Lazaro: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References
Gene name - lazaro
Cytological map position- 79E4
Function - enzyme
Keywords - phototransduction, lipid metabolism, phospholipids
Symbol - laza
FlyBase ID: FBgn0037163
Genetic map position - 3L
Classification - phosphate phosphohydrolase
Cellular location - surface transmembrane
During receptor-activated cell signaling cascades, it is important that the resynthesis of biochemical substrates is tightly coupled to their consumption by enzymatic reactions. This is particularly necessary in the context of neuronal signaling cascades, where at high rates of receptor stimulation, significant depletion of substrates might otherwise occur. An essential step in Drosophila phototransduction is the hydrolysis of phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2) by phospholipase Cβ (PLCβ) to generate a second messenger that opens the light-activated channels TRP and TRPL. Although the identity of this messenger remains unknown, recent evidence has implicated diacylglycerol kinase (DGK), encoded by rdgA, as a key enzyme that regulates levels of the second messenger, mediating both amplification and response termination within photoreceptor cells. This study demonstrates that lazaro (laza: from the Spanish novel Lazarillo de Tormes, in which Lazaro is the boy servant of a blind man and helps him to see) encodes a lipid phosphate phosphohydrolase (LPP) that functions during phototransduction. Whereas RdgA converts DAG to phosphatidic acid (PA) by adding phosphate residues to glycerol, Laza carries out the reverse reaction by removing the phosphate residue. In other words, DGK and LPP operate as a kinase/phosphatase pair that controls the level of PA. The activity of Laza laza rescues degeneration in the blind mutant rdgA and works synergistically with it to regulate amplification and response termination during phototransduction. Analysis of retinal phospholipids revealed a reduction in PA levels and an associated reduction in phosphatidylinositol (PI) levels. Together these results demonstrate the contribution of PI depletion to the rdgA phenotype and provide evidence that depletion of PI and its metabolites might be a key signal for TRP channel activation in vivo (Garcia-Murillas, 2006). Similar results have been obtained by (Kwon and Montell, 2006)
Calcium influx through plasma membrane channels regulates a range of functions in both adult and developing neurons, including the transduction of a number of sensory modalities, learning and memory, and the navigation of dendrites during neuronal development. A number of different ion channels mediate calcium influx that subserves these functions; one major family are the TRP proteins. Several classes of TRP channels have been described, and members of all classes mediate key physiological processes in neurons. Despite the importance of TRP proteins in neuronal function, the signaling mechanisms that regulate channel activity in vivo remain controversial and poorly understood. A common theme underpinning the regulation of several classes of TRP channels is the role of PI(4,5)P2 and/or its metabolites. When heterologously expressed, TRPC, TRPV, and TRPM channels are all reported to be modulated by changes in PI(4,5)P2 levels during signaling. However, the role of PI(4,5)P2 depletion in the activation of endogenous TRP channels remains limited by the lack of suitable in vivo models (Garcia-Murillas, 2006).
Drosophila phototransduction is a well-established model system for the analysis of calcium influx triggered by G protein-coupled phosphoinositide hydrolysis (Hardie, 2001). In photoreceptors, the absorption of a photon of light by rhodopsin triggers PLCβ activity. This signaling cascade triggers the opening of at least two classes of plasma membrane channels, Trp and Trpl, resulting in the influx of calcium into the cell. Despite intense investigation, the mechanism by which PLCβ activity results in Trp and Trpl opening remains unclear. The essential role of PI(4,5)P2 hydrolysis in phototransduction is well-established; null mutants in norpA, which lack eye-enriched PLCβ, show no response to light. The hydrolysis of PI(4,5)P2 generates the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 is insufficient for activation of Trp and Trpl in vivo, and the IP3 receptor does not appear to be essential for phototransduction. However, recent studies have suggested that lipid second messengers generated by PLCβ activity might play a role in Drosophila Trp and Trpl activation. Application of both DAG and PI(4,5)P2 has been reported to modulate heterologously overexpressed Trpl channels (Estacion, 2001), and polyunsaturated fatty acids (Chyb, 1999), metabolites of DAG, can activate Trp and Trpl (Garcia-Murillas, 2006).
In most eukaryotic cells, DAG is phosphorylated by DGK to generate PA. In Drosophila, rdgA mutants that lack eye-enriched DGK show severe retinal degeneration that can be rescued by trp mutants. The retinal degeneration phenotype of rdgA has recently been shown to be modulated by light, Gq, and PLCβ (Georgiev, 2005), all of which are key elements required for PI(4,5)P2 hydrolysis during phototransduction. Electrophysiologically, rdgA mutant photoreceptors show constitutive Trp channel activity as well as defects in amplification and response termination. Thus, the analysis of the rdgA mutant strongly suggests that in vivo the tight regulation of PI(4,5)P2-derived lipid metabolites is essential for normal Trp channel activation. While the analysis of rdgA demonstrates the key role of DGK in this process, the biochemical and molecular basis for its requirement is unclear. In particular, it has thus far not been possible to identify the specific lipid messenger(s) responsible for the abnormal Trp channel activation in rdgA. One possibility is that reduced DGK activity results in an elevation of DAG and/or its metabolites in the face of ongoing PI(4,5)P2 hydrolysis. Alternatively, it is possible that a reduction in levels of PA, the product of DGK, is a key determinant of the rdgA phenotype. In addition to being a potential second messenger in its own right, PA can also be recycled to resynthesize PI(4,5)P2. PA can bind to a number of proteins and in some cases regulate their activity. These include two molecules involved in PI turnover: type I phosphatidylinositol 4 phosphate 5 kinase (type I PIPkin) in mammalian cells and Opi1p, an ER resident transcription factor in yeast. Opi1p, when bound to PA, translocates to the nucleus and stimulates the transcription of genes regulating phosphatidylinositol (PI) biosynthesis. Thus, it is likely that PA levels would be tightly regulated and linked to PI turnover in vivo. It also raises the possibility that a depletion of PI and consequently PI(4,5)P2 might underlie the rdgA phenotype (Garcia-Murillas, 2006).
As part of a study to understand the biochemical basis of the rdgA phenotype, additional enzymes encoded in the Drosophila genome were examined that might regulate lipid turnover during PI(4,5)P2 hydrolysis. An immediate possibility is that lipid phosphate phosphohydrolases (LPPs) might act in synergy with DGK in controlling the balance of DAG and PA. LPPs were originally designated as type II PA phosphatases (PAP), since their activity was distinct from the type I PAP activity involved in glycerolipid biosynthesis. However, to date it is unclear whether LPPs can metabolise PA, an intracellular bioactive lipid generated during PI(4,5)P2 hydrolysis. This hypothesis was tested and the endogenous LPP that functions during Drosophila phototransduction was identified. The biochemical consequence is described of the concerted action of DGK and LPP during phototransduction and its implications for the mechanism of Trp channel activation (Garcia-Murillas, 2006).
During receptor-activated cell signaling cascades, it is important that the resynthesis of biochemical substrates is tightly coupled to their consumption by enzymatic reactions. This is particularly necessary in the context of neuronal signaling cascades, where at high rates of receptor stimulation, significant depletion of substrates might otherwise occur. For example, in bright daylight, Drosophila photoreceptors are able to successfully detect ~106 photons s−1 without significant loss of sensitivity. In the context of a G protein-coupled phosphoinositide signaling cascade, the hydrolysis of the substrate PI(4,5)P2, a minor membrane phospholipid, needs to be coupled to its resynthesis by the sequential phosphorylation of a cellular pool of PI. Thus, an adequate rate of PI(4,5)P2 resynthesis requires among other things the maintenance of this pool of PI. PI is synthesized in the ER by the condensation of inositol and CDP-DAG by PI synthase. Previous studies have suggested the importance of a constant supply of inositol generated from IP3 to maintain ongoing G protein-coupled PLC signaling in neurons. However, the role, if any, for the recycling of DAG to supply CDP-DAG required for PI synthesis has thus far not been addressed. In addition to being an allosteric activator of PKC, PA can be recycled into PI resynthesis by the sequential action of CDP-DAG synthase and PI synthase. In the present study, it was found found that (1) in rdgA3 photoreceptors, levels of total PA are reduced and that (2) this reduction is enhanced by overexpressing LPP. Together with previous findings that DAG levels are not elevated in the rdgA, these observations demonstrate the critical role of PA levels generated by DGK in the rdgA phenotype. However, the findings that elevation of PA levels via the patch pipette does not suppress constitutive Trp channel activity and that elevation of PA levels using the cds1 mutant did not rescue rdgA3 strongly suggest that PA does not directly influence Trp channel activity or contribute to the degeneration phenotype (Garcia-Murillas, 2006).
