Our studies showed that Xic2 did not exhibit the phosphoforms 2 modification in

5 and 3 hour time points, the Xic2 F123A point mutant was significantly reduced for ubiquitination compared to wildtype Xic2, suggesting that PCNA binding to Xic2 plays an important role in Xic2 ubiquitination. We also noted a moderate effect on Xic2 turnover, To explore buy AP24534 the role of PCNA in Xic2 turnover further, we depleted PCNA from the Xenopus extract and stud ied Xic2 turnover, The results showed that in the absence of PCNA, Xic2 ubiquitination and turnover were inhibited, again suggesting that PCNA plays an important role in Xic2 proteolysis, The addition of Cdt2, but not Skp2, promotes the turnover of Xic2 Past studies have indicated that many substrates of the ubiquitin pathway that require PCNA and PCNA bind ing for their proteolysis are frequently targeted for ubiquitination by the CRL4Cdt2 ubiquitin ligase, CRL4Cdt2 has been shown to ubiquitinate several sub strates in a PCNA dependent manner including Xic1, p21, and Cdt1, Because our studies suggest that the ubiquitination of Xic2 is dependent upon PCNA, we examined whether Xic2 could associate with Cdt2, the substrate binding component of CRL4.

Surprisingly, using a GST pull down assay, our studies indicated that Xic2 did not readily bind to in vitro translated Cdt2 compared to Xic1 and p21, To further ex plore a possible role for CRL4Cdt2 in Xic2 turnover, we supplemented the extract AT7519 844442-38-2 with Cdt2 to determine whether this could promote the turnover of Xic2.

Our previous studies have shown that for Xic1 turnover, Cdt2 is limiting in the egg extract, Our studies showed that the addition of unlabeled in vitro translated Cdt2 promoted the degradation of Xic2 compared to the addition of unprogrammed FDA approved Akt 阻害剤 reticulocyte lysate, while the addition of in vitro translated Skp2 did not, Moreover, the concurrent addition of both the CDK inhibitor p27 to prevent Xic2 phosphorylation and Cdt2, significantly promoted the turnover of Xic2, beyond the promotion observed by adding either component indi vidually, These studies suggest that Cdt2 plays a role in Xic2 ubiquitination and that Xic2 may only asso ciate tightly with Cdt2 in the context of replicating DNA as has been proposed for Xenopus Cdt1, Xic2 phosphorylation by CDK2 inhibits its proteolysis during interphase Upon examination of Xic2 in the presence of XSC in the Xenopus LSS, it is apparent that Xic2 is simultaneously targeted for phosphorylation and proteolysis.

However, the proteolysis of Xic2 in the interphase extract is not highly efficient, especially when compared to the prote olysis of Xic1 under the same conditions, We hypothesized that perhaps the phosphorylation of Xic2 may be negatively regulating the turnover of Xic2. A well characterized active kinase in the Xenopus inter phase extract is CDK2 cyclin E and to explore whether CDK2 activity may inhibit Xic2 turnover, we ex amined the effect of CDK2 inhibitors on Xic2 stability.