Insulin-stimulated GLUT4 translocation requires the CAP-dependent activation of TC10

The stimulation of glucose uptake by insulin in muscle and adipose tissue requires translocation of the GLUT4 glucose transporter protein from intracellular storage sites to the cell surface. Although the cellular dynamics of GLUT4 vesicle trafficking are well described, the signalling pathways that link the insulin receptor to GLUT4 translocation remain poorly understood. Activation of phosphatidylinositol-3-OH kinase (PI(3)K) is required for this trafficking event, but it is not sufficient to produce GLUT4 translocation. We previously described a pathway involving the insulin-stimulated tyrosine phosphorylation of Cbl, which is recruited to the insulin receptor by the adapter protein CAP. On phosphorylation, Cbl is translocated to lipid rafts. Blocking this step completely inhibits the stimulation of GLUT4 translocation by insulin. Here we show that phosphorylated Cbl recruits the CrkII-C3G complex to lipid rafts, where C3G specifically activates the small GTP-binding protein TC10. This process is independent of PI(3)K, but requires the translocation of Cbl, Crk and C3G to the lipid raft. The activation of TC10 is essential for insulin-stimulated glucose uptake and GLUT4 translocation. The TC10 pathway functions in parallel with PI(3)K to stimulate fully GLUT4 translocation in response to insulin.     

Elevated levels of diacylglycerol and decreased phorbol ester sensitivity in ras-transformed fibroblasts

Diacylglycerol (DG) plays a central role in phospholipid metabolism and is an endogenous activator of protein kinase C. We have suggested that constitutive activation of this kinase is one mechanism by which oncogenes transform cells. The ras-encoded proteins are similar to regulatory G-proteins and are candidates for the unknown G-protein that modulates phosphatidylinositol (PI) turnover. Differences in polyphosphoinositide metabolism have been reported for ras-transformed cells. But because these experiments were performed on confluent cultures of established cell lines, the differences are difficult to attribute to ras transformation. Here we show that exponentially growing NIH 3T3 fibroblasts recently transformed by Ha-ras or Ki-ras possess elevated DG concentrations without significant alterations in the levels of other polyphosphoinositide metabolites. The basal phosphorylation of protein kinase C substrate of relative molecular mass (Mr) 80,000 (80K) is significantly increased in all the ras-transformed cell lines. Surprisingly, however, further phosphorylation of this protein on addition of phorbol ester was greatly reduced. Ha-ras cells also show less binding of phorbol ester than control cells, suggesting that elevation of DG causes partial down-regulation in addition to activation of protein kinase C.     

Structural insight into filament formation by mammalian septins

Septins are GTP-binding proteins that assemble into homo- and hetero-oligomers and filaments. Although they have key roles in various cellular processes, little is known concerning the structure of septin subunits or the organization and polarity of septin complexes. Here we present the structures of the human SEPT2 G domain and the heterotrimeric human SEPT2-SEPT6-SEPT7 complex. The structures reveal a universal bipolar polymer building block, composed of an extended G domain, which forms oligomers and filaments by conserved interactions between adjacent nucleotide-binding sites and/or the amino- and carboxy-terminal extensions. Unexpectedly, X-ray crystallography and electron microscopy showed that the predicted coiled coils are not involved in or required for complex and/or filament formation. The asymmetrical heterotrimers associate head-to-head to form a hexameric unit that is nonpolarized along the filament axis but is rotationally asymmetrical. The architecture of septin filaments differs fundamentally from that of other cytoskeletal structures.