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In rabbit peritoneal neutrophils prelabeled with [3H] lyso platelet-activating factor, a protein kinase C inhibitor, staurosporine (> 1 microM), increased [3H]phosphatidylethanol ([3H]PEt) level in the presence of ethanol in a concentration- and time-dependent manner, providing evidence for staurosporine activation of phospholipase D (PLD). The staurosporine activation of the enzyme absolutely required both extracellular calcium and cytochalasin B, and was almost completely inhibited by pretreatment of the cells with pertussis toxin (IAP). In a reconstituted system where the purified Gi1 had been incorporated into phospholipid vesicles, staurosporine activated GTPase activity of Gi1 in a concentration-dependent fashion, with a maximal 4-5-fold effect. ADP-ribosylation by IAP of Gi1 in vesicles significantly suppressed the staurosporine activation. As with the GTPase activity of Gi1, GTPase activities of other purified IAP-sensitive G proteins, such as Gi2 and G(o), were significantly stimulated by staurosporine, but the cholera toxin substrate Gs was appreciably less sensitive to the staurosporine stimulation. The staurosporine activation of GTPase was also observed in rabbit neutrophil membranes from control cells, but not in membranes from IAP-treated neutrophils. From these results, we conclude that the staurosporine activation of PLD in rabbit neutrophils is attributed to the direct activation of an IAP-sensitive G protein in a similar manner to receptors occupied by agonists. By contrast, staurosporine failed to activate phosphoinositide-specific phospholipase C (PI-PLC) under the conditions in which it activated PLD, indicating that there exists a PLD activation pathway independent of PI-PLC. Furthermore, it was found that N-acetyl-beta-glucosaminidase release from the granules of intact neutrophils was evoked by staurosporine to almost the same extent as by fMLP (100 nM), but O2- generation was not affected. These results suggest a possibility that PLD pathway plays an important role in enzyme release, but is not sufficient for O2- generation, in rabbit peritoneal neutrophils.

Phosphatidic acid (PA), which can be produced by phospholipase D (PLD), is involved in various signaling events, such as cell proliferation, survival, and migration. However, the molecular mechanisms that link PA to cell migration are largely unknown. Here, we show that PA binds to the tyrosine kinase Fer and enhances its ability to phosphorylate cortactin, a protein that promotes actin polymerization. We found that a previously unknown lipid-binding module in Fer adjacent to the F-BAR [Fes-Cdc42-interacting protein 4 (CIP4) homology (FCH) and bin-amphiphysin-Rvs] domain mediated PA binding. We refer to this lipid-binding domain as the FX (F-BAR extension) domain. Overexpression of Fer enhanced lamellipodia formation and cell migration in a manner dependent on PLD activity and the PA-FX interaction. Thus, the PLD-PA pathway promotes cell migration through Fer-induced enhancement of actin polymerization.

We have characterized a soluble pertussis toxin (PT)-sensitive GTP-binding protein (G-protein) present in mouse mastocytoma P-815 cells. 65% of total ADP-ribosylation of PT substrate having a molecular mass of 40 kDa on SDS-polyacrylamide gel electrophoresis in cell homogenate was detected in the supernatant after centrifugation at 100,000 x g for 90 min. [32P]ADP-ribosylation of cytosolic PT substrate was significantly enhanced on the addition of exogenous beta gamma complex. The molecular mass of the cytosolic PT substrate was estimated to be about 80 kDa on an Ultrogel AcA 44 column, but the beta gamma complex was not detected in the cytosol by using the anti-beta gamma complex antibody. Furthermore, the cytosolic PT substrate was found to have some unique properties: [35S]GTP gamma S binding was not inhibited by GDP and [32P]ADP-ribosylation was not affected by GTP gamma S treatment. Only after the cytosolic PT substrate had been mixed with exogenous beta gamma complex, did it copurify with exogenous beta gamma complex by several column chromatographies including an Octyl-Sepharose CL-4B column. The PT substrate was identified as Gi2 alpha by Western blot analysis and peptide mapping with S. aureus V8 protease. These results suggest that Gi2 alpha without beta gamma complex exists with an apparent molecular mass of about 80 kDa in the cytosolic fraction of P-815 cells.

In the present study, we examined the ability of detergents to stimulate and solubilize phospholipase D (PLD) of a particulate fraction of rat brain. PLD activity was assayed by measuring the [3H]choline produced from the exogenous substrate dipalmitoyl phosphatidyl[3H]choline (dipalmitoyl [3H]PC). In the absence of detergents, PLD activity was not detectable. Of the detergents examined, Triton X-100 was found to markedly enhance PLD activity, whereas other detergents including sodium deoxycholate, sodium cholate, CHAPS and Lubrol-PX caused only a small, if any increase in activity. Enhancement by Triton X-100 was maximal at 0.1-0.2% (w/v) and decreased at higher concentrations. The optimal pH was 7.1-7.3. Both Ca2+ and Mg2+ inhibited enzyme activity stimulated by Triton X-100 in a concentration-dependent manner. Triton X-100 effectively solubilized PLD from the particulate fraction of rat brain; more than 70% of the activity of the particulate fraction was extracted by 0.5-1.0% (w/v) Triton X-100. Furthermore, when the PLD activities in brains of three different species (rat, rabbit and bovine) were measured under optimal conditions, the activities were found to differ greatly. PLD activity was highest in rabbit brain, followed by rat and bovine brains; the activity in bovine brain was extremely low compared to the activities in rat and rabbit brains. We conclude that Triton X-100 is potentially useful for the purification of PLD and that rabbit and rat brains are the preferred sources.

