Akt2 phosphorylates Synip to regulate docking and fusion of GLUT4-containing vesicles

Abstract

We have identified an unusual potential dual Akt/protein kinase B consensus phosphorylation motif in the protein Synip (RxKxRS97xS99). 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.

Synip Arrests Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE)-dependent Membrane Fusion as a Selective Target Membrane SNARE-binding Inhibitor

“…It has been proposed that synip is dissociated from SNAREs when its serine 99 residue is phosphorylated by Akt2, but the physiological role of this phosphorylation is being debated (32,61). Nevertheless, it is conceivable that an insulininduced phosphorylation may destabilize the t-SNARE-synip interaction to permit the entry of the v-SNARE.…”

“…In solution, synip binds to syntaxin-4 monomer and appears to inhibit the SNARE assembly (32,33). However, it is unclear how synip regulates SNARE assembly and membrane fusion in the membrane environment.…”

“…One of the SNARE regulators in GLUT4 exocytosis is synip, a soluble factor expressed in insulin-responsive tissues (32,33). It has been suggested that synip binds to the syntaxin-4 monomer and negatively regulates GLUT4 exocytosis (33).…”

Ins (endocytosis) and outs (exocytosis) of GLUT4 trafficking

“…Several syntaxin 4 binding partners have been identified including one member of the Munc18 family (Munc18c), Synip and Tomosyn [47,50,51]. Although the potential role of Tomosyn has not been well studied, the evidence supporting a critical role for either Munc18c and/or Synip has remained controversial.…”

“…Further studies have suggested that insulin induces a specific Akt 2 dependent Synip phosphorylation on serine 99 is required for the dissociation of Synip from syntaxin4 and thereby allowing for the assembly of the ternary SNARE-complex [51]. However, this model has been recently challenged as another group has reported that mutation of serine 99 had no significant effect on insulin-stimulated GLUT4 translocation [68].…”