Ceramide promotes calpain-mediated proteolysis of protein kinase C beta in murine polymorphonuclear leukocytes.
Ceramide has been recognized as an important second messenger in intracellular signaling. We demonstrate here that ceramide promotes the down-regulation of protein kinase C (PKC) activity in phorbol ester-stimulated murine polymorphonuclear leukocytes (PMNs). As reported previously, treatment of PMNs with phorbol ester caused a translocation of PKC from the cytosolic to the membrane fractions. When PMNs were pretreated with cell-permeable ceramide analogue, C2-ceramide, the membrane-associated PKC activity was rapidly down-regulated by phorbol ester stimulation. E64-d, a potent inhibitor of calpain which proteolyzes PKC, eliminated the rapid down-regulation of PKC activity. By hydroxyapatite column chromatography and Western blotting, the predominant PKC isoform was PKC beta with a small amount of PKC alpha in murine PMNs. We found that ceramide strikingly promoted calpain-mediated proteolysis of PKC beta in vitro. Ceramide was also shown to inhibit [3H]phorbol 12,13-dibutyrate(PDBu) binding to PKC beta. Moreover, we show that ceramide stimulates PKC beta autophosphorylation. These results suggest that ceramide directly activates PKC beta and promotes calpain-mediated proteolysis in murine PMNs.
Tanabe
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Department of Microbiology, Yamanashi Medical University, Japan. ftanabe@res.yamanashi-med.ac.jp
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D000818:Animals; D002154:Calpain / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D015536:Down-Regulation; D007527:Isoenzymes / Q000378:metabolism*; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D064546:Protein Kinase C beta; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology
D015853:Cysteine Proteinase Inhibitors; D007527:Isoenzymes; C064769:N-acetylsphingosine; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D002154:Calpain; D007930:Leucine; C108192:aloxistatin; D013110:Sphingosine