**1. Introduction**

Cell and tissue homeostasis is essential for normal development of an organism. When this is altered, metabolic dysfunctions and disease are prone to occur. Therefore, the maintenance of an appropriate balance in the activation / inhibition of the different metabolic pathways and cell signaling systems is simply vital.

Many lipids, including simple sphingolipids, are known to regulate cell activation and metabolism (Gomez-Munoz et al., 1992; Gomez-Munoz, 1998; Gomez-Munoz, 2004; Gomez-Munoz, 2006; Hannun & Obeid, 2008; Chen et al., 2011; Hannun & Obeid, 2011) . Some of them, including sphingosine, ceramides and their phosphorylated forms, sphingosine 1 phosphate (S1P) and ceramide 1-phosphate (C1P) have been described as crucial regulators of key processes that are essential for normal development, and have also been involved in the establishment and progression of different diseases (Gangoiti et al., 2008a; Arana et al., 2010). In particular, ceramides can induce cell growth arrest and cause apoptosis, when they are generated (Hannun et al., 1986; Kolesnick, 1987; Kolesnick & Hemer, 1990; Merrill & Jones, 1990; Merrill, 1991; Hannun, 1994; Kolesnick & Golde, 1994; Hannun & Obeid, 1995; Hannun, 1996; Spiegel & Merrill, 1996; Merrill et al., 1997; Kolesnick et al., 2000; Hannun & Obeid, 2002; Merrill, 2002). Nonetheless, although in general, ceramides are negative signals for cell survival, in neurons they can induce cell growth (Goodman & Mattson, 1996; Ping & Barrett, 1998; Brann et al., 1999; Song & Posse de Chaves, 2003; Plummer et al., 2005). Also, ceramides play important roles in the regulation of cell differentiation, inflammation, tumor development (Okazaki et al., 1990; Mathias et al., 1991; Dressler et al., 1992; Hannun, 1994; Kolesnick & Golde, 1994; Hannun & Obeid, 1995; Gomez-Munoz, 1998; Menaldino et al., 2003), bacterial and viral infections, and ischemia-reperfusion injury(Gulbins & Kolesnick, 2003). More recently, ceramides have been associated with insulin resistance through activation of protein phosphatase 2A and the subsequent dephosphorylation and inactivation of protein kinase B (PKB) (Schmitz-Peiffer, 2002; Adams et al., 2004; Stratford et al., 2004), and toll-like receptor 4 (TLR4)-dependent induction of inflammatory cytokines, a fact essential for TLR4-dependent insulin resistance (Holland et al., 2011).

Concerning ceramide generation, there are three different mechanisms by which these molecules can be synthesized in cells. Ceramides can be generated by i) *de novo* synthesis, which takes place in the endoplasmic reticulum (ER), ii) by the action of different sphingomyelinases (SMases) in the plasma membrane, lysosomes, or mitochondria, and iii) by reacylation of sphingosine, a pathway known as the salvage or recycled pathway (Hannun & Obeid, 2011). The biosynthetic and degradative pathways of ceramide are shown in figure 1, where further products of ceramide metabolism are also indicated.

