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ADP-ribosylation factor (Arf) family consisting of six family members, Arf1–Arf6, belongs to Ras superfamily and orchestrates vesicle trafficking under the control of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins. It is well established that brefeldin A, a potent inhibitor of ArfGEFs, blocks cytokine secretion from activated T cells, suggesting that the Arf pathway plays important roles in T cell functions. In this study, because Arf1 and Arf6 are the best-characterized members among Arf family, we established T lineage–specific Arf1-deficient, Arf6-deficient, and Arf1/6 double-deficient mice to understand physiological roles of the Arf pathway in the immune system. Contrary to our expectation, Arf deficiency had little or no impact on cytokine secretion from the activated T cells. In contrast, the lack of both Arf1 and Arf6, but neither Arf1 nor Arf6 deficiency alone, rendered naive T cells susceptible to apoptosis upon TCR stimulation because of imbalanced expression of Bcl-2 family members. We further demonstrate that Arf1/6 deficiency in T cells alleviates autoimmune diseases like colitis and experimental autoimmune encephalomyelitis, whereas Ab response under Th2-polarizing conditions is seemingly normal. Our findings reveal an unexpected role for the Arf pathway in the survival of T cells during TCR-induced activation and its potential as a therapeutic target in the autoimmune diseases.

Many plasma membrane proteins enter cells by clathrin-independent endocytosis (CIE). Rab family small GTPases play pivotal roles in CIE and following intracellular trafficking of cargo proteins. Here, we provide evidence that TBC1D24, which contains an atypical Rab GAP domain, facilitates formation of tubular recycling endosomes (TREs) that are a hallmark of the CIE cargo trafficking pathway in HeLa cells. Overexpression of TBC1D24 in HeLa cells dramatically increased TREs loaded with CIE cargo proteins, while deletion of TBC1D24 impaired TRE formation and delayed the recycling of CIE cargo proteins back to the plasma membrane. We also found that TBC1D24 binds to Rab22A, through which TBC1D24 regulates TRE-mediated CIE cargo recycling. These findings provide insight into regulatory mechanisms for CIE cargo trafficking.

Disordered expression and distribution of plasma membrane proteins at the cell surface leads to diverse malignant phenotypes in tumors, including cell invasion. The ubiquitin-specific protease TRE17/USP6, an oncogene identified in Ewing sarcoma, is highly expressed in several cancers and locally aggressive tumor-like lesions. We have previously demonstrated that TRE17 regulates the trafficking of plasma membrane proteins that enter cells via clathrin-independent endocytosis (CIE); TRE17 prevents CIE cargo proteins from being targeted to lysosomes for degradation by deubiquitylating them. However, functional insights into the effects of TRE17-mediated CIE cargo trafficking on cell invasion remain unknown. Here, we show that increased expression of TRE17 enhances invasiveness of the human sarcoma cell line HT-1080 by elevating the cell surface levels of the membrane glycoprotein CD147, which plays a central role in tumor progression. We demonstrate overexpression of TRE17 decreases ubiquitylated CD147, which is accompanied by suppression of CD147 transport to lysosomes, resulting in the stabilization and increase of cell surface-localized CD147. On the other hand, we show knockdown of TRE17 decreases cell surface CD147, which is coupled with reduced production of matrix metalloproteinases, the enzymes responsible for extracellular matrix degradation. Furthermore, we demonstrate that inhibition of CD147 by a specific inhibitor alleviated the TRE17-promoted tumor cell invasion. We therefore propose a model for the pathogenesis of TRE17-driven tumors in which TRE17 increases CD147 at the cell surface by preventing its lysosomal degradation, which in turn enhances matrix metalloproteinase synthesis and matrix degradation, thereby promoting tumor cell invasion.

Asthma is a chronic inflammatory disease of the airways associated with excess production of Th2 cytokines and lung eosinophil accumulation. This inflammatory response persists in spite of steroid administration that blocks autocrine/paracrine loops of inflammatory cytokines, and the detailed mechanisms underlying asthma exacerbation remain unclear. Here, we show that asthma exacerbation is triggered by airway macrophages through a prion-like cell-to-cell transmission of extracellular particulates, including ASC protein, that assemble inflammasomes and mediate IL-1β production. OVA-induced allergic asthma and associated IL-1β production were alleviated in mice with small GTPase Arf6-deficient macrophages. The extracellular ASC specks were slightly engulfed by Arf6–/– macrophages, and the IL-1β production was reduced in Arf6–/– macrophages compared with that in WT macrophages. Furthermore, pharmacological inhibition of the Arf6 guanine nucleotide exchange factor suppressed asthma-like allergic inflammation in OVA-challenged WT mice. Collectively, the Arf6-dependent intercellular transmission of extracellular ASC specks contributes to the amplification of allergic inflammation and subsequent asthma exacerbation.

The small GTPase Arf6 regulates many cellular processes, including cytoskeletal remodeling, receptor endocytosis, and pathogen phagocytosis. Arf6 silencing in neutrophil (PMN)-like cells is well-known to inhibit chemotactic peptide-mediated activation of phospholipase D, the oxidative burst, and β2 integrin-dependent adhesion. In conditional knockout (cKO) mice, the migration to inflammatory sites of Arf6-deficient PMNs was diminished and associated with reduced cell surface expression of β2 integrins. In this study we assessed the impact of Arf6 depletion on the functions and gene expression profile of PMNs isolated from the mouse air pouch. Numerous genes involved in response to oxygen levels, erythrocyte and myeloid differentiation, macrophage chemotaxis, response to chemicals, apoptosis, RNA destabilization, endosome organization, and vesicle transport were differentially expressed in PMNs cKO for Arf6. Lpar6 and Lacc-1 were the most up-regulated and down-regulated genes, respectively. The deletion of Arf6 also decreased Lacc-1 protein level in PMNs, and silencing of Arf6 in THP-1 monocytic cells delayed LPS-mediated Lacc-1 expression. We report that fMLP or zymosan-induced glycolysis and oxygen consumption rate were both decreased in air pouch PMNs but not in bone marrow PMNs of Arf6 cKO mice. Reduced oxygen consumption correlated with a decrease in superoxide and ROS production. Deletion of Arf6 in PMNs also reduced phagocytosis and interfered with apoptosis. The data suggest that Arf6 regulates energy metabolism, which may contribute to impaired phagocytosis, ROS production, and apoptosis in PMN-Arf6 cKO. This study provides new information on the functions and the inflammatory pathways influenced by Arf6 in PMNs.

