TBC1D1 interacting proteins, VPS13A and VPS13C, regulate GLUT4 homeostasis in C2C12 myotubes

Sharon C Hook,Hadi Al-Hasani,Alexandra Chadt,Elaine C Thomas,Shosei Kishida,Kate J Heesom,Jeremy M Tavaré

doi:10.1038/s41598-020-74661-1

Abstract

Proteins involved in the spaciotemporal regulation of GLUT4 trafficking represent potential therapeutic targets for the treatment of insulin resistance and type 2 diabetes. A key regulator of insulin- and exercise-stimulated glucose uptake and GLUT4 trafficking is TBC1D1. This study aimed to identify proteins that regulate GLUT4 trafficking and homeostasis via TBC1D1. Using an unbiased quantitative proteomics approach, we identified proteins that interact with TBC1D1 in C2C12 myotubes including VPS13A and VPS13C, the Rab binding proteins EHBP1L1 and MICAL1, and the calcium pump SERCA1. These proteins associate with TBC1D1 via its phosphotyrosine binding (PTB) domains and their interactions with TBC1D1 were unaffected by AMPK activation, distinguishing them from the AMPK regulated interaction between TBC1D1 and AMPKα1 complexes. Depletion of VPS13A or VPS13C caused a post-transcriptional increase in cellular GLUT4 protein and enhanced cell surface GLUT4 levels in response to AMPK activation. The phenomenon was specific to GLUT4 because other recycling proteins were unaffected. Our results provide further support for a role of the TBC1D1 PTB domains as a scaffold for a range of Rab regulators, and also the VPS13 family of proteins which have been previously linked to fasting glycaemic traits and insulin resistance in genome wide association studies.

Highlights

Skeletal muscle is a major sink for plasma glucose in the body, with insulin- and exercise-stimulated contraction being its principle physiological s timuli[1]
In the current study we show that the TBC1D1 phosphotyrosine binding (PTB) domains stably associate with several Rab regulatory proteins including MICAL1 and EHBP1L1, the calcium pump SERCA1 and VPS13A and VPS13C
AMPKα1, as a subset of proteins, identified to interact with the TBC1D1 PTB domains through SILAC-based quantitative proteomics on GFP-trap based immunoprecipitations from C2C12 myotubes stably expressing a GFP-tagged construct consisting of the two PTB domains of TBC1D1 (GFP-PTB1 + 2; Fig. 1a)[33]

Summary

Introduction

Skeletal muscle is a major sink for plasma glucose in the body, with insulin- and exercise-stimulated contraction being its principle physiological s timuli[1]. These stimuli activate distinct signalling pathways, both culminating in the mobilisation or translocation of intracellular stores of GLUT4 glucose transporters, sequestered within GLUT4 storage vesicles (GSV), to the cell surface to facilitate removal of glucose from the blood. Impaired glucose disposal, caused by defective plasma membrane translocation of GLUT4, has been linked to insulinresistance and type 2 d iabetes[2,3] Individuals with these diseases have preserved exercise-stimulated glucose uptake due to activation of insulin-independent signalling p athways[4]. Coverage: percentage of the protein sequence covered by identified peptides. # PSMs (peptide spectrum matches): total number of identified peptide sequences (includes those redundantly identified). #

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