Phosphatidic Acid Synthesis Research Articles

Clickable Substrate Mimics Enable Imaging of Phospholipase D Activity.

Chemical imaging techniques have played instrumental roles in dissecting the spatiotemporal regulation of signal transduction pathways. Phospholipase D (PLD) enzymes affect cell signaling by producing the pleiotropic lipid second messenger phosphatidic acid via hydrolysis of phosphatidylcholine. It remains a mystery how this one lipid signal can cause such diverse physiological and pathological signaling outcomes, due in large part to a lack of suitable tools for visualizing the spatial and temporal dynamics of its production within cells. Here, we report a chemical method for imaging phosphatidic acid synthesis by PLD enzymes in live cells. Our approach capitalizes upon the enzymatic promiscuity of PLDs, which we show can accept azidoalcohols as reporters in a transphosphatidylation reaction. The resultant azidolipids are then fluorescently tagged using the strain-promoted azide–alkyne cycloaddition, enabling visualization of cellular membranes bearing active PLD enzymes. Our method, termed IMPACT (Imaging Phospholipase D Activity with Clickable Alcohols via Transphosphatidylation), reveals pools of basal and stimulated PLD activities in expected and unexpected locations. As well, we reveal a striking heterogeneity in PLD activities at both the cellular and subcellular levels. Collectively, our studies highlight the importance of using chemical tools to directly visualize, with high spatial and temporal resolution, the subset of signaling enzymes that are active.

Nuclear Localization of Diacylglycerol Kinase Alpha in K562 Cells Is Involved in Cell Cycle Progression.

Phosphatidylinositol (PI) signaling is an essential regulator of cell motility and proliferation. A portion of PI metabolism and signaling takes place in the nuclear compartment of eukaryotic cells, where an array of kinases and phosphatases localize and modulate PI. Among these, Diacylglycerol Kinases (DGKs) are a class of phosphotransferases that phosphorylate diacylglycerol and induce the synthesis of phosphatidic acid. Nuclear DGKalpha modulates cell cycle progression, and its activity or expression can lead to changes in the phosphorylated status of the Retinoblastoma protein, thus, impairing G1/S transition and, subsequently, inducing cell cycle arrest, which is often uncoupled with apoptosis or autophagy induction. Here we report for the first time not only that the DGKalpha isoform is highly expressed in the nuclei of human erythroleukemia cell line K562, but also that its nuclear activity drives K562 cells through the G1/S transition during cell cycle progression. J. Cell. Physiol. 232: 2550-2557, 2017. © 2016 Wiley Periodicals, Inc.

Phosphorylation of the glycerol 3‐phosphate acyltransferase Gpt2 regulates the timing of TAG mobilization upon growth resumption

