Sphingolipids serve as potential reservoirs for bioactive lipids and are now included with the well-established mediators of signal transduction such as diacylglycerol, phosphatidylinositides, and eicosanoids, all derived from glycerolipids.¹⁶ Furthermore, sphingolipid metabolism has clearly been shown to be involved in the regulation of cell growth, differentiation, and programmed cell death.^1–6 Indeed, there have been over 4000 studies conducted on sphingolipids during the past five years, and a majority have been directed towards understanding the emerging role of ceramide as a cellular bioregulator. Thus, with the demonstration that ceramide mediates various signaling cascades, there is now a necessity for defining direct targets of ceramide and specific mechanisms regulated by ceramide. The search for potential direct targets for ceramide action led to the identification of several candidate ceramide-regulated enzymes such as ceramide-activated protein kinases (CAPK), cathepsin D, and a key set of targets, serine/threonine protein phosphatases.^7–12

Two serine/threonine phosphatases have been shown to be ceramide responsive in vitro and in vivo, protein phosphatase-1 (PP1) and protein phosphatase 2A (PP2A).^{10,11,13–16} These protein phosphatases are now collectively termed ceramide-activated protein phosphatases (CAPPs).This Chapter will discuss the relevant mechanisms by which CAPP is activated by ceramide in vitro and its emerging role in regulating various cascades of signal transduction through modulation of specific phospho-targets.

Identification of a Ceramide-Activated Protein Phosphatase (CAPP)

Based on biochemical parameters, Ser/Thr protein phosphatases were initially divided into two classes, type 1 protein phosphatases (PP1) and type 2 protein phosphatases (PP2A).¹⁷ The PP1 class is inhibited by two heat-stable proteins termed inhibitor 1 (I-1) and inhibitor 2 (I-2) whereas type 2 or PP2 class phosphatases are insensitive to these proteins, but inhibited by the small t-antigen of SV40.^17–19 Type 2 phosphatases can be further subdivided into spontaneously active (PP2A), Ca⁺²-dependent (PP2B) and Mg⁺²-dependent (PP2C) classes.^17,19 In the past few years, many novel serine/threonine phosphatases such as protein phosphatase-V and -X have been identified that do not fit into any of these classes.²⁰

Both PP1 and PP2A are composed of a catalytic subunit bound by one or more regulatory subunits.^17–19 These subunits regulate subcellular localization, substrate specificity, enzyme activity, and in some cases cofactor binding.^17–19 The catalytic and regulatory subunits are also regulated by covalent modifications such as phosphorylation and methylation.^18,19 Thus, regulation of these protein phosphatases is very complex requiring coordination of phosphorylation, methylation, subunit expression, and as discussed in this Chapter, lipid second messengers.

Originally, ceramide was shown to increase the activity of a serine/threonine protein phosphatase(s) in crude cytosolic extracts.^10,11 This in vitro activation was achieved by D-erythro-ceramide, but not by the biologically inactive dihydro-ceramide.^10,11 From this initial observation, a predominant ceramide-activated protein phosphatase was shown to exist in rat brain,²¹ and this phosphatase was identified as a member of the 2A (PP2A) family of serine/threonine phosphatases.^10,11 This conclusion was based on studies demonstrating that pre-treatment with okadaic acid inhibited the ability of ceramide to activate a serine/threonine phosphatase in T9 glioma cell extracts.^10,11 To conclusively demonstrate that PP2A is activated by ceramide, a novel chromatographic approach was utilized to purify the major CAPP activity from rat brain to near homogeneity.²¹ This approach used hydrophobic interaction chromatography on phenyl Sepharose followed by anion-exchange on MonoQ.²¹ CAPP purified in this manner was found to be composed of three major bands on SDS-PAGE which co-migrated with the three subunits of heterotrimeric PP2A.²¹ Immunological studies then identified CAPP as heterotrimeric (AB'C and ABαC) and heterodimeric (AC) PP2A species.²¹ Further demonstrating that PP2A was a form of CAPP, heterotrimeric PP2A (AB'C) and heterodimeric PP2A (AC) purified from rabbit skeletal muscle were shown to be activated by ceramide directly and in a specific manner.²¹ These results were further supported by the co-elution of CAPP with PP2A on size-exclusion chromatography and by their immunologic identity.²¹ These studies clearly demonstrated that PP2A was indeed a form of CAPP and provided a convenient and efficient method for isolating the PP2A species of CAPP to allow for in-depth biochemical analysis.²¹

Another eluting species of ceramide-activated phosphatases was also noted, and further studies showed that this was PP1, a closely-related serine/threonine phosphatase.^13,14 In vitro studies showed that purified PP1 responds to ceramide in vitro with similar specificities as PP2A,^13,14 thus establishing PP1 as a second CAPP.