Natural ceramides tipically have long *N*-acyl chains ranging from 16 to 26 carbons in length (Merrill, 2002; Pettus et al., 2003a; Merrill et al., 2005), and some times longer in tissues such as skin. Many studies have used a short-chain analog (N-acetylsphingosine, or C2-ceramide) in experiments with cells in culture because it can be incorporated into cells more easily and rapidly than long-chain ceramides. Of note, although C2-ceramide was suggested not to occur *in vivo*, recent studies demonstrated that C2-ceramide does exist in mammalian tissues. In particular, C2-ceramide was found in rat liver cells (Merrill et al., 2001; Van Overloop et al., 2007), and brain tissue (Van Overloop et al., 2007). Ceramide generation is also relevant because this sphingolipid is the precursor of important bioactive molecules that can also regulate cellular functions. For instance, stimulation of ceramidases results in generation of sphingosine (Fig. 1), which was first described as a physiological inhibitor of protein kinase C (PKC) (Hannun et al., 1986). There are numerous reports in the scientific literature showing that PKC is inhibited by exogenous addition of sphingosine to cells in culture. Moreover, Merrill and co-workers demonstrated that addition of the ceramide synthase inhibitor fumonisin B1 to J774.A1 macrophages to increase the levels of endogenous sphingoid bases, also inhibited protein kinase C (Smith et al., 1997). Further work showed that sphingosine can affect the activity of other important enzymes that are involved in the regulation of metabolic or cell signaling pathways such as the Mg2+ dependent form of phosphatidate phosphohydrolase (Jamal et al., 1991; Gomez-Munoz et al., 1992), phospholipase D (PLD) (Natarajan et al., 1994), or diacylglycerol kinase (DAGK) (Sakane et al., 1989; Yamada et al., 1993). Sphingosine, in turn, can be phosphorylated by the action of sphingosine kinases to generate S1P, which is a potent mitogenic agent and can also inhibit apoptosis in many cell types (Olivera & Spiegel, 1993; Wu et al., 1995; Spiegel et al., 1996; Spiegel & Merrill, 1996; Spiegel & Milstien, 2002; Spiegel & Milstien, 2003). More recently, we demonstrated that S1P stimulates cortisol (Rabano et al., 2003) and aldosterone secretion (Brizuela et al., 2006) in cells of the zona fasciculata or zona glomerulosa, respectively, of bovine adrenal glands, suggesting that S1P plays an important role in the regulation of steroidogenesis.

A major metabolite of ceramide in cells is ceramide-1-phosphate (C1P), which is formed directly through phosphorylation of ceramide by the action of ceramide kinase (CerK) (Fig. 1). There is increasing evidence suggesting that C1P can regulate cell proliferation and apoptosis (Reviewed in (Gomez-Munoz, 1998; Gomez-Munoz, 2004)), and Chalfant and coworkers have implicated C1P in inflammatory responses (Reviewed in (Chalfant & Spiegel, 2005; Lamour & Chalfant, 2005)). In addition, Shayman's group demonstrated that C1P plays a key role in phagocytosis (Hinkovska-Galcheva & Shayman; Hinkovska-Galcheva et al., 1998; Hinkovska-Galcheva et al., 2005).

The aim of the present chapter is to review and update recent progress on the regulation of cell survival and inflammation by C1P.

which takes place in the endoplasmic reticulum (ER), ii) by the action of different sphingomyelinases (SMases) in the plasma membrane, lysosomes, or mitochondria, and iii) by reacylation of sphingosine, a pathway known as the salvage or recycled pathway (Hannun & Obeid, 2011). The biosynthetic and degradative pathways of ceramide are shown in figure 1, where further products of ceramide metabolism are also indicated.

Natural ceramides tipically have long *N*-acyl chains ranging from 16 to 26 carbons in length (Merrill, 2002; Pettus et al., 2003a; Merrill et al., 2005), and some times longer in tissues such as skin. Many studies have used a short-chain analog (N-acetylsphingosine, or C2-ceramide) in experiments with cells in culture because it can be incorporated into cells more easily and rapidly than long-chain ceramides. Of note, although C2-ceramide was suggested not to occur *in vivo*, recent studies demonstrated that C2-ceramide does exist in mammalian tissues. In particular, C2-ceramide was found in rat liver cells (Merrill et al., 2001; Van Overloop et al., 2007), and brain tissue (Van Overloop et al., 2007). Ceramide generation is also relevant because this sphingolipid is the precursor of important bioactive molecules that can also regulate cellular functions. For instance, stimulation of ceramidases results in generation of sphingosine (Fig. 1), which was first described as a physiological inhibitor of protein kinase C (PKC) (Hannun et al., 1986). There are numerous reports in the scientific literature showing that PKC is inhibited by exogenous addition of sphingosine to cells in culture. Moreover, Merrill and co-workers demonstrated that addition of the ceramide synthase inhibitor fumonisin B1 to J774.A1 macrophages to increase the levels of endogenous sphingoid bases, also inhibited protein kinase C (Smith et al., 1997). Further work showed that sphingosine can affect the activity of other important enzymes that are involved in the regulation of metabolic or cell signaling pathways such as the Mg2+ dependent form of phosphatidate phosphohydrolase (Jamal et al., 1991; Gomez-Munoz et al., 1992), phospholipase D (PLD) (Natarajan et al., 1994), or diacylglycerol kinase (DAGK) (Sakane et al., 1989; Yamada et al., 1993). Sphingosine, in turn, can be phosphorylated by the action of sphingosine kinases to generate S1P, which is a potent mitogenic agent and can also inhibit apoptosis in many cell types (Olivera & Spiegel, 1993; Wu et al., 1995; Spiegel et al., 1996; Spiegel & Merrill, 1996; Spiegel & Milstien, 2002; Spiegel & Milstien, 2003). More recently, we demonstrated that S1P stimulates cortisol (Rabano et al., 2003) and aldosterone secretion (Brizuela et al., 2006) in cells of the zona fasciculata or zona glomerulosa, respectively, of bovine adrenal glands, suggesting that S1P plays an

