Insulin-stimulated Phosphorylation of the Rab GTPase-activating Protein TBC1D1 Regulates GLUT4 Translocation

Abstract

Insulin stimulates the translocation of the glucose transporter GLUT4 from intracellular locations to the plasma membrane in adipose and muscle cells. Prior studies have shown that Akt phosphorylation of the Rab GTPase-activating protein, AS160 (160-kDa Akt substrate; also known as TBC1D4), triggers GLUT4 translocation, most likely by suppressing its Rab GTPase-activating protein activity. However, the regulation of a very similar protein, TBC1D1 (TBC domain family, member 1), which is mainly found in muscle, in insulin-stimulated GLUT4 translocation has been unclear. In the present study, we have identified likely Akt sites of insulin-stimulated phosphorylation of TBC1D1 in C2C12 myotubes. We show that a mutant of TBC1D1, in which several Akt sites have been converted to alanine, is considerably more inhibitory to insulin-stimulated GLUT4 translocation than wild-type TBC1D1. This result thus indicates that similar to AS160, Akt phosphorylation of TBC1D1 enables GLUT4 translocation. We also show that in addition to Akt activation, activation of the AMP-dependent protein kinase partially relieves the inhibition of GLUT4 translocation by TBC1D1. Finally, we show that the R125W variant of TBC1D1, which has been genetically associated with obesity, is equally inhibitory to insulin-stimulated GLUT4 translocation, as is wild-type TBC1D1, and that healthy and type 2 diabetic individuals express approximately the same level of TBC1D1 in biopsies of vastus lateralis muscle. In conclusion, phosphorylation of TBC1D1 is required for GLUT4 translocation. Thus, the regulation of TBC1D1 resembles that of its paralog, AS160.Insulin stimulates glucose transport into adipose and muscle cells by increasing the amount of the GLUT4 glucose transporter at the cell surface by a process termed GLUT4 translocation (1, 2). Unstimulated adipocytes and myotubes sequester GLUT4 in intracellular compartments. Insulin activates signaling cascades that lead to the trafficking of specialized GLUT4 vesicles to the cell membrane and fusion of the vesicles therewith. A key signaling pathway for GLUT4 translocation proceeds from the insulin receptor through the activation of the protein kinase Akt. One Akt substrate that connects signaling to GLUT4 trafficking is the Rab GTPase-activating protein (GAP) 3 known as AS160. There is now considerable evidence for the following scheme (2, 3): under basal conditions, AS160 acts as a brake on GLUT4 translocation by maintaining one or more Rab proteins required for translocation in their inactive GDP state; in response to insulin, Akt phosphorylates AS160 and thereby suppresses its GAP activity; as a consequence, the elevation of the GTP form of the Rab proteins occurs, leading to the increased docking and subsequent fusion of the GLUT4 vesicles at the plasma membrane.More recently, we and others have characterized a paralog of AS160 known as TBC1D1 (4 -7). Overall, TBC1D1 is 47% identical to AS160, with the GAP domain being 79% identical (4). Its GAP domain has the same Rab specificity as the GAP domain of...

The Adaptor Protein APPL2 Inhibits Insulin-Stimulated Glucose Uptake by Interacting With TBC1D1 in Skeletal Muscle

“…Consistent with a previous study (27), we found that adenovirus-mediated expression of TBC1D1 did not affect insulin-evoked Akt phosphorylation (data not shown) but led to a significant inhibition of insulinstimulated glucose uptake compared with cells expressing luciferase control (Fig. 5F).…”

“…Of note, a previous study showed that Ser-229 of mouse TBC1D1 (equivalent to Ser-235 on human TBC1D1) within this region is potentially phosphorylated by Akt (27). We confirmed that insulin stimulated TBC1D1 phosphorylation at Ser-235 in a time-dependent manner in both EDL muscle of C57 mice and C2C12 myotubes (Fig.…”

“…Upon insulin stimulation, activated Akt induces phosphorylation of both TBC1D1 and TBC1D4 at multiple sites flanking the second PTB domain (26)(27)(28)35,36). Substitution of these phosphorylation sites with nonphosphorylatable alanine in TBC1D1 and TBC1D4 abolishes insulin-induced GLUT4 translocation to the plasma membrane in both adipocytes and muscle cells (25)(26)(27)(36)(37)(38). Although the precise mechanisms by Figure 6-APPL2 suppresses insulin-elicited phosphorylation of TBC1D1 at Thr-590 in skeletal muscle.…”

“…Several previous studies have shown that insulininduced Akt activation leads to direct TBC1D1 phosphorylation on Thr-596 (equivalent to Thr-590 in mouse TBC1D1) (6,(26)(27)(28), which in turn promotes the transport of GLUT4 to the plasma membrane by activating Rab-GTPases (6,(26)(27)(28). To investigate the effects of APPL2 on insulin-elicited phosphorylation of TBC1D1, we injected APPL2 Tg and APPL2 KO mice intraperitoneally with insulin.…”

Insulin and AMPK regulate FA translocase/CD36 plasma membrane recruitment in cardiomyocytes via Rab GAP AS160 and Rab8a Rab GTPase

“…It is worth noting that a close homolog of AS160, Rab GAP TBC1D1, was reported to function in GLUT4 translocation in adipocytes ( 47 ) and muscle cells ( 48 ). This homolog has substrate specifi city similar to that of AS160 ( 49 ) and could also be involved in CD36 traffi cking.…”