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Highlights • Ahnak−/− mice were more resistant to the pulmonary metastasis of B16F10 cells. • Transcriptomic analyses revealed that PCSK9 was a candidate for lung metastasis. • The lung metastasis was suppressed in Scgb1a1-Cre/PCSK9fl/fl mice. • Ahnak-TGFβ-PCSK9 axis regulates pulmonary metastasis of B16F10 cells., Previously we demonstrated that Ahnak mediates transforming growth factor-β (TGFβ)-induced epithelial-mesenchymal transition (EMT) during tumor metastasis. It is well-known that circulating tumor cells (CTCs) invade the vasculature of adjacent target tissues before working to adapt to the host environments. Currently, the molecular mechanism by which infiltrated tumor cells interact with host cells to survive within target tissue environments is far from clear. Here, we show that Ahnak regulates tumor metastasis through PCSK9 expression. To validate the molecular function of Ahnak in metastasis, B16F10 melanoma cells were injected into WT and Ahnak knockout (Ahnak−/−) mice. Ahnak−/− mice were more resistant to the pulmonary metastasis of B16F10 cells compared to wild-type (WT) mice. To investigate the host function of Ahnak in recipient organs against metastasis of melanoma cells, transcriptomic analyses of primary pulmonary endothelial cells from WT or Ahnak−/− mice in the absence or presence of TGFβ stimulation were performed. We found PCSK9, along with several other candidate genes, was involved in the invasion of melanoma cells into lung tissues. PCSK9 expression in the pulmonary artery was higher in WT mice than Ahnak−/− mice. To evaluate the host function of PCSK9 in lung tissues during the metastasis of melanoma cells, we established lung epithelial cell-specific tamoxifen-induced PCSK9 conditional KO mice (Scgb1a1-Cre/PCSK9fl/fl). The pulmonary metastasis of B16F10 cells in Scgb1a1-Cre/PCSK9fl/fl mice was significantly suppressed, indicating that PCSK9 plays an important role in the metastasis of melanoma cells. Taken together, our data demonstrate that Ahnak regulates metastatic colonization through the regulation of PCSK9 expression.

Background and aims Oxidative stress contributes to development of diabetic nephropathy. We implicated SH3YL1 in oxidative stress-induced inflammation and examined whether SH3YL1 could be used as a new biomarker of diabetic nephropathy. Methods and results In this study, we investigated the relationship between plasma level of SH3YL1 and diabetic nephropathy in patients with type 2 diabetes. In addition, we examined the physiological role of SH3YL1 in db/db mice and cultured podocytes. Plasma SH3YL1 concentration was significantly higher in patients with diabetes than in controls, even in normoalbuminuric patients, and was markedly increased in the macroalbuminuria group. Plasma SH3YL1 level was positively correlated with systolic blood pressure, HOMA-IR, postprandial blood glucose, plasma level of retinol binding protein 4 (RBP 4), and urinary albumin excretion (UAE) and was inversely correlated with BMI. Regression analysis showed that plasma level of RBP 4, UAE, and BMI were the only independent determinants of plasma SH3YL1 concentration. In db/db mice, plasma and renal SH3YL1 levels were significantly increased in mice with diabetes compared with control mice. In cultured podocytes, high glucose and angiotensin II stimuli markedly increased SH3YL1 synthesis. Conclusion These findings suggest that plasma level of SH3YL1 offers a promising new biomarker for diabetic nephropathy.

Several lines of evidence indicated that generation of NADPH oxidase (Nox)-mediated reactive oxygen species are associated with neuronal inflammation, leading to Parkinson's disease (PD). Novel benzylidene-1-methyl-2-thioxoimidazolidin-one derivatives as Nox inhibitors were designed and synthesized in order to increase blood-brain barrier (BBB) permeability to target Nox in brain cells. In lucigenin chemiluminescence assay, eight compounds showed excellent inhibition activity against NADPH oxidases and parallel artificial membrane permeability assay (PAMPA) identified compound 11 with high passive permeability. To validate the effect of compound 11 on neuronal inflammation, we tested the regulatory activity of compound 11 in lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines in BV-2 microglial cells and LPS-mediated microglial migration. Treatment of BV2 cells with compound 11 resulted in suppressed production of pro-inflammatory cytokines and migration activity of BV2 cells in response to LPS. To evaluate the therapeutic efficacy of compound 11 in PD animal model, compound 11 was applied to MPTP-induced PD mouse model. Oral administration of compound 11 (30 mg/kg/daily, 4 weeks) into the mice resulted in suppression of dopaminergic neuronal death in substantia nigra (SN) and in striatum as well as inhibition of microglial migration into SN. These results implicate compound 11 as a novel therapeutic agent for the treatment of PD.