Glycerol‐3‐phosphate acyltransferases (GPATs) catalyze the first step of glycerol‐3‐phosphate acylation at the sn‐1 position producing lyso‐phosphatidic acid. This is the committed and rate limiting step in de‐novo synthesis of phosphatidic acid, the key intermediate in the glycerophospholipids and triacylglycerols (TAG) biosynthetic pathways. Two GPATs have been identified in S cerevisiae, Gpt2 and Sct1. Gpt2 (but not Sct1) becomes essential when cells are grown with oleic acid as the only carbon source. While oleic acid induced large accumulation of lipid droplets (LDs) in wild‐type cells, cells lacking GPT2 failed to accumulate these storage compartments of TAG and other neutral lipids. This lead to a growth defect and significant reduction of cell viability. We were intrigued by the fact that the intracellular distribution of Gpt2 and its phosphorylation status changed under these growth conditions compared to growth on glucose. We identified several phosphoresidues in Gpt2 by mass spectrometry analysis, localized to a serine rich region in the carboxy‐terminus of the protein. To investigate the role of phosphorylation in the regulation of Gpt2, the phosphorylation status of the protein was manipulated by introducing Ser to Ala or to Asp mutations in the GPT2 locus. Importantly, all phosphorylation mutants were expressed at physiological levels and could support life in the absence of Sct1, indicating these mutants are functional. During characterization studies of these mutants, we have discovered that changes in the phosphorylation status of Gpt2 affect its ER distribution, with the protein concentrating at points of contact with lipid droplets in actively growing cells. Staining of neutral lipids with BODIPY™ 493/503 or Nile red, indicated that specific serine to alanine mutations result in larger lipid droplets compare to those in wild type cells. We determined that this phenotype was indeed accompanied by a significant increase in TAG levels, mostly notorious during exponential growth. The differences in LD size and TAG accumulation became milder during stationary phase. SDS‐PAGE analysis of wild type Gpt2 indicated that the phosphorylation pattern of the protein changes in stationary phase, with the appearance of a faster migrating band that resembles that observed for the S to A mutants. Interestingly, this second band vanishes upon growth resumption, coinciding with TAG mobilization and lipolysis. Microscopy inspection of S to A phosphomutant strains showed a clear delay in LD size reduction up to 5 hours after resuspension of 5‐days stationary phase cells in fresh medium. This was also accompanied by a delay in TAG consumption. These results highlight the role of Gpt2 in coordinating synthesis and glycerolipid turnover in response to nutrients. Altogether, it appears that phosphorylation regulates Gpt2 interaction with lipid droplets, allowing TAG mobilization upon growth resumption from a quiescent state.Support or Funding InformationThis work has been financially supported by NSERC (to V.Z. and B.S.) and the Austrian Science Fund (FWF; Project P21251 to K.A.)

Lipid sensing by mTOR complexes via de novo synthesis of phosphatidic acid

mTOR, the mammalian target of rapamycin, integrates growth factor and nutrient signals to promote a transformation from catabolic to anabolic metabolism, cell growth, and cell cycle progression. Phosphatidic acid (PA) interacts with the FK506-binding protein-12-rapamycin-binding (FRB) domain of mTOR, which stabilizes both mTOR complexes: mTORC1 and mTORC2. We report here that mTORC1 and mTORC2 are activated in response to exogenously supplied fatty acids via the de novo synthesis of PA, a central metabolite for membrane phospholipid biosynthesis. We examined the impact of exogenously supplied fatty acids on mTOR in KRas-driven cancer cells, which are programmed to utilize exogenous lipids. The induction of mTOR by oleic acid was dependent upon the enzymes responsible for de novo synthesis of PA. Suppression of the de novo synthesis of PA resulted in G1 cell cycle arrest. Although it has long been appreciated that mTOR is a sensor of amino acids and glucose, this study reveals that mTOR also senses the presence of lipids via production of PA.

Nuclear DGKα regulates cell cycle progression in K562 cells

The existence of an independent nuclear inositide pathway distinct from the cytoplasmic one has been demonstrated in different physiological systems and in diseases (1). Phosphatidylinositols (PIs) play an important role in nuclear function regulation and behave differently from their counterparts in the cytoplasm. The autonomous nuclear PI cycle in eukaryotic cells is involved in different regulation processes, from cell proliferation to differentiation and many others (2). At nuclear level an array of kinases and phosphatases can modulate PIs. Among these, Diacylglycerol Kinases (DGKs) are a class of phosphotransferases that phosphorylate diacylglycerol (DAG) and induce the synthesis of phosphatidic acid. We Investigated DGKα localization and function in human erythroleukemia cell line K562. Synchronization experiments at different cell cycle checkpoints showed an important expression of DGKα in the nuclear fraction of this cell model, slightly peaking at G2/M. This suggested that DGKα might have a function in nuclear signaling. In particular, nuclear DGKα expression can modulate cell cycle progression, leading to changes in the phosphorylated status of the Retinoblastoma protein (pRb), thus, regulating G1/S transition: DGKα silencing or downregulation leads to impaired G1/S transition and its overexpression leads to S phase progression. The molecular mechanism by which nuclear DGKα controls pRb phosphorylation and therefore cell cycle regulation in K562 cell line are still unclear. Further studies are needed to better understand the role of DGKα in relation to other pivotal PIs involved in cell cycle regulation in the hematopoietic system.