A major metabolite of ceramide in cells is ceramide-1-phosphate (C1P), which is formed directly through phosphorylation of ceramide by the action of ceramide kinase (CerK) (Fig. 1). There is increasing evidence suggesting that C1P can regulate cell proliferation and apoptosis (Reviewed in (Gomez-Munoz, 1998; Gomez-Munoz, 2004)), and Chalfant and coworkers have implicated C1P in inflammatory responses (Reviewed in (Chalfant & Spiegel, 2005; Lamour & Chalfant, 2005)). In addition, Shayman's group demonstrated that C1P plays a key role in phagocytosis (Hinkovska-Galcheva & Shayman; Hinkovska-Galcheva et

The aim of the present chapter is to review and update recent progress on the regulation of

important role in the regulation of steroidogenesis.

al., 1998; Hinkovska-Galcheva et al., 2005).

cell survival and inflammation by C1P.

Fig. 1. Biosynthesis of simple sphingolipids in mammalian cells. Ceramide is the central core of sphingolipid metabolism. It can be produced by *de novo* synthesis through the concerted action of serine palmitoyltransferase and dihydroceramide synthase or by degradation of sphingomyelin (SM) through sphingomyelinase (SMase) activation. Ceramides can also be generated through metabolism of more complex sphingolipids. Phosphorylation of ceramide by ceramide kinase gives rise to ceramide-1-phosphate. The reverse reaction is catalyzed by ceramide-1-phosphate phosphatase, or by lipid phosphate phosphatases. Alternatively, ceramide can be degraded by ceramidases to form sphingosine, which can, in turn, be phosphorylated to sphingosine-1-phosphate by sphingosine kinases. The reverse reaction is catalyzed by sphingosine-1-phosphate phosphatases, or by lipid phosphate phosphatases. Sphingosine-1-phosphate lyase breaks down Sphingosine-1-phosphate to hexadecenal and ethanolamine phosphate, both of which can be recycled back to generate phosphatidylethanolamine. Sphingomyelin *N*deacylase generates sphingosylphosphorylcholine, also known as lysosphingomyelin.

