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...

Vasopressin-activated calcium-mobilizing (VACM-1) protein is a cul-5 gene product that forms complexes with a subclass of ubiquitin E3 ligases involved in proteasomal protein degradation. The expression of VACM-1 cDNA in the T47D breast cancer cell line inhibits growth and decreases phosphorylation of mitogen activated protein kinase. Factors that regulate expression or stability of VACM-1 protein have not been identified, however. In our search to identify drugs/substances that may control VACM-1 protein expression, we examined the effects of resveratrol (trans-3,5,4'-trihydroxystilbene), a natural component in the human diet which inhibits tumor initiation and promotion. CMV vector and VACM-1 cDNA stably transfected T47D breast cancer-derived cells were treated with resveratrol and cell growth and VACM-1 protein concentrations were measured. Since the cellular mechanism of resveratrol-dependent inhibition of cell growth also involves the regulation of estrogen receptors, the effect of 17-β-estradiol and resveratrol on ERα levels and on cell growth was examined in control and in VACM-1 cDNA transfected cells. Our results demonstrate that antiproliferative effect of resveratrol observed in the control T47D cancer cells was significantly enhanced in VACM-1 cDNA transfected T47D cells. Western blot results indicated that resveratrol increased VACM-1 protein concentration. Finally, treatment with resveratrol for 24 and 48 h attenuated 17-β-estradiol induced increase in cell growth both in control and in VACM-1 cDNA transfected cells. The effect was significantly higher in the VACM-1 cDNA transfected cells when compared to controls. These results indicate that the antiproliferative effect of resveratrol may involve induction of VACM-1/cul5.

Angiogenesis is essential for cancer metastasis, thus the discovery and characterization of molecules that inhibit this process is important. Thalidomide is a teratogenic drug which is known to inhibit angiogenesis and effectively inhibit cancer metastasis, yet the specific cellular targets for its effect are not well known. We discovered that CUL5 (previously identified as VACM-1), a scaffold protein in E3 ligase complexes, is involved in thalidomide-dependent inhibition of endothelial cell growth. Our results show that in human endothelial cells (HUVEC), thalidomide-dependent decrease in cell growth was associated with decreased nuclear localization of CUL5. In HUVEC transfected with anti-VACM-1 siRNA, thalidomide failed to decrease cell growth. Previously it was established that the antiproliferative effect of CUL5 is inhibited in rat endothelial cells (RAMEC) transfected with mutated CUL5 which is constitutively modified by NEDD8, a ubiquitin-like protein. In this study, the antiproliferative response to thalidomide was compromised in RAMEC expressing mutated CUL5. These results suggest that CUL5 protein is involved in the thalidomide-dependent regulation of cellular proliferation in vitro. Consequently, CUL5 may be an important part of the mechanism for thalidomide-dependent inhibition of cellular proliferation, as well as a novel biomarker for predicting a response to thalidomide for the treatment of disorders such as multiple myeloma and HIV infection.

Vasopressin‐activated calcium‐mobilizing (VACM‐1) receptor, a cul‐5 gene product, has been shown to inhibit cellular growth in the T47D breast cancer cell line through a mechanism that involves MAPK phosphorylation and regulation of estrogen receptor (ERα) concentration. As a cul5 gene product, VACM‐1 protein is involved in ubiquitin ligase dependent protein degradation, but factors that regulate its expression and degradation have not been identified. In our search to identify factors/drugs that control VACM‐1 expression, we studied the effects of resveratrol (3,5,4′‐trihydroxy‐trans‐stilbene) which inhibits cellular functions associated with tumor initiation, promotion, and progression by a mechanism that involves inhibition of MAPK phosphorylation, ERα activation and cell cycle arrest. In our study, control and VACM‐1 cDNA transfected T47D breast cancer cells were treated with reservatol and cell growth and VACM‐1 levels were measured. Our data demonstrate that the dose and time dependent effect of resveratol on cellular proliferation is significantly enhanced in cells transfected with VACM‐1 cDNA. Further, our results indicate that expression of VACM‐1 protein is induced by resveratrol. Consequently, these results indicate that the antiproliferative effect of VACM‐1 can be further enhanced by resveratol. This work was supported by a grant from NCI (R15CA104014) and by Dept of Biology.