Cell cycle-dependent phosphorylation of centrosomes: localization of phosphopeptide specific antibodies to the...

The microtubule nucleation capacity of the centrosome increases dramatically as cells progress from interphase into mitosis. The increase in nucleation capacity of the centrosome correlates with the cell cycle-dependent localization of the mitotic protein monoclonal-2 (MPM-2) phosphoepitope-specific antibody to the mitotic centrosome. Therefore, the phosphorylation state of centrosomal components may regulate the microtubule nucleation capacity of this organelle during mitosis. Neither the identity of the MPM-2 kinase(s) nor all of the MPM-2-reactive phosphoproteins associated with the centrosome have been fully elucidated. Only recently have the characteristics of the MPM-2 epitope site been defined, and we used this information to prepare polyclonal antibodies against synthetic phosphopeptides containing potential MPM-2 epitopes derived from the sequences of two MPM-2-reactive proteins, topoisomerase II, and microtubule associated protein 1B (MAP1B). We demonstrate that these phosphopeptide-specific antibodies also localize to the centrosome in a cell cycle-dependent fashion. Thus, polyclonal antibodies have been generated against defined phosphopeptides that reiterate many of the immunofluorescence staining properties exhibited by the MPM-2 antibody. These new phosphopeptide-specific antibodies will provide additional probes to examine the phosphorylation of centrosomal components and the functional consequences of their phosphorylation during mitosis.