Lysophosphatidic acid (LPA) is a bioactive lipid growth factor which is present in high levels in serum and platelets. LPA binds to its specific G-protein-coupled receptors, including LPA1 to LPA6, thereby regulating various physiological functions, including cancer growth, angiogenesis, and lymphangiogenesis. Our previous study showed that LPA promotes the expression of the lymphangiogenic factor vascular endothelial growth factor (VEGF)-C in prostate cancer (PCa) cells. Interestingly, LPA has been shown to regulate the expression of calreticulin (CRT), a multifunctional chaperone protein, but the roles of CRT in PCa progression remain unclear. Here we investigated the involvement of CRT in LPA-mediated VEGF-C expression and lymphangiogenesis in PCa. Knockdown of CRT significantly reduced LPA-induced VEGF-C expression in PC-3 cells. Moreover, LPA promoted CRT expression through LPA receptors LPA1 and LPA3, reactive oxygen species (ROS) production, and phosphorylation of eukaryotic translation initiation factor 2α (eIF2α). Tumor-xenografted mouse experiments further showed that CRT knockdown suppressed tumor growth and lymphangiogenesis. Notably, clinical evidence indicated that the LPA-producing enzyme autotaxin (ATX) is related to CRT and that CRT level is highly associated with lymphatic vessel density and VEGF-C expression. Interestingly, the pharmacological antagonist of LPA receptors significantly reduced the lymphatic vessel density in tumor and lymph node metastasis in tumor-bearing nude mice. Together, our results demonstrated that CRT is critical in PCa progression through the mediation of LPA-induced VEGF-C expression, implying that targeting the LPA signaling axis is a potential therapeutic strategy for PCa.

The uptake of cholesterol carried by low-density lipoprotein (LDL) is tightly controlled in the body. Macrophages are not well suited to counteract the cellular consequences of excess cholesterol leading to their transformation into “foam cells,” an early step in vascular plaque formation. We have uncovered and characterized a novel mechanism involving phospholipase D (PLD) in foam cell formation. Utilizing bone marrow-derived macrophages from genetically PLD deficient mice, we demonstrate that PLD2 (but not PLD1)-null macrophages cannot fully phagocytose aggregated oxidized LDL (Agg-Ox-LDL), which was phenocopied with a PLD2-selective inhibitor. We also report a role for PLD2 in coupling Agg-oxLDL phagocytosis with WASP, Grb2, and Actin. Further, the clearance of LDL particles is mediated by both CD36 and PLD2, via mutual dependence on each other. In the absence of PLD2, CD36 does not engage in Agg-Ox-LDL removal and when CD36 is blocked, PLD2 cannot form protein–protein heterocomplexes with WASP or Actin. These results translated into humans using a GEO database of microarray expression data from atheroma plaques versus normal adjacent carotid tissue and observed higher values for NFkB, PLD2 (but not PLD1), WASP, and Grb2 in the atheroma plaques. Human atherectomy specimens confirmed high presence of PLD2 (mRNA and protein) as well as phospho-WASP in diseased arteries. Thus, PLD2 interacts in macrophages with Actin, Grb2, and WASP during phagocytosis of Agg-Ox-LDL in the presence of CD36 during their transformation into “foam cells.” Thus, this study provides new molecular targets to counteract vascular plaque formation and atherogenesis.

ACAP3 (ArfGAP with coiled-coil, ankyrin repeat and pleckstrin homology domains 3) belongs to the ACAP family of GAPs (GTPase-activating proteins) for the small GTPase Arf (ADP-ribosylation factor). However, its specificity to Arf isoforms and physiological functions remain unclear. In the present study, we demonstrate that ACAP3 plays an important role in neurite outgrowth of mouse hippocampal neurons through its GAP activity specific to Arf6. In primary cultured mouse hippocampal neurons, knockdown of ACAP3 abrogated neurite outgrowth, which was rescued by ectopically expressed wild-type ACAP3, but not by its GAP activity-deficient mutant. Ectopically expressed ACAP3 in HEK (human embryonic kidney)-293T cells showed the GAP activity specific to Arf6. In support of this observation, the level of GTP-bound Arf6 was significantly increased by knockdown of ACAP3 in hippocampal neurons. In addition, knockdown and knockout of Arf6 in mouse hippocampal neurons suppressed neurite outgrowth. These results demonstrate that ACAP3 positively regulates neurite outgrowth through its GAP activity specific to Arf6. Furthermore, neurite outgrowth suppressed by ACAP3 knockdown was rescued by expression of a fast cycle mutant of Arf6 that spontaneously exchanges guanine nucleotides on Arf6, but not by that of wild-type, GTP- or GDP-locked mutant Arf6. Thus cycling between active and inactive forms of Arf6, which is precisely regulated by ACAP3 in concert with a guanine-nucleotide-exchange factor(s), seems to be required for neurite outgrowth of hippocampal neurons.

Platelet and megakaryocyte endocytosis is important for loading certain granule cargo (ie, fibrinogen [Fg] and vascular endothelial growth factor); however, the mechanisms of platelet endocytosis and its functional acute effects are understudied. Adenosine 5'-diphosphate-ribosylation factor 6 (Arf6) is a small guanosine triphosphate-binding protein that regulates endocytic trafficking, especially of integrins. To study platelet endocytosis, we generated platelet-specific Arf6 knockout (KO) mice. Arf6 KO platelets had less associated Fg suggesting that Arf6 affects αIIbβ3-mediated Fg uptake and/or storage. Other cargo was unaffected. To measure Fg uptake, mice were injected with biotinylated- or fluorescein isothiocyanate (FITC)-labeled Fg. Platelets from the injected Arf6 KO mice showed lower accumulation of tagged Fg, suggesting an uptake defect. Ex vivo, Arf6 KO platelets were also defective in FITC-Fg uptake and storage. Immunofluorescence analysis showed initial trafficking of FITC-Fg to a Rab4-positive compartment followed by colocalization with Rab11-positive structures, suggesting that platelets contain and use both early and recycling endosomes. Resting and activated αIIbβ3 levels, as measured by flow cytometry, were unchanged; yet, Arf6 KO platelets exhibited enhanced spreading on Fg and faster clot retraction. This was not the result of alterations in αIIbβ3 signaling, because myosin light-chain phosphorylation and Rac1/RhoA activation were unaffected. Consistent with the enhanced clot retraction and spreading, Arf6 KO mice showed no deficits in tail bleeding or FeCl3-induced carotid injury assays. Our studies present the first mouse model for defining the functions of platelet endocytosis and suggest that altered integrin trafficking may affect the efficacy of platelet function.

