ABF1 Ser-720 is a predominant phosphorylation site for casein kinase II of Saccharomyces cerevisiae.
ABF1 is a multifunctional phosphoprotein that binds specifically to yeast origins of replication and to transcriptional regulatory sites of a variety of genes. We isolated a protein kinase from extracts of Saccharomyces cerevisiae on the basis of its ability to specifically phosphorylate the ABF1 protein. Physical and biochemical properties of this kinase identify it as casein kinase II (CKII). The purified kinase has a high affinity for the ABF1 substrate as indicated by a relatively low Km value. Furthermore, when incubated with ABF1 and anti-ABF1 antibodies, the kinase forms an immunocomplex active in the phosphorylation of ABF1. Biochemical and genetic mapping localized a major site for phosphorylation at Ser-720 near the C terminus of the ABF1 protein. This serine is embedded within a domain enriched for acidic amino acid residues. A Ser-720 to Ala mutation abolishes phosphorylation by CKII in vitro. The same mutation also abolishes phosphorylation of this site in vivo, suggesting that CKII phosphorylates Ser-720 in vivo as well. Although three CKII enzymes, yeast, sea star, and recombinant human, utilize casein as a substrate with similar efficiencies, only the yeast enzyme efficiently phosphorylates the ABF1 protein. This suggests that ABF1 is a specific substrate of the yeast CKII and that this specificity may reside within one of the beta regulatory subunits of the enzyme. Thus, phosphorylation of ABF1 by yeast CKII may prove to be a useful system for studying targeting mechanisms of CKII to a physiological substrate.
Upton
T
T
Department of Microbiology, University of Connecticut Medical School, Farmington 06030, USA.
Wiltshire
S
S
Francesconi
S
S
Eisenberg
S
S
D047390:Casein Kinase II; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009154:Mutation; D010449:Peptide Mapping; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D013379:Substrate Specificity; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
C061886:ABF1 protein, S cerevisiae; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D012694:Serine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases