Recent investigations have identified a new signal transduction pathway, termed the sphingomyelin pathway, that may mediate the action of tumor necrosis factor (TNF)-alpha and interleukin 1 (IL-1) Beta. This pathway is initiated by hydrolysis of plasma membrane sphingomyelin to ceramide by the action of a neutral sphingomyelinase. Ceramide functions as a second messenger stimulating a Ser/Thr proteins kinase termed ceramide- activated protein kinase to transduce the signal. Three lines of evidence support this notion. Firstly, the sphingomyelin pathway is activated within seconds to minutes by TNF in HL-60 cells and by IL-1 in human dermal fibroblasts and mouse EL4 T helper cells. Secondly, cell- permeable ceramide analogs bypass receptor activation and directly mimic cytokine action. Thirdly, the effects of TNF and IL-1 on this cascade have been reconstituted in cell-free extracts indicating tight coupling of this pathway to the respective receptors. This grant proposal focusses on the elucidation of the role of ceramide-activated protein kinase in this signaling cascade. Until now, this activity has only been defined as a Mg2+ dependent, membrane-bound activity capable of phosphorylating a peptide derived from this amino acid sequenced surrounding Thr669 of the epidermal growth factor receptor. This is the same site recognized by mitogen-activated protein (MAP) kinases (also known as extracellular signal-regulated protein kinases). Hence, ceramide-activated protein kinase may belong to an emerging family of proline-directed Ser/Thr protein kinases, which includes MAP and cdc2 kinases. These kinases recognize substrates containing the minimal motif, X-Thr/Ser-Pro-X, where the phosphoacceptor site is followed on the carboxyl-terminus by a proline residue and X can be any amino acid. This grant application has three goals: (1) To define the structural determinants for substrates recognition by ceramide-activated protein kinase and for ceramide stimulation, and determine the intracellular localization of enzyme activity; (2) to purify ceramide-activated protein kinase; and (3) to define how known antagonist of TNF action might act upon the sphingomyelin pathway and, in particular, ceramide-activated protein kinase. It is our belief that ceramide-activated protein kinase represents a new target for pharmacologic intervention in TNF and IL-1 action. Hence, the title of this application has been changed to reflect this emphasis. Hopefully, these studies will begin the process necessary for testing this hypothesis.
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