Impact of tyrosine phosphorylation of Syntaxin 4 and Munc18c on GLUT4 translocation

Black, Hannah Lucy (2016) Impact of tyrosine phosphorylation of Syntaxin 4 and Munc18c on GLUT4 translocation. PhD thesis, University of Glasgow.

Abstract

Insulin is an important regulator of glucose homeostasis. Insulin stimulation of fat and muscle cells results in the rapid translocation of the glucose transporter GLUT4 to the plasma membrane from its intracellular stores, allowing uptake of glucose into the cells from the blood. This tethering, docking and fusion event is driven by the formation of a SNARE complex at the plasma membrane consisting of the t-SNAREs Syntaxin4 (Sx4) and SNAP23 and the v-SNARE VAMP2. The formation of this complex is regulated by the SM protein Munc18c. It has been shown that Sx4 is phosphorylated at residues Y115 and Y251 following insulin stimulation; however the effects of these phosphorylation events have yet to be studied. In addition phosphorylation of Y521 in Munc18c is also increased following insulin stimulation. This study aimed to first elucidate the effects of Sx4 phosphorylation on SNARE protein interactions and GLUT4 trafficking, then to begin to examine the impact of phosphorylation of both Sx4 and Munc18c.

I have used an in vitro approach to assess the affect Sx4 phosphorylation has on SNARE complex assembly. I have shown, using phospho-mimetic recombinant proteins, that the phosphorylation state of Sx4 affects the rate of SNARE complex assembly and its binary interactions with VAMP2 and SNAP23. Moreover, that this may be due to a conformational change in the protein. I have also shown, for the first time, that it is likely that Sx4 can be phosphorylated on Y115 and Y251 simultaneously. In addition I have used a HA-GLUT4-GFP expressing HeLa cell line to show that expression of phospho- mimetic Sx4 increases translocation of GLUT4 to the PM under basal conditions. Finally, I have begun to investigate the implications of both Sx4 and Munc18c phosphorylation on their in vitro interactions. This data provides insights into the direct regulation of membrane trafficking proteins by the insulin-signalling pathway. Increased understanding of the regulation of GLUT4 translocation could help to develop future therapies for type 2 diabetes, where GLUT4 trafficking is impaired.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Phosphorylation, Syntaxin 4, Munc18c, GLUT4, insulin, trafficking.
• Subjects: Q Science > QH Natural history > QH301 Biology; Q Science > QH Natural history > QH345 Biochemistry
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Funder's Name: Biotechnology and Biological Sciences Research Council (BBSRC)
• Supervisor's Name: Gould, Professor Gwyn
• Date of Award: 2016
• Depositing User: Dr Hannah Black
• Unique ID: glathesis:2016-8181
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 16 May 2017 07:48
• Last Modified: 16 Nov 2017 11:14
• URI: https://theses.gla.ac.uk/id/eprint/8181