How might reduced levels of photoreceptor PA contribute to the rdgA phenotype? In this study, two observations provide an insight into this question. (1) The cds1 mutant that elevates photoreceptor PA levels while reducing PI and PIP levels enhances rather than suppressed degeneration in rdgA3. (2) The reductions in PA level are associated with a change in PI levels in rdgA3 retinae, and PI levels undergo a dramatic reduction when PA levels were further reduced by LPP overxpression. Taken together, it is likely that these findings reflect the requirement for a constant supply of PA generated by rdgA so that CDP-DAG can be generated by CDP-DAG synthase to be used as a substrate for PI synthesis. The finding that transcript levels of PI synthase in photoreceptors are directly correlated with PA levels suggests one mechanism by which PA might regulate PI resynthesis linked to the level of ongoing PI(4,5)P2 hydrolysis. In this analysis, colocalization of the enzyme within the cell body in a pattern highly reminiscent of the ER within these cells was found. This membrane compartment is the site at which the resynthesis of PI(4,5)P2 is initiated by the enzyme CDP-DAG synthase using PA as the substrate. In photoreceptors, DGK (encoded by rdgA) is also localized on the submicrovillar cisternae thought to be a specialization of the ER. Thus, three key enzymes that together contribute to the regulation of cellular PA and generation of PI appear to be localized to the ER, consistent with a role for PA in stimulating resynthesis of PI (Garcia-Murillas, 2006).
Since the sequential phosphorylation of PI is a major route of PI(4,5)P2 generation, changes in PI levels could impact on photoreceptor PI(4,5)P2 resynthesis. To test this possibility, the effect of laza activity was analyzed in rdgB, a mutant with reduced rates of PI(4,5)P2 resynthesis at the rhabdomeral plasma membrane (Hardie, 2001). The effect was analyzed of altering PA levels by both overexpressing LPP as well as using laza mutants to remove the endogenous LPP that functions during phototransduction and it was found that the retinal degeneration phenotype of rdgB can be modulated by levels of LPP activity in the retina. Together with biochemical data on the effects of altered PA (the substrate of Laza) levels in photoreceptors, these results suggest a role for PA levels in regulating the supply of PI to be transported to the plasma membrane for PI(4,5)P2 resynthesis during phototransduction. How might PA levels modulate the rdgB phenotype? It has previously been shown that PA does not bind to and alter phosphatidylinositol-transfer protein (PITP) function in vitro. One obvious mechanism is that the level of PI (a key substrate that is transported by PITP) is altered by changes in laza activity. An alternative mechanism is suggested by the finding that in vitro PA binds to and stimulates type I phosphatidylinositolphosphate kinase (PIPkin) activity. The finding that reduced type 1 PIPkinase activity (sktlΔ20/sktlΔ1-1) enhances the rate of degeneration in rdgA3 supports this mechanism. Thus, there are at least three mechanisms by which PA derived from PI(4,5)P2 can stimulate PI(4,5)P2 resynthesis: (1) as a unique substrate for CDP-DAG synthesis, (2) as a transcriptional regulator of PI synthase, (3) as an allosteric regulator of type I PIPkin. In the present study, evidence is provided in support of these three mechanisms in photoreceptors. It is therefore proposed that the generation of PA by DGK during PLCβ-mediated signaling provides a key signal to regulate the resynthesis of PI(4,5)P2 (Garcia-Murillas, 2006).
Given the critical role of PA during phototransduction, LPP was identified as an enzyme that might work in conjunction with DGK to regulate PA levels. LPP was originally designated as a type II PA phosphatase (PAP) (Jamal, 1991; Kai, 1996) and shows remarkable substrate promiscuity in vitro (reviewed in Brindley, 1998). When overexpressed in cell culture models, LPPs are able to dephosphorylate extracellular lipids presented to intact cells (Jasinska, 1999 and Roberts, 1998) and localize mainly to the plasma membrane (Alderton, 2001; Burnett, 2003; Jasinska, 1999), with residues involved in catalysis facing externally (Zhang, 2000). These findings have led to the idea that LPPs are ectoenzymes catalyzing the hydrolysis of extracellular bioactive lipid, although, to date, the in vivo substrate of any LPP has not been identified. In particular, it is unclear whether LPPs can metabolise PA generated by hydrolysis of PI(4,5)P2. During this study, the enzymatic activity was analyzed of a number of LPP gene products during Drosophila phototransduction. Overexpressing four different Drosophila LPPs individually can exacerbate the phenotype of hypomorphic mutants in DGK. Significantly, the effects of LPP overexpression are evident only in the sensitized background of DGK loss of function, providing strong evidence that LPP can function as antagonists of DGK. The finding that the reduced levels of PA in rdgA3 retinae are further reduced on LPP overexpression provides biochemical support for the genetic data. Further, it was also demonstrate that laza22 retinae show light-induced accumulation of PA. This finding supports the idea that laza regulates PA levels during phototransduction. Together, these findings provide compelling evidence that in vivo DGK and LPP operate as a kinase/phosphatase pair that controls the level of PA. The finding that overexpression of LPP in flies lacking both DGK and PLCβ in their photoreceptors (norpAP24, rdgA3) did not result in retinal degeneration strongly supports the conclusion that in vivo LPP can dephosphorylate PA generated from PI(4,5)P2 hydrolysis by the sequential enzymatic activity of PLCβ and DGK. This study identifies an in vivo substrate of LPP and also demonstrates a role for LPP in regulating the DAG/PA balance during G protein-coupled PI(4,5)P2 signaling (Garcia-Murillas, 2006).
During this study, it was found that when overexpressed LPPs encoded by a number of different gene products, including CG11426, laza, wun, and wun2, are all able to antagonize DGK function, reminiscent of the ability of all three isoforms of vertebrate LPPs (LPP1, LPP2, and LPP3) to dephosphorylate PA (Roberts, 1998). In contrast, it was found, using loss-of-function analysis, that the only LPP that is able to functionally antagonise DGK and hence suppress degeneration in rdgA is the eye-enriched LPP laza. It has recently been shown that when overexpressed, LPPs can form oligomers (Burnett, 2004). It is possible that high levels of expression result in the assembly of oligomers that lack substrate specificity (Garcia-Murillas, 2006).
A common but poorly resolved theme underlying the signaling mechanisms regulating TrpC channel activation is the role of PI(4,5)P2 or its metabolites in activation. Recently, a number of studies have shown that a range of lipids generated by PI(4,5)P2 hydrolysis can activate both vertebrate and invertebrate TrpC channels when applied exogenously to overexpressed channels (reviewed in Hardie, 2003). However, to date, the activation of endogenous TrpC channels has been studied in only two model systems: activation of Trp and Trpl during Drosophila phototransduction and the activation of TrpC2 channels in neurons of the mouse vomeronasal organ. In both of these systems, the inactivation of DGK (using the rdgA mutant in Drosophila and pharmacology in the mouse study; Lucas, 2003; Raghu, 2000) results in a sustained activation of TrpC channels in the face of ongoing PI(4,5)P2 hydrolysis. While these findings are consistent with a role for DGK in regulating response termination, they have thus far not allowed a definitive analysis of the messenger of activation. In the mouse vomeronasal organ, the finding that TrpC2-like channels can be activated by exogenous DAG (Lucas, 2003) implies that the build-up of this messenger is the biochemical basis of abnormal TrpC channel activation when DGK is inhibited. However, a build-up of DAG in the vomeronasal organ during DGK inhibition has yet to be demonstrated. A previous study showed that in Drosophila photoreceptors of the rdgA mutant, there was no elevation of DAG (Inoue, 1989), while demonstrating that light-induced PA levels were reduced. In the present study, it was also demonstrated that in addition to a reduction in retinal PA levels, there is also a reduction in the level of PI, the precursor for PI(4,5)P2 resynthesis, and an associated change in the levels of PI synthase, a key enzyme for PI resynthesis. These findings raise the possibility that the reduced level of PI might also result in a reduction in the rate of PI(4,5)P2 resynthesis. The finding that reduction in type I PIP kinase activity enhances degeneration in rdgA3 supports this hypothesis. Previous studies have shown that both rdgB photoreceptors as well as wild-type photoreceptors (in zero extracellular calcium) show prolonged activation of Trp channels following a response to light (Hardie, 2001). In addition, Trpl channels expressed in S2 cells have been reported to be inhibited by PI(4,5)P2 (Estacion, 2001). Taken together with the findings reported in this paper, it is proposed that an imbalance between the rate of PI(4,5)P2 hydrolysis by PLCβ and its resynthesis from PA might underlie the excessive activation of endogenous Trp channels in photoreceptors of the rdgA mutant (Garcia-Murillas, 2006).