A Chemoenzymatic Strategy for Imaging Cellular Phosphatidic Acid Synthesis.

Phosphatidic acid (PA) is a potent lipid secondary messenger, the synthesis of which is tightly regulated in both space and time. Established tools for detecting PA involve ex vivo analysis and do not provide information on the subcellular locations where this lipid is synthesized. Here, a chemoenzymatic strategy for imaging sites of cellular PA synthesis by phospholipase D (PLD) enzymes is reported. PLDs were found to be able to catalyze phospholipid head-group exchange with alkynols to generate alkyne-labeled PA analogues within cells. Subsequent fluorophore tagging through Cu-catalyzed azide-alkyne cycloaddition enabled both visualization by fluorescence microscopy and quantification by HPLC. Our studies revealed several intracellular sites of PLD-mediated PA synthesis. We envision applications of this approach to dissect PA-dependent signaling pathways, image PLD activity in disease, and remodel intracellular membranes with new functionality.

A Chemoenzymatic Strategy for Imaging Cellular Phosphatidic Acid Synthesis

AbstractPhosphatidic acid (PA) is a potent lipid secondary messenger, the synthesis of which is tightly regulated in both space and time. Established tools for detecting PA involve ex vivo analysis and do not provide information on the subcellular locations where this lipid is synthesized. Here, a chemoenzymatic strategy for imaging sites of cellular PA synthesis by phospholipase D (PLD) enzymes is reported. PLDs were found to be able to catalyze phospholipid head‐group exchange with alkynols to generate alkyne‐labeled PA analogues within cells. Subsequent fluorophore tagging through Cu‐catalyzed azide–alkyne cycloaddition enabled both visualization by fluorescence microscopy and quantification by HPLC. Our studies revealed several intracellular sites of PLD‐mediated PA synthesis. We envision applications of this approach to dissect PA‐dependent signaling pathways, image PLD activity in disease, and remodel intracellular membranes with new functionality.

Organelle dynamics: Sizing mitochondria with phospholipids.

The lipid composition of the mitochondrial membrane, in particular, synthesis of phosphatidic acid, regulates mitochondrial size by modulating fusion and fission cycles.

Phosphatidic acid induces EHD3-containing membrane tubulation and is required for receptor recycling

EHD3 is localized on the tubular structures of early endosomes, and it regulates their trafficking pathway. However, the regulatory mechanism of EHD3-containing tubular structures remains poorly understood. An in vitro liposome co-sedimentation assay revealed that EHD3 interacted with phosphatidic acid through its helical domain and this interaction induced liposomal tubulations. Additionally, inhibiting phosphatidic acid synthesis with diacylglycerol kinase inhibitor or lysophosphatidic acid acyltransferase inhibitor significantly reduced the number of EHD3-containing tubules and impaired their trafficking from early endosomes. These results suggest that EHD3 and phosphatidic acid cooperatively regulate membrane deformation and trafficking from early endosomes.

Phosphatidic acid synthesis, octadecanoic pathway and fatty acids content as lipid markers of exogeneous salicylic acid-induced elicitation in wheat.

Activators of plant defence responses against pathogens are a potential alternative to fungicides, and the well-known resistance inducer salicylic acid (SA) protects wheat (Triticum aestivum L.) against powdery mildew. The aim of our work was to investigate through biochemical and molecular approaches whether lipid metabolism alteration could be considered as a characteristic feature of induced resistance in wheat upon SA infiltration. Expression levels of lox, PI-PLC2 and ltp genes encoding for a lipoxygenase (LOX), a phospholipase C2 and a lipid-transfer protein, respectively, were investigated. Increase of phosphatidic acid (PA) content 48h after SA infiltration in wheat leaves, upregulation of PI-PLC2 gene expression and increased diacylglycerol content were recorded, indicating the involvement of the PLC pathway in the PA synthesis. The wheat octadecanoid pathway was shown to be highly responsive to SA infiltration through simultaneous increases in lox gene expression and LOX activity, as well as a reduction in the content of linolenic acid. Changes in several FA contents and increases of the ltp gene expression were also recorded during the latest hours after SA infiltration. The status of lipid metabolism, as well as the connections between its components as markers of SA-induced resistance in wheat, are discussed.

