Purification and tissue‐specific expression of casein kinase from the lactating guinea‐pig mammary gland

Abstract

A serinc-specific casein kinase, an integral membrane protein of the lactating guinea-pig mammary gland, has been purified from a Golgi-enriched membrane fraction, using a combination of sucrose gradient centrifugation and chromatography on ATP-agarose. The enzyme comprises a polypeptide of estimated M , 74000 as judged by sodium dodecyl sulphate/polyacrylamidc gel electrophoresis, compared with a monomer M , of 50 000 as determined by sucrose gradient centrifugation in the presence of500 mM NaCl and 0.1 %, Triton X-1 00. Kinetic studics show that the purified enzyme exhibits kinetic constants distinctly different from the rabbit reticulocyte casein kinases I and 11, whilst polyclonal antisera raised against the mammary gland enzyme did not cross-react with soluble liver or reticulocyte protein kinase activities. Immunoblotting and immunocytochemical analyses demonstrate the mammary gland enzyme's apparently unique location in lactating mammary gland tissuc. Comparative studies with polyclonal antisera raised against bovine galactosyltransferase, show that casein kinase and galactosyltransferase have a similar intracellular localisation in the lactating mammary gland as judged by immunocytochemistry at the light level, but that casein kinase was unique to mammary gland whereas galactosyltransferase could be found in other tissues. The results extended our earlier observations which suggest a Colgi location for casein kinase, and demonstrate that future studies using this enzyme may well prove advantageous for the study of intracellular mechanisms involved in the biogenesis of organelles, i n this instance the Golgi apparatus.We have previously dcscribed the presence in the lactating guinea-pig mainmary gland of a serine-specific casein kinasc, an integral membrane protein located in a Golgi-enriched membrane fraction [l]. Similar enzyme activities have been described in Golgi-enriched fractions isolated from mammary glands of othcr species [2-41. Other casein kinase activities isolated from diverse tissues and cells have also been described in the literature (see [S]). These are CAMP-independcnt and arc classified on the basis of their elution from DEAE-cellulose a s casein kinases 1 and 11; they differ from each other with respect to iiucleotide triphosphate substrate. Casein kinase 11, utilises both ATP and GTP [6] whikt casein kinase I has a preference for ATP. In additioi. cascin kinase I1 but not casein kiiiase I is inhibited by h e p r i n [7].Studies on the phosphorylation of guinea-pig caseins using lactating mammary gland explants in culture [8] or Xrnopus oocytes microinjected with milk protein mRNA [9] demonstrate that each contain partially processed unphosphorylatcd caseins sequestered within the secretory pathway, but that only those secreted by the guinea-pig mammary explants, not by the oocyte, are phosphorylated. These observations suggest that casein kinase is not an enzyme common to the secretory pathway of all cells, and may therefore be expressed in a tissue-specific manner.To determine whet...

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

“…It seems less likely, however, that phosphoproteins secreted into the extracellular environment of cells, such as the regulatory peptides and MGP, are phosphorylated by the same enzyme that phosphorylates milk and saliva proteins. Antibodies specific for the Golgi-associated protein kinase of lactating breast tissue strongly label the Golgi complex of lactating breast tissue, as expected, but fail to label the Golgi in a variety of other tissues including liver, spleen, and kidney (Moore et al, 1985). The possible existence of a different protein kinase isozyme that phosphorylates proteins secreted into the extracellular environment of cells is also supported by the observation that such secreted proteins are only partially phosphorylated at target serine residues.…”

“…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.…”

“…The specificity of this Golgi-associated protein kinase is quite different from that found for casein kinases I and I1 (Meggio et al, 1988), which are cytosolic enzymes that are not known to phosphorylate casein or other secreted proteins in vivo. The Golgi-associated protein kinase of lactating breast tissue is a membrane-bound protein of 74 kDa apparent size (Moore et al, 1985).…”

Purification of Golgi casein kinase from bovine milk

“…This resulted in the assumption that GCK is an integral Golgi membrane protein [25,26]. In our initial experiments, GCK activity was solubilized from lactating mammary tissue using 1 % Triton X-100 as previously described [6,[9][10][11].…”

“…An attempt to purify GCK from mammary Golgi fractions by ATPaffinity chromatography was reported, and a 70 kDa protein was claimed to represent the enzyme [25,26]. Surprisingly, immunocytochemical studies suggested that the 70 kDa protein is expressed only in mammary epithelial cells [25], a finding inconsistent with the possible general expression of GCK. This protein was, however, poorly characterized and its identity as GCK was not confirmed.…”