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.

The Epidermal Growth Factor Receptor Ligand Amphiregulin Protects From Cholestatic Liver Injury and Regulates Bile Acids Synthesis.

Intrahepatic accumulation of bile acids (BAs) causes hepatocellular injury. Upon liver damage, a potent protective response is mounted to restore the organ's function. Epidermal growth factor receptor (EGFR) signaling is essential for regeneration after most types of liver damage, including cholestatic injury. However, EGFR can be activated by a family of growth factors induced during liver injury and regeneration. We evaluated the role of the EGFR ligand, amphiregulin (AREG), during cholestatic liver injury and regulation of AREG expression by BAs. First, we demonstrated increased AREG levels in livers from patients with primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). In two murine models of cholestatic liver injury, bile duct ligation (BDL) and alpha-naphthyl-isothiocyanate (ANIT) gavage, hepatic AREG expression was markedly up-regulated. Importantly, Areg-/- mice showed aggravated liver injury after BDL and ANIT administration compared to Areg+/+ mice. Recombinant AREG protected from ANIT and BDL-induced liver injury and reduced BA-triggered apoptosis in liver cells. Oral BA administration induced ileal and hepatic Areg expression, and, interestingly, cholestyramine feeding reduced postprandial Areg up-regulation in both tissues. Most interestingly, Areg-/- mice displayed high hepatic cholesterol 7 α-hydroxylase (CYP7A1) expression, reduced serum cholesterol, and high BA levels. Postprandial repression of Cyp7a1 was impaired in Areg-/- mice, and recombinant AREG down-regulated Cyp7a1 mRNA in hepatocytes. On the other hand, BAs promoted AREG gene expression and protein shedding in hepatocytes. This effect was mediated through the farnesoid X receptor (FXR), as demonstrated in Fxr-/- mice, and involved EGFR transactivation. Finally, we show that hepatic EGFR expression is indirectly induced by BA-FXR through activation of suppressor of cytokine signaling-3 (SOC3). Conclusion: AREG-EGFR signaling protects from cholestatic injury and participates in the physiological regulation of BA synthesis.

Phospholipase D and the Mitogen Phosphatidic Acid in Human Disease: Inhibitors of PLD at the Crossroads of Phospholipid Biology and Cancer.

Lipids are key building blocks of biological membranes and are involved in complex signaling processes such as metabolism, proliferation, migration, and apoptosis. Extracellular signaling by growth factors, stress, and nutrients is transmitted through receptors that activate lipid-modifying enzymes such as the phospholipases, sphingosine kinase, or phosphoinositide 3-kinase, which then modify phospholipids, sphingolipids, and phosphoinositides. One such important enzyme is phospholipase D (PLD), which cleaves phosphatidylcholine to yield phosphatidic acid and choline. PLD isoforms have dual role in cells. The first involves maintaining cell membrane integrity and cell signaling, including cell proliferation, migration, cytoskeletal alterations, and invasion through the PLD product PA, and the second involves protein-protein interactions with a variety of binding partners. Increased evidence of elevated PLD expression and activity linked to many pathological conditions, including cancer, neurological and inflammatory diseases, and infection, has motivated the development of dual- and isoform-specific PLD inhibitors. Many of these inhibitors are reported to be efficacious and safe in cells and mouse disease models, suggesting the potential for PLD inhibitors as therapeutics for cancer and other diseases. Current knowledge and ongoing research of PLD signaling networks will help to evolve inhibitors with increased efficacy and safety for clinical studies.

Lack of effective translational regulation of PLD expression and exosome biogenesis in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that is difficult to treat since cells lack the three receptors (ES, PR, or HER) that the most effective treatments target. We have used a well-established TNBC cell line (MDA-MB-231) from which we found evidence in support for a phospholipase D (PLD)-mediated tumor growth and metastasis: high levels of expression of PLD, as well as the absence of inhibitory miRs (such as miR-203) and 3'-mRNA PARN deadenylase activity in these cells. Such findings are not present in a luminal B cell line, MCF-7, and we propose a new miR•PARN•PLD node that is not uniform across breast cancer molecular subtypes and as such TNBC could be pharmacologically targeted differentially. We review the participation of PLD and phosphatidic acid (PA), its enzymatic product, as new "players" in breast cancer biology, with the aspects of regulation of the tumor microenvironment, macrophage polarization, regulation of PLD transcripts by specific miRs and deadenylases, and PLD-regulated exosome biogenesis. A new signaling miR•PARN•PLD node could serve as new biomarkers for TNBC abnormal signaling and metastatic disease staging, potentially before metastases are able to be visualized using conventional imaging.

Down-regulation of MicroRNAs (MiRs) 203, 887, 3619 and 182 Prevents Vimentin-triggered, Phospholipase D (PLD)-mediated Cancer Cell Invasion.

