It is well recognized that the interaction of Syntaxin4 as t-SNARE and VAMP2 as v-SNARE is necessary for insulin-stimulated GLUT4 translocation in adipose tissue and skeletal muscle (Foster and Klip, 2000
). For example, overexpression of the Syntaxin4 cytoplasmic domain inhibits insulin-stimulated GLUT4 translocation (Olson et al., 1997
). This inhibition was specific for the VAMP2 binding domain within Syntaxin4 because deletion of this region did not result in inhibition of insulin-stimulated GLUT4 translocation (Olson et al., 1997
). Thus, Syntaxin4 and VAMP2 binding is one of the necessary steps for GLUT4 translocation. However, it is still unknown about precise mechanism how insulin controls Syntaxin4–VAMP2 interaction directly. In this regard, Synip is a Syntaxin4 specific binding protein that regulates the interaction between Syntaxin4 and VAMP2 in an insulin-dependent manner (Min et al., 1999
). Insulin causes Synip dissociation from Syntaxin4, and this dissociation allows VAMP2 to bind with Syntaxin4 because Synip and VAMP2 use the same binding site on Syntaxin4 (Min et al., 1999
). However, it has not yet been determined how insulin causes Synip dissociation from Syntaxin4. Therefore, we attempted to identify the Synip dissociation mechanism to understand how insulin regulates the Syntaxin4–VAMP2 interaction and docking/fusion step of GLUT4-containing vesicle.
PI3 kinase–independent pathway regulated by CAP/Cbl/TC10 molecules is recently reported to regulate the final steps of GLUT4 exocytosis by recruiting Exo70 to the plasma membrane (Inoue et al., 2003
). However, as shown in B, CAP-sorbin (Baumann et al., 2000
), which has a dominant-negative effect for the CAP/Cbl/TC10 pathway, did not affect Synip dissociation from Syntaxin4. Furthermore, our data strongly suggested that the PI3 kinase–Akt pathway specifically regulates Synip–Syntaxin4 interaction and that the PI3 kinase–atypical PKC pathway is not likely involved (, C–G). Furthermore, our siRNA experiments strongly suggested that Akt2 but not Akt1 regulates Synip–Syntaxin4 interaction ().
The next question is why Akt2 alone is involved in the regulation of Synip–Syntaxin4 interaction? What kind of mechanism does determine the Akt specificity in this pathway? As shown in A, Synip seemed to have a consensus motif including the 99th serine residue as Akt phosphorylation site. Therefore, we tested the possibility of whether or not Akt phosphorylates the 99th serine of Synip. We have observed that Akt2, but not Akt1, specifically phosphorylates Synip at serine 99 residue after insulin stimulation ( B). However, because it was formally possible that this was an indirect phosphorylation event, we synthesized the WT-Synip peptide, the S99F Synip mutant, the S97F/S99F Synip double mutant, or a scramble peptide including the serine 99 and performed in vitro phosphorylation experiments with recombinant activated forms of Akt1, Akt2, and Akt3. Surprisingly, only Akt2 was capable of directly phosphorylating the WT-Synip peptide ( C). Although serine 97 is also in an appropriate context as an Akt1 substrate, neither Akt1, Akt2, nor Akt3 was capable of phosphorylating the S99F Synip mutant, the S97F/S99F Synip double mutant, or a scramble peptide. Moreover, the similar substrate selectivity was observed in the case of in vitro phosphorylation using full-length WT-Synip and S99F-Synip ( D). Currently, there are more than 20 reported substrates for Akt, yet none of them have been shown to display Akt isoform specificity. It is generally assumed that either Akt and/or substrate spatial compartmentalization is the primary determinant of substrate selectivity. Although our data do not exclude this mechanism, our data indicate the presence of at least one unusual consensus site that displays relative Akt2 specificity in a physiologically regulated manner.
As Synip is a Syntaxin4 binding protein, its functional activity is assumed to exist at the plasma membrane. Although insulin-stimulated Akt2 translocates to the plasma membrane (Hanada et al., 2004
), Akt1 was reported to undergo nuclear localization after growth factor stimulation (Pekarsky et al., 2000
). As shown in E, insulin stimulation resulted in a specific increase in Akt2 but not Akt1 association with Synip. Whether or not the Synip–Akt2 interaction occurs through direct or indirect interaction, these results are consistent with the specificity and subcellular compartmentalization of Akt1 and Akt2.
Our data also provide a physiological consequence for Akt2-specific Synip phosphorylation. As shown in A, the dephosphorylated Synip binds Syntaxin4, whereas Akt2 phosphorylated Synip displayed significantly less binding compared with nonphosphorylated Synip ( B). Furthermore, S99F-Synip binding to Syntaxin4 was refractory to insulin stimulation and was unaffected by the presence or absence of active Akt2 ( B). Consistent with these results, only Synip, which has an Akt2 phosphorylation site, was permissive for insulin-stimulated glucose uptake and GLUT4 translocation, whereas the nonphosphorylatable mutant S99F was inhibitory. Thus, our data demonstrate that insulin-stimulated Akt2-dependent phosphorylation of Synip on serine residue 99 results in reduced binding interactions between Synip and Syntaxin4. These data are consistent with the requirement of Akt2 for insulin-stimulated glucose uptake and insulin sensitivity in culture cells, mice, and humans (Cho et al., 2001a
; Bae et al., 2003
; George et al., 2004
In summary, we have identified Synip as the first specific Akt2 substrate. Akt2-dependent phosphorylation at serine 99 directly modulates the interaction of Synip with Syntaxin4, suggesting a direct mechanism linking Akt2 function with the t-SNARE–mediated docking/fusion of GLUT4 cargo vesicles.