Ser2 is the autophosphorylation site in the beta subunit from bicistronically expressed human casein kinase-2 and from native rat liver casein kinase-2 beta.
Human casein kinase-2 (CK-2) subunits alpha and beta 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 beta 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 Km and Vmax. In parallel, native rat liver CK-2 beta 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 beta subunit to the faster migrating dephosphorylated polypeptide.
Boldyreff
B
B
Institut für Humangenetik, Universität des Saarlandes, Homburg, Germany.
James
P
P
Staudenmann
W
W
Issinger
O G
OG
D000595:Amino Acid Sequence; D000818:Animals; D047388:Casein Kinases; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism*
D010446:Peptide Fragments; D011994:Recombinant Proteins; D012694:Serine; D011494:Protein Kinases; D047388:Casein Kinases