ABSTRACT
Manic episodes are one of the major diagnostic symptoms in a spectrum of neuropsychiatric disorders that include schizophrenia, obsessive-compulsive disorder and bipolar disorder (BD). Despite a possible association between BD and the gene encoding phospholipase Cγ1 (PLCG1), its etiological basis remains unclear. Here, we report that mice lacking phospholipase Cγ1 (PLCγ1) in the forebrain (Plcg1f/f; CaMKII) exhibit hyperactivity, decreased anxiety-like behavior, reduced depressive-related behavior, hyperhedonia, hyperphagia, impaired learning and memory and exaggerated startle responses. Inhibitory transmission in hippocampal pyramidal neurons and striatal dopamine receptor D1-expressing neurons of Plcg1-deficient mice was significantly reduced. The decrease in inhibitory transmission is likely due to a reduced number of γ-aminobutyric acid (GABA)-ergic boutons, which may result from impaired localization and/or stabilization of postsynaptic CaMKII (Ca2+/calmodulin-dependent protein kinase II) at inhibitory synapses. Moreover, mutant mice display impaired brain-derived neurotrophic factor-tropomyosin receptor kinase B-dependent synaptic plasticity in the hippocampus, which could account for deficits of spatial memory. Lithium and valproate, the drugs presently used to treat mania associated with BD, rescued the hyperactive phenotypes of Plcg1f/f; CaMKII mice. These findings provide evidence that PLCγ1 is critical for synaptic function and plasticity and that the loss of PLCγ1 from the forebrain results in manic-like behavior.
Subject(s)
Bipolar Disorder/enzymology , Bipolar Disorder/genetics , Phospholipase C gamma/metabolism , Prosencephalon/enzymology , Animals , Bipolar Disorder/parasitology , Brain-Derived Neurotrophic Factor/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Hippocampus/enzymology , Hippocampus/metabolism , Mice , Neuronal Plasticity/physiology , Neurons/enzymology , Neurons/metabolism , Phospholipase C gamma/deficiency , Phospholipase C gamma/genetics , Prosencephalon/pathology , Pyramidal Cells/metabolism , Receptor, trkB/metabolism , Receptors, Dopamine D1 , Synapses/enzymology , Synapses/pathology , Synaptic Transmission/physiology , gamma-Aminobutyric Acid/metabolismABSTRACT
Emerging evidence suggests that aberrant O-GlcNAcylation is associated with tumorigenesis. Many oncogenic factors are O-GlcNAcylated, which modulates their functions. However, it remains unclear how O-GlcNAcylation and O-GlcNAc cycling enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), affect the development of cancer in animal models. In this study, we show that reduced level of OGA attenuates colorectal tumorigenesis induced by Adenomatous polyposis coli (Apc) mutation. The levels of O-GlcNAcylation and O-GlcNAc cycling enzymes were simultaneously upregulated in intestinal adenomas from mice, and in human patients. In two independent microarray data sets, the expression of OGA and OGT was significantly associated with poor cancer-specific survival of colorectal cancer (CRC) patients. In addition, OGA heterozygosity, which results in increased levels of O-GlcNAcylation, attenuated intestinal tumor formation in the Apc(min/+) background. Apc(min/+) OGA(+/-) mice exhibited a significantly increased survival rate compared with Apc(min/+) mice. Consistent with this, Apc(min/+) OGA(+/-) mice expressed lower levels of Wnt target genes than Apc(min/+). However, the knockout of OGA did not affect Wnt/ß-catenin signaling. Overall, these findings suggest that OGA is crucial for tumor growth in CRC independently of Wnt/ß-catenin signaling.
ABSTRACT
Epidermal growth factor (EGF) is synthesized in the pancreas and diabetic animals have low levels of EGF. However, the role of EGF in regulating the major function of the pancreas, insulin secretion, has not been studied. Here, we show that EGF rapidly increased insulin secretion in mouse pancreatic islets, as well as in a pancreatic beta-cell line. These events were dependent on a Ca(2+) influx and phospholipase D (PLD) activity, particularly PLD2, as determined using pharmacological blockers and molecular manipulations such as over-expression and siRNA of PLD isozymes. In addition, EGF also increased plasma insulin levels and mediated glucose lowering in normal and diabetic mice. Here, for the first time, we provide evidence that EGF is a novel secretagogue that regulates plasma glucose levels and a candidate for the development of therapeutics for diabetes.
