[show abstract][hide abstract] ABSTRACT: PRIP, phospholipase C (PLC)-related but catalytically inactive protein is a protein with a domain organization similar to PLC-δ1. We have reported that PRIP interacts with the catalytic subunits of protein phosphatase 1 and 2A (PP1c and PP2Ac), depending on the phosphorylation of PRIP. We also found that Akt was precipitated along with PRIP by anti-PRIP antibody from neuronal cells. In this article, we summarize our current reach regarding the interaction of PRIP with Akt and protein phosphatases, in relation to the cellular phospho-regulations. PP1 and PP2A are major members of the protein serine/threonine phosphatase families. We have identified PP1 and PP2A as interacting partners of PRIP. We first investigated the interaction of PRIP with two phosphatases, using purified recombinant proteins. PRIP immobilized on beads pulled-down the catalytic subunits of both PP1 and PP2A, indicating that the interactions were in a direct manner, and the binding of PP1 and PP2A to PRIP were mutually exclusive. Site-directed mutagenesis experiments revealed that the binding sites for PP1 and PP2A on PRIP were not identical, but in close proximity. Phosphorylation of PRIP by protein kinase A (PKA) resulted in the reduced binding of PP1, but not PP2A. Rather, the dissociation of PP1 from PRIP by phosphorylation accompanied the increased binding of PP2A in in vitro experiments. This binding regulation of PP1 and PP2A to PRIP by PKA-dependent phosphorylation was also observed in living cells treated with forskolin or isoproterenol. These results suggested that PRIP directly interacts with the catalytic subunits of two distinct phosphatases in a mutually exclusive manner and the interactions are regulated by phosphorylation, thus functioning as a scaffold to regulate the activities and subcellular localizations of both PP1 and PP2A in phospho-dependent cellular signaling.
[show abstract][hide abstract] ABSTRACT: Upon starvation, cells undergo autophagy, an intracellular bulk-degradation process, to provide the required nutrients. Here, we observed that phospholipase C-related catalytically inactive protein (PRIP) binds to microtubule-associated protein 1 light chain 3 (LC3), a mammalian autophagy-related initiator that regulates the autophagy pathway. Then, we examined the involvement of PRIP in the nutrient depletion-induced autophagy pathway. Enhanced colocalization of PRIP with LC3 was clearly seen in nutrient-starved mouse embryonic fibroblasts under a fluorescent microscope, and interaction of the proteins was revealed by immunoprecipitation experiments with an anti-LC3 antibody. Under starvation conditions, there were more green fluorescent protein fused-LC3 dots in mouse embryonic fibroblasts from PRIP-deficient mice than in fibroblasts from wild type cells. The formation of new dots in a single cell increased, as assessed by time-lapse microscopy. Furthermore, the increase in autophagosome formation in PRIP-deficient cells was notably inhibited by exogenously overexpressed PRIP. Taken together, PRIP is a novel LC3-binding protein that acts as a negative modulator of autophagosome formation.
Biochemical and Biophysical Research Communications 02/2013; · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: Phospholipase C-related, but catalytically inactive protein (PRIP) was first identified as a novel inositol 1,4,5-triphosphate binding protein. The PRIP-1 subtype is expressed predominantly in the central nervous system and binds directly to the GABA type A receptor (GABA(A)-R) β-subunit and several other proteins involved in the trafficking of GABA(A)-Rs to the plasma membrane. We found that the PRIP-1 knockout mouse showed an epileptic phenotype, confirmed by electroencephalogram. These ictal seizures were completely suppressed by diazepam (DZP), but the interictal discharges could not be abolished. We studied the electrophysiological properties of GABAergic transmission in hippocampal CA1 pyramidal neurons, using a slice patch-clamp technique. There was no difference in the effect of up to 1 μM DZP on the amplitude and frequency of miniature inhibitory postsynaptic currents between PRIP-1 knockout neurons versus wild-type neurons. In contrast, the amplitude of the tonic GABA current in PRIP-1 knockout neurons was markedly reduced compared with that in wild-type neurons. Consequently, the effect of DZP on PRIP-1 knockout mice was reduced. Dysfunction of extrasynaptic GABAergic transmission probably is involved in the epileptic phenotype of PRIP-1 knockout mice.
