Biochimica et Biophysica Acta 1781 (2008) 467–476

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta

j ourna l homepage: www.e lsev ie r.com/ locate /bba l ip

Review

Sphingosine 1-phosphate, lysophosphatidic acid and growth factorsignaling and termination

Nigel J. Pyne ⁎, Susan PyneCell Biology Group, SIPBS, University of Strathclyde, 27 Taylor Street, Glasgow, G4 0NR, Scotland, UK

⁎ Corresponding author. Tel.: +44 141 548 2659.E-mail address: n.j.pyne@strath.ac.uk (N.J. Pyne).

1388-1981/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.bbalip.2008.05.004

A B S T R A C T

A R T I C L E I N F O

Article history:

Sphingosine 1 phosphate (S Received 18 February 2008Received in revised form 27 April 2008Accepted 12 May 2008Available online 17 June 2008

Keywords:Sphingosine 1-phosphateLysophospatidic acidPDGFNGFGrowth factorLipid phosphate phosphataseSphingosine kinasePhospholipase DIntegrative signalling

1P) and lysophosphatidic acid (LPA) are bioactive lipid phosphates that bind tocell surface G-protein coupled receptors (GPCR) and, in addition, exhibit intracellular actions. We havesummarised herein, an important functional interaction between lipid phosphate GPCR and receptor tyrosinekinases (RTK) that enables growth factors to spatially regulate effectors, thereby governing the nature of thebiological response. For instance, we describe how the formation of functional complexes between the S1P1receptor and PDGFβ receptor may effectively re-programme platelet-derived growth factor from a mitogenicto a migratory stimulus. This is achieved by integration of RTK- and GPCR-specific signals that results inspatial regulation of a cytoplasmic retained pool of extracellular signal regulated kinase-1/2 linked to myosinlight chain kinase, myosin light chain phosphorylation and migration. We therefore suggest that the lipidphosphate receptor is a major determinant in regulating growth factor-dependent biology. Growth factorscan also increase S1P inside cells, and we discuss the concept of spatial/temporal aspects ofcompartmentalised intracellular signaling of S1P in relation to defined interactions between, for instance,sphingosine kinase, phospholipase D1 and lipid phosphate phosphatases and regulation of cell survival.

© 2008 Elsevier B.V. All rights reserved.

1. Receptor tyrosine kinase–lipid phosphate receptorcomplex signaling

Signals transmitted by receptor tyrosine kinases (RTK) andG-proteincoupled receptors (GPCR) are integrated to enhance growth factorstimulation of cellular responses [1]. The key component of thismodel isthat agents that disruptGPCR function (e.g. pertussis toxin (PTX)) reducethe growth factor-stimulated activation of extracellular signal regulatedkinase-1/2 (ERK-1/2), of which there are now several examples in theliterature [2–6]. In the context of lipid phosphate receptors, this placesthe GPCR parallel with the RTK while activated Gi functions down-stream from the RTK. We have termed this type of cross talk betweenRTK (e.g. platelet-derivedgrowth factor beta (PDGFβ) receptor) and lipidphosphate receptor (e.g. sphingosine 1-phosphate 1 receptor, S1P1)‘integrative signaling’ (Figs. 1a and 2). This mechanism is distinct fromgrowth factor (e.g. PDGF)-dependent activation of sphingosine kinase(catalyses synthesis of sphingosine 1-phosphate (S1P)) and subsequentrelease of S1P, which then functions in an autocrine manner at S1Preceptors (e.g. S1P1), termed here ‘sequential signaling’ [7] (Fig. 1b). Incontrast with integrative signaling, both the GPCR and activated G-protein function down-stream from the RTK in the sequential signalingmechanism. The major experimental problem here is that agents that

l rights reserved.

disrupt GPCR function do not distinguish between the twomechanisms.However, we describe herein a number of experimental findings thatdistinguish the two models. This said, there is no doubt that bothmechanisms exist and that, in certain cell types, might function inconcert. An example of where this might be appropriate is where theconstitutive activity of S1P1 is too low to enhance PDGF signaling in thecomplex, and might therefore, require PDGF-dependent release ofligand, S1P, via a sphingosine kinase 1 (SK1)-dependent mechanism toactivate the S1P1 receptor in the complex. In addition, such mechanisticlinkage between PDGFβ receptor–S1P1 receptor complexes and SK1might be extended to other possible RTK–S1P1 receptor complexes, suchas for IGFI and IGFII receptors.

1.1. The S1P1 receptor–PDGFβ receptor signaling complex

Lee et al. [8] were the first to identify that the S1P1 receptor bindsS1P. It is now known that there are five closely related GPCR termedS1P1–5 which exhibit high affinity for S1P [9]. We previously reportedthat the endogenous PDGFβ receptor and S1P1 receptor exist in afunctional complex in airway smooth muscle (ASM) cells and mouseembryonic fibroblasts (MEF) [6,10]. A recombinant complex can alsobe formed by over-expression of these receptors in HEK 293 cells [5].The amount of endogenous or recombinant PDGFβ receptor–S1P1receptor complex present in cells is not increased by PDGF or S1P[5,6,10], suggesting that all the available PDGFβ receptor forms acomplex with S1P1. We consider that the PDGFβ receptor and not the

Fig. 1. Schematic showing the comparison between the (a) integrative and (b) sequential signaling models. ⁎ denotes constitutive activation of S1P1.

468 N.J. Pyne, S. Pyne / Biochimica et Biophysica Acta 1781 (2008) 467–476

S1P1 receptor is limiting for formation of the complex based on threefindings. First over-expression of recombinant S1P1 against a back-ground of endogenous PDGFβ receptor/S1P1 receptor does notincrease the amount of complex that can be formed, suggesting thatthe limiting factor is indeed the PDGFβ receptor. Second, administra-tion of SB649146 (which binds to S1P1 receptor) to ASM cellsexpressing endogenous PDGFβ receptor–S1P1 receptor complexresults in exclusive binding of SB649146 to a putative pool of freeS1P1 receptor that is in equilibrium with S1P1 receptor that forms acomplex with PDGFβ receptor and which thereby disrupts PDGFsignaling by mass action (see later). Therefore, S1P1 receptor isunlikely to be limiting for formation of the complex as there appearsto be a pool of free S1P1 receptor. Third, treatment of HEK 293 cellswith PTX completely eliminates PDGF stimulation of ERK-1/2, sug-gesting that all of the available PDGFβ receptor signals to ERK-1/2 in amanner that is contingent on it being in a complex with S1P1 receptor[5]. This is confirmed by the finding that anti-sense mediated down-regulation of endogenous S1P1 in ASMcells eliminates PDGF-stimulatedactivation of ERK-1/2 [6].

The close proximity association between the PDGFβ receptor andthe S1P1 receptor permits the use of activated G-protein subunits(made available by the constitutively active or S1P-stimulated S1P1receptor) by the PDGFβ receptor to enhance signal transmission fromthis receptor in response to PDGF. One of the first events in thesignaling integration is the PDGF-stimulated tyrosine phosphorylationof endogenous Giα [5], which may keep the α and Gβγ subunits apartand thereby allow Gβγ subunits to remain functionally active forlonger. This tyrosine phosphorylation event appears to be the nexus ofthe signal integration between the two receptor classes and seques-tration of Gβγ using the over-expressed C-terminal tail of GRK2 (CT-GRK2) reduces both PDGF- and S1P-stimulation of ERK-1/2 [11]. Instudies where we have over-expressed Giα in HEK 293 cells, less than1% of the Giα is tyrosine phosphorylated in response to PDGF,suggesting that only a small fraction of Giα is used by the PDGFβreceptor–S1P1 receptor complex [5]. Indeed, one might expectlimitation of the amount of Giα used set against the background ofother GPCRs expressed in HEK 293 cells that might interact with Giα.There are some other examples of RTK being able to catalysephosphorylation of Giα. For instance, the insulin receptor tyrosinekinase phosphorylates Giα, with one molecule of alpha subunit beingphosphorylated by one molecule of the insulin receptor beta subunit[12,13].

The PDGF-stimulated tyrosine phosphorylation of Giα is coin-cident with the recruitment of c-Src to the PDGFβ receptor–S1P1receptor complex [11]. We suggest that the recruitment of c-Src in

response to PDGF and S1P is β-arrestin-dependent and does notinvolve Gi. Support for this model was evident from studies showingthat β-arrestin I is associated with the PDGF receptor–S1P1 receptorcomplex and that this is not increased by PDGF [5]. Once recruited tothe complex, c-Src is then activated in a Giβγ-dependent manner, asevidenced by results demonstrating that CT-GRK2 and c-Src inhibitorsreduce activation of ERK-1/2 by PDGF or S1P [11]. The PDGF-inducedactivation of c-Src is also reduced by treating ASM cells with PTX [4],an effect which has also been shown by Spiegel and colleagues infibroblast [14].

This model is consonant with previous studies by Lefkowitz andcolleagues, who have demonstrated that GPCR and RTK can utiliseβ-arrestin-dependent pathways that do not involve G-protein toregulate ERK-1/2 e.g. parathyroid hormone, angiotensin II, β-adrener-gic agonists and IGF-1 [15–18]. This represents a distinctmechanism toconventional Giβγ-regulation of c-Src and ERK-1/2 that can operate forother GPCR and RTK and which also provides a link between thesereceptor classes [19,20].

