Complementary regulation of TBC1D1 and AS160 by growth factors, insulin and AMPK activators

AS160 (Akt substrate of 160 kDa) and TBC1D1 are related RabGAPs (Rab GTPase-activating proteins) implicated in regulating the trafficking of GLUT4 (glucose transporter 4) storage vesicles to the cell surface. All animal species examined contain TBC1D1, whereas AS160 evolved with the vertebrates. TBC1D1 has two clusters of phosphorylated residues, either side of the second PTB (phosphotyrosine-binding domain). Each cluster contains a 14-3-3-binding site. When AMPK (AMP-activated protein kinase) is activated in HEK (human embryonic kidney)-293 cells, 14-3-3s bind primarily to pSer²³⁷ (where pSer is phosphorylated serine) in TBC1D1, whereas 14-3-3 binding depends primarily on pThr⁵⁹⁶ (where pThr is phosphorylated threonine) in cells stimulated with IGF-1 (insulin-like growth factor 1), EGF (epidermal growth factor) and PMA; and both pSer²³⁷ and pThr⁵⁹⁶ contribute to 14-3-3 binding in cells stimulated with forskolin. In HEK-293 cells, LY294002 inhibits phosphorylation of Thr⁵⁹⁶ of TBC1D1, and promotes phosphorylation of AMPK and Ser²³⁷ of TBC1D1. In vitro phosphorylation experiments indicated regulatory interactions among phosphorylated sites, for example phosphorylation of Ser²³⁵ prevents subsequent phosphorylation of Ser²³⁷. In rat L6 myotubes, endogenous TBC1D1 is strongly phosphorylated on Ser²³⁷ and binds to 14-3-3s in response to the AMPK activators AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside), phenformin and A-769662, whereas insulin promotes phosphorylation of Thr⁵⁹⁶ but not 14-3-3 binding. In contrast, AS160 is phosphorylated on its 14-3-3-binding sites (Ser³⁴¹ and Thr⁶⁴²) and binds to 14-3-3s in response to insulin, but not A-769662, in L6 cells. These findings suggest that TBC1D1 and AS160 may have complementary roles in regulating vesicle trafficking in response to insulin and AMPK-activating stimuli in skeletal muscle.