Consequences of tyrosine phosphorylation of Syntaxin4 and Munc18c on GLUT4 trafficking

Glucose homeostasis is regulated by the opposing actions of insulin and glucagon; insulin facilitates rapid glucose uptake from the blood stream to muscle and adipose cells. These tissues express the facilitative glucose transporter (GLUT4), which in response to insulin is translocated from intracellular compartments to the plasma membrane (PM) allowing glucose entry to cells. GLUT4 undergoes a complex intracellular trafficking itinerary, including recycling to and from the PM, but in the absence of insulin is mainly stored in GLUT4 storage vesicles (GSVs). When insulin binds its receptor, a complicated signalling cascade is initiated which results in the tethering, docking and fusion of GSVs to the PM. Fusion of the GSVs with the plasma membrane is mediated by SNARE proteins. The formation of a SNARE complex composed of Syntaxin4 (Sx4) and SNAP23 localised to the PM and VAMP2 on GSVs is a key event; the formation of this complex is in turn regulated by the Sec1/Munc18 protein, Munc18c. It has been shown that insulin stimulation leads to phosphorylation of Sx4 at two sites (Y115 and Y251) and Munc18c at Y521. The stoichiometry and order of phosphorylation are not yet known, and the biological consequences of this action to be uncovered. Therefore, the current study sought to ascertain the functional consequences of SNARE protein phosphorylation on GLUT4 trafficking. The approaches used include in vitro assessment of recombinant SNARE proteins and studies in vivo using 3T3-L1 adipocytes. The results of this study confirmed that tyrosine 115 and 251 of syntaxin4 are phosphorylated in response to insulin in 3T3-L1 adipocytes; however, phosphorylation of Munc18c was ambiguous. Subsequently, phosphomimetic recombinant SNARE proteins were expressed and ternary SNARE complex formation was successfully recapitulated in vitro and the complex found to be SDS and heat resistant. Phosphomimetic syntaxin4 mutants showed increased formation of SNARE complexes, notably double phosphomimetic mutant. Moreover, binary interactions with other SNAREs (SNAP23 and VAMP2) revealed tighter binding and higher affinity of the double phosphomimetic syntaxin4 compared to wild-type syntaxin4. Additional spectroscopic evidences suggest these differences are due to conformational changes and syntaxin4 mutants are more likely to be in the open form especially the double phosphomimetic mutant. In order to translate in vitro findings into cell models, in vivo intervention tools were generated including phospho-specific antibodies against phosphorylated residues in syntaxin4 and lentivirus particles for 3T3-L1 adipocytes infection. Antibodies validated on recombinant proteins phosphorylated using recombinant insulin receptor tyrosine kinase indicated they are functional and specific. Efforts dedicated towards optimizing working conditions for phospho-specific antibodies using adipocyte lysates were extensively examined, yet signals detected were generally found to be non-specific. A non-intrusive protein-protein interaction protocol, proximity ligation assay, was used to detect insulin-stimulated phosphorylation in adipocytes. A positive signal was detected confirming antibodies functionality in this assay; further work will be required to optimize this. Lentiviruses were used to over-express phosphomimetic mutants of syntaxin4 in 3T3-L1 adipocytes and Hela cells. The functionality of the transfected syntaxin4 mutants was assessed by measuring basal and insulin-stimulated glucose uptake. Infected native 3T3-L1 adipocytes showed a trend towards an increase in glucose uptake under basal conditions with no effect observed on the maximal insulin-stimulated rate of glucose transport. We speculate this may reflect the presence of the endogenous Sx4 molecules masking any effects of mutant over-expression thus Sx4 knockout cell lines was considered as an alternative experimental system. Sx4 overexpression rescued insulin-stimulated glucose uptake in Sx4 knockout 3T3-L1 cells and was significantly enhanced in cells expressing the double phosphomimetic mutant. Finally, our work showed that the native Munc18c native mouse gene sequence expressed poorly in bacteria; hence we used a gene-enhanced sequence that was found to express well and purify effectively. CD spectroscopy showed similar structures of expressed Munc18c using either sequences. Phosphomimetic Munc18c (Y521E) binds with higher affinity both wild type and double phosphomimetic syntaxin4 compared to wild type Munc18c. The data presented in this study suggest strongly that phosphorylation influences GLUT4 trafficking by altering the frequency and affinity of SNARE protein interactions. Such findings enrich knowledge about the mechanism of GLUT4 trafficking thus ultimately could help in understanding type 2 diabetes.