The phosphorylation sites of ribosomal P proteins from Saccharomyces cerevisiae cells by endogenous CK-2, PK60S and RAP protein kinases.
The phosphorylation sites of ribosomal acidic proteins (P proteins) from Saccharomyces cerevisiae were studied in vivo and in vitro by using CK-2, PK60S and RAP protein kinases. The three enzymes phosphorylate the last serine residues located in a highly conserved carboxyl end of the polypeptide chains. This was established by two-dimensional analysis of tryptic phosphopeptides from 32P-labelled proteins YP1 alpha, YP1 beta, YP2 alpha and YP2 beta, and by kinetic studies of the protein kinases with synthetic peptides corresponding to the fragments of endogenous ribosomal acidic polypeptides. In experiments with both endogenous P proteins and synthetic peptides as substrates protein kinase PK60S demonstrated unusual substrate specificity. In contrast to CK-2 and RAP protein kinases, PK60S phosphorylates predominantly two of the four P proteins, YP1 alpha and YP2 beta, with kinetic constants dependent on the primary structure of the N-terminal region of the polypeptide containing the target residue. The neutral amino acid, alanine, at position 3 in the peptide AAEESDDD (polypeptide fragments of YP1 beta and YP2 alpha) decreases the K(m) value more than 10-fold by comparison with the basic lysine residue at the same position in the peptide AKEESDDD (polypeptide fragments of YP1 alpha and YP2 beta).
Boguszewska
A
A
Department of Molecular Biology, Maria Curie-Skłodowska University, Lublin, Poland.
Szyszka
R
R
Grankowski
N
N
D000595:Amino Acid Sequence; D001665:Binding Sites; D047390:Casein Kinase II; D004586:Electrophoresis; D005656:Fungal Proteins / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism*
D005656:Fungal Proteins; D012269:Ribosomal Proteins; D011494:Protein Kinases; C083550:60S ribosomal protein kinase; C099264:RAP kinase; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases