| Abstract: |
Recent investigations identified a new signal transduction pathway, termed the sphingomyelin pathway, which may mediate the action of tumor necrosis factor (TNF)α and interleukin-1β (Mathias, S., Younes, A., Kan, C., Orlow, I., Joseph, C., and Kolesnick, R. N. (1993) Science 259, 519-522). This pathway is initiated by hydrolysis of sphingomyelin to ceramide by a neutral sphingomyelinase and stimulation of a ceramide-activated Ser/Thr protein kinase. Recent investigations demonstrated that kinase activity is proline- directed, recognizing substrates in which the phosphoacceptor site is followed by a proline residue. Until now, the kinase has been defined only as a membrane-bound activity capable of phosphorylating a peptide derived from the sequence surrounding Thr669 of the epidermal growth factor receptor. In the present studies, the kinase was quantitatively extracted from membrane with detergent and separated from protein kinase C by anion-exchange chromatography and isoelectric focusing. Ceramide-activated protein kinase was resolved as an exclusively membrane-bound, 97-kDa protein with a pI of 7.05. Kinase activity toward the epidermal growth factor receptor peptide co- purified with activity toward a generic proline-directed substrate, myelin basic protein. Kinase activity was reconstituted by a denaturation- renaturation procedure and demonstrated activity toward self (autophosphorylation) and exogenous substrate (myelin basic protein). Autophosphorylation occurred exclusively on serine residues. These activities were enhanced to 7-fold of control by ceramide and TNFα. These investigations provide additional evidence for a role for ceramide-activated protein kinase in signal transduction for TNFα. |
| Keywords: |
signal transduction; controlled study; human cell; cell line; enzyme activation; tumor cells, cultured; enzyme activity; autophosphorylation; phosphorylation; leukemia, promyelocytic, acute; enzyme phosphorylation; tumor necrosis factor alpha; kinetics; enzyme analysis; protein-serine-threonine kinases; substrate specificity; cell membrane; protein kinase c; protein folding; molecular weight; protein kinase; electrophoresis, polyacrylamide gel; tumor necrosis factor; detergents; protein denaturation; cell strain hl 60; enzyme isolation; membrane enzyme; human; priority journal; article; support, non-u.s. gov't; support, u.s. gov't, p.h.s.
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