Mammalian LPPs (lipid phosphate phosphatases) are integral membrane proteins that belong to a superfamily of lipid phosphatases/phosphotransferases. They have broad substrate specificity in vitro, dephosphorylating PA (phosphatidic acid), S1P (sphingosine 1-phosphate), LPA (lysophosphatidic acid) etc. Their physiological role may include the attenuation of S1P- and LPA-stimulated signalling by virtue of an ecto-activity (i.e. dephosphorylation of extracellular S1P and LPA), thereby limiting the activation of LPA- and S1P-specific G-protein-coupled receptors at the cell surface. However, our recent work suggests that an intracellular action of LPP2 and LPP3 may account for the reduced agonist-stimulated p42/p44 mitogen-activated protein kinase activation of HEK-293 (human embryonic kidney 293) cells. This may involve a reduction in the basal levels of PA and S1P respectively and the presence of an early apoptotic phenotype under conditions of stress (serum deprivation). Additionally, we describe a model whereby LPP2, but not LPP3, may be functionally linked to the phospholipase D1-derived PA-dependent recruitment of sphingosine kinase 1 to the perinuclear compartment. We also consider the potential regulatory mechanisms for LPPs, which may involve oligomerization. Lastly, we highlight many aspects of the LPP biology that remain to be fully defined.
Mammalian LPPs (lipid phosphate phosphatases) are integral membrane proteins that display broad substrate specificity in vitro, catalysing the dephosphorylation of lipid phosphates such as S1P (sphingosine 1-phosphate), LPA (lysophosphatidic acid), PA (phosphatidic acid) and C1P (ceramide 1-phosphate) in a Mg2+-independent and N-ethylmaleimide-insensitive manner [1,2]. Three mammalian LPP isoforms have been cloned, termed LPP1 (which also exists in a spliced variant form, LPP1a), LPP2 and LPP3 [3–7]. The LPPs are predicted to have six transmembrane domains with the active site facing the extracellular side of the plasma membrane or the luminal side of intracellular membranes [8,9]. A three-domain conserved catalytic motif exists (C1, KXXXXXXRP; C2, SXH; C3, -RXXXXXHXXXD; comprising residues within the second and third extramembrane loops and also proximal to the third extramembrane loop), placing LPPs into a phosphatase superfamily  where several absolutely conserved residues are essential for catalytic activity.
The related S1P-specific phosphatases (SPP1 and SPP2) also exhibit the same C1–C2–C3 catalytic motif although the C2 domain has a minor substitution (which may provide their substrate specificity) and these enzymes have two additional predicted transmembrane regions towards the C-terminus [11,12]. These ER (endoplasmic reticulum) proteins are believed to be involved in the generation of sphingosine for ceramide synthesis . The relative levels of S1P and ceramide contribute to growth/survival and apoptosis respectively [14,15]. Overexpression of SPP reduces cell survival due to an increased ceramide/S1P ratio [11,13], whereas small interfering RNA reduction of SPP1 expression increases both intracellular and extracellular S1P, resulting in an increased resistance of cells to agonist-induced apoptosis and chemotherapeutic drugs .
A highly related motif also exists in a family of recently identified LPRs/PRGs (lipid phosphatase-related proteins/plasticity-related genes), which have an extended C-terminal hydrophilic domain when compared with LPPs. LPR3/PRG1, which increases during brain development, was suggested to protect neurites from LPA-induced growth cone collapse due to the dephosphorylation of extracellular LPA , whereas LPR1/PRG3 promoted neurite outgrowth but had no catalytic activity . However, residues that are critical for catalytic activity are absent from the conserved motif of LPR3/PRG1 and the catalytic activity of LPR3/PRG1 is a matter of debate . Alternative roles for LPRs/PRGs may be to act as a ‘sink’ for lipid phosphates and/or have ‘receptor-like’ activity  and/or as regulators of LPPs (see below).
SMSs (sphingomyelin synthases), which catalyse the interconversion of phosphatidylcholine and ceramide with sphingomyelin and DG (diacylglycerol) by transfer of the phosphocholine headgroup, and CCS2 (type 2 candidate sphingomyelin synthase) also have similar motifs. However, SMSs have the same C2 and C3 domains but a differing C1 domain, whereas CCS2 lacks critical residues for catalysis and has no C1 domain. Collectively, these five groups of related proteins have been suggested to form a larger family of LPTs (lipid phosphatases/phosphotransferases) .