Journal of Pharmacology and Experimental Therapeutics 11/2011; 340(3):520-8. · 3.89 Impact Factor
[show abstract][hide abstract] ABSTRACT: PRIP (phospholipase C-related, but catalytically inactive protein) is a novel protein isolated in this laboratory. PRIP-deficient mice showed increased serum gonadotropins, but decreased gonadal steroid hormones. This imbalance was similar to that for the cause of bone disease, such as osteoporosis. In the present study, therefore, we analyzed mutant mice with special reference to the bone property. We first performed three-dimensional analysis of the femur of female mice. The bone mineral density and trabecular bone volume were higher in mutant mice. We further performed histomorphometrical assay of bone formation parameters: bone formation rate, mineral apposition rate, osteoid thickness, and osteoblast number were up-regulated in the mutant, indicating that increased bone mass is caused by the enhancement of bone formation ability. We then cultured primary cells isolated from calvaria prepared from both genotypes. In mutant mice, osteoblast differentiation, as assessed by alkaline phosphatase activity and the expression of osteoblast differentiation marker genes, was enhanced. Moreover, we analyzed the phosphorylation of Smad1/5/8 in response to bone morphogenetic protein, with longer phosphorylation in the mutant. These results indicate that PRIP is implicated in the negative regulation of bone formation.
Journal of Biological Chemistry 07/2011; 286(35):31032-42. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Phospholipase C-related inactive protein (PRIP) plays important roles in trafficking to the plasma membrane of GABA(A) receptor, which is involved in the dominant inhibitory neurotransmission in the spinal cord and plays an important role in nociceptive transmission. However, the role of PRIP in pain sensation remains unknown. In this study, we investigated the phenotypes of pain behaviors in PRIP type 1 knockout (PRIP-1 (-/-)) mice. The mutant mice showed hyperalgesic responses in the second phase of the formalin test and the von Frey test as compared with those in wild-type mice. In situ hybridization studies of GABA(A) receptors revealed significantly decreased expression of γ2 subunit mRNA in the dorsal and ventral horns of the spinal cord in PRIP-1 (-/-) mice, but no difference in α1 subunit mRNA expression. β2 subunit mRNA expression was significantly higher in PRIP-1 (-/-) mice than in wild-type mice in all areas of the spinal cord. On the other hand, the slow decay time constant for the spontaneous inhibitory current was significantly increased by treatment with diazepam in wild-type mice, but not in PRIP-1 (-/-) mice. These results suggest that PRIP-1 (-/-) mice exhibit the changes of the function and subunits expression of GABA(A) receptor in the spinal cord, which may be responsible for abnormal pain sensation in these mice.
[show abstract][hide abstract] ABSTRACT: The GABA(A) receptor, a pentamer composed predominantly of alpha, beta, and gamma subunits, mediates fast inhibitory synaptic transmission. We have previously reported that phospholipase C-related inactive protein (PRIP) is a modulator of GABA(A) receptor trafficking and that knockout (KO) mice exhibit a diazepam-insensitive phenotype in the hippocampus. The alpha subunit affects diazepam sensitivity; alpha1, 2, 3, and 5 subunits assemble with any form of beta and the gamma2 subunits to produce diazepam-sensitive receptors, whereas alpha4 or alpha6/beta/gamma2 receptors are diazepam-insensitive. Here, we investigated how PRIP is implicated in the diazepam-insensitive phenotype using cerebellar granule cells in animals expressing predominantly the alpha6 subunit. The expression of alpha1/beta/gamma2 diazepam-sensitive receptors was decreased in the PRIP-1 and 2 double KO cerebellum without any change in the total number of benzodiazepine-binding sites as assessed by radioligand-binding assay. Since levels of the alpha6 subunit were increased, the alpha1/beta/gamma2 receptors might be replaced with alpha6 subunit-containing receptors. Then, we further performed autoradiographic and electrophysiologic analyses. These results suggest that the expression of alpha6/delta receptors was decreased in cerebellar granule neurons, while that of alpha6/gamma2 receptors was increased. PRIP-1 and 2 double KO mice exhibit a diazepam-insensitive phenotype because of a decrease in diazepam-sensitive (alpha1/gamma2) and increase in diazepam-insensitive (alpha6/gamma2) GABA(A) receptors in the cerebellar granule cells.
