Phosphorylation of VACM-1/Cul5 by protein kinase A regulates its neddylation and antiproliferative effect.

Expression of the VACM-1/cul5 gene in endothelial and in cancer cell lines in vitro inhibits cellular proliferation and decreases phosphorylation of MAPK. Structure-function analysis of the VACM-1 protein sequence identified consensus sites specific for phosphorylation by protein kinases A and C and and a Nedd8 protein modification site. Mutations at the site in VACM-1/Cul5 ((S730A)VACM-1) sequence resulted in increased cellular growth and the appearance of a Nedd8-modified VACM-1/Cul5. The aim of this study was to examine if phosphorylation of VACM-1/Cul5 controls its neddylation status, phosphorylation by and ultimately growth. Our results indicate that in vitro transfection of rat adrenal medullary endothelial cells with anti-VACM-1-specific small interfering RNA oligonucleotides decreases endogenous VACM-1 protein concentration and increases cell growth. Western blot analysis of cell lysates immunoprecipitated with an antibody directed against a duanyu1529-specific phosphorylation site and probed with anti-VACM-1-specific antibody showed that duanyu1529-dependent phosphorylation of VACM-1 protein was decreased in cells transfected with (S730A)VACM-1 cDNA when compared with the cytomegalovirus-transfected cells. This change was associated with increased modification of VACM-1 protein by Nedd8. Induction of activity with forskolin reduced modification of VACM-1 protein by Nedd8. Finally, rat adrenal medullary endothelial cells transfected with (S730A)VACM-1/cul5 cDNA and treated with phorbol 12-myristate 13-acetate (10 and 100 nm) to induce activity grew significantly faster than the control cells. These results suggest that the antiproliferative effect of VACM-1/Cul5 is dependent on its posttranslational modifications and will help in the design of new anticancer therapeutics that target the Nedd8 pathway.

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