| Abstract: |
Recent investigations have identified a signaltransduction system involving sphingomyelin and derivatives. In this paradigm, sphingomyelin hydrolysis by a sphingomyelinese generates ceramide, which may be converted to the protein kinase C inhibitor sphingosine or to ceramide 1-phosphate. Ceramide may have second-messenger function because it induces epidermal growth factor receptor phosphorylation, presumably on Thr-669 in A-431 cells. The present studies describe a kinase that may mediate ceramide action. With a 19-amino acid epidermal growth factor receptor peptide containing Thr-669, a membrane-bound activity that phosphoryelated the peptide was detected in A-431 cells. Activity was linearly related to ATP (0.3-300 μM) and peptide concentration (0.02-1 mg/ml), possessed a physiologic pH optimum (pH 7.0-7.4), and was Mg22+-dependent. Other cations-Ca2+, Mn2+, and Zn2+ - were ineffective. Natural and synthetic ceramide induced time- and concentration-dependent enhancement of kinase activity. Ceramide (0.5 μM) increased kinase activity 2-fold by 30 s, and activity remained elevated for at least 15 min. As little as 0.001 μM ceramide was effective, and 1 μ ceramide induced maximal phosphorylation. Sphingosin was similarly effective. Because tumor necrosis factor (TNF) α rapidly induces sphingomyelin hydrolysis to ceramide during monocytic differentiation of HL-60 cells, its effects on kinase activity were assessed. Kinase activity was increased 1.5-fold at 5 nun and 2-fold at 2 hr in membranes derived from TNF-stimulated cells. The effective concentration range was 3 pM-30 nM TNF. Exogenous ceramide induced a similar effect. In sum, these studies demonstrate the existence of an unusual Mg2+-denendent ceramide-activated protein kinase that may Mediate some aspects of TNF-α function. |
| Keywords: |
signal transduction; human cell; carcinoma, squamous cell; protein kinases; cell line; receptor, epidermal growth factor; enzyme activation; leukemia, promyelocytic, acute; amino acid sequence; molecular sequence data; tumor necrosis factor alpha; kinetics; cell membrane; peptides; sphingosine; phosphates; protein kinase; carcinoma cell; tumor necrosis factor; ceramide; ceramides; magnesium; phosphopeptides; cell strain hl 60; phosphorus radioisotopes; human; priority journal; article; support, non-u.s. gov't; support, u.s. gov't, p.h.s.
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