title: Substrate specificity studies on a calf thymus nuclear phosphoprotein kinase.
abstract: 1. The protein kinase activity associated with the phosphoprotein fraction of calf thymus nuclei has been examined for its ability to phosphorylate exogenous phosphoprotein substrates. beta-Casein and phosvitin are both efficient phosphate acceptors. Phosphorylation of alpha s1 and beta-caseins was shown, directly, to occur at threonyl-49 and threonyl-41 respectively. Both threonyl residues occur within the sequence Thr-Glu-Asp. 2. alpha s1-Casein becomes a much better substrate for the kinase when partially dephosphorylated. A similar response is shown by a phosphopeptide containing the alpha s1-casein phosphate cluster and is due to a new phosphorylation site becoming available. Efficient phosphorylation of beta-casein requires that the phosphate cluster (residues 15-19) be intact and results are presented which are consistent with there being a similar requirement for phosphorylation of the site created in alpha s1-casein by partial dephosphorylation. 3. Comparison of genetic variants of beta-casein as phosphate acceptors for the kinase shows that the presence of lysyl residues close to the phosphorylation site markedly depresses the rate of phosphorylation. Maleylation of beta-casein increases the rate of phosphorylation for all of the variants tested, although to varying extents. Treatment of maleylated beta-casein with trypsin to remove the N-terminal phosphopeptide inhibits phosphorylation by the kinase. 4. The structural determinants of beta-casein allowing it to be efficiently phosphorylated by the kinase are concluded to be the presence of a sequence surrounding the phosphorylation site, which is rich in acidic amino acid residues and from which basic residues are absent. The acidic phosphate cluster of beta-casein also promotes phosphorylation either by interacting directly with the enzyme or through its influence on the conformation of beta-casein.
mesh_terms: D000818:Animals; D047388:Casein Kinases; D002364:Caseins / Q000378:metabolism; D002417:Cattle; D002467:Cell Nucleus / Q000201:enzymology*; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008298:Maleates / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology*; D014357:Trypsin
chemical_list: D002364:Caseins; D008298:Maleates; D011494:Protein Kinases; D047388:Casein Kinases; D010749:Phosphoprotein Phosphatases; D014357:Trypsin