In Vitro Regulation of CAPP by Ceramide

Several studies have now shown that short chain (cell permeable) ceramides activate PP2A and the catalytic subunit of PP1 (PP1c) in vitro.^10,14,21,22 D-e-C₂-ceramide activated heterotrimeric PP2A (AB'C) up to 3.5-fold with an EC₅₀ of approximately 5 μM and to a lesser extent (up to 2.5-fold) the heterodimeric form (AC) of PP2A.^10,21 Chain length studies on heterotrimeric PP2A demonstrated that ceramides possessing hexanoyl, decanoyl, and myristoyl, but not stearoyl acyl chains also activated the phosphatase.^10,21

The longer chain ceramides failed to activate due to solubility problems in delivering those hydrophobic ceramides (see below). Activation of heterotrimeric PP2A was ceramide-specific, as closely related lipids had no effect on PP2A activity.^10,21 Moreover, dihydro-C₂-ceramide (which lacks the trans 4–5 double bond found in ceramide) did not activate PP2A in vitro, but actually inhibited PP2A activity.^10,21 These findings were particularly important for several reasons. First, they demonstrated chain-length selectivity in the activation of PP2A. Second, dihydroceramide is a metabolic precursor of ceramide, thus establishing metabolic specificity of ceramide in activation of CAPP. Finally, dihydroceramides had been shown not to mimic the cellular activities of ceramide in inducing apoptosis, cell cycle arrest, or differentiation.²³ Thus, these corresponding specificities in cellular action and in vitro activation of CAPP suggested CAPP as a relevant cellular target for ceramide.

To further demonstrate that this activation was specific, the stereospecificity of activation of these phosphatases by ceramides was examined. Thus, the stereoisomers of the naturally occurring D-erytho-C₂ ceramide (2S, 3R) were synthesized. These were its enantiomer, L-erytho-C₂ ceramide (2R, 3S), and the two diastereoisomers in the threo conformation, D-threo-C₂ ceramide and L-threo-C₂ ceramide, which differ from erythro in that the C-2 configuration relative to C-3 is in a cis-conformation unlike erythro in which the groups are in a trans-conformation. PP2A demonstrated some stereospecificity with the short chain ceramides in that only the D- and L-erythro forms of C₂-ceramide activated PP2A and not the D- and L-threo forms.^10,21

PP1 was also activated by short chain ceramides in vitro. In these studies, D-e-C₆- and D-e-C₂-ceramide activated PP1c in a dose-dependent manner (Kishikawa K, Chalfant CE, Lee JY, Hannun YA. 2001; unpublished findings). PP1 showed similar N-acyl chain length dependencies (as PP2A) since the N-acetyl, hexanoyl, and decanoyl, but not stearoyl ceramides activated PP1 in vitro (Kishikawa K, Chalfant CE, Lee JY, Hannun YA. 2001; unpublished findings). Similar to PP2A, the activation was specific as again closely related lipids as well as the inactive form, dihydro-C6-ceramide, failed to activate PP1 (Kishikawa K, Chalfant CE, Lee JY, Hannun YA. 2001; unpublished findings). PP1 also demonstrated stereospecificity with D-erythro-C₆-ceramide activating to the greatest extent compared to its stereoisomers (Kishikawa K, Chalfant CE, Lee JY, Hannun YA. 2001; unpublished findings).

A major problem that hampered further evaluation of the biochemical regulation of CAPP in vitro had been the lack of an effect of long chain (natural) ceramides. Since long chain neutral lipids are insoluble in aqueous solutions, delivering long chain ceramides in vitro and in vivo is difficult and limited. To overcome this problem, long chain ceramides were solubilized with 2% dodecane at a final reaction concentration of 0.02%.¹³ Dodecane alone exerted an inhibitory effect on phosphatase activity, but solubilized D-e-C₁₈ ceramide clearly activated the PP1α catalytic subunit (PP1αc), PP1γ catalytic subunit (PP1γc), PP2Ac, and PP2A trimer in a dose-dependent manner.¹³ This activation was examined for specificity by long chain ceramides using stereoisomers of D-e-C₁₈ ceramide.¹³ Only D-e-C₁₈ ceramide activated each phosphatase. These observations are significant for at least three reasons.¹³ First, activation of CAPP by natural ceramides was demonstrated. Second, specificity for ceramide was clearly demonstrated for the long chain ceramide, and the stereochemical specificity became better defined with the long chain isomers than with the short chain ones. Third, a specific ceramide-binding/interaction site was now suggested to be present on the catalytic subunit of at least PP1 and PP2A since they respond directly to ceramide. Since these observations, unsaturated natural ceramides dispersed in aqueous buffer by sonication have been shown to activate both PP1c and PP2Ac without the need for dodecane solubilization (Chalfant CE, Hannun YA. 2001; unpublished findings).

