However, combined observations of the high molecular mass complex, association of the full length protein in transfected cells and the ability of the CTD to form a tetramer in vitro, suggest that NEDD1 may function as an oligomer in vivo. This study also confined the Pazufloxacin mesylate region responsible for the NEDD1/ c-tubulin interaction to residues 599�C660 of NEDD1. Indeed, this region of NEDD1 does not localize to the centrosome, but since it binds to c-tubulin, is able to prevent c-tubulin from localizing to the centrosome. In a recent study which Mangafodipir trisodium showed that phosphorylation of NEDD1 by Cdk1 and Plk1 is important for its interaction with c-tubulin, four Plk1 phosphorylation sites were identified. Mutation of all of these sites in combination reduced the binding of NEDD1 and c-tubulin, although it was not abolished. Since our data show that recombinant unphosphorylated NEDD1 CTD can bind c-tubulin, this suggests that Plk1-mediated phosphorylation is not essential but may be required for either formation of a tight complex, or for interaction of full-length NEDD1 with c-tubulin. Initial mutations in NEDD1 suggested that residues that are not strongly predicted to adopt ana-helical conformation might provide a binding surface for c-tubulin. Indeed, multiple mutations within residues 636�C642 of NEDD1 caused a significant reduction in binding to c-tubulin both in vivo and in vitro. These mutants are still helical and tetrameric in solution, suggesting that the mutated residues are important for direct binding of c-tubulin and do not abrogate binding by disrupting the structure of the CTD. The mutation of residues outside this region had minimal effect on c-tubulin binding. However, whilst specific residues within the CTD create a docking site for ctubulin, the entire region is required for the interaction, presumably by contributing to the conformation of the protein. In order to test the functional importance of these mutants, the localization of c-tubulin to the centrosome was assessed in transfected cells.Due to the presence of endogenous NEDD1 interfering with localization studies, the NEDD1 CTD construct was used for these studies, because it displays a dominant-negative effect in keeping c-tubulin away from the centrosome, overriding any endogenous NEDD1.