Breast cancer is a leading cause of morbidity and mortality among women. Metastasis is initiated after epithelial-mesenchymal-transition (EMT). We have found a connection between EMT markers and the expression of four microRNAs (miRs) mediated by the signaling enzyme phospholipase D (PLD). Low aggressive MCF-7 breast cancer cells have low endogenous PLD enzymatic activity and cell invasion, concomitant with high expression of miR-203, -887, and -3619 (that decrease PLD2 translation and a luciferase reporter) and miR-182 (targeting PLD1) that are, therefore, "tumor-suppressor-like" miRs. The combination miR-887+miR-3619 abolished >90% of PLD enzymatic activity. Conversely, post-EMT MDA-MB-231 cells have low miR expression, high levels of PLD1/2, and high aggressiveness. The latter was reversed by ectopically transfecting the miRs, which was negated by silencing miRs with specific siRNAs. We determined that the molecular mechanism is that E-cadherin triggers expression of the miRs in pre-EMT cells, whereas vimentin dampens expression of the miRs in post-EMT invasive cells. This novel work identifies for the first time a set of miRs that are activated by a major pre-EMT marker and deactivated by a post-EMT marker, boosting the transition from low invasion to high invasion, as mediated by the key phospholipid metabolism enzyme PLD.

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.

Adenovirus entry from the apical surface of polarized epithelia is facilitated by the host innate immune response.

Prevention of viral-induced respiratory disease begins with an understanding of the factors that increase or decrease susceptibility to viral infection. The primary receptor for most adenoviruses is the coxsackievirus and adenovirus receptor (CAR), a cell-cell adhesion protein normally localized at the basolateral surface of polarized epithelia and involved in neutrophil transepithelial migration. Recently, an alternate isoform of CAR, CAREx8, has been identified at the apical surface of polarized airway epithelia and is implicated in viral infection from the apical surface. We hypothesized that the endogenous role of CAREx8 may be to facilitate host innate immunity. We show that IL-8, a proinflammatory cytokine and a neutrophil chemoattractant, stimulates the protein expression and apical localization of CAREx8 via activation of AKT/S6K and inhibition of GSK3ß. Apical CAREx8 tethers infiltrating neutrophils at the apical surface of a polarized epithelium. Moreover, neutrophils present on the apical-epithelial surface enhance adenovirus entry into the epithelium. These findings suggest that adenovirus evolved to co-opt an innate immune response pathway that stimulates the expression of its primary receptor, apical CAREx8, to allow the initial infection the intact epithelium. In addition, CAREx8 is a new target for the development of novel therapeutics for both respiratory inflammatory disease and adenoviral infection.

Two sites of action for PLD2 inhibitors: The enzyme catalytic center and an allosteric, phosphoinositide biding pocket.

Phospholipase D (PLD) has been implicated in many physiological functions, such as chemotaxis and phagocytosis, as well as pathological functions, such as cancer cell invasion and metastasis. New inhibitors have been described that hamper the role of PLD in those pathologies but their site of action is not known. We have characterized the biochemical and biological behavior of the PLD1/2 dual inhibitor 5-Fluoro-2-indolyl des-chlorohalopemide (FIPI), and the specific PLD2 inhibitor, N-[2-[1-(3-Fluorophenyl)-4-oxo-1,3,-8-triazaspiro[4.5]dec-8-yl]ethyl]-2-naphthalenecarboxamide (NFOT), and found that both FIPI and NFOT are mixed-kinetics inhibitors. Mutagenesis studies indicate that FIPI binds at S757 of PLD2, which is within the HKD2 catalytic site of the enzyme, whereas NFOT binds to PLD2 at two different sites, one being at S757/S648 and another to an allosteric site that is a natural site occupied by PIP2 (R210/R212). This latter site, along with F244/L245/L246, forms a hydrophobic pocket in the PH domain. The mechanism of action of FIPI is a direct effect on the catalytic site (and as such inhibits both PLD1 and PLD2 isoforms), whereas PLD2 affects both the catalytic site (orthosteric) and blocks PIP2 binding to PLD2 (allosteric), which negates the natural enhancing role of PIP2. Moreover, NFOT prevents cell invasion of cancer cells, which does not occur in cells overexpressing PLD2-F244A/L245A/L246A, or PLD2-R210A/R212A, or PLD2-S757/S648 mutants. This study provides new specific knowledge of enzyme regulation and mechanisms of activation and inhibition of PLD2 that are necessary to understand its role in cell signaling and to develop new inhibitors for cancer cell invasion and metastasis.

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.

Phospholipase D in cell signaling: from a myriad of cell functions to cancer growth and metastasis.

Phospholipase D (PLD) enzymes play a double vital role in cells: they maintain the integrity of cellular membranes and they participate in cell signaling including intracellular protein trafficking, cytoskeletal dynamics, cell migration, and cell proliferation. The particular involvement of PLD in cell migration is accomplished: (a) through the actions of its enzymatic product of reaction, phosphatidic acid, and its unique shape-binding role on membrane geometry; (b) through a particular guanine nucleotide exchange factor (GEF) activity (the first of its class assigned to a phospholipase) in the case of the mammalian isoform PLD2; and (c) through protein-protein interactions with a wide network of molecules: Wiskott-Aldrich syndrome protein (WASp), Grb2, ribosomal S6 kinase (S6K), and Rac2. Further, PLD interacts with a variety of kinases (PKC, FES, EGF receptor (EGFR), and JAK3) that are activated by it, or PLD becomes the target substrate. Out of these myriads of functions, PLD is becoming recognized as a major player in cell migration, cell invasion, and cancer metastasis. This is the story of the evolution of PLD from being involved in a large number of seemingly unrelated cellular functions to its most recent role in cancer signaling, a subfield that is expected to grow exponentially.

