VACM‐1/cul5 Function Is Regulated by Its Posttranslational Modification Status

Abstract

VACM‐1 is a cul‐5 gene product, and when expressed in endothelial and in cancer cells in vitro, it inhibits cellular proliferation and MAPK activated signaling. Structure‐function analysis of VACM‐1 protein sequence identified consensus sites specific for phosphorylation by protein kinases PKA and PKC. Mutations at the PKA specific site resulted in increased cellular growth and an appearance of a larger Mr protein on Western blot, suggesting posttranslational modification of VACM‐1/cul5. The aim of our study was to examine if modification of VACM‐1/cul5 by PKC dependent phosphorylation is under the control of PKA dependent phosphorylation and if these changes can affect the biological activity of VACM‐1 protein. Our results indicate that when PKA phosphorylation site is mutated, VACM‐1/cul5 is phosphorylated by PKC. PMA, a drug which increases PKC activity, induced growth in the endothelial cells transfected with mutated VACM‐1/cul5 when compared to the wt VACM‐1 transfected cells. Similarly, Gö6983, an inhibitor of PKC, significantly decreased cell growth in cells transfected with mutated VACM‐1 when compared to the wt VACM‐1 cDNA transfected cells. These results suggest that biological activity of VACM‐1 is dependent on its phosphorylation by PKA and PKC. This work was supported by a grant from NCI (R15CA104014) and by Dept of Chemistry.