Journal of Neurochemistry 07/2010; 114(1):302-10. · 3.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: Potentiation of inhibitory gamma-aminobutyric acid subtype A (GABA(A)) receptor function is involved in the mechanisms of anesthetic action. The present study examined the immobilizing action of the volatile anesthetic isoflurane in mice with double knockout (DKO) of phospholipase C-related inactive protein (PRIP)-1 and -2. Both of these proteins play important roles in the expression of GABA(A) receptors containing the gamma2 subunit on the neuronal cell surface. Immunohistochemistry for GABA(A) receptor subunits demonstrated reduced expression of gamma2 subunits in the spinal cord of the DKO mice. Immunohistochemistry also revealed up-regulation of the alpha1 and beta3 subunits even though there were no apparent differences in the immunoreactivities for the beta2 subunits between wild-type and DKO mice. The tail-clamp method was used to evaluate the anesthetic/immobilizing effect of isoflurane and the minimum alveolar concentration (MAC) was significantly lower in DKO mice compared with wild-type controls (1.07+/-0.01% versus 1.36+/-0.04% atm), indicating an increased sensitivity to isoflurane in DKO mice. These immunohistochemical and pharmacological findings suggest that reduced expression of the GABA(A) receptor gamma2 subunit affects the composition and function of spinal GABA(A) receptors and potentiates the immobilizing action of isoflurane.
[show abstract][hide abstract] ABSTRACT: Orofacial movements are regulated by D(1)-like dopamine receptors interacting with additional mechanisms. Phospholipase C-related catalytically inactive protein (PRIP) regulates cell surface expression of GABA(A) receptors containing a gamma2 subunit. Mutant mice with double knockout of PRIP-1 and PRIP-2 were used to investigate aspects of GABAergic regulation of orofacial movements and interactions with D(1) mechanisms. Vertical jaw movements, tongue protrusions and movements of the head and vibrissae were reduced in PRIP-1/2 double knockouts. The GABA(A)ergic agent diazepam reduced movements of the head and vibrissae; these effects were unaltered in PRIP-1/2 double knockouts. The D(1)-like agonist SKF 83959 induced vertical jaw movements, incisor chattering, and movements of the head and vibrissae that were unaltered in PRIP-1/2 double knockouts. However, SKF 83959-induced tongue protrusions were reduced in PRIP-1/2 double knockouts. PRIP-mediated regulation of GABA(A)ergic receptor mechanisms influences topographically distinct aspects of orofacial movement and interacts with D(1) receptor systems.
[show abstract][hide abstract] ABSTRACT: A well-known protein module regulating molecular interactions is the pleckstrin homology (PH) domain whose best-characterised ligand is phosphoinositide. In the present study, we analysed the PH domain from PRIP (phospholipase C-related but catalytically inactive protein, comprising types 1 and 2) regarding phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] binding employing a variety of binding assays. The PH domains prepared from PRIP-1 and -2 showed similar binding profiles to soluble ligands in vitro and showed similar plasma membrane localisation to that of PLC-delta1; however, the PH domain with the N-terminal extension of PRIP-1 but not PRIP-2 showed even distribution throughout the cytoplasm, indicating that the N-terminal extension of PRIP-1 inhibited binding to PtdIns(4,5)P(2) present in the plasma membrane. A chimeric molecule of PLC-delta1 PH domain with the N-terminal extension of PRIP-1 exhibited similar localisation to PRIP-1 PH domain with the N-terminal extension. Binding assay to liposomes containing various concentrations of PtdIns(4,5)P(2) revealed that the PH domain of PLC-delta1 bound steeply to the maximum, even at a concentration of 1.2 mol%, whereas the PH domains from PRIP-1 and -2 bound depending on the concentration up to 5 mol%. We also performed binding experiments using saponin-permeabilised PC12 cells. PH domains from PRIP increased the binding to cells preincubated with the brain cytosol extract in the presence of ATP, during which PtdIns(4,5)P(2) were probably synthesised. The binding of PH domain with the following EF hand motifs showed Ca(2+)-dependent binding. These results indicate that the PH domain of PRIP binds to PtdIns(4,5)P(2) present in the plasma membrane, depending on the concentrations of the lipid ligand and Ca(2+), suggesting that PRIP might play physiological roles in events involved in the changes of these parameters, probably including Ins(1,4,5)P(3).
