Open AccessSer2 is the autophosphorylation site in the β subunit from bicistronically expressed human casein kinase‐2 and from native rat liver casein kinase‐2β
Author(s) -
BOLDYREFF Brigitte,
JAMES Peter,
STAUDENMANN Werner,
ISSINGER OlafGeorg
Publication year - 1993
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1993.tb18404.x
Subject(s) - autophosphorylation , casein kinase 2 , biochemistry , casein kinase 2, alpha 1 , recombinant dna , phosphorylation , casein kinase 1 , protein subunit , serine , phosphatase , microbiology and biotechnology , kinase , biology , enzyme , chemistry , protein kinase a , cyclin dependent kinase 2 , gene
Human casein kinase‐2 (CK‐2) subunits α and β were bicistronically expressed in bacteria. The recombinant holoenzyme shared all investigated properties with the native CK‐2 from mammalian sources (rat liver, Krebs II mouse ascites tumour cells). Contrary to recombinant human CK‐2 produced by self‐assembly in vitro , the bicistronically expressed β subunit was autophosphorylated during formation of the holoenzyme in bacteria. Electrospray ionisation mass spectrometry (ESI) revealed Ser2 (second amino acid, first serine) as the only phosphate acceptor site. Kinetic data obtained with either the phosphorylated or the unphosphorylated form of CK‐2 did not differ significantly, suggesting that the autophosphorylation had no influence on the kinetic parameters K m and V max . In parallel, native rat liver CK‐2β subunit was shown to incorporate 0.1 mol phosphate/mol holoenzyme, which suggests that the enzyme is already heavily phosphorylated. ESI analysis also revealed Ser2 as the only phosphorylated residue at the amino terminus. In the case of both recombinant human CK‐2 and native rat liver CK‐2, treatment with alkaline phosphatase readily reversed the phosphorylated form of the β subunit to the faster migrating dephosphorylated polypeptide.