NOX1 (NADPH oxidase) similar to phagocyte NADPH oxidase, is expressed mainly in the colon epithelium and it is responsible for host defense against microbial infections by generating ROS (reactive oxygen species). NOX1 is activated by two regulatory cytosolic proteins that form a hetero-dimer, Noxo1 (NOX organizer 1) and Noxa1 (NOX activator 1). The interaction between Noxa1 and Noxo1 is critical for activating NOX1. However no structural studies for interaction between Noxa1 and Noxo1 has not been reported till date. Here, we studied the inter-molecular interaction between the SH3 domain of Noxa1 and Noxo1 using pull-down assay and NMR spectroscopy. 15N/13C-labeled SH3 domain of Noxa1 has been purified for hetero-nuclear NMR experiments (HNCACB, CBCACONH, HNCA, HNCO, and HSQC). TALOS analysis using backbone assignment data of the Noxa1 SH3 domain showed that the structure primarily consists of β-sheets. Data from pull-down assay between the Noxo1 and Noxa1 showed that the SH3 domains (Noxa1) is responsible for interaction with Noxo1 C-terminal tail harboring proline rich region (PRR). The concentration-dependent titration of the Noxo1 C-terminal tail to Noxa1 shows that Noxo1 particularly in the RT loop: Q407*, H408, S409, A412*, G414*, E416, D417, L418, and F420; n-Src loop: C430, E431*, V432*, A435, W436, and L437; and terminal region: I447; F448*, F452* and V454 interact with Noxa1. Our results will provide a detailed understanding for interaction between Noxa1 and Noxo1 at the molecular level, providing insights into their cytoplasmic activity-mediated functioning as well as regulatory role of C-terminal tail of Noxo1 in the NOX1 complex.

We report the synthesis of novel 3-substituted 5-benzylidene-1-methyl-2-thiohydantoins 3, and their biological evaluation using NADPH oxidase (NOX) 1 and 4. Based on structural and pharmacophore analyses of known inhibitors such as hydroxypyrazole 2, we envisioned interesting 2-thiohydantoin compounds, 3-substituted 5-benzylidene-1-methyl-2-thiohydantoins 3 that would be expected to well match the structural features in 2. Efficient synthesis of eighteen target compounds 3 were achieved through the synthetic pathway of 4→11→3, established after consideration of several plausible synthetic pathways. The inhibitory activities of compounds 3 against NOX 1 and 4 were measured, with some of the target compounds showing similar or higher activities compared with reference 2; in particular, compounds 3bz, 3cz, and 3ez were found to be promising inhibitors of both NOX 1 and 4 with modest isozyme selectivities, which highlights the significance of the 2-thiohydantoin substructure for inhibition of NOX 1 and 4. This marks the first time these compounds have been applied to the inhibition of NOX enzymes.

We have previously reported that Ahnak-mediated TGFβ signaling leads to down-regulation of c-Myc expression. Here, we show that inhibition of Ahnak can promote generation of induced pluripotent stem cells (iPSC) via up-regulation of endogenous c-Myc. Consistent with the c-Myc inhibitory role of Ahnak, mouse embryonic fibroblasts from Ahnak-deficient mouse (Ahnak(-/-) MEF) show an increased level of c-Myc expression compared with wild type MEF. Generation of iPSC with just three of the four Yamanaka factors, Oct4, Sox2, and Klf4 (hereafter 3F), was significantly enhanced in Ahnak(-/-) MEF. Similar results were obtained when Ahnak-specific shRNA was applied to wild type MEF. Of note, expressionof Ahnak was significantly induced during the formation of embryoid bodies from embryonic stem cells, suggesting that Ahnak-mediated c-Myc inhibition is involved in embryoid body formation and the initial differentiation of pluripotent stem cells. The iPSC from 3F-infected Ahnak(-/-) MEF cells (Ahnak(-/-)-iPSC-3F) showed expression of all stem cell markers examined and the capability to form three primary germ layers. Moreover, injection of Ahnak(-/-)-iPSC-3F into athymic nude mice led to development of teratoma containing tissues from all three primary germ layers, indicating that iPSC from Ahnak(-/-) MEF are bona fide pluripotent stem cells. Taken together, these data provide evidence for a new role for Ahnak in cell fate determination during development and suggest that manipulation of Ahnak and the associated signaling pathway may provide a means to regulate iPSC generation.