Overexpression of LPP enhances degeneration in rdgA: To test the ability of LPP to dephosphorylate PA generated during PI(4,5)P2 hydrolysis, the rdgA mutant was used. In rdgA3, a hypomorphic allele, photoreceptors look ultrastructurally normal on eclosion and degenerate with time when grown in laboratory incubator conditions. Transgenic flies were generated and whether overexpression of one of the Drosophila LPPs, CG11426 (encoding an LPP - closely linked to laza), affected the phenotype of rdgA, was tested. When CG11426 was overexpressed in rdgA3 using Rh1-GAL4, there was obvious degeneration of the rhabdomeres in newly eclosed flies. Over a period of 72 hr post-eclosion, rdgA3+ LPP photoreceptors degenerated more rapidly than rdgA3 alone. By contrast, when CG11426 was overexpressed in otherwise wild-type photoreceptors, there was no detectable effect on photoreceptor ultrastructure. Similar results were obtained with three other Drosophila LPPs, wun, wun2, and laza. These results provide strong genetic evidence that LPP can functionally antagonise the DGK activity encoded by rdgA (Garcia-Murillas, 2006).
The enhancement of rdgA3 by LPPs suggests that these enzymes can dephosphorylate PA in vivo. This was tested by measuring retinal PA levels using liquid chromatography followed by mass spectrometry, comparing rdgA3 with rdgA3 + CG11426. It was found that levels of PA in rdgA3 retinae were ~60% of wild-type. Importantly, this reduction was dramatically enhanced in rdgA3+ LPP: these retinae had ~20% of total wild-type PA levels. These results demonstrate that PA levels in rdgA3 are reduced and that when overexpressed, LPP enhances this reduction in vivo (Garcia-Murillas, 2006).
Retinal degeneration in rdgA3 can be completely blocked by norpAP24, a strong hypomorph in PLCβ essential for phototransduction, suggesting that the enhancement of rdgA by LPP is most likely mediated by PA derived from PI(4,5)P2. To confirm this idea, CG11426 was overexpressed in norpAP24, rdgA3. This analysis revealed normal rhabdomeres in norpAP24, rdgA3 + LPP and shows that the enhancement of rdgA3 by LPP requires ongoing light-induced PI(4,5)P2 hydrolysis. These results demonstrate that LPP can antagonize DGK function, most likely by dephosphorylating PA generated by PI(4,5)P2 hydrolysis (Garcia-Murillas, 2006).
Changes in retinal PA in rdgA are associated with depletion of PI: While analysis of laza genetic interaction with rdgA demonstrate the concerted action of rdgA and laza in regulating phosphatidic acid levels during phototransduction, they raise the question of how PA levels are linked to abnormal Trp channel activity seen in rdgA. One possibility is that PA directly regulates Trp channels, although it has previously been reported that supplementation of PA during whole-cell recording failed to suppress constitutive Trp activity in rdgA. To test the possibility that there might be changes in other phospholipid classes that could account for the rdgA phenotype, a lipidomic analysis was performed studying three other classes of phospholipids, including PI, phosphatidylcholine (PC), and phosphatidylethanolamine (PE). Since the reduction in absolute levels of phospholipids presumably partly reflects the ongoing degeneration process and associated loss of membranes, the levels of each phospholipid was expressed as a fraction of the level of PC + PE that are major building blocks of membranes. This analysis revealed a significant reduction in the levels of PI in rdgA3 that was further enhanced on overexpression of LPP. These findings demonstrate that the reductions in PA levels during phototransduction are associated with a reduction in at least one other phospholipid class, namely PI, and raise the possibility that reduction in photoreceptor PI levels might contribute to the rdgA phenotype (Garcia-Murillas, 2006).
Mutants in cds enhance degeneration of rdgA3: To test the role of PI depletion in the rdgA phenotype, mutants in CDP-DAG synthase (cds1), the enzyme that converts PA to CDP-DAG, were used. cds1 has been shown to affect the rate of PI(4,5)P2 resynthesis during the light response (Hardie, 2001). In addition, it has been found that cds1 mutants show reductions in the abundance of the two molecular species that constitute the major fraction of PI in wild-type photoreceptors. rdgA3; cds1 double mutants were generated and the rate of degeneration was compared to rdgA3 and cds1. It was found that cds1 enhances the rate of degeneration in rdgA3. To understand the biochemical basis of this degeneration, phospholipid levels were analyzed comparing rdgA3 retinae with those from rdgA3; cds1. This study revealed that although PA and DAG levels were not different in the two genotypes, there were significant changes in the levels of PI and PIP, suggesting that reduced levels of PI and PIP may contribute to the degeneration phenotype of rdgA3 (Garcia-Murillas, 2006).
PA regulates levels of PI synthase transcripts: An immediate metabolic fate of PA is conversion to PI by the sequential activity of CDP-DAG synthase and PI synthase. Thus, it is likely that the reduction of PI levels in rdgA3 and rdgA3 + LPP are partly explained by the reduced levels of PA available as substrate for CDP-DAG synthase. However, recently it has been shown that in yeast PA can transcriptionally regulate the levels of PI synthase (Loewen, 2004). To test whether this was also the case in Drosophila photoreceptors, RT-PCR was used to compare levels of PI synthase (CG9245- CDP-diacylglycerol-inositol 3-phosphatidyltransferase) transcript in wild-type retinae with those from rdgA3 and rdgA3 + LPP. This analysis revealed that CG9245 transcript levels in rdgA3 were reduced and were virtually obliterated in RNA from rdgA3 + LPP retinae. Thus, the levels of PI synthase transcript are directly correlated with the levels of retinal PA (Garcia-Murillas, 2006).
laza modulates the degeneration of rdgB: Since biochemical analysis strongly indicated that PA levels generated during phototransduction are linked to PI resynthesis, it was of interest to ask whether this might also impact on the PI(4,5)P2 resynthesis. To test this in vivo, the rdgB mutant, defective in the Drosophila homolog of PI transfer protein, was used. Loss-of-function mutants in rdgB show (1) light-dependent retinal degeneration and (2) a reduced rate of PI(4,5)P2 resynthesis during the light response. LPP was overexpressed in rdgB mutants and rdgBKS222 was compared with rdgBKS222 + LPP. This analysis showed that overexpression of LPP enhances degeneration in rdgBKS222. Conversely, loss-of-function mutants in laza were able to slow the rate of degeneration of rdgBKS222 (Garcia-Murillas, 2006).
sktl enhances degeneration in rdgA3: To test the role of reduced PI(4,5)P2 synthesis consequent to the reduced PA and PI levels in rdgA, the effect was analyzed of reduced type 1 PIP kinase activity on the rdgA phenotype. For this mutants were used in sktl that encode one of the two type I PIP kinases in Drosophila. Since null mutants in sktl are cell-lethal in photoreceptors, a heteroallelic combination of sktlΔ20 (a null allele) and sktlΔ1-1 (a hypomorphic insertion in the upstream region of the sktl gene) were used. sktlΔ20/sktlΔ1-1 itself does not show any degeneration on the timescale of the experiments described. rdgA3; sktlΔ20/sktlΔ1-1 photoreceptors were generated and the rate of degeneration was compared to that of the rdgA3 single mutant. This analysis revealed that sktlΔ20/sktlΔ1-1 enhances the rate of degeneration in rdgA3 (Garcia-Murillas, 2006).