Although several lines of evidence have shown that the small GTPase ADP-ribosylation factor 6 (Arf6) plays pivotal roles in cancer progression of several types of cancers, little is known about the functions of Arf6 in tumor microenvironment. We demonstrated that Arf6 in vascular endothelial cells (VECs) plays a crucial role in tumor angiogenesis and growth using endothelial cell-specific Arf6 conditional knockout mice into which B16 melanoma and Lewis lung carcinoma cells were implanted. It was also found that Arf6 in VECs positively regulates hepatocyte growth factor (HGF)-induced β1 integrin recycling, which is a critical event for tumor angiogenesis by promoting cell migration. Importantly, pharmacological inhibition of HGF-induced Arf6 activation significantly suppresses tumor angiogenesis and growth in mice, suggesting that Arf6 signaling would be a potential target for anti-angiogenic therapy. In this manuscript, we summarize the multiple roles of Arf6 in cancer progression, particularly in cancer cell invasion/metastasis and our recent findings on tumor angiogenesis, and discuss a possible approach to develop innovative anti-cancer drugs.

A switch in the response of commissural axons to the repellent Slit is critical for ensuring that they cross the ventral midline only once. However, the underlying mechanisms remain to be elucidated. We found that both endocytosis and recycling of Robo1 receptor are crucial for modulating Slit sensitivity in vertebrate commissural axons. Robo1 endocytosis and its recycling back to the cell-surface maintained the stability of axonal Robo1 during Slit stimulation. We identified Arf6 guanosine triphosphatase and its activators, cytohesins, as previously unknown components in Slit-Robo1 signalling in vertebrate commissural neurons. Slit-Robo1 signalling activated Arf6. The Arf6-deficient mice exhibited marked defects in commissural axon midline-crossing. Our data showed that a Robo1 endocytosis-triggered and Arf6-mediated positive-feedback strengthens Slit response in commissural axons upon their midline crossing. Furthermore, cytohesin-Arf6 pathways modulated this self-enhancement of Slit response before and after midline crossing, resulting in a switch that reinforced robust regulation of axon midline crossing. Our study provides insights into endocytic trafficking-mediated mechanisms for spatiotemporally controlled axonal responses and uncovers new players in the midline switch in Slit responsiveness of commissural axons.

Lysophosphatidic acid (LPA) is a bioactive lipid growth factor which is present in high levels in serum and platelets. LPA binds to its specific G-protein-coupled receptors, including LPA

The GTPase-activating protein (GAP) specific to the small GTPase Arf6, ACAP3, is known to regulate morphogenesis of neurons in vitro. However, physiological significance of ACAP3 in the brain development in vivo remains unclear. Here, we show that ACAP3 is involved in neuronal migration in the developing cerebral cortex of mice. Knockdown of ACAP3 in the developing cortical neurons of mice in utero significantly abrogated neuronal migration in the cortical layer, which was restored by ectopic expression of wild type of ACAP3, but not by its GAP-inactive mutant. Furthermore, morphological changes of neurons during migration in the cortical layer were impeded in ACAP3-knocked-down cortical neurons. These results provide evidence that ACAP3 plays a crucial role in migration of cortical neurons by regulating their morphological change during development of cerebral cortex.

HGF and its receptor c-Met are critical molecules in various biological processes. Others and we have previously shown that the small GTPase Arf6 plays a pivotal role in HGF signaling in hepatocytes. However, the molecular mechanism of how Arf6 regulates HGF signaling is unclear. Here, we show that Arf6 plays an important role in HGF-stimulated hepatocyte proliferation and liver regeneration through the phosphatidylinositol 4,5-bisphosphate (PIP2)-producing enzyme PIP5K1A. We find that knockdown of Arf6 and PIP5K1A in HepG2 cells inhibits HGF-stimulated proliferation, Akt activation, and generation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and its precursor PIP2. Interestingly, PIP5K1A is recruited to c-Met upon HGF stimulation in an Arf6 activity-dependent manner. Finally, we show that hepatocyte proliferation and liver regeneration after partial hepatectomy are suppressed in Pip5k1a knockout mice. These results provide insight into the molecular mechanism for HGF-stimulated hepatocyte proliferation and liver regeneration: Arf6 recruits PIP5K1A to c-Met and activates it upon HGF stimulation to produce PIP2 and subsequently PIP3, which in turn activates Akt to promote hepatocyte proliferation, thereby accelerating liver regeneration after liver injury.

The earlier step of cutaneous wound healing process, re-epithelialization of the wounded skin, is triggered by a variety of growth factors. However, molecular mechanisms through which growth factors trigger skin wound healing are less understood. Here, we demonstrate that hepatocyte growth factor (HGF)/c-Met signaling-induced expression of the small G protein Arf6 mRNA in keratinocytes is essential for the skin wound healing. Arf6 mRNA expression was dramatically induced in keratinocytes at the wounded skin, which was specifically suppressed by the c-Met inhibitor. Wound healing of the skin was significantly delayed in keratinocyte-specific Arf6 conditional knockout mice. Furthermore, Arf6 deletion from keratinocytes remarkably suppressed HGF-stimulated cell migration and peripheral membrane ruffle formation, but did not affect skin morphology and proliferation/differentiation of keratinocytes. These results are consistent with the notion that Arf6 expressed in skin keratinocytes through the HGF/c-Met signaling pathway in response to skin wounding plays an important role in skin wound healing by regulating membrane dynamics-based motogenic cellular function of keratinocytes.

Rab small GTPases, well-known regulators of membrane trafficking pathways in eukaryotic cells, comprise approximately 60 different members in mammals. During the past decade, our understanding of the functions of mammalian Rab32 subfamily members (Rab32 and Rab38) have deepened, especially on the biogenesis of lysosome-related organelles, such as melanosomes, and the protection mechanisms against several pathogenic microbial infections. Endosome-mediated membrane trafficking by Rab32 subfamily members plays pivotal roles in these events. In this review, we provide an overview of the regulatory mechanisms of mammalian Rab32-family members in endosomal trafficking, especially focusing on their GEF, GAP and effector molecules, and describe the latest findings on physiological and pathological functions regulated by these molecules.

The small GTPase ADP-ribosylation factor 6 (Arf6) plays pivotal roles in a wide variety of cellular events, including exocytosis, endocytosis, actin cytoskeleton reorganization and phosphoinositide metabolism, in various types of cells. To control such a wide variety of actions of Arf6, activation of Arf6 could be precisely controlled by its activators, guanine nucleotide exchange factors (GEFs), in spatial and temporal manners. In this manuscript, we summarize and discuss the characteristics of previously identified GEFs specific to Arf6 and activation machineries of Arf6.

Growth cone motility and morphology, which are critical for axon guidance, are controlled through intracellular events such as actin cytoskeletal reorganization and vesicular trafficking. The membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] has been implicated in regulation of these cellular processes in a diverse range of cell types. The main kinases involved in the production of PI(4,5)P2 are the type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family, which consist of three isozymes, α, β and γ. Here, we demonstrate the involvement of PIP5Kβ in growth cone dynamics. Overexpression of a lipid kinase-deficient mutant of PIP5Kβ (PIP5Kβ-KD) in mouse dorsal root ganglion (DRG) neurons stimulated axon elongation and increased growth cone size, whereas wild-type PIP5Kβ tended to show opposite effects. Furthermore, PIP5Kβ-KD inhibited growth cone collapse of DRG neurons induced by semaphorin 3A (Sema3A). These results provide evidence that PIP5Kβ negatively regulates axon elongation and growth cone size and is involved in the cellular signaling pathway for Sema3A-triggered repulsion in DRG neurons.

