Depletion of phosphatidylinositol 4-phosphate at the Golgi translocates K-Ras to mitochondria.

Ras proteins are small GTPases localized to the plasma membrane (PM), which regulate cellular proliferation, apoptosis and differentiation. After a series of post-translational modifications, H-Ras and N-Ras traffic to the PM from the Golgi via the classical exocytic pathway, but the exact mechanism of K-Ras trafficking to the PM from the ER is not fully characterized. ATP5G1 (also known as ATP5MC1) is one of the three proteins that comprise subunit c of the F0 complex of the mitochondrial ATP synthase. In this study, we show that overexpression of the mitochondrial targeting sequence of ATP5G1 perturbs glucose metabolism, inhibits oncogenic K-Ras signaling, and redistributes phosphatidylserine (PtdSer) to mitochondria and other endomembranes, resulting in K-Ras translocation to mitochondria. Also, it depletes phosphatidylinositol 4-phosphate (PI4P) at the Golgi. Glucose supplementation restores PtdSer and K-Ras PM localization and PI4P at the Golgi. We further show that inhibition of the Golgi-localized PI4-kinases (PI4Ks) translocates K-Ras, and PtdSer to mitochondria and endomembranes, respectively. We conclude that PI4P at the Golgi regulates the PM localization of PtdSer and K-Ras.This article has an associated First Person interview with the first author of the paper.

D-series Resolvins activate Phospholipase D in phagocytes during inflammation and resolution.

A successful acute inflammatory response results in the elimination of infectious agents by neutrophils and monocytes, followed by resolution and repair through tissue-resident and recruited macrophages. Resolvins (D-series and E-series) are pro-resolving lipid mediators involved in resolution and tissue repair, whose intracellular signaling remains of interest. Here, we report that D-series resolvins (RvD1- RvD5) activate phospholipase D (PLD), a ubiquitously expressed membrane lipase enzyme activity in modulating phagocyte functions. The mechanism for PLD-mediated actions of Resolvin-D5 (RvD5) in polarizing macrophages (M1-like toward M2-like) was found to be two-pronged: (a) RvD5 inhibits post-transcriptional modifications, by miRs and 3'exonucleases that process PLD2 mRNA, thus increasing PLD2 expression and activity; and (b) RvD5 enhances PLD2-S6Kinase signaling required for membrane expansion and efferocytosis. In an in vivo model of second organ reflow injury, we found that RvD5 did not reduce lung neutrophil myeloperoxidase levels in PLD2-/- mice compared to WT and PLD1-/- mice, confirming a novel role of PLD2 as the isoform in RvD5-mediated resolution processes. These results demonstrate that RvD5-PLD2 are attractive targets for therapeutic interventions in vascular inflammation such as ischemia-reperfusion injury and cardiovascular diseases.

A non-mitotic role for Aurora kinase A as a direct activator of cell migration upon interaction with PLD, FAK and Src.

Timely activation of Aurora kinase A (AURA, also known as AURKA) is vital for centrosome formation and the progression of mitosis. Nonetheless, it is still unclear if and when other cellular functions are activated by AURA. We report here that Src phosphorylates and activates AURA at T288, and AURA also activates focal adhesion kinase (FAK, also known as PTK2), leading to initiation of cell movement. An additional and new way by which AURA is regulated, is by phospholipase D2 (PLD2), which causes AURA activation. In addition, AURA phosphorylates PLD, so both proteins engage in a positive reinforcement loop. AURA and PLD2 form a protein­protein complex and colocalize to cytoplasmic regions in cells. The reason why PLD activates AURA is because of the production of phosphatidic acid by the lipase, which binds directly to AURA, with the region E171­E211 projected to be a phosphatidic-acid-binding pocket. Furthermore, this direct interaction with phosphatidic acid enhances tubulin polymerization and cooperates synergistically with AURA, FAK and Src in yielding a fully effectual cellular migration. Thus, Src and FAK, and PLD and phosphatidic acid are new upstream regulators of AURA that mediate its role in the non-mitotic cellular function of cell migration.

