AS160 (Akt substrate of 160kDa) 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 pSer(237) (where pSer is phosphorylated serine) in TBC1D1, whereas 14-3-3 binding depends primarily on pThr(596). (where pThr is phosphorylated threonine) in cells stimulated with IGF-1 (insulin-like growth factor 1), EGF (epidermal growth factor) and PMA; and both pSer (237) and pThr(596). contribute to 14-3-3 binding in cells stimulated with forskolin. In HEK-293 cells, LY294002 inhibits phosphorylation of Thr(596) of TBC1D1, and promotes phosphorylation of AMPK and Set 211 of TBC1D1. In vitro phosphorylation experiments indicated regulatory interactions among phosphorylated sites, for example phosphorylation of Ser(235) prevents subsequent phosphorylation of Ser(237). In rat L6 myotubes, endogenous TBC1D1 is strongly phosphorylated on Ser(237) and binds to 14-3-3s in response to the AMPK activators AICAR (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside), phenformin and A-769662, whereas insulin promotes phosphorylation of Thr(596) but not 14-3-3 binding. In contrast, AS 160 is phosphorylated on its 14-3-3-binding sites (Ser(341) and Thr(642)) and binds to 14-3-3s in response to insulin, but not A-769662, in L6 cells. These findings suggest that TBC1D1 and AS 160 may have complementary roles in regulating vesicle trafficking in response to insulin and AMPK-activating stimuli in skeletal muscle.