Phosphorylation of p97(VCP) and p47 in vitro by p34(cdc2) kinase

The hexameric ATPase p97/yeast Cdc48p has been implicated in a number of ce llular events that are regulated during mitosis, including homotypic membra ne fusion, spindle pole body function, and ubiquitin-dependent protein degr adation. p97/Cdc48p contains two conserved consensus p34(cdc2) kinase phosp horylation sites within its second ATP binding domain. This domain is likel y to play a role in stabilising the hexameric form of the protein. We there fore investigated whether p97 could be phosphorylated by p34(cdc2) kinase i n vitro, and whether phosphorylation might influence the oligomeric status of p97, Monomeric, but not hexameric, p97 was phosphorylated by p34(cdc2) k inase, as was the p97-associated protein p47, However, phosphorylation by p 34(cdc2) kinase did not impair subsequent re-hexamerisation of p97, implyin g that the phosphorylated residue(s) are not critical for interaction betwe en p97 monomers. Moreover, p97 within both interphase and mitotic cytosols was almost exclusively hexameric, suggesting that the activity of p97 is no t regulated during mitosis by influencing the extent of oligomerisation.