Lefkowitz and colleagues have demonstrated that the mechanismused by β-arrestin to regulate ERK-1/2 signaling involves GRK5/6and is therefore different compared with its action in desensitisingG-protein-dependent regulation of adenylyl cyclase, which involvesGβγ and GRK2. This is exemplified by studies on the β-adrenergicreceptor (β2AR) signaling system [17]. In this case, activation of ERK-1/2 by the β2AR expressed in HEK-293 cells was resolved into twodistinct mechanistic pathways, one involving a Gs/Gi-protein kinase A(PKA) switch, while the second involves β-arrestins. G-protein-dependent activation was rapid and transient and was blocked byPTX andwas insensitive to siRNA-mediated elimination of endogenousβ-arrestins. β-arrestin-dependent activation of ERK-1/2 was slower inonset but more sustained. It was insensitive to PTX and H-89 andsensitive to siRNA-mediated elimination of β-arrestin 1 or -2. More-over, over-expression of GRK5 or GRK6 resulted in the formation ofstable receptor-β-arrestin complexes on endosomes, and increasedagonist-stimulated phosphorylation of ERK1/2 on endosomes. Incontrast, GRK2 membrane translocation, which requires Gβγ releaseafter G-protein activation, was ineffective. These findings elegantlyshow that the β2AR can activate ERK-1/2 via a GRK5/6-β-arrestin-dependent pathway, which is independent of G-protein coupling. Inthe context of PDGFβ receptor signaling, it is therefore interesting tonote that Freedman and colleagues have demonstrated that GRK5 hasan important role in enhancing PDGFβ receptor-stimulated c-Src acti-vation in aortic smooth muscle cells [21]. In summary, our proposedmodel for PDGFβ receptor–S1P1 receptor complex signaling mayinvolve an integrated Giβγ/c-Src/β-arrestin-dependent regulation of

Fig. 2. Schematic showing the sequence of molecular events involvedwhen PDGF binds to the PDGFβ receptor–S1P1 receptor complex leading to spatial regulation of ERK-1/2 and cellmigration. ⁎ denotes constitutive activation of S1P1.

469N.J. Pyne, S. Pyne / Biochimica et Biophysica Acta 1781 (2008) 467–476

ERK-1/2 that combines the two models proposed by Lefkowitz andcolleagues for GPCR signaling.

Although the constitutively active S1P1 receptor stimulates Gi, thisis not translated into activation of ERK-1/2 (i.e. basal ERK-1/2 activationstatus is low under these conditions), unless PDGF or S1P engage theirrespective receptors in the complex and promote recruitment of c-Srcto the complex [11]. This constraint on ERK-1/2 activation is veryimportant and a key element for the model to be consistent with theexperimental findings. c-Src is most likely recruited to the complex inresponse to PDGF through binding to tyrosine phosphates on thePDGFβ receptor, resultant from the PDGF-dependent activation of theintrinsic PDGFβ receptor kinase. As mentioned earlier β-arrestin I(which also functions to load GPCR complexes into clathrin coated pitsprior to endosome formation) may also play an accessory functionalbinding role. This is based on evidence showing that the over-expression of the clathrin binding domain of β-arrestin I (aminoacids 319–418), which competes with endogenous β-arrestin I forclathrin, reduced the PDGF- and S1P-induced activation of ERK-1/2[11]. Moreover, c-Src can bind to β-arrestin and can thereby act asa functional link to ERK-1/2 activation [22,23]. We do not knowwhether β-arrestin I can activate c-Src, or is simply involved inrecruitment of c-Src to the PDGFβ receptor–S1P1 receptor complex.Clearly, the constitutive association of β-arrestin I with the PDGFβreceptor–S1P1 receptor complex does not enable breaching of a c-Srcactivation threshold required for stimulation of the ERK-1/2 pathwayas basal ERK-1/2 activation is very low in the cells and is not increasedby over-expression of S1P1 receptor. However, the recruitment ofc-Src to the PDGFβ receptor–S1P1 receptor complex in response toPDGF, by operating via a possible two site binding interaction (bindingto tyrosine phosphates on the PDGFβ receptor and via interactionwith β-arrestin), appears to increase the amount of PDGFβ receptor–S1P1 receptor complex with associated c-Src sufficiently to enablebreaching of the Giβγ-regulated c-Src activation threshold requiredfor ERK-1/2 activation.

We have also shown that S1P increases the fraction of PDGFβreceptor–S1P1 receptor complexes associated with β-arrestin I [5].Therefore, this increase might explain why S1P promotes recruitment ofc-Src to the PDGFβ receptor–S1P1 receptor complex. Moreover, thestabilisationof S1P1 by S1Pwill increase the amountof activatedGi,whichin combination with the recruitment of c-Src to β-arrestin I, might thenbreach the Giβγ-regulated Src activation threshold required for stimula-tion of the ERK-1/2 pathway in response to S1P [11]. In addition, thePDGFβ receptor participates in enhancing the S1P-stimulation of ERK-1/2.With respect to formation of RTK-GPCR complexes, Akekawatchai andcolleagues have described a similar molecular arrangement in which thechemokineGPCR, CXCR4 and IGF-1R andG-protein subunits Giα2 andGβare constitutively associated in a complex in breast cancer MDA-MB-231cells [24].

What happens after c-Src is recruited to the PDGFβ receptor–S1P1receptor complex and activated by Giβγ in response to PDGF? Part ofthe answer to this is that c-Src catalyses the tyrosine phosphorylationof Grb-2 associated binder, Gab1 [11,25], which is then followed byrecruitment of phosphoinositide 3-kinase 1a (PI3K1a) to tyrosinephosphorylated Gab1 (which is pre-associated with Grb-2) [11,25].Dynamin II binding to the Grb-2-tyrosine phosphorylated Gab1scaffold is PI3K-dependent and functions to ‘pinch off’ endocyticvesicles that contain the PDGFβ receptor–S1P1 receptor complex,resulting in internalization of the complex [6,11,25]. ERK-1/2 is thenrecruited to endosomal PDGFβ receptor–S1P1 receptor complexes andis activated [6,11]. The biological imperative that is met by formingintegrative complexes between the S1P1 receptor and PDGFβ receptoris that this enables the ligands for these receptors to spatially regulateERK-1/2. In this case, this leads to activation and retention of asubstantial pool of ERK-1/2 in the cytoplasm/plasma membrane andregulation of cell migration (see later). Indeed, we have establishedthat MLC20 phosphorylation, which plays a key role in regulating cellmigration is increased by PDGF in an ERK-1/2-dependent manner inASM cells [26]. Therefore, we have proposed that the S1P1 receptor

470 N.J. Pyne, S. Pyne / Biochimica et Biophysica Acta 1781 (2008) 467–476

might function to re-programme PDGF from a mitogenic to amigratory stimulus.

Lefkowitz and colleagues have also shown that β-arrestin-mediatedsignaling to ERK-1/2 results in cytoplasmic retention of activated ERK-1/2[27]due to interactionof activatedERK-1/2withβ-arrestin inendosomes.Thus, the PDGFβ receptor might use the β-arrestin machinery providedby the S1P1 receptor to retain activatedERK-1/2 in the cytoplasm, therebylocalising activated ERK-1/2 close to MLCK and MLC20 and enablingregulation of cell migration. This migratory signaling mechanismcontrasts with the mitogenic effects of PDGF, the latter which results innuclear translocation of activated ERK-1/2.

2. Pharmacological manipulation of the PDGFβ receptor–S1P1

receptor complex

SB649146 is a compound that has unusual properties and func-tions as a protean agonist of the S1P1 receptor. SB649146 has exquisitespecificity for the S1P1 receptor and decreases the S1P-, SEW2871(selective S1P1 receptor agonist) and PDGF-induced stimulation of theERK-1/2 pathway and reduces ASM cell and MEF migration inresponse to PDGF [26]. SB649146 functions by reducing the con-stitutive activity of the S1P1 receptor in the complex with the PDGFβreceptor [26]. It does not interact directly with the PDGFβ receptor.Therefore, SB649146 effectively reduces the amount of active Gi

subunits that are available for use by the PDGFβ receptor when thelatter has engaged PDGF [26]. As mentioned earlier, the constitutivelyactive S1P1 receptor can enhance PDGF signaling. Direct support forthis was obtained using mutant recombinant S1P1 receptors (R120Aand E121A), which are deficient in their ability to bind S1P [28]. Intheory, mutagenesis of key amino acids in the binding pocket of S1P1may not affect the ability of this receptor to undergo equilibriumtransition to the constitutively active form. In accordance with theproposed model, both over-expressed mutant S1P1 receptors enhancePDGF-stimulated ERK-1/2 activation to a level comparable with thewild type recombinant S1P1 receptor in HEK 293 cells co-expressingrecombinant PDGFβ receptor [26].

SB649146 functions as a protean agonist (see [29,30] for details onprotean agonism) because, by definition, it can act as an inverseagonist, competitive antagonist or partial agonist depending upon theassay used to measure its effects. Thus, SB649146 reduces constitutivebasal S1P1 receptor-stimulated GTPγS binding (inverse agonism),inhibits S1P-stimulated activation of ERK-1/2 (competitive antagon-ism) and, on its own weakly stimulates the ERK-1/2 pathway (partialagonism) [26]. However, all of these actions of SB649146 (and itsability to reduce PDGF signaling) can be explained by a simple one sitebinding model in which the S1P1 receptors exists in multipleconformational states that are in equilibrium and are either associatedor not with the PDGFβ receptor (Fig. 3). In the proposed model, theS1P1 receptor exists in an inactive conformation (R) and at least two

Fig. 3. The effect of protean agonism of the R2 S1P1 receptor transition state withSB649146 and the consequential effect on the engagement of R1 S1P1 receptor transitionstate with the PDGFβ receptor signaling system.

constitutively active conformations (e.g. R1 and R2) in equilibrium. R1and R2 are defined as having different efficacy in terms of coupling toGi. R1 S1P1 receptor has high efficacy while R2 S1P1 receptor has lowefficacy for coupling to Gi. The PDGFβ receptor interacts with the highefficacy R1 form of the S1P1 receptor, while the R2 S1P1 receptor isdissociated from the PDGFβ receptor. SB649146 binds exclusively toR2 S1P1 receptor and stabilises this receptor conformation resulting inlow efficacy stimulation of the ERK-1/2 pathway (partial agonism)presumably because SB649146 can also recruit c-Src to R2 S1P1receptor. The binding of SB649146 to R2 S1P1 receptor will also reducethe amount of R1 S1P1 receptor that is associated with the PDGFβreceptor bymass action. Therefore, the binding of SB649146 to R2 S1P1receptor and thus, transition of high efficacy R1 S1P1 receptor to lowefficacy R2 S1P1 receptor for Gi coupling by mass action explains howSB649146 can reduce basal S1P1 receptor-stimulated G-proteinactivation (inverse agonism) and decrease PDGF stimulation of theERK-1/2 pathway. It is important to note that SB649146 does notcompletely eliminate constitutive activation of the S1P1 receptor inGTPγS assays [26]. Indeed, the reduction in GTPγS binding saturateswith increasing concentration of SB649146 and there is residual Gi

activation (~25% of the total GTPγS binding) that might be a con-sequence of stabilisation of low efficacy R2 S1P1 receptor by SB649146.