Summary In the present study, we investigated the involvement of protein kinase C (PKC) in antigen (Ag, DNP-Ascaris suum)-induced phospholipase D (PLD) activation of rat peritoneal mast cells. Phorbor myristate acetate (PMA) as well as Ag activated PLD as inferred by phosphatidylethanol (PEt) production. PKC inhibitors, staurosporine and H-7, however, failed to suppress PMA-stimulated PLD activation, suggesting that PLD activation by PMA is independent of PKC. By contrast, Ag-stimulated PLD activity was significantly reduced by staurosporine and slightly by H-7. Surprisingly, the inhibitors inhibited Ag-stimulated phospholipase C (PLC), correlated to the inhibition of PLD. These observations lead us to conclude that in Ag-stimulated mast cells 1, 2-diacylglycerol (DG) formed by PLC directly or indirectly stimulates PLD, independently of PKC.

Stimulation by fMet-Leu-Phe analogs of GTPase activity in plasma membranes from rabbit neutrophils was compared with the stimulation of degranulation in intact neutrophils. All four formyl peptides examined (fMet-Leu-Phe-Phe, fMet-Leu-Phe, fNle-Leu-Phe, and fVal-Leu-Phe) were full agonists for both responses. Their ED50 values for the two responses correlated well, although those for GTPase stimulation were uniformly about tenfold greater. The specific antagonist tBoc-Phe-Leu-Phe-Leu-Phe competitively inhibited both GTPase activity and degranulation stimulated by fMet-Leu-Phe; its K values were similar for the two responses. Pertussis toxin treatment, in contrast, inhibited the maximal stimulation of both responses by fMet-Leu-Phe with minimal shift in ED50. The inhibitory actions of tBoc-Phe-Leu-Phe-Leu-Phe and pertussis toxin on GTPase activity thus paralleled the effects on degranulation. These observations substantiate the hypothesis that a guanine nucleotide-binding protein that is a pertussis toxin substrate couples the formyl peptide receptors to physiological function in the neutrophil.

The remarkably versatile phospholipid, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], plays crucial roles in signal transduction, actin cytoskeleton reorganization, clathrin-dependent endocytosis, and regulation of membrane morphology. In mammalian cells, PI(4,5)P(2) is synthesized predominantly by phosphatidylinositol 4-phosphate [PI(4)P] 5-kinase (PIP5K) through phosphorylation of PI(4)P at the D-5 position of the inositol ring. PIP5K is composed of three isoforms, PIP5Kalpha, beta, and gamma, and three splicing variants of the gamma isozyme. Although the PIP5Kgamma splicing variant PIP5Kgamma661 appears to be very specifically activated by talin, which plays a crucial role in focal adhesion formation, and the adaptor complex AP-2, the regulation of activities of other PIP5K isozymes is not fully understood at present. To understand the activation mechanism and the physiological function specific to each PIP5K isozyme, it is required to identify a specific activator of each PIP5K isozyme. This chapter describes common assays used to measure interaction and activation of PIP5K isozymes by activators thus far identified. In addition, procedures for preparation of PIP5K isozymes and activators are described.

Phosphatidylinositol-4,5-bisphosphate (PIP(2)) is an abundant phospholipid that contributes to second messenger formation and has also been shown to contribute to the regulation of cytoskeletal dynamics in all eukaryotic cells. Although the alpha, beta, and gamma isoforms of phosphatidylinositol-4-phosphate-5-kinase I (PIP5KI) all synthesize PIP2, mammalian cells usually contain more than one PIP5KI isoform. This raises the question of whether different isoforms of PIP5KI fulfill different functions. Given the speculated role of PIP(2) in platelet and megakaryocyte actin dynamics, we analyzed murine megakaryocytes lacking individual PIP5KI isoforms. PIP5KIgamma(-/-) megakaryocytes exhibited plasma membrane blebbing accompanied by a decreased association of the membrane with the cytoskeleton. This membrane defect was rescued by adding back wild-type PIP5KIgamma, but not by adding a catalytically inactive mutant or a splice variant lacking the talin-binding motif. Notably, both PIP5KIbeta- and PIP5KIgamma(-/-) cells had impaired PIP(2) synthesis. However, PIP5KIbeta-null cells lacked the membrane-cytoskeleton defect. Furthermore, overexpressing PIP5KIbeta in PIP5KIgamma(-/-) cells failed to revert this defect. Megakaryocytes lacking the PIP5KIgamma-binding partner, talin1, mimicked the membrane-cytoskeleton defect phenotype seen in PIP5KIgamma(-/-) cells. These findings demonstrate a unique role for PIP5KIgamma in the anchoring of the cell membrane to the cytoskeleton in megakaryocytes, probably through a pathway involving talin. These observations further demonstrate that individual PIP5KI isoforms fulfill distinct functions within cells.

The remarkably versatile phospholipid, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ], plays crucial roles in signal transduction, actin cytoskeleton reorganization, clathrin-dependent endocytosis, and regulation of membrane morphology. In mammalian cells, PI(4,5)P 2 is synthesized predominantly by phosphatidylinositol 4-phosphate [PI(4)P] 5-kinase (PIP5K) through phosphorylation of PI(4)P at the D-5 position of the inositol ring. PIP5K is composed of three isoforms, PIP5Kα, β, and γ, and three splicing variants of the γ isozyme. Although the PIP5Kγ splicing variant PIP5Kγ661 appears to be very specifically activated by talin, which plays a crucial role in focal adhesion formation, and the adaptor complex AP-2, the regulation of activities of other PIP5K isozymes is not fully understood at present. To understand the activation mechanism and the physiological function specific to each PIP5K isozyme, it is required to identify a specific activator of each PIP5K isozyme. This chapter describes common assays used to measure interaction and activation of PIP5K isozymes by activators thus far identified. In addition, procedures for preparation of PIP5K isozymes and activators are described.

