npg Axin/TNKS/KIF3A complex regulates GLUT4 translocation 1254 Figure 7 Akt dependence of insulin-induced accumulation of the Axin/TNKS/KIF3A complex. (A) Inhibition of Akt attenuates insulin-induced complex formation. 3T3-L1 cells were serum starved for 4 h and then incubated with 1.8 µM Akt inhibitor VIII (inh) and 200 nM insulin (ins) for 30 min, followed by IP with anti-TNKS antibody. The bar graphs (on the right) show fold in- creases of indicated protein on lanes 2-5 (normalized to lane 2). The precipitated TNKS and the co-precipitated proteins were analyzed by immunoblotting using specific antibodies as indicated. (B) Inhibition of Akt antagonizes the effect of insulin on TNKS PARsylation and ubiquitination. Insulin treatment decreases PARsylation and ubiquitination of TNKS (lane 3 compared to lane 2), which was abrogated by the Akt inhibitor. (C) A simplified model showing that the Axin/TNKS complex is connected to GLUT4 vesicles trafficking by KIF3A. Axin, TNKS and KIF3A form a ternary complex that is associated with the TGN. In the absence of insulin, TNKS and Axin are degraded after poly-ADP-ribosylation and ubiquitination. Insulin treatment prevents the PARsylation of TNKS and Axin in an Akt-dependent manner to increase the abundance of the Axin/TNKS/KIF3A com- plex, in that Axin acts to enhance the complex. It has been shown that insulin-activated Akt phosphorylates AS160 that is a GTPase-activating protein for Rabs; upon phosphorylation, AS160 can no longer exert its inhibitory effect on Rabs, ultimately allowing GSV to translocate to the cell surface [37-42]. ing a functional linkage of the Axin/TNKS complex to glucose transport. It is worthy to note that insulin signaling diverges to intersect with GLUT4 trafficking via multiple routes. Both PI3K-dependent and PI3K-independent pathways operate in the insulin-induced GLUT4 translocation [1, 4]. Genetic or pharmacologic disruption of Akt inhibits only about two-thirds of the signaling to GLUT4 [8, 35, Cell Research | Vol 22 No 8 | August 2012