No abstract

Purified variants of asl casein and p 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 I1 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 I1 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 p caseins, the phosphorylated peptides observed with casein kinase I were different from those obtained with casein kinase 11. With the asl caseins, the same phosphorylated peptide was observed with both protein kinases. The phosphorylation sites in the aSl and p 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 p-A2 casein and Ser-41 in aSl-A casein, suggesting that this protein kinase recognized the primary sequence Glu-X-Ser. Thr-41 in p-A2 casein and Thr-49 in aSl-A casein were the primary sites phosphorylated by casein kinase 11. The recognition determinants for casein kinase I1 appeared to be Thr-Glu-Asp. When endogenous phosphate was removed from the caseins, the rate of phosphorylation of / 3 casein was diminished by greater than six-fold indicating that negative charge near the phosphorylation site facilitated phosphorylation.Two CAMP-independent protein kinases which modify casein have been highly purified from rabbit reticulocytes by ion-exchange chromatography as previously described by Hathaway and Traugh [l]. These enzymes have been designated casein kinase I and casein kinase I1 in order of elution from DEAEcellulose. Casein kinase I is a single polypeptide chain of molecular weight 37 000 and preferentially uses ATP. Casein kinase I1 is a tetramer ( M , 130000) and utilizes both ATP and GTP. Enzymes similar to casein kinase I1 have also been highly purified from rat liver [2] and Novikoff ascites tumor cells [3].Little is known about the mechanism of action or the biological role of the CAMP-independent protein kinases from reticulocytes, although several translational initiation factors have been identified as endoAbbreviation. Cyclic AMP or CAMP, adenosine 3': 5'-monoEnzymes. Chymotrypsin (EC 3.4.21.1); acid phosphatase (EC phosphate. 3.1.3.2).genous substrates [4 -91. Initiation factors eIF-4B and eIF-5 have been shown to be phosphorylated by casein kinase I while initiation factors eIF-2, eIF-3, eIF-4B and eIF-5 are modified by casein kinase I1 [4]. In these studies the purified casein variants have...

No abstract

Two milk proteins, beta-casein and alpha-lactalbumin, were compared as substrates for casein kinase from bovine mammary gland. beta-Casein could be rephosphorylated after removal of its phosphate groups, whereas alpha-lactalbumin was an effective substrate after the protein had been reduced and carboxymethylated. The native proteins could not be phosphorylated. Magnesium2+, Ca2+, and Mn2+ stimulated phosphorylation of the modified proteins. Calcium2+ was the most effective cation for alpha-lactalbumin and Mn2+ for beta-casein. Michaelis constants were 144 microM for alpha-lactalbumin in the presence of Ca2+ and 142 microM for beta-casein in the presence of Mn2+; however, the maximum velocity for alpha-lactalbumin was three times that of beta-casein. After phosphorylation with [gamma-32P] ATP, partial hydrolysis showed that only serine residues were phosphorylated in both proteins. Chymotryptic peptides of phosphorylated alpha-lactalbumin and tryptic peptides of phosphorylated beta-casein were examined by HPLC and selected peptides were analyzed for amino acid content. Comparison of the analyses with sequence data showed that serine at position 47 in alpha-lactalbumin is the major site of phosphate incorporation. Dephosphorylated beta-casein was only partially rephosphorylated. However, the sites identified correspond to the phosphorylated residues in native beta-casein, namely, serine at position 35 and the cluster of four serines between residues 15 and 20.