Modulation by Sphingosine of Phosphorylation of Substrate Proteins by Protein Kinase C in Nuclei from Cow Mammary Gland

Abstract

ABSTRACT. Protein kinase C (PKC) is an enzyme activated by diacylglycerols such as 1-oleoyl-2-acetyl-sn-glycerol (OAG), phospholipids (in particular phosphatidylserine; PS) and Ca 2+ , which regulate a wide variety of intracellular functions by phosphorylating multiple substrate proteins and enzymes. The effect of sphingosine, the backbone moiety of sphingolipids, on PKC activity and phosphorylation of endogenous proteins catalyzed by PKC was investigated in nuclei of cow mammary gland. Sphingosine inhibited nuclear PKC activity when lysine-rich histone was used as the substrate. The sphingosine inhibition of the PKC activity was reversed by the excess addition of PS, but not by OAG or Ca 2+ . Several nuclear proteins, including 56-kDa, 43-kDa, 38-kDa and 36-kDa proteins, were shown to be substrates for PKC. Of the substrate proteins, the 38-kDa and 36-kDa proteins were identified as annexin I, the Ca 2+ /phospholipid-binding protein; the 56-kDa and 43-kDa proteins have not yet been identified. Sphingosine inhibited phosphorylation of the 56-kDa p rotein and the 36-kDa annexin I, whereas it enhanced that of the 43-kDa protein. The 38-kDa annexin I species was unaffected by sphing osine. As with the PKC activity, inhibition by sphingosine of phosphorylation of the 56-kDa protein and 36-kDa annexin I was reversed b y the excess addition of PS, but not by OAG or Ca 2+. In addition, by the excess addition of PS and not by OAG or Ca 2+ , the sphingosineenhanced phosphorylation of the 43-kDa protein was reversed and returned to near the level in the absence of sphingosine. It is suggested that sphingosine is involved in the regulation of PKC-dependent phosphorylation in the nucleus by modulating the association of PKC or its substrates, particularly annexin I, with membrane phospholipids in cow mammary gland. KEY WORDS: annexin I, cow mammary gland, phosphorylation, protein kinase C, sphingosine.J. Vet. Med. Sci. 66(10): 1237-1242, 2004 Protein kinase C (PKC) is a phosphotransferase activated by diacylglycerols, phosphatidylserine (PS) and Ca 2+, and plays an essential role in transmembrane signal transduction [28,36]. Responding to extracellular signals such as hormones, diacylglycerols are transiently produced from phosphatidylinositol-4,5-bisphosphate in the plasma membrane. The diacylglycerols produced increase the affinity of PKC for Ca 2+ as well as for PS, thereby activating PKC. The activated PKC phosphorylates substrate proteins particularly in the plasma membrane, and the phosphorylated proteins regulate diverse biological functions.Other than in the plasma membrane, it has recently been found that PKC is resident in the cell nucleus as well as in other intracellular organelles, or the enzyme is translocated from the cytosol to the nucleus [4,7,20,31,32,42]. In addition, accumulating evidence suggests that a wide variety of lipids, including diacylglycerols, phospholipids, cholesterols, gangliosides and sphingolipids, are distributed in the nucleus and act as second messengers for PKC-dependent phosphorylati...

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