CDK1-mediated phosphorylation of the RII alpha regulatory subunit of PKA works as a molecular switch that promotes dissociation of RII alpha from centrosomes at mitosis

Protein kinase A regulatory subunit RII alpha is tightly bound to centrosom al structures during interphase through interaction with the A-kinase ancho ring protein AKAP450, but dissociates and redistributes from centrosomes at mitosis. The cyclin B-p34(cdc2) kinase (CDK1) has been shown to phosphoryl ate RII alpha on T54 and this has been proposed to alter the subcellular lo calization of RII alpha. We have made stable transfectants from an RII alph a -deficient leukemia cell line (Reh) that expresses either wild-type or mu tant RII alpha (RII alpha (T54E)). When expressed, RII alpha detaches from centrosomes at mitosis and dissociates from its centrosomal location in pur ified nucleus-centrosome complexes by incubation with CDK1 in vitro. By con trast, centrosomal RII alpha (T54E) is not redistributed at mitosis, remain s mostly associated with centrosomes during all phases of the cell cycle an d cannot be solubilized by CDK1 in vitro. Furthermore, RII alpha is solubil ized from particular cell fractions and changes affinity for AKAP450 in the presence of CDK1. D and V mutations of T54 also reduce affinity for the N- terminal RII-binding domain of AKAP450, whereas small neutral residues do n ot change affinity detected by surface plasmon resonance. In addition, only RII alpha (T54E) interacts with AKAP450 in a RIPA-soluble extract from mit otic cells. Finally, microtubule repolymerization from mitotic centrosomes of the RIIa(T54E) transfectant is poorer and occurs at a lower frequency th an that of RII alpha transfectants. Our results suggest that T54 phosphoryl ation of RII alpha by CDK1 might serve to regulate the centrosomal associat ion of PKA during the cell cycle.