The small GTPase RhoA regulates a wide spectrum of cellular functions including transformation and cytoskeletal reorganization. A large number of proteins have been identified as targets of RhoA, but their specific roles in these processes are not clear. Phospholipase D (PLD) was shown to be one such target several years ago; more recent work from our laboratory and others has demonstrated that of the two mammalian PLD isozymes, PLD1 but not PLD2 is activated by RhoA and this activation proceeds through direct binding both in vitro and in vivo. In this study, using a series of RhoA mutants, we have defined a PLD1-specific interacting site on RhoA composed of the residues Asn41, Trp58 and Asp76, using the yeast two-hybrid system, co-immunoprecipitation, and a PLD in vivo assay. The results further substantiate our previous finding that RhoA activates PLD1 through direct interaction. These mutants were then used to investigate the role of PLD1 in the cytoskeletal reorganization stimulated by RhoA signaling. Our results show that PLD1 is not required for the RhoA-mediated stress fiber and focal adhesion formation. The lack of importance of PLD1 signaling in RhoA-mediated cytoskeletal reorganization is further supported by the observation that PLD1 depletion using an shRNA approach and tetracycline-induced overexpression of the wild-type and the catalytically inactive mutant of PLD1 in stable cell lines do not alter stress fiber and focal adhesion formation.

The membrane phospholipid phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P(2)] is a critical signal transducer in eukaryotic cells. However, the physiological roles of the type I phosphatidylinositol phosphate kinases (PIPKIs) that synthesize PI(4,5)P(2) are largely unknown. Here, we show that the alpha isozyme of PIPKI (PIPKIalpha) negatively regulates mast cell functions and anaphylactic responses. In vitro, PIPKIalpha-deficient mast cells exhibited increased degranulation and cytokine production after Fcepsilon receptor-I cross-linking. In vivo, PIPKIalpha(-/-) mice displayed enhanced passive cutaneous and systemic anaphylaxis. Filamentous actin was diminished in PIPKIalpha(-/-) mast cells, and enhanced degranulation observed in the absence of PIPKIalpha was also seen in wild-type mast cells treated with latrunculin, a pharmacological inhibitor of actin polymerization. Moreover, the association of FcepsilonRI with lipid rafts and FcepsilonRI-mediated activation of signaling proteins was augmented in PIPKIalpha(-/-) mast cells. Thus, PIPKIalpha is a negative regulator of FcepsilonRI-mediated cellular responses and anaphylaxis, which functions by controlling the actin cytoskeleton and dynamics of FcepsilonRI signaling. Our results indicate that the different PIPKI isoforms might be functionally specialized.

Temporal lobe epilepsy (TLE) is the most common form of epilepsy, affecting ∼1-2% of the population. Seizure events resulting from TLE are characterized by aberrant hippocampal mossy fiber sprouting and plastic responses that affect brain function. Seizure susceptibility is modulated by the enzyme tissue plasminogen activator (tPA), the normal physiological role of which includes promotion of synaptic reorganization in the mossy fiber pathway by initiating a proteolytic cascade that cleaves extracellular matrix components and influences neurite extension. tPA is concentrated at and selectively secreted from growth cones during excitatory events. However, the mechanisms underlying tPA release during seizure-induced synaptogenesis are not well understood. We examine here potential roles for the signaling enzyme phospholipase D1 (PLD1), which promotes regulated exocytosis in non-CNS cell types, and which we previously demonstrated increases in expression in hippocampal neurons during seizure-induced mossy fiber sprouting. We now show that overexpression of wild-type PLD1 in cultured neurons promotes tPA release and tPA-dependent neurite extension, whereas overexpression of an inactive PLD1 allele or pharmacological inhibition of PLD1 inhibits tPA release. Similarly, viral delivery of wild-type PLD1 into the hippocampus facilitates tPA secretion and mossy fiber sprouting in a seizure-inducing model, whereas the inactive PLD1 allele inhibits tPA release and elicits blunted and abnormal mossy fiber extension similar to that observed for tPA-/-mice. Together, these findings secretion and thus mossy fiber extension in the setting of elevated suggest that PLD1 functions endogenously to regulate tPA-/-neuronal stimulation, such as that seen in TLE.

Bin1/M-amphiphysin-II is an amphiphysin-II isoform highly expressed in transverse tubules of adult striated muscle and is implicated in their biogenesis. Bin1 contains a basic unique amino-acid sequence, Exon10, which interacts with certain phosphoinositides such as phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), to localize to membranes. Here we found that Exon10 also binds to the src homology 3 (SH3) domain of Bin1 itself, and hence blocks the binding of the SH3 domain to its canonical PxxP ligands, including dynamin. This blockage was released by addition of PI(4,5)P(2) in vitro or in cells overexpressing phosphatidylinositol 4-phosphate 5-kinase. The Exon10-binding interface of the Bin1 SH3 domain largely overlapped with its PxxP-binding interface. We also show that the PLCdelta pleckstrin homology domain, another PI(4,5)P(2)-binding module, cannot substitute for Exon10 in Bin1 function in transverse tubule formation, and suggest the importance of the dual biochemical properties of Exon10 in myogenesis. Our results exemplify a novel mechanism of SH3 domain regulation, and suggest that the SH3-mediated protein-protein interactions of Bin1 are regulated by Exon10 so that it may only occur when Bin1 localizes to certain submembrane areas.

