Syntaxin4 Interacting Protein (Synip) Binds Phosphatidylinositol (3,4,5) Triphosphate

Tsugumichi Saito,Masatomo Mori,Koshi Hashimoto,Jeffrey E Pessin,Masanobu Yamada,Aya Osaki,Tetsurou Satoh,Shuichi Okada,Atsushi Nohara,Yuko Tagaya,Shinsuke Oh-I,Takafumi Tsuchiya,Hiroki Takahashi,Frederick G Hamel

doi:10.1371/journal.pone.0042782

Tsugumichi Saito, Masatomo Mori + Show 12 more

Open Access

https://doi.org/10.1371/journal.pone.0042782

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Journal: PloS one	Publication Date: Aug 3, 2012
Citations: 26	License type: CC BY 4.0

Affiliation: Gunma University, Albert Einstein College of Medicine

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.

Highlights

Insulin stimulates the tyrosine autophosphorylation of the insulin receptor (IR)-b subunit activating its tyrosine kinase activity that phosphorylates the insulin receptor substrate (IRS)-1 protein [1,2]
We demonstrate that the syntaxin interacting protein (Synip) WW domain displays an atypical binding to PIP3 and that this interaction localizes Synip to the plasma membrane
To determine if Synip binding was selective for PIP3 or could bind other phosphatidylinositides that have been implicated in insulin-stimulated glucose transporter isoform 4 (Glut4) translocation [23], we compared the interaction of GST-Synip with PI, phosphatidylinositol phosphate (PIP), PIP2 and PIP3

Summary

Introduction

Insulin stimulates the tyrosine autophosphorylation of the insulin receptor (IR)-b subunit activating its tyrosine kinase activity that phosphorylates the insulin receptor substrate (IRS)-1 protein [1,2]. 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]. A GST pull-down assay was performed by mixing the CHO cell or 3T3L1 adipocytes lysate containing Flag-tagged Synip WT or Synip DWW stimulated with or without insulin to glutathione sepharose beads coupled with the purified GST-tagged syntaxin 4 protein.

Results

Conclusion