The apparent paradox here is how low level activation of Gi bystabilisation of R2 S1P1 receptor with SB649146 results in PTX-sensitive stimulation of ERK-1/2, while high level constitutive Gi

activation by R1 S1P1 receptor does not activate ERK-1/2. This can bereconciled by the possibility that SB649146 might promote Gi-independent β-arrestin I/c-Src recruitment to R2 S1P1, which at thelow level of G-protein activation can be translated into weak ERK-1/2stimulation, although this requires formal testing. In the absence ofSB649146, the R1 S1P1 conformation appears to predominate anddespite exhibiting higher efficacy Gi activation, this receptor con-formation does not stimulate the ERK-1/2 pathway unless PDGF or S1Pengages the PDGFβ receptor–R1 S1P1 receptor complex to promoterecruitment of c-Src to the complex, whereupon c-Src is activated by aGβγ-dependent mechanism above threshold with consequentialsignal relay to ERK-1/2. In this case the activation of ERK-1/2 inducedby PDGF or S1P will theoretically greatly exceed (for PDGF due to theincreased Gi activation induced by R1 S1P1, the PDGFβ receptor-stimulated tyrosine phosphorylation of Giα and determinants on thePDGFβ receptor that contribute to recruitment of c-Src, while for S1P,due to amplification of S1P1 receptor signaling by PDGFβ receptor)that which can be induced by SB649146 on its own and this is indeedobserved experimentally and is the basis of the inhibition of PDGF/S1Psignaling to ERK-1/2 by SB649146. In our view these results collective-ly argue strongly for a model in which regulation of β-arrestin I and Gi

by the S1P1 receptor are mechanistically separated and that β-arrestinI binding to the R1 S1P1 receptor complex is G-protein-independent.There is no restriction for equilibrium transition of R1 to R2 to passthrough an intermediate inactive R form.

In someways, the effect of SB649146 on the S1P1 receptor is similarto the so-called biased agonism observed for β2AR agonists thatdemonstrate a bias for stabilising GPCR-β-arrestin active conforma-tions and which results in activation of ERK-1/2, while at the sametime are inverse agonists for β2AR coupled G-protein signaling [31].SB649146 considerably reduces the amount of the S1P1 receptorconformation that exhibits high efficacy for G-protein coupling andthis might be achieved by stabilising a receptor conformation thatexhibits low G-protein coupling efficacy, but which might retainability to bind β-arrestin.

The proposed equilibrium transition model also predicts thatSB649146 will cause dissociation of the PDGFβ receptor–R1 S1P1receptor complex. We know that SB649146 reduces the PDGF-stimulated endocytosis of the PDGFβ receptor–S1P1 receptor complexand this explains how it can block activation of ERK-1/2 in response toPDGF [26]. The pool of S1P1 receptor that is internalised with the

471N.J. Pyne, S. Pyne / Biochimica et Biophysica Acta 1781 (2008) 467–476

PDGFβ receptor in common endocytic vesicles in response to PDGF[26] may therefore, correspond to the R1 conformation. SB649146 alsostimulates the internalization of a pool of the S1P1 receptor [26].However, this occurs without accompanying PDGFβ receptor, andtherefore, this second pool of S1P1 receptormay indeed, correspond tothe R2 conformation.

We have not been able to demonstrate the presence of a sequentialmechanism for PDGF and S1P in HEK 293 cells, ASM cells or MEF basedon the failure of dominant negative sphingosine kinase 1 andsphingosine kinase inhibitors to reduce the PDGF-stimulated activationof ERK-1/2 [5,6,10]. The integrative model imposes strict adherence tointernalisation of both receptors (as they are in a complex) to commonendocytic vesicles in response to PDGF and S1P, and this is indeed,observed experimentally [6,26]. The demonstration that S1P can alsopromote internalisation of the PDGFβ receptor and S1P1 receptor incommon endocytic vesicles [6] indicates that cross talk between PDGFand S1P is bidirectional. If a sequential mechanismwas operative in theabsence of complex formation between the receptors, then one wouldexpect exogenous S1P to induce internalisation of only S1P1 receptorand not PDGFβ receptor, as S1P would bypass the PDGFβ receptor.Finally, S1P does not increase PDGFβ receptor tyrosine phosphoryla-tion, thereby ruling out PDGF release and transactivation of the PDGFβreceptor via this mechanism [5]. Indeed, we propose that S1Ppreferentially binds to the R1 conformation of the S1P1 receptor inthe complex with the PDGFβ receptor. This is based on several lines ofevidence. First, over-expression of PDGFβ receptor enhances S1P-stimulation of ERK-1/2 in HEK 293 cells [32]. Second, PDGFβ receptorkinase inhibitors (e.g. tyrphostin AG1296) reduce S1P stimulation ofERK-1/2 in ASM cells. Third, S1P induces internalization of the PDGFβreceptor and S1P1 receptor, which are co-localised in the sameendocytic vesicles [6]. The binding of SB649146 to R2 therefore explainsits ability to reduce S1P stimulation of ERK-1/2 mediated by R1 S1P1.

These findings raise the issue ofwhether S1P1 can signal to ERK-1/2in the absence of PDGFβ receptor. Clearly there is a casewhere this canhappen as binding of SB649146 to the putative R2 S1P1 receptor andactivation of ERK-1/2 are independent of PDGFβ receptor in ASM cells[26]. Moreover, we have demonstrated that S1P can stimulate ERK-1/2,albeit weakly in HEK 293 cells that are PDGFβ receptor null [5,32].However, whether the significant activity of S1P1, in awider context, isdirected via cross talk with RTK in terms of enhancing ERK-1/2activation remains a question that needs to be more fully evaluatedin the future. There are studies suggesting that regulation of MEFmigration in response to PDGF does not require S1P1 [33]. However,others have clearly implicated a role of S1P1 in regulating PDGF-stimulated Rac activation [7], which is required for cell migration, bothgroups using S1P1−/− MEFs. We have demonstrated in MEFs, whichexpress endogenous PDGFβ receptor–S1P1 receptor complexes, thatSB649146 reduces PDGF-stimulated activation of ERK-1/2 and migra-tion, thereby implicating a role for the complex inMEFs [10]. However,whether the PDGFβ receptor–S1P1 receptor complex is also involved inregulating effectors such a Rac, which are also linked to migration,requires future study.

3. RGS12 and interaction with PDGFβ receptor–S1P1 receptorcomplex

Since the PDGFβ receptor can form a functional signaling complexwith the S1P1 receptor, we considered the possibility that GTPaseaccelerating proteins (RGS proteins), which regulate GPCR-mediatedsignaling might modulate PDGFβ receptor-mediated signal transmis-sion. In this regard, we have demonstrated that over-expressed RGS12PDZ/PTB domain N-terminus or RGS12 PTB domain reduced the S1P-and PDGF-induced activation of ERK-1/2 [unpublished data and 34].We further demonstrated that the RGS12 PDZ/PTB domain N-terminusand RGS12 PDZ domain can form a complex with the PDGFβ receptor[34]. RGS12 may contain auto-inhibitory domains that prevent the

RGS12 PDZ/PTB domain N-terminus from interacting efficientlywith the PDGFβ receptor. As such, agonists may therefore regulateRGS12 by relieving the inhibitory constraint on the PDZ/PTB domainN-terminus. There is evidence to support a role for PDZ domains inregulating RTK internalization. For instance, Lazar et al. [35] showedthat NHERF contains a PDZ domain that interacts with the EGFreceptor and prevents internalization of the EGF receptor. Further-more, Maudsley et al. [36] demonstrated that NHERF interacts withthe PDGF receptor via a high affinity PDZ domain and that NHERFenhances activation of ERK-1/2 by PDGF. Therefore, it is conceivablethat RGS12 via its PDZ domain might interact with a PDZ bindingsequence in the PDGFβ receptor that normally binds another reg-ulatory protein, to prevent ERK-1/2 activation in response to PDGF orS1P. In this model, RGS12 would function as a competitive inhibitordisplacing NHERF and thereby reducing both PDGF- and S1P-stimulation of the ERK-1/2 pathway.

We know that the PDGFβ receptor contains a PDZ binding sequencethat can bind PDZ domain containing proteins (e.g. RGS12). We havefound what may represent a weak PDZ binding sequence (EKSLALL) inthe 3rd cytoplasmic loop of S1P1. This is clearly important as this mayalso represent a means by which a PDZ domain containing proteinmight tether the PDGFβ receptor with S1P1 receptor.

