Characterization of a proline-directed casein kinase from bovine brain.
A messenger-independent ser/thr casein kinase, p45 casein kinase (p45 CK), was purified to homogeneity from bovine brain. The enzyme is specific for ATP with a Km value of 3.50 microM, one of the lowest values identified for protein kinases, p45 casein kinase is active over broad NaCl concentrations from 30 to 300 mM. The enzyme activity is inhibited by polylysine, spermine, transition metal ions, ADP, and AMP. The kinase completely lost its activity in the presence of 1 mM p-chloromercuric benzoate in a reaction that is reversed by 1 mM dithiothreitol. The enzyme prefers serine over threonine in its substrate bradykinin, the Vmax/Km ratio for the serine peptide (RPPGFSPFR) being 7.5-fold higher than for the threonine analog (RPPGFTPFR). Assays, performed by utilizing synthetic peptides, suggest that p45 CK prefers serine/threonine residues with a proline residue immediately carboxy-terminal to the site of phosphorylation. Distinction between p45 CK and other protein kinases found to contain a proline residue within their substrate recognition sites can be made based on phosphorylation site specificity and chromatographic and biochemical behavior. It is concluded that p45 CK is a proline-directed protein kinase recognizing the sequence X-Ser/Thr-Pro-X or Ser/Thr-Pro-X.
Angelov
I
I
Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D047388:Casein Kinases; D002364:Caseins / Q000378:metabolism; D002417:Cattle; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010774:Phosvitin / Q000378:metabolism; D011392:Proline; D011494:Protein Kinases / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity
D002364:Caseins; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D010774:Phosvitin; D011392:Proline; D011494:Protein Kinases; D047388:Casein Kinases; D017346:Protein Serine-Threonine Kinases