[show abstract][hide abstract] ABSTRACT: A mechanism for regulating the strength of synaptic inhibition is enabled by altering the number of GABA(A) receptors available at the cell surface. Clathrin and adaptor protein 2 (AP2) complex-mediated endocytosis is known to play a fundamental role in regulating cell surface GABA(A) receptor numbers. Very recently, we have elucidated that phospholipase C-related catalytically inactive protein (PRIP) molecules are involved in the phosphorylation-dependent regulation of the internalization of GABA(A) receptors through association with receptor beta subunits and protein phosphatases. In this study, we examined the implications of PRIP molecules in clathrin-mediated constitutive GABA(A) receptor endocytosis, independent of phospho-regulation. We performed a constitutive receptor internalization assay using human embryonic kidney 293 (HEK293) cells transiently expressed with GABA(A) receptor alpha/beta/gamma subunits and PRIP. PRIP was internalized together with GABA(A) receptors, and the process was inhibited by PRIP-binding peptide which blocks PRIP binding to beta subunits. The clathrin heavy chain, mu2 and beta2 subunits of AP2 and PRIP-1, were complexed with GABA(A) receptor in brain extract as analyzed by co-immunoprecipitation assay using anti-PRIP-1 and anti-beta2/3 GABA(A) receptor antibody or by pull-down assay using beta subunits of GABA(A) receptor. These results indicate that PRIP is primarily implicated in the constitutive internalization of GABA(A) receptor that requires clathrin and AP2 protein complex.
Journal of Neurochemistry 06/2007; 101(4):898-905. · 3.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: The K+ Cl- cotransporter KCC2 plays an important role in chloride homeostasis and in neuronal responses mediated by ionotropic GABA and glycine receptors. The expression levels of KCC2 in neurons determine whether neurotransmitter responses are inhibitory or excitatory. KCC2 expression is decreased in developing neurons, as well as in response to various models of neuronal injury and epilepsy. We investigated whether there is also direct modulation of KCC2 activity by changes in phosphorylation during such neuronal stressors. We examined tyrosine phosphorylation of KCC2 in rat hippocampal neurons under different conditions of in vitro neuronal stress and the functional consequences of changes in tyrosine phosphorylation. Oxidative stress (H2O2) and the induction of seizure activity (BDNF) and hyperexcitability (0 Mg2+) resulted in a rapid dephosphorylation of KCC2 that preceded the decreases in KCC2 protein or mRNA expression. Dephosphorylation of KCC2 is correlated with a reduction of transport activity and a decrease in [Cl-]i, as well as a reduction in KCC2 surface expression. Manipulation of KCC2 tyrosine phosphorylation resulted in altered neuronal viability in response to in vitro oxidative stress. During continued neuronal stress, a second phase of functional KCC2 downregulation occurs that corresponds to decreases in KCC2 protein expression levels. We propose that neuronal stress induces a rapid loss of tyrosine phosphorylation of KCC2 that results in translocation of the protein and functional loss of transport activity. Additional understanding of the mechanisms involved may provide means for manipulating the extent of irreversible injury resulting from different neuronal stressors.
Journal of Neuroscience 03/2007; 27(7):1642-50. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: The subunit composition of GABA(A) receptors is known to be associated with distinct physiological and pharmacological properties. Previous studies that used phospholipase C-related inactive protein type 1 knock-out (PRIP-1 KO) mice revealed that PRIP-1 is involved in the assembly and/or the trafficking of gamma2 subunit-containing GABA(A) receptors. There are two PRIP genes in mammals; thus the roles of PRIP-1 might be compensated partly by those of PRIP-2 in PRIP-1 KO mice. Here we used PRIP-1 and PRIP-2 double knock-out (PRIP-DKO) mice and examined the roles for PRIP in regulating the trafficking of GABA(A) receptors. Consistent with previous results, sensitivity to diazepam was reduced in electrophysiological and behavioral analyses of PRIP-DKO mice, suggesting an alteration of gamma2 subunit-containing GABA(A) receptors. The surface numbers of diazepam binding sites (alpha/gamma2 subunits) assessed by [3H]flumazenil binding were reduced in the PRIP-DKO mice as compared with those of wild-type mice, whereas the cell surface GABA binding sites (alpha/beta subunits, assessed by [3H]muscimol binding) were increased in PRIP-DKO mice. The association between GABA(A) receptors and GABA(A) receptor-associated protein (GABARAP) was reduced significantly in PRIP-DKO neurons. Disruption of the direct interaction between PRIP and GABA(A) receptor beta subunits via the use of a peptide corresponding to the PRIP-1 binding site reduced the cell surface expression of gamma2 subunit-containing GABA(A) receptors in cultured cell lines and neurons. These results suggest that PRIP is implicated in the trafficking of gamma2 subunit-containing GABA(A) receptors to the cell surface, probably by acting as a bridging molecule between GABARAP and the receptors.