#### **2. Biosynthesis of ceramide 1-phosphate. The essential role of ceramide kinase**

At present, the only enzyme known to produce C1P in mammalian cells is ceramide kinase (CerK). This enzyme was first observed in brain synaptic vesicles (Bajjalieh et al., 1989), and was later found in human leukemia HL-60 cells (Kolesnick & Hemer, 1990). Cerk was first reported to be confined to the microsomal membrane fraction, but more recent studies indicate that it is mainly located in the cytosol (Mitsutake et al., 2004). These contradictory observations may arise from the different degrees of enzyme expression in different cell types, and it may also be possible that subcellular localization of this enzyme varies depending on cell metabolism. In this connection, Van Veldhoven and co-workers found that tagged forms of human CerK (FLAG-HsCerK and EGFP-HsCerK fusions), upon expression in Chinese Hamster Ovary (CHO) cells, were mainly localized to the plasma membrane, whereas no evidence for association with the ER was observed (Van Overloop et al., 2006). These findings are in agreement with those of Boath et al. (Boath et al., 2008) who showed that ceramides are not phosphorylated at the ER but must be transported to the Golgi apparatus for phosphorylation by CerK. When C1P is synthesized, it traffics from the Golgi network along the secretory pathway to the plasma membrane, where it can be backexchanged into the extracellular environment and then bind to acceptor proteins such as albumin or lipoproteins (Boath et al., 2008). These observations are consistent with published work by Chalfant's group (Lamour et al., 2007), and it was demonstrated that CerK utilizes ceramide transported to the trans-Golgi apparatus by ceramide transport protein (CERT). In fact, downregulation of CERT by RNA interference resulted in strong inhibition of newly synthesized C1P, suggesting that CERT plays a critical role in C1P formation. However, Boat et al (Boath et al., 2008) reported that the transport of ceramides to the vicinity of CerK is not dependent upon CERT intervention. The reason for such discrepancy is unknown at the present time, but it is possible that the different experimental approaches used in those studies rendered different results. Specifically, whilst Lamour and co-workers used siRNA technology to inhibit CERT (Lamour et al., 2007), Boath and coworkers utilized pharmacological inhibitors (Boath et al., 2008). Also, it might be possible that different cell types may have different subcellular distribution of CerK, and / or that expression of this enzyme activity is not the same in all cell types.

With regards to the regulation of CerK, its ability to move intracellularly from one compartment to another and the dependency on cations (mainly Ca2+ ions) for activity seem to be well established. More recently, CerK has been proposed to be regulated by phosphorylation/dephosphorylation processes (Baumruker et al., 2005), and that it can be myristoylated at its N-terminus, a feature that is related to targeting proteins to membranes. Nonetheless, cleavage of the myristoylated moiety did not affect the intracellular localization of the enzyme. In addition, both CerK location and activity seem to require the integrity of its PH domain, which actually includes the myristoylation site, as deletion of this domain abolishes both the specific subcellular localization of the enzyme, as well as its activity (Baumruker et al., 2005).

Although CerK is thought to be the only enzyme for production of C1P, it was reported that bone marrow-derived macrophages (BMDM) from CerK-null mice (CerK-/-) still had significant levels of C1P (Boath et al., 2008). This observation suggests that there are other metabolic pathways, at least in mammals, capable of generating C1P independently of CerK.

At present, the only enzyme known to produce C1P in mammalian cells is ceramide kinase (CerK). This enzyme was first observed in brain synaptic vesicles (Bajjalieh et al., 1989), and was later found in human leukemia HL-60 cells (Kolesnick & Hemer, 1990). Cerk was first reported to be confined to the microsomal membrane fraction, but more recent studies indicate that it is mainly located in the cytosol (Mitsutake et al., 2004). These contradictory observations may arise from the different degrees of enzyme expression in different cell types, and it may also be possible that subcellular localization of this enzyme varies depending on cell metabolism. In this connection, Van Veldhoven and co-workers found that tagged forms of human CerK (FLAG-HsCerK and EGFP-HsCerK fusions), upon expression in Chinese Hamster Ovary (CHO) cells, were mainly localized to the plasma membrane, whereas no evidence for association with the ER was observed (Van Overloop et al., 2006). These findings are in agreement with those of Boath et al. (Boath et al., 2008) who showed that ceramides are not phosphorylated at the ER but must be transported to the Golgi apparatus for phosphorylation by CerK. When C1P is synthesized, it traffics from the Golgi network along the secretory pathway to the plasma membrane, where it can be backexchanged into the extracellular environment and then bind to acceptor proteins such as albumin or lipoproteins (Boath et al., 2008). These observations are consistent with published work by Chalfant's group (Lamour et al., 2007), and it was demonstrated that CerK utilizes ceramide transported to the trans-Golgi apparatus by ceramide transport protein (CERT). In fact, downregulation of CERT by RNA interference resulted in strong inhibition of newly synthesized C1P, suggesting that CERT plays a critical role in C1P formation. However, Boat et al (Boath et al., 2008) reported that the transport of ceramides to the vicinity of CerK is not dependent upon CERT intervention. The reason for such discrepancy is unknown at the present time, but it is possible that the different experimental approaches used in those studies rendered different results. Specifically, whilst Lamour and co-workers used siRNA technology to inhibit CERT (Lamour et al., 2007), Boath and coworkers utilized pharmacological inhibitors (Boath et al., 2008). Also, it might be possible that different cell types may have different subcellular distribution of CerK, and / or that

**2. Biosynthesis of ceramide 1-phosphate. The essential role of ceramide** 

expression of this enzyme activity is not the same in all cell types.

activity (Baumruker et al., 2005).