S6K is a morphogenic protein with a mechanism involving Filamin-A phosphorylation and phosphatidic acid binding.

Change of cell shape in vivo plays many roles that are central to life itself, such as embryonic development, inflammation, wound healing, and pathologic processes such as cancer metastasis. Nonetheless, the spatiotemporal mechanisms that control the concerted regulation of cell shape remain understudied. Here, we show that ribosomal S6K, which is normally considered a protein involved in protein translation, is a morphogenic protein. Its presence in cells alters the overall organization of the cell surface and cell circularity [(4π × area)/(perimeter)(2)] from 0.47 ± 0.06 units in mock-treated cells to 0.09 ± 0.03 units in S6K-overexpressing macrophages causing stellation and arborization of cell shape. This effect was partially reversed in cells expressing a kinase-inactive S6K mutant and was fully reversed in cells silenced with small interference RNA. Equally important is that S6K is itself regulated by phospholipids, specifically phosphatidic acid, whereby 300 nM 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA), but not the control 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), binds directly to S6K and causes an ∼ 2.9-fold increase in S6K catalytic activity. This was followed by an increase in Filamin A (FLNA) functionality as measured by phospho-FLNA (S(2152)) expression and by a subsequent elevation of actin nucleation. This reliance of S6K on phosphatidic acid (PA), a curvature-inducing phospholipid, explained the extra-large perimeter of cells that overexpressed S6K. Furthermore, the diversity of the response to S6K in several unrelated cell types (fibroblasts, leukocytes, and invasive cancer cells) that we report here indicates the existence of an underlying common mechanism in mammalian cells. This new signaling set, PA-S6K-FLNA-actin, sheds light for the first time into the morphogenic pathway of cytoskeletal structures that are crucial for adhesion and cell locomotion during inflammation and metastasis.

The molecular basis of leukocyte adhesion involving phosphatidic acid and phospholipase D.

Defining how leukocytes adhere to solid surfaces, such as capillary beds, and the subsequent migration through the extracellular matrix, is a central biological issue. We show here that phospholipase D (PLD) and its enzymatic reaction product, phosphatidic acid (PA), regulate cell adhesion of immune cells (macrophages and neutrophils) to collagen and have defined the underlying molecular mechanism in a spatio-temporal manner that coincides with PLD activity timing. A rapid (t½ = 4 min) and transient activation of the PLD1 isoform occurs upon adhesion, and a slower (t½ = 7.5 min) but prolonged (>30 min) activation occurs for PLD2. Importantly, PA directly binds to actin-related protein 3 (Arp3) at EC50 = 22 nm, whereas control phosphatidylcholine did not bind. PA-activated Arp3 hastens actin nucleation with a kinetics of t½ = 3 min at 300 nm (compared with controls of no PA, t½ = 5 min). Thus, PLD and PA are intrinsic components of cell adhesion, which reinforce each other in a positive feedback loop and react from cues from their respective solid substrates. In nascent adhesion, PLD1 is key, whereas a sustained adhesion in mature or established focal points is dependent upon PLD2, PA, and Arp3. A prolonged adhesion could effectively counteract the reversible intrinsic nature of this cellular process and constitute a key player in chronic inflammation.

A GEF-to-phospholipase molecular switch caused by phosphatidic acid, Rac and JAK tyrosine kinase that explains leukocyte cell migration.

