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Signal transduction through lipid second messengers
Signal transduction through lipid second messengers Sarah Spiegel*, This review emphasizes sphingolipid
second
David Foster? and Richard Kolesnickz
the generation
messengers,
The role of the phosphatidylinositol phospholipase
of glycerolipid
and
and their molecular targets. transfer protein and
D in signal transmission, and the structures
of the 1,2-diacylglycerol and calcium-binding sites of protein kinase C are discussed. Further, ceramide signaling through protein kinases and the role of cross-talk in the signaling of apoptosis and inflammation are addressed.
Addresses *Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, DC 20007, USA *Department of Biological Sciences, Hunter College of City University of New York, 695 Park Ave, New York, NY 10021, USA Xaboratory of Signal Transduction, Memorial Sloan-Kettering Cancer Center, New York NY 10021, USA Current Opinion in Cell Biology 1996, 8:159-l
67
0 Current Biology Ltd ISSN 0955-0674 Abbreviations CAPK ceramide-activated protein kinase CAPP ceramide-activated protein phosphatase DAG 1,2-diacylglycerol IL interleukin LPS lipopolysaccharide LPA lysophosphatidic acid MAPK mitogen-activated protein kinase NGF nerve growth factor PA phosphatidic acid PC phosphatidylcholine PDGF platelet-derived growth factor PH pleckstrin homology PI phosphatidylinositol PIP* PI 4,5-bisphosphate PITP phosphatidylinositol transfer protein PKC protein kinase C PLC phospholipase C PLD phospholipase D SH Src homology SPP sphingosine 1 -phosphate TNF tumor necrosis factor
Introduction Ceramide, the second messenger of the sphingomyelin pathway, like its more widely studied counterpart in the phosphoinositide pathway, 1,2-diacylglycerol (DAG), exists in mammalian cells at the level of l-Z% with respect to total phospholipid concentration [l-4]. Ceramide and DAG not only serve as second messengers, but also as the backbone of all phospholipids. As structural lipids, they are found in bound and free forms distributed throughout all cellular membranes. In response to the activation of cell surface receptors, the levels of the free lipids increase 1.4-2.0-fold above control levels [l-4]. These quantities grossly underestimate the increase in the signaling pools, which are generally considered to contain little or no second messenger in the absence
of stimulus. Unfortunately, conventional assays cannot distinguish between the structural and signaling pools of these lipids and measure stimulated elevations over the entire cell. Signal generation is followed by signal amplification, an observation which helps to explain how small transient signals generated at the cell surface can induce pleiotropic biological responses. The study of lipid messengers has been greatly aided by the use of synthetic lipid analogs which usually retain stereospecific activation but sacrifice potency [la]. Recent investigations have begun to clarify both the mechanisms for generating this class of messengers and their direct molecular targets. A synthesis is emerging that recognizes the integration of lipid second messengers and protein kinase cascades. In this regard, many lipid second messengers have an inherent ability to cross bilayers and connect compartments that are normally separated under resting conditions. Here, we review some exciting findings of the past year, and emphasize mechanisms for generating lipid second messengers and identifying their molecular targets.
Signaling via glycerolipid-derived messengers Phospholipase
second
C
Although a role for DAG as a second messenger of the phosphoinositide pathway is well established [ 1,2], many questions remain unanswered regarding its generation and its ability to activate protein kinase C (PKC). The phosphoinositide-specific phospholipases C (PLCs), which hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP*) to generate DAG, fall into three groups: p, y and 6 [S]. All three PLC isoforms are single polypeptides containing two regions of high homology, designated X and Y, preceded by a pleckstrin homology (PH) domain. In addition, PLC-)I has multiple Src homology (SH)Z domains and a single SH3 domain. Two mechanisms have been described for PLC activation: PLC-fi activation through serpentine receptors is mediated via heterotrimeric G proteins, whereas PLC-y binds to receptor tyrosine kinases or receptor-associated tyrosine kinases via its SH2 domains and is activated by phosphorylation. PLC-p activation occurs either via GTP-bound CL subunits of the Gq class or via By subunits. Gqa interacts with a region distal to the Y domain of PLC-fl whereas the @y units bind to an amino-terminal segment, perhaps via the PH domain. Thus, structural elements of PLC-B determine G-protein interactions. Further, the sensitivity of distinct PLC-B isozymes to a and &r subunits differs, undoubtedly contributing to the quality of the ultimate response. With respect to PLC-y activation, the sites that bind PLC-y on the epidermal growth factor, platelet-derived growth factor (PDGF), nerve growth factor (NGF) and fibroblast
160
Cell regulation
growth factor receptors have been mapped. Further, all of these receptor kinases are now known to phosphorylate the same tyrosine residues on PLC-y and thereby increase its enzymatic activity [S]. A major finding involves the recognition of the role of phosphatidylinositol transfer protein (PITP) in signaling through either PLC-fl or PLC-y [6,7”]. Although PITP transfers phosphatidylinositol (PI) and not the substrate of PLC, PIP2, PITP was found to be necessary for sustained PLC signaling in reconstitution assays using streptolysin 0 permeabilized cells. Presumably, PITP allows refilling of the pool of PIP2 by presenting PI to PI 4-kinase. It has also been suggested that PITP potentiates phospholipase D (PLD) activity by increasing the cellular content of PIP2, which is a putative PLD cofactor [8].
Phospholipase
D
PLD hydrolyzes phosphatidylcholine (PC) to phosphatidic acid (PA) and choline in response to various extracellular stimuli [8,9]. PA has been implicated as a lipid second messenger in the regulation of protein kinases, GTPase-activating proteins, PI kinases, adenylate cyclase and other signaling molecules [lo]; however, direct effects of PA have not been established and many effects are probably mediated by the PA metabolites DAG and lysophosphatidic acid (LPA) [l,ll]. PLD has also been implicated in membrane trafficking and vesicular transport [12-141, in which processes acidic phospholipids may facilitate membrane budding and/or fusion [ 151. Two biochemically distinguishable PLD activities have been characterized; one is dependent upon the small GTPase Arf and upon PIP2 [16**,17], and another is stimulated by oleate [18]. At late stages of purification, the Arf-dependent PLD activity is lost, presumably due to loss of cofactors [14,15,16”]. Recently, a gene required for meiosis in &c&romyces cermisiae [19**] was isolated and shown to have sequence homology to a castor bean PLD [ZO]. A human homolog has been cloned which possesses PLD activity that is dependent upon Arf and PIP2 but not upon oleate [Zl”]. A second PLD gene has now been cloned from a murine source. Substantial progress has been made in characterizing mechanisms of PLD activation. PLD activation by v-Src depends upon a GTPase cascade containing Ras [ZZ”] and Ral [23”]. Ral constitutively associates with PLD through Ral’s novel amino terminus, but Ral does not activate PLD alone [23”]. Although it is not known whether Ral-associated PLD depends upon Arf, as the Arf-dependent PLD does, the Ral-associated activity does depend upon PIP2 [23”]. Evidence also implicates Rho in PLD activation [24,25] suggesting a complex interplay of multiple small GTPases.
Protein kinase C
PKC is the only clearly defined target for DAG [2]. PKC consists of isoforms which share a common requirement for phospholipid and which are divided into three groups: conventional PKCs (cPKCs IX, pl, g2 and y), new PKCs (nPKCs 6, E, o and p) and atypical PKCs (aPKCs < and A) [l]. DAG, or its analog phorbol ester, activates cPKCs and most nPKC isoforms but not aPKCs. Calcium is a cofactor expressly required for the cPKC forms, increasing their affinity for acidic phospholipids. Recently, lysophosphatidylcholine (LPC) and free fatty acid (FFA), the products of hydrolysis of PC by phospholipase AZ, have been shown to enhance DAG-induced activation of some PKC isoforms (LPC and FFA co-activate all cPKCs, and FFA also activates PKCE). LPC and FFA are inactive alone. In contrast, FFA inhibits DAG-induced PKCG activation. These studies demonstrate cooperativity between lipid classes in PKC activation.