The tumor suppressor PTEN plays an essential role in regulating signaling pathways involved in cell growth and apoptosis and is inactivated in a wide variety of tumors. In this study, we have identified a protein, referred to as PICT-1 (protein interacting with carboxyl terminus 1), that binds to the C terminus of PTEN and regulates its phosphorylation and turnover. Down-regulation of PICT-1 in MCF7 cells by RNA interference enhances the degradation of PTEN with a concomitant decrease in its phosphorylation. PTEN C-terminal tumor-associated mutants, which are highly susceptible to protein degradation, have lost the ability to bind to PICT-1 along with their reduced phosphorylation, suggesting that their rapid turnover results from impaired binding to PICT-1. Our results identify PICT-1 as a PTEN-interacting protein that promotes the phosphorylation and stability of PTEN. These findings suggest a novel molecular mechanism underlying the turnover of PTEN, which also provides an explanation for the loss of PTEN function due to C-terminal mutations.

The signaling pathway that triggers morphological differentiation of PC12 cells is mediated by extracellular signal-regulated kinase (ERK), the classic mitogen-activated protein (MAP) kinase. However, mediators of the pathway downstream of ERK have not been identified. We show here that phospholipase D2 (PLD2), which generates the pleiotropic signaling lipid phosphatidic acid (PA), links ERK activation to neurite outgrowth in nerve growth factor (NGF)-stimulated PC12 cells. Increased expression of wild type PLD2 (WT-PLD2) dramatically elongated neurites induced by NGF stimulation or transient expression of the active form of MAP kinase-ERK kinase (MEK-CA). The response was activity-dependent, because it was inhibited by pharmacological suppression of the PLD-mediated PA production and by expression of a lipase-deficient PLD2 mutant. Furthermore, PLD2 was activated by MEK-CA, whereas NGF-stimulated PLD2 activation and hypertrophic neurite extension were blocked by an MEK-specific inhibitor. Taken together, these results provide evidence that PLD2 functions as a downstream signaling effector of ERK in the NGF signaling pathway, which leads to neurite outgrowth by PC12 cells.

a Department of Pharmacology, Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan b Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Tokyo 192-0397, Japan c Laboratory of Molecular Pharmacology, Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, Kagawa 769-2193, Japan d Division of Biochemistry, BEX Co. Ltd., Tokyo 173-0004, Japan e Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8120, Japan

The tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) plays essential roles in regulating signalling pathways involved in cell growth and apoptosis, and is inactivated in a wide variety of tumours. The role of PTEN as a tumour suppressor has been firmly established; however, the mechanism(s) by which its function and activity are regulated remains elusive. Here, we summarize recent progress in research directed towards trying to understand the molecular basis of regulatory mechanisms for PTEN. We also describe our novel finding that a tumour suppressor candidate protein binds to extreme C-terminal region of PTEN and regulates PTEN protein turnover.

Stimulation of the neuronal cell adhesion molecule L1 in cerebellar granule neurons (CGNs) enhances neurite outgrowth and this response is inhibited by the primary alcohol ethanol. Because primary alcohols suppress the formation of the signaling lipid phosphatidic acid (PA) by phospholipase D (PLD), this observation prompted us to investigate whether PLD plays a role in the L1-mediated neurite outgrowth in CGNs. In the cerebellum of postnatal day 8 mice, PLD2 protein was abundantly expressed, while PLD1 expression was not detected. The L1-stimulated neurite outgrowth was inhibited by primary alcohols and by overexpression of lipase-deficient PLD2. Increases in cellular PA levels by direct PA application or overexpression of wild-type PLD2 mimicked the L1-dependent stimulation of neurite outgrowth. Furthermore, it was found that L1 stimulation in CGNs increased PLD activity concomitantly with phosphorylation of extracellular signal-regulated kinase (ERK), both of which were inhibited by the MAP kinase-ERK kinase (MEK) inhibitor. These results provide evidence that PLD2 functions as a downstream signaling molecule of ERK to mediate the L1-dependent neurite outgrowth of CGNs, a mechanism that may be related to alcohol-related neurodevelopmental disorders.

Drosophila PTEN (dPTEN) plays indispensable roles in the development of Drosophila melanogaster by controlling cell size and number. Although three potential spliced forms of dPTEN have been isolated, functional distinction among these forms remains elusive. In this study, we demonstrate that all spliced forms of dPTEN dephosphorylate phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3); however, PI(3,4,5)P3-dependent activation of Drosophila Akt is suppressed specifically by one of three spliced forms, dPTEN3. Further, dPTEN3 dramatically changes its expression during the Drosophila development, while the other forms are expressed throughout the development. Our results suggest that dPTEN3 is the predominant spliced form that participates in PI(3,4,5)P3-mediated signaling pathways.

Leukotriene B(4) (LTB(4)) is a potent lipid mediator involved in host defense and inflammatory responses. It causes chemotaxis, generation of reactive oxygen species, and degranulation. However, only little is known of the molecular mechanisms by which LTB(4) induces these biological activities. To analyze the intracellular signaling pathways to mediate lysosomal enzyme release through the cloned LTB(4) receptor (BLT1), we transfected BLT1 to rat basophilic leukemia cells (RBL-2H3). LTB(4) dose-dependently released beta-hexosaminidase, and the release was mostly inhibited when the cells were pretreated with pertussis toxin, indicating that the degranulation is mediated by G(i) proteins. LTB(4) activated phosphatidylinositol 3-kinase (PI3-K) through G(i), and inhibition of PI3-K by wortmannin or LY290042 inhibited degranulation. Granulocytes from PI3-Kgamma-deficient mice showed reduced LTB(4)-induced degranulation, suggesting that this isozyme of PI3-K is involved in the degranulation. LTB(4) also caused calcium release from intracellular stores and calcium influx from the outside milieu through G(i), but only the calcium influx is critical for the lysosomal enzyme release. Calcium influx and PI3-K activation are both downstream events of G(i), since they were inhibited by pertussis toxin. These two events are in essence independent each other, because calcium depletion did not affect PI3-K, and inhibition of PI3-K did not attenuate calcium influx significantly. Thus, our results have clearly shown that LTB(4) binds BLT1 and activates G(i)-like protein, and both PI3-Kgamma activation and a sustained calcium elevation by calcium influx are necessary for enzyme release in these cells.