Physiological roles of LPPs
LPP activity at the cell surface
The subcellular localization of LPPs is the key to their physiological role. Isoform-selective antibodies and epitope-tagged antibodies have been used to show the plasma membrane location of LPP1, LPP2 and LPP3 [3,20–25], with LPP1 and LPP3 being detected in detergent-resistant membranes and caveolae [22,23] and differentially localized to the apical (LPP1) and basolateral (LPP3) membranes of the polarized Madin–Darby canine kidney cells . Thus LPPs have the potential to influence physiological processes, such as proliferation/survival, apoptosis, differentiation, migration etc., by limiting the availability of the lipid agonists LPA and S1P at their respective GPCRs (G-protein-coupled receptors; LPA1–LPA4 and S1P1–S1P5) . Ecto-LPP activity (i.e. dephosphorylation of exogenously added substrate) against LPA and S1P has been reported in cells where LPP1 has been overexpressed [5,20,21,27] and in preadipocytes where LPP1 is endogenously expressed . Similarly, cells overexpressing LPP2 or LPP3 exhibit ecto-LPP activity against LPA [20,29,30], whereas extracellular S1P can be degraded by cells overexpressing LPP3 . Notably, platelets, which endogenously express LPP1, exhibit a substantial proportion of their total cellular LPP activity as ecto-LPP activity and this regulates their acute responsiveness to LPA. LPP1 redistributes to the platelet surface upon platelet activation and acute responses to LPA, such as shape change and aggregation, are increased in the presence of an LPP1-specific inhibitor, supporting the physiological relevance of ecto-LPP activity of LPP1 . Endogenous LPP may also play a role in limiting chronic LPA responsiveness by reducing the bulk concentration of extracellular LPA. This is exemplified in preadipocytes where extracellular LPA has a half-life of approx. 2–3 h . Interestingly, human ovarian carcinoma samples exhibit reduced levels of LPP1 mRNA compared with normal ovarian surface epithelial cells , which may contribute to the elevated LPA levels in ascites from ovarian cancer patients.
However, the plasma LPA concentration and composition are not significantly different in transgenic LPP1 mice when compared with wild-type mice . Additionally, in LPP1-transfected Rat2 fibroblasts, only a small proportion of total cellular LPP activity can be measured as ecto-LPP activity and the bulk concentration of extracellular LPA is not significantly reduced under acute conditions . Despite this, LPA-stimulated responses including the activation of PLD (phospholipase D) and p42/p44 MAPK (mitogen-activated protein kinase) are reduced in Rat2 cells overexpressing LPP1 . Similarly, we have shown that ecto-LPP activity against exogenous LPA and S1P is minimal in HEK-293 cells (human embryonic kidney 293 cells) that separately stably overexpress LPP1, LPP2 or LPP3 and that this does not correlate with either the extent of LPP overexpression or the loss of LPA- or S1P-stimulated p42/p44 MAPK activation . In contrast, immortalized embryonic fibroblasts derived from wild-type mice and LPP1 transgenic mice show no difference in LPA-stimulated p42/p44 MAPK activation . The differences in these observations regarding LPP1 might be accounted for by its more moderate level of overexpression in cells derived from LPP1 transgenic mice. Significantly, however, thrombin-stimulated p42/p44 MAPK activation was also reduced in HEK-293 cells overexpressing either LPP1 or LPP2 , suggesting that ecto-LPP activity might not account for the reduced responsiveness of these cells and that an intracellular mechanism may be involved. More recently, we have shown that activation of p42/p44 MAPK in response to the dephosphorylation-resistant thio-LPA analogue 2S-OMPT (1-O-oleoyl-2-O-methylglyceryl-3-phosphothionate) was reduced in LPP2- or LPP3-overexpressing HEK-293 cells . Since 2S-OMPT has been shown to act at LPA receptors and is resistant to LPP-catalysed dephosphorylation [36,37], this observation also supports an intracellular action of overexpressed LPP2 and LPP3 in the reduction of the LPA-dependent stimulation of p42/p44 MAPK in serum-deprived HEK-293 cells. A partial intracellular action of LPP1, in addition to its possible extracellular action, was also observed for the modulation of LPA-stimulated nuclear factor κB activation and interleukin-8 secretion in bronchial epithelial cells . Thus intracellular mechanisms might also contribute to the loss of acute agonist-stimulated responses in LPP-overexpressing cells (see below).
