). Serum-starved or epidermal growth factor (EGF; 100 ng/ml)-stimulated cells were fractionated into high density microsomal, cytosolic, plasma membrane/ER, and high speed pellet fractions, which were immunoblotted with affinity-purified anti-SKIP antibodies.…”

“…We also determined the SKIP localization in subcellular fractions of serum-starved and EGF-stimulated U87 cells, isolated using a simplified differential centrifugation that enables enrichment of fractions with markers for the plasma membrane/ER, endosomes and clathrin-coated vesicles (high density microsomes), and intracellular membranes comprising clathrin-coated vesicles and other membranes in the high speed pellet (24). The supernatant designated the cytosol contained all elements that did not sediment at 177,000 ϫ g (Fig.…”

Exercise does not alter subcellular localization, but increases phosphorylation of insulin-signaling proteins in human skeletal muscle

“…IRS protein localization within the cell may influence different cellular functions and in doing so provide a level of biological specificity. Although the subcellular localization of IRS proteins is not absolutely defined, these proteins have been identified with the plasma membrane, in the nucleus (18,26), and in a high-speed pellet (HSP) fraction, which suggests an association with the cytoskeleton (2,8). Furthermore, in response to various stimuli, such as insulin (1-3, 7), insulin-like growth factor I (IGF-I; see Ref.…”