Ceramide-binding and activation defines protein kinase c-Raf as a ceramide-activated protein kinase.

Abstract

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...

The sphingolipid salvage pathway in ceramide metabolism and signaling

“…Extensive studies using these short-chain ceramides have been performed to examine the biological aspects of ceramide, revealing intracellular ceramide targets as well as various ceramide signaling pathways [51][52][53][54][55]. Importantly, recent studies also shed light on the metabolic fate of short-chain ceramides [18,22,56] (Fig.…”

“…Successfully, some ceramide targets have been identified, including PKCζ [52], kinase suppressor of Ras [78], cRaf [51], cathepsin D [79], and CAPPs composed of protein phosphatase 1 (PP1) and protein phosphate 2A (PP2A) [80][81][82]. CAPPs belong to the family of Ser/Thr protein phosphatases, and ceramide has been shown to activate CAPPs leading to the dephosphorylation of various proteins including, Bax, Bcl-2, PKCα, Rb protein, SR protein, and Akt, ultimately modulating/mediating various responses of cells [29,83].…”

Recent advances in the immunobiology of ceramide

“…TNFα and ceramide induced activation of ERK1/2 and involves KSR and Raf-1. BAD enables ceramide-mediated apoptotic signaling via Ras and Raf-1 (Huwiler et al, 1996;Yan and Polk, 2001;Basu et al, 1998). Bax has been demonstrated to be a critical regulator of ceramide-induced apoptotic pathway, upstream of cytochrome c release.…”

“…Ceramide has been implicated in diverse signaling cascades that involve protein kinases viz. SAPK, JNK (Verheij et al, 1996), PKC ζ (Bourbon et al, 1992), kinase suppressor of Ras (KSR) (Zhang et al, 1997), Raf (Huwiler et al, 1996), double stranded RNA-dependent protein kinase (PKR) as well as protein phosphatases such as PP2A and PP1 (Ruvolo et al, 2001;Chalfant et al, 1999). Ceramide-induced apoptosis usually involves the SAPK/JNK signaling pathway (Verheij et al, 1996).…”

Fas/CD95/Apo-I activates the acidic sphingomyelinase via Caspases

“…The regulatory mechanisms of sphingomyelinase activation by the Fas receptor are completely unknown; however, recent data suggest that reaper triggered release of ceramide depends on the function of caspases (Pronk et al, 1996), implying that sphingomyelinases and ceramide are downstream targets of caspases. Ceramide has been shown to stimulate a variety of enzymes including a phosphatase (Joseph et al, 1993), a ceramide activated proline-directed protein kinase (Dobrowsky et al, 1993), Jun-N-terminal kinase (Verheij et al, 1996;Westwick et al, 1995), Raf-K (Huwiler et al, 1996) and tyrosine phosphorylation (Ji et al, 1995).…”