Intracellular LPP activity
Cellular levels of PA are reduced and those of DG increased in some, but not all, cell types where LPPs have been overexpressed. For example, the relative amounts of PA and DG were altered when LPP1 was overexpressed in endothelial cells , but not in embryonic fibroblasts derived from LPP1 transgenic mice, although PMA-stimulated DG levels were increased . Conversely, DG levels were reduced and PA levels increased in embryonic fibroblasts derived from LPP3 knockout mice . Such changes may occur at specific subcellular locations. Indeed, LPP3 has been localized with PLD2 in caveolin-enriched detergent-resistant microdomains, where LPP3, but not LPP1, metabolizes PLD2-derived PA . Local concentrations of PA, which indirectly affect local concentrations of phosphoinositides, and the ratio of PA/LPA are important factors in facilitating membrane curvature that is required for endocytosis , and GPCR endocytosis is required for the activation of p42/p44 MAPK . Additionally, PA is involved in the functioning of Raf , an upstream kinase in the p42/p44 MAPK cascade. Therefore a reduction in local concentrations of PA might account for the loss of agonist-stimulated p42/p44 MAPK activation in HEK-293 cells that overexpress LPPs (see above) [20,35].
We have obtained evidence that is consistent with an important role for LPP2 and LPP3 in regulating intracellular pools of PA and S1P, both of which are substrates of LPPs in vitro. Intracellular actions of S1P that promote cell survival may include the inhibition of caspase-3 and caspase-7  and the mobilization of intracellular stores of Ca2+ . Additionally, SK1 (sphingosine kinase 1) and PLD1 have been shown to protect cells from apoptosis [14,44]. Therefore a reduction in intracellular pools of PA and S1P may promote an apoptotic status in cells that have been subjected to cellular stress (serum deprivation). This, in turn, might oppose agonist-stimulated p42/p44 MAPK activation, which is critical for cell survival .
Overexpression of LPP2, but not LPP3, in HEK-293 cells reduced basal intracellular PA and increased DG . In contrast, overexpression of LPP3, but not LPP2, reduced intracellular S1P (produced from exogenous sphingosine added to serum-deprived HEK-293 cells) in the absence of any reduction in SK activity or effect upon sphingosine uptake by the cells . Therefore the net reduction in S1P may be due to increased dephosphorylation of S1P by LPP3 . Interestingly, when we examined the subcellular distribution of GFP (green fluorescent protein)-tagged SK1 in LPP2- and LPP3-overexpressing HEK-293 cells, we observed that GFP-tagged SK1 was constitutively co-localized with LPP2 and LPP3. Therefore, although both LPP2 and LPP3 co-localize with SK1, only LPP3 reduces intracellular S1P levels .
HEK-293 cells overexpressing LPP2 or LPP3 were shown to more readily adopt an apoptotic phenotype upon serum starvation compared with vector-transfected cells . This was evident as an increase in caspase-3/caspase-7 activity of cell lysates derived from LPP-overexpressing cells when compared with vector-transfected cells. In addition, serum-deprived LPP2- and LPP3-overexpressing HEK-293 cells exhibited increased internucleosomal DNA fragmentation, suggesting the onset of early stage apoptotic events. In contrast, no significant DNA laddering was evident in serum-deprived vector-transfected cells. The onset of the apoptotic state appears to be dependent upon serum deprivation as non-confluent LPP2- or LPP3-overexpressing HEK-293 cells grown in the presence of serum are viable, actively proliferate and do not undergo apoptosis. In contrast, vector-transfected cells remain capable of growth (measured as protein content) on third day after serum withdrawal, whereas growth is arrested in LPP2- and LPP3-overexpressing cells, where protein content is reduced. The latter is indicative of impaired cell survival and consistent with cell death .