4. The Trk A receptor–LPA1 receptor signaling complex

There are other examples of RTK–lipid phosphate receptorsignaling. Indeed, we reported that treatment of PC12 cells with PTXreduced the NGF-dependent activation of the ERK-1/2 pathway [37].We concluded that Trk A receptor (which binds NGF) may use a GPCR-mediated pathway to stimulate the ERK-1/2 pathway. This wassupported by the finding that over-expressed β-arrestin I, which isinvolved in GPCR signaling, enhanced the NGF-induced activation ofERK-1/2 and is associated with Trk A receptor [37]. We have furtherdemonstrated that the constitutively active LPA1 receptor providesGβγ subunits which are used by the Trk A receptor to enhanceactivation of ERK-1/2 in response to NGF [38,39]. This is based on thefinding that the over-expression of the CT-GRK-2 peptide, whichsequesters Gβγ subunits, reduced the NGF-induced activation of ERK-1/2 [38,39]. In contrast, the stimulation of the LPA1 receptor with LPAresulted in a predominant Giα2-mediated Trk A-independent activa-tion of ERK-1/2 [38,39]. Therefore, constitutively active LPA1 receptorbehaves differently from receptor that has engaged LPA, and we haveargued that constitutively active LPA1 forms a functional complex withTrk A receptor, while there may be an additional pool of LPA1 receptorthat binds LPA and which is dissociated from Trk A (see later). Thismodel is supported by data showing that the Trk A receptor is co-immunoprecipitated with the myc-tagged LPA1 receptor from PC12cell lysates (using anti-myc tag antibody) and that LPA inducesdissociation of the complex [39]. Further evidence implicating theLPA1 receptor was obtained by the demonstration that anti-sensemediated down-regulation of the LPA1 receptor reduced NGF- andLPA-stimulated activation of ERK-1/2 [38]. Finally, Ki16425, a selectiveprotean agonist of the LPA1 receptor reduced LPA- and NGF-stimula-tion of ERK-1/2, and weakly stimulated ERK-1/2 on its own [39].Indeed, the treatment of cells with Ki16425 inhibited NGF-stimulatedneurite outgrowth [39], thereby suggesting that the Trk A-constitu-tively active LPA1 receptor complex regulates neuronal differentiation.

5. Pharmacological manipulation of the Trk A receptor–LPA1

receptor signaling complex

As with S1P1, the LPA1 receptor may exist in multiple constitutivelyactive conformations. We have therefore proposed the followingmodel to explain how Ki16425 can reduce NGF signaling [39]. R1 isdesignated with high efficacy while R2 has low efficacy for coupling toGi (Fig. 4). The R1 conformation of the LPA1 receptor is associated with

Fig. 4. The effect of protean agonism of the R2 LPA1 receptor transition state withKi16425 and the consequential effect on the engagement of R1 LPA1 receptor transitionstate with the Trk A receptor signaling system.

472 N.J. Pyne, S. Pyne / Biochimica et Biophysica Acta 1781 (2008) 467–476

the Trk A receptor, while the R2 is dissociated from the Trk A receptor.Ki16425 binds exclusively to R2 and stabilises this receptor conforma-tion to stimulate ERK-1/2 with low efficacy (there is likely to be arequirement for Gi-independent recruitment of a regulatory protein(s)that functions to enhance Gi-mediated signaling to ERK-1/2). This willreduce the amount of R1 by mass action, thereby decreasing the NGF-induced activation of ERK-1/2. There are differences with the modelthat describes the S1P1 receptor and PDGFβ receptor interaction. First,Trk A receptor kinase inhibitors have no effect on the LPA-inducedactivation of ERK-1/2. Second, LPA induces dissociation of the Trk Areceptor–LPA1 receptor complex. These data can be explained by athird binding conformation, in which LPA binds to R3 (Gi couplingefficacy NR2) andwhich is in equilibriumwith R1 (which does not bindLPA) and R2. We also propose that R3 is not associated with the Trk Areceptor. In order for LPA to induce dissociation of the Trk A receptor–R1 LPA1 receptor complex, the amount of R3 that binds LPA must berelatively high (determined by the equilibrium constant for transitionof R to R3). Binding of Ki16425 exclusively to R2 will therefore alsoreduce LPA-induced activation of ERK-1/2 bymass actionmediated viaR3 LPA1. We have concluded from these studies that the constitutivelyactive LPA1 receptor functions to enhance NGF signaling, while LPAbinding to a different conformation of LPA1 might effectivelyantagonises NGF via equilibrium transition of LPA1. The positive effectof LPA1 receptor on neuronal stem cell migration, survival and neuriteoutgrowth is well documented [40,41] and protean agonism mayexplain these effects in the context of a functional Trk A–GPCR axis.

We conclude that proximity interaction between lipid phosphatereceptors and RTK allows programming of integrated spatial signalingin cells. Moreover, our findings with selective protean agonists of lipidphosphate receptors not only invoke a major paradigm shift in ourunderstanding of how lipid phosphates and growth factors act, butrequires re-evaluation of how drugs that act on lipid phosphatereceptors might function to alleviate disease.

6. Sphingosine kinases and intracellular S1P

An intracellular role for S1P is supported by the sub-cellular local-ization of enzymes that are involved in its synthesis and degradation.S1P is produced by the sphingosine kinase (SK)-catalysed phosphor-ylation of sphingosine. SK activity is localised in the cytoplasmalthough it can associatewithmembranes and this varies between celltypes. Two isoforms, SK1 and SK2, have been identified, which differin their structure, biochemical properties and regulation [reviewed in[42]]. Additionally, multiple spliced variant forms of each isoformexist, differing in their N terminal regions [reviewed in [43]]. Whilehaving little effect on enzyme kinetics, these variants display differentsub-cellular localization [42–44]. For example, endogenous SK1localizes to the cytoplasm, plasma membrane and vesicles in HUVECsand N-terminal variants of recombinant human SK1a, SK1b and SK1c,exhibit cytoplasmic, cytoplasmic/plasmamembrane and vesicular dis-

tributions, respectively [45]. Other reported locations of SK1 includethe endoplasmic reticulum, perinuclear regions, including the Golgiapparatus and cytoskeleton-associated [44]. Redistribution of SK1 tothe plasmamembrane occurs in response to agonist stimulation due tophosphorylation by protein kinase C or ERK-1/2, by association withphospholipids such as phosphatidylserine and phosphatidic acid(which also redistributes SK1 to the Golgi apparatus) or by associationwith calcium–calmodulin, which is one of a number of proteins thathave been identified to interact with SK1 [44]. The plasma membranedisposition of SK1 has been linked with an autocrine/sequentialfunction for S1P. This involves the synthesis of S1P in response toextracellular stimuli, subsequent release of S1P from the cell and/orpartition into extracellular facing plasma membrane micro-domainsand proximity binding of S1P to cell surface S1P receptors to initiate‘inside out’ signaling. Intuitively, the plasmamembrane localization ofSK1 places the enzyme in close proximity to the extracellular milieuwhere it may be able to regulate ‘inside out’ signaling [46]. This issupported by the fact that extracellular stimuli that use S1P in asequential mechanism promote translocation of SK1 from thecytoplasm to the plasma membrane in, for instance, EGF-stimulatedMCF-7 cells [47] and antigen-stimulated mast cells [48]. Additionally,S1P can be synthesised extracellularly following release of SK1 [45].Redistribution of SK1 may therefore, facilitate down-stream signalingby producing S1P in a spatial/temporal manner.

Distribution of SK2 is between the cytosolic and nuclear compart-ments and varies between cell types. A reduction in nuclear SK2 canbe observed at low cellular confluence [49], upon serum withdrawal(which promotes association with the endoplasmic reticulum) [50]and following protein kinase D-catalysed phosphorylation of the SK2nuclear localisation sequence [51]. As with SK1, N-terminal variantsdiffer in their distribution. For example, recombinant SK2a and SK2bwere detected in the nucleus/cytoplasm and cytoplasmic vesicles,respectively, in HUVECs [45].

Therefore, there may bemultiple compartmentalized pools of S1P. Astrong a priori reason for believing that intracellular S1P might beconfined to discrete compartmentalized pools comes from a work oncAMP, where functional scaffold complexes of AKAP, PKA, EPAC anddistinct cAMPphosphodiesterase isoforms are localized to different sub-cellular compartments in cells [52]. In this case, the arrangement ofprotein complexes functions to match cAMP diffusion rates with cAMPbinding affinity and to therefore enhance kinetically the probability ofcAMP binding to protein targets. Such compartmentalization enables asingle second-messenger molecule to regulate different cellular out-comes dependent on the placement of these protein complexes in closeproximity with compartment specific effectors. Therefore, we believe itimportant to undertake further research into the regulation ofcompartmentalized intracellular S1P signaling.

Therehavebeensuggested roles for intracellular S1P in the contextofcalcium signaling and regulation of proliferation [53,54]. However, themajor reason for the lack of advance to date is the failure to identifyintracellular receptors/targets of S1P and thereby to appreciate thespatial and temporal aspects of S1P formation and degradation. In ouropinion, there are two important questions related to defining theintracellular function of S1P. These are: (i) how is the synthesis anddegradation of S1P regulated in a spatial (relates specifically tocompartmentalised pools of S1P linked to regulation of distinctbiological response) and temporal manner?; and (ii) what are theintracellular targets of S1P and can definition of these targets helpfurther our overall understanding of the spatial/temporal aspect ofintracellular S1P signaling?

7. New intracellular functions for SK1 and S1P

We have shown that activation of PLD1 results in localisation, inpart, of GFP-SK1 to the Golgi apparatus, where PLD1 is expressed[55,56]. This was demonstrated in CHO cells over-expressing a

Fig. 5. Schematic of the interplay between PLD1-derived PA, SK1, LPP2 and LPP3 thatpotentially regulates spatial/temporal dynamics of intracellular S1P signaling.

473N.J. Pyne, S. Pyne / Biochimica et Biophysica Acta 1781 (2008) 467–476

doxycycline-inducible PLD1. The localisation of SK1 to the Golgiapparatus could also be induced by PMA (protein kinase C activator)and antigen (in mast cells) which stimulates PLD1. Further character-ization of the interaction between SK1 and PLD1 revealed that SK1binds to PLD1-derived phosphatidic acid (PA) through a region withinthe C-terminal of SK1. At this time, it is not established whether PLD1induction causes recruitment of SK1 from cytoplasmic vesicles to theGolgi apparatus or whether PLD1 effectively traps SK1 at the Golgiapparatus, and thereby prevents trafficking of SK1 on cytoplasmicvesicles. The important point of these findings in the context of thecurrent research is that the interaction between PLD1-derived PAderived from PLD1 and SK1 is confined to a defined intracellularcompartment and therefore provides persuasive evidence for anintracellular role of SK1/S1P. SK1 has been implicated in regulating cellsurvival, as has PLD-derived PA [57]. Therefore we believe that defectsin the interaction between SK1 and PA derived from PLD1, andablation of the Golgi apparatus S1P pool may lead to an apoptoticphenotype. This possibility is supported by our findings on the role oflipid phosphate phosphatases in regulating intracellular SK1 traffick-ing [56].