Regulation of Yeast Glycerol‐3‐Phosphate Acyltransferase Gpt2p/Gat1p Through Phosphorylation

Glycerol‐3‐phosphate acyltransferases (GPAT) catalyze the acylation of glycerol‐3‐phosphate, the first and rate limiting reaction in de novo synthesis of phosphatidic acid (PA). GPAT redundancy led to the hypothesis that isoenzymes are forming different PA pools which supply different pathways. In the model organism S. cerevisiae two GPATs have been identified, Gpt2 and Sct1. Most recently we demonstrated the important role of Gpt2 (but not Sct1) in cells challenged by oleic acid as the only carbon source. Whereas growth in oleic acid normally induces lipid particle accumulation, cells lacking GPT2 failed to accumulate these neutral lipids storage compartments, exhibited a strong growth defect, and cell viability was reduced. The fact that the intracellular distribution of Gpt2 and its phosphorylation status changed under this growth condition attracted our interest. We have identified 3 highly conserved phosphorylation sites in Gpt2 by mass spectrometry, localized to a Ser rich region in the carboxy‐end. Ser to Ala triple mutations as well as deletion of the Ser rich region, were introduced in the yeast genome to investigate the role of phosphorylation in the regulation of Gpt2. Importantly, both Gpt2 mutant forms retain enzymatic activity sufficient to support life of cells lacking Sct1. Microscopic inspection of phosphomutants revealed changes in lipid droplet formation. In line, cells overproducing these mutants exacerbated this phenotype, suggesting that lack of Gpt2 phosphorylation leads to abnormal accumulation of lipid droplets and carbon source consumption. The role of the Gpt2 serine‐rich carboxy‐end in mediating protein‐protein interactions is currently investigated

Special issue: from membranes to pathologies.

Lipin 2 is a phosphatidic acid phosphatase (PAP) responsible for the penultimate step of triglyceride synthesis and dephosphorylation of phosphatidic acid (PA) to generate diacylglycerol. The lipin family of PA phosphatases is composed of lipins 1–3, which are members of the conserved haloacid dehalogenase superfamily. Although genetic alteration of LPIN2 in humans is known to cause Majeed syndrome, little is known about the biochemical regulation of its PAP activity. Here, in an attempt to gain a better general understanding of the biochemical nature of lipin 2, we have performed kinetic and phosphorylation analyses. We provide evidence that lipin 2, like lipin 1, binds PA via the electrostatic hydrogen bond switch mechanism but has a lower rate of catalysis. Like lipin 1, lipin 2 is highly phosphorylated, and we identified 15 phosphosites. However, unlike lipin 1, the phosphorylation of lipin 2 is not induced by insulin signaling nor is it sensitive to inhibition of the mammalian target of rapamycin. Importantly, phosphorylation of lipin 2 does not negatively regulate either membrane binding or PAP activity. This suggests that lipin 2 functions as a constitutively active PA phosphatase in stark contrast to the high degree of phosphorylation-mediated regulation of lipin 1. This knowledge of lipin 2 regulation is important for a deeper understanding of how the lipin family functions with respect to lipid synthesis and, more generally, as an example of how the membrane environment around PA can influence its effector proteins.

Lipin 2 Binds Phosphatidic Acid by the Electrostatic Hydrogen Bond Switch Mechanism Independent of Phosphorylation