This heterogeneity in lipid activation, coupled with evidence that PKC isoforms occupy specific subcellular compartments [ 1,2], suggests that specific intracellular signaling mechanisms will eventually be deciphered. In this regard, it has been proposed that the PKCc( isoform directly phosphorylates and activates the proto-oncoprotein Rafl to initiate signaling via the mitogen-activated protein kinase (MAPK) cascade [26]. Although this proposal has been disputed, in many cell systems PKC activation does lead to Rafl activation [2]. Other direct targets for activation by PKC have been described, including MARCKS, myogenin, lamin B and P-glycoprotein [2]; however, a comprehensive pathway for cellular activation by PKC remains to be defined.
Recent investigations provided substantive information on the structural elements of PKC that mediate lipid activation [27]. The lipid-binding moieties of cPKCs had long been known to reside in two conserved modular domains (Cl and C2). Resolution of the crystal structure of the second cysteine-rich region of the Cl domain of PKCy bound to phorbol ester revealed that the phorbol ester binding site is formed by p sheets, contained in the top half of the Cl domain, that are pulled apart to form a cavity [28”]. Insertion of phorbol ester into this site does not alter the conformation of this domain but rather results in a surface that is continuously hydrophobic, perhaps allowing for efficient interactions with membranes. The lower half of the domain contains the metal-binding sites and appears to be responsible for maintaining the appropriate conformation of the domain. PKC isoforms that contain Cl regions but do not bind phorbol ester lack a particular conserved proline residue [27].
Sprang and coworkers [29**] have recently resolved the crystal structure of the C2 domain of synaptotagmin, an enzyme which, like PKC, binds to acidic membranes in
Signal transductionthrough lipid second messengers Spiegel, Foster and Kolesnick
a Ca2+-dependent manner. This domain is composed of a four-stranded fl sheet which contains a Ca2+-binding site constructed of a set of aspartate residues which are conserved in PKC. Caz+ binding may better orient a region of aromatic amino acids for hydrophobic interaction with membranes, and a region of basic residues for interaction with acidic phosphohpids. Caz+-independent PKCs may substitute a positively charged amino acid for Ca2+, allowing for a similar orientation of this active site. The interaction of these domains of PKC with membranes results in tight binding of PKC to membranes (i.e. in PKC translocation), in removal of the auto-inhibitory pseudosubstrate domain from the active site, and PKC activation. Activation of PKCs in specific membranes also appears to involve a group of proteins collectively known as receptors for activated protein kinase C (RACKS) [30]. RACK1 has been cloned [31**]. These studies have begun to unravel the mystery of the mechanism by which lipid second messengers initiate signaling. Lysophosphatidic acid
Recently, second messenger function has been ascribed to LPA, which is released from activated cells, especially platelets, and elicits diverse biological responses in different systems including platelet aggregation, cell growth [ 11,321 and neurite retraction [11,33]. LPA may also play a role in inflammatory and proliferative responses to injury [ 111. LPA binds to a 38-40 kDa receptor which couples, via distinct G proteins, to multiple independent effector pathways. The a subunit of Gi directly inhibits adenylate cyclase, whereas the &u dimer is thought to activate Ras via an intermediary protein tyrosine kinase. The LPA receptor, via a mechanism involving Gq and stimulation of PLC, also activates Rho, resulting in stimulation of focal adhesion kinase and phosphorylation and reorganization of cytoskeletal proteins [34’].
Sphingolipid-derived
second messengers
Sphingolipid metabolism
Because details of the metabolism of sphingolipids are not as well known as are details of glycerolipid metabolism, this section starts with a brief overview of this topic (Fig. 1) [35]. The sphingolipid biosynthetic pathway begins with the condensation of serine with palmitoylCoA to form 3-ketodihydrosphingosine or 3-ketosphinganine, a reaction catalyzed by serine palmitoyl transferase. 3-ketodihydrosphingosine is then reduced by an NADPHdependent reductase to dihydrosphingosine (sphinganine); acylation with fatty acyl CoA to produce dihydroceramide follows. Recent evidence suggests that dihydroceramide is converted directly to ceramide, the precursor of all complex sphingolipids, by the introduction of a trans-4,5 double bond [35]. The transfer of a phosphorylcholine head group from phosphatidylcholine to ceramide by sphingomyelin synthase yields sphingomyelin. Hydrolysis of sphingomyelin, catalyzed by sphingomyelinase, a sphingomyelin-specific PLC, yields ceramide and phosphorylcholine. Cleavage of the amide-
161
linked fatty acid from ceramide to form sphingosine is catalyzed by ceramidases. Sphingosine can be metabolized further, via a pathway involving sphingosine kinase catalyzed phosphorylation, to sphingosine l-phosphate (SPP), and SPP can be cleaved to ethanolamine phosphate and trans-Z-hexadecanal by a specific lyase located in the endoplasmic reticulum. Ceramide as a second messenger
The past two years have seen the emergence of sphingolipids as second messengers [3,4]. The sphingomyelin pathway is a ubiquitous, evolutionarily conserved signaling system analogous to more well defined systems such as the CAMP and phosphoinositide pathways. Sphingomyelin is a phospholipid preferentially concentrated in the outer layer of the plasma membrane of mammalian cells, and was originally considered to be a structural element of that compartment, providing a rigid barrier to the environment. It is now clear that sphingomyelin hydrolysis by sphingomyelinase initiates pleiotropic cellular responses. Activation of sphingomyelinase has been linked to several cell surface receptors (the 55 kDa tumor necrosis factor [TNF] receptor, the 80 kDa interleukin [IL]-1 receptor, the 75 kDa NGF receptor, and Fas) as an immediate event, and may be a primary mediator of their biological function [3,4]. Alternatively, ceramide may be generated as a downstream event in the response pattern for some receptors (those of Vitamin D3, progesterone, and CD28). In this latter category, ceramide generation may nevertheless be critical in order to allow the ultimate biological response to proceed to completion. Classes of sphingomyelinase
There are two classes of sphingomyelinase, defined by their optimal pHs, that may signal cellular activation [36**]: an acidic isoform (pH optimum -pH5), and two forms with neutral pH optima. One of the neutral isoforms is a Mgz+-dependent membrane-bound enzyme, and the other is a Mgz+-independent cytosolic enzyme. Although the acidic sphingomyelinase was originally considered to reside exclusively in lysosomes, Sandhoff and coworkers [37] demonstrated that a fraction is targeted to compartments other than lysosomes. Furthermore, Kronke and coworkers [36”] suggested that activation of acidic sphingomyelinase occurs in endosomes subsequent to receptor internalization and acidification. Stimuli linked to activation of neutral sphingomyelinase include TNFa, IL-lb, Vitamin D3, NGF, ionizing radiation and Fas, whereas activation of acidic sphingomyelinase has been associated with TNFa, Fas and CD28 (reviewed in [35]). Kronke and coworkers [36”] used mutants of the cytoplasmic domain of the 55 kDa TNF receptor to show that different receptor domains link to these distinct sphingomyelinases. A membrane-proximal region of the cytoplasmic domain linked the neutral sphingomyelinase to the MAPK cascade, whereas the carboxyl terminus of the TNF receptor connected acidic sphingomyelinase to NFKB activation. These authors proposed that activation
162
Cell regulation
Figure 1
0
Cop + &ASH 0 CH20H
3-Ketosphinganine
tNHs
I
Sphi”g~CHzOH +NH,
@
Fat!y acyl-CoA I
&CH*OH NH D-m
0
t
OH CH20P03-H2
4_
@
OH
ATP
CH20H NH
6 cCeramide
l-phosphate
4
Fatly acyl-CoA
Sphingosine
+NH3
0
-=?i+ PI PC 0 DG
OH
CH20P03-CH*CH2rj(CH3)3 0
NH
A Hexadecanal
CHO
I;Hs/\
CH20P0s-H2
Phosphoefhanolamine
Metabolism of sphingolipids. The enzymes catalyzing the numbered reactions are: (1) serine palmitoyltransferase; (2) 3-ketosphinganine reductase; (3) ceramide synthase; (4) dihydroceramide saturase; (5) ceramide kinase; (6) ceramide 1-phosphate phosphatase [79,60]; (7) sphingomyelin synthase; (8) sphingomyelinase; (9) ceramidase; (10) sphingosine kinase; (11) sphingosine l-phosphate phosphatase [al]; (12) sphingosine lyase. DG represents 1,2-diacylglycerol.