The crystal structure determination of tautomeric products produced by the alkylation of 1-(2-pyridinyl)-3-phenyl-4-propyl-1H-5-hydroxypyrazole 2 was investigated. Treatment of 2 with isopropyloxycarbonyloxymethyl iodide and potassium carbonate under phase-transfer conditions affords two major products out of three possible O-, N-, and C-alkylated tautomers. The tautomeric structures of O-alkylated 3a and N-alkylated 3c were elucidated by means of NMR spectroscopic investigations and confirmed by single crystal X-ray analysis. The single crystal structures of alkylated compounds provide clear difference between the tautomeric pyrazole and pyrazolone ring systems in terms of bond lengths and torsional angles, moreover, conformational changes between two tautomers.

Testosterone is an endocrine hormone with functions in reproductive organs, anabolic events, and skin homeostasis. We report here that GPRC6A serves as a sensor and mediator of the rapid action of testosterone in epidermal keratinocytes. The silencing of GPRC6A inhibited testosterone-induced intracellular calcium ([Ca(2+)]i) mobilization and H2O2 generation. These results indicated that a testosterone-GPRC6A complex is required for activation of Gq protein, IP3 generation, and [Ca(2+)]i mobilization, leading to Duox1 activation. H2O2 generation by testosterone stimulated the apoptosis of keratinocytes through the activation of caspase-3. The application of testosterone into three-dimensional skin equivalents increased the apoptosis of keratinocytes between the granular and stratified corneum layers. These results support an understanding of the molecular mechanism of testosterone-dependent apoptosis in which testosterone stimulates H2O2 generation through the activation of Duox1.

Background Allergic rhinitis (AR) and asthma are 2 entities of allergic airway diseases that frequently occur together, which is referred to as united airways . In contrast to this general concept, we hypothesized that innate immunity of the upper and lower airways is respectively distinctive, because the immunologic conditions of the nasal and lung mucosa as well as the functions of the immune cells within their epithelia are different. Objective We wanted to identify distinctive mechanisms of innate immunity in the nose and lung mucosa, which are responsible for house dust mite (HDM)–induced AR and allergic asthma (AA), respectively. Methods We constructed a mouse model of AR or AA induced by sensitization and consequent provocation with HDM extracts. Results HDM-derived β-glucans, rather than LPS, were proven to be essential to activating innate immunity in the nasal mucosa and triggering AR, which depended on Toll-like receptor 2 (TLR2), but not on TLR4; however, the LPS/TLR4 signaling axis, rather than β-glucans/TLR2, was critical to HDM-induced AA. These differences were attributed to the specific role of β-glucans and LPS in inducing the surface expression of TLR2 and TLR4 and their translocation to lipid rafts in nasal and bronchial epithelial cells, respectively. We also showed that dual oxidase 2–generated reactive oxygen species mediate both β-glucan–induced TLR2 activation and LPS-induced TLR4 activation. Conclusions We describe a novel finding of distinctive innate immunity of the nose and lungs, respectively, which trigger AR and AA, by showing the critical role of HDM-induced TLR activation via dual oxidase 2–mediated reactive oxygen species.

Small ubiquitin-like modifier 1 (SUMO1) is a member of the superfamily of ubiquitin-like proteins. Despite its structural similarity with ubiquitin, SUMO1 does not seem to play any role in protein degradation and its precise biological function is poorly understood. During our studies on heat-shock responses, we found that heat-shock stress increased SUMO1 conjugation in a dose-dependent manner. Intriguingly, SUMO1 conjugation resulted in decrease of intracellular ROS generation and protection cells from death under heat-shock stress. We showed that NADPH oxidase 2 (NOX2) is a target protein of sumoylation by SUMO1 using immunoprecipitation and is colocalized with SUMO1 at plasma membrane. Additionally, we demonstrated that the attenuation in intracellular ROS generation resulted from inhibition of NADPH oxidase complex (NOX) activity. These results suggested that SUMO1 plays an important role in modulation of NOX activity required for ROS generation.

