Insulin stimulates the translocation of the glucose transporter GLUT4 from intracellular vesicles to the plasma membrane. In the present study we have conducted a comprehensive proteomic analysis of affinity-purified GLUT4 vesicles from 3T3-L1 adipocytes to discover potential regulators of GLUT4 trafficking. In addition to previously identified components of GLUT4 storage vesicles including the insulin-regulated aminopeptidase insulin-regulated aminopeptidase and the vesicle soluble N-ethylmaleimide factor attachment protein (v-SNARE) VAMP2, we have identified three new Rab proteins, Rab10, Rab11, and Rab14, on GLUT4 vesicles. We have also found that the putative Rab GTPase-activating protein AS160 (Akt substrate of 160 kDa) is associated with GLUT4 vesicles in the basal state and dissociates in response to insulin. This association is likely to be mediated by the cytosolic tail of insulinregulated aminopeptidase, which interacted both in vitro and in vivo with AS160. Consistent with an inhibitory role of AS160 in the basal state, reduced expression of AS160 in adipocytes using short hairpin RNA increased plasma membrane levels of GLUT4 in an insulin-independent manner. These findings support an important role for AS160 in the insulin regulated trafficking of GLUT4.Glucose transport into mammalian muscle and fat cells is an important step in insulin action and is critical for the maintenance of glucose homeostasis within the body (1). In mammalian muscle and fat cells, insulin stimulation activates a phosphorylation cascade, which in turn causes intracellular vesicles that contain the glucose transporter GLUT4, 4 to translocate to the plasma membrane (PM) and fuse (2, 3). In the basal state GLUT4 is distributed between the endosomal system, the trans-Golgi network (TGN), and a GLUT4 storage vesicle (GSV) compartment that is highly insulin-responsive (4 -6).The protein kinase Akt is activated in response to insulin and plays a critical role in GLUT4 translocation (1, 7). However, the link between the insulin signaling pathway and GLUT4 translocation is not fully understood. The insulin-dependent movement of GLUT4 vesicles to the PM is an Akt-independent process, and this is followed by an Aktdependent step likely involving the docking and fusion of vesicles with the PM (7-9). The mechanism by which Akt controls the docking and fusion of GLUT4 vesicles with the PM is not known. However, it was previously shown that a Rab GTPase-activating protein (RabGAP) known as AS160 is phosphorylated by Akt in response to insulin (10). How AS160 functions in GLUT4 trafficking and its cognate Rab proteins are not known. The role of a variety of Rab proteins in GLUT4 trafficking including Rab3d, Rab4, Rab5, and Rab11 has been examined (11-16). However, although these Rab proteins may participate in some aspects of GLUT4 trafficking, no compelling evidence for specific involvement in the insulin-regulated trafficking of GLUT4 has been found.In this study we describe four key findings that add to our understanding of GLUT4 traffic...

The proteome is a dynamic system in which each protein has interconnected properties - dimensions - that together contribute to the phenotype of a cell. Measuring these properties has proved challenging owing to their diversity and dynamic nature. Advances in mass spectrometry-based proteomics now enable the measurement of multiple properties for thousands of proteins, including their abundance, isoform expression, turnover rate, subcellular localization, post-translational modifications and interactions. Complementing these experimental developments are new data analysis, integration and visualization tools as well as data-sharing resources. Together, these advances in the multidimensional analysis of the proteome are transforming our understanding of various cellular and physiological processes.

Translocation of the insulin-regulated glucose transporter GLUT4 to the cell surface is dependent on the phosphatidylinositol 3-kinase/Akt pathway. The RabGAP (Rab GTPase-activating protein) AS160 (Akt substrate of 160 kDa) is a direct substrate of Akt and plays an essential role in the regulation of GLUT4 trafficking. We have used liquid chromatography tandem mass spectrometry to identify several 14-3-3 isoforms as AS160-interacting proteins. 14-3-3 proteins interact with AS160 in an insulin-and Akt-dependent manner via an Akt phosphorylation site, Thr-642. This correlates with the dominant negative effect of both the AS160 T642A and the AS160 4P mutants on insulin-stimulated GLUT4 translocation. Introduction of a constitutive 14-3-3 binding site into AS160 4P restored 14-3-3 binding without disrupting AS160-IRAP (insulin-responsive amino peptidase) interaction and reversed the inhibitory effect of AS160 4P on GLUT4 translocation. These data show that the insulin-dependent association of 14-3-3 with AS160 plays an important role in GLUT4 trafficking in adipocytes.The insulin-regulated glucose transporter GLUT4 3 is mainly expressed in muscle and adipose tissue and plays an important role in glucose homeostasis. Its translocation from intracellular compartments to the cell surface in response to insulin is a multistep process involving intracellular sorting, vesicular transport to the cell surface along cytoskeletal elements, reorganization of cortical actin, and finally docking, priming, and fusion of so-called GLUT4 storage vesicle with the cell surface (1-4). Although several players in each step are known, the exact nodes between signaling and GLUT4 trafficking still remain elusive. The phosphatidylinositol 3-kinase/Akt pathway has been shown to play an essential role in GLUT4 translocation. One of its many substrates, the Akt substrate of 160 kDa, AS160, contains a Rab GTPase-activating domain, but its cognate in vivo Rab has not yet been identified (5, 6).There is accumulating evidence in the recent literature that this RabGAP plays an important role in insulin-triggered GLUT4 translocation. Insulin treatment of adipocytes results in phosphorylation of AS160 at six separate sites, and this appears to be mediated by Akt (5, 6). Overexpression of an AS160 mutant, in which four of these phosphorylation sites have been mutated to alanines (AS160 4P ), inhibits insulin-stimulated GLUT4 translocation to the cell surface in adipocytes. This effect is dependent on an intact RabGAP domain because simultaneous disruption of the putative GAP domain in AS160 4P overcame this inhibitory effect on GLUT4 translocation (6, 7). This observation indicates that the GAP activity of this protein has an important role in GLUT4 trafficking, thus providing one of the first links between signaling and membrane trafficking in this process. More recent evidence suggests that AS160 may inhibit GLUT4 trafficking under basal conditions because knockdown of AS160 using RNA interference resulted in insulin-independent translocation of GL...