Two physiological substrate‐specific casein kinases are present in the bovine mammary gland

Brooks, Charles L.

doi:10.1016/0014-5793(89)80167-x

Cited by 7 publications

(4 citation statements)

References 13 publications

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“…One explanation for the two phases of casein phosphorylation and the differential effects of Ca# + depletion would be the presence of two casein kinases differing in Ca# + sensitivity, with the Ca# + -independent kinase acting at an earlier stage of the secretory pathway than the Ca# + -dependent kinase. Independent evidence in favour of this explanation comes from earlier work which demonstrated two biochemically distinct casein kinase activities in bovine mammary gland Golgi vesicles, one of which was Ca# + -independent and the other Ca# + -dependent [38]. Functional dissection of the Golgi complex by treatment of mouse mammary cells with brefeldin A has also suggested the possible presence of two distinct casein kinases [29].…”

Section: Discussionmentioning

confidence: 94%

Characterization of the effects of Ca2+ depletion on the synthesis, phosphorylation and secretion of caseins in lactating mammary epithelial cells

Duncan

Burgoyne

1996

Biochemical Journal

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We have examined the effects of depleting lumenal Ca2+ on the synthesis, phosphorylation and secretion of caseins in lactating mouse mammary cells by using inhibitors of the endoplasmic reticulum Ca(2+)-ATPase or the ionophore ionomycin in the absence of external Ca2+. Treatment with these drugs resulted in a transient increase in the cytosolic Ca2+ concentration due to Ca2+ mobilization. Protein synthesis over a 1 h period was substantially inhibited by Ca2+ depletion, but in a pulse-chase protocol secretion of pre-synthesized proteins was unaffected by Ca2+ depletion. Analysis of polysome profiles showed that Ca2+ depletion resulted in a loss of polysomes, consistent with an inhibition of initiation of protein synthesis. Neither treatment with Ca(2+)-ATPase inhibitors to deplete endoplasmic reticulum Ca2+ nor treatment with ionomycin/EGTA had any effect on an early phase of phosphorylation of alpha- or beta/gamma-caseins, but Ca2+ depletion resulted in a decrease in a late phase of casein phosphorylation. These results indicate that lumenal Ca2+ is required to maintain protein synthesis in lactating mammary cells but is not required for protein secretion, and that Ca2+ accumulation in the Golgi cisternae is required for a late but not for an early phase of casein phosphorylation.

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Section: Discussionmentioning

confidence: 94%

Characterization of the effects of Ca2+ depletion on the synthesis, phosphorylation and secretion of caseins in lactating mammary epithelial cells

Duncan

Burgoyne

1996

Biochemical Journal

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“…Resolution of two activities was also found when freshly prepared whey fraction was immediately separated by ion-exchange chromatography (results not shown), arguing against the generation of two forms by limited proteolysis during the purification protocol. It is significant that previous work has indirectly suggested the existence of two GCKs in mammary tissue based on differing substrate specificities and cofactor requirements [36,37]. In addition, dissection of the secretory pathway in mouse lactating mammary epithelial cells using brefeldin A has suggested the presence of two GCK activities located in either cis-or trans-Golgi compartments with different substrate specificities [7].…”

Section: Discussionmentioning

confidence: 99%

Purification of Golgi casein kinase from bovine milk

DUNCAN¹,

WILKINSON²,

Burgoyne³

2000

Biochem. J.

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Caseins and many other secretory proteins are phosphorylated during their transport through the secretory pathway by a protein kinase present within Golgi compartments. Molecular analysis of the Golgi casein kinase (GCK) has not been possible since it has not been purified to homogeneity or been cloned. Previous attempts have been made to purify GCK activity from mammary gland Golgi fractions, but these have not resulted in extensive purification of the enzyme. In the present study, we have demonstrated that substantial amounts of GCK activity, assayed using a specific peptide substrate, can be detected as a soluble form in bovine milk, and we have used milk as a source for purification. A purification protocol was established that allowed>80000-fold purification to a specific activity of GCK (approx. 700 nmoles/min per mg of protein) far higher than previously achieved. These findings cast doubts on previous claims for purification of GCK activity. In addition, ion-exchange chromatography resolved two closely eluting peaks of activity, suggesting the existence of two related, but distinct, GCK activities.

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“…Resolution of two activities was also found when freshly prepared whey fraction was immediately separated by ion-exchange chromatography (results not shown), arguing against the generation of two forms by limited proteolysis during the purification protocol. It is significant that previous work has indirectly suggested the existence of two GCKs in mammary tissue based on differing substrate specificities and cofactor requirements [36,37]. In addition, dissection of the secretory pathway in mouse lactating mammary epithelial cells using brefeldin A has suggested the presence of two GCK activities located in either cis-or transGolgi compartments with different substrate specificities [7].…”

Section: Discussionmentioning

confidence: 99%

Purification of Golgi casein kinase from bovine milk

Duncan

Wilkinson

Burgoyne

2000

Biochemical Journal

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Caseins and many other secretory proteins are phosphorylated during their transport through the secretory pathway by a protein kinase present within Golgi compartments. Molecular analysis of the Golgi casein kinase (GCK) has not been possible since it has not been purified to homogeneity or been cloned. Previous attempts have been made to purify GCK activity from mammary gland Golgi fractions, but these have not resulted in extensive purification of the enzyme. In the present study, we have demonstrated that substantial amounts of GCK activity, assayed using a specific peptide substrate, can be detected as a soluble form in bovine milk, and we have used milk as a source for purification. A purification protocol was established that allowed > 80000-fold purification to a specific activity of GCK (approx. 700nmoles/min per mg of protein) far higher than previously achieved. These findings cast doubts on previous claims for purification of GCK activity. In addition, ion-exchange chromotography resolved two closely eluting peaks of activity, suggesting the existence of two related, but distinct, GCK activities.

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Two physiological substrate‐specific casein kinases are present in the bovine mammary gland

Cited by 7 publications

References 13 publications

Characterization of the effects of Ca2+ depletion on the synthesis, phosphorylation and secretion of caseins in lactating mammary epithelial cells

Characterization of the effects of Ca2+ depletion on the synthesis, phosphorylation and secretion of caseins in lactating mammary epithelial cells

Purification of Golgi casein kinase from bovine milk

Purification of Golgi casein kinase from bovine milk

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