Transcript levels for all five genes encoding lipid phosphate phosphohydrolases were compared in total RNA from wild-type heads with those from soD (a mutant that lacks eyes) using semiquantitative RT-PCR. This analysis revealed that only laza transcripts are enriched in the eye (Garcia-Murillas, 2006).
The finding that LPP can regulate intracellular PA levels in Drosophila photoreceptors raises questions on where the enzyme is localized within these cells. The subcellular localization of GFP-tagged laza was examined in Drosophila photoreceptors between 0–12 hr after eclosion. Under these conditions, expression of laza-GFP in the plasma membrane of either the rhabdomere or the cell body could not be detected. Rather, the protein appears to be punctate and distributed throughout the cell body. The pattern of distribution showed considerable overlap to that of an antibody directed to the ER retention signal KDEL. These results suggest that under these conditions the overexpressed enzyme is localized to the endoplasmic reticulum or a compartment thereof. Similar results were obtained with CG11426. Despite concerted efforts, no antibody was raised that allows study of the distribution of the endogenous protein. However, to test whether laza-GFP encodes a functional protein whose distribution might report that of the endogenous enzyme, attempts were made to rescue the phenotype of laza22 using the laza-GFP construct used in this study. This analysis revealed that laza-GFP was able to rescue the retinal degeneration phenotype of laza22 restoring wild-type rhabdomere ultrastructure (Garcia-Murillas, 2006).
The Drosophila phototransduction cascade serves as a paradigm for characterizing the regulation of sensory signaling and TRP channels in vivo. Activation of these channels requires phospholipase C (PLC) and may depend on subsequent production of diacylglycerol (DAG) and downstream metabolites. DAG could potentially be produced through a second pathway involving the combined activities of a phospholipase D (PLD)and a phosphatidic acid (PA) phosphatase (PAP). However, a role for a PAP in the regulation of TRP channels has not been described. This study reports the identification of a PAP, referred to as Lazaro (Laza). Mutations in laza caused a reduction in the light response and faster termination kinetics. Loss of laza suppresses the severity of the phenotype caused by mutation of the DAG kinase, RDGA, indicating that Laza functions in opposition to RDGA. The retinal degeneration resulting from overexpression of the PLD is suppressed by elimination of Laza. These data demonstrate a requirement for a PLD/PAP-dependent pathway for achieving the maximal light response. The genetic interactions with both rdgA and Pld indicate that Laza functions in the convergence of both PLC- and PLD-coupled signaling in vivo (Kwon, 2006).
To identify new proteins that are involved in phosphoinositide (PI) signaling and participate in Drosophila phototransduction, focus was placed on a gene (CG11440), which was identified in a microarray screen for genes expressed predominately in the adult eye. CG11440 mRNA is expressed in the adult eye at a concentration 15-fold higher than in the rest of the head and encodes a 334 amino acid protein that is >35% identical to known lipid phosphate phosphatases. As with other lipid phosphate phosphatases (LPPs), CG11440 is characterized by multiple transmembrane segments and three conserved domains that form the catalytic domain. An LPP would be predicted to catalyze the reverse reaction promoted by RDGA (Kwon, 2006).
To address a potential function for the CG11440 (referred to as lazaro, laza) in phototransduction, a P element insertion line, GE27043 (GenExel), was obtained which was inserted in the 5′ untranslated region (lazaGE). A null mutation in laza was created by genetically mobilizing the P element, because excisions of P elements are frequently associated with deletions of the flanking genomic DNA. Pools of genomic DNA were screened by PCR and two lines (laza1 and laza2) were obtained that deleted the first ATG and additional portions of the coding region. In addition, one revertant line was retained in which the P element excised precisely (lazaRE) (Kwon, 2006).
Antibodies were raised against the C-terminal region of Laza and a protein of 42 kDa (predicted molecular weight of 37 kDa) was identified that was not detected in any of the laza mutants (lazaGE, laza1, laza2) or in flies containing lazaGE in trans with a deficiency chromosome, Df(3L)ED230, that removed laza. The 42 kDa protein was restored in lazaRE as well as in laza1 flies in which a wild-type CG11440 genomic transgene was introduced. These data indicated that expression of the Laza protein was disrupted in lazaGE, laza1, and laza2. Protein levels of other phototransduction proteins were unchanged in each of the lines studied (Kwon, 2006).
To evaluate whether the Laza protein was expressed in the retina, a biochemical approach was used because the antibodies were ineffective for immunostaining. Laza was detected exclusively in extracts from wild-type heads but not bodies. Moreover, the Laza protein was absent in head extracts prepared from a mutant lacking eyes (sine oculis, so) and was greatly reduced in the heads of a mutant (glass), which were missing photoreceptor cells specifically. Thus, Laza was expressed primarily in photoreceptor cells (Kwon, 2006).
The requirement for laza for phototransduction was evaluated by performing electroretinogram (ERG) recordings, which assay the summed responses of all retinal cells to light. Exposure of wild-type flies to light results in a corneal negative receptor potential, which decays to baseline after cessation of the light stimulus. The amplitude of the light response was reduced nearly 40% in lazaGE flies and 3-fold in laza1 and laza2 flies. These reductions in ERG amplitudes were not due to background mutations because they were observed in lazaGE/Df flies and the lazaGE phenotype reverted to wild-type upon precise excision of the P element. Furthermore, the wild-type laza+ transgene rescued the reduction in the ERG amplitude. Although it was difficult to formally exclude that the laza mutation had no effects on the overall physiology of the photoreceptor cells, the reduced ERG amplitude in laza flies did not appear to be due to retinal degeneration because 7-day-old adults did not display retinal degeneration and the ERGs were performed on flies <7 days old. As is the consequence of most mutations that affect phototransduction, in older flies (≥15 days old) the laza mutation resulted in retinal degeneration, which was light-dependent. Nevertheless, the decreased ERG responsiveness in the laza flies was not age dependent (Kwon, 2006).
To assess whether the decreased amplitude in laza was more likely a reflection of a lower sensitivity to light or a decreased maximal obtainable amplitude, the ERG responses was measured at three different intensities. Over the 130-fold intensity range tested, both wild-type and laza flies showed a linear intensity-responsive relationship when the amplitudes were plotted against the logs of the light intensities. However, in the laza mutant, the plot is shifted down. If there was a reduction strictly in the sensitivity of the light response, the lines generated from the wild-type and laza intensity-responsive relationships should have been parallel. This was not the case, because the amplitude of the laza response increased less sharply than in wild-type. These data are most consistent with a defect in the maximum light response in the mutant; however, the data do not allow assessment of whether or not there is a defect in the sensitivity to light. If laza operates in a pathway downstream of PLD, then there would be expected to be overlaps in the pld and laza mutant phenotypes. Interestingly, in the pld null mutant, there is also a defect in the maximum achievable ERG amplitude (Kwon, 2006).
Mutations in rdgA, which encodes a diacylglycerol (DAG) kinase, prolong termination kinetics. To test whether elimination of laza might accelerate deactivation kinetics, consistent with the predicted biochemical function, the time required for an 80% return to the baseline after cessation of the photoresponse was measured. The termination of the photoresponse was approximately 4-fold faster in the laza1 and laza2 mutant flies than in wild-type. Introduction of the genomic rescue transgene in the laza1 mutant background restored the deactivation kinetics to that of wild-type (Kwon, 2006).
The apparent increase in the speed of termination of the photoresponse in the laza mutants may have been due to the smaller ERG amplitude. Therefore, wild-type flies were exposed to a lower light intensity so that the amplitudes of the wild-type and laza ERGs were comparable. Under these conditions, the time required for 80% termination of the photoresponse was 2-fold shorter in the laza1 and laza2 mutant flies than in wild-type. Thus, the more rapid termination in laza flies was the reverse of that observed in rdgA flies (Kwon, 2006).
