Specific Casein Phosphorylation by a Casein Kinase from Lactating Bovine Mammary Gland

A preparation which contains protein kinase activity capable of rephosphorylating dephosphorylated αs1 and β‐caseins has been prepared from lactating bovine mammary gland. This activity is localised in the Golgi fraction and in this respect is similar to that obtained previously from rat tissue. It differs from the rat preparation in being unable to rephosphorylate dephosphorylated ϰ‐casein. Progressive dephosphorylation of αs1 and β‐caseins increases the rate of their rephosphorylation in the presence both of Ca2+ and of Mg2+. For β‐casein significant non‐specific phosphorylation occurs in the presence of Mg2+ while in the presence of Ca2+ the level of phosphorylation is very low. Comparison of the products of the action of this kinase on partially dephosphorylated αs1 and β‐caseins indicates that seryl residues grouped with pre‐existing phosphoseryl residues are phosphorylated preferentially in the presence of Mg2+, while the phosphorylation of isolated single seryl residues is promoted by Ca2+. Digests of αs1‐casein, prepared by treatment with cyanogen bromide, have been fractionated and the three major peptides, separated. After dephosphorylation two of these, which between them contain all of the phosphate of αs1‐casein, are rephosphorylated at sites occupied in the native protein by phosphoseryl residues, i.e., at positions 46, 48, 75 and 115 of the αs1‐casein molecule. Evidence is presented which shows that this specificity of action also operates when intact αs1‐casein is used as phosphate acceptor. It is concluded that the enzyme responsible for specific phosphorylation acts through recognition of an amino acid sequence and does not require the provision of a specific structural conformation in the protein substrate.