Lipid phosphate phosphatases (LPP) belong to a family of enzymesthat are Mg2+ independent and N-ethylmaleimide-insensitive phos-phatases that display broad substrate specificity in vitro, catalyzingthe dephosphorylation of several lipid phosphates (e.g. PA, S1P, LPAand ceramide 1-phosphate (C1P)) [58]. LPPs have the potential to acton both extracellular and intracellular phosphorylated lipid sub-strates. The LPPs are integral membrane proteins that have sixtransmembrane domains with the catalytic site predicted, by model-ling of monomers, to face the extracellular side of the plasma mem-brane and shown experimentally to face the luminal side of theendoplasmic reticulum for LPP3 [59,60]. Four mammalian LPPisoforms have been cloned (LPP1, LPP1a, LPP2 and LPP3 [61–64]).While each has been detected at the plasmamembrane using isoform-selective [56,59,65–67] or epitope-tag antibodies [56,68–70], intra-cellular pools of LPP2 and LPP3 have also been identified [56,65,66].LPP1 and LPP3 have been identified in caveolae [66,67] whereasdifferential extraction of LPP1 and LPP3 suggests that these isoformslocalise to distinct lipid rafts [71]. In MDCK cells, LPP1 and LPP3 arelocalised to the apical and basolateral membrane respectively, guidedby specific sorting signals within the N-terminal region of LPP1 andthe second cytoplasmic region of LPP3 [72].

In the process of obtaining more information about the interactionbetween SK1 and PLD1-derived PA, we surprisingly found that over-expressed LPP2, which reduces intracellular PA levels, is co-localisedwith SK1 on cytoplasmic vesicles and may prevent localisation of SK1to the Golgi apparatus upon PLD1 induction in CHO cells [56]. Weproposed that LPP2 might reduce PA in the vicinity of PLD1 therebyreleasing SK1 from the ‘PLD1 trap’ or preventing trafficking of SK1 toPA derived PLD1 in the Golgi apparatus. We also found that over-expressed LPP3 reduces intracellular S1P, and is co-localised with SK1at the Golgi apparatus of CHO cells in which PLD1 is induced [56].Therefore, it is possible that LPP3 removes S1P from the Golgiapparatus pool. If the active site of LPPs faces the lumen of intracellularcompartments then presumably access to substrates generated byenzymes at the cytoplasmic facewould require transbilayermovementof these substrates.

Having identified a spatial/compartmentalised interactionbetween LPP2/3 and SK1 inside cells, we evaluated the functionalconsequence of the action of LPP2 and LPP3. In this regard, we havefound that cells over-expressing LPP2 and LPP3 are more prone tostress-induced apoptosis [56]. This was evident from an increasedintranucleosomal DNA laddering in LPP over-expressing serumdeprived cells and increased caspase 3/7-catalysed cleavage of PARP.Indeed, over-expression of LPP2 and LPP3 also reduced S1P- and LPA-stimulated ERK-1/2 activation and this was reversed by pre-treatingserum deprived cells with the caspase 3/7 inhibitor, Ac-DEVD-CHO.

The LPP-dependent activation of caspase-3/7 may have been aconsequence of reduced survival signals including intracellular PA(significantly reduced by LPP2) and intracellular S1P (significantlyreduced by LPP3). Indeed, caspase 7 activation accompanied by cellcycle arrest and apoptosis was observed to be a consequence ofknockdown of endogenous SK1 [73]. The interaction betweenintracellular SK1/S1P and PLD1/PA is shown in Fig. 5. It is alsoimportant to note that there are several other studies, which reportthat LPPs can induce apoptosis. For instance, over-expression of LPP1[74] or LPP3 [75] in ovarian cancer cell lines increases the rate ofapoptosis whereas there was no effect upon the cell cycle. In bothcases, a ‘bystander effect’was evident, i.e. the LPP over-expressing cellswere able to reduce the ability of co-cultured non-over-expressingcells to form colonies, suggesting an ecto-LPP catalysed degradation ofextracellular LPA, a survival signal.

In addition to LPPs, the role of S1P lyase [76] and S1P phosphatase[77,78] are important aspects in defining overall spatial S1P signalingin defined intracellular compartments.

8. Role of LPPs in terminating RTK signaling and migration

The only known form of regulation of LPPs to date is at thetranscriptional level. For example, EGF increased mRNA for LPP3 inHeLa cells [63] whereas mRNA for LPP1 was increased by androgen inadenocarcinoma cells [79]. We have shown that moderate increases inLPP1 expression reduce LPA- and PDGF-activation of ERK-1/2 andMEFmigration [80]. The LPP1-dependent reduction in PDGF-stimulatedmigration results from down-regulation of typical PKC isoform(s),which are normally required for PDGF-induced activation of ERK-1/2and migration [80]. Indeed, a role for PKC in regulating cell migrationhas been demonstrated before. For instance, Campbell and Trimble[81] have shown that PKCβII is required for PDGF-induced ERK-1/2activation and migration of vascular smooth muscle cells andYamamura et al. [82] have shown that down-regulation of PKCαinhibits the migration of endothelial cells. Therefore, relatively minorincreases in the expression level of LPP1 might represent a hetero-logous desensitisation mechanism for attenuating PKC-mediatedsignaling and regulation of cell migration.

However, these results contrast with those of Yue et al. [83], whereno inhibition of PDGF-stimulated ERK-1/2 activation was observed inMEF derived from LPP1 transgenic mice. A key difference between thetwo studies is the number of gene copies of LPP1, being ×2 [83] and×20 [80], respectively. This translates into significant differences inLPP1 activities (LPP activity: control: 12.5±1.54 nmol/min/mg protein;

474 N.J. Pyne, S. Pyne / Biochimica et Biophysica Acta 1781 (2008) 467–476

×2: 21.5±3.8 nmol/min/mg; ×20: 41.47±6.9 nmol/min/mg protein,n=3–5 (means±SEM) using 50 μM PA as substrate). It is thereforeconceivable that more profound increases in LPP activity have effectson PKC-mediated signaling that abrogate PDGF-stimulated MEFmigration.

Nonetheless, using Rat2 fibroblasts, Brindley et al. [84] reported thata similar 4-fold over-expression of LPP1 activity reduced PDGF-stimulated PA formation, consistent with our findings in as much asthey extend the action of LPP1 to growth factors. However, LPP1 over-expression in Rat2 fibroblastswaswithout effect upon PDGF-stimulatedERK-1/2 andmigration [84], although it is unknownwhether typical PKCisoforms play a role in regulating PDGF-stimulated migration in thesecells.

Consistent with our interpretation of LPP actions down-stream ofreceptor activation [80], Brindley et al. [84] reported a reduction inLPA-stimulated migration in MEF derived from ×2 LPP1 transgenicmice and upon LPP1 over-expression in Rat2 fibroblasts. This effectwas independent of ecto-LPP activity yet required the catalytic activityof LPP1. In Rat2 fibroblasts, the attenuation of LPA-stimulatedmigration by LPP1 over-expression was due to reduced PLD2 activityalthough reductions in LPA-stimulated activation of ERK-1/2 and Rhoand basal activities of Rac and Cdc42 were also affected. Moreover, theeffect on LPA-induced migration appears to be mediated via an LPP1-dependent release of tissue inhibitor of matrix metalloproteinase(TIMP-2). Indeed, knockdown of TIMP-2 in LPP1 over-expressing cellsincreased LPA-induced migration (Brindley, unpublished conferencecommunication). However, there are still major questions to beaddressed here, because TIMP-2 has been demonstrated to modulatePDGF responses in a number of cell types [85,86].

We have also observed a reduction in S1P-induced activation ofERK-1/2 inMEF derived from LPP1 transgenic mice [80]. As mentionedabove, we have previously demonstrated that both S1P and PDGF acton PDGFβ receptor–S1P1 receptor complexes in these cells [10], sug-gesting that LPP1 can function to terminate integrative signalingthrough this complex.

LPPs expressed at the plasma membrane exhibit an ecto-activity,i.e. catalyse the dephosphorylation of extracellular substrates, whichmay limit the actions of S1P and LPA at their respective receptors and/or participate in the uptake of these lysolipids, thereby influencingcellular function [87,88]. One of the best examples of ecto-activity isthat of the Drosophila LPP homologues, wunen and wunen-2.Migration of primordial germ cells is by repulsion away from cellsexpressing wun/wun2 (e.g. cells of the central nervous system andposterior midgut) and towards the highest phospholipid levels. Thus,germ cells are guided away from the midline and towards the regionwhere somatic gonadal precursors are specified [89]. Wun/Wun2activity also governs death of mis-migrating germ cells [89] and polecell survival is determined by competition for a common extracellularphospholipid substrate between pole cells and somatic cells [90].

