RESUMO
Two alpha-type CK2-activated PKAs (CK2-aPKAIalpha and CK2-aPKAIIalpha) were biochemically characterized in vitro using GST-HBV core fusion protein (GST-Hcore) and GST-Hcore157B as phosphate acceptors. It was found that (i), in the absence of cAMP, these two CK2-aPKAs phosphorylated both Ser-170 and Ser-178 on GST-Hcore and Hcore157B; (ii) this phosphorylation was approx. 4-fold higher than their phosphorylation by cAMP-activated PKAs; and (iii) suramin effectively inhibited the phosphorylation of Hcore157B by CK2-aPKAIIalpha through its direct binding to Hcore157B in vitro. These results suggest that high phosphorylation of HBV-CP by two CK2-aPKAs, in the absence of cAMP, may be involved in the pregenomic RNA (pgRNA) encapsidation and DNA-replication in HBV-infected cells.
Assuntos
Caseína Quinase II/metabolismo , Vírus da Hepatite B/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas do Core Viral/metabolismo , Animais , Antineoplásicos/farmacologia , Bovinos , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico , Ativação Enzimática , Genoma Viral/fisiologia , Hepatite B/enzimologia , Humanos , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Viral/metabolismo , Serina/metabolismo , Suramina/farmacologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologiaRESUMO
The physiological significance of the casein kinase 2 (CK2)-mediated phosphorylation of type II cAMP-dependent protein kinase (PKAIIalpha) and free type II regulatory (R) subunit (RIIalpha) on their activities was mainly investigated in vitro. In these experiments, [gamma-32P]GTP was used as a phosphate donor for the CK2-mediated phosphorylation of free RIIalpha and PKAIIalpha (bovine heart) in vitro. It was found that: (i). CK2 phosphorylated only threonine (Thr)-residues of free RIIalpha and phosphorylated preferentially Thr-residues of the R subunit (RIIalpha) of PKAIIalpha (PKA RIIalpha) in vitro; (ii). this phosphorylation was selectively inhibited by quercetin (an CK2 inhibitor); and (iii). the phosphorylation of free RIIalpha by CK2 resulted in the reduction of its suppressive effect on the activity (phosphorylation of histone H2B) of the catalytic (C) subunit and in the reduction of its ability to form a complex with the C subunit in vitro. As expected, the activity of PKAIIalpha was approx. 3.5-fold enhanced after its R subunit was fully phosphorylated by CK2 in vitro. cAMP synergistically stimulated the activity of PKAIIalpha phosphorylated by CK2 in vitro. These results strongly suggest that CK2 may be a protein kinase responsible for the activation of PKAIIalpha through specific phosphorylation of its R subunit at the cellular level.