Lipin 2 is a phosphatidic acid phosphatase (PAP) responsible for the penultimate step of triglyceride synthesis and dephosphorylation of phosphatidic acid (PA) to generate diacylglycerol. The lipin family of PA phosphatases is composed of lipins 1-3, which are members of the conserved haloacid dehalogenase superfamily. Although genetic alteration of LPIN2 in humans is known to cause Majeed syndrome, little is known about the biochemical regulation of its PAP activity. Here, in an attempt to gain a better general understanding of the biochemical nature of lipin 2, we have performed kinetic and phosphorylation analyses. We provide evidence that lipin 2, like lipin 1, binds PA via the electrostatic hydrogen bond switch mechanism but has a lower rate of catalysis. Like lipin 1, lipin 2 is highly phosphorylated, and we identified 15 phosphosites. However, unlike lipin 1, the phosphorylation of lipin 2 is not induced by insulin signaling nor is it sensitive to inhibition of the mammalian target of rapamycin. Importantly, phosphorylation of lipin 2 does not negatively regulate either membrane binding or PAP activity. This suggests that lipin 2 functions as a constitutively active PA phosphatase in stark contrast to the high degree of phosphorylation-mediated regulation of lipin 1. This knowledge of lipin 2 regulation is important for a deeper understanding of how the lipin family functions with respect to lipid synthesis and, more generally, as an example of how the membrane environment around PA can influence its effector proteins.

SEC14 phospholipid transfer protein is involved in lipid signaling-mediated plant immune responses in Nicotiana benthamiana.

We previously identified a gene related to the SEC14-gene phospholipid transfer protein superfamily that is induced in Nicotiana benthamiana (NbSEC14) in response to infection with Ralstonia solanacearum. We here report that NbSEC14 plays a role in plant immune responses via phospholipid-turnover. NbSEC14-silencing compromised expression of defense–related PR-4 and accumulation of jasmonic acid (JA) and its derivative JA-Ile. Transient expression of NbSEC14 induced PR-4 gene expression. Activities of diacylglycerol kinase, phospholipase C and D, and the synthesis of diacylglycerol and phosphatidic acid elicited by avirulent R. solanacearum were reduced in NbSEC14-silenced plants. Accumulation of signaling lipids and activation of diacylglycerol kinase and phospholipases were enhanced by transient expression of NbSEC14. These results suggest that the NbSEC14 protein plays a role at the interface between lipid signaling-metabolism and plant innate immune responses.

A late liver stage rodent malaria parasite (left) is surrounded by a parasitophorous vacuole membrane (red) and has a complex apicoplast (green). Parasites lacking genes for apicoplast-targeted phosphatidic acid synthesis are attenuated (right). For furth

A late liver stage rodent malaria parasite (left) is surrounded by a parasitophorous vacuole membrane (red) and has a complex apicoplast (green). Parasites lacking genes for apicoplast-targeted phosphatidic acid synthesis are attenuated (right). For furth

Enzymes involved in plastid‐targeted phosphatidic acid synthesis are essential for Plasmodium yoelii liver‐stage development

Malaria parasites scavenge nutrients from their host but also harbour enzymatic pathways for de novo macromolecule synthesis. One such pathway is apicoplast-targeted type II fatty acid synthesis, which is essential for late liver-stage development in rodent malaria. It is likely that fatty acids synthesized in the apicoplast are ultimately incorporated into membrane phospholipids necessary for exoerythrocytic merozoite formation. We hypothesized that these synthesized fatty acids are being utilized for apicoplast-targeted phosphatidic acid synthesis, the phospholipid precursor. Phosphatidic acid is typically synthesized in a three-step reaction utilizing three enzymes: glycerol 3-phosphate dehydrogenase, glycerol 3-phosphate acyltransferase and lysophosphatidic acid acyltransferase. The Plasmodium genome is predicted to harbour genes for both apicoplast- and cytosol/endoplasmic reticulum-targeted phosphatidic acid synthesis. Our research shows that apicoplast-targeted Plasmodium yoelii glycerol 3-phosphate dehydrogenase and glycerol 3-phosphate acyltransferase are expressed only during liver-stage development and deletion of the encoding genes resulted in late liver-stage growth arrest and lack of merozoite differentiation. However, the predicted apicoplast-targeted lysophosphatidic acid acyltransferase gene was refractory to deletion and was expressed solely in the endoplasmic reticulum throughout the parasite life cycle. Our results suggest that P. yoelii has an incomplete apicoplast-targeted phosphatidic acid synthesis pathway that is essential for liver-stage maturation.

