title: Renaturation and tumor necrosis factor-alpha stimulation of a 97-kDa ceramide-activated protein kinase.
abstract: Recent investigations identified a new signal transduction pathway, termed the sphingomyelin pathway, which may mediate the action of tumor necrosis factor (TNF) alpha and interleukin-1 beta (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 TNF alpha. These investigations provide additional evidence for a role for ceramide-activated protein kinase in signal transduction for TNF alpha.
authors.affiliation: Laboratory of Signal Transduction, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
authors.forename: J
authors.initials: J
authors.lastname: Liu
authors.forename: S
authors.initials: S
authors.lastname: Mathias
authors.forename: Z
authors.initials: Z
authors.lastname: Yang
authors.forename: R N
authors.initials: RN
authors.lastname: Kolesnick
mesh_terms: D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D008970:Molecular Weight; D010766:Phosphorylation; D011489:Protein Denaturation; D017510:Protein Folding; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D003902:Detergents; D014409:Tumor Necrosis Factor-alpha; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C