The kinase Raf-1 can be activated by treatment of cells with mitogens and by the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) (reviewed in refs 1,2). Activated Raf-1 triggers a protein kinase cascade by direct phosphorylation of MAP kinase kinase, resulting in phosphorylation of ternary complex factor and Jun by MAP kinase. Here we investigate the molecular mechanism and biological consequences of PKC alpha-mediated Raf-1 activation in NIH3T3 fibroblasts. PKC alpha directly phosphorylates and activates Raf-1 both in vitro and in vivo. PKC alpha induces Raf-1 phosphorylation at several sites, including a serine residue at position 499. Mutation of serine at this position or at residue 259 does not abrogate Raf-1 stimulation by a combination of Ras plus the src tyrosine kinase Lck, but severely impedes Raf-1 activation by PKC alpha. Consistent with such a direct interaction is the observation that Raf-1 and PKC alpha cooperate in the transformation of NIH3T3 cells. The Ser499 phosphorylation site is necessary for this synergism.

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Interleukin 1 is the prototype of an inflammatory cytokine, and evidence suggests that it uses the sphingomyelin pathway and ceramide production to trigger mitogen-activated protein kinase (MAPK) activation and subsequent gene expression required for acute inflammatory processes. To identify downstream signaling targets of ceramide, a radioiodinated photoaffinity labeling analog of ceramide ([1251]3-trifluoromethyl-3-(m-iodophenyl)diazirineceramide) was employed. It is observed that ceramide specifically binds to and activates protein kinase c-Raf, leading to a subsequent activation of the MAPK cascade. Ceramide does not bind to any other member of the MAPK module nor does it bind to protein kinase C-c. These data identify protein kinase c-Raf as a specific molecular target for interleukin 1,8-stimulated ceramide formation and demonstrate that ceramide is a lipid cofactor participating in regulation of c-Raf activity.Interleukin 1 (IL-1) is a major product of activated monocytes and is also released by many cell types when exposed to an inflammatory environment. The biological activities of IL-1 are initiated by binding to two types of IL-1 receptors, designated IL-1 receptors type I and type II. Although the cytoplasmic portions of both IL-1 receptors do not contain kinase domains or motifs homologous to any other known signaling pathway, rapid intracellular protein phosphorylation occurs in response to IL-1 stimulation. The nature of the primary signal delivered by IL-1 receptor activation is poorly understood and controversial (1), but it is clear that IL-1 does stimulate protein kinase activities in a wide variety of different cell types. IL-1 and tumor necrosis factor a have been shown to induce rapid mitogen-activated protein kinase (MAPK) activation in fibroblasts, U937 cells, KB cells, and mesangial cells (2-6). Furthermore, both cytokines employ the sphingomyelin signaling pathway to generate ceramide and to stimulate a putative ceramide-activated serine/threonine protein kinase (7-9). Lipid second messengers are increasingly recognized as important mediators of extracellular signals, and ceramide generated by the action of neutral and acidic sphingomyelinases has been implicated in a variety of physiological processes, like effects on cell growth, differentiation, and apoptosis (8, 9).Rat renal mesangial cells are a well-defined IL-i-responsive cell type that is involved in most pathological processes of the renal glomerulus (10, 11). Resting mesangial cells do not produce any inflammatory mediator constitutively but require a triggering by invading immune cells. Three prominent features of mesangial cells evolve as a result of the crosstalk with invading neutrophils and macrophages, as follows: increased mediator production, increased matrix synthesis, and increased mesangial cell proliferation (10, 11). IL-1 has been reported to induce the expression of a specific type IV collagenase, a group II phospholipase A2, eicosanoids, an inducible nitric oxide synthase, and a variety of chemokines in...

MiR-148a sensitized chemotherapy-sensitive oesophageal cancer cell lines to cisplatin and, to a lesser extent, to 5-flurouracil and attenuated resistance in chemotherapy-resistant variants. Further experimental and clinical studies to investigate the exact mechanisms involved are warranted.