4D, lanes 3 and 4) or S509 (S509A) (Fig. 4D, lanes 5 and 6) was mutated to A, suggesting that the up-regulation effect of HBx on WT AIB1 is mainly the result of its inhibition of Fbw7α function. It is known that full-length AIB1 contains five functional domains: basic-helix-loop-helix domain; serine/threonine (S/T) domain; receptor interaction domain (RID);

CBP/P300 interaction domain; and histone acetyltransferase (HAT) domain (Fig. 5A). To identify which domains of AIB1 interact with Fbw7α or HBx, each of these five domains of AIB1 was coexpressed with Fbw7α or HBx in 293T cells, and Co-IP assays were performed. Western blotting analysis revealed that AIB1 interacted with HBx through its S/T and HAT domains (Fig. 5B) and interacted with Fbw7α through its S/T and RID domains (Fig. 5C). Because the S/T domain of AIB1 not only interacted with Fbw7α, selleck chemicals but also interacted with HBx,

it is possible that HBx inhibits the interaction between AIB1 and Fbw7α through the S/T domain. To test this hypothesis, Fbw7α was selleck kinase inhibitor coexpressed with full-length AIB1, S/T domain, or RID of AIB1 alone or in combination with HBx in 293T cells; then, Co-IP assays were performed. In the presence of HBx, the amount of Fbw7α protein coimmunoprecipitated with full-length AIB1, as well as the S/T domain of AIB1, was significantly reduced (Fig. 5D, lane 9 versus lane 8, and lane 11 versus lane 10), whereas the amount of Fbw7α protein coimmunoprecipitated with RID of AIB1 was comparable in the absence or presence of HBx, as expected (Fig. 5D, lane 13 versus lane 12), because RID could not interact with HBx. Taken together, these data indicate that HBx inhibits the Fbw7α-mediated ubiquitination and degradation of AIB1 by competitively inhibiting Suplatast tosilate the interaction between Fbw7α and the S/T domain of AIB1. It has been reported that AIB1 plays an important role in the transactivation process mediated by transcription factors, such as NF-κB and AP-1, which can also be activated by HBx.10, 12 Thus, we hypothesized that HBx and AIB1 can

cooperatively promote the transactivation activities of these transcription factors. To test this hypothesis, we cotransfected HBx and AIB1 with p65 or c-jun together with NF-κB reporter or AP-1 reporter into HepG2 cells, respectively. Compared to control transfection, the NF-κB reporter activities induced by HBx and AIB1 alone were 3- and 2-fold, respectively, whereas it was induced more than 5-fold by the coexpression of HBx and AIB1 (Fig. 6A). Similarly, the AP-1 reporter activities induced by HBx and AIB1 were less than 15- and 2-fold, respectively, whereas the coexpression of HBx and AIB1 dramatically increased AP-1 reporter activity more than 30-fold (Fig. 6B). These results suggest that HBx cooperates with AIB1 to promote the activities of NF-κB and AP-1.