If laza functions in the regulation of the photoresponse by catalyzing the PI-cycle reaction that is the reciprocal of that catalyzed by RDGA, then it is plausible that introduction of laza into an rdgA background would suppress the retinal degeneration observed in rdgA flies. To test this hypothesis, the laza1 mutation was combined with the rdgABS12 mutation and tested for suppression of the retinal degeneration. Wild-type flies contain compound eyes consisting of ~800 repetitive units, referred to ommatidia, each of which contains seven photoreceptor cells in any given cross-section. Each photoreceptor cell includes a microvillar structure, the rhabdomere, which is the site of action of phototransduction. Wild-type flies contain seven rhabdomeres in all ommatidial cross-sections regardless of their age. In contrast, the number and size of the rhabdomeres is dramatically reduced even in 1-day-old rdgA flies as a result of constitutive activation of the TRP channel (Kwon, 2006).
Of significance here, the laza1 mutation suppresses the degeneration-associated rdgABS12. At 1 day posteclosion, the average number and size of the rhabdomeres was significantly increased. By 3 days posteclosion, the double-mutant flies retained at least as many rhabdomeres as 1-day-old rdgABS12 flies. However, by 7 days after eclosion, the rdgABS12;;laza1 flies also displayed severe retinal degeneration. Consistent with the partial suppression of retinal degeneration by the laza mutation, the ERG response in the double-mutant flies was also partially restored. Whereas there was virtually no light response in 1-day-old rdgABS12 flies, a small ERG was restored in the rdgABS12;;laza1 flies. The suppression of rdgA may not have been complete because of the presence of seven other PAPs encoded in the Drosophila genome, including wunen, wunen 2, CG11425, CG11426, CG11437, CG11438, and CG12746, which could potentially be expressed in the photoreceptor cells (Kwon, 2006).
Overexpression of phospholipase D (PLD) causes light-dependent retinal degeneration that is suppressed by mutation of the TRP channel. Because PLD converts phosphatidylcholine (PC) to PA, the retinal degeneration could result from an elevation in phosphatidylinositol 4,5-bisphosphate (PIP2) production. Alternatively, an increase in production of PA could lead to higher DAG levels, an increase that causes degeneration as a result of higher levels of TRP channel activation. Indeed, it has been shown in mammalian cell culture systems that DAG can be generated from PC by the sequential reactions promoted by PLD and a PA phosphatase. These observations raise the interesting possibility that Laza may function in a common pathway with the PLD. To test this idea, whether the retinal degeneration caused by overexpression of PLD was suppressed by the laza mutation was examined. One-day-old Pld-overexpression flies (Rh1-Pld) displayed early stages of degeneration and were typically missing one rhabdomere per ommatidium. By two days posteclosion, the Pld-overexpression flies displayed clear retinal degeneration; the numbers and sizes of the rhabdomeres were greatly reduced. The degeneration gradually progressed until nearly all of the rhabdomeres disappeared in 7-day-old Rh1-Pld flies. In contrast, no degeneration was detected in 1-day-old Rh1-Pld;laza1 flies, and the retinal degeneration was significantly reduced in the 2-day-old Rh1-Pld;laza1 relative to the Pld-overexpression flies. Suppression was detected in 3-day-old flies; however, by 7 days posteclosion, the differences between Rh1-Pld and Rh1-Pld;laza1 were not significant. These data indicated that the severity of the retinal degeneration in Rh1-Pld flies is delayed by introduction of the laza1 mutation (Kwon, 2006).
The observation that mutations in laza suppress the degeneration resulting from loss-of-function mutations in rdgA provides genetic evidence that Laza functions opposite to RDGA in the phosphoinositide cycle, consistent with its predicted biochemical function. Furthermore, the faster termination kinetics in laza is the converse of that reported in rdgA mutant flies. These results suggest that the slower response kinetics in wild-type flies is due to Laza-dependent reduction in PA and production of DAG. The DAG or downstream metabolites, such as polyunsaturated fatty acids (PUFAs), may in turn slow down the termination kinetics as a result of prolonged activation of the channels. Several mammalian TRPC channels are activated by DAG, and Drosophila TRP and TRPL have been reported to be activated by PUFAs. The findings in the current report support the model that DAG or downstream metabolites, such as PUFAs, activate the TRPC channels in fly photoreceptor cells. These include the smaller ERG responses and faster termination kinetics in laza mutant flies. Interestingly, a TRPC-like current in rabbit artery myocytes is suppressed by inhibitors of either PLD or PA phosphatases (Kwon, 2006).
PA is generated in fly photoreceptor cells from two sources. The first involves the well-established PLC-dependent hydrolysis of the PIP2 to produce DAG and phosphorylation by DAG kinase. The second is cleavage of PC to generate PA through activity of PLD. Overexpression of PLD results in retinal degeneration, and it has been proposed that this degeneration results from the subsequent conversion of excess PA to DAG, which in turn leads to overstimulation of the TRPC channels. An indication that Laza may function in a PLD-mediated pathway leading to production of DAG is that the phenotypes resulting from loss-of-function mutations in either the pld or laza are similar. In both cases, there is reduced responsiveness to light. More direct genetic support that Laza functions in a PC/PA/DAG pathway is that elimination of Laza suppresses the retinal degeneration resulting from overexpression of PLD. However, the possiblity that the PLD-dependent production of PA also couples to an alternative Laza-independent pathway cannot be excluded (Kwon, 2006).
It is proposed that the DAG formed through the PLD/Laza pathway, that is, the product of Laza enzymatic function, primes the phototransduction cascade to increase the responsiveness to light. Given the prior evidence that PUFAs, which are formed from DAG, lead to activation of TRP channels, the additional DAG formed through activity of Laza may be necessary for a maximal light response. It is not known whether or not Laza is expressed in the rhabdomeres or cell bodies; however, it is noteworthy that the PLD and RDGA are both in the cell bodies, indicating that lipid metabolism in the cell bodies contributes to the photoresponse. The activity of the PLD has been suggested to be light independent. Nevertheless, given that no light response is observed in the norpA mutant, it cannot be excluded that PLD activity may be indirectly linked to the activity of norpA. It will be of interest to address the nature of the signals that contribute to PLD activation in vivo (Kwon, 2006).
Several LPPs are expressed in the adult retina, and overexpression of each enhanced rdgA3, raising the question of which endogenous LPP functions during phototransduction. To test this, loss-of-function mutants in wun, wun2, and laza were analyzed. Loss-of-function mutants in laza were generated by transposon mutagenesis. A number of mutant alleles were isolated; two of these, laza15 and laza22, are described. laza22 is a 703 bp deletion that removes the presumed start codon and the first 28 amino acids of the encoded protein. laza15 is a larger deletion of 2.996 kb that removes the entire open reading frame. Both laza15 and laza22 leave adjacent genes physically intact and give homozygous viable adults. RT-PCR analysis revealed that the transcript encoding laza was completely absent in laza15 and laza22, and transcript levels of the other four LPPs were unaffected (Garcia-Murillas, 2006).
To test the potential role of laza in phototransduction, retinal ultrastructure was examined in laza15 and laza22 using electron microscopy. Essentially similar results were obtained with both alleles and are henceforth presented for only laza22, the smaller deletion. On eclosion, laza22 photoreceptors show normal ultrastructure. However, when grown in bright light, they undergo progressive vesiculation and degeneration of the rhabdomeres that is quite severe by 5 days post-eclosion. Such degeneration was not seen when laza22 flies were grown in dim light. The observation that laza mutants show light-dependent retinal degeneration strongly suggests that this gene product functions during phototransduction (Garcia-Murillas, 2006).
Levels of retinal phospholipids from laza22, comparing samples from flies grown in dark with those grown in bright light. This analysis revealed an ~2.5-fold increase in PA levels when laza22 were grown in light compared to those grown in dark as well as wild-type flies grown in light. By contrast, there were no significant changes in the levels of PC, PE, PI, and DAG. These results demonstrate that light-induced PA levels increase in flies lacking the LPP activity encoded by laza (Garcia-Murillas, 2006).