9. Molecular structure/function of LPPs

Previous observations have indicated that LPP may undergooligomerisation. Indeed, Siess and Hofstetter reported that the Stokesradius of LPP is consistent with a hexameric arrangement of subunits[91] whereas Wunen forms dimers [92]. However, current topogra-phical modelling of LPPs is based on a monomeric arrangement. Thus,we examined whether LPPs are oligomers and which would thereforenecessitate a reappraisal of the modelling. We studied the simplestmodel in which dimers are formed and organised in an oligomericarrangement. In this model, it is possible that the C1, C2 and C3domains in each LPP monomer form two competent catalytic sites bydomain swapping between the monomers (e.g. C1 from monomer A,conferring substrate binding, is shared with C2 and C3 frommonomerB, conferring catalytic activity, and vice versa). Therefore, we createdcatalytically deficient myc-tagged R127K LPP1 (C1 mutagenesis) and

FLAG-tagged H223L LPP1 (C3 mutagenesis) mutants. If dimerisationinvolves domain swapping between the monomers to form two cat-alytic sites then co-expression of the two catalytic deficient LPP1forms, mutated in C1 and C3 domains, respectively, should theoreti-cally form one competent catalytic site and reconstitute ~50% of theWT LPP1 activity at equivalent expression level. However, when thesemutants were co-expressed (and co-immunoprecipitated with anti-FLAG antibody), we detected negligible LPP1 activity [93]. Signifi-cantly, the two LPP mutants can still form oligomers suggesting thatcatalytic activity is not required for oligomerisation. This is in contrastwith Wunen where catalytic activity is required for dimerisation, butnot for in vivo function [92]. Additionally, we established that acatalytically inactive FLAG-tagged H223L LPP1 mutant can form anoligomer with wild type catalytically active myc-tagged LPP1 and thatwild type LPP1 activity is preserved in the oligomer [93]. This suggeststhat the catalytic sites are able to function independently of each otherin an oligomeric arrangement. The established localization of some ofthese enzymes to detergent resistant or insoluble membrane domainsraises the possibility that these interactions are indirect. However, wedemonstrated that β-methylcyclodextran (to disrupt lipid rafts) didnot inhibit oligomerisation of LPP1/3 [93].

10. Conclusion

There have been many advances in lipid phosphate biology overthe past 5 years, including improved understanding of how lipidphosphates are made and removed, their action at cognate receptors,interaction with growth factors, and their role in a vast array of bio-logical processes such as angiogenesis, immune regulation and mod-ulation and neuronal survival and differentiation. The use of knockouttechnology and study of disease processes has also defined importantroles for these lipid phosphates. These advances will be progressedfurther in the next 5 years to unravel the biological role of S1P and LPA.However, it is our opinion that there is a need for new impetus into theinvestigation of the spatial and temporal aspects of intracellularsignaling for both LPA and S1P and the identification of intracellularS1P and LPA targets. This will enable a full appreciation of the im-portance of extracellular versus intracellular function in a given cell.Once the intracellular action of lipid phosphates are understood, wecan use (with further advances on the cognate GPCR and cross talkwith other receptors) systems biology approaches to define the fullspectrum of actions and integrated functions of S1P and LPA in singlecells to whole organisms in both health and disease.

Acknowledgements

We wish to thank Drs Gabor Tigyi, Viswanathan Natarajan andNicholas Ktistakis for their collaboration. This work was supported bygrants from Biotechnology and Biological Sciences Research Council.

References

[1] C. Waters, S. Pyne, N.J. Pyne, The role of G-protein coupled receptors and associatedproteins in receptor tyrosine kinase signal transduction, Semin. Cell Dev. Biol.15 (2004)309–323.

[2] L.M. Luttrell, T. Van Biesen, B.E. Hawes, W.J. Koch, K. Touhara, R.J. Lefkowitz,G(betagamma) subunits mediate mitogen-activated protein kinase activation bythe tyrosine kinase insulin-like growth factor 1 receptor, J. Biol. Chem. 270 (1995)16495–16498.

[3] Y.V. Fedorov, N.C. Jones, B.B. Olwin, Regulation of myogenesis by fibroblast growthfactors requires beta-gamma subunits of pertussis toxin-sensitive G proteins, Mol.Cell. Biol. 18 (1998) 5780–5787.

[4] A.-M. Conway, S. Rakhit, S. Pyne, N.J. Pyne, Platelet-derived-growth-factorstimulation of the p42/p44 mitogen-activated protein kinase pathway in airwaysmoothmuscle: role of pertussis-toxin-sensitive G-proteins, c-Src tyrosine kinasesand phosphoinositide 3-kinase, Biochem. J. 337 (1999) 171–177.

[5] F. Alderton, S. Rakhit, K.C. Kong, T. Palmer, B. Sambi, S. Pyne, N.J. Pyne, Tethering ofthe platelet-derived growth factor beta receptor to G-protein-coupled receptors: a

475N.J. Pyne, S. Pyne / Biochimica et Biophysica Acta 1781 (2008) 467–476

novel platform for integrative signaling by these receptor classes in mammaliancells, J. Biol. Chem. 276 (2001) 28578–28585.

[6] C. Waters, B. Sambi, K.C. Kong, D. Thompson, S.M. Pitson, S. Pyne, S.N.J. Pyne,Sphingosine 1-phosphate and platelet-derived growth factor (PDGF) act via PDGFβreceptor-sphingosine 1-phosphate receptor complexes in airway smooth musclecells, J. Biol. Chem. 278 (2003) 6282–6290.

[7] J.P. Hobson, H.S. Rosenfeldt, L.S. Barak, A. Olivera, S. Poulton, M. Caron, S. Milstien,S. Spiegel, Role of sphingosine 1-phosphate receptor EDG-1 in PDGF-induced cellmotility, Science 291 (2001) 1800–1803.

[8] M.-J. Lee, J.R. Van Brocklyn, S. Thangada, C.H. Liu, A.R. Hand, R. Menzeleev, S.Spiegel, T. Hla, Sphingosine-1-phosphate as a ligand for the G protein-coupledreceptor EDG-1, Science 279 (1998) 1552–1555.

[9] J. Chun, E.G. Goetzl, T. Hla, Y. Igarashi, K.R. Lynch, W. Moolenaar, S. Pyne, G. Tigyi,IUPHAR XXXIV Lysophospholipid receptor nomenclature, Pharmacol. Rev. 54 (2002)265–269.

[10] J.S. Long, V. Natarajan, G. Tigyi, S. Pyne, N.J. Pyne, The functional PDGFβ receptor–S1P1 receptor signaling complex is involved in regulating migration of mouseembryonic fibroblasts in response to platelet derived growth factor, ProstaglandinsOther Lipid Mediat. 80 (2006) 74–80.

[11] C. Waters, M. Connell, S. Pyne, N.J. Pyne, c-Src is involved in regulating signaltransmission from PDGFβ receptor-GPCR signal complexes in mammalian cells,Cell. Signal. 17 (2005) 263–277.

[12] R.M. O'Brien, M.D. Houslay, G. Milligan, K. Siddle, The insulin receptor tyrosylkinase phosphorylates holomeric forms of the guanine nucleotide regulatoryproteins Gi and Go, FEBS Lett. 212 (1987) 281–288.

[13] J. Krupinski, R. Rajaram, M. Lakonishok, J.L. Benovic, R.A. Cerione, Insulin-dependent phosphorylation of GTP-binding proteins in phospholipid vesicles,J. Biol. Chem. 263 (1988) 12333–12341.

[14] H.M. Rosenfeldt, J.P. Hobson, M. Maceyka, A. Olivera, V.E. Nava, S. Milstien, S. Spiegel,EDG-1 links the PDGF receptor to Src and focal adhesion kinase activation leading tolamellipodia formation and cell migration, FASEB J. 15 (2001) 2649–2659.

[15] D. Gesty-Palmer, M. Chen, E. Reiter, S. Ahn, C.D. Nelson, S. Wang, A.E. Eckhardt, C.L.Cowan, R.F. Spurney, R.J. Lefkowitz, Distinct beta-arrestin- and G protein-dependent pathways for parathyroid hormone receptor-stimulated ERK1/2activation, J. Biol. Chem. 281 (2006) 10856–10864.

[16] S.K. Shenoy, R.J. Lefkowitz, Angiotensin II-stimulated signaling through G proteinsand beta-arrestin, Sci. STKE (2005) 311.

[17] S.K. Shenoy, M.T. Drake, C.D. Nelson, D.A. Houtz, K. Xiao, S. Madabushi, E. Reiter, R.T.Premont, O. Lichtarge, R.J. Lefkowitz, Beta-arrestin-dependent, G protein-indepen-dent ERK1/2 activation by the beta2 adrenergic receptor, J. Biol. Chem. 281 (2006)1261–1273.

[18] S. Dalle, W. Ricketts, T. Imamura, P. Vollenweider, J.M. Olefsky, Insulin and insulin-like growth factor I receptors utilize different G protein signaling components,J. Biol. Chem. 276 (2001) 15688–15695.

[19] T. van Biesen, B.E. Hawes, D.K. Luttrell, K.M. Krueger, K. Touhara, E. Porfiri, M.Sakaue, L.M Luttrell, R.J. Lefkowitz, Receptor-tyrosine-kinase- and G beta gamma-mediated MAP kinase activation by a common signalling pathway, Nature 376(1995) 781–784.

[20] L.M. Luttrell, B.E. Hawes, T. Van Biesen, D.K. Luttrell, T.J. Lansing, R.J. Lefkowitz, Roleof c-Src tyrosine kinase in G-protein coupled receptor- and Gbetagamma subunit-mediated activation of mitogen activated protein kinases, J. Biol. Chem. 271 (1996)19443–19450.

[21] J.H. Wu, R. Goswami, X. Cai, S.T. Exum, X. Huang, L. Zhang, L. Brian, R.T. Premont, K.Peppei, N.J. Freedman, Regulation of the platelet derived growth factor receptor-beta by G-protein coupled-receptor kinase-5 in vascular smooth muscle involvesthe phosphatase Shp2, J. Biol. Chem. 281 (2006) 37758–37772.

[22] L.M. Luttrell, S.S. Ferguson, Y. Daaka, W.E. Miller, S. Maudsley, G.J. Della Rocca, F.Lin, H. Kawakatsu, K. Owada, D.K. Luttrell, M.G. Caron, R.J. Lefkowitz, Beta-arrestin-dependent formation of beta2 adrenergic receptor-Src protein kinase complexes,Science 283 (1999) 650–651.

[23] Q. Wang, R. Lu, J. Zhao, L.E. Limbird, Arrestin serves as a molecular switch, linkingendogenousalpha2-adrenergic receptor to SRC-dependent, but not SRC-independent,ERK activation, J. Biol. Chem. 281 (2006) 25948–25955.

[24] C. Akekawatchai, J.D. Holland, M. Kochetkova, J.C. Wallace, S.H. McColl,Transactivation of CXCR4 by IGF-1R in human MDA-MB-231 breast cancerepithelial cells, J. Biol. Chem. 280 (2005) 39701–39708.