Selective CAPP Substrates in Cells

Since initial studies showed that the specificity for CAPP activation in vitro closely resembled the specificity for various cellular activities of ceramide such as apoptosis,^{5,10,11,13,14,24,25} CAPP emerged as a key candidate for mediating cellular activities of ceramide through dephosphorylation of key substrates. Several criteria for establishing a CAPP substrate are proposed: 1) exogenous ceramides induce the dephosphorylation of the particular phospho-substrate; 2) inhibitors of serine/threonine protein phosphatase block this effect; 3) exogenous dihydro-ceramide should not affect the phosphorylation state of the particular phospho-substrate; and 4) the target phospho-protein should act as an in vitro substrate for CAPP. Using these criteria, several targets for CAPP have been identified by multiple groups (Fig. 1).

Figure 1

Schematic of known in vivo substrates of ceramide-activated protein phosphatases.

Bcl-2

Recently, several key apoptotic factors have been shown to be regulated by phosphorylation/dephosphorylation via serine/threonine protein phosphatases. For example, Bcl-2 phosphorylation on serine 70 has been demonstrated to be necessary for its protective/survival function, and dephosphorylation of Bcl-2 serine 70 leads to BAD/Bcl-2 heterodimerization, effectively inhibiting the function of Bcl-2.^32,33 Interestingly, serine 70 is a direct target for phosphorylation by PKCα and dephosphorylation by PP2A. Recently, May and co-workers demonstrated increased mitochondrial PP2A activity and specific dephosphorylation of mitochondrial Bcl-2 in response to ceramide.^32,33 Again, okadaic acid was able to inhibit this effect, further implicating the PP2A form of CAPP in this ceramide effect.^32,33 Thus, PP2A has been demonstrated to dephosphorylate both a Bcl-2 kinase (PKCα) and Bcl-2 in response to ceramide, suggesting a possible signaling complex. Furthermore, this report validated the signal transduction sequence for ceramide since other studies had placed ceramide formation somewhere between the action of upstream and downstream caspases, with Bcl-2 acting downstream of ceramide in that same pathway.^34,35 Thus, ceramide regulates the phosphorylation of Bcl-2 and its anti-apoptotic function via CAPP, clearly placing ceramide generation upstream of Bcl-2 (Fig. 2).^33–36

Figure 2

Schematic of TNFα signaling in apoptosis. The schematic depicts a general scheme of TNFα signaling demonstrating the function of Bcl-2 and PKCα downstream of ceramide generation. PP2A is shown to dephosphorylate both Bcl-2 and (more...)

Role of Endogenous Ceramide in Regulating CAPP Activity

One of the main difficulties in validating CAPP in cells had been the absence of studies demonstrating a dependence on the generation of endogenous ceramide for the dephosphorylation of a particular phospho-protein. As discussed previously in the Chapter, B-SMase treatment was shown to induce activation of PP2A leading to the dephosphorylation of c-JUN, thus demonstrating the ability of endogenous ceramide to exert specific functions.²⁸ Although this study showed the ability of endogenous ceramide to activate CAPP in cells, a physiological role for endogenous ceramide in regulating CAPP in cells was not established. In order to establish a role for CAPP in signal transduction, studies are needed that utilize relevant agonists (e.g., TNFα) and demonstrate a necessary role for the generation of ceramide to activate CAPP. Indeed, two such models of CAPP activation have been defined recently. First, fumonisin B₁, and inhibitor of Co-A dependent ceramide synthase and the generation of de novo ceramide, was shown to block dephosphorylation of PKCα by protein phosphatase 2A in response to TNFα in L929 cells (Fig. 3).²⁷ Second, the same inhibitor of de novo ceramide generation also blocked the dephosphorylation of SR proteins in response to Fas activation (Fig. 3).¹⁵ These two reports were milestones validating the existence of ceramide-activated protein phosphatase in cells and demonstrating the necessity of generating endogenous ceramide in order to activate CAPP. Furthermore, these studies demonstrate that de novo ceramide can activate both PP1 and PP2A forms of CAPP.

Figure 3

Schematic of serine/threonine protein phosphatases activated by de novo ceramide.