Phospholipase D2 (PLD2) is a cell-signaling molecule that bears two activities: a guanine-nucleotide exchange factor (GEF) and a lipase that reside in the PX/PH domains and in two HKD domains, respectively. Upon cell stimulation, the GEF activity yields Rac2-GTP and the lipase activity yields phosphatidic acid (PA). In the present study, we show for the first time that these activities regulate one another. Upon cell stimulation, both GEF and lipase activities are quickly (within ∼3 min) elevated. As soon as it is produced, PA positively feeds back on the GEF and further activates it. Rac2-GTP, on the other hand, is inhibitory to the lipase activity. PLD2 would remain downregulated if it were not for the contribution of the tyrosine kinase Janus kinase 3 (JAK3), which restores lipase action (by phosphorylation at Y415). Conversely, the GEF is inhibited upon phosphorylation by JAK3 and is effectively terminated by this action and by the increasing accumulation of PA at >15 min of cell stimulation. This PA interferes with the ability of the GEF to bind to its substrate (Rac2-GTP). Thus, both temporal inter-regulation and phosphorylation-dependent mechanisms are involved in determining a GEF-lipase switch within the same molecule. Human neutrophils stimulated by interleukin-8 follow a biphasic pattern of GEF and lipase activation that can be explained by such an intramolecular switch. This is the first report of a temporal inter-regulation of two enzymatic activities that reside in the same molecule with profound biological consequences in leukocyte cell migration.

A new signaling pathway (JAK-Fes-phospholipase D) that is enhanced in highly proliferative breast cancer cells.

The products of the oncogene Fes and JAK3 are tyrosine kinases, whose expressions are elevated in tumor growth, angiogenesis, and metastasis. Phosphatidic acid, as synthesized by phospholipase D (PLD), enhances cancer cell survival. We report a new signaling pathway that integrates the two kinases with the lipase. A new JAK3-Fes-PLD2 axis is responsible for the highly proliferative phenotype of MDA-MB-231 breast cancer cells. Conversely, this pathway is maintained at a low rate of expression and activity levels in untransformed cells such as MCF10A. We also deciphered the inter-regulation that exists between the two kinases (JAK3 and the oncogene Fes) and between these two kinases and the lipase (PLD2). Whereas JAK3 and Fes marginally activate PLD2 in non-transformed cells, these kinases greatly enhance (>200%) PLD activity following protein-protein interaction through the SH2 domain and the Tyr-415 residue of PLD2. We also found that phosphatidic acid enhances Fes activity in MDA-MB-231 cells providing a positive activation loop between Fes and PLD2. In summary, the JAK3, Fes and PLD2 interactions in transformed cells maintain PLD2 at an enhanced level that leads to abnormal cell growth. Modulating this new JAK3-Fes-PLD2 pathway could be important to control the highly invasive phenotype of breast cancer cells.

Phospholipase D2 (PLD2) is a guanine nucleotide exchange factor (GEF) for the GTPase Rac2.

We have discovered that the enzyme phospholipase D2 (PLD2) binds directly to the small GTPase Rac2, resulting in PLD2 functioning as a guanine nucleotide exchange factor (GEF), because it switches Rac2 from the GDP-bound to the GTP-bound states. This effect is large enough to be meaningful (∼72% decrease for GDP dissociation and 300% increase for GTP association, both with PLD2), it has a half-time of ∼7 min, is enhanced with increasing PLD2 concentrations, and compares favorably with other known GEFs, such as Vav-1. The PLD2-Rac2 protein-protein interaction is sufficient for the GEF function, because it can be demonstrated in vitro with just recombinant proteins without lipid substrates, and a catalytically inactive lipase (PLD2-K758R) has GEF activity. Apart from this function, exogenous phosphatidic acid by itself (300 pM) increases GTP binding and enhances PLD2-K758R-mediated GTP binding (by ∼34%) but not GDP dissociation. Regarding the PLD2-Rac2 protein-protein association, it involves, for PLD2, residues 263-266 within a Cdc42/Rac interactive binding region in the PH domain, as well as the PX domain, and it involves, for Rac2, residue N17 within its Switch-1 region. PLD2's GEF function is demonstrated in living cells, because silencing PLD2 results in reduced Rac2 activity, whereas PLD2-initiated Rac2 activation enhances cell adhesion, chemotaxis, and phagocytosis. There are several known GEFs, but we report that this GEF is harbored in a phospholipase. The benefit to the cell is that PLD2 brings spatially separated molecules together in a membrane environment, ready for fast intracellular signaling and cell function.

