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 (PKA and PKC) and a Nedd8 protein modification site. Mutations at the PKA-specific 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 PKA-dependent phosphorylation of VACM-1/ Cul5 controls its neddylation status, phosphorylation by PKC, 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 PKAspecific phosphorylation site and probed with anti-VACM-1-specific antibody showed that PKA-dependent phosphorylation of VACM-1 protein was decreased in cells transfected with S730A VACM-1 cDNA when compared with the cytomegalovirustransfected cells. This change was associated with increased modification of VACM-1 protein by Nedd8. Induction of PKA 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 PKC 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.VACM-1 (vasopressin-activated calcium-mobilizing) protein (1), now identified as a cul5 gene product (2-4), is a 780-amino acid protein with a calculated M r of 91 kDa. Transfection of various cell lines with VACM-1/cul5 cDNA attenuates cellular growth by a mechanism that involves inhibition of cAMP production, decreased phosphorylation of MAPK, 3 and a decrease in nuclear localization of early growth response gene (egr-1) product (5-7). In vivo, VACM-1/Cul5 protein expression is specific to established endothelial cells (8) but is absent in sprouting capillaries (9), suggesting its involvement in the regulation of endothelium-specific growth. Interestingly, the human homolog of VACM-1 differs only in six amino acids from the rabbit VACM-1 and has been proposed to be a candidate for a tumor suppressor (2). Although expression of VACM-1/cul5 cDNA in a cancer-derived cell line, T47D, decreased nuclear concentration of estrogen receptor, ER␣, and inhibited cellular growth (6), the precise mechanism by which VACM-1/Cul5 may regulate cell growth is not known. Like other cullins, however, VACM-1/Cul5 may serve as scaffold protein that allows the assembly of E3 ubiquitin ligase comp...

Expression of VACM‐1/cul‐5 gene in endothelial and in cancer cell lines in vitro inhibits cellular proliferation and decreases phosphorylation of mitogen‐activated protein kinase (MAPK). 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 in VACM‐1 (S730AVACM‐1) sequence resulted in increased cellular growth of rat endothelial cells, RAMEC. The aim of our study was to examine if PKC activity also regulates VACM‐1 dependent cell growth. To induce PKC activity, cells were treated with Phorbol 12‐myristate 13‐acetate (PMA) for 16 hours and growth was monitored. PKC specific phosphorylation of VACM‐1 was examined using anti‐PKC substrate specific antibody. Our results show that cells transfected with S730AVACM‐1 cDNA and treated with PMA for 16 hours grew significantly faster than control cells treated with PMA. Further, Western blot analysis of cell lysates from both groups probed with anti‐PKC substrate specific antibody revealed a higher level of VACM‐1 phosphorylation in S730AVACM‐1 cDNA transfected cells when compared to controls. These results suggest that the antiproliferative effect of VACM‐1/cul5 is dependent on its posttranslational phosphorylation by PKA and PKC.

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.