The apparent onset of an apoptotic state of serum-starved LPP2- and LPP3-overexpressing HEK-293 cells correlates with a reduced ability to respond to GPCR agonists . For example, LPA- and S1P-stimulated phosphorylation of p42/p44 MAPK is attenuated in serum-starved LPP2- and LPP3-overexpressing HEK-293 cells . Significantly, this loss of responsiveness to LPA and S1P is restored following the pretreatment of serum-deprived LPP2- and LPP3-overexpressing HEK-293 cells with the caspase-3/caspase-7 inhibitor Ac-DEVD-CHO (where Ac-DEVD stands for N-acetyl-Asp-Glu-Val-Asp and CHO for Chinese hamster ovary). This is consistent with other findings showing that the activation of caspase-3 leads to proteolytic inactivation of Raf, an upstream regulator of p42/p44 MAPK . The putative action of caspase-3 on a common signalling intermediate (such as Raf) in the p42/p44 MAPK cascade might account for the heterologous inhibitory effect of LPP2 and LPP3 with respect to receptor stimulation by, for example, LPA, S1P and thrombin . The apoptotic action of LPP2 and LPP3 is supported by the fact that DG and sphingosine, the products of LPP-dependent dephosphorylation of PA and S1P respectively, have been implicated in inducing apoptosis. For instance, PKC (protein kinase C), which is activated by DG, is involved in the promotion of apoptosis in certain cell types, while sphingosine activates PKCδ to promote apoptosis . We therefore suggest that LPP2 and LPP3 might act on specific small intracellular pools of PA and S1P respectively that regulate the apoptotic status of the cell. These pools of PA and S1P appear to be accessed by the LPPs under conditions of cellular stress (e.g. serum deprivation) that may lead to the attenuation of agonist-stimulated p42/p44 MAPK signalling.
LPPs and SK1
We have recently shown that SK1, which catalyses the formation of intracellular S1P, contains a PA-binding domain in the C-terminal region . Moreover, SK1 is recruited to PLD1-derived PA in a discrete perinuclear compartment (partially co-localizing with the endosomal protein EEA1 (early endosomal antigen 1) and with the Golgi protein mannosidase 2) upon induction of PLD1 expression or agonist stimulation . Therefore we examined the potential link between PLD1-derived PA, SK1 and LPP2 or LPP3. To do this, a PLD1-inducible CHO cell line was used in which SK1 becomes co-localized with induced PLD1 in the perinuclear compartment . Transient transfection of the PLD1-inducible CHO cell line with FLAG-tagged LPP2 and GFP-tagged SK1 showed that these proteins are constitutively co-localized in cytoplasmic vesicles. Similarly, transient transfection of the cells with FLAG-tagged LPP3 and GFP-tagged SK1 resulted in their co-localization in cytoplasmic vesicles. However, a significant difference between the FLAG–LPP2/GFP–SK1- and FLAG–LPP3/GFP–SK1-transfected cells was observed upon PLD1 induction. Treatment of FLAG–LPP3/GFP–SK1-transfected cells with doxycycline (to induce PLD1 expression) resulted in the translocation of both GFP–SK1 and FLAG–LPP3 to the perinuclear compartment . This result supports our previous observation that PLD1-derived PA regulates the subcellular distribution of SK1 . Additionally, it suggests that LPP3 does not dephosphorylate PLD1-derived PA. In contrast, doxycycline-induced expression of PLD1 in the FLAG–LPP2/GFP–SK1 co-expressing cells was without effect upon their subcellular localization, i.e. FLAG–LPP2 and GFP–SK1 remained co-localized in cytoplasmic vesicles and the expected PLD1-induced translocation of GFP–SK1 to the perinuclear compartment was prevented . This result is consistent with a model in which LPP2 might remove PLD1-derived PA and thereby prevent the recruitment of SK1 to the perinuclear compartment. Significantly, we also demonstrated that endogenously expressed LPP2 and LPP3 in CHO cells are predominantly localized in cytoplasmic vesicles throughout the cell, with some accumulation in the perinuclear compartment . Treatment with PMA, which has previously been shown to activate PLD1 in these cells , increased the accumulation of endogenous LPP3 in the perinuclear compartment, consistent with the observed redistribution of recombinant LPP3 (and GFP–SK1) in response to PLD1 induction (see above). However, endogenous LPP2 did not accumulate in the perinuclear compartment in either control or PMA-treated cells, which is consistent with the lack of effect of PLD1 induction upon the subcellular localization of recombinant LPP2 (see above) . As yet, the mechanism underlying the redistribution of LPP3 to the perinuclear compartment remains unknown. However, others have also reported a perinuclear/ER distribution of endogenous LPP3 in HEK-293 cells and platelets [22,31]. These results begin to establish a functional link between PLD1-derived PA and LPP2, where their relative activities could contribute to the subcellular localization of SK1 (1).
