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

Abstract

1. A preparation which contains protein kinase activity capable of rephosphorylating dephosphorylated @,I and P-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 x-casein.2. Progressive dephosphorylation of asl and P-caseins increases the rate of their rephosphorylation in the presence both of Ca2+ and of Mg2+. For 8-casein significant non-specific phosphorylation occurs in the presence of Mg2+ while in the presence of Ca2+ the level of phosphorylation is very low.3. Comparison of the products of the action of this kinase on partially dephosphorylated aSl and B-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+.4. Digests of aSl-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 aS1-casein, are rephosphorylated at sites occupied in the native protein by phosphoseryl residues, i. e., at positions 46, 48, 75 and 115 of the a,l-casein molecule. Evidence is presented which shows that this specificity of action also operates when intact aSl-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.Determination of the sequences of amino acid residues occurring in the three major components of bovine casein, @,I, P and x-caseins, has defined accurately the locations of the phosphate residues in these proteins [1,2]. Altogether some 14 seryl residues are phosphorylated and a prominent feature of the primary structures of the a ,~ and p-caseins is a region of homology which includes a cluster of four phosphoseryl residues. In aSl-casein this extends over residues 63 -70 thus : Glu-Ser(P)-Ile-Ser(P)-Ser(P)-Ser-(P)-Glu-Glu, a sequence which is repeated in residues 14-21 of /?-casein with the substitution of leucine for isoleucine. In addition to these a,l-casein contains four phosphoseryl residues located at positions 46, 48, 75 and 11 5 and P-casein contains one located at position 35. %-Casein contains only one phosphoseryl residue. It has been shown by Turkington and Topper [3] that in mouse mammary glands at least a significant proportion of the phosphate of casein is incorporated after polypeptide synthesis rather than by incorporation of phosphorylated amino acids, and more recently Bingham and coworkers [4,5] have detected in the Golgi apparatus of lactating rat mammary gland a protein kinase for which dephosphorylated bovine caseins were found to act as better phosphat...

Conserved phosphorylation of serines in the Ser‐X‐Glu/Ser(P) sequences of the vitamin K‐dependent matrix Gla protein from shark, lamb, rat, cow, and human

“…A protein kinase has been isolated from the Golgi fraction of breast tissue from lactating guinea pigs (Moore et al, 1985) and cows (MacKinlay et al, 1977), which, in studies carried out using peptide substrates, proved to be specific for serine residues in Ser-X-Glu/Ser(P) sequences (Meggio et al, 1988). This enzyme appears to be the best candidate for the protein kinase that phosphorylates the proteins that are secreted into milk, namely the caseins and PP3.…”

Characterisation of a Membrane‐Bound Serine‐Specific Casein Kinase Isolated from Lactating Guinea‐Pig Mammary Gland

“…Guinea-pig caseins were prepared as described previously [8], and either used directly as substrate, or dephosphorylated using potato acid phosphatase at a final concentration of 2 units/ml essentially as described by Bingham and coworkers [I 81. Dephosphorylated casein was recovered according to the method of Mackinlay et al [19]. This procedure resulted in complete ( 2 95 %) dephosphorylation of guinea-pig caseins as judged by dephosphorylation of a mixture of 32P-labelled caseins (20000 counts/min) which had been synthesized and secreted by guinea-pig mammary gland explants in culture, in the presence of 100 pg total guinea-pig casein carrier isolated from milk.…”

Casein kinase activity in rat mammary gland Golgi vesicles

“…Although the detailed structure of these micelles has not been fully elucidated, it is known that the presence of Ca2+ and the phosphorylation of certain serine residues in caseins are prerequisites for micelle formation [2 -61. In the mammary gland secretory cell, this phosphorylation occurs during passage of the caseins from their sites of synthesis on rough endoplasmic reticulum to the alvaeolar lumen, most probably within cisternae and vesicles of the Golgi apparatus Identification and characterization of the protein kinase(s) responsible for the physiological phosphorylation of caseins has been complicated by the fact that Golgi-associated protein kinase activity accounts for only a small proportion of the total protein kinase activity of lactating mammary tissue [8,91. Nevertheless, Bingham and co-workers [lo -151 have reported that Golgi vesicle preparations from both rat and bovine mammary glands contain protein kinases capable of phosphorylating bovine caseins, and a similar kinase activity has been demonstrated in Golgi vesicles isolated from guinea pig mammary glands [16].…”