[25] S. Rakhit, S. Pyne, N.J. Pyne, The platelet-derived growth factor receptorstimulation of p42/p44 mitogen-activated protein kinase in airway smoothmuscle involves a G-protein-mediated tyrosine phosphorylation of Gab1, Mol.Pharmacol. 58 (2000) 413–420.

[26] C. Waters, J. Long, I. Gorshkova, Y. Fujiwara, M. Connell, K. Belmonte, G. Tigyi, V.Natarajan, S. Pyne, N.J. Pyne, Cell migration activated by platelet-derived growthfactor receptor is blocked by an inverse agonist of the sphingosine 1-phosphatereceptor-1, FASEB J. 20 (2006) 509–511.

[27] W.E. Miller, R.J. Lefkowitz, Expanding roles for beta arrestins as scaffolds andadaptors in GPCR signaling and trafficking, Curr. Opin. Cell Biol. 13 (2001) 139–145.

[28] A.B. Parrill, D. Wang, D.L. Bautista, J. Van Brocklyn, Z. Lorinez, D.J. Fischer, D.L.Baker, K. Liliom, S. Spiegel, G. Tigyi, Identification of EDG1 receptor residues thatrecognise sphingosine 1-phosphate, J. Biol. Chem. 275 (2000) 39379–39384.

[29] T. Kenakin, Inverse, protean, and ligand-selective agonism: matters of receptorconformation, FASEB J. 15 (2001) 598–611.

[30] F. Gbahou, A. Rouleau, S. Morisset, R. Parmentier, S. Crochet, J.S. Lin, X. Ligneau, J.Tardivel-Lacombe, H. Stark, W. Schunack, C.R. Ganellin, J.C. Schwartz, J.M. Arrang,Protean agonism at histamine H3 receptors in vivo and in vitro, Proc. Natl. Acad. Sci.U. S. A. 100 (2003) 11086–11091.

[31] M.T. Drake, J.D. Violin, E.J. Whalen, J.W. Wisler, S.K. Shenoy, R.J. Lefkowitz, Beta-arrestin-biased agonism at the beta2 adrenergic receptor, J. Biol. Chem. 283 (2008)5669–5676.

[32] S. Pyne, N.J. Pyne, Sphingosine 1-phosphate signaling and termination at lipidphosphate receptors, Biochim. Biophys. Acta 1582 (2002) 121–131.

[33] M.J. Kluk, C. Colmont, M.T.Wu, T. Hla, Platelet-derived growth factor (PDGF)-inducedchemotaxis does not require the G protein-coupled receptor S1P1 in murineembryonic fibroblasts and vascular smooth muscle cells, FEBS Lett. 533 (2003)25–28.

[34] B.S. Sambi, M.D. Hains, C.M. Waters, M.C. Connell, F.S. Willard, A.J. Kimple, S. Pyne,D.P. Siderovski, N.J. Pyne, The effect of RGS12 on PDGFβ receptor signaling to ERK-1/2 in mammalian cells, Cell. Signal. 18 (2005) 971–981.

[35] C.S. Lazar, C.M. Cresson, D.A. Lauffenburger, G.N. Gill, The Na+/H+ exchangerregulatory factor stabilises epidermal growth factor receptors at the cell surface,Mol. Biol. Cell 15 (2004) 5470–5480.

[36] S.Maudsley, A.M. Zamah, N. Rahman, J.T. Blitzer, L.M. Luttrell, R.J. Lefkowitz, R.A. Hall,Platelet-derivedgrowth factor receptorassociationwithNa+/H+ exchanger regulatoryfactor potentiates receptor activity, Mol. Cell. Biol. 20 (2000) 8352–8863.

[37] S. Rakhit, S. Pyne, N.J. Pyne, Nerve growth factor stimulation of p42/p44 mitogen-activated protein kinase in PC12 cells: role of G(i/o), G protein-coupled receptorkinase 2, β-arrestin I, and endocytic processing, Mol. Pharmacol. 60 (2001) 63–70.

[38] N. Moughal, C. Waters, B. Sambi, S. Pyne, N.J. Pyne, Nerve growth factor signalinginvolves interaction between the Trk A receptor and LPA receptor 1 systems:nuclear translocation of the LPA receptor 1 and Trk A receptors in pheochromo-cytoma 12 cells, Cell. Signal. 16 (2004) 127–136.

[39] N.A. Moughal, C.A. Waters, W.J. Valentine, M.C. Connell, J.C. Richardson, G. Tigyi, S.Pyne, N.J. Pyne, Protean agonism of the lysophosphatidic acid receptor-1 withKi16425 reduces nerve growth factor-induced neurite outgrowth in PC12 cells, J.Neurochem. 98 (2006) 1920–1929.

[40] X. Ye, I. Ishii, M.A. Kingsbury, J. Chun, Lysophosphatidic acid as a novel cell survival/apoptotic factor, Biochim. Biophys. Acta 1585 (2002) 108–113.

[41] Y. Fujiwara, A. Sebok, S. Meakin, T. Kobayashi, K. Murakami-Murofishi, G. Tigyi,Cyclic phosphatidic acid elicits neurotrophin-like actions in embryonic hippo-campal neurones, J. Neurochem. 87 (2003) 1272–1283.

[42] T.A. Taha, Y.A. Hannun, L.M. Obeid, Sphingosine kinase: biochemical and cellularregulation and role in disease, J. Biochem. Mol. Biol. 39 (2006) 113–131.

[43] R. Alemany, C.J. van Koppen, K. Danneberg, M. ter Braak, D. Meyer zu Heringdorf,Regulation and functional roles of sphingosine kinases, Naunyn SchmiedebergsArch. Pharmacol. 374 (2007) 413–428.

[44] B.W. Wattenberg, S.M. Pitson, D.M. Raben, The sphingosine and diacylglycerolkinase superfamily of signaling kinases: localisation as a key to signaling function,J. Lipid Res. 47 (2006) 1128–1139.

[45] K. Venkataraman, S. Thangada, J. Michaud, M.L. Oo, Y. Ai, Y-M. Lee, M. Wu, N.S.Parikh, F. Khan, R.L. Proia, T. Hla, Extracellular export of SK1a contributes to thevascular S1P gradient, Biochem. J. 397 (2006) 461–471.

[46] S. Spiegel, S. Milstien, Functions of the multifaceted family of sphingosine kinasesand some close relatives, J. Biol. Chem. 282 (2007) 2125–2129.

[47] S. Sarker, M. Maceyka, N.C. Hait, S.W. Paugh, H. Senkala, S. Milstien, S. Spiegel,Sphingosine kinase 1 is required for migration, proliferation and survival of MCF-7human breast cancer cells, FEBS Lett. 579 (2005) 5313–5317.

[48] A.J. Melendez, A.K. Khaw, Dichotomy of Ca2+ signals triggered by differentphospholipid pathways in antigen stimulation of human mast cells, J. Biol. Chem.277 (2002) 17255–17262.

[49] N. Igarashi, T. Okada, S. Hayashi, T. Fujita, S. Jahangeer, S. Nakamura, Sphingosinekinase 2 is a nuclear protein and inhibits DNA synthesis, J. Biol. Chem. 278 (2003)46832–46839.

[50] M. Maceyka, H. Sankala, N.C. Hait, H. Le Stunff, H. Liu, R. Toman, C. Collier, M.Zhang, L. Satin, A.H. Merrill Jr., S. Milstien, S. Spiegel, SphK1 and SphK2,sphingosine kinase isoenzymes with opposing functions in sphingolipid metabo-lism, J. Biol. Chem. 280 (2005) 37118–37129.

[51] G. Ding, H. Sonoda, H. Yu, T. Kajimoto, S.K. Goparaju, S. Jahangeer, T. Okada, S.Nakamura, Protein kinase D mediated phosphorylation and nuclear export ofsphingosine kinase 2, J. Biol. Chem. 282 (2007) 27493–27502.

[52] M.J. Lynch, E.V. Hill, M.D. Houslay, Intracellular targeting of phosphodiesterase-4underpins compartmentalised cAMP signaling, Curr. Top. Dev. Biol. 75 (2006)225–259.

[53] C.E. Chalfant, S. Spiegel, Sphingosine 1-phosphate and ceramide 1-phosphate:expanding roles in cell signaling, J. Cell Sci. 118 (2005) 4605–4612.

[54] D. Meyer zu Heringdorf, Lysophospholipid receptor-dependent and -independentcalcium signaling, J. Cell. Biochem. 92 (2004) 937–948.

[55] C. Delon, M. Manifava, E. Wood, D. Thompson, S. Krugmann, S. Pyne, K.T. Ktistakis,Sphingosine kinase 1 is an intracellular effector of phosphatidic acid, J. Biol. Chem.279 (2004) 44763–44774.

[56] J. Long, P.I. Darroch, K.F. Wan, K.C. Kong, N.T. Ktistakis, N.J. Pyne, S. Pyne, Regulationof cell survival by lipid phosphate phosphatases involves the modulation ofintracellular phosphatidic acid and sphingosine 1-phosphate pools, Biochem. J.391 (2005) 25–32.

[57] Y. Nozawa, Roles of phospholipase D in apoptosis and pro-survival, Biochim.Biophys. Acta 1585 (2002) 77–86.

[58] D.N. Brindley, D. English, C. Pilquil, C. Buri, Z.C. Ling, Lipid phosphate phosphatasesregulate signal transduction through glycerolipids and sphingolipids, Biochim.Biophys. Acta 1582 (2002) 33–44.

[59] Q.X. Zhang, C.S. Pilquil, J. Dewald, L.G. Berthiaume, D.N. Brindley, Identification ofstructurally important domains of lipid phosphate phosphatase-1: implicationsfor its sites of action, Biochem. J. 345 (2000) 181–184.