Model of LPP2 and LPP3 in the regulation of second messenger lipids, SK1 translocation and cell survival or apoptosis
Regulation of LPPs
The regulation of LPPs is yet to be defined. To date, there is no direct evidence of agonist-dependent phosphorylation (or dephosphorylation) of LPPs although LPP activity was reported to redistribute into caveolae in response to the PKC activator, PMA . Regulation of LPPs by protein–protein association may occur . For example, sequences similar to the caveolin-1-binding domain consensus sequence were identified in LPP1, LPP2 and LPP3, although LPP activity failed to co-immunoprecipitate with caveolin-1 . Alternatively, LPP activity may be regulated by oligomerization. Conventional purification strategies indicated a native molecular mass between 53 and 83 kDa, which is significantly larger than the 32–35 kDa finally determined by cloning [3,4,8]. Similarly, we have used gel filtration of LPP activity isolated from HEK-293 cells that separately overexpress LPP isoforms to show that multimeric forms of LPP may be present and that these are associated with increased activity . In addition, we have isolated both homomeric and heteromeric complexes of Myc- and FLAG-epitope-tagged LPPs from transiently transfected HEK-293 cells by their separate immunoprecipitation with either anti-Myc-epitope or anti-FLAG-epitope antibodies. LPP complexes are not detected when extracts from cells that have been separately transfected with either Myc-tagged LPP or FLAG-tagged LPP are simply combined (J.S. Long and S. Pyne, unpublished work). Interestingly, epitope-tagged mLPP1 (murine LPP1) and hLPP3 (human LPP3) have been reported to homodimerize, but not heterodimerize, when expressed in Drosophila S2 cells . Similarly, Wun (a Drosophila homologue of LPP) homodimerizes but cannot heterodimerize with Wun2, mLPP1 or hLPP3. Dimerization was dependent upon the C-terminal 35 amino acids of Wun and was abolished by mutation of Asp248 (a required residue for catalytic activity) to threonine . However, dimerization was not believed to be required for in vivo activity of Wun since the inclusion of a C-terminal trimeric Myc tag, which abolished the disruptive effect of ectopic expression of Wun on germ cell migration, did not prevent dimerization . Nevertheless, oligomerization in mammalian cells may provide a mechanism for the activation of LPPs in response to agonists and/or contribute to the enhanced LPP activity in caveolae. Additionally, regulation by association with members of the LPR/PRG family in cell types where these proteins are co-expressed with LPPs is yet to be addressed.
Despite intense efforts, there are many aspects of LPP biology that remain unclear including their precise physiological roles. These might include (i) the modulation of LPA release, (ii) uptake of extracellular lipids and (iii) regulation of adhesion and development (reviewed in ), in addition to the proposed ectoactivity and modulation of intracellular second messenger lipids, described above. Further development of knockout and transgenic animal models and the study of related proteins in bacteria and yeast [2,19] are expected to advance our understanding of this intriguing group of proteins. To date, LPP1 knockout mice have not been reported whereas transgenic LPP1 mice exhibit no difference in plasma LPA concentration or composition compared with wild-type mice but have defects in fur growth and spermatogenesis . LPP2 knockout mice have no obvious phenotype and are viable . Lastly, knockout of LPP3 is embryonic lethal with mice exhibiting defects in vasculogenesis and patterning in early development . Interestingly, Wnt signalling appears to be negatively regulated by LPP3 in a manner that is independent of its catalytic activity and which remains to be defined . This raises further questions that need to be addressed for this interesting subset of the LPT family.
Cellular Information Processing: A Focus Topic at BioScience2005, held at SECC Glasgow, U.K., 17–21 July 2005. Edited by F. Antoni (Edinburgh, U.K.), C. Cooper (Essex, U.K.), M. Cousin (Edinburgh, U.K.), A. Morgan (Liverpool, U.K.), M. Murphy (Cambridge, U.K.), S. Pyne (Strathclyde, U.K.) and M. Wakelam (Birmingham, U.K.).
type 2 candidate sphingomyelin synthase
green fluorescent protein
cells, human embryonic kidney 293 cells
lipid phosphate phosphatase
lipid phosphatase-related protein/plasticity-related gene
mitogen-activated protein kinase
protein kinase C
sphingosine kinase 1
S1P-specific phosphatase, 2S-OMPT, 1-O-oleoyl-2-O-methyl-glyceryl-3-phosphothionate