The carboxy-terminal region of the TBC1D4 (AS160) RabGAP mediates protein homodimerization

Abstract

TBC1D4 (also known as AS160) is a Rab·GTPase-activating protein (RabGAP) which functions in insulin signaling. TBC1D4 is critical for translocation of glucose transporter 4 (GLUT4), from an inactive, intracellular, vesicle-bound site to the plasma membrane, where it promotes glucose entry into cells. The TBC1D4 protein is structurally subdivided into two N-terminal phosphotyrosine-binding (PTB) domains, a C-terminal catalytic RabGAP domain, and a disordered segment in between containing potential Akt phosphorylation sites. Structural predictions further suggest that a region C-terminal to the RabGAP domain adopts a coiled-coil motif. We show that C-terminal region (CTR) region is largely α-helical and mediates TBC1D4 RabGAP dimerization. RabGAP catalytic activity and thermal stability appear to be independent of CTR-mediated dimerization.

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

“…Interestingly, the purified protein appeared to be mostly in an oligomeric state as indicated by an apparent M r in size exclusion chromatography about 3 times higher than predicted from the amino acid sequence. This is consistent with a previous study that showed dimerization of the related TBC1D4 GAP domain through a ϳ100 amino acid C-ter-minal region predicted to adopt a coiled-coil motif (21). A similar C-terminal sequence is also found in TBC1D1, which might contribute to oligomerization of the protein.…”

“…A similar C-terminal sequence is also found in TBC1D1, which might contribute to oligomerization of the protein. However, oligomer formation has not been found to have an impact on the GAP activity of the truncated domain (21), implicating other possible functions except involvement in catalysis. Further experiments are required to compare the enzymatic properties of the high M r form and the low M r form of full-length TBC1D1.…”

“…Interestingly, a naturally occurring mutation of an arginine residue (R125W) located in the first PTB domain of TBC1D1 has been linked to familial obesity in humans and impaired glucose transport in mice through an unclear mechanism (5,6,20). Recent studies have investigated the structure and function of the C-terminal GAP domains of TBC1D1 and TBC1D4 in vitro (13,17,21,22). Although the overall three-dimensional structure of the GAP domains were similar to that of the previously described yeast homolog Gyp1p (11,23), large differences were described for their catalytic activities and interfaces potentially involved in substrate recognition and enzymatic specificity (11,22).…”

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

“…Coimmunoprecipitations with HA-and FLAG-tagged TBC1D4 constructs show that both N-terminal PTB domains together with the 130 amino-acid-spanning C-terminal tail are responsible for TBC1D4 oligomerization. This is in agreement with the previous studies that indicated possible proteinprotein interactions through the C-terminal region (29) and the PTB domains (21) of TBC1D4. Interestingly, mapping of tryptic sites within TBC1D4 using partial digests indicates that the regions flanking two phosphorylation targets in TBC1D4, Ser 348 and Ser 577 , where the former is presumably involved in 14-3-3 binding (8,9), are highly susceptible to proteolysis and thus likely more prominently exposed to the solvent than other regions of the protein.…”

Cooperative actions of Tbc1d1 and AS160/Tbc1d4 in GLUT4-trafficking activities

“…Indeed, Tbc1d1 reportedly interacts through PTB domains with the cytoplasmic domain of the insulin-responsive aminopeptidase, and the resulting association is influenced by phosphorylation of Tbc1d1 by Akt or AMPK (22). Furthermore, recent biochemical studies have revealed that AMPK containing the ␣1, but not the ␣2, isoform of the catalytic subunit form a complex with Tbc1d1 via PTB domains (23). Although dimerization of AS160 via its C-terminal GAP domain is reported to be unrelated to GAP catalytic activity (24), it is still possible that PTB domainmediated heterodimerization and associations with other proteins, such as IRAP and AMPK, can modulate the functional balances between the two RabGAPs.…”