To test the role of endogenous wun and wun2 in phototransduction, the phenotype was examined of loss-of-function mutants in these genes. No signs of rhabdomeral vesiculation and degeneration were seen in the case of either wun or wun2, suggesting that the LPP encoded by these genes do not participate directly in phototransduction (Garcia-Murillas, 2006).
If endogenous laza is a biochemical antagonist of DGK, an immediate prediction of the findings on overexpression of LPP in rdgA is that loss of the endogenous enzyme will have the opposite effect, i.e., suppress degeneration. To test this hypothesis, rdgA3; laza22 double mutants were generated and their phenotype studied. When grown in dim light, rdgA3 undergoes degeneration that is enhanced by growing flies in bright light (Georgiev, 2005). In dim light, rdgA3; laza22 degenerate at almost the same rate as rdgA3, suggesting that laza is probably not active in antagonizing DGK during low rates of PI(4,5)P2 hydrolysis. However, when grown in bright light, rdgA3; laza22 showed a dramatic reduction in the rate of degeneration compared to rdgA3 under the same conditions; laza22 heterozygotes slowed the degeneration to a rate intermediate between rdgA3 and rdgA3; laza22. These results clearly demonstrate that the endogenous LPP encoded by laza functions during phototransduction and works in synergy with the DGK encoded by rdgA (Garcia-Murillas, 2006).
Whether the concerted activity of rdgA and laza impacts on Trp and Trpl function during the light response was also tested. To do this, responses to light regulates response termination during phototransduction were analyzed in wild-type, rdgA3, and rdgA3; laza22 double mutants. The rate of deactivation of the light response was compared following a 1 s stimulus of light. Under these conditions, rdgA3 mutants show a pronounced decrease in the rate of deactivation. This defect in deactivation was substantially rescued in the rdgA3; laza22 double mutant. In this study, no clear electrophysiological phenotype was found for the laza22 single mutant using either ERG or whole-cell recording. The effect of overexpressing laza was also studied in rdgA3 photoreceptors. Since rdgA3 + laza photoreceptors show rhabdomeral degeneration which itself could affect deactivation kinetics, rdgA3/+ photoreceptors were used for this analysis. rdgA3/+ photoreceptors do not show retinal degeneration over the time periods used in this study; however, they do show a small but distinct defect in deactivation. This defect was enhanced by overexpression of laza. Overexpression of laza in wild-type photoreceptors did not have any effect on deactivation kinetics. Together, these findings suggest that the combined activity of DGK (encoded by rdgA) and LPP (encoded by laza) can regulate the deactivation of Trp and Trpl during the light response (Garcia-Murillas, 2006).
To test the role of endogenous wun and wun2 in phototransduction, double mutants of these were generated with rdgA3 and their rates of degeneration were studied under bright light illumination. Neither wun nor wun2 were able to slow the rate of degeneration in rdgA3, suggesting that the endogenous LPP encoded by these genes does not participate directly in phototransduction (Garcia-Murillas, 2006).
Phosphatidic acid (PA), lysophosphatidic acid, ceramide 1-phosphate (C1P), and sphingosine 1-phosphate (S1P) are lipid mediators generated by phospholipases, sphingomyelinases, and lipid kinases. The major pathway for degradation of these lipids is dephosphorylation catalyzed by members of two classes (types 1 and 2) of phosphohydrolase activities (PAPs). cDNAs encoding two type 2 PAPs, PAP-2a and -2b, have been expressed by transient transfection and shown to catalyze hydrolysis of PA, C1P, and S1P. This study reports the cloning and expression of a third type 2 PAP enzyme (288 amino acids), PAP-2c, which exhibits 54 and 43% sequence homology to PAPs 2a and 2b. Expression of HA epitope-tagged PAP-2a, -2b, and 2c in HEK293 cells produced immunoreactive proteins and increased membrane-associated PAP activity. Sf9 insect cells contain very low endogenous PAP activity. Recombinant expression of the three PAP enzymes using baculovirus vectors produces dramatic increases in membrane-associated Mg2+-independent, N-ethylmaleimide-insensitive PAP activity. Expression of PAP-2a but not PAP-2b or -2c resulted in high levels of cell surface PAP activity in intact insect cells. Kinetic analysis of PAP-2a, -2b, and -2c activity against PA, lysophosphatidic acid, C1P, and S1P presented in mixed micelles of Triton X-100 revealed differences in substrate specificity and susceptibility to inhibition by sphingosine, Zn2+, and propranol (Roberts, 1998).
Lipid phosphate phosphohydrolase (LPP)-1 cDNA was cloned from a rat liver cDNA library. It codes for a 32-kDa protein that shares 87% and 82% amino acid sequence identities with putative products of murine and human LPP-1 cDNAs, respectively. Membrane fractions of rat2 fibroblasts that stably expressed mouse or rat LPP-1 exhibit higher specific activities for phosphatidate dephosphorylation compared with vector controls. Increases in the dephosphorylation of lysophosphatidate, ceramide 1-phosphate, sphingosine 1-phosphate and diacylglycerol pyrophosphate were similar to those for phosphatidate. Rat2 fibroblasts expressing mouse LPP-1 cDNA showed increases in the hydrolysis of exogenous lysophosphatidate, phosphatidate and ceramide 1-phosphate compared with vector control cells. Recombinant LPP-1 is located partially in plasma membranes with its C-terminus on the cytosolic surface. Lysophosphatidate dephosphorylation is inhibited by extracellular Ca2+ and this inhibition is diminished by extracellular Mg2+. Changing intracellular Ca2+ concentrations did not alter exogenous lysophosphatidate dephosphorylation significantly. Permeabilized fibroblasts showed relatively little latency for the dephosphorylation of exogenous lysophosphatidate. LPP-1 expression decreased the activation of mitogen-activated protein kinase and DNA synthesis by exogenous lysophosphatidate. The product of LPP-1 cDNA is concluded to act partly to degrade exogenous lysophosphatidate and thereby regulate its effects on cell signalling (Jasinska, 1999).
Lipid phosphate phosphatase-1 (LPP-1) dephosphorylates exogenous lysophosphatidate and thereby regulates the activation of lysophosphatidate receptors and cell division. Mutation of seven amino acids in three conserved domains of mouse LPP-1 abolishes its activity. A glycosylation site was demonstrated between conserved Domains 1 and 2. LPP-1 is expressed in the plasma membrane, and the present results demonstrate the active site to be located on the outer surface (Zhang, 2000).
Sphingosine 1-phosphate, lysophosphatidic acid, and phosphatidic acid bind to G-protein-coupled receptors to stimulate intracellular signaling in mammalian cells. Lipid phosphate phosphatases (1, 1a, 2, and 3) are a group of enzymes that catalyze de-phosphorylation of these lipid agonists. It has been proposed that the lipid phosphate phosphatases exhibit ecto activity that may function to limit bioavailability of these lipid agonists at their receptors. This study shows that the stimulation of the p42/p44 mitogen-activated protein kinase pathway by sphingosine 1-phosphate, lysophosphatidic acid, and phosphatidic acid, all of which bind to G(i/o)-coupled receptors, is substantially reduced in human embyronic kidney 293 cells transfected with lipid phosphate phosphatases 1, 1a, and 2 but not 3. This was correlated with reduced basal intracellular phosphatidic acid and not ecto lipid phosphate phosphatase activity. These findings were supported by results showing that lipid phosphate phosphatases 1, 1a, and 2 also abrogate the stimulation of p42/p44 mitogen-activated protein kinase by thrombin, a peptide G(i/o)-coupled receptor agonist whose bioavailability at its receptor is not subject to regulation by the phosphatases. Furthermore, the lipid phosphate phosphatases have no effect on the stimulation of p42/p44 mitogen-activated protein kinase by other agents that do not use G-proteins to signal, such as serum factors and phorbol ester. Therefore, these findings show that the lipid phosphate phosphatases 1, 1a, and 2 may function to perturb G-protein-coupled receptor signaling per se rather than limiting bioavailability of lipid agonists at their respective receptors (Alderton, 2001).