Activation of receptor tyrosine kinases leads to the formation of two different types of plasma membrane structures: peripheral ruffles and dorsal ruffles. Although the formation of both ruffle types requires activation of the small GTPase Rac, the difference in kinetics suggests that a distinct regulatory mechanism operates for their ruffle formation. DOCK1 and DOCK5 are atypical Rac activators and are both expressed in mouse embryonic fibroblasts (MEFs). We found that although PDGF-induced Rac activation and peripheral ruffle formation were coordinately regulated by DOCK1 and DOCK5 in MEFs, DOCK1 deficiency alone impaired dorsal ruffle formation in MEFs. Unlike DOCK5, DOCK1 bound to phosphatidic acid (PA) through the C-terminal polybasic amino acid cluster and was localized to dorsal ruffles. When this interaction was blocked, PDGF-induced dorsal ruffle formation was severely impaired. In addition, we show that phospholipase D, an enzyme that catalyzes PA synthesis, is required for PDGF-induced dorsal, but not peripheral, ruffle formation. These results indicate that the phospholipase D-PA axis selectively controls dorsal ruffle formation by regulating DOCK1 localization.

Phosphatidylinositol (PI) 4,5-bisphosphate (PIP2), generated by PI 4-phosphate 5-kinase (PIP5K), regulates many critical cellular events. PIP2 is also known to mediate plasma membrane localization of the Toll/IL-1 receptor domain-containing adaptor protein (TIRAP), required for the MyD88-dependent Toll-like receptor (TLR) 4 signaling pathway. Microglia are the primary immune competent cells in brain tissue, and TLR4 is important for microglial activation. However, a functional role for PIP5K and PIP2 in TLR4-dependent microglial activation remains unclear. Here, we knocked down PIP5Kα, a PIP5K isoform, in a BV2 microglial cell line using stable expression of lentiviral shRNA constructs or siRNA transfection. PIP5Kα knockdown significantly suppressed induction of inflammatory mediators, including IL-6, IL-1β, and nitric oxide, by lipopolysaccharide. PIP5Kα knockdown also attenuated signaling events downstream of TLR4 activation, including p38 MAPK and JNK phosphorylation, NF-κB p65 nuclear translocation, and IκB-α degradation. Complementation of the PIP5Kα knockdown cells with wild type but not kinase-dead PIP5Kα effectively restored the LPS-mediated inflammatory response. We found that PIP5Kα and TIRAP colocalized at the cell surface and interacted with each other, whereas kinase-dead PIP5Kα rendered TIRAP soluble. Furthermore, in LPS-stimulated control cells, plasma membrane PIP2 increased and subsequently declined, and TIRAP underwent bi-directional translocation between the membrane and cytosol, which temporally correlated with the changes in PIP2. In contrast, PIP5Kα knockdown that reduced PIP2 levels disrupted TIRAP membrane targeting by LPS. Together, our results suggest that PIP5Kα promotes TLR4-associated microglial inflammation by mediating PIP2-dependent recruitment of TIRAP to the plasma membrane. Background: Phosphoinositides are involved in regulating TLR4 signaling. Results: PIP5Kα knockdown in BV2 microglial cells inhibits LPS-induced inflammatory responses, PIP2 increase, and TIRAP translocation to the plasma membrane. Conclusion: PIP5Kα-derived PIP2 facilitates TLR4-mediated microglial inflammatory responses through recruitment of TIRAP to the plasma membrane. Significance: Regulation of PIP5Kα-dependent PIP2 pool may modulate TLR4-associated immune function in microglia.

Garcinia vilersiana is a traditional medicinal plant in Vietnam. The petroleum ether extract of stem bark of Garcinia vilersiana (GVE) was prepared to evaluate its potential to activate Nrf2, a transcription factor of antioxidant and detoxifying enzymes. Exposure of mouse macrophage RAW264.7 cells to GVE (0.625-2.5 µg/ml) resulted in a significant activation of Nrf2, as evaluated by nuclear accumulation of this transcription factor, and increased antioxidant response element (ARE) binding activity in a time- and concentration-dependent manner. As a result, GVE caused ARE-dependent up-regulation of heme oxygenase-1 (HO-1) in the cells. These results suggest that GVE contains components that have the ability to activate the Nrf2/ARE/HO-1 signaling pathway, leading to cellular protection.

Phospholipase D (PLD), which produces the lipid messenger phosphatidic acid (PA), has been implicated in superoxide generation and degranulation in neutrophils. The basis for this conclusion is the observation that primary alcohols, which interfere with PLD-catalyzed PA production, inhibit these neutrophil functions. However, off-target effects of primary alcohols cannot be totally excluded. Here, we generated PLD(-/-) mice in order to reevaluate the involvement of PLD in and investigate the molecular mechanisms of these neutrophil functions. Surprisingly, N-formyl-methionyl-leucyl-phenylalanine (fMLP) induced these functions in PLD(-/-) neutrophils, and these functions were suppressed by ethanol. These results indicate that PLD is dispensable for these neutrophil functions and that ethanol nonspecifically inhibits them, warning against the use of primary alcohols as specific inhibitors of PLD-mediated PA formation. The calcium ionophore ionomycin and the membrane-permeative compound 1-oleoyl-2-acetyl-sn-glycerol (OADG) synergistically induced superoxide generation. On the other hand, ionomycin alone induced degranulation, which was further augmented by OADG. These results demonstrate that conventional protein kinase C (cPKC) is crucial for superoxide generation, and a Ca(2+)-dependent signaling pathway(s) and cPKC are involved in degranulation in mouse neutrophils.