Mucosal epithelia are in direct contact with microbes, which range from beneficial symbionts to pathogens. Accordingly, hosts must have a conflicting strategy to combat pathogens efficiently while tolerating symbionts. Recent progress has revealed that dual oxidase (DUOX) plays a key role in mucosal immunity in organisms that range from flies to humans. Information from the genetic model of Drosophila has advanced our understanding of the regulatory mechanism of DUOX and its role in mucosal immunity. Further investigations of DUOX regulation in response to symbiotic or non-symbiotic bacteria and the in vivo consequences in host physiology will give a novel insight into the microbe-controlling system of the mucosa.

The mechanism of angiotensin II (Ang II)-induced superoxide production was investigated with HEK293 or Chinese hamster ovary cells reconstituted with the angiotensin type 1 receptor (AT(1)R) and NADPH oxidase (either Nox1 or Nox2) along with a pair of adaptor subunits (either NOXO1 with NOXA1 or p47(phox) with p67(phox)). Ang II enhanced the activity of both Nox1 and Nox2 supported by either adaptor pair, with more effective activation of Nox1 in the presence of NOXO1 and NOXA1 and of Nox2 in the presence of p47(phox) and p67(phox). Expression of several AT(1)R mutants showed that interaction of the receptor with G proteins but not that with beta-arrestin or with other proteins (Jak2, phospholipase C-gamma1, SH2 domain-containing phosphatase 2) that bind to the COOH-terminal region of AT(1)R, was necessary for Ang II-induced superoxide production. The effects of constitutively active alpha subunits of G proteins and of various pharmacological agents implicated signaling by a pathway comprising AT(1)R, Galpha(q/11), phospholipase C-beta, and protein kinase C as largely, but not exclusively, responsible for Ang II-induced activation of Nox1 and Nox2 in the reconstituted cells. A contribution of Galpha(12/13), phospholipase D, and phosphatidyl-inositol 3-kinase to Ang II-induced superoxide generation was also suggested, whereas Src and the epidermal growth factor receptor did not appear to participate in this effect of Ang II. In reconstituted cells stimulated with Ang II, Nox2 exhibited a more sensitive response than Nox1 to the perturbation of protein kinase C, phosphatidylinositol 3-kinase, or the small GTPase Rac1.

Phospholipase C-gamma1 (PLC-gamma1), which generates two second messengers, namely, inositol-1, 4, 5-trisphosphate and diacylglycerol, is implicated in growth factor-mediated chemotaxis. However, the exact role of PLC-gamma1 in integrin-mediated cell adhesion and migration remains poorly understood. In this study, we demonstrate that PLC-gamma1 is required for actin cytoskeletal organization and cell motility through the regulation of Pyk2 and paxillin activation. After fibronectin stimulation, PLC-gamma1 directly interacted with the cytoplasmic tail of integrin beta1. In PLC-gamma1-silenced cells, integrin-induced Pyk2 and paxillin phosphorylation were significantly reduced and PLC-gamma1 potentiated the integrin-induced Pyk2/paxillin activation in its enzymatic activity-dependent manner. In addition, specific knock-down of PLC-gamma1 resulted in a failure to form focal adhesions dependent on fibronectin stimulation, which appeared to be caused by the suppression of Pyk2 and paxillin phosphorylation. Interestingly, PLC-gamma1 potentiated the activations of Rac, thus integrin-induced lamellipodia formation was up-regulated. Consequently, the strength of cell-substratum interaction and cell motility were profoundly up-regulated by PLC-gamma1. Taken together, these results suggest that PLC-gamma1 is a key player in integrin-mediated cell spreading and motility achieved by the activation of Pyk2/paxillin/Rac signaling.

