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

Protein kinase A regulatory subunit RIIα is tightly bound to centrosomal structures during interphase through interaction with the A-kinase anchoring protein AKAP450, but dissociates and redistributes from centrosomes at mitosis. The cyclin B-p34cdc2 kinase (CDK1) has been shown to phosphorylate RIIα on T54 and this has been proposed to alter the subcellular localization of RIIα. We have made stable transfectants from an RIIα-deficient leukemia cell line (Reh) that expresses either wild-type or mutant RIIα (RIIα(T54E)). When expressed, RIIα detaches from centrosomes at mitosis and dissociates from its centrosomal location in purified nucleus-centrosome complexes by incubation with CDK1 in vitro. By contrast, centrosomal RIIα(T54E) is not redistributed at mitosis, remains mostly associated with centrosomes during all phases of the cell cycle and cannot be solubilized by CDK1 in vitro. Furthermore, RIIα is solubilized 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 not change affinity detected by surface plasmon resonance. In addition, only RIIα(T54E) interacts with AKAP450 in a RIPA-soluble extract from mitotic cells. Finally, microtubule repolymerization from mitotic centrosomes of the RIIα(T54E) transfectant is poorer and occurs at a lower frequency than that of RIIα transfectants. Our results suggest that T54 phosphorylation of RIIα by CDK1 might serve to regulate the centrosomal association of PKA during the cell cycle.