We compared, by immunoblotting and immunohistochemical staining, the distribution of the alpha subunit of Go in human normal brain tissue with that in human brain tumours of neuroectodermal origin. The ten tumour samples included seven high-grade astrocytomas (grades III and IV) and three low-grade astrocytomas (grade II). Immunoreactivity of high-grade astrocytomas with the alpha subunit of Go (Go alpha) antibody was lower than that of low-grade astrocytomas and normal white matter. In the immunohistochemical study, neoplastic cells in the astrocytomas were found to contain little or no significant Go alpha, although nonlesional white matter was strongly stained with Go alpha antibody. This study suggests that Go alpha antibody may assist in defining the boundary of an astrocytoma, and the difference in Go content may affect the alteration in the physical properties of the membrane, which exhibit, in part, the character of the transformation.

We investigated the interaction of ADP-ribosylation factor (Arf) with the secretory granules in rat parotid acinar cells. The 20.5-kDa small-molecular-mass GTP-binding protein in the cytosolic fraction of rat parotid acinar cells was identified as ADP-ribosylation factor1 by using a pan-Arf monoclonal antibody and isotype-specific polyclonal antibodies for Arf proteins 1, 3, 5, and 6. Incubation of the cytosolic fraction with isolated secretory granule membranes in the presence of GTPγS resulted in the translocation of Arf1 from the cytosolic fraction to the secretory granule membranes. The translocation was not observed in the presence of GDPβS in place of GTPγS, indicating that the process is GTP-dependent. The immunoelectron microscopy experiment confirmed Arf1 is translocated to the secretory granules. A prior treatment of the granule membranes with trypsin inhibited the translocation of Arf1 at 2 mM Mg 2+ , but had no effect in the absence of Mg 2+ (condition of spontaneous conversion of Arf-GDP to Arf-GTP). Thus, the trypsin-sensitive nucleotide exchange activity for Arf1 is probably associated with the secretory granule membranes. These results demonstrate Arf1 translocates to the secretory granules in rat parotid acinar cells.

Activation of phospholipase D (PLD) is involved in receptor-mediated signal transduction responses. Signaling from PLD to a downstream molecule(s) appears to be mediated by the PLD product phosphatidic acid (PA). A target molecule(s) of PA, however, has not yet been identified. The present study sought to define such a target molecule(s) of PA. In bovine brain cytosol, proteins with apparent molecular weights of 29,000 (p29) and 32,000 (p32) were prominently phosphorylated in the presence of PA, but not in its absence, indicating that there is a PA-regulated protein kinase (PARK) in bovine brain that phosphorylates p29 and p32. One of these substrates, p29, was purified to near homogeneity. Its partial amino acid sequence was determined and found to be identical to that of a known brain-specific 25-kDa protein (p25). The purified p29 was also readily recognized by and immunoprecipitated with an anti-p25 antibody. These results suggest that p29 is very similar to or identical with p25. Using the purified p29 as a substrate, PARK was purified to near homogeneity. The purified PARK had an apparent molecular weight of 80,000, was strongly recognized by an antiprotein kinase C (PKC)alpha antibody, and was activated by phosphatidylserine (PS) as well as PA. The PA- and PS-stimulated PARK activity was extremely augmented by the presence of 1 microM free Ca2+. In the presence of 1 mM EGTA, phorbol 12-myristate 13-acetate activated PARK synergistically with PA or PS. Similar results were obtained with the purified recombinant PKCalpha. From these results, it is suggested that the PARK activity purified might be attributed to PKCalpha. In p25-depleted bovine brain cytosol, which was prepared by treatment of bovine brain cytosol with the anti-p25 antibody, PA-dependent phosphorylation of p29, but not p32, was almost completely eliminated. When PKCalpha in bovine brain cytosol was depleted by its precipitation with the anti-PKCalpha antibody, neither p29 nor p32 in this PKCalpha-depleted cytosol was phosphorylated in the presence of PA. These results indicate that in bovine brain cytosol PA activates PKCalpha, which, in turn, phosphorylates p29, which may be identical with p25.

We have purified a protein that binds phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] using beads bearing a PtdIns(3,4,5)P3 analogue. This protein, with a molecular mass of 43 kDa, was termed PtdIns(3,4,5)P3-binding protein. The partial amino acid sequences were determined and a full-length cDNA encoding the protein was isolated from bovine brain cDNA library. The clone harbored an open reading frame of 373 amino acids which contained one zinc finger motif similar to that of ADP-ribosylation-factor GTPase-activating protein and two pleckstrin homology domains. The entire sequence was 83% similar to centaurin alpha, another PtdIns(3,4,5)P3-binding protein. The protein bound PtdIns(3,4,5)P3 with a higher affinity than it did inositol 1,3,4,5-tetrakisphosphate, phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3-phosphate suggesting that the binding to PtdIns(3,4,5)P3 was specific. The binding activity was weaker in the mutants with a point mutation in the conserved sequences in each pleckstrin homology domain. Introduction of both mutations abolished the activity. These results suggest that this new binding protein binds PtdIns(3,4,5)P3 through two pleckstrin domains present in the molecule.