of acidic sphingomyelinase occurred in response to DAG generated via a PC-specific PLC. However, the existence of such an enzyme remains uncertain. These studies suggest compartmentalization of signaling for these different sphingomyelinases. In this regard, Linardic and Hannun [38*] have localized one TNF-responsive pool of sphingomyelin specifically to the inner surface of the plasma membrane. Now that knockout mice for the acidic sphingomyelinase have become available [39,40], it is likely that these signaling pathways will be clarified in the near future. Targets for ceramide action
A number of direct targets for ceramide action have now been identified, including a ceramide-activated protein kinase (CAPK) [41], a ceramide-activated protein phosphatase (CAPP) [42], the putative guanine-nucleotide exchange factor Vav [43] and the PKC< isofoim [44”,45].
CAPK is a 97 kDa plasma membrane bound serine/threonine protein kinase with a preference for the sequence X-Ser/Thr-Leu-Pro-X in which the phosphoacceptor site (serine or threonine) is amino-terminal to a proline residue, and X may be any amino acid [46]. Substrate phosphorylation by CAPK and CAPK autophosphorylation are enhanced by nearly tenfold upon elevation of cellular ceramide levels with either ceramide analogs or TNFa. CAPP is a member of the protein phosphatase 2A class of Ser/Thr protein phosphatases which are expressed as heterotrimers with distinct functional domains [47]. Catalytic activity resides within the C subunit, whereas sensitivity to ceramide is conferred by the B subunit. Increased availability of ceramide may trigger CAPP translocation from the cytoplasm to membranes, where dephosphorylation of phosphoprotein substrates would presumably occur. CAPP activity has been closely correlated with growth inhibition in numerous systems.
Signal transductionthrough lipid second messengers
Ceramide may also stimulate the guanine nucleotide exchange activity of the proto-oncoprotein Vav, a putative activator for Ras in hematopoietic cells [43].
of inflammation
Foster and Kolesnick
163
Figure 2
Finally, PKC<, an atypical PKC which is insensitive to phorbol ester or DAG, may also be a direct target for ceramide stimulation [44”,45]. Treatment with either TNFa or ceramide enhanced PKC< phosphorylation and activity in U937 and NIH3T3 cells, and ceramide increased PKC< activity in h-o. PKC< activation via ceramide may link the TNF receptor to NFKB. Signaling
Spiegel,
6 Sphingomyelin SMase
and apoptosis via ceramide
The most comprehensive data on second messenger function of ceramide have been published for TNFa [1,2]. Ceramide appears to be a primary mediator of both the inflammatory and the apoptotic responses to TNFa (Fig. 2). With respect to the inflammatory response, ceramide stimulates CAPK to phosphorylate Rafl on Thr269, enhancing Rafl activity. This initiates signaling thorough the MAPK cascade [48.*], and results in activation of cytosolic phospholipase AZ and release of arachidonic acid (R Schatzman, R Heller, RA Kolesnick, unpublished data). Lipopolysaccharide (LPS), which mediates the effects of Gram-negative bacteria on mammalian systems, mimics many of the early actions of TNFa and IL-la. Molecular modeling of carbons l-3 of the reducing saccharide of the lipid A moiety of LPS and its associated fatty acids revealed close structural similarity to ceramide [49**]. LPS also activates CAPK in a CD14-dependent manner, suggesting that LPS may coopt the second messenger function of ceramide in order to initiate inflammatory responses. With regards to apoptosis, TNFa induces rapid hydrolysis of sphingomyelin to ceramide in all cells in which it initiates apoptosis, and cell-permeable analogs of ceramide mimic this initiation of apoptosis in a stereospecific manner. Analogs of other lipid second messengers, including analogs of DAG, arachidonic acid and PA, are ineffective. Studies published within the past year provide strong evidence that ceramide also signals Fas-mediated apoptosis [50,51]. Ceramide-mediated apoptosis extends beyond apoptosis induced by cytokine receptors. Ionizing radiation appears to directly target membranes, resulting in hydrolysis of sphingomyelin to ceramide within seconds [52”]. This event may initiate an interphase form of apoptotic cell death. Further, the chemotherapeutic drug daunorubicin may initiate apoptosis by a mechanism involving ceramide synthesis, by activation of ceramide synthase [53”]. Ceramide synthesis appears to be obligatory, as fumonisin Br, a fungal inhibitor of ceramide synthase, blocked daunorubicin-induced ceramide synthesis and apoptosis [53”]. In total, these studies suggest that ceramide acts as a generic signal for several forms of apoptosis. Preliminary investigations suggest that the stress-activated protein kinase/c-Jun kinase signaling system, and not the MAPK cascade, is a downstream effector
Raf-1
MEKKl
MEK
SEKl
9 MAPK
SAPK
0
9
CPLA,
c-Jun
9 AA
9 Inflammation
Apoptosis 0 1996 Current Opinion m CM Biology
Signaling systems engaged by ceramide. Activation of the 55 kDa TNF receptor stimulates neutral and acidic sphingomyelinase (SMase) isoforms, resulting in ceramide generation. Ceramide can stimulate CAPK in the plasma membrane to phosphorylate and activate Raf-1, initiating signaling though the MAPK cascade. Alternatively, ceramide, by an unknown mechanism, appears to initiate signaling through the stress-activated protein kinase/Jun kinase cascade in cells in which it mediates
an apoptotic
cytosolic phospholipase
response.
MEK, MAPWERK
As; AA, arachidonic
kinase 1 ; SEKl , SAPK/ERK kinase.
kinase; cPLA2,
acid; MEKKl,
kinase-1 ; SAPK, stress-activated
MEK protein
system for the initiation of apoptosis by diverse stresses ([54**,55]; M Verheij et al., personal communication; see Note added in proof). Sphingosine and SPP as mitogenie second messengers
Although originally proposed as negative regulators of PKC [56], sphingosine and derivatives may play alternative signaling roles. In this regard, sphingosine induced a mitogenic effect that was stereospecific and independent of PKC inhibition in 3T3 fibroblasts [57,58]. Further, fumonisin Br increased both the level of sphingoid bases and cell growth in these cells. It has been proposed that this ability to increase sphingoid base levels mediates fumonisin-induced carcinogenesis [.59]. Sphingosine and SPP may also regulate T-cell proliferation [60]. Growth inhibition by ISP-l/myriocin, a potent immunosuppressant which inhibits serine palm-
164
Cellregulation
itoyltransferase and consequently sphingoid base synthesis, was reversed by exogenous sphingosines or SPP [60]. Moreover, certain growth factors, such as PDGF, induced a rapid, transient elevation in sphingosine and SPP levels in fibroblasts [61], arterial smooth muscle cells [62] and glomerular mesangial cells [63*-l, and pharmacological inhibition of the generation of these sphingolipids reduced PDGF-induced cellular proliferation [61,63**]. Thus, mitogenic and apoptotic responses may be regulated, in part, through different sphingolipid second messenger molecules. In this regard, recent evidence suggests that regulation of ceramidase may determine whether signals flow via SPP or via ceramide [63**], perhaps altering the balance between these mitogenic and apoptotic lipid signals. Although these mitogenic effects appear widespread, in some cell types sphingosine inhibits cell growth, perhaps via PKC inhibition [56]. Sphingosine may even be an endogenous mediator of apoptosis [64], although its ready acylation to produce ceramide in most systems confounds this issue. Sphingosine and SPP uniformly stimulate an increase in the levels of PA, a putative mitogenic signal. Ceramide, in contrast, decreases PA levels by inhibiting PLD activation [6.5]. This latter effect may play a role in cellular senescence, during which endogenous ceramide levels are high [66]. Sphingosine and SPP mobilize calcium from internal sources via an inositol-trisphosphate-independent pathway [67,68*]. The response of calcium to SPP has many hallmarks of a receptor-mediated event, including rapidity, reversibility, and specificity. As the endoplasmic reticulum contains the kinase which converts sphingosine to SPP, all of the elements sufficient for regulated calcium signaling via these lipids appear to be topologically compartmentalized [68*]. A sphingosinephosphorylcholine-gated calcium channel, with unique pharmacological and electrophysiological properties, has recently been characterized, and may represent the SPP-gated calcium channel [69]. Downstream signaling for SPP is beginning to be deciphered. SPP engages the MAPK cascade in 3T3 fibroblasts [70’] and activates the transcription factor activator protein-l (AP-1) [71*]. Thus, AP-1 may be the site at which signals carried by sphingolipid-derived messengers such as ceramide and SPP merge with the glycerophospholipid-derived second messengers, such as DAG.