It is well established that growth-factor-induced reactive oxygen species (ROS) act as second messengers in cell signaling. We have previously reported that betaPix, a guanine nucleotide exchange factor for Rac, interacts with NADPH oxidase 1 (Nox1) leading to EGF-induced ROS generation. Here, we report the identification of the domains of Nox1 and betaPix responsible for the interaction between the two proteins. GST pull-down assays show that the PH domain of betaPix binds to the FAD-binding region of Nox1. We also show that overexpression of the PH domain of betaPix results in inhibition of superoxide anion generation in response to EGF. Additionally, NADPH oxidase Organizer 1 (NoxO1) is shown to interact with the NADPH-binding region of Nox1. These results suggest that the formation of the complex consisting of Nox1, betaPix, and NoxO1 is likely to be a critical step in EGF-induced ROS generation.

Phospholipase C-gamma1 (PLC-gamma1) plays pivotal roles in cellular growth and proliferation. Upon the stimulation of growth factors and hormones, PLC-gamma1 is rapidly phosphorylated at three known sites; Tyr771, Tyr783 and Tyr1254 and its enzymatic activity is up-regulated. In this study, we demonstrate for the first time that Grb2, an adaptor protein, specifically interacts with tyrosine-phosphorylated PLC-gamma1 at Tyr783. The association of Grb2 with PLC-gamma1 was induced by the treatment with epidermal growth factor (EGF). Replacement of Tyr783 with Phe completely blocked EGF-induced interaction of PLC-gamma1 with Grb2, indicating that tyrosine phosphorylation of PLC-gamma1 at Tyr783 is essential for the interaction with Grb2. Interestingly, the depletion of Grb2 from HEK-293 cells by RNA interference significantly enhanced increased EGF-induced PLC-gamma1 enzymatic activity and mobilization of the intracellular Ca2+, while it did not affect EGF-induced tyrosine phosphorylation of PLC-gamma1. Furthermore, overexpression of Grb2 inhibited PLC-gamma1 enzymatic activity. Taken together, these results suggest Grb2, in addition to its key function in signaling through Ras, may have a negatively regulatory role on EGF-induced PLC-gamma1 activation.

A fundamental question that applies to all organisms is how barrier epithelia efficiently manage continuous contact with microorganisms. Here, we show that in Drosophila an extracellular immune-regulated catalase (IRC) mediates a key host defense system that is needed during host-microbe interaction in the gastrointestinal tract. Strikingly, adult flies with severely reduced IRC expression show high mortality rates even after simple ingestion of microbe-contaminated foods. However, despite the central role that the NF-kappaB pathway plays in eliciting antimicrobial responses, NF-kappaB pathway mutant flies are totally resistant to such infections. These results imply that homeostasis of redox balance by IRC is one of the most critical factors affecting host survival during continuous host-microbe interaction in the gastrointestinal tract.

We have recently shown that phospholipase C-gamma (PLC-gamma) is activated by the central repeated units (CRUs) of the AHNAK protein in the presence of arachidonic acid. Here we demonstrate that four central repeated units (4 CRUs) of AHNAK act as a scaffolding motif networking PLC-gamma and PKC-alpha. Specifically, 4 CRUs of AHNAK bind and activate PKC-alpha, which in turn stimulates the release of arachidonic acid near where PLC-gamma1 is localized. Moreover, 4 CRUs of AHNAK interacted with PLC-gamma and the concerted action of 4 CRUs with arachidonic acid stimulated PLC-gamma activity. Stimulation of NIH3T3 cells expressing 4 CRUs of AHNAK with phorbol 12-myristate 13-acetate resulted in the increased generation of total inositol phosphates (IP(T)) and mobilization of the intracellular calcium. Phorbol 12-myristate 13-acetate-dependent generation of IP(T) was completely blocked in NIH3T3 cells depleted of PLC-gamma1 by RNA interference. Furthermore, bradykinin, which normally stimulated the PLC-beta isozyme resulting in the generation of a monophasic IP(T) within 30 s in NIH3T3 cells, led to a biphasic pattern for generation of IP(T) in NIH3T3 cells expressing 4 CRUs of AHNAK. The secondary activation of PLC is likely because of the scaffolding activity of AHNAK, which is consistent with the role of 4 CRUs as a molecular linker between PLC-gamma and PKC-alpha.

