Phosphorylation of centrin during the cell cycle and its role in centrioleseparation preceding centrosome duplication

Once during each cell cycle, mitotic spindle poles arise by separation of n ewly duplicated centrosomes. Ne report here the involvement of phosphorylat ion of the centrosomal protein centrin in this process. We show that centri n is phosphorylated at serine residue 170 during the G(2)/M phase of the ce ll cycle. Indirect immunofluorescence staining of HeLa cells using a phosph ocentrin-specific antibody reveals intense labeling of mitotic spindle pole s during prophase and metaphase of the cell division cycle, with diminished staining of anaphase and no staining of telophase and interphase centrosom es. Cultured cells undergo a dramatic increase in centrin phosphorylation f ollowing the experimental elevation of PKA activity, suggesting that this k inase can phosphorylate centrin tn vivo. Surprisingly, elevated PKA activit y also resulted intense phosphocentrin antibody labeling of interphase cent rosomes and in the concurrent movement of individual centrioles apart from one another, Taken together, these results suggest that centrin phosphoryla tion signals the separation of centrosomes at prophase and implicates centr in phosphorylation in centriole separation that normally precedes centrosom e duplication.