As shown in Fig. takes on an important part on growth and antiapoptosis, whereas Akt2 functions primarily like a regulator of glucose rate of metabolism (Cho et al., 2001b; Bae et al., 2003). For example, Akt1?/? mice have reduced body size but relatively normal glucose homeostasis, whereas Akt2?/? mice displayed insulin-resistant glucose metabolism in liver and muscle mass (Cho et al., 2001a). However, the insulin resistance is definitely relatively slight and becomes significantly more pronounced in conjunction with the loss of Akt1 (Jiang et al., 2003). Consistent YS-49 with a primary part for Akt2, a family with severe insulin resistance and overt diabetes was mapped to a single point mutation in Akt2 (George et al., 2004). Therefore, there seems to exist intracellular transmission specificity and some payment mechanism for the rules of glucose rate of metabolism between Akt1 and Akt2. It is well recorded that Akt is definitely involved in the regulation of glucose rate of metabolism by inhibiting glycogen synthesis through the inhibition of glycogen synthesis kinase 3 (GSK3) activity (Mix et al., 1995; Coghlan et al., 2000; Doble and Woodgett, 2003). However, how Akt regulates glucose uptake (muscle mass and adipose cells), its association with peripheral insulin resistance, and the molecular basis for the apparent Akt2 YS-49 specificity is still unfamiliar. Peptide substrate mapping studies have identified the preferred Akt1 phosphorylation consensus site as RxRxxS/T (Alessi et al., 1996). Currently, over 20 substrates for Akt have been identified; however, none of these substrates has been reported to exhibit Akt isoform selectivity. Therefore, at present, the molecular basis for the physiologic specificities of Akt isoform function remains a fundamental issue that has yet to be resolved. In this regard, we previously recognized Synip like a Syntaxin4 interacting protein (Min et al., 1999). Under YS-49 the basal Rabbit Polyclonal to Androgen Receptor conditions, Synip was constitutively bound to Syntaxin4 and prevented the connection of Syntaxin4 with both SNAP23 (synaptosome-associated proteins of 23 kD) and VAMP2 (vesicle-associated membrane protein 2; Min et al., 1999). Insulin treatment resulted in a dissociation of the SynipCSyntaxin4 complex allowing for the assembly of a fusogenic Syntaxin4CSNAP23CVAMP2 complex necessary for glucose transporter 4 (GLUT4) translocation (Min et al., 1999). With this paper, we now demonstrate that Synip is definitely a desired Akt2-specific substrate with an unusual dual consensus phosphorylation site. The specific Akt2-dependent phosphorylation of serine 99 is essential for the insulin-stimulated dissociation of Synip from Syntaxin4, translocation, and plasma membrane fusion of GLUT4-comprising vesicles. Results Analysis of insulin signal-regulating SynipCSyntaxin4 connection Insulin stimulates the translocation of GLUT4 proteins from intracellular storage sites to the plasma YS-49 membrane. To day, two major insulin-mediated signal transduction pathways have been implicated in the rules of this process (Saltiel and Pessin, 2003). The insulin activation and/or focusing on of the type 1A phosphatidylinositol 3 (PI3) kinase generate PI3, 4, 5P3 in the plasma membrane (Okada et al., 1994). PI3, 4, 5P3 recruits and/or activates phosphoinositide-dependent kinase 1 that serves as an immediate upstream kinase for Akt and the atypical protein kinase C isoforms and (Bellacosa et al., 1991; Belham et al., 1999). The plasma membrane translocation of GLUT4 requires the specific connection of the plasma membrane t-SNARE proteins Syntaxin4 and SNAP23 with the v-SNARE protein VAMP2 in GLUT4-comprising cargo vesicles (Pessin et al., 1999). To determine the specific insulin signaling pathway responsible for the dissociation of Synip from Syntaxin4, we treated cells with numerous pharmacological agents.