Substrate Specificity Studies on a Calf Thymus Nuclear Phosphoprotein Kinase

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Abstract
The protein kinase activity associated with the phosphoprotein fraction of calf thymus nuclei has been examined for its ability to phosphorylate exogenous phosphoprotein substrates. β‐Casein and phosvitin are both efficient phosphate acceptors. Phosphorylation of αs1 and β‐caseins was shown, indirectly, to occur at threonyl‐49 and threonyl‐41 respectively. Both threonyl residues occur within the sequence Thr‐Glu‐Asp. αs1‐Casein becomes a much better substrate for the kinase when partially dephosphorylated. A similar response is shown by a phosphopeptide containing the αs1‐casein phosphate cluster and is due to a new phosphorylation site becoming available. Efficient phosphorylation of β‐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 αs1‐casein by partial dephosphorylation. Comparison of genetic variants of β‐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 β‐ casein increases the rate of phosphorylation for all of the variants tested, although to varying extents. Treatment of maleylated β‐casein with trypsin to remove the N‐terminal phosphopeptide inhibits phosphotylation by the kinase. The structural determinants of β‐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 β‐casein also promotes phosphorylation either by interacting directly with the enzyme or through its influence on the conformation of β‐casein.