Yeast surviving factor Svf1 as a new interacting partner, regulator and in vitro substrate of protein kinase CK2.
Since Svf1 is phosphoprotein, we investigated whether it was a substrate for protein kinase CK2. According to the amino acid sequence Svf1 harbours 20 putative CK2 phosphorylation sites. Here, we have reported cloning, overexpression, purification and characterization of yeast Svf1 as a substrate for three forms of yeast CK2. Svf1 serves as a substrate for both the recombinant CK2alpha (Km 0.35 microM) and CK2alpha' (Km 0.18 microM) as well as CK2 holoenzyme (Km 1.1 microM). Different Km values argue that CK2beta(beta') subunit has an inhibitory effect on the activity of both CK2alpha and CK2alpha' towards surviving factor Svf1. Reconstitution of alpha'2betabeta' isoform of CK2 holoenzyme shows that beta/beta' subunits have regulatory effect depending on the kind of CK2 catalytic subunit. This effect was not observed in the case of alpha2betabeta' isoform, which may be due to interaction between Svf1 and regulatory CK2beta subunit (shown by co-immunoprecipitation experiments). Interactions between CK2 subunits and Svf1 protein may have influence on ATP as well as ATP-competitive inhibitors (TBBt and TBBz) binding. CK2 phosphorylates up to six serine residues in highly acidic peptide K199EVIPESDEEESSADEDDNEDEDEESGDSEEESGSEEESDSEEVEITYED248 of the Svf1 protein in vitro. Presented data may help to elucidate the role of protein kinase CK2 and Svf1 in the regulation of cell survival pathways.
Masłyk
Maciej
M
Department of Molecular Biology, Environmental Protection Institute, John Paul II Catholic University of Lublin, Kraśnicka Av.102, 20-718 Lublin, Poland.
Kochanowicz
Elzbieta
E
Zieliński
Rafał
R
Kubiński
Konrad
K
Hellman
Ulf
U
Szyszka
Ryszard
R
D000595:Amino Acid Sequence; D047390:Casein Kinase II / Q000378:metabolism*; D002470:Cell Survival; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; C504373:Svf1 protein, S cerevisiae; D047390:Casein Kinase II