With regards to the regulation of CerK, its ability to move intracellularly from one compartment to another and the dependency on cations (mainly Ca2+ ions) for activity seem to be well established. More recently, CerK has been proposed to be regulated by phosphorylation/dephosphorylation processes (Baumruker et al., 2005), and that it can be myristoylated at its N-terminus, a feature that is related to targeting proteins to membranes. Nonetheless, cleavage of the myristoylated moiety did not affect the intracellular localization of the enzyme. In addition, both CerK location and activity seem to require the integrity of its PH domain, which actually includes the myristoylation site, as deletion of this domain abolishes both the specific subcellular localization of the enzyme, as well as its

Although CerK is thought to be the only enzyme for production of C1P, it was reported that bone marrow-derived macrophages (BMDM) from CerK-null mice (CerK-/-) still had significant levels of C1P (Boath et al., 2008). This observation suggests that there are other metabolic pathways, at least in mammals, capable of generating C1P independently of CerK.

**kinase** 

Specifically, formation of C16-C1P, which is a major species of C1P in cells, was not abolished in CerK-/- BMDM. Two alternative pathways for generation of C1P in cells might be: i) acylation of S1P by a putative acyl transferase that would catalyze the formation of a N-linked fatty acid in the S1P moiety to form C1P, and ii) cleavage of sphingomyelin (SM) by the action of a D-type SMase (SMase D), which would generate choline and C1P in an analogous manner to that of phospholipase D acting on phosphatidylcholine to produce choline and phosphatidic acid (PA). However, work from our own lab (Gomez-Munoz et al., 1995a) and that of others (Boath et al., 2008) demonstrated that acylation of S1P to form C1P does not occur in mammalian cells. Also, formation of C1P by the action of a putative SMase D has not yet been reported for mammalian cells. SMase D is a major component of the venom of a variety of arthropods including spiders of the gender *Loxosceles* (the brown recluse spider), such as *L. reclusa*. SMase D is also present in the toxins of some bacteria including *Corynebacterium pseudotuberculosis*, or *Vibrio damsela* (Truett & King, 1993). The bites of this spider result in strong inflammatory responses and may lead to renal failure, and occasionally lead to death (Lee & Lynch, 2005). Although we found no evidence for an analogous activity of SMase D in rat fibroblasts (Gomez-Munoz et al., 1995a), this possibility should be explored in more detail using different types of cells; so it is possible that SMase D may still be the cause for C1P generation in selective tissues.

Concerning regulation, mammalian CerK was demonstrated to be highly dependent on Ca2+ ions for activity (Van Overloop et al., 2006). More recently, it has been shown that treatment of human lung adenocarcinoma A549 cells and Chinese hamster ovary cells (CHO) with orthovanadate, a potent inhibitor of tyrosine phosphatases, increased CerK expression potently (Tada et al., 2010), suggesting a possible regulation of CerK by phosphorylation/dephosphorylation processes on tyrosine residues. Also, it has been suggested that CerK expression can be regulated through activation of Toll-like receptor 4 (TLR-4) by agonists such as the bacterial toxin lipopolysaccharide (Rovina et al., 2010).

The cloning of CerK (Sugiura et al., 2002) opened a new avenue of research that led to determination of important structural properties of this enzyme. The protein sequence has 537 amino acids with two protein sequence motifs, an N-terminus pleckstrin homology (PH) domain, and a C-terminal region containing a Ca2+/calmodulin binding domain. Using sitedirected mutagenesis, it was found that leucine 10 in the PH domain is essential for the catalytic activity of CerK (Kim et al., 2005). In addition, it was reported that the interaction between the PH domain of CERK and phosphatidylinositol 4,5-bisphosphate regulates the plasma membrane targeting and the levels of C1P (Kim et al., 2006). CERK also contains the five conserved sequence stretches (C1-C5) that are specific for lipid kinases (Reviewed in (Baumruker et al., 2005)).