Subject(s)
Blood Glucose/metabolism , Diabetes Mellitus, Experimental/metabolism , Epidermal Growth Factor/pharmacology , Insulin/metabolism , Animals , Calcium/metabolism , Cells, Cultured , Enzyme Activation/drug effects , Epidermal Growth Factor/blood , Insulin/blood , Insulin Secretion , Islets of Langerhans/drug effects , Islets of Langerhans/metabolism , Male , Mice , Mice, Inbred ICR , Phospholipase D/metabolismABSTRACT
It has been shown that when CFTR and NHE3 are co-expressed on the apical membrane of the A6-NHE3 cell monolayers, the two transporters interact via a shared regulatory complex composed of NHERF2, ezrin, and PKA. We observe here that co-expression of NHE3 reduced both PKA-dependent apical CFTR expression and its activation once in place by approximately 50%. To analyze the role of NHERF2 in this process, we transfected NHE3 expressing and non-expressing A6 monolayers with NHERF2 cDNA in which its binding domains had been deleted. When only CFTR is expressed on the apical membrane, deletion of any of the NHERF2 binding domains inhibited both PKA-dependent apical CFTR expression and its activation, while when NHE3 was co-expressed with CFTR PDZ2 deletion was without effect on CFTR sorting and activity. This suggests that when the PDZ2 domain is "sequestered" by interacting with NHE3 it can no longer participate in CFTR functional expression.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cytoskeletal Proteins/metabolism , Sodium-Hydrogen Exchangers/metabolism , Animals , Binding Sites/genetics , Blotting, Western , Cell Line , Colforsin/pharmacology , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/physiology , Mutation , Nephrons/cytology , Nephrons/metabolism , Protein Binding/drug effects , Protein Transport/drug effects , Sodium-Hydrogen Exchanger 3 , Sodium-Hydrogen Exchangers/genetics , Sodium-Hydrogen Exchangers/physiology , Transfection , Xenopus laevisABSTRACT
Phospholipase C-gamma1(PLC-gamma1) is known to play an essential role in various cellular responses, such as proliferation and tumorigenesis, and PLC-gamma1-specific inhibitors are commonly employed to investigate the mechanism of the PLC-gamma1-mediated signaling pathway. In this study, we developed a single chain antibody fragment (scFv) as a blocker for PLC-gamma1 mediated signaling. scFv, designated F7-scFv, specifically bound to PLC-gamma1 with high affinity (K(d)=1.9x10(-8) M) in vitro. F7-scFv also bound to PLC-gamma1 in vivo and altered the distribution pattern of PLC-gamma1 from the cytoplasm to the intracellular aggregates, where F7-scFv was localized. Moreover, F7-scFv interrupted the EGF-induced translocation of PLC-gamma1 from the cytosol to the membrane ruffle and attenuated EGF-induced inositol phosphates generation and intracellular calcium mobilization. These results indicate that F7-scFv blocks EGF-induced PLC-gamma1 activation by causing sequestering of PLC-gamma1 into intracellular aggregates, and may therefore be useful in studies of the PLC-gamma1-mediated signaling pathway.
