The tankyrase (TRF1-interacting ankyrin-related ADP-ribose polymerase, TNKS) protein family consists of two members, tankyrase 1 (TNKS1) and tankyrase 2 (TNKS2), which share 85% identity in amino-acid se- quences [19]. Both proteins contain a poly-ADP-ribosyl polymerase (PARP) domain. TNKS1/2 have been shown to interact with insulin-responsive aminopeptidase (IRAP) [20], which is one of the integral membrane proteins in GLUT4 vesicles [21] and may play impor- tant roles in the maintenance and sorting of insulin- responsive GLUT4 vesicles [22, 23]. It was reported that TNKS1 enhanced insulin-stimulated exocytosis of GLUT4 [24]. TNKS1-deficient mice exhibit various metabolic changes including abnormality in glucose metabolism [25], and TNKS2−/− mice show growth re- tardation [26]. Unfortunately, double-knockout mice are embryonically lethal [19], which has hindered the analy- sis of the combined roles of both TNKS in glucose ho- meostasis. Possibility exists that TNKS1 and TNKS2 are functionally redundant to each other, which may explain the uncompromised overall glucose intake in TNKS1- deficient mice [25]. Evidence in support of a possible role of TNKS1 in controlling glucose homeostasis also includes that TNKS1 is a candidate gene for diabetes in humans, based on its location in the chromosomal locus of 8p23.1 that contains susceptibility genes for a subset of monogenic forms of type 2 diabetes [27]. The role of TNKS2 in glucose homeostasis is not clear. Axin was originally identified as a key inhibiting factor in the Wnt pathway by serving as a central scaf- fold for the assembly of the degradation complex of β-catenin [28]. We have identified additional pathways regulated by Axin that are stress-related, including the JNK cascade [29] and DNA damage response [30-32]. Moreover, Axin interacts with DCAP (Drosophila ho- molog of CAP), a component of the glucose transport regulatory complex, suggesting that Axin may play a role in insulin-mediated glucose transport [33]. Func- tional linkage between Axin and TNKS came from one study showing that the PARP activity of TNKS medi- ates PARsylation of Axin and subsequently enhances its ubiquitination and degradation [34]. Down-regulation of Axin by TNKS elevates the level of β-catenin, leading to the stimulation of Wnt signaling [34]. We have identified, by yeast two-hybrid screens, a series of signaling molecules that interact with Axin, in- cluding Daxx, HIPK2 and Pirh2 that are key regulators of the tumor suppressor p53 [30-32]. Among the other clones was the one that encodes tankyrase 2 (TNKS2). To elucidate the biological function of the interaction be- tween Axin and TNKS2, we carried out another round of yeast two-hybrid screen using TNKS2 as the bait. As a www.cell-research.com | Cell Research Hui-Ling Guo et al . npg 1247 result, we identified KIF3A, a kinesin motor protein, as a novel TNKS2-interacting protein. Here we report that Axin, TNKS and KIF3A form a ternary complex that is required for insulin-stimulated GLUT4 redistribution to the cell surface to increase glucose uptake. Results Axin forms a ternary complex with TNKS2 and KIF3A To search for molecular cues for the physiological function of the Axin/TNKS interaction, a yeast two- hybrid screen was carried out using the ankyrin domain (residues 648-937) of TNKS2 as the bait. Interestingly, 19 identified candidate clones encode KIF3A, an es- sential component of the motor protein kinesin-2. The C-terminal fragment of aa 494-CT is common to all the 19 overlapping KIF3A clones identified. To confirm this interaction, we performed immunoprecipitation (IP) experiments. As shown in Figure 1A, KIF3A was co- precipitated with TNKS by the antibody for endogenous TNKS. We then investigated whether Axin may also bind to KIF3A. Indeed, KIF3A appeared in the immuno- precipitate of Axin, indicating that Axin can also interact with KIF3A (Figure 1B). The TNKS/KIF3A and Axin/ KIF3A interactions were also supported by IP using over-expressed tagged proteins in 293T cells (Supple- mentary information, Figure S1A). Furthermore, we em- ployed in vitro pull-down assays to examine the direct interaction between TNKS2 and KIF3A, and between Axin and KIF3A. GST-KIF3A could be pulled down by His-tagged TNKS2 (Figure 1C) and by His-tagged Axin (Figure 1D). To address whether Axin, TNKS2 and KIF3A could co-exist in the same complex, we carried out a two-step co-IP, and found that they could form a ternary complex in the cell (Figure 1E). When the three proteins were co-expressed, Axin and KIF3A could be simultaneously detected in the precipitate of FLAG- tagged TNKS2 (Supplementary information, Figure S1B). Notably, over-expression of Axin could increase the amount of KIF3A co-precipitated with TNKS2 (Supplementary information, Figure S1B). This sug- gests that Axin could promote the association between TNKS2 and KIF3A. We then knocked down Axin by infecting adipocytes with adenovirus-siAxin, and carried out co-IP using the anti-TNKS antibody. As predicted, co-precipitated KIF3A was decreased (Supplementary information, Figure S1C). All these results suggest that, within the ternary complex, each component simultane- ously binds to the other two partners, with Axin directly interacting with both TNKS2 and KIF3A, TNKS2 with Axin and KIF3A, and KIF3A with Axin and TNKS2.