The Role of Diacylglycerol Kinase ζ and Phosphatidic Acid in the Mechanical Activation of Mammalian Target of Rapamycin (mTOR) Signaling and Skeletal Muscle Hypertrophy

The activation of mTOR signaling is essential for mechanically induced changes in skeletal muscle mass, and previous studies have suggested that mechanical stimuli activate mTOR (mammalian target of rapamycin) signaling through a phospholipase D (PLD)-dependent increase in the concentration of phosphatidic acid (PA). Consistent with this conclusion, we obtained evidence which further suggests that mechanical stimuli utilize PA as a direct upstream activator of mTOR signaling. Unexpectedly though, we found that the activation of PLD is not necessary for the mechanically induced increases in PA or mTOR signaling. Motivated by this observation, we performed experiments that were aimed at identifying the enzyme(s) that promotes the increase in PA. These experiments revealed that mechanical stimulation increases the concentration of diacylglycerol (DAG) and the activity of DAG kinases (DGKs) in membranous structures. Furthermore, using knock-out mice, we determined that the ζ isoform of DGK (DGKζ) is necessary for the mechanically induced increase in PA. We also determined that DGKζ significantly contributes to the mechanical activation of mTOR signaling, and this is likely driven by an enhanced binding of PA to mTOR. Last, we found that the overexpression of DGKζ is sufficient to induce muscle fiber hypertrophy through an mTOR-dependent mechanism, and this event requires DGKζ kinase activity (i.e. the synthesis of PA). Combined, these results indicate that DGKζ, but not PLD, plays an important role in mechanically induced increases in PA and mTOR signaling. Furthermore, this study suggests that DGKζ could be a fundamental component of the mechanism(s) through which mechanical stimuli regulate skeletal muscle mass.

The Coffin-Lowry Syndrome-Associated Protein RSK2 Regulates Neurite Outgrowth through Phosphorylation of Phospholipase D1 (PLD1) and Synthesis of Phosphatidic Acid

More than 80 human X-linked genes have been associated with mental retardation and deficits in learning and memory. However, most of the identified mutations induce limited morphological alterations in brain organization and the molecular bases underlying neuronal clinical features remain elusive. We show here that neurons cultured from mice lacking ribosomal S6 kinase 2 (Rsk2), a model for the Coffin-Lowry syndrome (CLS), exhibit a significant delay in growth in a similar way to that shown by neurons cultured from phospholipase D1 (Pld1) knock-out mice. We found that gene silencing of Pld1 or Rsk2 as well as acute pharmacological inhibition of PLD1 or RSK2 in PC12 cells strongly impaired neuronal growth factor (NGF)-induced neurite outgrowth. Expression of a phosphomimetic PLD1 mutant rescued the inhibition of neurite outgrowth in PC12 cells silenced for RSK2, revealing that PLD1 is a major target for RSK2 in neurite formation. NGF-triggered RSK2-dependent phosphorylation of PLD1 led to its activation and the synthesis of phosphatidic acid at sites of neurite growth. Additionally, total internal reflection fluorescence microscopy experiments revealed that RSK2 and PLD1 positively control fusion of tetanus neurotoxin insensitive vesicle-associated membrane protein (TiVAMP)/VAMP-7 vesicles at sites of neurite outgrowth. We propose that the loss of function mutations in RSK2 that leads to CLS and neuronal deficits are related to defects in neuronal growth due to impaired RSK2-dependent PLD1 activity resulting in a reduced vesicle fusion rate and membrane supply.

Abstract 2470: Identification and evaluation of small molecule inhibitors of lysophosphatidic acid acyltransferase-beta.