With regards to substrate specificity, it was reported that phosphorylation of ceramide by CERK is stereospecific (Wijesinghe et al., 2005). The latter report also showed that a minimum of a 12-carbon acyl chain was required for normal CERK activity, whereas the short-chain ceramide analogues C8-ceramide, C4-ceramide, or C2-ceramide were poor substrates for CERK. It was concluded that CERK phosphorylates only the naturally occurring D-erythro-ceramides (Wijesinghe et al., 2005). However, C2-ceramide has been shown to also be a good substrate for CerK, especially when albumin is used as a carrier, and that C2-ceramide can be converted to C2-C1P within cells (Van Overloop et al., 2007). This raises the possibility that C2-C1P is also a natural sphingolipid, capable of eliciting important biologic effects, as previously demonstrated (i.e. stimulation of cell proliferation (Gomez-Munoz et al., 1995a)). These observations suggested that substrate presentation is an important factor when testing CerK activity and that the use of different vehicles may result in different outcomes. Also, it should be borne in mind that CerK expression may not be the same in all cell types. The importance of CERK in cell signaling was emphasized in experiments using specific small interfering RNA (siRNA) to silence the gene encoding for CerK. Downregulation of CerK blocked the response of the enzyme to treatment with ATP, the calcium ionophore A23187, or interleukin 1-betta (Pettus et al., 2003b; Chalfant & Spiegel, 2005), and led to a potent inhibition of arachidonic acid release and PGE2 formation in A549 lung adenocarcinoma cells. The relevance of CerK in cell biology was also highlighted in studies using CerK null mice; specifically, a potent reduction in the amount of neutrophils in the blood and spleen of these animals compared to their wild type counterparts was observed, whereas de amount of leukocytes, other than neutrophils, was increased in those mice. These observations suggested an important role of CerK in neutrophil biology (Graf et al., 2008). In addition to CerK, a ceramide kinase-like (CERKL) protein was identified in human retina (Tuson et al., 2004), and this was subsequently cloned (Bornancin et al., 2005). However, CERKL failed to phosphorylate ceramide or other related lipids, under conditions commonly used to measure CERK activity. Therefore, the role of this protein in cell biology is unclear at the present time.

CerK has also been reported to exist in dicotyledonous plants, where it was associated to the regulation of cell survival (Bi et al., 2011). Also, it has been recently found that a conserved cystein motif is critical for rice CerK activity and function (Bi et al., 2011). However, no reports on the possible existence of Cerk in monocot plants are available at the present time.

## **3. Catabolism of ceramide 1-phosphate**

From the above discussion, it should be apparent that C1P is a bioactive metabolite, capable of altering cell metabolism rapidly and potently. So, the existence of enzymes capable of degrading C1P seemed to be feasible for regulation of C1P levels. The identification of a specific C1P phosphatase in rat brain (Shinghal et al., 1993), and hepatocytes (Boudker & Futerman, 1993), together with the existence of CerK suggested that ceramide and C1P are interconvertible in cells. C1P phosphatase is enriched in brain synaptosomes and liver plasma membrane fractions, and appeared to be distinct from PA phosphohydrolase, the phosphatase that hydrolyzes PA. Nonetheless, C1P can also be converted to ceramide by the action of a PA phosphohydrolase that is specifically located in the plasma membrane of cells (Waggoner et al., 1996). The latter enzyme belongs to a family of at least three mammalian lipid phosphate phosphatases (LPPs) (Brindley & Waggoner, 1998). LPPs have recently been shown to regulate cell survival by controlling the levels of intracellular PA and S1P pools (Long et al., 2005), and also to regulate leukocyte infiltration and airway inflammation (Zhao et al., 2005). Dephosphorylation of C1P might be a way of terminating its regulatory effects, although the resulting formation of ceramide could potentially be detrimental for cells. Controlling the levels of ceramide and C1P by the coordinated action of CERK and C1P phosphatases, may be of crucial importance for the metabolic or signaling pathways that are regulated by these two sphingolipids. It could be speculated that another possibility for degradation of C1P might be its deacylation to S1P, which could then be cleaved by lyase activity to render a fatty aldehyde and ethanolamine phosphate (Merrill & Jones, 1990), or to