[72] and SPP (A Hall, S Spiegel, unpublished data) induce actin stress fiber formation and stimulate focal adhesion kinase in Swiss 3T3 fibroblasts. The importance of sphingolipid metabolites in cytoskeletal dynamics will be an exciting area of future research. Although many studies indicate an intracellular site of action of SPP, some of its biological effects when added exogenously, such as inhibition of motility and invasiveness, may be due to extracellular effects on cell surface receptors [73’]. SPP is stored in high concentrations in human platelets, is released upon activation by physiological stimuli, and may play a role in platelet aggregation [73*]. Gi/Go-coupled receptors may be directly or indirectly involved, as some responses to SPP are pertussis toxin sensitive [74].
Cross-talk between glycerolipids sphingolipids
and
The roles of ceramide and DAG as pro-apoptotic and anti-apoptotic stimuli, respectively, have become apparent over the past year [75,76’]. Phorbol esters, previously shown to be anti-apoptotic in diverse systems, antagonize ceramide-mediated apoptosis [77]. Although the PKC isozymes involved have yet to be identified, it is clear that DAG inhibits ceramide-induced apoptosis at numerous sites, including the sphingomyelinase and downstream of this enzyme. Furthermore, stage 1 and 2 tumor promoters and even non-tumor-promoting phorbol esters possess this capability. This effect of DAG may represent more than a result of pharmacological manipulation, as the radioprotective effect of basic fibroblast growth factor in bovine aortic endothelial cells and in oivo may involve this mechanism [78]. Although ceramide elevation induces apoptosis in many cells, when it is combined with DAG elevation differentiated function and proliferation often ensue. In these instances, it is likely that DAG abrogates the ceramide-initiated signals leading to apoptosis while allowing signals for other events to proceed. More detailed information on the elements of apoptotic and anti-apoptotic signaling would appear necessary to designate the exact sites of cross-talk. Nevertheless, costimulated cellular activation may be a primary mechanism determining the penultimate biological responses.
Note added in proof The paper referred to in the text as M Verheij et a/., personal communication, has now been accepted for publication [&?I.
Acknowledgements A potentially important effect of SPP is its interference with PDGF-stimulated actin filament assembly/disassembly, resulting in marked inhibition of cell spreading and in chemotaxis towards PDGF in human arterial smooth muscle cells [63**]. However, sphingosine
We
would
like
to
thank
communicating
results
research
ROI
of Health NIH, for
grants (NIH).
BE243 Tobacco
CA57400
from
J Engebrecht,
prior GM43880
David
Foster
the American
Research.
and CA42385
M
to publication.
Richard
and
Frohman, Sarah
CA61774
from
was supported Cancer
Society,
Kolesnick
was
and
Spiegel the
Morris
for
was supported
AJ
by
National
by grants
Institutes
CA46677
from
and 3075
from
the Council
supported
by
NIH
the
grants
Signal transduction through lipid second messengers Spiegel,
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Liu J, Mathias S, Yang 2, Kolesnick RN: Renaturstion and TNFa stimulation of a 97 kDa oeramide-activated protein klnase. J Biot Chem 1994,269:3047-3052.
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Bornfeldt KE, Graves LM, Raines EW, lgaraahi Y, Waymsn G, Yamamura S, Yatomi Y, Sidhu JS, Krebs EG, Hakomori S, Ross R: Sph~~~~ne-I-ph~p~te inhibits PDGF-endued chemotexls of human arterial smooth muscle cells: spatial and temporal modulation of PDGF chemotactic signal transduction. J Cell Biol 1995, 130:193-206.
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Wolff RA, Dobrowsky TR, Biilawska, A, Hannun YA: Role of ceremide-activated protein phosphatese in ceramide-medlated signal transduction. J Viol Chem 1994, 269:19605-l 9609.
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Yao B. Zhano Y. Delikat S. Mathias S. Basu S. Kolesnick R: Ceramlde-a&hated protein kinase is a Raf kinase. Nature 1995,378:307-310. These investigations define a mechanism which links ceramide signaling to the MAPK pathway.
..
Joseph CK, Wright SD, Bornmann WG, Randolph JT, Kumar ER, Bittman R, Liu J, Kolesnick RN: Bacterial lipopolysaccharide has structural similarity to ceramide and stimulates ceramideactivated protein klnase in myeloid cells. J Biol Chem 1994, 269:17606-l 7610. These investigations show that the iipid A moiety of LPS contains a ceramide-like stru~ure, and suggest that LPS mimics TNF and IL-1 action by taking on the second messenger function of ceramide.
Coroneos E, Martinez M, McKenna S, Kester M: Differential regulation of sphlngomyelinase and ceramidase activity by growth factor and cytoldnes: lrnpli~~~ for cellular prol~er~ion and diffe~n~a~~ J Biot Chem 1995, 270:23305-23309. These investigations suggest that the pleiotropic responses induced by growth factors, and by cytokines involved in cell growth and survival, might be a consequence of regulation of the levels of distinct sphingolipid-derived second messengers. 64.
Ohta H, Sweeney EA, Maaamune A, Yatomi Y, Hakomori S, lgarashi Y: Induction of apoptosls by sphi~o~~ In human leukemfc HL-60 cells: e possible endogenous modulator of apoptotic DNA fragmentation occurring during phorbol esterinduced differentiation. Cancer Res 1995, 55:691-697.
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Gonez MA, Martin A, O’Brien L, Brindley DN: Cell-permeable ceramides inhibit the stimulation of DNA synthesls and phospholipase D activity by phosphstldate and lysophospha~d~e in rat fibroblasts. J Biol Chem 19Q4, 269:6937-8943.
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Venabie ME, Blobe GC, Obeid LM: Identification
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Cifone MC, De Maria R, Roncaioli P, Rippo MR, Azuma M, Lewis LL, Santoni A, Testi R: Apoptotic signaling through CD95(Fas/Apo-1) activates an acidic sphingomyelinase. J Exp Med 1993,177:1547-l 552. Gulbins E, Bissonette R, Mahboubi A, Martin S, Nishioka W, Brunner T, Baier G, Bitter~~h-Baier G, Byrd C, Lang F et el.: Fas-induced apoptosis is mediated by a ceremide-initiated signaling pathway. immunity 1995, 2:341-351.
52. ..
