Figure 6 Insulin stabilizes the Axin/TNKS/KIF3A complex by suppressing TNKS PARsylation activity. (A) Insulin treatment increases protein levels of TNKS and Axin. Differentiated 3T3- L1 adipocytes were treated with or without 200 nM insulin for 30 min after 4 h serum deprivation, and were harvested with SDS sample buffer for western blotting with antibodies for en- dogenous TNKS and Axin. The bar graphs show basal (lane 1) and insulin-stimulated (lane 2) TNKS and Axin protein levels (normalized to lane 1) determined from immunoblotting. (B) PARsylation and ubiquitination of TNKS (left panel) and Axin (right panel) were decreased after insulin treatment. Adipocytes were serum starved for 4 h, and then incubated with DMEM supplemented with or without 200 nM insulin for 30 min in the presence of 20 µM MG132. The endogenous TNKS proteins were immunoprecipitated with anti-TNKS, followed by analysis of their levels of ADP-ribosylation and ubiquitination using anti- poly-ADP-ribose (PAR) and anti-ubiquitin antibodies, respectively. (C) Insulin treatment elevates the abundance of the Axin/TNKS/ KIF3A complex. Adipocytes were serum-depleted for 4 h, then added with or without insulin to a final concentration of 200 nM, and incubated for 30 min before lysis. The endogenous proteins were immunoprecipitated with anti-TNKS antibody, followed by immunoblotting with individual antibodies to determine levels of each protein in the complex, quantified by densitometry after nor- malizing to their respective levels from the unstimulated cells. www.cell-research.com | Cell Research Hui-Ling Guo et al . npg 1253 the abundance of these proteins and on their interaction in cells treated with or without insulin by IP and western blotting analysis. As can be seen from Figure 7A, while insulin treatment increased the protein levels of Axin and TNKS, and the co-IP of KIF3A with TNKS, Akt inhibitor VIII (for both Akt1 and Akt2) abrogated such increases. Mechanistically, Akt acts to suppress PARsylase activ- ity of TNKS, because treatment with the Akt inhibitor reversed the effect of insulin on PARsylation and sub- sequent ubiquitination of TNKS (Figure 7B). Moreover, we carried out co-IP experiments to check if insulin treatment could increase the association of the complex with microtubules and GLUT4 vesicles. Increased levels of TNKS were detected in the GLUT4 immunoprecipi- tate (Supplementary information, Figure S11), and tubu- lin and KIF3A were more abundantly co-present in the TNKS immunoprecipitate (Supplementary information, Figure S12), from insulin-treated cells. Discussion In the present study, we have demonstrated that Axin, TNKS and KIF3A form a multimeric complex that mediates insulin–stimulated GLUT4 translocation and glucose uptake. Remarkably, both Axin and TNKS are directly linked to KIF3A, a critical component of KIF3 known to be necessary for GLUT4 translocation. We have also provided multiple lines of evidence to demon- strate that this complex is directly involved in glucose transport by promoting GLUT4 translocation, at least in part from the Golgi origin, to the surface membrane in response to insulin. First of all, although normally fed adult TNKS2−/− mice show little change in blood glu- cose levels, the normally fed young TNKS2−/− mice have significantly higher blood glucose levels compared to the wildtype littermates. Second, adult re-fed TNKS2−/− mice display a significant increase in blood glucose lev- els. GTT and ITT assays also show that the decline of blood glucose in TNKS2−/− mice was slower following injection of excessive glucose or insulin. When Axin, TNKS and KIF3A were knocked down individually by siRNA in 3T3-L1 adipocytes, insulin-induced GLUT4 translocation and glucose uptake were obviously im- paired. We knocked down TNKS and KIF3 together and found that the effect on glucose uptake was similar to that of KIF3A single knockdown, suggesting that TNKS and KIF3A work in conjunction to regulate insulin- stimulated glucose uptake. Third, immunofluorescence staining experiments revealed co-localization of the complex components with GLUT4 as well as with the Golgi apparatus in the perinuclear region. TNKS has been shown to interact with IRAP [20], further suggest-