Isoform-specific AMPK association with TBC1D1 is reduced by a mutation associated with severe obesity

Abstract

AMP-activated protein kinase (AMPK) is a key regulator of cellular and systemic energy homeostasis which achieves this through the phosphorylation of a myriad of downstream targets. One target is TBC1D1 a Rab-GTPase-activating protein that regulates glucose uptake in muscle cells by integrating insulin signalling with that promoted by muscle contraction. Ser237 in TBC1D1 is a target for phosphorylation by AMPK, an event which may be important in regulating glucose uptake. Here, we show AMPK heterotrimers containing the α1, but not the α2, isoform of the catalytic subunit form an unusual and stable association with TBC1D1, but not its paralogue AS160. The interaction between the two proteins is direct, involves a dual interaction mechanism employing both phosphotyrosine-binding (PTB) domains of TBC1D1 and is increased by two different pharmacological activators of AMPK (AICAR and A769962). The interaction enhances the efficiency by which AMPK phosphorylates TBC1D1 on its key regulatory site, Ser237. Furthermore, the interaction is reduced by a naturally occurring R125W mutation in the PTB1 domain of TBC1D1, previously found to be associated with severe familial obesity in females, with a concomitant reduction in Ser237 phosphorylation. Our observations provide evidence for a functional difference between AMPK α-subunits and extend the repertoire of protein kinases that interact with substrates via stabilisation mechanisms that modify the efficacy of substrate phosphorylation.

AKT and AMP-activated protein kinase regulate TBC1D1 through phosphorylation and its interaction with the cytosolic tail of insulin-regulated aminopeptidase IRAP

“…The R125W coding variant in TBC1D1 has been linked to an increased risk for familial obesity in humans (5,6). Modeling studies based on related PTB domain structures suggested that the R125W exchange might affect certain ligand binding properties of the first PTB domain in TBC1D1 and AMPK interaction (35,36). However, in this study we found no evidence that addition of excess PTB domains alters the GAP activity of TBC1D1 in vitro.…”

Chemical biology probes of mammalian GLUT structure and function

“…This could involve an inhibition of endocytosis as observed in cardiomyocytes, as this would allow a slow build-up of cell-surface GLUT4 even when exocytosis occurs at the slow basal rate. Studies on phosphorylation of signalling intermediates, such as TBC1D1 and TBC1D4, suggest signalling convergence of insulin and AMPK signalling [ 80 , 86 ], but the kinetics of GLUT4 translocation suggest signalling from insulin and AMPK can have divergent effects on GLUT4 traffic. Changes in the AMP/ATP ratio that result from stimulation of the ‘exercise’ signalling pathway may have effects on endocytosis that are not present following an insulin treatment and are possibly independent of TBC1D1 and TBC1D4.…”

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

“…We and others have previously demonstrated that the PTB domains of TBC1D1 bind to 14-3-3 proteins 33,[36][37][38] , IRAP 30,33,35 , AMPK complexes containing α1 but not α2 catalytic subunits 33 and APPL2 65 . Using a differentiated in vitro muscle cell model and intact mouse quadriceps muscle, we extend this repertoire of validated binding proteins to include VPS13A, VPS13C, EHBP1L1, MICAL1 and SERCA1.…”

“…Mouse model. Transgenic MCK-3xFLAG-Tbc1d1 mice with muscle-specific overexpression of Tbc1d1 were generated as previously described 33 and kept in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals, and all experiments were approved by the Ethics Committee of the State Ministry of Agriculture, Nutrition and Forestry (State of North Rhine-Westphalia, Germany). Male mice between 10 and 14 weeks were sacrificed by cervical dislocation, quadricep muscle dissected and directly snap-frozen in liquid nitrogen.…”

“…Molecular biology. Constructs for lentiviral expression of full-length TBC1D1 and TBC1D1 PTB1 + 2, amino acids 1-381 based on secondary structure prediction 68 , were cloned as previously described 33 into pXLG3. Also cloned into pXLG3 were HA-GLUT4 and HA-GLUT1, both with a HA-tag in the first exofacial loop, from pQB125-HA-GLUT4 85 and pCDNA3-HA-GLUT1 86 respectively.…”