In addition to inhibiting matrix metalloproteinases, tissue inhibitor of metalloproteinase-1 (TIMP-1) is involved in the regulation of cell growth and survival. To determine its mechanism of action, we investigated effects of TIMP-1 on cell proliferation and survival and signaling pathways induced by TIMP-1 in the human breast carcinoma T-47D cell line. Treatment of T-47D cells with TIMP-1 strongly inhibited apoptosis induced by serum deprivation, but did not affect cell proliferation. TIMP-1 induced phosphorylation of Akt and extracellular signal-regulated protein kinases (ERKs), but pertussis toxin and specific inhibitors of Src family tyrosine kinases, protein tyrosine kinases, and phosphatidylinositol-3 kinase (PI3 kinase) blocked the ability of TIMP-1 to activate Akt and ERKs as well as the anti-apoptotic effect of TIMP-1. We found that TIMP-1 enhanced the kinase activities of c-Src and PI3 kinase and that this enhancement was inhibited by pertussis toxin. Inhibition of ERK activation, however, resulted in a slight decrease of the TIMP-1-induced anti-apoptotic effect. These findings demonstrate that the ability of TIMP-1 to inhibit apoptosis in T-47D cells is mediated by the sequential activation of pertussis toxin-sensitive G protein, c-Src, PI3 kinase, and Akt.

Salicylates and nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in a variety of cancer cells, including those of colon, prostate, breast, and leukemia. We examined the effects of sodium salicylate (NaSal) on reactive oxygen species (ROS) production and the association of these effects with apoptotic tumor cell death. We demonstrate that NaSal mediates ROS production followed by a decrease in mitochondrial membrane potential (deltapsi(m)), release of cytochrome c, and activation of caspase-9 and caspase-3. However, expression of Bcl-2 or Bcl-x(L) prevents ROS production and subsequent loss of deltapsi(m), thereby inhibiting apoptotic cell death. The presence of ROS scavengers and an inhibitor of NADPH oxidase or expression of a dominant negative form of Rac1 blocks ROS production, deltapsi(m) collapse, and the subsequent activation of caspases. These observations indicate that NaSal mediates ROS production critical in the triggering of apoptotic tumor cell death through a Rac1-NADPH oxidase-dependent pathway. Our data collectively imply that NaSal-induced ROS are key mediators of deltapsi(m) collapse, which leads to the release of cytochrome c followed by caspase activation, culminating in tumor apoptosis.

The melanocortin receptors, MC3R and MC4R, are G protein-coupled receptors that are involved in regulating energy homeostasis. Using a luciferase reporter gene under the transcriptional control of a cAMP- responsive element (CRE), the coupling efficiency of the MC4R and MC3R to G-proteins was previously shown to be different. MC4R exhibited only 30-50% of the maximum activity induced by MC3R. To assess the role of the different MC3R and MC4R domains in G-protein coupling, several chimeric MC3R/MC4R receptors were constructed. The relative luciferase activities, which were assessed after transfecting the chimeric receptors into HEK 293T cells, showed that the i3 (3rd intracellular) loop domain has an essential role in the differential signaling of MC3R and MC4R. To reveal which amino acid residue was involved in the MC4R-specific signaling in the i3 loop, a series of mutant MC4Rs was constructed. Reporter gene analysis showed that single mutations of Arg(220) to Ala and Thr(232) to either Val or Ala increased the relative luciferase activities, which suggests that these specific amino acids, Arg(220) and Thr(232), in the i3 loop of MC4R play crucial roles in G-protein coupling and the subtype-specific signaling pathways. An examination of the inositol phosphate (IP) levels in the cells transfected with either MC3R or MC4R after being exposed to the melanocortin peptides revealed significant stimulation of IP production by MC3R but no detectable increase in IP production was observed by MC4R. Furthermore, none of the MC4R mutants displayed melanocortin peptide-stimulated IP production. Overall, this study demonstrated that MC3R and MC4R have distinct signaling in either the cAMP- or the inositol phospholipid-mediated pathway with different conformational requirements.

