Substrate specificity characterization of a cdc2-like protein kinase purified from bovine brain.
A serine/threonine kinase from bovine brain has been purified (Lew, J., Beaudette, K. N., Litwin, C. M. E., and Wang, J. H. (1992) J. Biol. Chem. 267, 13383-13390) and found to consist of a 33-kDa catalytic subunit having high sequence homology to p34cdc2 and cdk2 (Lew, J., Winkfein, R. J., Paudel, H., and Wang, J. H. (1992) J. Biol. Chem. 267, 25922-25926). Substrate specificity determinants for this cdc2-like kinase were examined using synthetic peptide substrates derived from the in vitro p34cdc2 phosphorylation sites of histone H1. The peptide P-K-T-P-K-K-A-K-K-L was found to be an excellent substrate for the bovine cdc2-like kinase, having a Km value in the micromolar range. Important determinants for efficient substrate phosphorylation of this peptide were found both within the proposed substrate consensus motif (S/T-P-X-K/R) of p34cdc2 kinase and outside of this sequence. In addition to the absolute requirement for a proline residue immediately COOH-terminal to the phosphorylatable residue (+1) and a basic residue at the +3 position, a basic amino acid at the +2 position was greatly preferred over an acidic amino acid. A proline residue at the -2 position and a cluster of basic amino acids further COOH-terminal to the consensus motif were also found to be important for substrate binding. HeLa cell p34cdc2 kinase displays similar specificity to that of the bovine cdc2-like kinase, as the additional determinants outside of the consensus motif that contribute to the efficient phosphorylation of the histone peptide by this novel enzyme also appear to be important for p34cdc2-catalyzed phosphorylation.
Beaudette
K N
KN
Department of Medical Biochemistry, Faculty of Medicine, University of Calgary, Alberta, Canada.
Lew
J
J
Wang
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D016203:CDC2 Protein Kinase / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D006367:HeLa Cells; D006657:Histones / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D013379:Substrate Specificity
D006657:Histones; D010446:Peptide Fragments; D016203:CDC2 Protein Kinase