Ncogene. It has lately been documented that TRAF6 exhibits E3 ligase activity, and it may possibly catalyze substrate ubiquitination. 25,26 In an effort to recognize the mechanism underlying TRAF6induced ma lignant progression in cancer cells, we sought to explore whether TRAF6 triggers cancer cell proliferation by affecting the ubiquiti nation of specific substrates. As a serinethreonine protein kinase, AKT plays a key function in multiple cancer processes. 27,28 Activated AKT could stimulate cancer cell proliferation and cell migration and influence cell cycle progression. Although the precise mechanism was unknown, it was reported that activation of AKT was normally ac companied by TRF6 overexpression in cancers.15,29 Hence, we speculated that TRAF6 may well contribute towards the malignant behavior of human cancers by way of affecting AKT ubiquitination. Our data showed that TRAF6 could effectively catalyze the JYL 1421 Technical Information ubiquitination of AKT in cancer cells. As the intact RING domain of TRAF6 in con junction together with the E2 Ubconjugating enzyme is necessary for its E3 ligaseactivity,anE3ligasedeficientTRAF6C70Amutantinwhich the extremely critical Cys residue in its RING domain was mutated to Ala (TRAF6 C70A), was applied in our study to exclude a possi ble indirect effect of TRAF6 on AKT ubiquitination. In contrast toSHI et al.TRAF6 wt, TRAF6 mut showed no influence on AKT ubiquitination, indicating that TRAF6 straight induced the ubiquitination of AKT. The role of AKT signaling in cancer development has been effectively documented. 30 Aberrant activation of AKT signaling has been extensively implicated in a lot of cancers. 27,28,30 Despite the fact that it truly is well-known that AKT activity is regulated through phosphoryla tion, some other types of posttranslational modifications, for example ubiquitination, SUMOylation, acetylation, and m6A mRNA methylation, have also been reported to market AKT activity and function. 31,32 Not too long ago, it was reported that AKT ubiquitina tion is correlated with its phosphorylation level, suggesting that ubiquitination represents a novel posttranslational modification that plays a important part in AKT activation. 33 Consistent with these reports, our information indicated that, along with ubiquitination, the ectopic expression of TRAF6 wt but not TRAF6 E3ligasedefi cient mut could also substantially facilitate AKT phosphorylation. Additionally, the reconstitution of TRAF6 wt, but not TRAF6 mut, directly contributes for the proliferation, migration, and marked G 0G1 to S phase transition in cancer cells. This result supports that AKT ubiquitination appears to be as equally essential as AKT phosphorylation and highlighted the vital role of TRAF6medi ated AKT ubiquitination and subsequent phosphorylation in the malignant progression of cancer cells. However, AKT ubiquitina tion is just not the only kind of posttranslational modification that could market AKT phosphorylationactivation. Further studies are necessary to detect whether or not TRAF6 impacts other kinds of posttranslational modifications of AKT. In summary, our findings indicate that TRAF6mediated AKT ubiquitination and phosphorylation play vital roles in the course of the malignant progression of tumors. Our study also gives evidence that TRAF6 may be a possible therapeutic target in cancer.AC K N OW L E D G M E N T S This function was supported by grants in the National Nature Science FoundationofChina(grantnos.81772871and81472518),National KeyR DProgramofChina(2017YFC0840110),andtheInnovation Fund for Doctoral System of Concurrent Inhibitors targets Shanghai J.
