Protein phosphorylation in chick kidney. Response to parathyroid hormone, cyclic AMP, calcium, and phosphatidylserine.
The regulation of endogenous protein phosphorylation by parathyroid hormone (PTH) was investigated using confluent monolayer cultures of chick kidney cells. Homogenates and subcellular fractions of PTH (bovine 1-34)-treated cells were subjected to an endogenous protein phosphorylation assay using ((gamma- 32P]ATP in the presence or absence of 2.0 microM cAMP or 0.5 mM Ca2+ with 25 micrograms/ml of phosphatidylserine and reactions terminated with sodium dodecyl sulfate. In other experiments, cultures were incubated in a phosphate-free 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered saline containing 50 muCi/ml of [32P]PO4 and incubations were terminated with sodium dodecyl sulfate. Protein phosphorylation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Cyclic AMP stimulated 32P incorporation into proteins having molecular weights of 17,000, 22,000, 35,000, 42,000, 54,000, 75,000, 80,000, 120,000, and 143,000. Calcium-phosphatidylserine stimulated the phosphorylation of proteins of 20,000, 52,000, 58,000, 60,000, and 143,000. The protein phosphorylation patterns in cultured kidney cells and freshly isolated kidney tissue were quite similar. Treatment of cultured cells with 5-50 ng/ml of PTH resulted in stimulated phosphorylation of the 35,000 and 42,000 dalton proteins as assessed by endogenous phosphorylation in homogenates. In intact cells incubated with [32P]PO4, PTH stimulated most noticeably the phosphorylation of the 35,000-dalton protein. Based on studies with cultured and fresh kidney cells, the majority of the substrate proteins for cAMP and calcium-dependent protein kinases were located in the cytoplasm with the exception of the 42,000-dalton protein which was located in the brush-border-plasma membrane fraction. The cytoplasmic cAMP-dependent protein kinase activity was responsible for the majority of PTH-stimulated protein phosphorylation.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002478:Cells, Cultured; D002645:Chickens; D000242:Cyclic AMP / Q000494:pharmacology*; D004789:Enzyme Activation; D007668:Kidney / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010718:Phosphatidylserines / Q000494:pharmacology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology
D010281:Parathyroid Hormone; D010718:Phosphatidylserines; D011506:Proteins; D000242:Cyclic AMP; D011494:Protein Kinases; D002118:Calcium