Journal of Neuroscience 03/2007; 27(7):1692-701. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: GABAA receptors are a family of ligand-gated ion channels that are pentamer composed predominantly of α, β and γ subunits. They are the major target of the endogenous inhibitory neurotransmitter (GABA, γ-aminobutyric acid) and maintain the majority of fast inhibitory ion currents in the central nervous system, in addition to being drug targets for benzodiazepines, barbiturates, alcohols, neurosteroids, and some anesthetics. Moreover, modifications in GABAA receptor function are crucial in central nervous system diseases such as anxiety disorders, sleep disturbances, and seizure disorders. Therefore it is very important to understand the molecular mechanisms underlying the maintenance of functional inhibitory synapses including GABAA receptors. We have first isolated PRIP (phospholipase C-related, but catalytically inactive protein) as a novel inositol 1,4,5-trisphosphate binding protein, and subsequently found GABARAP (GABAA receptor associated protein) as one of binding partners that binds to γ2 subunit of the receptor and thus is implicated in the clustering and trafficking of the receptor to synaptic membrane. Further studies revealed that PRIP binds β subunit of the receptors and PP1c (catalytic subunit of protein phosphatase 1). These findings have prompted us to explore the possible involvement of PRIP in modulation of GABAA receptor signaling. Here we summarize our current understanding regarding how PRIP is involved in the modification of GABAA receptor signaling on the basis of the characteristics of these interacting molecules.
Journal of Oral Biosciences 01/2007; 49(2):105-112.
[show abstract][hide abstract] ABSTRACT: Brain-derived neurotrophic factor (BDNF) modulates several distinct aspects of synaptic transmission, including GABAergic transmission. Exposure to BDNF alters properties of GABA(A) receptors and induces changes in the expression level at the cell surface. Although phospholipase C-related inactive protein-1 (PRIP-1) plays an important role in GABA(A) receptor trafficking and function, its role in BDNF-dependent modulation of these receptors, together with the role of PRIP-2, was investigated using neurons cultured from PRIP double knock-out mice. The BDNF-dependent inhibition of whole cell GABA-evoked currents observed in wild type neurons was not detected in neurons cultured from knock-out mice. Instead, a gradual increase in GABA-evoked currents in these neurons correlated with a gradual increase in phosphorylation of GABA(A) receptor beta3 subunit in response to BDNF. To characterize the specific role(s) that PRIP plays as components of underlying molecular machinery, we examined the recruitment of protein phosphatase(s) to GABA(A) receptors. We demonstrate that PRIP associates with phosphatases as well as with beta subunits. PRIP was found to colocalize with GABA(A) receptor clusters in cultured neurons and with recombinant GABA(A) receptors when co-expressed in HEK293 cells. Importantly, a peptide mimicking a domain of PRIP involved in binding to beta subunits disrupted the co-localization of these proteins in HEK293 cells and potently inhibited the BDNF-mediated attenuation of GABA(A) receptor currents in wild type neurons. Together, the results suggest that PRIP plays an important role in BDNF-dependent regulation of GABA(A) receptors by mediating the specific association between beta subunits of these receptors with protein phosphatases.