We have recently shown that phospholipase C-gamma (PLC-gamma) is activated by tau, a neuronal cell-specific microtubule-associated protein, in the presence of arachidonic acid. We now report that non-neuronal tissues also contain a protein that can activate PLC-gamma in the presence of arachidonic acid. Purification of this activator from bovine lung cytosol yielded several proteins with apparent molecular sizes of 70-130 kDa. They were identified as fragments derived from an unusually large protein (approximately 700 kDa) named AHNAK, which comprises about 30 repeated motifs each 128 amino acids in length. Two AHNAK fragments containing one and four of the repeated motifs, respectively, were expressed as glutathione S-transferase fusion proteins. Both recombinant proteins activated PLC-gamma1 at nanomolar concentrations in the presence of arachidonic acid, suggesting that an intact AHNAK molecule contains multiple sites for PLC-gamma activation. The role of arachidonic acid was to promote a physical interaction between AHNAK and PLC-gamma1, and the activation by AHNAK and arachidonic acid was mainly attributable to reduction in the enzyme's apparent Km toward the substrate phosphatidylinositol 4,5-bisphosphate. Our results suggest that arachidonic acid liberated by phospholipase A2 can act as an additional trigger for PLC-gamma activation, constituting an alternative mechanism that is independent of tyrosine phosphorylation.

Signal transduction across cell membranes often involves the activation of both phosphatidylinositol (PI)-specific phospholipase C (PLC) and phosphoinositide 3-kinase (PI 3-kinase). Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a substrate for both enzymes, is converted to phosphatidylinositol 3,4, 5-trisphosphate (PI(3,4,5)P3) by the action of PI 3-kinase. Here, we show that PI(3,4,5)P3 activates purified PLC-gamma isozymes by interacting with their Src homology 2 domains. Furthermore, the expression of an activated catalytic subunit of PI 3-kinase in COS-7 cells resulted in an increase in inositol phosphate formation, whereas platelet-derived growth factor-induced PLC activation in NIH 3T3 cells was markedly inhibited by the specific PI 3-kinase inhibitor LY294002. These results suggest that receptors coupled to PI 3-kinase may activate PLC-gamma isozymes indirectly, in the absence of PLC-gamma tyrosine phosphorylation, through the generation of PI(3,4,5)P3.

In the accompanying paper (Chung, J.-K., Sekiya, F., Kang, H.-S., Lee, C., Han, J.-S., Kim, S. R., Bae, Y. S., Morris, A. J., and Rhee, S. G. (1997) J. Biol. Chem. 272, 15980-15985), synaptojanin is identified as a protein that inhibits phospholipase D (PLD) activity stimulated by ADP-ribosylation factor and phosphatidylinositol 4, 5-bisphosphate (PI(4,5)P2). Here, the purification from rat brain cytosol of another PLD-inhibitory protein that is immunologically distinct from synaptojanin is described, and this protein is identified as clathrin assembly protein 3 (AP3) by peptide sequencing and immunoblot analysis. AP3 binds both inositol hexakisphosphate and preassembled clathrin cages with high affinity. However, neither inositol hexakisphosphate binding nor clathrin cage binding affected the ability of AP3 to inhibit PLD. AP3 also binds to PI(4,5)P2 with low affinity. But the PI(4,5)P2 binding was not responsible for PLD inhibition, because the potency and efficacy of AP3 as an inhibitor of PLD were similar in the absence and presence of PI(4,5)P2. A bacterially expressed fusion protein, glutathione S-transferase-AP3 (GST-AP3), also inhibited PLD with a potency equal to that of brain AP3. The inhibitory effect of AP3 appeared to be the result of direct interaction between AP3 and PLD because PLD bound GST-AP3 in an in vitro binding assay. Using GST fusion proteins containing various AP3 sequences, we found that the sequence extending from residues Pro-290 to Lys-320 of AP3 is critical for both inhibition of and binding to PLD. The fact that AP3 is a synapse-specific protein indicates that the AP3-dependent inhibition of PLD might play a regulatory role that is restricted to the rapid cycling of synaptic vesicles.