Evidence that keratinocyte microvesicle particles carrying Platelet-activating factor mediate the widespread multi-organ damage associated with intoxicated thermal burn injury.

Thermal burn injuries can result in significant morbidity and mortality. The combination of ethanol intoxication with thermal burn injury results in increased morbidity through an exaggerated inflammatory response involving many organs. Recent studies have linked involvement of the lipid mediator Platelet-activating factor (PAF) in the pathology associated with intoxicated thermal burn injury (ITBI). The present studies tested the roles of PAF and the elevated levels of subcellular microvesicle particles (MVP) generated in response to ITBI in the subsequent multi-organ toxicity. First, thermal burn injury of HaCaT keratinocytes preincubated with ethanol resulted in augmented MVP release, which was blocked by inhibiting the PAF-generating enzyme cytosolic phospholipase A2 and the PAF receptor (PAFR). Second, ITBI of mice resulted in increased pro-inflammatory cytokine production and neutrophilic inflammation in multiple organs which were not present in mice deficient in PAFRs nor the MVP-generating enzyme acid sphingomyelinase (aSMase). Moreover, the increased bacterial translocation from the gut to mesenteric lymph nodes previously reported in murine ITBI was also dependent upon PAFR and aSMase. MVP released from ITBI-treated keratinocytes contained high levels of PAFR agonistic activity. Finally, use of topical aSMase inhibitor imipramine following ITBI attenuated the widespread organ inflammatory response of ITBI, suggesting a potential therapeutic for this condition. These studies provide evidence for PAF-enriched MVP generated in skin, which then act upon the gut PAFR resulting in bacterial translocation as the mechanism for the multi-organ dysfunction associated with ITBI. Inasmuch as aSMase inhibitors are widely available, these studies could result in effective treatments for ITBI.

Evidence for the involvement of keratinocyte-derived microvesicle particles in the photosensitivity associated with xeroderma pigmentosum type A deficiency.

Photosensitivity can be due to numerous causes. The photosensitivity associated with deficiency of xeroderma pigmentosum type A (XPA) has been previously shown to be associated with excess levels of the lipid mediator platelet-activating factor (PAF) generated by the keratinocyte. As PAF has been reported to trigger the production of subcellular microvesicle particles (MVP) due to the enzyme acid sphingomyelinase (aSMase), the goal of these studies was to discern if PAF and aSMase could serve as therapeutic targets for the XPA deficiency photosensitivity. HaCaT keratinocytes lacking XPA generated greater levels of MVP in comparison to control cells. Mice deficient in XPA also generated enhanced MVP levels in skin and in plasma in response to UV radiation. Use of a genetic strategy with mice deficient in both XPA and PAF receptors revealed that these mice generated less MVP release as well as decreased skin erythema and cytokine release compared to XPA knockout mice alone. Finally, the aSMase inhibitor imipramine blocked UV-induced MVP release in HaCaT keratinocytes, as well as XPA knockout mice. These studies support the concept that the photosensitivity associated with XPA involves PAF- and aSMase-mediated MVP release and provides a potential pharmacologic target in treating this form of photosensitivity.

Myotubularin-related proteins regulate KRAS function by controlling plasma membrane levels of polyphosphoinositides and phosphatidylserine.

