Substrate specificity studies on a calf thymus nuclear phosphoprotein kinase.
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.
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
D002364:Caseins; D008298:Maleates; D011494:Protein Kinases; D047388:Casein Kinases; D010749:Phosphoprotein Phosphatases; D014357:Trypsin