Modulation by sphingosine of substrate phosphorylation by protein kinase C in bovine mammary gland.
The effect of sphingosine on the phosphorylation of endogenous proteins by protein kinase C (PKC) was investigated in bovine mammary gland. Several proteins were shown to be substrates for PKC in both cytosolic and total particulate fractions by phosphorylation in the absence or presence of 1-oleoyl-2-acetyl-sn-glycerol, phosphatidylserine (PS) and Ca2+. At concentrations of 83 microM or less, sphingosine inhibited phosphorylation of several substrates for PKC in both fractions. Phosphorylation of cytosolic 36 kDa, 21 kDa and particulate 36 kDa proteins was particularly sensitive to sphingosine. Cytosolic 97 kDa phosphorylation (which was enhanced by Ca2+ alone) was also sensitive to sphingosine. The inhibition was reversed by excess addition of lipid cofactors, particularly PS, but not by Ca2+. At higher concentrations (167 and 417 microM), in addition to the inhibition seen at lower concentrations, sphingosine stimulated phosphorylation of several proteins, including cytosolic 19 kDa and particulate 53 kDa, which were not detected in the absence of sphingosine. The sphingosine-induced phosphorylation disappeared with excess addition of PS, but not with addition of Ca2+. The results point toward the importance of the interaction of sphingosine with membrane phospholipids in the signal transduction pathway mediated by PKC-dependent phosphorylation in bovine mammary gland.
Katoh
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National Institute of Animal Health, Hokkaido Branch Laboratory, Sapporo, Japan.
D000818:Animals; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D003600:Cytosol / Q000201:enzymology; D004075:Diglycerides / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008321:Mammary Glands, Animal / Q000201:enzymology / Q000378:metabolism*; D008970:Molecular Weight; D010718:Phosphatidylserines / Q000494:pharmacology; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D013110:Sphingosine / Q000494:pharmacology*; D013347:Subcellular Fractions / Q000378:metabolism
D004075:Diglycerides; D010718:Phosphatidylserines; D010750:Phosphoproteins; C038237:1-oleoyl-2-acetylglycerol; D011493:Protein Kinase C; D013110:Sphingosine