Phospholipase D (PLD) is believed to play an important role in cell signal transduction: PLD catalyzes the hydrolysis primarily of phosphatidylcholine (PC) to produce phosphatidic acid that may serve as a lipid second messenger. Although the mechanism of PLD activation has not yet been fully understood, a member of the low molecular weight GTP-binding protein (small G protein) superfamily, ADP-ribosylation factor (ARF), has been identified as a PLD-activating factor. In addition to ARF, we found that RhoA, another member of the small G proteins, activated rat brain PLD, and that ARF and RhoA synergistically stimulated the enzyme activity. When proteins of bovine brain cytosol were subjected to anion exchange column chromatography and then reconstituted with rat brain PLD partially purified from the membranes, fractions eluted at 60 mM NaCl, where ARF was not detected, activated the enzyme in a guanosine 5′- O -(3-thiotriphosphate)-dependent manner. This PLD-stimulating activity seemed to be attributed to a small G protein RhoA. Evidence provided includes the findings that: (1) the partially purified preparation of the PLD-activating factor by subsequent column chromatographies contained a 22 kDa substrate for botulinum C3 exoenzyme ADP-ribosyltransferase; (2) the 22 kDa protein strongly reacted with anti-RhoA antibody; (3) the treatment of the partially purified PLD-activating factor with C3 exoenzyme and NAD together, but not individually, significantly inhibited the PLD-stimulating activity; and (4) recombinant isoprenylated RhoA activated the PLD. On the contrary, recombinant nonisoprenylated RhoA failed to activate the PLD. Interestingly, the partially purified PLD-activating factor and ARF synergistically activated rat brain PLD, and recombinant isoprenylated RhoA could substitute for the partially purified preparation. These results conclude that rat brain PLD is regulated by RhoA in concert with ARF, and that the post-translational modification of RhoA is essential for its function as the PLD activator.

An activator of rat brain phospholipase D (PLD) that is distinct from the already identified PLD activator, ADP-ribosylation factor (ARF), was partially purified from bovine brain cytosol by a series of chromatographic steps. The partially purified preparation contained a 22-kDa substrate for Clostridium botulinum C3 exoenzyme ADP-ribosyltransferase, which strongly reacted with anti-rhoA p21 antibody, but not with anti-rac1 p21 or anti-cdc42Hs p21 antibody. Treatment of the partially purified PLD-activating factor with both C3 exoenzyme and NAD significantly inhibited the PLD-stimulating activity. These results suggest that rhoA p21 is, at least in part, responsible for the PLD-stimulating activity in the preparation. Recombinant isoprenylated rhoA p21 expressed in and purified from Sf9 cells activated rat brain PLD in a concentration- and GTP gamma S (guanosine 5'-O-(3-thiotriphosphate))-dependent manner. In contrast, recombinant non-isoprenylated rhoA p21 (fused to glutathione S-transferase) expressed in Escherichia coli failed to activate the PLD. This difference cannot be explained by a lower affinity of non-isoprenylated rhoA p21 for GTP gamma S, as the rates of [35S]GTP gamma S binding were very similar for both recombinant preparations and the GTP gamma S-bound form of non-isoprenylated rhoA p21 did not induce PLD activation. Interestingly, recombinant isoprenylated rhoA p21 and ARF synergistically activated rat brain PLD; a similar pattern was seen with the partially purified PLD-activating factor. The synergistic activation was inhibited by C3 exoenzyme-catalyzed ADP-ribosylation of recombinant isoprenylated rhoA p21 in a NAD-dependent manner. Inhibition correlated with the extent of ADP-ribosylation. These findings suggest that rhoA p21 regulates rat brain PLD in concert with ARF, and that isoprenylation of rhoA p21 is essential for PLD regulation in vitro.

Rat T lymphocyte alloantigen 6.1 (RT6.1), which was synthesized as the fusion protein with a maltose-binding protein in Escherichia coli, displayed NAD(+)-dependent auto-ADP-ribosylation in addition to an enzyme activity of NAD+ glycohydrolase. Such ADP-ribosylation of RT6.1 was also observed in lymphocytes isolated from rat tissues as follows. When intact rat lymphocytes expressing RT6.1 mRNA were incubated with [alpha-32P]NAD+, its radioactivity was incorporated into a cell surface protein with the M(r) of 31,000. The radiolabeled 31-kDa protein was released from the cell surface by treatment of the cells with phosphatidylinositol-specific phospholipase C and immunoprecipitated with anti-RT6.1 antiserum. The radioactivity incorporated into the 31-kDa protein was recovered as 5'-[32P]AMP upon incubation with snake venom phosphodiesterase and also removed by NH2OH treatment. These results suggested that the NAD(+)-dependent modification of the 31-kDa protein was due to ADP-ribosylation of glycosylphosphatidylinositol-anchored RT6.1 at an arginine residue. When intact lymphocytes, in which RT6.1 had been once modified by [32P]ADP-ribosylation, were further incubated in the absence of NAD+, there was reduction of the radioactivity in the [32P]ADP-ribosylated RT6.1. The reduced radioactivity was recovered from the incubation medium as [32P]ADP-ribose. This reduction was effectively inhibited by the addition of ADP-ribose to the reaction mixture. Moreover, readdition of NAD+ caused the ADP-ribosylation of RT6.1 again. Thus, the ADP-ribosylation of RT6.1 appeared to proceed reversibly in intact rat lymphocytes.