Abstract Abstract: Lysophosphatidic acid acyltransferase-beta (LPAAT-β) catalyzes the production of phosphatidic acid (PA) from lysophosphatidic acid (LPA). PA is a lipid cofactor that contributes to the activation of c-Raf, BRAF, mTOR and PKC-ζ. LPAAT-β expression is a prognostic factor in gynecologic malignancies and is being investigated as a therapeutic target in a variety of tumor types. We previously reported that knockdown of LPAAT-β by siRNA led to a significant inhibition of both anchorage-dependent and -independent growth in pancreatic cancer cell lines. Having thus established LPAAT-β as a valid therapeutic target in pancreatic cancer, we screened small molecule libraries for compounds which block LPAAT-β enzymatic activity in vitro. Methods: We utilized a 96-well plate screening assay for LPAAT-β enzymatic activity modified from Aguado and Campbell, J Biol Chem 273(7):4096-4105, 1998. Briefly, compounds were introduced into a mixture containing full-length, recombinant, baculovirus-produced LPAAT-β (Blue Sky Biotechnology), oleoyl-LPA, and oleoyl coenzyme A. The ability of the compound to inhibit the enzyme activity was assayed by the measurement of the reaction of the thiol group of the released coenzyme A with the DTNB, resulting in an increase in the optical density of the sample when read at an absorbance of 413 nm. To rule out non-specific compounds identified from the DTNB plate assay, we confirmed activity using a secondary thin layer chromatography (TLC) assay that directly measures the ability of LPAAT-β to catalyze the synthesis of PA from oleoyl-LPA and a fluorochrome-labeled (NBD) palmitoyl coenzyme A. Results: We screened a total of ten libraries containing 11204 compounds, and achieved a 1.2% hit rate for inhibition of >50% at the tested concentration (100μM for nine libraries; 10μM for one library). Further testing identified several compounds with IC50s ranging from 0.1-15 μM. One hit identified with an IC50 of 100 nM was chosen for further investigation and for synthesis of analogs to optimize potency and undertake SAR. We verified the activity of this hit and its analogs against LPAAT-β by determining their inhibitory activity of the synthesis of NBD-labeled PA in a TLC assay. Finally, we demonstrated that the best analog from this group inhibited the proliferation of several pancreatic cancer cell lines. Conclusions: We have developed a library of compounds which show inhibitory activity against LPAAT-β in a screening assay, and have confirmed their ability to inhibit LPAAT-β directly in a TLC assay which monitors the production of PA. These compounds show low micromolar activity against LPAAT-β in vitro, and micromolar activity against the proliferation of pancreatic cancer cell lines in culture. Citation Format: Michelle A. Blaskovich, Harsukh Gevariya, Nicholas J. Lawrence, Saïd Sebti, Gregory Springett. Identification and evaluation of small molecule inhibitors of lysophosphatidic acid acyltransferase-beta. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2470. doi:10.1158/1538-7445.AM2013-2470

The Coffin‐Lowry syndrome‐associated protein RSK2 regulates neurite outgrowth through phosphorylation of PLD1 and synthesis of phosphatidic acid, a membrane curving fusogenic lipid.

Most of the mutations associated with mental retardation (MR) induce limited visible morphological alterations in brain organization and the molecular bases underlying the neuronal clinical features remain elusive. We show here that neurons cultured from mice deficient for ribosomal S6 kinase 2 (Rsk2), a model for the Coffin‐Lowry syndrome (CLS), exhibit a significant delay in development in a pattern very similar to that shown by neurons cultured from phospholipase D1 (Pld1) knockout mice. We found that gene silencing of Pld1 or Rsk2 as well as acute pharmacological inhibition of PLD1 or RSK2 strongly impaired neuronal growth factor (NGF)‐induced neurite outgrowth from PC12 cells. NGF triggered RSK2‐dependent phosphorylation of PLD1 leading to the synthesis of phosphatidic acid at the site of neurite growth. NGF‐induced neurite outgrowth and VAMP‐7 vesicle exocytosis was severely inhibited in PC12 cells silenced for RSK2 expression. However, expression of a phosphomimetic PLD1 mutant or provision of lysophosphatidic acid, an inverted cone‐shaped lipid that favors membrane curvature were able to rescue the growth defect in RSK2 silenced cells, revealing that PLD1 is the major target of RSK2 in neurite formation. We propose that the loss of function mutations in RSK2 that lead to CLS and neuronal deficits are related to defects in neuronal growth due to impaired RSK2‐dependent PLD1 activity.