KRAS is a small GTPase, ubiquitously expressed in mammalian cells, that functions as a molecular switch to regulate cell proliferation and differentiation. Oncogenic mutations that render KRAS constitutively active occur frequently in human cancers. KRAS must localize to the plasma membrane (PM) for biological activity. KRAS PM binding is mediated by interactions of the KRAS membrane anchor with phosphatidylserine (PtdSer), therefore, depleting PM PtdSer content abrogates KRAS PM binding and oncogenic function. From a genome-wide siRNA screen to search for genes that regulate KRAS PM localization, we identified a set of phosphatidylinositol (PI) 3-phosphatase family members: myotubularin-related (MTMR) proteins 2, 3, 4 and 7. Here we show that knockdown of MTMR 2/3/4/7 expression disrupts KRAS PM interactions. The molecular mechanism involves depletion of PM PI 4-phosphate (PI4P) levels, which in turn disrupts the subcellular localization and operation of oxysterol-binding protein related protein (ORP) 5, a PtdSer lipid transfer protein that maintains PM PtdSer content. Concomitantly, silencing MTMR 2/3/4/7 expression elevates PM levels of PI3P and reduces PM and total cellular levels of PtdSer. In summary we propose that the PI 3-phosphatase activity provided by MTMR proteins is required to generate PM PI for the synthesis of PM PI4P, which in turn, promotes the PM localization of PtdSer and KRAS.

Identification of the catalytic site of phospholipase D2 (PLD2) newly described guanine nucleotide exchange factor activity.

We have demonstrated that phospholipase D2 (PLD2) is a guanine nucleotide exchange factor (GEF) for Rac2 and determined the PLD2 domains and amino acid site(s) responsible for its GEF activity. Experiments using GST fusion proteins or GST-free counterparts, purified proteins revealed that the PX domain is sufficient to exert GEF activity similar to full-length PLD2. The PLD2-GEF catalytic site is formed by a hydrophobic pocket of residues Phe-107, Phe-129, Leu-166, and Leu-173, all of which are in the PX domain. A nearby Arg-172 is also important in the overall activity. PX mutants altering any of those five amino acids fail to have GEF activity but still bind to Rac2, while their lipase activity was mostly unaffected. In addition to the PX domain, a region in the pleckstrin homology domain (Ile-306-Ala-310) aids in the PX-mediated GEF activity by providing a docking site to hold Rac2 in place during catalysis. We conclude that PLD2 is a unique GEF, with the PX being the major catalytic domain for its GEF activity, whereas the pleckstrin homology domain assists in the PX-mediated activity. The physiological relevance of this novel GEF in cell biology is demonstrated here in chemotaxis and phagocytosis of leukocytes, as the specific PX and PH mutants abolished cell function. Thus, this study reveals for the first time the catalytic site that forms the basis for the mechanism behind the GEF activity of PLD2.

Phospholipase D2 (PLD2) shortens the time required for myeloid leukemic cell differentiation: mechanism of action.

Cell differentiation is compromised in acute leukemias. We report that mammalian target of rapamycin (mTOR) and S6 kinase (S6K) are highly expressed in the undifferentiated promyelomonocytic leukemic HL-60 cell line, whereas PLD2 expression is minimal. The expression ratio of PLD2 to mTOR (or to S6K) is gradually inverted upon in vitro induction of differentiation toward the neutrophilic phenotype. We present three ways that profoundly affect the kinetics of differentiation as follows: (i) simultaneous overexpression of mTOR (or S6K), (ii) silencing of mTOR via dsRNA-mediated interference or inhibition with rapamycin, and (iii) PLD2 overexpression. The last two methods shortened the time required for differentiation. By determining how PLD2 participates in cell differentiation, we found that PLD2 interacts with and activates the oncogene Fes/Fps, a protein-tyrosine kinase known to be involved in myeloid cell development. Fes activity is elevated with PLD2 overexpression, phosphatidic acid or phosphatidylinositol bisphosphate. Co-immunoprecipitation indicates a close PLD2-Fes physical interaction that is negated by a Fes-R483K mutant that incapacitates its Src homology 2 domain. All these suggest for the first time the following mechanism: mTOR/S6K down-regulation→PLD2 overexpression→PLD2/Fes association→phosphatidic acid-led activation of Fes kinase→granulocytic differentiation. Differentiation shortening could have a clinical impact on reducing the time of return to normalcy of the white cell counts after chemotherapy in patients with acute promyelocytic leukemia.

