Cyclic nucleotide-independent protein kinases from rabbit reticulocytes. Site-specific phosphorylation of casein variants

Purified variants of α s1 casein and β casein have been used to examine the substrate‐specificity of two adenosine‐3′:5′‐monophosphate‐independent protein kinase activities purified from rabbit reticulocytes. These enzymes, identified as casein kinase I and II were stimulated by monovalent cations. Casein kinase I had a pH optimum between 6.8 and 8.0, used ATP as the phosphate donor and preferentially phosphorylated serine. Casein kinase II had optimal activity at pH 8.5 to 10, utilized both ATP and GTP in the phosphotransferase reaction and modified predominantly threonine residues. The chymotryptic peptides of the phosphorylated casein variants were analyzed by thin‐layer electrophoresis and ascending chromatography and the phosphorylated peptides were identified by autoradiography. In the β caseins, the phosphorylated peptides observed with casein kinase I were different from those obtained with casein kinase II. With the α s1 caseins, the same phosphorylated peptide was observed with both protein kinases. The phosphorylation sites in the α s1 and β caseins have been assigned on the basis of the amino acid analyses of the phosphorylated chymotryptic peptides and the phosphoserine and phosphothreonine determinations in conjunction with the known primary sequences. Casein kinase I phosphorylated Ser‐22 in β‐A 2 casein and Ser‐41 in α s1 ‐A casein, suggesting that this protein kinase recognized the primary sequence Glu‐X‐Ser. Thr‐41 in β‐A 2 casein and Thr‐49 in α s1 ‐A casein were the primary sites phosphorylated by casein kinase II. The recognition determinants for casein kinase II appeared to be Thr‐Gou‐Asp. When endogenous phosphate was removed from the caseins, the rate of phosphorylation of β casein was diminished by greater than six‐fold indicating that negative charge near the phosphorylation site facilitated phosphorylation.