AKT and AMPK site-specific phosphorylation on TBC1D4 biological activity

TBC1D4 is a 160 kDa multi-domain containing RabGAP (Rab GTPase-activating protein) and a downstream target of the insulin and contraction-activated kinases AKT and AMPK. While phosphorylation of TBC1D4 has been linked to GLUT4 translocation from storage vesicles (GSVs) to the cell surface, its impact on TBC1D4 enzymatic function is not well understood as previous studies mostly investigated the truncated GAP domain lacking the phosphorylation sites. In the present study, we expressed and purified recombinant full-length TBC1D4 using the baculovirus system. Size exclusion chromatography and co-immunoprecipitation experiments revealed that full-length TBC1D4 forms oligomers of ~600 kDa. Moreover, full-length TBC1D4 displayed similar substrate specificity but had a markedly higher specific GAP activity towards Rab10 compared to the truncated GAP domain. Using high-resolution mass spectrometry, we mapped 19 Ser/Thr phosphorylation sites in TBC1D4.