Evidence for two CRIB domains in phospholipase D2 (PLD2) that the enzyme uses to specifically bind to the small GTPase Rac2.

Phospholipase D (PLD) and small GTPases are vital to cell signaling. We report that the Rac2 and the PLD2 isoforms exist in the cell as a lipase-GTPase complex that enables the two proteins to elicit their respective functionalities. A strong association between the two molecules was demonstrated by co-immunoprecipitation and was confirmed in living cells by FRET with CFP-Rac2 and YFP-PLD2 fluorescent chimeras. We have identified the amino acids in PLD2 that define a specific binding site to Rac2. This site is composed of two CRIB (Cdc42-and Rac-interactive binding) motifs that we have named "CRIB-1" and "CRIB-2" in and around the PH domain in PLD2. Deletion mutants PLD2-ΔCRIB-1/2 negate co-immunoprecipitation with Rac2 and diminish the FRET signal in living cells. The PLD2-Rac2 association was further confirmed in vitro using affinity-purified recombinant proteins. Binding was saturable with an apparent K(d) of 3 nm and was diminished with PLD2-ΔCRIB mutants. Furthermore, PLD2 bound more efficiently to Rac2-GTP than to Rac2-GDP or to a GDP-constitutive Rac2-N17 mutant. Increasing concentrations of recombinant Rac2 in vitro and in vivo during cell adhesion inhibit PLD2. Conversely, Rac2 activity is increased in the presence of PLD2-WT but not in PLD2-ΔCRIB. We propose that in activated cells PLD2 affects Rac2 in an initial positive feedback, but as Rac2-GTP accumulates in the cell, this constitutes a "termination signal" leading to PLD2 inactivation.

Phosphatidic acid is a leukocyte chemoattractant that acts through S6 kinase signaling.

Phosphatidic acid (PA) is a pleiotropic lipid second messenger in mammalian cells. We report here that extracellular PA acts as a leukocyte chemoattractant, as membrane-soluble dioleoyl-PA (DOPA) elicits actin polymerization and chemotaxis of human neutrophils and differentiated proleukemic HL-60 cells. We show that the mechanism for this involves the S6 kinase (S6K) signaling enzyme. Chemotaxis was inhibited >90% by the S6K inhibitors rapamycin and bisindolylmaleimide and by S6K1 silencing using double-stranded RNA. However, it was only moderately ( approximately 30%) inhibited by mTOR siRNA, indicating the presence of an mTOR-independent mechanism for S6K. Exogenous PA led to robust time- and dose-dependent increases in S6K enzymatic activity and Thr(421)/Ser(424) phosphorylation, further supporting a PA/S6K connection. We also investigated whether intracellular PA production affects cell migration. Overexpression of phospholipase D2 (PLD2) and, to a lesser extent, PLD1, resulted in elevation of both S6K activity and chemokinesis, whereas PLD silencing was inhibitory. Because the lipase-inactive PLD2 mutants K444R and K758R neither activated S6K nor induced chemotaxis, intracellular PA is needed for this form of cell migration. Lastly, we demonstrated a connection between extracellular and intracellular PA. Using an enhanced green fluorescent protein-derived PA sensor (pEGFP-Spo20PABD), we showed that exogenous PA or PA generated in situ by bacterial (Streptomyces chromofuscus) PLD enters the cell and accumulates in vesicle-like cytoplasmic structures. In summary, we report the discovery of PA as a leukocyte chemoattractant via cell entry and activation of S6K to mediate the cytoskeletal actin polymerization and leukocyte chemotaxis required for the immune function of these cells.

Blocking K-Ras Interaction With the Plasma Membrane Is a Tractable Therapeutic Approach to Inhibit Oncogenic K-Ras Activity.

