Abstract
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 CKα (Km 0.35μM) and CK2α′ (Km 0.18μM) as well as CK2 holoenzyme (Km 1.1μM). Different Km values argue that CK2β(β′) subunit has an inhibitory effect on the activity of both CK2α and CK2α′ towards surviving factor Svf1. Reconstitution of α′2 ββ′ isoform of CK2 holoenzyme shows that ββ′ subunits have regulatory effect depending on the kind of CK2 catalytic subunit. This effect was not observed in the case of α2ββ′ isoform, which may be due to interaction between Svf1 and regulatory CK2β 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 K199 EVIPESDEEESSADEDDNEDEDEESGDSE EESGSEEESDSEEVEITYED248 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.
Original language | English (US) |
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Pages (from-to) | 61-69 |
Number of pages | 9 |
Journal | Molecular and Cellular Biochemistry |
Volume | 312 |
Issue number | 1-2 |
DOIs | |
State | Published - May 2008 |
Externally published | Yes |
Keywords
- ATP-competitive inhibitors
- Apoptosis
- Masss pectrometry
- Phosphorylation
- Protein kinase CK2
- Svf1
- Svf1-CK2β interaction
- Yeast
ASJC Scopus subject areas
- Molecular Biology
- Clinical Biochemistry
- Cell Biology