Mitosis-specific Phosphorylation and Subcellular Redistribution of the RIIα Regulatory Subunit of cAMP-dependent Protein Kinase

doi:10.1074/jbc.273.51.34594

Mitosis-specific Phosphorylation and Subcellular Redistribution of the RIIα Regulatory Subunit of cAMP-dependent Protein Kinase*

From ^‡INSERM Unité 427, UniversitéRené Descartes, Faculté des Sciences Pharmaceutiques et Biologiques de Paris, F-75270 Paris Cedex 06, France, ^**Institute of Medical Biochemistry, University of Oslo, N-0317 Oslo, Norway,^‖Medizinische Universität Klinik, Institut für Klinische Biochemie und Pathobiochemie, Josef-Schneider Straße 2, D-97080 Würzburg, Germany, and ^¶Centre de Recherches de Biochimie Macromoléculaire, CNRS, BP 5051, 1919 Route de mende, 34033 Montpellier cedex 1, France

Abstract

Phosphorylation of the RII regulatory subunits of cyclic AMP-dependent protein kinases (PKAs) was examined during the HeLa cell cycle. Three RIIα isoforms of 51, 54, and 57 kDa were identified by RIIα immunodetection and labeling with 8-azido[³²P]cAMP in different cell cycle phases. These isoforms were characterized as different phosphorylation states by the use of selective PKA and cyclin-directed kinase inhibitors. Whereas RIIα autophosphorylation by PKA caused RIIα to shift from 51 to 54 kDa, phosphorylation of RIIα by one other or a combination of several kinases activated during mitosis caused RIIα to shift from 51 to 57 kDa. In vivo incorporation of [³²P]orthophosphate into mitotic cells and RIIα immunoprecipitation demonstrated that RIIα was hyperphosphorylated on a different site than the one phosphorylated by PKA. Deletion and mutation analysis demonstrated that the cyclin B-p34^cdc2 kinase (CDK1) phosphorylated human recombinant RIIα in vitro on Thr⁵⁴. Whereas RIIα was associated with the Golgi-centrosomal region during interphase, it was dissociated from its centrosomal localization at metaphase-anaphase transition. Furthermore, particulate RIIα from HeLa cell extracts was solubilized following incubation with CDK1 in vitro. Our results suggest that at the onset of mitosis, CDK1 phosphorylates RIIα, and this may alter its subcellular localization.

Footnotes

↵* This work was supported by Association Française pour la Recherche sur le cancer Grant ARC 2071 (to G. K. and M. Y.) and by INSERM, the Norwegian Research Council (NFR), the Norwegian Cancer Society, the Novo Nordisk Research Foundation, and the Anders Jahre’s Foundation (to K. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ To whom correspondence should be addressed: INSERM Unité 427, Université René Descartes, Faculté des Sciences Pharmaceutiques et Biologiques de Paris, 4 Avenue de l’Observatoire, F-75270 Paris Cedex 06, France. Tel.: 33 1 44 07 39 91; Fax: 33 1 44 07 39 92; E-mail: keryer{at}pharmacie.univ-paris5.fr.
↵2 K. Taskén, R. Kopperud, A. E. Christensen, L. H. Dybdahl, B. Fauske, B. T. Gjertsen, R. Solberg, V. Hansson, T. Jahnsen, and S. O. Døskeland, manuscript in preparation.
Abbreviations:

PKA

cyclic AMP-dependent protein kinases

CFK1

cyclin B-p34^cdc2 kinase

PAGE

polyacrylamide gel electrophoresis

Ab

antibody

PBS

phosphate-buffered saline

GST

glutathioneS-transferase

Pipes

1,4-piperazinediethanesulfonic acid.
- Received August 12, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.