Mitosis-specific phosphorylation and subcellular redistribution of the RIIα 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 RII alpha isoforms of 51, 54, and 57 kDa were identified by RII alpha immunodetection and labeling with 8-azido[P-32]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 RII alpha autophosphorylation by PKA caused RII alpha to shift from 51 to 54 kDa, phosphorylation of RII alpha by one other or a combination of several kinases activated during ;mitosis caused RII alpha to shift from 51 to 57 kDa. In vivo incorporation of [P-32]orthophosphate into mitotic cells and RII alpha immunoprecipitation demonstrated that RII alpha was hyperphosphorylated on a different site than the one phosphorylated by PRA, Deletion and mutation analysis demonstrated that the cyclin B-p34(cdc2) kinase (CDK1) phosphorylated human recombinant RII alpha in vitro on Thr(54). Whereas RII alpha was associated with the Golgi-centrosomal region during interphase, it was dissociated from its centrosomal localization at metaphase-anaphase transition. Furthermore, particulate RII alpha from HeLa cell extracts was solubilized following incubation with CDK1 in vitro, Our results suggest that at the onset of mitosis, CDK1 phosphorylates RII alpha, and this may alter its subcellular localization.