AKT/AMPK-mediated phosphorylation of TBC1D4 disrupts the interaction with insulin-regulated aminopeptidase

Samaneh Eickelschulte,Sonja Hartwig,Ben Leiser,Stefan Lehr,Viola Joschko,Manopriya Chokkalingam,Alexandra Chadt,Hadi Al-Hasani

doi:10.1016/j.jbc.2021.100637

Abstract

TBC1D4 is a 160 kDa multidomain Rab GTPase-activating protein (RabGAP) and a downstream target of the insulin- and contraction-activated kinases AKT and AMPK. Phosphorylation of TBC1D4 has been linked to translocation of GLUT4 from storage vesicles (GSVs) to the cell surface. However, its impact on enzymatic activity is not well understood, as previous studies mostly investigated the truncated GAP domain lacking the known phosphorylation sites. In the present study, we expressed and purified recombinant full-length TBC1D4 using a baculovirus system. Size-exclusion chromatography and coimmunoprecipitation experiments revealed that full-length TBC1D4 forms oligomers of ∼600 kDa. Compared with the truncated GAP domain, full-length TBC1D4 displayed similar substrate specificity, but had a markedly higher specific GAP activity toward Rab10. Using high-resolution mass spectrometry, we mapped 19 Ser/Thr phosphorylation sites in TBC1D4. We determined Michaelis–Menten kinetics using in vitro phosphorylation assays with purified kinases and stable isotope-labeled γ-[18O4]-ATP. These data revealed that Ser324 (KM ∼6 μM) and Thr649 (KM ∼25 μM) were preferential sites for phosphorylation by AKT, whereas Ser348, Ser577, Ser595 (KM ∼10 μM), Ser711 (KM ∼79 μM), and Ser764 were found to be preferred targets for AMPK. Phosphorylation of TBC1D4 by AKT or AMPK did not alter the intrinsic RabGAP activity, but did disrupt interaction with insulin-regulated aminopeptidase (IRAP), a resident protein of GSVs implicated in GLUT4 trafficking. These findings provide evidence that insulin and contraction may regulate TBC1D4 function primarily by disrupting the recruitment of the RabGAP to GLUT4 vesicles.

Highlights

In skeletal muscle, stimulation by both insulin and contraction triggers an increase in glucose uptake from the blood stream through a rapid and reversible redistribution of GLUT4 from GLUT4 transporter storage vesicles (GSVs) to the cell surface [1, 2]
The Ras-related proteins in brain (Rab) GTPase-activating proteins (RabGAPs) TBC1D1 and TBC1D4 are downstream targets of AKT and AMPK kinases, and phosphorylation of these two proteins is associated with enhanced translocation of GLUT4 to the plasma membrane in response to insulin and contraction [5, 6]
We further investigated structural features of TBC1D4 complexes by limited proteolysis using low concentration of trypsin and analysis of partial digestion products by mass spectrometry

Summary

Introduction

Stimulation by both insulin and contraction triggers an increase in glucose uptake from the blood stream through a rapid and reversible redistribution of GLUT4 from GLUT4 transporter storage vesicles (GSVs) to the cell surface [1, 2]. The Rab GTPase-activating proteins (RabGAPs) TBC1D1 and TBC1D4 ( known as AS160) are downstream targets of AKT and AMPK kinases, and phosphorylation of these two proteins is associated with enhanced translocation of GLUT4 to the plasma membrane in response to insulin and contraction [5, 6]. Previous studies suggested a point of convergence for AKT and AMPK signaling through elevated insulinstimulated phosphorylation of TBC1D4 after contraction [10, 11] and in particular AMPK activation [12,13,14]. Due to the fact that all previous studies utilized truncated GAP domain of TBC1D4, the molecular function of the phosphorylation sites remains largely unknown. Phosphorylation of specific sites in TBC1D4 may influence phosphorylation of other sites, which may explain in part the insulin-sensitizing effect of muscle contraction

Objectives

Results

Conclusion