Yamada, E. et al. Akt2 phosphorylates Synip to regulate docking and fusion of GLUT4-containing vesicles. J. Cell Biol. 168, 921-928

Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi, Gunma, 371-8511, Japan.
The Journal of Cell Biology (Impact Factor: 9.83). 04/2005; 168(6):921-8. DOI: 10.1083/jcb.200408182
Source: PubMed


We have identified an unusual potential dual Akt/protein kinase B consensus phosphorylation motif in the protein Synip (RxKxRS(97)xS(99)). Surprisingly, serine 97 is not appreciably phosphorylated, whereas serine 99 is only a specific substrate for Akt2 but not Akt1 or Akt3. Although wild-type Synip (WT-Synip) undergoes an insulin-stimulated dissociation from Syntaxin4, the Synip serine 99 to phenylalanine mutant (S99F-Synip) is resistant to Akt2 phosphorylation and fails to display insulin-stimulated Syntaxin4 dissociation. Furthermore, overexpression of WT-Synip in 3T3L1 adipocytes had no effect on insulin-stimulated recruitment of glucose transporter 4 (GLUT4) to the plasma membrane, whereas overexpression of S99F-Synip functioned in a dominant-interfering manner by preventing insulin-stimulated GLUT4 recruitment and plasma membrane fusion. These data demonstrate that insulin activation of Akt2 specifically regulates the docking/fusion step of GLUT4-containing vesicles at the plasma membrane through the regulation of Synip phosphorylation and Synip-Syntaxin4 interaction.

