Substrate specificity and effect on GLUT4 translocation of the Rab GTPase-activating protein Tbc1d1

Abstract

Insulin stimulation of the trafficking of the glucose transporter GLUT4 to the plasma membrane is controlled in part by the phosphorylation of the Rab GAP (GTPase-activating protein) AS160 (also known as Tbc1d4). Considerable evidence indicates that the phosphorylation of this protein by Akt (protein kinase B) leads to suppression of its GAP activity and results in the elevation of the GTP form of a critical Rab. The present study examines a similar Rab GAP, Tbc1d1, about which very little is known. We found that the Rab specificity of the Tbc1d1 GAP domain is identical with that of AS160. Ectopic expression of Tbc1d1 in 3T3-L1 adipocytes blocked insulin-stimulated GLUT4 translocation to the plasma membrane, whereas a point mutant with an inactive GAP domain had no effect. Insulin treatment led to the phosphorylation of Tbc1d1 on an Akt site that is conserved between Tbc1d1 and AS160. These results show that Tbc1d1 regulates GLUT4 translocation through its GAP activity, and is a likely Akt substrate. An allele of Tbc1d1 in which Arg(125) is replaced by tryptophan has very recently been implicated in susceptibility to obesity by genetic analysis. We found that this form of Tbc1d1 also inhibited GLUT4 translocation and that this effect also required a functional GAP domain.

Disruption of the AMPK–TBC1D1 nexus increases lipogenic gene expression and causes obesity in mice via promoting IGF1 secretion

“…Furthermore, metformin and AICAR could also significantly decrease IGF1 secretion rates in these hepatocytes expressing hTBC1D1 or hTBC1D1 Ser237Ala mutant proteins. Interestingly, the expression of a GAP-deficient TBC1D1 mutant (20) could not restore IGF1 secretion rates, suggesting that the GAP activity of TBC1D1 is required for IGF1 secretion. We next investigated which Rab(s) downstream of TBC1D1 mediates IGF1 secretion by downregulating four known in vitro TBC1D1 substrates, namely Rab2a, Rab8a, Rab10, and Rab14 (20).…”

“…Interestingly, the expression of a GAP-deficient TBC1D1 mutant (20) could not restore IGF1 secretion rates, suggesting that the GAP activity of TBC1D1 is required for IGF1 secretion. We next investigated which Rab(s) downstream of TBC1D1 mediates IGF1 secretion by downregulating four known in vitro TBC1D1 substrates, namely Rab2a, Rab8a, Rab10, and Rab14 (20). Knockdown of Rab8a caused a significant decrease in IGF1 secretion rates in WT primary hepatocytes, whereas down-regulation of Rab2a, Rab10, and Rab14 had no significant effect (SI Appendix, Fig.…”

Crystal Structures of Human TBC1D1 and TBC1D4 (AS160) RabGTPase-activating Protein (RabGAP) Domains Reveal Critical Elements for GLUT4 Translocation

“…8A). Cells expressing wild-type or catalytically inactive mouse R941K (human R854K) TBC1D1 served as positive and dominant negative controls, respectively, and numbers of cells positive for insulin-stimulated GLUT4 translocation were determined (23).…”

Thrifty Tbc1d1 and Tbc1d4 proteins link signalling and membrane trafficking pathways

“…The GAP domains are 79 % identical, and, in this issue of the Biochemical Journal, Roach et al [10] demonstrate that the Rabs identified as substrates (from the panel of Rabs that they tested) are essentially identical. Furthermore, Tbc1d1 has also been demonstrated to be phosphorylated by insulin signalling through activated Akt [10]. The domain architectures of Tbc1d1 and Tbc1d4 are also similar.…”

“…The ability of the protein to facilitate retention of GLUT4 vesicles in their basal location would then be lost and the cell would translocate more GLUT4 to the cell surface and consequently take up more glucose in the absence of any insulin stimulus. Roach et al [10] have demonstrated that Tbc1d1 reverses insulin action on GLUT4 trafficking and that insulin action suppresses Tbc1d1 activity. Therefore active Tbc1d1 would normally suppress peripheral glucose uptake and inactive Tbc1d1 would promote peripheral glucose uptake.…”