36]. Since the Axin/TNKS/KIF3A complex is regulated in an AKT-dependent manner, the partial effect of the TNKS2 knockout on glucose disposal is therefore not unexpected. In particular, we have provided evidence supporting a functional overlap between TNKS1 and TNKS2, as shown in glucose uptake assays. Based on the data from our current study and from others, it is clear that in the absence of insulin, TNKS proteins poly-ADP-ribosylate themselves and Axin, leading to their degradation. Insulin signaling sup- presses the PARsylase activity of TNKS in an Akt- dependent manner to stabilize the Axin/TNKS/KIF3A complex. By virtue of its simultaneous interaction with both TNKS and KIF3A, Axin serves as a scaffold to facilitate the formation of the ternary complex in the presence of insulin, which may promote the complex to associate with GLUT4 vesicles to enable their ultimate translocation to the PM (Figure 7C). The observation that treatment with the TNKS PARsylase inhibitor XAV939 could increase the complex formation, GLUT4 translocation, and insulin-stimulated glucose uptake fur- ther underscores the importance of PARsylase activity in the regulation of glucose uptake. Together, we have uncovered a functional linkage between the Axin/TNKS complex and the kinesin motor for GLUT4 transloca- tion in response to insulin, in which the PARsylase ac- tivity plays a negative role in controlling the stability of the translocation complex. However, it remains unclear how insulin-activated Akt signals to the Axin complex to trigger GLUT4 translocation to the cell surface. It has been shown that Akt can phosphorylate AS160, leading to derepression of the small G protein Rabs for GLUT4 trafficking [37-42]. The functional interrelationship be- tween the Axin/TNKS/KIF3A complex and Akt-mediat- ed regulation of AS160 awaits further investigation. Materials and Methods Plasmid constructions CMV2-FLAG-TNKS2 (human) was kindly provided by Dr Chi NW. It was tagged with three tandem HA epitopes fused to the N-terminus and cloned to pCMV5 vector. Full-length cDNA encoding mouse KIF3A was obtained from an EST clone (IMAGE No. 601205534, Open Biosystem). RNA interference and adenovirus infection Adenovirus vector pAdEasy (GFP) was used for expression of siRNA. mTNKS1: 5′-GAGATGCAGAGCACTATTC-3′; mT- NKS2: 5′-GGAAGGATGTAGTTGAAT A-3′; mAxin1: 5′-GGA- GAGAAGGAGATCAGTA-3′; mKIF3A: 5′-AGCTAAAATGT- GTGG CCTA-3′. 3T3-L1 cells were induced to differentiate into adipocytes and on day 6 were infected with individual adenovi- rus. After 48 h of infection, adipocytes were subjected into ex- periments. www.cell-research.com | Cell Research Hui-Ling Guo et al . npg 1255 Cell culture 293T and 3T3-L1 cells were maintained in Dulbecco’s modi- fied Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 IU penicillin and 100 mg/ ml streptomycin at 37 °C in a humidified incubator containing 5% CO2. Differentiation of 3T3-L1 cells was initiated at day 2 post- confluence by addition of 5 µg/ml of insulin, 1 µM dexamethasone (DEX), and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) for 2 d, followed by culturing in DMEM supplemented with 10% FBS and 5 µg/ml insulin for additional 2 d. The differentiated cells were then maintained in normal culture medium. In vivo PARsylation cells were serum starved in low glucose DMEM (1 g/l glucose, Hyclone) for 4 h, and then treated with 20 µM MG132 accom- panied with or without 200 nM insulin for 30 min. Cells were harvested with RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 µM leupeptin, 1 µM PMSF) supplemented with 1% SDS. Cleared cell lysates were diluted with RIPA buffer to reduce the SDS concen- tration to 0.2% and subjected to IP using the antibodies indicated and protein A/G beads overnight, followed by western blotting. Yeast two-hybrid screen Yeast two-hybrid screen of a mouse brain cDNA library was carried out using MATCHMAKER GAL4 Two-Hybrid System 3 (Clontech) according to the manufacturer’s instructions as de- scribed previously [31]. The sequence of aa 648-937 encompass- ing the ankyrin-repeat and SAM domain of human tankyrase 2 (TNKS2) was used as the bait. Antibodies and drugs Rabbit polyclonal anti-Axin antibodies C2b and C8b were raised as described previously [30]. Commercial antibodies used in this study include mouse anti-HA (F-7), anti-Myc (9E10), goat anti-Axin (S-20), rabbit anti-tankyrase1/2 (H350), goat anti- GLUT4 (C20), rabbit anti-TGN38 (M-290) and mouse anti-ubiq- uitin (P4D1) (Santa Cruz Biotechnology), rabbit anti-GLUT4 anti- body (ab654, Abcam), mouse anti-FLAG antibody (M2 antibody, Sigma), mouse anti-KIF3A (BD Transduction Laboratories), and rabbit anti-KIF3A antibody (Abcam). Bovine insulin,IBMX, and DEX were purchased from Sigma, and Akt inhibitors VIII from Calbiochem. Immunofluorescence staining 3T3-L1 cells were differentiated to adipocytes and plated on 22-mm2 coverslips in six-well plates. After serum starvation for 4 h, adipocytes were treated with or without 200 nM insulin for 30 min, followed by fixation as previously described [32]. Cells were then incubated with indicated primary antibodies in blocking solu- tion overnight at 4 ºC. Next, cells were washed four times with PBS containing 0.02% Triton X-100, 1.5% BSA and 1 mM NaN3, incubated with 4 µg/ml of bovine anti-goat Texas red, donkey anti-mouse Alexa Fluor 488, goat anti-rabbit Alexa Fluor 647 in blocking solution for 2 h. Cells were then washed and stained with DAPI (1 µg/ml) in PBS for 5 min. Glucose uptake assay 3T3-L1 cells grown in 12-well plates and were induced to