Bose R, Verheij M, Haimovitz-Friedman A, Scotto K, Fuks 2, Kolesnick R: Ceramlde synthase mediates daunorubicin-
of a defect
in the phospholipase D/diacylglycerol pathway in cellular senescence. J Biol Chem 1994, 269:26040-26044. 67.
ras
Haimovitx-Friedman A, Kan C-C, Ehleiter D, Persaud RS, Mcloughlin M, Fuks 2, Kolesnick RN: Ionizing radiation acts on cellular membranes to generate ceramide and Initiate apoptosis. J fxp Med 1994,180:525-535. These investigations propose that ionizing radiition may initiate a form of apoptosis by direct activation of membrane sphingomyelinase. 53. ..
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Mattie ME, Brooker G, Spiegel S: Sphingosine-1 -phosphate, a putabve second messenger, mob&es calcium from internal stores via an inosltol ~sph~ph~e-inde~n~nt pathway. J Biol Chem 1994, 269:3161-3188.
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Ghosh TK, Bian J, Gill DL: Sphingosine 1 -phosphate generated in the endoplasmic reticulum membrane activates release of stored calcium. J Biol . ., Chem . . 1994, 269:22626-22635. I Sphmgostne IS presenr In tne enaoplasmlc reticulum, where It may be con. varted to SPP and release stored calcium. 69.
Kindman LA, Kim S, McDonald TV, Gardner P: Characterlzetion of a novel intracellular sphingolipid-gated Caz+-permeable channel from rat basophilic leukemia cells. J Biol Chem 1994, 269:13086-l 3091.
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70. .
Wu J, Spiegel S, Sturgill TW: Sphingosine-1 -phosphate rapidly ectivetes the MAP kinase pathway by a G-protein dependent mechanism. J Biol Chem 1995,270:11484-1148%. This study shows that MAPK is a downstream effector for the mitogenic action of Sppl 71. .
Su Y, Rosenthal D, Smulson M, Spiegel S: Sphingoslne lphosphate, a novel signaling molecule, atimuiates DNA binding activity of AP-1 In quleacent 3T3 fibroblasts. J Viol Chem 1994, 269:16512-l 6517. This report suggests that SPP-induced DNA synthesisand cell divisionresult from activationof the tran~ript~ factor AP-1, linkingsignal tr~sdu~i~ by SPP to gene expression. 72.
Seufferlein T, Rozengurt E: Sphingoslne induces pl25FAK and pexiilin tyrosine phosphoryiatton, ectin stress fiber formation, and focel confect assembly in Swiss 3T3 ceils. J Biol Chem 1994, 269:2781 O-2761 Z
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Yatomi Y, Ruan F, Hakcmori S, lgarashi Y: Sphingosine-lphosphete: a pl~~-~~~ s~i~oitpid released from agonist-stimulated human platelets. Blood 1995, 88:193-202. This is the first demonstrationthat SPP can ba released from activated cells, suggesting that it may play a physiologicalrole in thrombosis, hemostasis and natural wound healing. 74.
75.
Goodemote KA, Mattie ME, Berger A, Spiegel S: involvement of a oartusata toxin sensitive G protein in the mitoQenic signaling f&ways of sphingostne-l -phosphate. J Biot &em 1QQ5, 2?0:10272-10277. Obeid LM, Linardic CM, Karolak LA, Hannun YA: Programmed cell death by ceramide. Science 1993, 259:1769-l 771.
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Jawis WD, Fomari FA Jr, Browning JL, Gewirtz DA, Koiesnick -.RN; Grant S: Attenuatton of ceramide-Induced apoptosls by digiyceride and pharmecoiogicei activetom of protein kinase C in human myeioid leukemia cells. J Biol Chem 1994, 288:31%85-31892. This paper details regulationof apoptosis via the sphing~yeiin and phosphoinositidesignaling systems; these two systems appear to coordinately regulate apoptosis in an opposing manner. 76. .
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JarvisWD, Grant S, Kolesnick RN: Ceramide and the induction of apoptosis. C/in Cancer Res 1996, 2:1-6.
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second
David Foster? and Richard Kolesnickz
the generation
messengers,
The role of the phosphatidylinositol phospholipase
of glycerolipid
and
and their molecular targets. transfer protein and
D in signal transmission, and the structures
of the 1,2-diacylglycerol and calcium-binding sites of protein kinase C are discussed. Further, ceramide signaling through protein kinases and the role of cross-talk in the signaling of apoptosis and inflammation are addressed.
Addresses *Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, DC 20007, USA *Department of Biological Sciences, Hunter College of City University of New York, 695 Park Ave, New York, NY 10021, USA Xaboratory of Signal Transduction, Memorial Sloan-Kettering Cancer Center, New York NY 10021, USA Current Opinion in Cell Biology 1996, 8:159-l
67
0 Current Biology Ltd ISSN 0955-0674 Abbreviations CAPK ceramide-activated protein kinase CAPP ceramide-activated protein phosphatase DAG 1,2-diacylglycerol IL interleukin LPS lipopolysaccharide LPA lysophosphatidic acid MAPK mitogen-activated protein kinase NGF nerve growth factor PA phosphatidic acid PC phosphatidylcholine PDGF platelet-derived growth factor PH pleckstrin homology PI phosphatidylinositol PIP* PI 4,5-bisphosphate PITP phosphatidylinositol transfer protein PKC protein kinase C PLC phospholipase C PLD phospholipase D SH Src homology SPP sphingosine 1 -phosphate TNF tumor necrosis factor
Introduction Ceramide, the second messenger of the sphingomyelin pathway, like its more widely studied counterpart in the phosphoinositide pathway, 1,2-diacylglycerol (DAG), exists in mammalian cells at the level of l-Z% with respect to total phospholipid concentration [l-4]. Ceramide and DAG not only serve as second messengers, but also as the backbone of all phospholipids. As structural lipids, they are found in bound and free forms distributed throughout all cellular membranes. In response to the activation of cell surface receptors, the levels of the free lipids increase 1.4-2.0-fold above control levels [l-4]. These quantities grossly underestimate the increase in the signaling pools, which are generally considered to contain little or no second messenger in the absence
of stimulus. Unfortunately, conventional assays cannot distinguish between the structural and signaling pools of these lipids and measure stimulated elevations over the entire cell. Signal generation is followed by signal amplification, an observation which helps to explain how small transient signals generated at the cell surface can induce pleiotropic biological responses. The study of lipid messengers has been greatly aided by the use of synthetic lipid analogs which usually retain stereospecific activation but sacrifice potency [la]. Recent investigations have begun to clarify both the mechanisms for generating this class of messengers and their direct molecular targets. A synthesis is emerging that recognizes the integration of lipid second messengers and protein kinase cascades. In this regard, many lipid second messengers have an inherent ability to cross bilayers and connect compartments that are normally separated under resting conditions. Here, we review some exciting findings of the past year, and emphasize mechanisms for generating lipid second messengers and identifying their molecular targets.
Signaling via glycerolipid-derived messengers Phospholipase
second
C
Although a role for DAG as a second messenger of the phosphoinositide pathway is well established [ 1,2], many questions remain unanswered regarding its generation and its ability to activate protein kinase C (PKC). The phosphoinositide-specific phospholipases C (PLCs), which hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP*) to generate DAG, fall into three groups: p, y and 6 [S]. All three PLC isoforms are single polypeptides containing two regions of high homology, designated X and Y, preceded by a pleckstrin homology (PH) domain. In addition, PLC-)I has multiple Src homology (SH)Z domains and a single SH3 domain. Two mechanisms have been described for PLC activation: PLC-fi activation through serpentine receptors is mediated via heterotrimeric G proteins, whereas PLC-y binds to receptor tyrosine kinases or receptor-associated tyrosine kinases via its SH2 domains and is activated by phosphorylation. PLC-p activation occurs either via GTP-bound CL subunits of the Gq class or via By subunits. Gqa interacts with a region distal to the Y domain of PLC-fl whereas the @y units bind to an amino-terminal segment, perhaps via the PH domain. Thus, structural elements of PLC-B determine G-protein interactions. Further, the sensitivity of distinct PLC-B isozymes to a and &r subunits differs, undoubtedly contributing to the quality of the ultimate response. With respect to PLC-y activation, the sites that bind PLC-y on the epidermal growth factor, platelet-derived growth factor (PDGF), nerve growth factor (NGF) and fibroblast
160
Cell regulation
growth factor receptors have been mapped. Further, all of these receptor kinases are now known to phosphorylate the same tyrosine residues on PLC-y and thereby increase its enzymatic activity [S]. A major finding involves the recognition of the role of phosphatidylinositol transfer protein (PITP) in signaling through either PLC-fl or PLC-y [6,7”]. Although PITP transfers phosphatidylinositol (PI) and not the substrate of PLC, PIP2, PITP was found to be necessary for sustained PLC signaling in reconstitution assays using streptolysin 0 permeabilized cells. Presumably, PITP allows refilling of the pool of PIP2 by presenting PI to PI 4-kinase. It has also been suggested that PITP potentiates phospholipase D (PLD) activity by increasing the cellular content of PIP2, which is a putative PLD cofactor [8].