Ras proteins are membrane-bound small GTPases that promote cell proliferation, differentiation, and apoptosis. Consistent with this key regulatory role, activating mutations of Ras are present in ∼19% of new cancer cases in the United States per year. K-Ras is one of the three ubiquitously expressed isoforms in mammalian cells, and oncogenic mutations in this isoform account for ∼75% of Ras-driven cancers. Therefore, pharmacological agents that block oncogenic K-Ras activity would have great clinical utility. Most efforts to block oncogenic Ras activity have focused on Ras downstream effectors, but these inhibitors only show limited clinical benefits in Ras-driven cancers due to the highly divergent signals arising from Ras activation. Currently, four major approaches are being extensively studied to target K-Ras-driven cancers. One strategy is to block K-Ras binding to the plasma membrane (PM) since K-Ras requires the PM binding for its signal transduction. Here, we summarize recently identified molecular mechanisms that regulate K-Ras-PM interaction. Perturbing these mechanisms using pharmacological agents blocks K-Ras-PM binding and inhibits K-Ras signaling and growth of K-Ras-driven cancer cells. Together, these studies propose that blocking K-Ras-PM binding is a tractable strategy for developing anti-K-Ras therapies.

Avicin G is a potent sphingomyelinase inhibitor and blocks oncogenic K- and H-Ras signaling.

K-Ras must interact primarily with the plasma membrane (PM) for its biological activity. Therefore, disrupting K-Ras PM interaction is a tractable approach to block oncogenic K-Ras activity. Here, we found that avicin G, a family of natural plant-derived triterpenoid saponins from Acacia victoriae, mislocalizes K-Ras from the PM and disrupts PM spatial organization of oncogenic K-Ras and H-Ras by depleting phosphatidylserine (PtdSer) and cholesterol contents, respectively,  at the inner PM leaflet. Avicin G also inhibits oncogenic K- and H-Ras signal output and the growth of K-Ras-addicted pancreatic and non-small cell lung cancer cells. We further identified that avicin G perturbs lysosomal activity, and disrupts cellular localization and activity of neutral and acid sphingomyelinases (SMases), resulting in elevated cellular sphingomyelin (SM) levels and altered SM distribution. Moreover, we show that neutral SMase inhibitors disrupt the PM localization of K-Ras and PtdSer and oncogenic K-Ras signaling. In sum, this study identifies avicin G as a new potent anti-Ras inhibitor, and suggests that neutral SMase can be a tractable target for developing anti-K-Ras therapeutics.

PLD2 has both enzymatic and cell proliferation-inducing capabilities, that are differentially regulated by phosphorylation and dephosphorylation.

Phospholipase D2 (PLD2) overexpression in mammalian cells results in cell transformation. We have hypothesized that this is due to an increase of de novo DNA synthesis. We show here that overexpression of PLD2-WT leads to an increased DNA synthesis, as measured by the expression levels of the proliferation markers PCNA, p27(KIP1) and phospho-histone-3. The enhancing effect was even higher with phosphorylation-deficient PLD2-Y179F and PLD2-Y511F mutants. The mechanism for this did not involve the enzymatic activity of the lipase, but, rather, the presence of the protein tyrosine phosphatase CD45, as silencing with siRNA for CD45 abrogated the effect. The two Y-->F mutants had in common a YxN consensus site that, in the phosphorylated counterparts, could be recognized by SH2-bearing proteins, such as Grb2. Even though Y179F and Y511F cannot bind Grb2, they could still find other protein partners, one of which, we have reasoned, could be CD45 itself. Affinity purified PLD2 is indeed activated by Grb2 and deactivated by CD45 in vitro. We concluded that phosphorylated PLD2, aided by Grb2, mediates lipase activity, whereas dephosphorylated PLD2 mediates an induction of cell proliferation, and the specific residues involved in this newly discovered regulation of PLD2 are Y(179) and Y(511).