Now the question can be raised whether ceramide generated by other pathways can activate CAPP, only specific forms of CAPP, or even CAPPs localized in specific cellular compartments. Further studies are needed to answer these questions, but the identification of these ceramide-dependent mechanisms now also allows for the investigation of mechanisms downstream of CAPP.

Thus, the recent availability of specific inhibitors of pathways for generating ceramide in response to exogenous agonists and molecular tools (such as the overexpression of enzymes that clear ceramide, or antisense constructs for enzymes that generate ceramide), should now allow the modulation of ceramide levels, thereby, determining their effects on signaling events. Based on this, the criteria for defining a particular phospho-target as a substrate for CAPP should be expanded, and the designation as a CAPP substrate should now require the additional criterion of demonstrating that specific inhibition of ceramide generation leads to subsequent inhibition of the dephosphorylation of the phospho-substrate in question.

General Mechanisms Regulated by Ceramide-Activated Protein Phosphatases

Studies using specific inhibitors of serine/threonine protein phosphatases have demonstrated a clear role for CAPPs in regulating key biological responses. These studies and observations are briefly discussed below along with their implications.

Regulation of mRNA Processing as a Specific Pathway Regulated by CAPP

Through examination of possible downstream mechanisms regulated by ceramide-responsive enzymes, a common theme of substrates for both CAPP and PKCζ was recognized in the alternative processing of pre-mRNA. Both PP1 and PKCζ have been demonstrated to regulate protein factors involved in the mechanism of alternative splicing.¹⁵ Specifically, in response to endogenous ceramide, PP1 dephosphorylated SR proteins, a family of factors that regulate constitutive and alternative processing of pre-mRNA.^{15,49,51–54,71–73} Dephosphorylation of SR proteins effectively inhibits many of their functions and has been reported to regulate alternative processing of pre-mRNA.^{49,51–54,71–73} On the other hand, ceramide has been shown to induce PKCζ to phosphorylate hnRNPA1, a known regulator of the alternative processing of pre-mRNA and antagonist of SR proteins.^7,74 Phosphorylation of hnRNP A1 induces its nuclear translocation and increases its RNA binding ability, thereby enhancing its function as an antagonist of SR proteins.^7,74,75 Therefore, ceramide, via the “cross-talk” of two direct targets, effectively diminishes the ability of SR proteins to regulate pre-mRNA processing by inhibiting activity directly (dephosphorylation) and indirectly (activation of an SR protein antagonist). Thus, based on these observations, we propose that ceramide may regulate the alternative splicing of genes that play an important role in cellular stress responses and apoptosis.

Indeed, examination of the literature reveals that the both Bcl-x and caspase 9 genes produced splicing variants with opposing functions.^76,77 The Bcl-x splice variant, Bcl-x(L) and the caspase 9 splice variant, caspase 9b, inhibit apoptosis in contrast to the pro-apoptotic splice variants, Bcl-x(s) and caspase 9.^76,77 Ceramide was found to regulate the alternative splicing of both Bcl-x and caspase 9, reducing the expression of the cell survival factors, caspase 9b and Bcl-x(L) while increasing the expression of the pro-apoptotic factors, Bcl-x(s) and caspase 9.⁷⁸ Ceramide-induced alternative splicing was blocked by inhibitors of serine-threonine protein phosphatases and of the de novo ceramide pathway, demonstrating a role for CAPP and endogenous ceramide in regulating this mechanism.⁷⁸ These observations were important in that a novel, direct, and specific mechanism mediated by a ceramide-activated protein phosphatase and endogenous ceramide has now been defined.

It is now obvious that regulation of sphingolipid pathways plays an important and complex role in regulating many cellular responses. Through careful examination of ceramide signaling, the various cascades of ceramide action are now being delineated (Fig. 4). This conclusion increases the need for intense investigation of sphingolipid-mediated mechanisms and with the emergence of new molecular and biochemical tools for sphingolipid metabolic enzymes, new mechanisms and targets mediated by ceramide-activated protein phosphatases and other ceramide-responsive enzymes will soon be discovered.

Figure 4

Pathways of signal transduction regulated by ceramide-activated protein phosphatases. The schematic depicts known pathways of signal transduction modulated by ceramide and ceramide-activated protein phosphatase. The diagram also depicts other candidate (more...)

Acknowledgments

This work was supported by a grant (to Y.A.H.) from the National Institutes of Health (CA87584 & GM43825), a National Research Service Award (to C.E.C.) from the National Institutes of Health (GM19953-02), and a VA Merit Review (to C.E.C.) from the Veteran's Administration.

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