Mitosis-specific phosphorylation and subcellular redistribution of the RIIalpha regulatory subunit of cAMP-dependent protein kinase.
Phosphorylation of the RII regulatory subunits of cyclic AMP-dependent protein kinases (PKAs) was examined during the HeLa cell cycle. Three RIIalpha isoforms of 51, 54, and 57 kDa were identified by RIIalpha 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 RIIalpha autophosphorylation by PKA caused RIIalpha to shift from 51 to 54 kDa, phosphorylation of RIIalpha by one other or a combination of several kinases activated during mitosis caused RIIalpha to shift from 51 to 57 kDa. In vivo incorporation of [32P]orthophosphate into mitotic cells and RIIalpha immunoprecipitation demonstrated that RIIalpha 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 RIIalpha in vitro on Thr54. Whereas RIIalpha was associated with the Golgi-centrosomal region during interphase, it was dissociated from its centrosomal localization at metaphase-anaphase transition. Furthermore, particulate RIIalpha from HeLa cell extracts was solubilized following incubation with CDK1 in vitro. Our results suggest that at the onset of mitosis, CDK1 phosphorylates RIIalpha, and this may alter its subcellular localization.
Keryer
G
G
INSERM Unité 427, Université René Descartes, Faculté des Sciences Pharmaceutiques et Biologiques de Paris, F-75270 Paris Cedex 06, France. keryer@pharmacie.univ-paris5.fr
Yassenko
M
M
Labbé
J C
JC
Castro
A
A
Lohmann
S M
SM
Evain-Brion
D
D
Taskén
K
K
D000345:Affinity Labels / Q000493:pharmacokinetics; D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal; D001386:Azides / Q000493:pharmacokinetics; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology*; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D008938:Mitosis; D016297:Mutagenesis, Site-Directed; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology; D014162:Transfection
D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D001386:Azides; D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; C017042:8-azidoadenosine-3',5'-monophosphate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D018844:Cyclin-Dependent Kinases