Differential effect of brefeldin A on phosphorylation of the caseins in lactating mouse mammary epithelial cells.
The major milk proteins, the caseins, contain multiple phosphorylation sites. Phosphorylation of the caseins is necessary to allow Ca2+ binding and aggregation of the caseins to form micelles. We have followed the phosphorylation of the caseins in isolated acini from lactating mouse mammary gland. Incubation of mammary cells with [32P]orthophosphate revealed that phosphorylation of newly synthesised caseins was complete within 20 minutes of synthesis. Extensive secretion of alpha-, beta- and gamma-caseins occurred over a 2 hour period. Activation of the regulated secretory pathway using ionomycin over the last hour resulted in a preferential increase in secretion of alpha- and gamma-caseins. Brefeldin A (BFA) inhibited protein secretion and synthesis in mammary cells in prolonged incubations. An examination of short-term treatments with BFA on 32P incorporation into the caseins revealed a differential effect of BFA in which the drug inhibited phosphorylation of beta- and gamma- but not alpha-caseins. These results suggest that phosphorylation of alpha-casein normally occurs in Golgi cisternae whereas that of beta- and gamma-caseins occurs in the trans-Golgi network. Phosphorylation of specific secretory proteins may, therefore, occur in different Golgi compartments.
Turner
M D
MD
Physiological Laboratory, University of Liverpool, UK.
Handel
S E
SE
Wilde
C J
CJ
Burgoyne
R D
RD
D000818:Animals; D020126:Brefeldin A; D002364:Caseins / Q000378:metabolism*; D003517:Cyclopentanes / Q000494:pharmacology*; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D005260:Female; D006056:Golgi Apparatus / Q000378:metabolism; D066298:In Vitro Techniques; D007774:Lactation / Q000187:drug effects / Q000378:metabolism*; D008321:Mammary Glands, Animal / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D051379:Mice; D010766:Phosphorylation / Q000187:drug effects; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*
D002364:Caseins; D003517:Cyclopentanes; D011500:Protein Synthesis Inhibitors; D020126:Brefeldin A