Hope College, Biology and Chemistry, Holland, MI 49423
THE SUBCELLULAR LOCALIZATION OF VACM-1/CUL 5 AND NEDD8/RUB1, L. R. Dabney, M. Burnatowska-Hledin*, Hope College, Departments of Biology and Chemistry, Holland, MI 49423, hledin@hope.edu
The vasopressin-activated calcium-mobilizing protein (VACM-1), also known as cul-5, attenuates cellular growth by down-regulating the phosphorylation of mitogen-activated protein kinase, production of cyclic adenosine monophosphate, and concentration of the early growth response in the nucleus. Cellular localization of VACM-1 is cell cycle-dependent; during G1/0 phase it localizes to the membranes, it disappears during S phase, and during M phase it localizes to the nuclear region and cytosol. A novel protein, neural precursor cell expressed, developmentally down-regulated 8 (NEDD8), modifies cullins through a process similar to ubiquitination. The subcellular localization and biological activity of VACM-1 may depend on its neddylation status, but whether NEDD8 expression is cell cycle dependent or it colocalizes with VACM-1 was previously unclear. This study aimed to determine whether NEDD8 expression is cell cycle dependent and if, in cells transfected with VACM-1 cDNA, its expression colocalized with that of the VACM-1 protein. Double-immunofluorescent staining experiments were performed using antibodies directed against the VACM-1 and NEDD8 proteins. Our results indicate that cellular localization of NEDD8 is cell cycle-dependent; during the G1/0 phase NEDD8 localizes to the nuclear region, and during the S and M phases it localizes to the cytosol. Thus, only during the M phase did VACM-1 and NEDD8 immunostaining show colocalization. Interestingly, during the M phase, NEDD8 is highly localized to the microtubules and the centrosomes. These results reveal NEDD8 and VACM-1 colocalization only occurs during the M phase of the cell cycle, and neddylation may be occurring to another protein.
L. R. Dabney is supported by NSF-URC grant No. 0629174
[Abstract (DOC)]