P300/CBP-associated factor (PCAF)-mediated acetylation of Fascin at lysine 471 inhibits its actin-bundling activity and tumor metastasis in esophageal cancer.

BACKGROUND: Fascin is crucial for cancer cell filopodium formation and tumor metastasis, and is functionally regulated by post-translational modifications. However, whether and how Fascin is regulated by acetylation remains unclear. This study explored the regulation of Fascin acetylation and its corresponding roles in filopodium formation and tumor metastasis. METHODS: Immunoprecipitation and glutathione-S-transferase pull-down assays were performed to examine the interaction between Fascin and acetyltransferase P300/CBP-associated factor (PCAF), and immunofluorescence was used to investigate their colocalization. An in vitro acetylation assay was performed to identify Fascin acetylation sites by using mass spectrometry. A specific antibody against acetylated Fascin was generated and used to detect the PCAF-mediated Fascin acetylation in esophageal squamous cell carcinoma (ESCC) cells using Western blotting by overexpressing and knocking down PCAF expression. An in vitro cell migration assay was performed, and a xenograft model was established to study in vivo tumor metastasis. Live-cell imaging and fluorescence recovery after photobleaching were used to evaluate the function and dynamics of acetylated Fascin in filopodium formation. The clinical significance of acetylated Fascin and PCAF in ESCC was evaluated using immunohistochemistry. RESULTS: Fascin directly interacted and colocalized with PCAF in the cytoplasm and was acetylated at lysine 471 (K471) by PCAF. Using the specific anti-AcK471-Fascin antibody, Fascin was found to be acetylated in ESCC cells, and the acetylation level was consequently increased after PCAF overexpression and decreased after PCAF knockdown. Functionally, Fascin-K471 acetylation markedly suppressed in vitro ESCC cell migration and in vivo tumor metastasis, whereas Fascin-K471 deacetylation exhibited a potent oncogenic function. Moreover, Fascin-K471 acetylation reduced filopodial length and density, and lifespan of ESCC cells, while its deacetylation produced the opposite effect. In the filipodium shaft, K471-acetylated Fascin displayed rapid dynamic exchange, suggesting that it remained in its monomeric form owing to its weakened actin-bundling activity. Clinically, high levels of AcK471-Fascin in ESCC tissues were strongly associated with prolonged overall survival and disease-free survival of ESCC patients. CONCLUSIONS: Fascin interacts directly with PCAF and is acetylated at lysine 471 in ESCC cells. Fascin-K471 acetylation suppressed ESCC cell migration and tumor metastasis by reducing filopodium formation through the impairment of its actin-bundling activity.

Regulation of the oncoprotein KLF8 by a switch between acetylation and sumoylation.

KLF8 regulates target genes by recruiting the p300 and PCAF co-activators via glutamines (Q) 118 and 248, the CtBP co-repressor to 86PVDLS90 or SUMO to lysine (K) 67. Here we examined how these interactions coordinate to regulate KLF8 transactivity. Mass spectrometry and immunoprecipitations determined that p300 and/or PCAF promoted KLF8 acetylation at K67, K93, and K95 and this acetylation was abolished in lysine-to-arginine (R) mutants. Treatment with HDAC inhibitors or expression of co-activators inhibited sumoylation at K67. K93R or K95R mutation exerted high levels of sumoylation while the acetylation mimetic mutations K93Q and K95Q blocked the sumoylation. Interestingly, CtBP promoted sumoylation at K67 of wild-type but not AVALF mutant KLF8, and KLF8 interaction with CtBP was inhibited by treatment with the HDAC inhibitors, ectopic expression of the co-activators, or K93Q or K95Q mutation. Promoter reporter assays showed that CtBP inhibited KLF8 transactivity which was rescued by PCAF or p300 expresson. Finally, KLF8-mediated cyclin D1 protein expression and cell cycle progression were significantly decreased in the K93R and K95R but increased in the K93Q, K95Q, K67R or K67Q mutant. Taken together, these results identified a novel mechanism by which co-activators promote KLF8 transactivity by competing with SUMO for K67 modification and by acetylating K93 and K95 to inhibit CtBP-induced K67 sumoylation.