Journal of Biological Chemistry 09/2006; 281(31):22180-9. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: PRIP, phospholipase C related, but catalytically inactive protein was first identified as a novel inositol 1,4,5-trisphosphate binding protein. It has a number of binding partners including protein phosphatase (PP1 and 2A), GABAA receptor associated protein, and the beta subunits of GABAA receptors, in addition to inositol 1,4,5-trisphosphate. The identification of these molecules led us to examine the possible involvement of PRIP in the phospho-regulation of the beta subunits of GABAA receptors using hippocampal neurons prepared from PRIP-1 and 2 double knock-out (DKO) mice. Experiments were performed with special reference to the dephosphorylation processes of the beta subunits. The phosphorylation of beta3 subunits by the activation of protein kinase A in cortical neurons of the control mice continued for up to 5 min, even after washing out of the stimulus, followed by a gradual dephosphorylation. That of DKO mice gradually increased in spite of the lower phosphorylation levels induced by the stimulation. There was little difference in the amount of cellular cyclic AMP and protein kinase A activity between the control and mutant mice, indicating that phosphatases such as PP1 and PP2A are primarily involved in the difference. The time course of PP1 activity changes in the vicinity of the receptors in control mice corresponded to the phosphorylation of PRIP, while that of the mutant mice decreased with the period of the incubation. This is a good agreement with the suggestion that PRIP binds to and inactivates PP1, which is regulated by the phosphorylation of PRIP at threonine 94. These results suggest that PRIP plays an important role in controlling the dynamics of GABAA receptor phosphorylation by through PP1 binding and, therefore, the efficacy of synaptic inhibition mediated by these receptors.
[show abstract][hide abstract] ABSTRACT: Investigation of chemically synthesized inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] analogs has led to the isolation of a novel binding protein with a molecular size of 130 kDa, characterized as a molecule with similar domain organization to phospholipase C-d1 (PLC-d1) but lacking the enzymatic activity. An isoform of the molecule was subsequently identified, and these molecules have been named PRIP (PLC-related, but catalytically inactive protein), with the two isoforms named PRIP-1 and -2. Regarding its ability to bind Ins(1,4,5)P3 via the pleckstrin homology domain, the involvement of PRIP-1 in Ins(1,4,5)P3-mediated Ca2+ signaling was examined using COS-1 cells overexpressing PRIP-1 and cultured neurons prepared from PRIP-1 knock-out mice. Yeast two hybrid screening of a brain cDNA library using a unique N-terminus as bait identified GABARAP (GABAA receptor associated protein) and PP1 (protein phosphatase 1), which led us to examine the possible involvement of PRIP in GABAA receptor signaling. For this purpose PRIP knock-out mice were analyzed for GABAA receptor function in relation to the action of benzodiazepines from the electrophysiological and behavioral aspects. During the course of these experiments we found that PRIP also binds to the b-subunit of GABAA receptors and PP2A (protein phosphtase 2A). Here, we summarize how PRIP is involved in Ins(1,4,5)P3-mediated Ca2+ signaling and GABAA receptor signaling based on the characteristics of binding molecules.
Molecules and Cells 01/2006; 20(3):305-14. · 2.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: 1 Bisphosphonates are inhibitors of tumor cell growth as well as of bone resorption by inducing cell apoptosis. However, little is known regarding the mechanisms by which the drug induces cell apoptosis. The aim of the present study was to determine the effect of alendronate, one of the nitrogen-containing bisphosphonates on the phoshoinositide 3-kinase (PI3K)-Akt-NFkappaB pathway, the major cell survival pathway. 2 The PI3K-Akt-NFkappaB pathway was activated in the osteosarcoma cell line MG-63 treated with tumor necrosis factor-alpha or insulin. Saos-2 was also used in some experiments. This was assessed by the production of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), increased PI3K activity, phosphorylation of Akt at serine 473 and threonine 308, increase in activity of the inhibitor of nuclear factor kappaB (IkappaB) kinase (IKK) and finally phosphorylation of IkappaB and its subsequent degradation. 3 Pretreatment with alendronate at 100 microM for 24 h prior to the stimulation with tumor necrosis factor-alpha or insulin partially inhibited the IkappaB phosphorylation and degradation. These events were more clearly observed in the presence of inhibitors of proteasomes, which are responsible for the degradation of IkappaB. The drug also partially inhibited the activity of IKK, but almost fully inhibited the phosphorylation of Akt and the production of PtdIns(3,4,5)P(3). 4 The inhibitory effect of alendronate on IkappaB phosphorylation and degradation was not attenuated by the exogenous addition of geranylgeraniol to replenish the cytosolic isoprenyl lipid substrate. 5 The present findings demonstrate that alendronate inhibited the PI3K-Akt-NFkappaB cell survival pathway at the point of PI3K activation, thus indicating the presence of new targets of alendronate.