476 N.J. Pyne, S. Pyne / Biochimica et Biophysica Acta 1781 (2008) 467–476

[60] D. Barilá, M. Plateroti, F. Nobili, A. Onetti Muda, T. Xie, G. Morimoto, G. Perozzi, TheDri 42 gene, whose expression is up-regulated during epithelial differentiation,encodes a novel endoplasmic reticulum resident transmembrane protein, J. Biol.Chem. 271 (1996) 29928–29936.

[61] M.Kai, I.Wada, S. Imai, F. Sakane,H.Kanoh, Identification and cDNAcloningof 35-kDaphosphatidic acid phosphatase (type 2) bound to plasma membranes. Polymerasechain reaction amplification of mouse H2O2-inducible hic53 clone yielded the cDNAencoding phosphatidic acid phosphatase, J. Biol. Chem. 271 (1996) 18931–18938.

[62] D.W. Leung, C.K. Tompkins, T.White,Molecular cloning of two alternatively splicedforms of human phosphatidic acid phosphatase cDNAs that are differentiallyexpressed in normal and tumour cells, DNA Cell Biol. 17 (1998) 377–385.

[63] M. Kai, I. Wada, S. Imai, F. Sakane, H. Kanoh, Cloning and characterization of twohuman isozymes of Mg2+-independent phosphatidic acid phosphatase, J. Biol.Chem. 272 (1997) 24572–24578.

[64] R. Roberts, V.A. Sciorra, A.J. Morris, Human type 2 phosphatidic acid phosphohy-drolases. Substrate specificity of the type 2a, 2b, and 2c enzymes and cell surfaceactivity of the 2a isoform, J. Biol. Chem. 273 (1998) 22059–22067.

[65] F.A. Alderton, P. Darroch, B. Sambi, A. McKie, I.S. Ahmed, N.J. Pyne, S. Pyne, G-protein-coupled receptor stimulation of the p42/p44 mitogen-activated proteinkinase pathway is attenuated by lipid phosphate phosphatases 1, 1a, and 2 inhuman embryonic kidney 293 cells, J. Biol. Chem. 276 (2001) 13452–13460.

[66] V.A. Sciorra, A.J. Morris, Sequential actions of phospholipase D and phosphatidicacid phosphohydrolase 2b generate diglyceride inmammalian cells, Mol. Cell. Biol.10 (1999) 3863–3876.

[67] M. Nanjundan, F. Possmayer, Pulmonary lipid phosphate phosphohydrolase inplasma membrane signaling platforms, Biochem. J. 358 (2001) 637–646.

[68] R. Jasinska, Q.X. Zhang, C.S. Pilquil, I. Singh, J. Xu, J. Dewald, D.A. Dillon, L.G.Bertiaume, G.M. Carman, D.W. Waggoner, D.N. Brindley, Lipid phosphatephosphohydrolase-1 degrades exogenous glycerolipid and sphingolipid phos-phate esters, Biochem. J. 340 (1999) 677–686.

[69] T. Ishikawa, M. Kai, I. Wada, H. Kanoh, Cell surface activities of the human type 2bphosphatidic acid phosphatase, J. Biochem. 127 (2000) 645–651.

[70] Y.-J. Jia, M. Kai, I. Wada, F. Sakane, H. Kanoh, Differential localisation of lipidphosphate phosphatases 1 and 3 to cell surface subdomains in polarised MDCKcells, FEBS Lett. 552 (2003) 240–246.

[71] M. Kai, F. Sakane, Y.J. Jia, S. Imai, S. Yasuda, H. Kanoh, Lipid phosphate phosphatases1 and 3 are localised in distinct lipid rafts, J. Biochem. 140 (2006) 677–686.

[72] Y.J. Jia,M. Kai, I.Wada, F. Sakane, H. Kanoh, Differential localisation of lipid phosphatephosphatases 1 and 3 to cell surface subdomains in polarised MDCK cells, FEBS Lett.552 (2003) 240–246.

[73] T.A. Taha, K. Kitatani, M. El-Alwani, J. Bielawski, Y.A. Hannun, L.M. Obeid, Loss ofsphingosine kinase 1 activates the intrinsic pathway of programmed cell death:modulation of sphingolipid levels and the induction of apoptosis, FASEB J. 20(2006) 482–484.

[74] J.L. Tanyi, Y. Hasegawa, R. Lapushin, A.J. Morris, J.K. Wolf, A. Berchuck, K. Lu, D.I.Smith, K. Kalli, L.C. Hartmann, K. McCune, D. Fishman, R. Broaddus, K.W. Cheng, E.N. Atkinson, J.M. Yamal, R.C. Bast, E.A. Felix, R.A. Newman, G.B. Mills, Role ofdecreased levels of lipid phosphate phosphatase-1 in accumulation of lysopho-sphatidic acid in ovarian cancer, Clin. Cancer Res. 9 (2003) 3534–3545.

[75] J.L. Tanyi, A.J. Morris, J.K. Wolf, X. Fang, Y. Hasegawa, R. Lapushin, N. Auersperg, Y.J.Sigal, R.A. Newman, E.A. Felix, E.N. Atkinson, G.B. Mills, The human lipid phosphatephosphatase-3 decreases the growth, survival and tumourigenesis of ovariancancer cells: validation of the lysophosphatidic acid signaling cascade as a targetfor therapy in ovarian cancer, Cancer Res. 63 (2003) 1073–1082.

[76] P. Bandhuvula, J.D. Saba, Sphingosine 1-phosphate lyase in immunity and cancer:silencing the siren, Trends Mol. Med. 13 (2007) 210–217.

[77] C. Ogawa, M. Kihara, M. Gokoh, Y. Igarashi, Identification and characterisation of anovel sphingosine 1-phosphate phosphohydrolase, hSPP2, J. Biol. Chem. 278(2002) 1268–1272.

[78] S.M. Mandala, R. Thornton, I. Galve-Roperh, S. Poulton, A. Peterson, A. Olivera, J.Bergstrom, M.B. Kurtz, S. Spiegel, Molecular cloning and characterisation of a lipidphosphohydrolase that degrades sphingosine 1-phosphate and induces cell death,Proc. Natl. Acad. Sci. 97 (2000) 7859–7864.

[79] W. Ulrix, J.V. Swinnen, W. Heynes, G. Verhoven, Identification of phosphatidic acidphosphatase type 2a isozyme as an androgen-regulated gene in human prostaticadenocarcinoma cell line LNCaP, J. Biol. Chem. 273 (1997) 4660–4665.

[80] J.S. Long, K. Yokoyama, G. Tigyi, N.J. Pyne, S. Pyne, Lipid phosphate phosphatase-1regulates lysophosphatidic acid- and platelet-derived growth factor-induced cellmigration, Biochem. J. 394 (2006) 495–500.

[81] M. Campbell, E.R. Trimble, Modification of PI3K- andMAPK-dependent chemotaxisin aortic vascular smooth muscle cells by protein kinase C beta II, Circ. Res. 96(2005) 197–206.

[82] S. Yamamura, P.R. Nelson, K.C. Kent, Role of protein kinase C in attachment,spreading and migration of human endothelial cells, J. Surg. Res. 63 (1996)349–354.

[83] J. Yue, K. Yokoyama, L. Balazs, D.L. Baker, D. Smalley, C. Pilquil, D.N. Brindley, G.Tigyi, Mice with transgenic expression of lipid phosphate phosphatase-1 displaymultiple organotypic deficits without alteration in circulating lysophosphatidatelevel, Cell. Signal. 16 (2004) 385–399.

[84] C. Pilquil, J. Dewald, A. Cherney, I. Gorshkova, G. Tigyi, D. English, V. Natarajan, D.N.Brindley, Lipid phosphate phosphatase-1 regulates lysophosphatidate-inducedfibroblast migration by controlling phospholipase D2-dependent phosphatidategeneration, J. Biol. Chem. 281 (2006) 38418–38429.

[85] M. Sasaki, M. Kashima, T. Ito, A. Watanabe, M. Sano, M. Kagaya, T. Shioya, M. Miura,Effect of heparin and related glycosaminoglycan on PDGF-induced lung fibroblastproliferation, chemotactic response and matrix metalloproteinase activity,Mediators Inflamm. 9 (2000) 85–91.

[86] J. Zhong, M.M. Gencay, L. Bubendorf, J.K. Burgess, H. Parson, B.W. Robinson, M.Tamm, J.L. Black, M. Roth, ERK-1/2 and p38MAP kinase control MMP-2,MT1-MMP,and TIMP action and affect cell migration: a comparison between mesotheliomaand mesothelial cells, J. Cell. Physiol. 207 (2006) 540–552.

[87] Y.J. Sigal, M.I. McDermott, A.J. Morris, Integral membrane lipid phosphatases/phosphotransferases: common structure and diverse functions, Biochem. J. 387(2005) 281–293.

[88] S. Pyne, K-C. Kong, P.I. Darroch, Lysophosphatidic acid and sphingosine 1-phosphatebiology: the role of lipid phosphate phosphatases, Semin. Cell Dev. Biol. 15 (2004)491–501.

[89] H. Sano, A.D. Renault, R. Lehmann, Control of lateral movement and germ cellelimination by the Drosophila melanogaster lipid phosphate phosphatases Wunenand Wunen-2, J. Cell Biol. 171 (2005) 675–683.

[90] K. Hanyu-Nakamura, S. Kobayashi, A. Nakamura, Germ cell autonomous Wunen2is required for germline development in Drosophila embryos, Development 131(2004) 4545–4553.

[91] E.A. Siess, M.M. Hofstetter, Identification of phosphatidate phosphohydrolasepurified from rat liver membranes on SDS-polyacrylamide gel electrophoresis,FEBS Lett. 381 (1996) 169–173.

[92] C. Burnett, P. Makridou, L. Hewlett, K. Howard, Lipid phosphate phosphatasesdimerise, but this interaction is not required for in vivo activity, BMC Biochemistry5 (2004) 2.

[93] J.S. Long, N.J. Pyne, S. Pyne, Lipid phosphate phosphatases form homo- and hetero-oligomers: catalytic competency, sub-cellular distribution and function, Biochem.J. 411 (2008) 371–377.