Recent evidence indicates that reactive oxygen species (ROS) may function as intracellular messengers in receptor signaling pathways. The possible role of ROS in epidermal growth factor (EGF) signaling was therefore investigated. Stimulation of A431 human epidermoid carcinoma cells with EGF resulted in a transient increase in the intracellular concentration of ROS, measured with the oxidation-sensitive fluorescent probe 2′,7′-dichlorofluorescin diacetate and laser-scanning confocal microscopy. The predominant ROS produced appeared to be H2O2, because the EGF-induced increase in fluorescence was completely abolished by incorporation of catalase into the cells by electroporation. The elimination of H2O2 by catalase also inhibited the EGF-induced tyrosine phosphorylation of various cellular proteins including the EGF receptor and phospholipase C-γ1. The dependence of H2O2 production on the intrinsic tyrosine kinase activity of the EGF receptor and the autophosphorylation sites located in its COOH-terminal tail was investigated. EGF failed to induce H2O2 generation in cells expressing a kinase-inactive EGF receptor. However, normal H2O2 generation was observed in cells expressing a mutant receptor from which the 126 COOH-terminal amino acids had been deleted to remove four (out of the total of five) autophosphorylation sites. These results suggest that EGF-induced H2O2 formation requires the kinase activity but probably not the autophosphorylation sites of the EGF receptor and that inhibition of protein tyrosine phosphatase activity by H2O2 may be required for EGF-induced protein tyrosine phosphorylation to be manifested.

Phospholipase C-gamma (PLC-gamma) isozymes are thought to be activated by receptor-induced tyrosine phosphorylation. Proteins that activate PLC-gamma1 have now been purified from bovine brain and identified as members of the tau family of microtubule-associated proteins. Activation of PLC-gamma by tau was enhanced in the presence of unsaturated fatty acids such as arachidonic acid, saturated fatty acids being ineffective. Maximal (15-20-fold) activation was apparent in the presence of 0.15 microM tau and 25 microM arachidonic acid (AA). The effect of tau and AA was specific to PLC-gamma isozymes in the presence of submicromolar concentrations of Ca2+ and was markedly inhibited by phosphatidylcholine. These results suggest that in cells that express tau, receptors coupled to cytosolic phospholipase A2 may activate PLC-gamma isozymes indirectly in the absence of tyrosine phosphorylation through the hydrolysis of phosphatidylcholine to generate AA.

Rat brain cytosol contains proteins that markedly inhibit the activity of partially purified brain membrane phospholipase D (PLD) stimulated by ADP-ribosylation factor (Arf) and phosphatidylinositol 4,5-bisphosphate (PIP). Sequential chromatography of the brain cytosol yielded four inhibitor fractions, which exhibited different kinetics to heat treatment at 70°C. Purification of the most heat-labile inhibitor to homogeneity yielded two preparations, which displayed apparent molecular masses of 150 kDa and 135 kDa, respectively, on SDS-polyacrylamide gels. Tryptic digests of the 150- and 135-kDa proteins yielded similar elution profiles on a C reverse-phase column, suggesting that the 135-kDa form is a truncated form of the 150-kDa form. Sequences of two tryptic peptides were determined. A data base search revealed no proteins with these sequences. The purified 150-kDa inhibitor negated the PLD activity stimulated by Arf, RhoA, or Cdc42. The concentration required for half-maximal inhibition was 0.4 nM. Concentration dependence on the 150-kDa inhibitor was not affected by changes in the concentrations of Arf, PIP, or phosphatidylcholine used in the assays, suggesting that the inhibition is not due to competition with the activators or substrate for PLD. The purified inhibitor did not affect the PIP-hydrolyzing activity of a phospholipase C isozyme that was measured with substrate vesicles of lipid composition identical with that used for the PLD assay. Thus, the mechanism of inhibition appears to be a specific allosteric modification of PLD rather than disruption of substrate vesicles.

Previously, we demonstrated that microinjection of phosphoinositide-specific phospholipase C gamma 1 (PLC gamma 1) and lipase-defective mutants of PLC gamma 1 induced G(0) growth arrested NIH 3T3 fibroblasts to enter S phase of the cell cycle. These experiments suggested that regions other than the catalytic domain of PLC gamma 1 may be responsible for inducing mitogenesis. To test other regions of PLC gamma 1 for DNA synthesis inducing activity, cDNA fragments encoding Src homology (SH) and pleckstrin homology (PH) domains were subcloned into the bacterial expression plasmid pGEX-2TK, and the GST fusion proteins were purified. The complete PLC gamma l SH domain peptide was found to induce DNA synthesis after microinjection into growth arrested fibroblasts. Peptides containing a single SH3 domain or two SH2 domains induced a partial response that was restored to full activity if they were co-injected. The PH domain peptide did not induce DNA synthesis. Thus, both SH3 and SH2 activity combine to give maximum DNA synthesis induction, demonstrating that non-catalytic structural domains of PLC gamma 1 have pronounced effects on mitogenic signaling pathways.