Subject(s)
Epidermal Growth Factor/pharmacology , Immunoglobulin Fragments/immunology , Immunoglobulin Fragments/pharmacology , Isoenzymes/antagonists & inhibitors , Isoenzymes/immunology , Type C Phospholipases/antagonists & inhibitors , Type C Phospholipases/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Antibody Affinity , Base Sequence , COS Cells , Calcium Signaling/drug effects , Cell Line , Enzyme Activation/drug effects , Enzyme-Linked Immunosorbent Assay , Fluorescein-5-isothiocyanate , Hybridomas/cytology , Hybridomas/immunology , Immunoglobulin Fragments/chemistry , Inositol Phosphates/metabolism , Isoenzymes/metabolism , Kinetics , Mice , Microscopy, Fluorescence , Molecular Sequence Data , Phospholipase C gamma , Protein Transport/drug effects , Thermodynamics , Type C Phospholipases/metabolismABSTRACT
Pancreatic beta cells are the source of insulin, which directly lowers blood glucose levels in the body. Our analyses of alpha(1D) gene-knockout (alpha(1D)(-/-)) mice show that the L-type calcium channel, alpha(1D), is required for proper beta cell generation in the postnatal pancreas. Knockout mice were characteristically slightly smaller than their littermates and exhibited hypoinsulinemia and glucose intolerance. However, isolated alpha(1D)(-/-) islets persisted in glucose sensing and insulin secretion, with compensatory overexpression of another L-type channel gene, alpha(1C). Histologically, newborn alpha(1D)(-/-) mice had an equivalent number of islets to wild-type mice. In contrast, adult alpha(1D)(-/-) mice showed a decrease in the number and size of islets, compared with littermate wild-type mice due to a decrease in beta cell generation. TUNEL staining showed that there was no increase in cell death in alpha(1D)(-/-) islets, and a 5-bromo-2' deoxyuridine-labeling (BrdU-labeling) assay illustrated significant reduction in the proliferation rate of beta cells in alpha(1D)(-/-) islets.
Subject(s)
Calcium Channels, L-Type/metabolism , Islets of Langerhans/cytology , Animals , Body Constitution , Calcium Channels, L-Type/genetics , Calcium Channels, L-Type/physiology , Cell Division , Deafness/etiology , Deafness/metabolism , Gene Expression , Glucose Intolerance/etiology , Glucose Intolerance/metabolism , Hyperinsulinism/etiology , Hyperinsulinism/metabolism , Mice , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
The possible involvement of phospholipase C (PLC) in the regulation of insulin secretion is not clearly understood and neither its isozymes expressed nor cellular localization in the pancreatic islets is known. By using specific monoclonal antibodies, we have investigated the expression and localization of eight different PLC isozymes, beta1, beta2, beta3, beta4, gamma1, gamma2, delta1, and delta2, in the pancreatic islets of adult mice. Immunohistochemical analysis carried out on paraffin embedded sections showed a distinct pattern of expression for each of the PLC isozymes. In the central part of the islets containing beta cells, a high level of beta4 and moderate levels of beta3 and gamma1 were expressed, whereas PLC-beta1 and -gamma1 were abundantly expressed in the exocrine pancreas. These results demonstrated the heterogeneity in expression of the phospholipase C isozymes in pancreatic islets. It is conceivable that these isozymes are coupled to different receptors and perform selective tasks in the regulation of insulin secretion for glucose homeostasis.
Subject(s)
Islets of Langerhans/enzymology , Type C Phospholipases/analysis , Animals , Antibodies, Monoclonal , Glucagon/analysis , Insulin/analysis , Islets of Langerhans/cytology , Isoenzymes/analysis , Isoenzymes/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Type C Phospholipases/immunologyABSTRACT
Extracellular ATP has been known to have many functions as a fast transmitter, and a co-transmitter, and to have morphogenic and mitogenic activity in neuronal cells. Although it was reported that ATP activates phospholipase D (PLD), the role of PLD versus the ATP function was unclear in neuronal cells. In this study, we investigated the role of PLD on the ATP-induced extracellular signal regulated protein kinase (ERK) activation and mitogenic effect in rat pheochromocytoma PC12 cells. In these cells ATP caused PLD2 activation and ERK phosphorylation, which was dramatically reduced by wild-type PLD2-overexpression but not by lipase-inactive-mutant PLD2-overexpression. The accumulation of phosphatidic acid (PA) by preincubating PC12 cells with propranolol (an inhibitor of PA phosphohydrolase) also decreased the ERK phosphorylation. Inhibition of phosphatases by okadaic acid or pervanadate completely blocked PLD2-dependent ERK dephosphorylation. In addition, ATP-stimulated thymidine incorporation was reduced by the overexpression of wild-type PLD2, but not by the overexpression of lipase-inactive-mutant PLD2. Okadaic acid pretreatment overcame the decrease of ATP-induced thymidine incorporation by PLD2 overexpression. Taken together, we suggest that PLD2 activity might play a negative role in ATP-induced ERK phosphorylation and mitogenic signal possibly through phosphatases.