Blood platelets play an essential role in ischemic heart disease and stroke contributing to acute thrombotic events by release of potent inflammatory agents within the vasculature. Lysophosphatidic acid (LPA) is a bioactive lipid mediator produced by platelets and found in the blood and atherosclerotic plaques. LPA receptors on platelets, leukocytes, endothelial cells, and smooth muscle cells regulate growth, differentiation, survival, motility, and contractile activity. Definition of the opposing pathways of synthesis and degradation that control extracellular LPA levels is critical to understanding how LPA bioactivity is regulated. Intact platelets and platelet membranes actively dephosphorylate LPA and the major enzyme responsible has been identified as lipid phosphate phosphatase 1 (LPP1). Localization of LPP1 to the platelet surface is increased by exposure to LPA. A novel receptor-inactive sn-3-substituted difluoromethylenephosphonate analog of phosphatidic acid that is a potent competitive inhibitor of LPP1 activity potentiates platelet aggregation and shape change responses to LPA and amplifies LPA production by agonist-stimulated platelets. These results identify LPP1 as a pivotal regulator of LPA signaling in the cardiovascular system. These findings are consistent with genetic and cell biological evidence implicating LPPs as negative regulators of lysophospholipid signaling and suggest that the mechanisms involve both attenuation of lysophospholipid actions at cell surface receptors and opposition of lysophospholipid production (Smyth, 2003).
Lipid phosphate phosphatases (LPPs) are integral membrane proteins with six transmembrane domains that act as ecto-enzymes dephosphorylating a variety of extracellular lipid phosphates. Using polarized MDCK cells stably expressing human LPP1 and LPP3, it was found that LPP1 is located exclusively at the apical surface whereas LPP3 is distributed mostly in the basolateral subdomain. A novel apical sorting signal was identified at the N-terminus of LPP1 composed of F(2)DKTRL(7). In the case of LPP3, a dityrosine motif present in the second cytoplasmic portion was identified as basolateral targeting signal. This work shows that LPP1 and LPP3 are equipped with distinct sorting signals that cause them to differentially localize to the apical vs. the basolateral subdomain, respectively (Jia, 2003).
Lipid phosphate phosphatase 1 (LPP-1) is presumed to regulate the balance between lipid phosphates and their dephosphorylated counterparts. The currently prevailing hypothesis based on in vitro studies proposes that LPP-1 should regulate phospholipid lipid growth factors and second messengers, including lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P), diacylglycerol (DAG), and phosphatidic acid (PA). To evaluate the role of LPP-1 in vivo, three transgenic lines were established. RT-PCR, Western blotting, and enzymatic activity measurement confirmed a copy number-dependent ubiquitous overexpression of LPP-1. PMA-stimulated PA production in immortalized LPP-1 fibroblasts led to an elevation in the accumulation of DAG without major changes in the phospholipid classes isolated from the liver. The LPP-1 phenotype showed reduced body size, birth weight, and abnormalities in fur growth, whereas histological abnormalities included significantly decreased number of hair follicles, disrupted hair structure, and a severely impaired spermatogenesis. Implantation of LPP-1 or wild-type embryos into pseudopregnant LPP-1 mothers yielded a reduced litter size. The plasma level of alanine-leucine aminotransferase was significantly elevated. Unexpectedly, plasma concentrations of the five major acyl-species of LPA were indistinguishable between wild-type and LPP-1 animals. In contrast with previous studies using plasmid-mediated overexpression in vitro, transgenic overexpression of LPP-1 did not affect ERK1/2 activation elicited by LPA, S1P, thrombin, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF), which was presumed to be a major signaling event regulated by LPP-1. Thus, transgenic overexpression of LPP-1 in mice elicited a number of unexpected phenotypic alterations without affecting several aspects of LPA signaling, which point to previously unappreciated mechanisms and roles of lipid phosphates in select organs (Yue, 2004).
LPA (lysophosphatidic acid), a potent bioactive phospholipid, elicits diverse cellular responses through activation of the G-protein-coupled receptors LPA1-LPA4. LPA-mediated signalling is partially regulated by LPPs (lipid phosphate phosphatases; LPP-1, -2 and -3) that belong to the phosphatase superfamily. This study addresses the role of LPPs in regulating LPA-mediated cell signalling and IL-8 (interleukin-8) secretion in HBEpCs (human bronchial epithelial cells). Reverse transcription-PCR and Western blotting revealed the presence and expression of LPP-1-3 in HBEpCs. Exogenous [3H]oleoyl LPA was hydrolysed to [3H]-mono-oleoylglycerol. Infection of HBEpCs with an adenoviral construct of human LPP-1 for 48 h enhanced the dephosphorylation of exogenous LPA by 2-3-fold compared with vector controls. Furthermore, overexpression of LPP-1 partially attenuated LPA-induced increases in the intracellular Ca2+ concentration, phosphorylation of IkappaB (inhibitory kappaB) and translocation of NF-kappaB (nuclear factor-kappaB) to the nucleus, and almost completely prevented IL-8 secretion. Infection of cells with an adenoviral construct of the mouse LPP-1 (R217K) mutant partially attenuated LPA-induced IL-8 secretion without altering LPA-induced changes in intracellular Ca2+ concentration, phosphorylation of IkappaB, NF-kappaB activation or IL-8 gene expression. These results identify LPP-1 as a key regulator of LPA signalling and IL-8 secretion in HBEpCs. Thus LPPs could represent potential targets in regulating leucocyte infiltration and airway inflammation (Zhao, 2005).
LPPs (lipid phosphate phosphatases) reduce the stimulation of the p42/p44 MAPK (p42/p44 mitogen-activated protein kinase) pathway by the GPCR (G-protein-coupled receptor) agonists S1P (sphingosine 1-phosphate) and LPA (lysophosphatidic acid) in serum-deprived HEK-293 cells. This can be blocked by pretreating HEK-293 cells with the caspase 3/7 inhibitor, Ac-DEVD-CHO [N-acetyl-Asp-Glu-Val-Asp-CHO (aldehyde)]. Therefore LPP2 and LPP3 appear to regulate the apoptotic status of serum-deprived HEK-293 cells. This was supported further by: (1) caspase 3/7-catalysed cleavage of PARP [poly(ADP-ribose) polymerase] was increased in serum-deprived LPP2-overexpressing compared with vector-transfected HEK-293 cells; and (2) serum-deprived LPP2- and LPP3-overexpressing cells exhibited limited intranucleosomal DNA laddering, which was absent in vector-transfected cells. Moreover, LPP2 reduces basal intracellular phosphatidic acid levels, whereas LPP3 decreases intracellular S1P in serum-deprived HEK-293 cells. LPP2 and LPP3 are constitutively co-localized with SK1 (sphingosine kinase 1) in cytoplasmic vesicles in HEK-293 cells. Moreover, LPP2 but not LPP3 prevents SK1 from being recruited to a perinuclear compartment upon induction of PLD1 (phospholipase D1) in CHO (Chinese-hamster ovary) cells. Taken together, these data are consistent with an important role for LPP2 and LPP3 in regulating an intracellular pool of PA and S1P respectively, that may govern the apoptotic status of the cell upon serum deprivation (Long, 2005).
LPPs (lipid phosphate phosphatases) are members of a family of enzymes that catalyse the dephosphorylation of lipid phosphates. The only known form of regulation of this family of enzymes is via de novo expression of LPP isoforms in response to growth factors. This study evaluated the effect of moderate increases in the expression of recombinant LPP1 on signal transduction by both G-protein-coupled receptors and receptor tyrosine kinases. Evidence is presented for a novel role of LPP1 in reducing PDGF (platelet-derived growth factor)- and lysophosphatidic acid-induced migration of embryonic fibroblasts. The overexpression of LPP1 inhibits cell migration by reducing the PDGF-induced activation of p42/p44 MAPK (mitogen-activated protein kinase). This appears to occur via a mechanism that involves the LPP1-induced down-regulation of typical PKC (protein kinase C) isoform(s), which are normally required for PDGF-induced activation of p42/p44 MAPK and migration. In this regard, DAG (diacylglycerol) levels are high and sustained in cells overexpressing LPP1, suggesting a dynamic interconversion of phosphatidic acid into DAG by LPP1. This may account for the effects of LPP1 on cell migration, as sustained DAG is known to down-regulate PKC isoforms in cells. Therefore the physiological changes in the expression levels of LPP1 might represent a heterologous desensitization mechanism for attenuating PKC-mediated signalling and regulation of cell migration (Long, 2006).