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    • "Synip was reported to bind to syntaxin to inhibit ternary SNARE complex, furthermore overexpression of synip in β-cell derived clonal cells resulted in the inhibition of glucose-induced insulin secretion [51]. Phosphorylation of Synip by Akt promotes the dissociation of synip from syntaxin, which allows vesicles to dock and fuse with the plasma membrane [52]. Thus, synip may link the inhibition of Akt activity with the suppression of fusions from previously docked granules. "
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    ABSTRACT: In glucose-induced insulin secretion from pancreatic β-cells, a population of insulin granules fuses with the plasma membrane without the typical docking process (newcomer granule fusions), however, its mechanism is unclear. In this study, we investigated the PI3K signaling pathways involved in the upregulation of newcomer granule fusions. Acute treatment with the class IA-selective PI3K inhibitors, PIK-75 and PI-103, enhanced the glucose-induced insulin secretion. Total internal reflection fluorescent microscopy revealed that the PI3K inhibitors increased the fusion events from newcomer granules. We developed a new system for transfection into pancreatic islets and demonstrated the usefulness of this system in order for evaluating the effect of transfected genes on the glucose-induced secretion in primary cultured pancreatic islets. Using this transfection system together with a series of constitutive active mutants, we showed that the PI3K-3-phosphoinositide dependent kinase-1 (PDK1)-Akt pathway mediated the potentiation of insulin secretion. The Akt inhibitor also enhanced the glucose-induced insulin secretion in parallel with the upregulation of newcomer granule fusions, probably via increased motility of intracellular insulin granules. These data suggest that the PI3K-PDK1-Akt pathway plays a significant role in newcomer granule fusions, probably through an alteration of the dynamics of the intracellular insulin granules.
    PLoS ONE 10/2012; 7(10):e47381. DOI:10.1371/journal.pone.0047381 · 3.23 Impact Factor
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    • "Synip binds syntaxin 4 in the basal state and inhibits the interaction between syntaxin 4 and the V-SNARE vesicle associated protein-2 (Vamp2) that is present in Glut4 containing vesicles [9]. Insulin stimulation results in the Akt2-mediated phosphorylation of Synip and its dissociation from syntaxin 4 [9], [10]. These events are thought to allow Glut4 vesicle Vamp2 access to syntaxin 4 and the binding of Vamp2 to syntaxin 4 generates a fusion competent complex [12]. "
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    ABSTRACT: The insulin responsive Glut4 transport vesicles contain the v-SNARE protein Vamp2 that associate with the plasma membrane t-SNARE protein Syntaxin 4 to drive insulin-stimulated Glut4 translocation in skeletal muscle and adipocytes. The syntaxin 4 interacting protein (Synip) binds to syntaxin 4 in the basal state and dissociates in the insulin-stimulated state allowing for the subsequent binding of Vamp2 containing Glut4 vesicles and fusion with the plasma membrane. In this study, we have found that Synip binds phosphatidylinositol 3,4,5-triphosphate (PIP3), but not phosphatidylinositol 3 phosphate (PIP) or phosphatidylinositol 3,4-biphosphate (PIP2) through the Synip WW domain as deletion of this domain (Synip ΔWW) failed to bind PIP3. Over-expressed Synip ΔWW in 3T3L1 adipocytes reduced the basal levels of Glut4 at the plasma membrane with no effect on the binding to syntaxin 4 in vitro. Subcellular fractionation demonstrated that the amount of Synip ΔWW at the PM was decreased in response to insulin in 3T3L1 adipocytes whereas the amount of Synip WT increased. These data suggest that in the presence of insulin, the dissociated Synip remains anchored to the plasma membrane by binding to PIP3.
    PLoS ONE 08/2012; 7(8):e42782. DOI:10.1371/journal.pone.0042782 · 3.23 Impact Factor
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    ABSTRACT: Glucose homeostasis depends on the ability of insulin to stimulate glucose uptake into both muscle and adipose tissue by promoting the translocation of glucose transporters (GLUT4) from intracellular sites to the plasma membrane (PM). In individuals with Type 2 diabetes the ability of insulin to stimulate glucose transport is impaired. The incidence of Type 2 diabetes is increasing worldwide, highlighting the need to understand the molecular basis of insulin-stimulated glucose uptake. GLUT4 translocation is a specialised example of vesicular trafficking. Within the context of vesicle trafficking, all eukaryotic cells contain a common set of conserved components responsible for the execution of membrane fusion. Central to this machinery are members of the SNARE (soluble NSF attachment protein receptor) family of proteins. The process of SNARE-mediated membrane fusion needs to be tightly regulated and the SNARE proteins are partially responsible for the specificity in communication between eukaryotic subcellular organelles. Other proteins such as the Sec1p/ Munc18 (SM) proteins were shown to be essential for SNARE-mediated membrane fusion. Several methods were used to test the ability of SNARE proteins to drive membrane fusion, and one of the most important methods described to date is the in vitro fusion assay used in this study. The first topic addressed in this thesis was related to the molecular interactions between the regulatory SM protein Munc18c and the SNARE proteins VAMP2 and syntaxin 4. The use of pull-down assays revealed the novel fact that Munc18c interacts not only with the t-SNARE syntaxin 4 but also with the v-SNARE VAMP2 via its SNARE motif. The SM:v-SNARE interaction was disrupted by the presence of syntaxin 4 revealing that these two SNARE proteins compete for binding to Munc18c. Next, the role of Munc18c in membrane fusion driven by four different versions of syntaxin 4 plus SNAP23 and VAMP2 liposomes, was investigated using the well-characterised in vitro fusion assay. Results suggested that Munc18c negatively regulates SNARE- mediated membrane fusion by inhibiting the formation of SNARE complexes. Interestingly, deletion of the first 36 amino acids of syntaxin 4 was not sufficient to suppress Munc18c negative regulation of fusion indicating that this inhibition might involve other interactions apart from the short N-terminal peptide of syntaxin 4. Finally, the role of phosphorylation in SNARE complex formation was assessed using several techniques such as site-directed mutagenesis, pull-down assays and radiolabelling studies. Data obtained revealed that both syntaxin 4 and Munc18c become phosphorylated in vitro by a recombinant cytoplasmic insulin receptor kinase (CIRK). Munc18c phosphorylated by CIRK was unable to bind syntaxin 4 in vitro. Furthermore, phosphomimetic mutations were also introduced on both proteins and pull-down assays indicated that phosphorylated Munc18c is unable to interact with both syntaxin 4 and VAMP2, whereas phosphomimetic mutations in syntaxin 4 did not affect the interaction with its cognate SNARE proteins and Munc18c. These results were very useful to further understand and confirm the importance of phosphorylation in SNARE complex formation. Collectively, these data suggest that Munc18c acts through different modes of interaction with its cognate SNARE proteins, and support a model in which Munc18c negatively regulates SNARE complex formation. However, this regulation might also be dependent on other factors such as phosphorylation upon insulin signalling.
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