important biologic effects, as previously demonstrated (i.e. stimulation of cell proliferation (Gomez-Munoz et al., 1995a)). These observations suggested that substrate presentation is an important factor when testing CerK activity and that the use of different vehicles may result in different outcomes. Also, it should be borne in mind that CerK expression may not be the same in all cell types. The importance of CERK in cell signaling was emphasized in experiments using specific small interfering RNA (siRNA) to silence the gene encoding for CerK. Downregulation of CerK blocked the response of the enzyme to treatment with ATP, the calcium ionophore A23187, or interleukin 1-betta (Pettus et al., 2003b; Chalfant & Spiegel, 2005), and led to a potent inhibition of arachidonic acid release and PGE2 formation in A549 lung adenocarcinoma cells. The relevance of CerK in cell biology was also highlighted in studies using CerK null mice; specifically, a potent reduction in the amount of neutrophils in the blood and spleen of these animals compared to their wild type counterparts was observed, whereas de amount of leukocytes, other than neutrophils, was increased in those mice. These observations suggested an important role of CerK in neutrophil biology (Graf et al., 2008). In addition to CerK, a ceramide kinase-like (CERKL) protein was identified in human retina (Tuson et al., 2004), and this was subsequently cloned (Bornancin et al., 2005). However, CERKL failed to phosphorylate ceramide or other related lipids, under conditions commonly used to measure CERK activity. Therefore, the

CerK has also been reported to exist in dicotyledonous plants, where it was associated to the regulation of cell survival (Bi et al., 2011). Also, it has been recently found that a conserved cystein motif is critical for rice CerK activity and function (Bi et al., 2011). However, no reports on the possible existence of Cerk in monocot plants are available at the present time.

From the above discussion, it should be apparent that C1P is a bioactive metabolite, capable of altering cell metabolism rapidly and potently. So, the existence of enzymes capable of degrading C1P seemed to be feasible for regulation of C1P levels. The identification of a specific C1P phosphatase in rat brain (Shinghal et al., 1993), and hepatocytes (Boudker & Futerman, 1993), together with the existence of CerK suggested that ceramide and C1P are interconvertible in cells. C1P phosphatase is enriched in brain synaptosomes and liver plasma membrane fractions, and appeared to be distinct from PA phosphohydrolase, the phosphatase that hydrolyzes PA. Nonetheless, C1P can also be converted to ceramide by the action of a PA phosphohydrolase that is specifically located in the plasma membrane of cells (Waggoner et al., 1996). The latter enzyme belongs to a family of at least three mammalian lipid phosphate phosphatases (LPPs) (Brindley & Waggoner, 1998). LPPs have recently been shown to regulate cell survival by controlling the levels of intracellular PA and S1P pools (Long et al., 2005), and also to regulate leukocyte infiltration and airway inflammation (Zhao et al., 2005). Dephosphorylation of C1P might be a way of terminating its regulatory effects, although the resulting formation of ceramide could potentially be detrimental for cells. Controlling the levels of ceramide and C1P by the coordinated action of CERK and C1P phosphatases, may be of crucial importance for the metabolic or signaling pathways that are regulated by these two sphingolipids. It could be speculated that another possibility for degradation of C1P might be its deacylation to S1P, which could then be cleaved by lyase activity to render a fatty aldehyde and ethanolamine phosphate (Merrill & Jones, 1990), or to

role of this protein in cell biology is unclear at the present time.

**3. Catabolism of ceramide 1-phosphate** 

sphingosine by the action of S1P phosphatases (Fig. 1). However, no C1P deacylases or lyases have so far been identified in mammalian tissues, suggesting that the only pathway for degradation of C1P in mammals is through phosphatase activity.