Thematic minireview series on phospholipase D and cancer.

Phospholipase D (PLD) signaling plays a critical role in cell growth and proliferation, vesicular trafficking, secretion, and endocytosis. At the cellular level, PLD and its reaction product, phosphatidate, interact with a large number of protein partners that are directly related to the actin cytoskeleton and cell migration. Cancer invasion and metastasis rely heavily on cellular motility, and as such, they have put PLD at center stage in cancer research. This minireview series highlights some of the molecular mechanisms that provide evidence for the emerging tumorigenic potential of PLD, the role of the microenvironment, and putative connections with inflammation. PLD represents a potential target for the rational development of therapeutics against cancer and other diseases.

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.

Role of c-Met/phosphatidylinositol 3-kinase (PI3k)/Akt signaling in hepatocyte growth factor (HGF)-mediated lamellipodia formation, reactive oxygen species (ROS) generation, and motility of lung endothelial cells.

Hepatocyte growth factor (HGF) mediated signaling promotes cell proliferation and migration in a variety of cell types and plays a key role in tumorigenesis. As cell migration is important to angiogenesis, we characterized HGF-mediated effects on the formation of lamellipodia, a pre-requisite for migration using human lung microvascular endothelial cells (HLMVECs). HGF, in a dose-dependent manner, induced c-Met phosphorylation (Tyr-1234/1235, Tyr-1349, Ser-985, Tyr-1003, and Tyr-1313), activation of PI3k (phospho-Yp85) and Akt (phospho-Thr-308 and phospho-Ser-473) and potentiated lamellipodia formation and HLMVEC migration. Inhibition of c-Met kinase by SU11274 significantly attenuated c-Met, PI3k, and Akt phosphorylation, suppressed lamellipodia formation and endothelial cell migration. LY294002, an inhibitor of PI3k, abolished HGF-induced PI3k (Tyr-458), and Akt (Thr-308 and Ser-473) phosphorylation and suppressed lamellipodia formation. Furthermore, HGF stimulated p47(phox)/Cortactin/Rac1 translocation to lamellipodia and ROS generation. Moreover, inhibition of c-Met/PI3k/Akt signaling axis and NADPH oxidase attenuated HGF- induced lamellipodia formation, ROS generation and cell migration. Ex vivo experiments with mouse aortic rings revealed a role for c-Met signaling in HGF-induced sprouting and lamellipodia formation. Taken together, these data provide evidence in support of a significant role for HGF-induced c-Met/PI3k/Akt signaling and NADPH oxidase activation in lamellipodia formation and motility of lung endothelial cells.

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.

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.

Enzymatically active Rho and Rac small-GTPases are involved in the establishment of the vacuolar membrane after Toxoplasma gondii invasion of host cells.

BACKGROUND: GTPases are the family of hydrolases that bind and hydrolyze guanosine triphosphate. The large Immunity-related GTPases and the small GTPase ADP-ribosylation factor-6 in host cells are known to accumulate on the parasitophorous vacuole membrane (PVM) of Toxoplasma gondii and play critical roles in this parasite infection, but these GTPases cannot explain the full extent of infection. RESULTS: In this research, RhoA and Rac1 GTPases from the host cell were found to accumulate on the PVM regardless of the virulence of the T. gondii strains after T. gondii invasion, and this accumulation was dependent on their GTPase activity. The real-time micrography of T. gondii tachyzoites invading COS-7 cells overexpressing CFP-RhoA showed that this GTPase was recruited to the PVM at the very beginning of the invasion through the host cell membrane or from the cytosol. Host cell RhoA and Rac1 were also activated after T. gondii tachyzoites invasion, which was needed for host cell cytoskeleton reorganization to facilitate intracellular pathogens invasion. The decisive domains for the RhoA accumulation on the PVM included the GTP/Mg2+ binding site, the mDia effector interaction site, the G1 box, the G2 box and the G5 box, respectively, which were related to the binding of GTP for enzymatic activity and mDia for the regulation of microtubules. The recruited CFP-RhoA on the PVM could not be activated by epithelial growth factor (EGF) and no translocation was observed, unlike the unassociated RhoA in the host cell cytosol that migrated to the cell membrane towards the EGF activation spot. This result supported the hypothesis that the recruited RhoA or Rac1 on the PVM were in the GTP-bound active form. Wild-type RhoA or Rac1 overexpressed cells had almost the same infection rates by T. gondii as the mock-treated cells, while RhoA-N19 or Rac1-N17 transfected cells and RhoA, Rac1 or RhoA + Rac1 siRNA-treated cells showed significantly diminished infection rates compared to mock cells. CONCLUSIONS: The accumulation of the RhoA and Rac1 on the PVM and the requisite of their normal GTPase activity for efficient invasion implied their involvement and function in T. gondii invasion.