A small GTPase, Arf6, is involved in cytokinesis by localizing to the Flemming body (the midbody). However, it remains unknown how Arf6 contributes to cytokinesis. Here, we demonstrate that Arf6 directly interacts with mitotic kinesin-like protein 1 (MKLP1), a Flemming body-localizing protein essential for cytokinesis. The crystal structure of the Arf6–MKLP1 complex reveals that MKLP1 forms a homodimer flanked by two Arf6 molecules, forming a 2:2 heterotetramer containing an extended β-sheet composed of 22 β-strands that spans the entire heterotetramer, suitable for interaction with a concave membrane surface at the cleavage furrow. We show that, during cytokinesis, Arf6 is first accumulated around the cleavage furrow and, prior to abscission, recruited onto the Flemming body via interaction with MKLP1. We also show by structure-based mutagenesis and siRNA-mediated knockdowns that the complex formation is required for completion of cytokinesis. A model based on these results suggests that the Arf6–MKLP1 complex plays a crucial role in cytokinesis by connecting the microtubule bundle and membranes at the cleavage plane.

p38 mitogen-activated protein (MAP) kinase plays an important role in neurite outgrowth. However, the underlying molecular mechanism(s) remains unclear. Here, we demonstrate that phospholipase D2 (PLD2) mediates p38 signaling in neurite outgrowth. Stimulation of rat pheochromocytoma PC12 cells with nerve growth factor activated PLD2 and augmented neurite outgrowth, both of which were inhibited by pharmacological suppression of p38. Overexpression of constitutively active MAP kinase kinase 6 (MKK6-CA) activated coexpressed PLD2 in PC12 and mouse neuroblastoma N1E-115 cells. Overexpression of wild-type PLD2 in these cells strongly augmented the neurite outgrowth induced by MKK6-CA, whereas lipase-deficient PLD2 suppressed it. These findings provide evidence that PLD2 functions as a downstream molecule of p38 in the neurite outgrowth signaling cascade.

OBJECTIVE Phosphatidylinositol-4-phosphate-5-kinase (PI4P5K) has been proposed to facilitate regulated exocytosis and specifically insulin secretion by generating phosphatidylinositol-4,5-bisphosphate (PIP2). We sought to examine the role of the α isoform of PI4P5K in glucohomeostasis and insulin secretion. RESEARCH DESIGN AND METHODS The response of PI4P5Kα−/− mice to glucose challenge and a type 2-like diabetes-inducing high-fat diet was examined in vivo. Glucose-stimulated responses and PI4P5Kα−/− pancreatic islets and β-cells were characterized in culture. RESULTS We show that PI4P5Kα−/− mice exhibit increased first-phase insulin release and improved glucose clearance, and resist high-fat diet-induced development of type 2-like diabetes and obesity. PI4P5Kα−/− pancreatic islets cultured in vitro exhibited decreased numbers of insulin granules docked at the plasma membrane and released less insulin under quiescent conditions, but then secreted similar amounts of insulin on glucose stimulation. Stimulation-dependent PIP2 depletion occurred on the plasma membrane of the PI4P5Kα−/− pancreatic β-cells, accompanied by a near-total loss of cortical F-actin, which was already decreased in the PI4P5Kα−/− β-cells under resting conditions. CONCLUSIONS Our findings suggest that PI4P5Kα plays a complex role in restricting insulin release from pancreatic β-cells through helping to maintain plasma membrane PIP2 levels and integrity of the actin cytoskeleton under both basal and stimulatory conditions. The increased first-phase glucose-stimulated release of insulin observed on the normal diet may underlie the partial protection against the elevated serum glucose and obesity seen in type 2 diabetes-like model systems.

The predominant pathway for phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P(2)) synthesis is thought to be phosphorylation of phosphatidylinositol 4-phosphate at the 5 position of the inositol ring by type I phosphatidylinositol phosphate kinases (PIPK): PIPKIalpha, PIPKIbeta, and PIPKIgamma. PIPKIgamma has been shown to play a role in PI(4,5)P(2) synthesis in brain, and the absence of PIPKIgamma is incompatible with postnatal life. Conversely, mice lacking PIPKIalpha or PIPKIbeta (isoforms are referred to according to the nomenclature of human PIPKIs) live to adulthood, although functional effects in specific cell types are observed. To determine the contribution of PIPKIalpha and PIPKIbeta to PI(4,5)P(2) synthesis in brain, we investigated the impact of disrupting multiple PIPKI genes. Our results show that a single allele of PIPKIgamma, in the absence of both PIPKIalpha and PIPKIbeta, can support life to adulthood. In addition, PIPKIalpha alone, but not PIPKIbeta alone, can support prenatal development, indicating an essential and partially overlapping function of PIPKIalpha and PIPKIgamma during embryogenesis. This is consistent with early embryonic expression of PIPKIalpha and PIPKIgamma but not of PIPKIbeta. PIPKIbeta expression in brain correlates with neuronal differentiation. The absence of PIPKIbeta does not impact embryonic development in the PIPKIgamma knock-out (KO) background but worsens the early postnatal phenotype of the PIPKIgamma KO (death occurs within minutes rather than hours). Analysis of PIP(2) in brain reveals that only the absence of PIPKIgamma significantly impacts its levels. Collectively, our results provide new evidence for the dominant importance of PIPKIgamma in mammals and imply that PIPKIalpha and PIPKIbeta function in the generation of specific PI(4,5)P(2) pools that, at least in brain, do not have a major impact on overall PI(4,5)P(2) levels.

Developmental timing in the nematode Caenorhabditis elegans is controlled by heterochronic genes, mutations in which cause changes in the relative timing of developmental events. One of the heterochronic genes, let-7, encodes a microRNA that is highly evolutionarily conserved, suggesting that similar genetic pathways control developmental timing across phyla. Here we report that the nuclear receptor nhr-25, which belongs to the evolutionarily conserved fushi tarazu-factor 1/nuclear receptor NR5A subfamily, interacts with heterochronic genes that regulate the larva-to-adult transition in C. elegans. We identified nhr-25 as a regulator of apl-1, a homolog of the Alzheimer's amyloid precursor protein-like gene that is downstream of let-7 family microRNAs. NHR-25 controls not only apl-1 expression but also regulates developmental progression in the larva-to-adult transition. NHR-25 negatively regulates the expression of the adult-specific collagen gene col-19 in lateral epidermal seam cells. In contrast, NHR-25 positively regulates the larva-to-adult transition for other timed events in seam cells, such as cell fusion, cell division and alae formation. The genetic relationships between nhr-25 and other heterochronic genes are strikingly varied among several adult developmental events. We propose that nhr-25 has multiple roles in both promoting and inhibiting the C. elegans heterochronic gene pathway controlling adult differentiation programs.