British Journal of Pharmacology 12/2005; 146(5):633-41. · 5.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: GABA(A) receptors mediate most of the fast inhibitory neurotransmission in the brain, and are believed to be composed mainly of alpha, beta, and gamma subunits. It has been shown that GABA(A) receptors interact with a number of binding partners that act to regulate both receptor function and cell surface stability. Here, we reveal that GABA(A) receptors interact directly with N-ethylmaleimide-sensitive factor (NSF), a critical regulator of vesicular dependent protein trafficking, as measured by in vitro protein binding and co-immunoprecipitation assays. In addition, we established that NSF interacts with residues 395-415 of the receptor beta subunits and co-localizes with GABA(A) receptors in hippocampal neurons. We also established that NSF can regulate GABA(A) receptor cell surface expression depending upon residues 395-415 in the beta3 subunit. Together, our results suggest an important role for NSF activity in regulating the cell surface stability of GABA(A) receptors.
Molecular and Cellular Neuroscience 11/2005; 30(2):197-206. · 3.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: Microtubule-associated protein (MAP) light chain 3 (LC3) is a human homologue of yeast Apg8/Aut7/Cvt5 (Atg8), which is essential for autophagy. MAP-LC3 is cleaved by a cysteine protease to produce LC3-I, which is located in cytosolic fraction. LC3-I, in turn, is converted to LC3-II through the actions of E1- and E2-like enzymes. LC3-II is covalently attached to phosphatidylethanolamine on its C terminus, and it binds tightly to autophagosome membranes. We determined the solution structure of LC3-I and found that it is divided into N- and C-terminal subdomains. Additional analysis using a photochemically induced dynamic nuclear polarization technique also showed that the N-terminal subdomain of LC3-I makes contact with the surface of the C-terminal subdomain and that LC3-I adopts a single compact conformation in solution. Moreover, the addition of dodecylphosphocholine into the LC3-I solution induced chemical shift perturbations primarily in the C-terminal subdomain, which implies that the two subdomains have different sensitivities to dodecylphosphocholine micelles. On the other hand, deletion of the N-terminal subdomain abolished binding of tubulin and microtubules. Thus, we showed that two subdomains of the LC3-I structure have distinct functions, suggesting that MAP-LC3 can act as an adaptor protein between microtubules and autophagosomes.
Journal of Biological Chemistry 08/2005; 280(26):24610-7. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: PRIP-1 was isolated as a novel inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] binding protein with a domain organization similar to phospholipase C-delta1 (PLC-delta1) but lacking the enzymatic activity. Further studies revealed that the pleckstrin homology (PH) domain of PRIP-1 is the region responsible for binding Ins(1,4,5)P3. In this study we aimed to clarify the role of PRIP-1 at the physiological concentration in Ins(1,4,5)P3-mediated Ca2+ signaling, as we had previously used COS-1 cells overexpressing PRIP-1 (Takeuchi et al., 2000, Biochem J 349:357-368). For this purpose we employed PRIP-1 knock out (PRIP-1-/-) mice generated previously (Kanematsu et al., 2002, EMBO J 21:1004-1011). The increase in free Ca2+ concentration in response to purinergic receptor stimulation was lower in primary cultured cortical neurons prepared from PRIP-1-/- mice than in those from wild type mice. The relative amounts of [3H]Ins(1,4,5)P3 measured in neurons labeled with [3H]inositol was also lower in cells from PRIP-1-/- mice. In contrast, PLC activities in brain cortex samples from PRIP-1-/- mice were not different from those in the wild type mice, indicating that the hydrolysis of Ins(1,4,5)P3 is enhanced in cells from PRIP-1-/- mice. In vitro analyses revealed that type1 inositol polyphosphate 5-phosphatase physically interacted with a PH domain of PRIP-1 (PRIP-1PH) and its enzyme activity was inhibited by PRIP-1PH. However, physical interaction with these two proteins did not appear to be the reason for the inhibition of enzyme activity, indicating that binding of Ins(1,4,5)P3 to the PH domain prevented its hydrolyzation. Together, these results indicate that PRIP-1 plays an important role in regulating the Ins(1,4,5)P3-mediated Ca2+ signaling by modulating type1 inositol polyphosphate 5-phosphatase activity through binding to Ins(1,4,5)P3.
Journal of Cellular Physiology 03/2005; 202(2):422-33. · 4.22 Impact Factor