Lipid phosphates are potent mediators of cell signaling and control processes including development, cell migration and division, blood vessel formation, wound repair, and tumor progression. Lipid phosphate phosphatases (LPPs) regulate the dephosphorylation of lipid phosphates, thus modulating their signals and producing new bioactive compounds both at the cell surface and in intracellular compartments. Knock-down of endogenous LPP2 in fibroblasts delays cyclin A accumulation and entry into S-phase of the cell cycle. Conversely, overexpression of LPP2, but not a catalytically inactive mutant, causes premature S-phase entry, accompanied by premature cyclin A accumulation. At high passage, many LPP2 overexpressing cells arrest in G(2)/M and the rate of proliferation declines severely. This is accompanied by changes in proteins and lipids characteristic of senescence. Additionally, arrested LPP2 cells contain decreased lysophosphatidate concentrations and increased ceramide. These effects of LPP2 activity were not reproduced by overexpression or knock-down of LPP1 or LPP3. This work identifies a novel and specific role for LPP2 activity and bioactive lipids in regulating cell cycle progression (Morris, 2006).
Search PubMed for articles about Drosophila Lazaro
Alderton, F., et al. (2001). G-protein-coupled receptor stimulation of the p42/p44 mitogen-activated protein kinase pathway is attenuated by lipid phosphate phosphatases 1, 1a, and 2 in human embryonic kidney 293 cells. J. Biol. Chem. 276(16): 13452-60. 11278307
Brindley, D. N. and Waggoner, D. W. (1998). Mammalian lipid phosphate phosphohydrolases. J. Biol. Chem. 273(38): 24281-4. 9733709
Burnett, C. and Howard, K. (2003). Fly and mammalian lipid phosphate phosphatase isoforms differ in activity both in vitro and in vivo. EMBO Rep. 4(8): 793-9. 12856002
Burnett, C., Makridou, P., Hewlett, L. and Howard, K. (2004). Lipid phosphate phosphatases dimerise, but this interaction is not required for in vivo activity. BMC Biochem. 5:2. 14725715
Chyb, S., Raghu, P. and Hardie, R. C. (1999). Polyunsaturated fatty acids activate the Drosophila light-sensitive channels TRP and TRPL. Nature 397(6716): 255-9. 9930700
Estacion, M., Sinkins, W. G. and Schilling, W. P. (2001). Regulation of Drosophila transient receptor potential-like (TrpL) channels by phospholipase C-dependent mechanisms. J. Physiol. 530(Pt 1): 1-19. 11136854
Garcia-Murillas, I., Pettitt, T., Macdonald, E., Okkenhaug, H., Georgiev, P., Trivedi, D., Hassan, B., Wakelam, M. and Raghu, P. (2006). lazaro encodes a lipid phosphate phosphohydrolase that regulates phosphatidylinositol turnover during Drosophila phototransduction. Neuron 49(4): 533-46. 16476663
Georgiev, P., Garcia-Murillas, I., Ulahannan, D., Hardie, R. C. and Raghu, P. (2005). Functional INAD complexes are required to mediate degeneration in photoreceptors of the Drosophila rdgA mutant. J Cell Sci. 118(Pt 7): 1373-84. 15755798
Hardie, R. C., et al. (2001). Calcium influx via TRP channels is required to maintain PIP2 levels in Drosophila photoreceptors. Neuron 30(1): 149-59. 11343651
Hardie, R. C. (2003). Regulation of TRP channels via lipid second messengers. Annu. Rev. Physiol. 65: 735-59. 12560473
Hooks, S. B., Ragan, S. P. and Lynch, K. R. (1998). Identification of a novel human phosphatidic acid phosphatase type 2 isoform. FEBS Lett. 427(2): 188-92. 9607309
Inoue, H., Yoshioka, T. and Hotta, Y. (1989). Diacylglycerol kinase defect in a Drosophila retinal degeneration mutant rdgA. J. Biol. Chem. 264(10): 5996-6000. 2538432
Jamal, Z., Martin, A., Gomez-Munoz, A. and Brindley, D. N. (1991). Plasma membrane fractions from rat liver contain a phosphatidate phosphohydrolase distinct from that in the endoplasmic reticulum and cytosol. J. Biol. Chem. 266(5): 2988-96. 1993672
Jasinska, R., et al. (1999). Lipid phosphate phosphohydrolase-1 degrades exogenous glycerolipid and sphingolipid phosphate esters. Biochem. J. 340: 677-86. 10359651
Jia, Y. J., et al. (2003). Differential localization of lipid phosphate phosphatases 1 and 3 to cell surface subdomains in polarized MDCK cells. FEBS Lett. 552(2-3): 240-6. 14527693
Kai, M., et al. (1996). Identification and cDNA cloning of 35-kDa phosphatidic acid phosphatase (type 2) bound to plasma membranes. Polymerase chain reaction amplification of mouse H2O2-inducible hic53 clone yielded the cDNA encoding phosphatidic acid phosphatase. J. Biol. Chem. 271(31): 18931-8. 8702556
Kwon, Y. and Montell, C. (2006). Dependence on the Lazaro phosphatidic acid phosphatase for the maximum light response. Curr. Biol. 16(7): 723-9. 16513351
Loewen, C. J., et al. (2004). Phospholipid metabolism regulated by a transcription factor sensing phosphatidic acid. Science 304(5677): 1644-7. 15192221
Long, J., et al. (2005). Regulation of cell survival by lipid phosphate phosphatases involves the modulation of intracellular phosphatidic acid and sphingosine 1-phosphate pools. Biochem. J. 391(Pt 1): 25-32. 15960610
Long, J. S., et al. (2006). Lipid phosphate phosphatase-1 regulates lysophosphatidic acid- and platelet-derived-growth-factor-induced cell migration. Biochem J. 394(Pt 2): 495-500. 16356167
Lucas, P., Ukhanov, K., Leinders-Zufall, T. and Zufall, F. (2003). A diacylglycerol-gated cation channel in vomeronasal neuron dendrites is impaired in TRPC2 mutant mice: mechanism of pheromone transduction. Neuron 40(3): 551-61. 14642279
Morris, K. E., Schang, L. M. and Brindley, D. N. (2006). Lipid phosphate phosphatase-2 activity regulates S-phase entry of the cell cycle in Rat2 fibroblasts. J. Biol. Chem. 281(14): 9297-306. 16467304
Raghu, P., Usher, K., Jonas, S., Chyb, S., Polyanovsky, A. and Hardie, R. C. (2000). Constitutive activity of the light-sensitive channels TRP and TRPL in the Drosophila diacylglycerol kinase mutant, rdgA. Neuron 26(1): 169-79. 10798401
Roberts, R., Sciorra, V. A. and Morris, A. J. (1998). Human type 2 phosphatidic acid phosphohydrolases. Substrate specificity of the type 2a, 2b, and 2c enzymes and cell surface activity of the 2a isoform. J. Biol. Chem. 273(34): 22059-67. 9705349
Smyth, S. S., et al. (2003). Lipid phosphate phosphatases regulate lysophosphatidic acid production and signaling in platelets: studies using chemical inhibitors of lipid phosphate phosphatase activity. J. Biol. Chem. 278(44): 43214-23. 12909631
Yue, J., Yokoyama, K., Balazs, L., Baker, D. L., Smalley, D., Pilquil, C., Brindley, D. N. and Tigyi, G. (2004). Mice with transgenic overexpression of lipid phosphate phosphatase-1 display multiple organotypic deficits without alteration in circulating lysophosphatidate level. Cell Signal. 16(3): 385-99. 14687668
Zhang, Q. X., et al. (2000). Identification of structurally important domains of lipid phosphate phosphatase-1: implications for its sites of action. Biochem. J. 345 Pt 2: 181-4. 10620492
Zhao, Y., et al. (2005). Lipid phosphate phosphatase-1 regulates lysophosphatidic acid-induced calcium release, NF-kappaB activation and interleukin-8 secretion in human bronchial epithelial cells. Biochem. J. 385(Pt 2): 493-502. 15461590
date revised: 1 October 2006
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