Phospholipase
D
PLD hydrolyzes phosphatidylcholine (PC) to phosphatidic acid (PA) and choline in response to various extracellular stimuli [8,9]. PA has been implicated as a lipid second messenger in the regulation of protein kinases, GTPase-activating proteins, PI kinases, adenylate cyclase and other signaling molecules [lo]; however, direct effects of PA have not been established and many effects are probably mediated by the PA metabolites DAG and lysophosphatidic acid (LPA) [l,ll]. PLD has also been implicated in membrane trafficking and vesicular transport [12-141, in which processes acidic phospholipids may facilitate membrane budding and/or fusion [ 151. Two biochemically distinguishable PLD activities have been characterized; one is dependent upon the small GTPase Arf and upon PIP2 [16**,17], and another is stimulated by oleate [18]. At late stages of purification, the Arf-dependent PLD activity is lost, presumably due to loss of cofactors [14,15,16”]. Recently, a gene required for meiosis in &c&romyces cermisiae [19**] was isolated and shown to have sequence homology to a castor bean PLD [ZO]. A human homolog has been cloned which possesses PLD activity that is dependent upon Arf and PIP2 but not upon oleate [Zl”]. A second PLD gene has now been cloned from a murine source. Substantial progress has been made in characterizing mechanisms of PLD activation. PLD activation by v-Src depends upon a GTPase cascade containing Ras [ZZ”] and Ral [23”]. Ral constitutively associates with PLD through Ral’s novel amino terminus, but Ral does not activate PLD alone [23”]. Although it is not known whether Ral-associated PLD depends upon Arf, as the Arf-dependent PLD does, the Ral-associated activity does depend upon PIP2 [23”]. Evidence also implicates Rho in PLD activation [24,25] suggesting a complex interplay of multiple small GTPases.
Protein kinase C
PKC is the only clearly defined target for DAG [2]. PKC consists of isoforms which share a common requirement for phospholipid and which are divided into three groups: conventional PKCs (cPKCs IX, pl, g2 and y), new PKCs (nPKCs 6, E, o and p) and atypical PKCs (aPKCs < and A) [l]. DAG, or its analog phorbol ester, activates cPKCs and most nPKC isoforms but not aPKCs. Calcium is a cofactor expressly required for the cPKC forms, increasing their affinity for acidic phospholipids. Recently, lysophosphatidylcholine (LPC) and free fatty acid (FFA), the products of hydrolysis of PC by phospholipase AZ, have been shown to enhance DAG-induced activation of some PKC isoforms (LPC and FFA co-activate all cPKCs, and FFA also activates PKCE). LPC and FFA are inactive alone. In contrast, FFA inhibits DAG-induced PKCG activation. These studies demonstrate cooperativity between lipid classes in PKC activation.
This heterogeneity in lipid activation, coupled with evidence that PKC isoforms occupy specific subcellular compartments [ 1,2], suggests that specific intracellular signaling mechanisms will eventually be deciphered. In this regard, it has been proposed that the PKCc( isoform directly phosphorylates and activates the proto-oncoprotein Rafl to initiate signaling via the mitogen-activated protein kinase (MAPK) cascade [26]. Although this proposal has been disputed, in many cell systems PKC activation does lead to Rafl activation [2]. Other direct targets for activation by PKC have been described, including MARCKS, myogenin, lamin B and P-glycoprotein [2]; however, a comprehensive pathway for cellular activation by PKC remains to be defined.
Recent investigations provided substantive information on the structural elements of PKC that mediate lipid activation [27]. The lipid-binding moieties of cPKCs had long been known to reside in two conserved modular domains (Cl and C2). Resolution of the crystal structure of the second cysteine-rich region of the Cl domain of PKCy bound to phorbol ester revealed that the phorbol ester binding site is formed by p sheets, contained in the top half of the Cl domain, that are pulled apart to form a cavity [28”]. Insertion of phorbol ester into this site does not alter the conformation of this domain but rather results in a surface that is continuously hydrophobic, perhaps allowing for efficient interactions with membranes. The lower half of the domain contains the metal-binding sites and appears to be responsible for maintaining the appropriate conformation of the domain. PKC isoforms that contain Cl regions but do not bind phorbol ester lack a particular conserved proline residue [27].
Sprang and coworkers [29**] have recently resolved the crystal structure of the C2 domain of synaptotagmin, an enzyme which, like PKC, binds to acidic membranes in
Signal transductionthrough lipid second messengers Spiegel, Foster and Kolesnick
a Ca2+-dependent manner. This domain is composed of a four-stranded fl sheet which contains a Ca2+-binding site constructed of a set of aspartate residues which are conserved in PKC. Caz+ binding may better orient a region of aromatic amino acids for hydrophobic interaction with membranes, and a region of basic residues for interaction with acidic phosphohpids. Caz+-independent PKCs may substitute a positively charged amino acid for Ca2+, allowing for a similar orientation of this active site. The interaction of these domains of PKC with membranes results in tight binding of PKC to membranes (i.e. in PKC translocation), in removal of the auto-inhibitory pseudosubstrate domain from the active site, and PKC activation. Activation of PKCs in specific membranes also appears to involve a group of proteins collectively known as receptors for activated protein kinase C (RACKS) [30]. RACK1 has been cloned [31**]. These studies have begun to unravel the mystery of the mechanism by which lipid second messengers initiate signaling. Lysophosphatidic acid
Recently, second messenger function has been ascribed to LPA, which is released from activated cells, especially platelets, and elicits diverse biological responses in different systems including platelet aggregation, cell growth [ 11,321 and neurite retraction [11,33]. LPA may also play a role in inflammatory and proliferative responses to injury [ 111. LPA binds to a 38-40 kDa receptor which couples, via distinct G proteins, to multiple independent effector pathways. The a subunit of Gi directly inhibits adenylate cyclase, whereas the &u dimer is thought to activate Ras via an intermediary protein tyrosine kinase. The LPA receptor, via a mechanism involving Gq and stimulation of PLC, also activates Rho, resulting in stimulation of focal adhesion kinase and phosphorylation and reorganization of cytoskeletal proteins [34’].