The small GTPase ADP-ribosylation factor 6 (ARF6) plays crucial roles in a wide variety of cell functions. To better understand the molecular mechanisms of ARF6-mediated signaling and cellular functions, we sought new ARF6-binding proteins in the mouse brain. We identified the signaling scaffold protein JNK-interacting protein 3 (JIP3), which is exclusively expressed in neurons, as a downstream effector of ARF6. Overexpression of a unique dominant negative mutant of ARF6, which was unable to interact with JIP3, and knockdown of JIP3 in mouse cortical neurons stimulated the elongation and branching of neurites. These results provide evidence that ARF6/JIP3 signaling regulates neurite morphogenesis.Structured summaryMINT-7892698: PIP5K gamma 661 (uniprotkb:O70161) physically interacts (MI:0915) with Arf6 (uniprotkb:P62331) by anti tag coimmunoprecipitation (MI:0007)MINT-7892333, MINT-7892573, MINT-7892594, MINT-7892629, MINT-7892644, MINT-7892522, MINT-7892716: Arf6 (uniprotkb:P62331) physically interacts (MI:0915) with JLP (uniprotkb:Q58A65) by anti tag coimmunoprecipitation (MI:0007)MINT-7892509: Arf6 (uniprotkb:P62331) physically interacts (MI:0915) with JIP3 (uniprotkb:Q9ESN9) by pull down (MI:0096)MINT-7892770: Arf6 (uniprotkb:P62331) binds (MI:0407) to JIP3 (uniprotkb:Q9ESN9) by pull down (MI:0096)MINT-7892755: Arf6 (uniprotkb:P62331) binds (MI:0407) to JLP (uniprotkb:Q58A65) by pull down (MI:0096)MINT-7892289, MINT-7892314: Arf6 (uniprotkb:P62331) physically interacts (MI:0915) with JLP (uniprotkb:Q58A65) by pull down (MI:0096)MINT-7892353, MINT-7892615, MINT-7892657, MINT-7892672, MINT-7892549, MINT-7892738: Arf6 (uniprotkb:P62331) physically interacts (MI:0915) with JIP3 (uniprotkb:Q9ESN9) by anti tag coimmunoprecipitation (MI:0007)

Background: To maintain good health, it is important to eliminate extra reactive oxygen generated in the body. Furthermore, ingesting foods containing antioxidants is beneficial. The oxygen radical absorbance capacity (ORAC) values for black raspberry extract (BRE), blueberry extract (BBE), and raspberry extract (RBE) are 62, 66, and 49 µM Trolox equivalents (TE)/g respectively. These values are higher than those for typical antioxidant foods that have been discovered so far (3–30 µM TE/g). Our aim was to find new functionality from the food with the high ORAC value. Therefore, we have prepared these four berry extracts and examined whether they have anti-aging effects and if those effects correlate with their antioxidant activities. Methods: We studied the following effects of 4 berry extracts: 1) lens cell protective effects; 2) effects against alopecia; 3) induction of uncoupling protein-1 (UCP1), a regulator of fat and energy consumption in adipocytes, and stimulation of irisin secretion from skeletal muscle cells; and 4) inhibitory effects on melanocyte tyrosinase activity. The evaluation method was based on below; 1) a -crystallin , type 17 collagen, heat shock protein 47 (HSP47), UCP1 and Irisin - mRNA by qRT-PCR, 2) the amount of the UCP1 and Irisin protein by ELISA. 3) Inhibition of tyrosinase activity was measured by dopachrome production using L-tyrosine. Results: In lens cells, a -crystallin mRNA expression was induced 1 hour after treatment of the cell with Blabina (a powdered formulation containing BRE) and BRE. The extracts of all four berry species promoted the growth of follicle dermal papilla cells by 3-20% in a concentration-dependent manner. These berry extracts were also discovered to markedly induce the expression of mRNAs of type 17 collagen and HSP47 in the hair follicle stem cell and elevate the expression levels of UCP1 mRNA and its protein in adipocytes in a concentration-dependent manner. BRE and Blabina inhibited 5a-reductase in follicle dermal papilla cells and tyrosinase activity in melanocytes at the concentrations which inhibited dopachrome production by at least 50%. Finally, Blabina was discovered to stimulate the irisin secretion from skeletal muscles. Conclusion: These results suggest that berry extracts, particularly BRE, have anti-aging effects through their higher antioxidant activities. Keywords: Anti-aging; antioxidant; alopecia; black raspberry; weight loss; oxygen radical, absorbance capacity; skin whitening

Since the original discovery and structural elucidation of the mammalian phospholipase D (PLD), its potential to play a role in the lipid signalling pathway has attracted considerable interest. Now, it is generally accepted that different PLD isozymes are likely to serve diverse functions in membrane trafficking, endocytosis, exocytosis, cell growth, differentiation and actin cytoskeletal organization. In addition, PLDs are known to play a key role in neurite outgrowth, especially axon outgrowth, in neuronal cells.

During chemotaxis, activation of the small guanosine triphosphatase Rac is spatially regulated to organize the extension of membrane protrusions in the direction of migration. In neutrophils, Rac activation is primarily mediated by DOCK2, an atypical guanine nucleotide exchange factor. Upon stimulation, we found that DOCK2 rapidly translocated to the plasma membrane in a phosphatidylinositol 3,4,5-trisphosphate–dependent manner. However, subsequent accumulation of DOCK2 at the leading edge required phospholipase D–mediated synthesis of phosphatidic acid, which stabilized DOCK2 there by means of interaction with a polybasic amino acid cluster, resulting in increased local actin polymerization. When this interaction was blocked, neutrophils failed to form leading edges properly and exhibited defects in chemotaxis. Thus, intracellular DOCK2 dynamics are sequentially regulated by distinct phospholipids to localize Rac activation during neutrophil chemotaxis.

Cells sense nutrients present in the extracellular environment and modulate the activities of intracellular signaling systems in response to nutrient availability. This study demonstrates that RalA and its activator RalGDS participate in nutrient sensing and are indispensable for activation of mammalian target of rapamycin complex 1 (mTORC1) induced by extracellular nutrients. Knockdown of RalA or RalGDS abolished amino acid- and glucose-induced mTORC1 activation, as judged by phosphorylation of S6 kinase and eukaryotic translation initiation factor 4E-binding protein 1. The amount of GTP-bound RalA increased in response to increased amino acid availability. In addition, RalA knockdown suppressed Rheb-induced S6 kinase phosphorylation, and the constitutively active form of RalA induced mTORC1 activation in the absence of Rheb. These results collectively suggest that RalGDS and RalA act downstream of Rheb and that RalA activation is a crucial step in nutrient-induced mTORC1 activation.

The three isoforms of PIP5KI (α, β, and γ) synthesize PI4,5P 2 (PIP 2 ) by phosphorylating PI4P. Therefore, it is not clear why platelets, like all eukaryotic cells, have more than one isoform. To test the hypothesis that PIP5KI isoforms have nonoverlapping functions, we generated a murine line containing a null mutation of PIP5KIβ and analyzed the effect on platelet signaling. PIP5KIβ-null mice had normal platelet counts. In contrast to platelets lacking PIP5KIα, platelets lacking PIP5KIβ exhibited impaired aggregation accompanied by disaggregation. Although platelets lacking PIP5KIβ had only a moderate deficiency of PIP 2 under basal conditions, they had a striking deficiency in PIP 2 synthesis and IP 3 formation after thrombin stimulation. We have also observed that platelets lacking both PIP5KIα and PIP5KIβ have a complete loss of thrombin-induced IP 3 synthesis even though they still contain PIP5KIγ, the predominant PIP5KI isoform in platelets. These results demonstrate that PIP5KIβ, like PIP5KIα, contributes to the rapid synthesis of a pool of PIP 2 that is required for second-messenger formation, whereas the pool of PIP 2 synthesized by PIP5KIγ does not contribute to this process. Additionally, we found that PIP5KIβ-null platelets failed to form arterial thrombi properly in vivo . Together, these data demonstrate that PIP5KIβ is required for rapid PIP 2 synthesis, second-messenger production, and stable platelet adhesion under shear in vivo . These results also demonstrate that after stimulation of a G protein-coupled receptor, IP 3 is completely derived from a rapidly synthesized discrete pool of PIP 2 synthesized by PIP5KIα and PIP5KIβ.

