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

Abstract

In skeletal muscle, the Rab GTPase-activating (GAP) protein TBC1D1 is phosphorylated by AKT and AMP-activated protein kinase (AMPK) in response to insulin and muscle contraction. Genetic ablation of Tbc1d1 or mutation of distinct phosphorylation sites impairs intracellular GLUT4 retention and GLUT4 traffic, presumably through alterations of the activation state of downstream Rab GTPases. Previous studies have focused on characterizing the C-terminal GAP domain of TBC1D1 that lacks the known phosphorylation sites, as well as putative regulatory domains. As a result, it has been unclear how phosphorylation of TBC1D1 would regulate its activity. In the present study, we have expressed, purified, and characterized recombinant full-length TBC1D1 in Sf9 insect cells via the baculovirus system. Full-length TBC1D1 showed RabGAP activity toward GLUT4-associated Rab8a, Rab10, and Rab14, indicating similar substrate specificity as the truncated GAP domain. However, the catalytic activity of the full-length TBC1D1 was markedly higher than that of the GAP domain. Although in vitro phosphorylation of TBC1D1 by AKT or AMPK increased 14-3-3 binding, it did not alter the intrinsic RabGAP activity. However, we found that TBC1D1 interacts through its N-terminal PTB domains with the cytoplasmic domain of the insulin-regulated aminopeptidase, a resident protein of GLUT4 storage vesicles, and this binding is disrupted by phosphorylation of TBC1D1 by AKT or AMPK. In summary, our findings suggest that other regions outside the GAP domain may contribute to the catalytic activity of TBC1D1. Moreover, our data indicate that recruitment of TBC1D1 to GLUT4-containing vesicles and not its GAP activity is regulated by insulin and contraction-mediated phosphorylation.

Thirty sweet years of GLUT4

“…Accordingly, it has been suggested, although not experimentally proven, that phosphorylation and/or 14-3-3 binding directly inhibits TBC1D4 GAP activity. A recent study has shown that the TBC1D4 ortholog TBC1D1 that is also phosphorylated by Akt and AMPK does not undergo any significant change in GAP activity following its phosphorylation (82). However, the stoichiometry of TBC1D1 phosphorylation was not carefully assessed in this study, so it is unclear what proportion of the total TBC1D1 pool that was under investigation was actually phosphorylated.…”

“…GSV binding-TBC1D4 binds to GSV cargo, including the cytosolic tails of IRAP and LRP1, and this interaction is reduced in response to insulin (43,84). In fact, phosphorylation of TBC1D1, determines its dissociation from IRAP rather than its inactivation (82). Assuming that, as with TBC1D1, phosphorylation of TBC1D4 does not regulate its GAP activity, then phosphorylation-dependent release of TBC1D1/TBC1D4 from GSVs may be the major mechanism for regulating the activity of this protein, enabling GTP loading of Rabs on GSVs.…”

“…It is notable that in this assay, the GAP activity of the TBC1D4 fragment used was relatively low compared with that observed for other GAPs. In a recent study examining TBC1D1 expressed in Sf9 cells, it was shown that the GAP activity of the full-length protein was markedly higher than that of the GAP domain alone (82). Hence, it would be fruitful to re-examine the Rab GTPase specificity of TBC1D4 using the full-length protein.…”

Improved exercise capacity in cyclophilin‐D knockout mice associated with enhanced oxygen utilization efficiency and augmented glucose uptake via AMPK‐TBC1D1 signaling nexus

“…AMPK phosphorylation at Thr172 has been reported during in vivo exercise (20) and during in vitro simulated exercise by electrical stimulation (21). Activated AMPK phosphorylates a Rab GTPase activating protein known as TBC1D1 that belongs to TBC1D-family at specific residues-Ser231, Ser660, and Ser700-leading to its inactivation as shown in mouse skeletal muscle (22)(23)(24).…”

Ablating the Rab‐GTPase activating protein TBC1D1 predisposes rats to high‐fat diet‐induced cardiomyopathy

“…Although the underlying mechanism is unknown, TBC1D1 may exert regulatory control over FABPpm trafficking in the heart via its downstream GAP activity, similar to AMPK-AS160-mediated regulation of GLUT4, as well as FAT/CD36 trafficking (Samovski et al 2012). In particular, TBC1D1 and AS160 display substrate specificity toward the Rab GTPase proteins Rab8a, Rab10 and Rab14 in the heart (Mafakheri et al 2018). Notably, research in HL-1 cardiomyocytes has established that the Rab GTPase protein Rab8a regulates FAT/CD36 membrane trafficking (Samovski et al 2012).…”