title: Protein phosphorylation in rat mammary acini and in cytosol preparations in vitro. Phosphorylation of acetyl-CoA carboxylase is unaffected by cyclic AMP.
abstract: Phosphorylation of soluble proteins in rat mammary acinar cells was investigated. When phosphorylation proceeded in intact cells, in the presence of [32P]Pi, the major non-casein phosphoproteins, including acetyl-CoA carboxylase, were unresponsive to incubation conditions that caused major increases in the intracellular concentration of cyclic AMP. The overall 32P specific radioactivity (c.p.m./microgram of protein) of acetyl-CoA carboxylase, assessed after affinity purification of the enzyme with avidin-Sepharose, was unchanged by incubation under such conditions. Furthermore, the distribution of 32P among tryptic phosphopeptides of the enzyme, resolved by reversed-phase h.p.l.c., was not altered by cyclic AMP-increasing treatments of the acinar cells. When cytosol fractions were incubated with [gamma-32P]ATP, some phosphoproteins responded to the addition of micromolar concentrations of dibutyryl cyclic AMP or cyclic AMP by undergoing an enhancement of phosphate incorporation. In these experiments in vitro, protein phosphatase activity did not make a major contribution to the net phosphorylation of individual phosphoproteins, and acetyl-CoA carboxylase was not prominent among the phosphoproteins identified after short (less than 1 min) incubations of cytosols with [gamma-32P]ATP. The resistance of protein phosphorylation to variations in the cyclic AMP concentration in intact mammary epithelial cells, demonstrated by this work, is one of several mechanisms that ensure the pleiotropic refractoriness of those cells to agents which normally cause a stimulation of adenylate cyclase activity in hormone-sensitive cells.
mesh_terms: D000103:Acetyl-CoA Carboxylase / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D000242:Cyclic AMP / Q000494:pharmacology*; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D066298:In Vitro Techniques; D008025:Ligases / Q000378:metabolism*; D008321:Mammary Glands, Animal / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D010446:Peptide Fragments; D011506:Proteins; D003994:Bucladesine; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D008025:Ligases; D000103:Acetyl-CoA Carboxylase