Synaptic vesicles (SVs) are retrieved by clathrin-mediated endocytosis at the nerve terminals. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] drives this event by recruiting the components of the endocytic machinery. However, the molecular mechanisms that result in local generation of PI(4,5)P2 remain unclear. We demonstrate here that AP-2 complex directly interacts with phosphatidylinositol 4-phosphate 5-kinase gamma661 (PIP5Kgamma661), the major PI(4,5)P2-producing enzyme in the brain. The beta2 subunit of AP-2 was found to bind to the C-terminal tail of PIP5Kgamma661 and cause PIP5Kgamma661 activation. The interaction is regulated by PIP5Kgamma661 dephosphorylation, which is triggered by depolarization in mouse hippocampal neurons. Finally, overexpression of the PIP5Kgamma661 C-terminal region in hippocampal neurons suppresses depolarization-dependent SV endocytosis. These findings provide evidence for the molecular mechanism through which PIP5Kgamma661 locally generates PI(4,5)P2 in hippocampal neurons and suggest a model in which the interaction trigger SV endocytosis.

The phosphoinositide kinase, phosphatidylinositol 4-phosphate 5-kinase (PIP5K), produces the versatile phospholipid phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)), through which PIP5K plays crucial roles in a wide variety of cell functions. So far, three PIP5K isozymes and splicing variants have been identified. We speculate that each PIP5K isozyme or splicing variant is activated in a tempo-spatially different manner, due to the existence of activators or recruiters specific to each isozyme: this tempo-spatially different activation of PIP5K produces PI4,5P(2) at different compartments of the cell at different times, which phenomenon may be responsible for the apparent multifunction of PI4,5P(2)/PIP5K. Accumulating evidence supports this notion that each PIP5K isozyme is activated by its specific activator and plays a crucial role in a unique cell function. In this article, we describe recent advances regarding the PIP5K isozyme-specific activation mechanisms and physiological functions.

The small G protein Arf6 and the GTPase dynamin2 (Dyn2) play key roles in clathrin-mediated endocytosis (CME). However, their functional relationship remains obscure. Here, we show that Arf6 functions as a downstream molecule of Dyn2 in CME. Wild type of Dyn2 overexpressed in HeLa cells markedly activates Arf6, while a GTPase-lacking Dyn2 mutant does not. Of the Arf6-specific guanine nucleotide exchange factors, EFA6A, EFA6B and EFA6D specifically interact with Dyn2. Furthermore, overexpression of dominant negative mutants or knockdown of EFA6B and EFA6D significantly inhibit Dyn2-induced Arf6 activation. Finally, overexpression of the binding region peptide of EFA6B for Dyn2 or knockdown of EFA6B and EFA6D significantly suppresses clathrin-mediated transferrin uptake. These results provide evidence for a novel Arf6 activation mechanism by Dyn2 through EFA6B and EFA6D in CME in a manner dependent upon the GTPase activity of Dyn2.

Anti-angiogenic drugs targeting vascular endothelial cell growth factor receptor have provided modest clinical benefit, in part, owing to the actions of additional angiogenic factors that stimulate tumour neoangiogenesis in parallel. To overcome this redundancy, approaches targeting these other signalling pathways are required. Here we show, using endothelial cell-targeted mice, that the small GTPase Arf6 is required for hepatocyte growth factor (HGF)-induced tumour neoangiogenesis and growth. Arf6 deletion from endothelial cells abolishes HGF-stimulated β1 integrin recycling. Pharmacological inhibition of the Arf6 guanine nucleotide exchange factor (GEF) Grp1 efficiently suppresses tumour vascularization and growth. Grp1 as well as other Arf6 GEFs, such as GEP100, EFA6B and EFA6D, regulates HGF-stimulated β1 integrin recycling. These findings provide insight into the mechanism of HGF-induced tumour angiogenesis and offer the possibility that targeting the HGF-activated Arf6 signalling pathway may synergize with existing anti-angiogenic drugs to improve clinical outcomes.

Proper neutrophil migration into inflammatory sites ensures host defense without tissue damage. Phosphoinositide 3-kinase (PI(3)K) and its lipid product phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) regulate cell migration, but the role of PtdIns(3,4,5)P3-degrading enzymes in this process is poorly understood. Here, we show that Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1), a PtdIns(3,4,5)P3 phosphatase, is a key regulator of neutrophil migration. Genetic inactivation of SHIP1 led to severe defects in neutrophil polarization and motility. In contrast, loss of the PtdIns(3,4,5)P3 phosphatase PTEN had no impact on neutrophil chemotaxis. To study PtdIns(3,4,5)P3 metabolism in living primary cells, we generated a novel transgenic mouse (AktPH–GFP Tg) expressing a bioprobe for PtdIns(3,4,5)P3. Time-lapse footage showed rapid, localized binding of AktPH–GFP to the leading edge membrane of chemotaxing ship1+/+AktPH–GFP Tg neutrophils, but only diffuse localization in ship1−/−AktPH–GFP Tg neutrophils. By directing where PtdIns(3,4,5)P3 accumulates, SHIP1 governs the formation of the leading edge and polarization required for chemotaxis.

The tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) regulates diverse cellular functions by dephosphorylating the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate (PIP3). Recent study revealed that PICT-1/GLTSCR2 bound to and stabilized PTEN protein in cells, implicating its roles in PTEN-governed PIP3 signals. In this study, we demonstrate that RNA interference-mediated knockdown of PICT-1 in HeLa cells down-regulated endogenous PTEN and resulted in the activation of PIP3 downstream effectors, such as protein kinase B/Akt. Furthermore, the PICT-1 knockdown promoted HeLa cell proliferation; however the proliferation of PTEN-null cells was not altered by the PICT-1 knockdown, suggesting its dependency on PTEN status. In addition, apoptosis of HeLa cells induced by staurosporine or serum-depletion was alleviated by the PICT-1 knockdown in the similar PTEN-dependent manner. Most strikingly, the PICT-1 knockdown in HeLa and NIH3T3 cells promoted anchorage-independent growth, a hallmark of tumorigenic transformation. Furthermore, PICT-1 was aberrantly expressed in 18 (41%) of 44 human neuroblastoma specimens, and the PICT-1 loss was associated with reduced PTEN protein expression in spite of the existence of PTEN mRNA. Collectively, these results suggest that PICT-1 plays a role in PIP3 signals through controlling PTEN protein stability and the impairment in the PICT-1–PTEN regulatory unit may become a causative factor in human tumor(s).