ADP-ribose moiety containing digoxigenin was transferred by pertussis toxin (IAP) to the alpha subunit of Gi (Gi alpha) from digoxigenin-conjugated NAD (DIG-NAD) in a beta gamma subunit-dependent manner. ADP-ribosylation of Gi alpha with DIG-NAD plus IAP was inhibited by native NAD. These results indicate that non-radiolabeled DIG-NAD also serves as the substrate for IAP-catalyzed ADP-ribosylation of G proteins. Using DIG-NAD and fluorescein isothiocyanate-labeled anti-digoxigenin antibody, IAP-sensitive G protein(s) was found to be exist in nuclei as well as plasma membranes of rat liver and HeLa cells. Thus, DIG-NAD is useful to identify pertussis toxin-substrate G proteins.

The existence and regulation of phospholipase D (PLD) activity in cell-free system from primary cultured cells of fetal rat frontal cortex were investigated. PLD activity was detectable only in the presence of Triton X-100. Other detergents examined (deoxycholate, taurocholate, CHAPS, Tween 20, sodium dodecyl sulfate) caused only a small increase in PLD activity. Triton X-100 enhanced PLD activity maximally at 0.1% (w/v) and reduced at higher concentrations. The optimal pH was about 7.2. Both Ca2+ and Mg2+ inhibited PLD activity in a dose-dependent manner. When comparing the primary cultured cells with adult rat frontal cortices, all of the results of the primary cultured cells were in agreement with those of the frontal cortices. Moreover, the apparent Km value of the enzyme in primary cultured cells for phosphatidyl-choline was the same as that in rat frontal cortex. These results suggest that the same kinds of PLD exist in the primary cultured cells and the rat frontal cortex, and that the primary cultured cells are a good experimental model for analyzing the mechanism of PLD in neuronal system.

MEDICAL SCIENCES Correction for “Loss of PIP5KIβ demonstrates that PIP5KI isoform-specific PIP2 synthesis is required for IP3 formation,” by Yanfeng Wang, Xinsheng Chen, Lurong Lian, Tang Tang, Timothy J. Stalker, Takehiko Sasaki, Lawrence F. Brass, John K. Choi, John H. Hartwig, and Charles S. Abrams, which appeared in issue 37, September 16, 2008, of Proc Natl Acad Sci USA (105:14064–14069; first published September 4, 2008; 10.1073/pnas.0804139105). The authors request that Yasunori Kanaho, Department of Physiological Chemistry, Graduate School of Comprehensive Human Sciences, Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Ten-nohdai, Tsukuba 305-8575, Japan, be added to the author list, between Takehiko Sasaki and Lawrence F. Brass, and be credited with contributing new reagents/analytic tools. The online version has been corrected. The corrected author and affiliation lines, and related footnotes, appear below.

Immunohistochemical localization of pertussis toxin-sensitive GTP-binding proteins in rat cerebellum was investigated using antibodies raised against purified G alpha o and synthetic peptides corresponding to specific sequences of G alpha i1, G alpha i2 and G alpha o was detected mostly in the molecular layer but not in the cell body of the Purkinje cells. G alpha i1 and G alpha i2 were found predominantly in the molecular layer and in addition in the cell body of the Purkinje cells. G alpha i3 was not detected in rat cerebellum. Immunoblot analysis demonstrated that in the cerebellum membrane G alpha 0 was most abundant and surprisingly the amount of G alpha i2 was much higher than that of G alpha i1. Thus, each pertussis toxin substrate was found to be expressed differently in rat cerebellum. The different patterns of expression imply that each subtype of GTP-binding proteins may have a specific function in modulating the neuronal activity in rat cerebellum.

1. 1. Phospholipase B-like activity was found in rabbit brain membranes. 2. 2. The activity was greatly enhanced by 0.025% (w/v) Triton X-100 and was inhibited by both Ca2+ and Mg2+. 3. 3. With increasing pH of the reaction mixture, the activity was augmented. 4. 4. The characteristics of the enzyme activity possibly suggest that phospholipase B in rabbit brain may be distinct from those previously reported.

As an approach to understanding the molecular basis of the pathophysiology of cerebral ischemia, we examined qualitative and quantitative changes in pertussis toxin substrates, Gi1 and G0, in the membrane of rat cerebral cortex after decapitation. Within 1 min after decapitation, the extent of pertussis toxin-catalyzed [32P]ADP ribosylation of the G proteins in the cerebral cortex membrane was significantly decreased and the magnitude of the decrease became slightly larger upon further incubation of the decapitated brain. Addition of guanine nucleotides, GTP and GDP, or the purified βγ subunits of transducin to the membranes of control and ischemic cerebral cortex stimulated [32P]ADP ribosylation of the G proteins. The stimulation of [32P]ADP ribosylation in the control situation by guanine nucleotides was almost to the same extent as that in ischemia. However, the stimulation by transducin βγ subunits was different; the control stimulation was greater than that in ischemia. In immunoblots probed with antibodies against Gi1α G0α and Tβ, the immunoreactivity of the corresponding proteins in ischemia was similar to that in control, suggesting that the amounts of G proteins were not changed in ischemia. These results suggest that ischemia accelerates the dissociation of α–GDP–βγ to α–GDP and free βγ and causes the denaturation of the dissociated α–GDP, thereby decreasing [32P]ADP ribosylation.

