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

Abstract

Serine-specific and threonine-specific casein kinase activities have been identified in a Golgi-enriched membrane fraction isolated from the lactating guinca-pig mammary gland. The serine-specific cascin kinase has been purified 2000-fold by affinity chromatography on ATP-agarose. The enzyme has an estimated M , of I00000 as determined by sucrose gradient centrifugation and phosphorylates the serine residues of dephosphorylated guinea-pig caseins A and B in a qualitatively and quantitatively identical manner to caseins A and B secreted by lactating mammary gland explants in organ culture. The enzyme also phosphorylates casein C at serine, but not threonine residues. Studies on the relative location of the enzyme within a Golgi-enriched membrane fraction show that it is an integral component of the membrane, either in the form of a transmembrane protein or exposed on the luminal side of the membrane. Although casein kinase activity is not associated with the endoplasmic reticulum, it remains to be proven whether it is truly a Golgi enzyme, since analysis of subcellular meinbrane components fractionated by sucrose gradient centrifugation shows that the particulate protein kinase activity of the lactating mammary gland does not cosediinent with galactosyl transferase, possibly a reflection of the heterogeneous nature of mammary gland Glogi apparatus. It seems likely that the serine-specific casein kinase activity described is responsible for the phosphorylation of caseins in the lactating guinea-pig mammary gland, and that this occurs after the sequestration of processed but unphosphorylated caseins within the lumen of the endoplasmic reticulum.Our understanding of the intracellular events involved in the synthesis, sequestration, post-translational modification and secretion of proteins has improved dramatically in recent years. This has been due to the development of mRNAdirected cell-free protein-synthesizing systems [I, 21, and the discovery that the injection of secretory protein mRNAs into Xerzopus oocytes results in sequestration and secretion of the translation product [3,4]. Both approaches demonstrate that the synthesis of membrane and secretory proteins is tightly coupled to sequestration and core glycosylation at the level of the endoplasmic reticulum, and that these mechanisms are common to many cell types [ 5 ] . Recent studies on the mechanisms of secretion using Xenopus oocytes also demonstrate that intracellular events subsequent to sequestration must be common to different cell types, and that the posttranslational addition of prosthetic groups, such as the glycosylation of known glycoproteins, is not required to ensure secretion [6].Milk proteins are synthesized and secreted in large amounts by the lactating mammary gland. A large proportion comprises a well characterised family of phosphoproteins, the caseins (see [7]). In the guinea-pig, three major caseins have been identified and partially characterised [8], though recent application of recombinant DNA technology has identified additional var...

Guinea‐pig casein A cDNA

“…However, additional potential sites appear to be unique to the guinea-pig sequence (residues 13,15,30,54, 132, 134, 136 and 203). Although it has yet to be established exactly which of the sites are phosphorylated in guinea-pig casein A, tryptic peptide maps of the 32P-labelled casein A secreted by explants, or of the casein A phosphorylated in vitro using purified guinea-pig mammary-gland-specific casein kinase, both suggest a complex (although identical) phosphorylation pattern [18], implicating the involvement of most, if not all, potential phosphorylation sites. …”

Analysis of a sub-proteome which co-purifies with and is phosphorylated by the Golgi casein kinase

“…While the biochemical characterization of G-CK with special reference to the definition of its substrate specificity was quite straightforward thanks to the availability of fairly active G-CK preparations from the Golgi apparatus of lactating mammary gland and the synthesis of appropriate phosphoacceptor peptide substrates, the elucidation of the primary structure of G-CK turned out to be a troublesome and frustrating task. In fact, despite the recurrent efforts of several laboratories [11,12,[19][20][21][22][23][24][25][26], G-CK could not be purified to homogeneity, a situation which has hindered up to now any reliable structural analysis of this elusive kinase. This prompted us to address the problem from a different angle, i.e.…”

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].…”

“…GCK has not yet been characterized at a molecular level. 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.…”