Sphingolipid-derived
second messengers
Sphingolipid metabolism
Because details of the metabolism of sphingolipids are not as well known as are details of glycerolipid metabolism, this section starts with a brief overview of this topic (Fig. 1) [35]. The sphingolipid biosynthetic pathway begins with the condensation of serine with palmitoylCoA to form 3-ketodihydrosphingosine or 3-ketosphinganine, a reaction catalyzed by serine palmitoyl transferase. 3-ketodihydrosphingosine is then reduced by an NADPHdependent reductase to dihydrosphingosine (sphinganine); acylation with fatty acyl CoA to produce dihydroceramide follows. Recent evidence suggests that dihydroceramide is converted directly to ceramide, the precursor of all complex sphingolipids, by the introduction of a trans-4,5 double bond [35]. The transfer of a phosphorylcholine head group from phosphatidylcholine to ceramide by sphingomyelin synthase yields sphingomyelin. Hydrolysis of sphingomyelin, catalyzed by sphingomyelinase, a sphingomyelin-specific PLC, yields ceramide and phosphorylcholine. Cleavage of the amide-
161
linked fatty acid from ceramide to form sphingosine is catalyzed by ceramidases. Sphingosine can be metabolized further, via a pathway involving sphingosine kinase catalyzed phosphorylation, to sphingosine l-phosphate (SPP), and SPP can be cleaved to ethanolamine phosphate and trans-Z-hexadecanal by a specific lyase located in the endoplasmic reticulum. Ceramide as a second messenger
The past two years have seen the emergence of sphingolipids as second messengers [3,4]. The sphingomyelin pathway is a ubiquitous, evolutionarily conserved signaling system analogous to more well defined systems such as the CAMP and phosphoinositide pathways. Sphingomyelin is a phospholipid preferentially concentrated in the outer layer of the plasma membrane of mammalian cells, and was originally considered to be a structural element of that compartment, providing a rigid barrier to the environment. It is now clear that sphingomyelin hydrolysis by sphingomyelinase initiates pleiotropic cellular responses. Activation of sphingomyelinase has been linked to several cell surface receptors (the 55 kDa tumor necrosis factor [TNF] receptor, the 80 kDa interleukin [IL]-1 receptor, the 75 kDa NGF receptor, and Fas) as an immediate event, and may be a primary mediator of their biological function [3,4]. Alternatively, ceramide may be generated as a downstream event in the response pattern for some receptors (those of Vitamin D3, progesterone, and CD28). In this latter category, ceramide generation may nevertheless be critical in order to allow the ultimate biological response to proceed to completion. Classes of sphingomyelinase
There are two classes of sphingomyelinase, defined by their optimal pHs, that may signal cellular activation [36**]: an acidic isoform (pH optimum -pH5), and two forms with neutral pH optima. One of the neutral isoforms is a Mgz+-dependent membrane-bound enzyme, and the other is a Mgz+-independent cytosolic enzyme. Although the acidic sphingomyelinase was originally considered to reside exclusively in lysosomes, Sandhoff and coworkers [37] demonstrated that a fraction is targeted to compartments other than lysosomes. Furthermore, Kronke and coworkers [36”] suggested that activation of acidic sphingomyelinase occurs in endosomes subsequent to receptor internalization and acidification. Stimuli linked to activation of neutral sphingomyelinase include TNFa, IL-lb, Vitamin D3, NGF, ionizing radiation and Fas, whereas activation of acidic sphingomyelinase has been associated with TNFa, Fas and CD28 (reviewed in [35]). Kronke and coworkers [36”] used mutants of the cytoplasmic domain of the 55 kDa TNF receptor to show that different receptor domains link to these distinct sphingomyelinases. A membrane-proximal region of the cytoplasmic domain linked the neutral sphingomyelinase to the MAPK cascade, whereas the carboxyl terminus of the TNF receptor connected acidic sphingomyelinase to NFKB activation. These authors proposed that activation
162
Cell regulation
Figure 1
0
Cop + &ASH 0 CH20H
3-Ketosphinganine
tNHs
I
Sphi”g~CHzOH +NH,
@
Fat!y acyl-CoA I
&CH*OH NH D-m
0
t
OH CH20P03-H2
4_
@
OH
ATP
CH20H NH
6 cCeramide
l-phosphate
4
Fatly acyl-CoA
Sphingosine
+NH3
0
-=?i+ PI PC 0 DG
OH
CH20P03-CH*CH2rj(CH3)3 0
NH
A Hexadecanal
CHO
I;Hs/\
CH20P0s-H2
Phosphoefhanolamine
Metabolism of sphingolipids. The enzymes catalyzing the numbered reactions are: (1) serine palmitoyltransferase; (2) 3-ketosphinganine reductase; (3) ceramide synthase; (4) dihydroceramide saturase; (5) ceramide kinase; (6) ceramide 1-phosphate phosphatase [79,60]; (7) sphingomyelin synthase; (8) sphingomyelinase; (9) ceramidase; (10) sphingosine kinase; (11) sphingosine l-phosphate phosphatase [al]; (12) sphingosine lyase. DG represents 1,2-diacylglycerol.
of acidic sphingomyelinase occurred in response to DAG generated via a PC-specific PLC. However, the existence of such an enzyme remains uncertain. These studies suggest compartmentalization of signaling for these different sphingomyelinases. In this regard, Linardic and Hannun [38*] have localized one TNF-responsive pool of sphingomyelin specifically to the inner surface of the plasma membrane. Now that knockout mice for the acidic sphingomyelinase have become available [39,40], it is likely that these signaling pathways will be clarified in the near future. Targets for ceramide action
A number of direct targets for ceramide action have now been identified, including a ceramide-activated protein kinase (CAPK) [41], a ceramide-activated protein phosphatase (CAPP) [42], the putative guanine-nucleotide exchange factor Vav [43] and the PKC< isofoim [44”,45].
CAPK is a 97 kDa plasma membrane bound serine/threonine protein kinase with a preference for the sequence X-Ser/Thr-Leu-Pro-X in which the phosphoacceptor site (serine or threonine) is amino-terminal to a proline residue, and X may be any amino acid [46]. Substrate phosphorylation by CAPK and CAPK autophosphorylation are enhanced by nearly tenfold upon elevation of cellular ceramide levels with either ceramide analogs or TNFa. CAPP is a member of the protein phosphatase 2A class of Ser/Thr protein phosphatases which are expressed as heterotrimers with distinct functional domains [47]. Catalytic activity resides within the C subunit, whereas sensitivity to ceramide is conferred by the B subunit. Increased availability of ceramide may trigger CAPP translocation from the cytoplasm to membranes, where dephosphorylation of phosphoprotein substrates would presumably occur. CAPP activity has been closely correlated with growth inhibition in numerous systems.
Signal transductionthrough lipid second messengers
Ceramide may also stimulate the guanine nucleotide exchange activity of the proto-oncoprotein Vav, a putative activator for Ras in hematopoietic cells [43].