Actin cytoskeletal reorganization and membrane trafficking are important for spine morphogenesis. Here we investigated whether the small GTPase, ADP-ribosylation factor 6 (ARF6), which regulates actin dynamics and peripheral vesicular trafficking, is involved in the regulation of spine formation. The developmental expression pattern of ARF6 in mouse hippocampus was similar to that of the post-synaptic density protein-95, and these molecules colocalized in mouse hippocampal neurons. Overexpression of a constitutively active ARF6 mutant in cultured hippocampal neurons decreased the spine density, whereas a dominant-negative ARF6 mutant increased the density. These results demonstrate a novel function for ARF6 as a key regulator of spine formation.

Cell division ends up with the membrane separation of two daughter cells, presumably by a membrane fusion that requires dynamic changes of the distribution and the composition of membrane lipids. We have previously shown that a membrane lipid phosphatidylethanolamine (PE) is exposed on the cell surface of the cleavage furrow during late cytokinesis and that this PE movement is involved in regulation of the contractile ring disassembly. Here we show that immobilization of cell surface PE by a PE-binding peptide blocks the RhoA inactivation in the late stage of cytokinesis. Phosphatidylinositol 4-phosphate 5-kinase (PIP5K), but not other RhoA effectors, is co-localized with RhoA in the peptide-treated cells. Indeed, PIP5K and its product phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) are localized to the cleavage furrow of normally dividing cells. Both overexpression of a kinase-deficient PIP5K mutant and microinjection of anti-PI(4,5)P(2) antibodies compromise cytokinesis by preventing local accumulation of PI(4,5)P(2) in the cleavage furrow. These findings demonstrate that the localized production of PI(4,5)P(2) is required for the proper completion of cytokinesis and that the possible formation of a unique lipid domain in the cleavage furrow membrane may play a crucial role in coordinating the contractile rearrangement with the membrane remodeling during late cytokinesis.

Mammalian phospholipase D (PLD), a multifunctional signaling enzyme, has been reported to facilitate neurite outgrowth in cultured neurons. However, two mammalian isoforms have been found, PLD1 and PLD2, and it has not been determined which isoform is involved, or whether this in vitro phenomenon is relevant to neurite extension in vivo. Using confocal microscopy, we demonstrate that the PLDs are expressed by different cell types in the mouse brain: PLD1 by neurons, and PLD2 by astrocytes. Moreover, using a model of experimentally induced hippocampal mossy fiber sprouting, both isoforms were observed to increase dramatically in expression level along tracts of mossy fiber spouting, supporting the proposal that PLD plays a role in this process. Given that the two isoforms undertake unique molecular functions in cultured cells, our findings suggest that in vivo PLD1 and PLD2 may modulate neuronal plasticity via different pathways and cell types.

Stimulation of the epidermal growth factor receptor (EGFR) produces membrane ruffles through the small G protein Rac1; however, the signaling pathway from EGFR to Rac1 has not yet been clarified. Here, we show that autophosphorylation of EGFR at tyrosine 992 is required for EGF-induced membrane ruffle formation in CHO cells. Signaling from the autophosphorylated tyrosine 992 appears to be mediated by phospholipase C (PLC) gamma 1. Furthermore, activation of Rac1 by EGF is inhibited by a PLC inhibitor. These results, taken together, suggest that autophosphorylation of EGFR at tyrosine 992 and the subsequent PLC gamma 1 activation transduce the signal to Rac1 to induce membrane ruffle formation.

Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) is absolutely required for the ADP-ribosylation factor-stimulated phospholipase D (PLD) activity. In the present study, partially purified rat brain PLD was found to be activated by another PLD activator, RhoA, when PIP 2 but not other acidic phospholipids, was included in vesicles comprising phosphatidylethanolamine (PE) and the PLD substrate phosphatidylcholine (PC) (PE/PC vesicles), demonstrating the absolute requirement of PIP 2 for the RhoA-stimulated PLD activation, too. It is interesting that the RhoA-dependent PLD activity in the partially purified preparation was drastically decreased after the preparation was incubated with and separated from PE/PC vesicles containing PIP 2 . The PLD activity was extracted by higher concentrations of NaCl from the vesicles containing PIP 2 that were incubated with and then separated from the partially purified PLD preparation. These results demonstrate that RhoA-dependent PLD binds to PE/PC vesicles with PIP 2 . The degree of binding of the RhoA-dependent PLD activity to the vesicles was totally dependent on the amount of PIP 2 in the vesicles and correlated well with the extent of the enzyme activation. Furthermore, it was found that a recombinant peptide of the pleckstrin homology domain of β-adrenergic receptor kinase fused to glutathione S-transferase, which specifically binds to PIP 2 , inhibited the PIP 2 -stimulated, RhoA-dependent PLD activity in a concentration-dependent manner. From these results, it is concluded that in vitro rat brain PLD translocates to the vesicles containing PIP 2 , owing to its specific interaction with PIP 2 , to access its substrate PC, thereby catalyzing the hydrolysis of PC. PLD appears to localize exclusively on plasma membranes of cells and tissues. An aminoglycoside, neomycin, that has high affinity for PIP 2 effectively extracted the RhoA-dependent PLD activity from rat brain membranes. This indicates that PIP 2 serves as an anchor to localize PLD on plasma membranes in vivo.

Phosphatidylinositol 4-phosphate 5-kinase (PIP-5kin) regulates actin cytoskeletal reorganization through its product phosphatidylinositol 4,5-bisphosphate. In the present study we demonstrate that PIP-5kin is essential for neurite remodeling, which is regulated by actin cytoskeletal reorganization in neuroblastoma N1E-115 cells. Overexpression of wild-type mouse PIP-5kin-alpha inhibits the neurite formation that is normally stimulated by serum depletion, whereas a lipid kinase-defective mutant of PIP-5kin-alpha, D266A, triggers neurite extension even in the presence of serum and blocks lysophosphatidic acid-induced neurite retraction. These results phenocopy those previously reported for the small GTPase RhoA and its effector p160 Rho-associated coiled coil-forming protein kinase (ROCK). However, the ROCK-specific inhibitor Y-27632 failed to block the inhibition by PIP-5kin-alpha of neurite extension, whereas D266A did block the neurite retraction induced by overexpression of ROCK. These results, taken together, suggest that PIP-5kin-alpha functions as a downstream effector for RhoA/ROCK to couple lysophosphatidic acid signaling to neurite retraction presumably through its product phosphatidylinositol 4,5-bisphosphate.