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 prot ein kinases (PKAs) was examined during the HeLa cell cycle. Three RII alpha isoforms of 51, 54, and 57 kDa were identified by RII alpha immunodetectio n 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, pho sphorylation 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 v ivo 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) phosphoryla ted human recombinant RII alpha in vitro on Thr(54). Whereas RII alpha was associated with the Golgi-centrosomal region during interphase, it was diss ociated 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 o nset of mitosis, CDK1 phosphorylates RII alpha, and this may alter its subc ellular localization.