In vitro effects of biscoclaurine alkaloids on cell morphology and the osmotic resistance of human erythrocyte membranes were examined in relationship to their incorporation into the membrane.All the alkaloids tested induced invaginational type of transformation and a change in resistance against hypotonic hemolysis as a function of the concentration, within a range of 10-5 M to 2 × 10-4 M, in the medium. Alkaloids also were incorporated dose-dependently into cells in the same concentration range; the major part of the incorporation was in the membrane fraction. The effects of the alkaloids on the membrane as wall es their affinity for the membrane, judged by the amount incorporated, decrease as follows : tetrandrine > cepharanthine > berbamine. Fangchinoline and isotetrandrine had an invaginating effect similar to that of cepharanthine. The shape-transforming effect of cepharanthine was antagonistic to that of snake venom phospholipase A2 which induced crenation of the cells by hydrolyzing the outer layer phospholipids of the lipid bilayer of the membrane. This alkaloid did not inhibit the hydrolytic action of the enzyme on the membrane. These results indicate that the biological action of the alkaloids on the erythrocyte membrane may be due to their interaction with the lipid bilayer of the membrane.

The addition of the chemotactic factor fMet-Leu-Phe to cell homogenates causes a decrease in the pertussis toxin catalyzed ADP-ribosylation of a 41 kDa protein. The fMet-Leu-Phe induced decrease is not abolished in homogenates prepared from phorbol 12-myristate 13-acetate treated neutrophils. This decreased ribosylation probably reflects a dissociation of the GTP-binding protein oligomer that is not followed by association, possibly because of the release of the alpha-subunit into the suspending medium. Furthermore, fMet-Leu-Phe stimulates the binding of radiolabelled guanylylimidodiphosphate to membrane preparations. Again, the stimulated binding of guanylylimidodiphosphate is not affected by treating the intact neutrophils with phorbol 12-myristate 13-acetate. In addition leukotriene B4, platelet activating factor and fMet-Leu-Phe activate a high-affinity GTPase in membrane preparations. The basal level of this GTPase activity is dramatically inhibited in membrane preparations isolated from cells treated with phorbol 12-myristate 13-acetate. On the other hand, the fMet-Leu-Phe stimulated component is only marginally reduced. The present findings suggest that PMA does not prevent receptor G-protein interaction.

Cepharanthine incorporated into rabbit platelets dose dependently, inhibited calcium influx as well as aggregation in response to collagen, and also inhibited arachidonate release in response to collagen and A23187 in the same concentration ranges. The latter action of cepharanthine was shown not to be due to the direct action on phospholipase A2 molecules but to the depression of susceptibility of substrate phospholipids to enzymatic hydrolysis. These depressed functions, as well as the inhibited aggregation, were almost restored by removing the bound drugs from the platelets. Arachidonate-induced aggregation and prostaglandin synthesis from externally added arachidonate were not suppressed by the addition of cepharanthine. These results suggest that cepharanthine physically changes the lipid properties and thereby inhibits the function of the calcium channel or the susceptibility of substrate phospholipids to enzymatic hydrolysis by phospholipase A2.

Treatment of normal, disc-shaped rabbit platelets with lysophosphatidylcholine and chlorpromazine induced respectively spine formation and spherical transformation. In similar concentration ranges to those in which they induced these morphological changes, the drugs suppressed a series of events triggered by thrombin: pseudopod formation, arachidonate release from the membrane phospholipids, and aggregation. Washing the drug-treated platelets reversed the morphological changes and abolished the inhibitory effect on aggregation. These observations suggest that amphiphilic drugs perturb the plasma membrane structure of platelets, inducing the membrane shape change and inhibiting the stimulus-induced aggregation.

The tissue distribution of pertussis toxin-sensitive GTP-binding proteins was examined using specific antibodies raised against the purified alpha-subunit of G0 from bovine brain or against synthetic peptides predicted from cDNAs for distinct Gi subtypes. GTP-binding proteins were partially purified from membrane fractions prepared from rabbit tissues including brain, heart, liver, lung, erythrocytes and neutrophils. Brain contained both G0 and Gi1. Gi1 was also found to be abundant in heart. All peripheral tissues contained readily detectable amounts of Gi2, whereas only barely detectable amounts of Gi2 were found in brain. Gi3 was found to be prominent in erythrocytes and exists as a minor component of G proteins in neutrophils and liver. Thus, Gi2 appears to be widely disseminated in peripheral rabbit tissues, while other pertussis toxin substrates are more limited in their distribution.

Platelet aggregation elicited by collagen or thrombin was inhibited by cepharanthine in a concentration-dependent manner. The in-hibition of thrombin-induced aggregation was correlated with the inhibition of arachidonate-release from the phospholipids of the membrane, notably from phosphatidylcholine.The normal disc shape of the washed platelets with a few pseudopods was transformed to a sphere due to treatment with cepharanthine at a concentra-tion which considerably inhibited aggregation. Washing the cepharanthine-treated platelets with a calcium-free solution changed the spherical shape back to the disc shape and abolished the inhibition of aggregation.These results suggest that cepharanthine disturbs the plasma membrane due to its incorporation into the membrane, which results in the change in shape of the membrane and the inhibition of aggregation.

Stimulation by N-formyl-Met-Leu-Phe (fMLP) of rabbit peritoneal neutrophils, in which phosphatidylcholine was preferentially labeled with l-O-[3H]octadecyl lyso platelet-activating factor, activated phospholipase D, resulting in the formation of [3H]PA from [3H]PC. A direct activator of GTP-binding proteins (G-proteins), NaF, also stimulated [3H]PA formation. fMLP-stimulated [3H]PA formation was inhibited by pertussis toxin (IAP) in a time- and dose-dependent manner. IAP also inhibited fMLP-stimulated IP3 formation, but the inhibition of IP3 formation was significantly greater than that of [3H]PA formation. These results indicate that activation of phospholipase D by fMLP in rabbit neutrophils is mediated by an IAP-sensitive G-protein that may be distinct from a phospholipase C-regulating protein.