of inflammation
Foster and Kolesnick
163
Figure 2
Finally, PKC<, an atypical PKC which is insensitive to phorbol ester or DAG, may also be a direct target for ceramide stimulation [44”,45]. Treatment with either TNFa or ceramide enhanced PKC< phosphorylation and activity in U937 and NIH3T3 cells, and ceramide increased PKC< activity in h-o. PKC< activation via ceramide may link the TNF receptor to NFKB. Signaling
Spiegel,
6 Sphingomyelin SMase
and apoptosis via ceramide
The most comprehensive data on second messenger function of ceramide have been published for TNFa [1,2]. Ceramide appears to be a primary mediator of both the inflammatory and the apoptotic responses to TNFa (Fig. 2). With respect to the inflammatory response, ceramide stimulates CAPK to phosphorylate Rafl on Thr269, enhancing Rafl activity. This initiates signaling thorough the MAPK cascade [48.*], and results in activation of cytosolic phospholipase AZ and release of arachidonic acid (R Schatzman, R Heller, RA Kolesnick, unpublished data). Lipopolysaccharide (LPS), which mediates the effects of Gram-negative bacteria on mammalian systems, mimics many of the early actions of TNFa and IL-la. Molecular modeling of carbons l-3 of the reducing saccharide of the lipid A moiety of LPS and its associated fatty acids revealed close structural similarity to ceramide [49**]. LPS also activates CAPK in a CD14-dependent manner, suggesting that LPS may coopt the second messenger function of ceramide in order to initiate inflammatory responses. With regards to apoptosis, TNFa induces rapid hydrolysis of sphingomyelin to ceramide in all cells in which it initiates apoptosis, and cell-permeable analogs of ceramide mimic this initiation of apoptosis in a stereospecific manner. Analogs of other lipid second messengers, including analogs of DAG, arachidonic acid and PA, are ineffective. Studies published within the past year provide strong evidence that ceramide also signals Fas-mediated apoptosis [50,51]. Ceramide-mediated apoptosis extends beyond apoptosis induced by cytokine receptors. Ionizing radiation appears to directly target membranes, resulting in hydrolysis of sphingomyelin to ceramide within seconds [52”]. This event may initiate an interphase form of apoptotic cell death. Further, the chemotherapeutic drug daunorubicin may initiate apoptosis by a mechanism involving ceramide synthesis, by activation of ceramide synthase [53”]. Ceramide synthesis appears to be obligatory, as fumonisin Br, a fungal inhibitor of ceramide synthase, blocked daunorubicin-induced ceramide synthesis and apoptosis [53”]. In total, these studies suggest that ceramide acts as a generic signal for several forms of apoptosis. Preliminary investigations suggest that the stress-activated protein kinase/c-Jun kinase signaling system, and not the MAPK cascade, is a downstream effector
Raf-1
MEKKl
MEK
SEKl
9 MAPK
SAPK
0
9
CPLA,
c-Jun
9 AA
9 Inflammation
Apoptosis 0 1996 Current Opinion m CM Biology
Signaling systems engaged by ceramide. Activation of the 55 kDa TNF receptor stimulates neutral and acidic sphingomyelinase (SMase) isoforms, resulting in ceramide generation. Ceramide can stimulate CAPK in the plasma membrane to phosphorylate and activate Raf-1, initiating signaling though the MAPK cascade. Alternatively, ceramide, by an unknown mechanism, appears to initiate signaling through the stress-activated protein kinase/Jun kinase cascade in cells in which it mediates
an apoptotic
cytosolic phospholipase
response.
MEK, MAPWERK
As; AA, arachidonic
kinase 1 ; SEKl , SAPK/ERK kinase.
kinase; cPLA2,
acid; MEKKl,
kinase-1 ; SAPK, stress-activated
MEK protein
system for the initiation of apoptosis by diverse stresses ([54**,55]; M Verheij et al., personal communication; see Note added in proof). Sphingosine and SPP as mitogenie second messengers
Although originally proposed as negative regulators of PKC [56], sphingosine and derivatives may play alternative signaling roles. In this regard, sphingosine induced a mitogenic effect that was stereospecific and independent of PKC inhibition in 3T3 fibroblasts [57,58]. Further, fumonisin Br increased both the level of sphingoid bases and cell growth in these cells. It has been proposed that this ability to increase sphingoid base levels mediates fumonisin-induced carcinogenesis [.59]. Sphingosine and SPP may also regulate T-cell proliferation [60]. Growth inhibition by ISP-l/myriocin, a potent immunosuppressant which inhibits serine palm-
164
Cellregulation
itoyltransferase and consequently sphingoid base synthesis, was reversed by exogenous sphingosines or SPP [60]. Moreover, certain growth factors, such as PDGF, induced a rapid, transient elevation in sphingosine and SPP levels in fibroblasts [61], arterial smooth muscle cells [62] and glomerular mesangial cells [63*-l, and pharmacological inhibition of the generation of these sphingolipids reduced PDGF-induced cellular proliferation [61,63**]. Thus, mitogenic and apoptotic responses may be regulated, in part, through different sphingolipid second messenger molecules. In this regard, recent evidence suggests that regulation of ceramidase may determine whether signals flow via SPP or via ceramide [63**], perhaps altering the balance between these mitogenic and apoptotic lipid signals. Although these mitogenic effects appear widespread, in some cell types sphingosine inhibits cell growth, perhaps via PKC inhibition [56]. Sphingosine may even be an endogenous mediator of apoptosis [64], although its ready acylation to produce ceramide in most systems confounds this issue. Sphingosine and SPP uniformly stimulate an increase in the levels of PA, a putative mitogenic signal. Ceramide, in contrast, decreases PA levels by inhibiting PLD activation [6.5]. This latter effect may play a role in cellular senescence, during which endogenous ceramide levels are high [66]. Sphingosine and SPP mobilize calcium from internal sources via an inositol-trisphosphate-independent pathway [67,68*]. The response of calcium to SPP has many hallmarks of a receptor-mediated event, including rapidity, reversibility, and specificity. As the endoplasmic reticulum contains the kinase which converts sphingosine to SPP, all of the elements sufficient for regulated calcium signaling via these lipids appear to be topologically compartmentalized [68*]. A sphingosinephosphorylcholine-gated calcium channel, with unique pharmacological and electrophysiological properties, has recently been characterized, and may represent the SPP-gated calcium channel [69]. Downstream signaling for SPP is beginning to be deciphered. SPP engages the MAPK cascade in 3T3 fibroblasts [70’] and activates the transcription factor activator protein-l (AP-1) [71*]. Thus, AP-1 may be the site at which signals carried by sphingolipid-derived messengers such as ceramide and SPP merge with the glycerophospholipid-derived second messengers, such as DAG.
[72] and SPP (A Hall, S Spiegel, unpublished data) induce actin stress fiber formation and stimulate focal adhesion kinase in Swiss 3T3 fibroblasts. The importance of sphingolipid metabolites in cytoskeletal dynamics will be an exciting area of future research. Although many studies indicate an intracellular site of action of SPP, some of its biological effects when added exogenously, such as inhibition of motility and invasiveness, may be due to extracellular effects on cell surface receptors [73’]. SPP is stored in high concentrations in human platelets, is released upon activation by physiological stimuli, and may play a role in platelet aggregation [73*]. Gi/Go-coupled receptors may be directly or indirectly involved, as some responses to SPP are pertussis toxin sensitive [74].
Cross-talk between glycerolipids sphingolipids
and
The roles of ceramide and DAG as pro-apoptotic and anti-apoptotic stimuli, respectively, have become apparent over the past year [75,76’]. Phorbol esters, previously shown to be anti-apoptotic in diverse systems, antagonize ceramide-mediated apoptosis [77]. Although the PKC isozymes involved have yet to be identified, it is clear that DAG inhibits ceramide-induced apoptosis at numerous sites, including the sphingomyelinase and downstream of this enzyme. Furthermore, stage 1 and 2 tumor promoters and even non-tumor-promoting phorbol esters possess this capability. This effect of DAG may represent more than a result of pharmacological manipulation, as the radioprotective effect of basic fibroblast growth factor in bovine aortic endothelial cells and in oivo may involve this mechanism [78]. Although ceramide elevation induces apoptosis in many cells, when it is combined with DAG elevation differentiated function and proliferation often ensue. In these instances, it is likely that DAG abrogates the ceramide-initiated signals leading to apoptosis while allowing signals for other events to proceed. More detailed information on the elements of apoptotic and anti-apoptotic signaling would appear necessary to designate the exact sites of cross-talk. Nevertheless, costimulated cellular activation may be a primary mechanism determining the penultimate biological responses.
Note added in proof The paper referred to in the text as M Verheij et a/., personal communication, has now been accepted for publication [&?I.
Acknowledgements A potentially important effect of SPP is its interference with PDGF-stimulated actin filament assembly/disassembly, resulting in marked inhibition of cell spreading and in chemotaxis towards PDGF in human arterial smooth muscle cells [63**]. However, sphingosine
We
would
like
to
thank
communicating
results
research
ROI
of Health NIH, for
grants (NIH).
BE243 Tobacco
CA57400
from
J Engebrecht,
prior GM43880
David
Foster
the American
Research.
and CA42385
M
to publication.
Richard
and
Frohman, Sarah
CA61774
from
was supported Cancer
Society,
Kolesnick
was
and
Spiegel the
Morris
for
was supported
AJ
by
National
by grants
Institutes
CA46677
from
and 3075
from
the Council
supported
by
NIH
the
grants
Signal transduction through lipid second messengers Spiegel,
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