We report a rapid and sensitive colorimetric approach to quantitate the amount of glucose transporters exposed at the surface of intact cells, using L6 muscle cells expressing GLUT4 containing an exofacial myc epitope. Unstimulated cells exposed to the surface 5 fmol GLUT4myc per mg protein. This value increased to 10 fmol/mg protein in response to insulin as 2-deoxyglucose (10 W WM) uptake doubled. The results are substantiated by immunofluorescent detection of GLUT4myc in unpermeabilized cells and by subcellular fractionation. We further show that wortmannin and the cytoskeleton disruptors cytochalasin D and latrunculin B completely blocked these insulin effects. The rapid quantitative assay described here could be of high value to study insulin signals and to screen for potential anti-diabetic drugs.z 1998 Federation of European Biochemical Societies.

Polo-like kinase-1 (Plk1) phosphorylates a number of mitotic substrates, but the diversity of Plk1-dependent processes suggests the existence of additional targets. Plk1 contains a specialized phosphoserine-threonine binding domain, the Polo-box domain (PBD), postulated to target the kinase to its substrates. Using the specialized PBD of Plk1 as an affinity capture agent, we performed a screen to define the mitotic Plk1-PBD interactome by mass spectrometry. We identified 622 proteins that showed phosphorylation-dependent mitosis-specific interactions, including proteins involved in well-established Plk1-regulated processes, and in processes not previously linked to Plk1 such as translational control, RNA processing, and vesicle transport. Many proteins identified in our screen play important roles in cytokinesis, where, in mammalian cells, the detailed mechanistic role of Plk1 remains poorly defined. We go on to characterize the mitosis-specific interaction of the Plk1-PBD with the cytokinesis effector kinase Rho-associated coiled-coil domain-containing protein kinase 2 (Rock2), demonstrate that Rock2 is a Plk1 substrate, and show that Rock2 colocalizes with Plk1 during cytokinesis. Finally, we show that Plk1 and RhoA function together to maximally enhance Rock2 kinase activity in vitro and within cells, and implicate Plk1 as a central regulator of multiple pathways that synergistically converge to regulate actomyosin ring contraction during cleavage furrow ingression.

Insulin plays a central role in the regulation of glucose homeostasis in part by stimulating glucose uptake and glycogen synthesis. The serine/threonine protein kinase Akt has been proposed to mediate insulin signaling in several processes. However, it is unclear whether Akt is involved in insulin-stimulated glucose uptake and which isoforms of Akt are responsible for each insulin action. We confirmed that expression of a constitutively active Akt, using an adenoviral expression vector, promoted translocation of glucose transporter 4 (GLUT4) to plasma membrane, 2-deoxyglucose (2-DG) uptake, and glycogen synthesis in both Chinese hamster ovary cells and 3T3-L1 adipocytes. Inhibition of Akt either by adenoviral expression of a dominant negative Akt or by the introduction of synthetic 21-mer short interference RNA against Akt markedly reduced insulin-stimulated GLUT4 translocation, 2-DG uptake, and glycogen synthesis. Experiments with isoform-specific short interference RNA revealed that Akt2, and Akt1 to a lesser extent, has an essential role in insulin-stimulated GLUT4 translocation and 2-DG uptake in both cell lines, whereas Akt1 and Akt2 contribute equally to insulinstimulated glycogen synthesis. These data suggest a prerequisite role of Akt in insulin-stimulated glucose uptake and distinct functions among Akt isoforms.

Physical exercise induces translocation of GLUT4 from an intracellular pool to the cell surface in skeletal muscles and increases glucose uptake via an insulin-independent pathway. However, the molecular mechanism remains to be identified. Some studies have suggested that bradykinin is locally released from contracting muscles and may be responsible for GLUT4 translocation and the increase of glucose transport in skeletal muscles. To determine whether bradykinin directly triggers GLUT4 translocation, we established L6 myotubes, 3T3-L1 adipocytes, and Chinese hamster ovary cells stably expressing c-myc epitope-tagged GLUT4 (GLUT4myc) and bradykinin B2 receptors. We found that bradykinin directly triggered GLUT4myc translocation and increased the rate of glucose uptake in a dose-dependent manner in these cells. The translocation with bradykinin occurred even after pretreatment with an islet-activating protein, wortmannin, and phorbol 12,13-dibutyrate. The signaling pathway does not seem to be mediated by Gi, phosphatidylinositol 3-kinase, or protein kinase C. It is insulin-independent and via trimeric G-protein Gq. Bradykinin is probably one of the factors responsible for exercise-stimulated glucose uptake in skeletal muscles.