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

¹ Institute of Medical Biochemistry, University of Oslo, PO Box 1112 Blindern, N-0317 Oslo, Norway
² Ruhr-Universität Bochum, Institut für Physiologische Chemie, Abt. Für Biochemie Supramolekularer Systeme, 44801 Bochum, Germany
³ Centre de Recherches de Biochimie Macromoléculaire, CNRS, BP 5051, 1919 Route de Mende, 34033 Montpellier Cedex 1, France
⁴ Institute for Nutrition Research, University of Oslo, PO Box 1046 Blindern, N-0317 Oslo, Norway
⁵ Institut Curie, Biologie du Cycle Cellulaire et de la Motilité, 75248 Paris Cedex 05, France.

*Author for correspondence (e-mail: cathrine.carlson{at}basalmed.uio.no)

Accepted June 10, 2001

Protein kinase A regulatory subunit RII 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-p34^cdc2 kinase (CDK1) has been shown to phosphorylate RII on T54 and this has been proposed to alter the subcellular localization of RII. We have made stable transfectants from an RII-deficient leukemia cell line (Reh) that expresses either wild-type or mutant RII (RII(T54E)). When expressed, RII 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 RII(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, RII 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 RII(T54E) interacts with AKAP450 in a RIPA-soluble extract from mitotic cells. Finally, microtubule repolymerization from mitotic centrosomes of the RII(T54E) transfectant is poorer and occurs at a lower frequency than that of RII transfectants. Our results suggest that T54 phosphorylation of RII by CDK1 might serve to regulate the centrosomal association of PKA during the cell cycle.

Key words: Centrosome, PKA, AKAP450, Mitosis, CDK1

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