npg Axin/TNKS/KIF3A complex regulates GLUT4 translocation 1248 Figure 1 Axin forms a ternary complex with TNKS and KIF3A. (A) TNKS interacts with KIF3A. Lysates of 3T3-L1 adipocytes were subjected to IP with control IgG or rabbit anti-TNKS poly- clonal antibody for endogenous TNKS. The immunoprecipitates along with total cell lysates were analyzed by immunoblotting (IB) separately with anti-KIF3A and anti-TNKS antibodies. (B) Axin binds KIF3A. The experiment was performed in the same way as in A, except that anti-Axin antibody was used for IP. (C, D) Interactions between TNKS2 and KIF3A (C) and between Axin and KIF3A (D) were examined by in vitro pull-down assays. His- tagged TNKS2 or Axin was incubated with GST or GST-tagged KIF3A for 4 h. The samples were then incubated with Ni-NTA beads to pull down His-tagged TNKS2 or Axin, followed by im- munoblotting with anti-His or anti-GST antibody. (E) Axin forms a ternary complex with TNKS2 and KIF3A. HA-TNKS2 and Myc- Axin were cotransfected with or without FLAG-KIF3A into 293T as indicated beneath TCL. A two-step co-IP was performed as outlined [31]. The FLAG antibody co-precipitated HA-TNKS2 and Myc-Axin only from the cells cotransfected with FLAG-KIF3A. The Axin/TNKS2/KIF3A complex is critical for GLUT4 translocation in response to insulin Since KIF3A has been shown to function in microtu- bule-associated GLUT4 translocation and TNKS1/2 in- teract with IRAP, an integral protein of GLUT4 vesicles, we set out to determine whether the Axin/TNKS/KIF3A complex participates in insulin-stimulated GLUT4 translocation. First, we carried out immunofluorescent staining in differentiated 3T3-L1 adipocytes as the cells express high levels of GLUT4. We found that Axin, TNKS and KIF3A are co-localized in the perinuclear region of adipocytes (Figure 2A), and that Axin and KIF3A are partially co-localized with the TGN marker TGN38 (Figure 2B). Importantly, TNKS, KIF3A and Axin show co-staining with GLUT4 (Figure 2C). These results indicate that, in the absence of insulin, the Axin/ TNKS/KIF3A complex co-localizes with GLUT4 (Figure 2C). In the presence of insulin, we observed a dramatic redistribution of GLUT4 to the cell surface (Figure 2C). However, insulin treatment did not enrich Axin, TNKS or KIF3A to the cell membrane (Figure 2A-2C, Supple- mentary information, Figure S2). We next determined the effects of knockdown of the Axin/TNKS/KIF3A complex on insulin-stimulated translocation of GLUT4 by immunofluorescence mi- crocopy (Figure 3A). The results show that depletion of each component of the Axin/TNKS/KIF3A complex impaired insulin-stimulated GLUT4 translocation, in line with the previous observation that microinjection of anti-KIF3 adaptor subunit (KAP) or anti-KIF3B an- tibody into adipocytes attenuated GLUT4 translocation [18]. Next, we prepared the plasma membrane fraction of untreated and insulin-stimulated adipocytes that were infected with siRNA-delivering adenoviruses targeting Axin (Figure 3B) or TNKS (Figure 3C) and assessed the relative amounts of the GLUT4 protein in the PM frac- tions and in the post-PM fractions (supernatants, clear of PM) by immunoblotting. The PM content of GLUT4 af- ter insulin stimulation was drastically decreased in Axin- or TNKS-knockdown cells compared to adipocytes ex- pressing control siRNA, whereas the overall expression of GLUT4 was not affected. Insulin receptor β-subunit (IR-β), which is located on the cell membrane, was de- tected as a marker for the prepared PM fractions. Double knockdown of TNKS1 and TNKS2 further decreased insulin-stimulated GLUT4 translocation to the PM than single knockdown (Supplementary information, Figure S4). The above data collectively indicate that the com- plex assembled by Axin, TNKS and KIF3A is important in promoting GLUT4 translocation in response to insu- lin. Cell Research | Vol 22 No 8 | August 2012