Subject(s)
Adenosine Triphosphate/metabolism , Mitogen-Activated Protein Kinases/metabolism , Mitosis/physiology , PC12 Cells/metabolism , Phospholipase D/metabolism , Adenosine Triphosphate/pharmacology , Animals , Cell Line , Enzyme Induction/drug effects , Mitosis/drug effects , Okadaic Acid/pharmacology , PC12 Cells/cytology , Phospholipase D/genetics , Phosphorylation/drug effects , Propranolol/pharmacology , Rats , Tetracycline/pharmacology , Thymidine/metabolismABSTRACT
Mammalian phospholipase C-beta isozymes are activated by a heterotrimeric GTP-binding protein linked to various cell surface receptors. Recent reports suggest that PDZ domain proteins play a significant role of PDZ-containing proteins in the regulation of mammalian PLC-beta isozymes. PDZ-containing proteins mediate the clustering of receptors and signaling molecules and thereby regulate agonist-induced signal transduction in polarized cells such as neuronal and epithelial cells. NORPA, a Drosophila PLC-beta, is known to be a component of a signaling complex that includes TRP and rhodopsin through interaction with INAD, a PDZ-containing protein. Mammalian PLC-beta1 and -beta2 isoforms interact with a PDZ-containing protein NHERF which is coupled to Trp4, a Ca(2+) channel. In addition, PLC-beta3 specifically interacts with E3KARP, another protein closely related to NHERF, through its C-terminal PDZ-binding motif. E3KARP up-regulates the PLC-beta3 activation coupled to muscarinic receptor. In this review, the role of signaling complexes mediated by PDZ-containing proteins in the regulation of PLC-beta isoforms will be discussed.
Subject(s)
Drosophila Proteins , Isoenzymes/physiology , Signal Transduction , Type C Phospholipases/physiology , Animals , Calcium Channels/metabolism , Cytoskeletal Proteins/physiology , Drosophila/physiology , Eye Proteins/metabolism , Isoenzymes/chemistry , Models, Biological , Phospholipase C beta , Phosphoproteins/physiology , Protein Structure, Tertiary , Sodium-Hydrogen Exchangers , TRPC Cation Channels , Type C Phospholipases/chemistry , Vision, OcularABSTRACT
Phospholipase D (PLD), phosphatidylinositol 3-kinase (PI3K), and Akt are known to be involved in cellular signaling related to proliferation and cell survival. In this report, we provide evidence that PLD links sphingosine 1-phosphate (S1P)-induced activation of the G protein-coupled EDG3 receptor to stimulation of PI3K and its downstream effector Akt in Chinese hamster ovary (CHO) cells. S1P stimulation of EDG3-overexpressing CHO cells but not vector-transfected cells induced activation of PLD, PI3K, and Akt in a time- and dose-dependent manner. Akt phosphorylation was prevented by the PI3K inhibitors wortmannin and LY294002 (2-(4-monrpholinyl)-8-phenyl-4H-1-benzopyran-4-one), indicating that Akt activation was dependent on PI3K. S1P-induced activation of PI3K and Akt was abrogated by 1-butanol, which inhibited S1P-induced accumulation of phosphatidic acid by serving as a phosphatidyl group acceptor in the transphosphatidylation reaction catalyzed by PLD, whereas both PI3K and Akt activation were not inhibited by 2-butanol without such reaction. Co-expression of wild-type PLD2 with myc-Akt resulted in increased Akt activation in response to S1P. In contrast, co-expression of a catalytically inactive mutant of PLD2 eliminated the S1P-induced Akt activation. The treatment of EDG3-expressing CHO cells with exogenous Streptomyces chromofuscus PLD, which caused an accumulation of phosphatidic acid, resulted in increases in PI3K activity and the phosphorylation of Akt, the latter of which was completely abolished by LY294002. Furthermore, S1P-induced membrane ruffling, which was dependent on PI3K and Rac, was inhibited by 1-butanol, but not by 2-butanol. These results demonstrate that PLD participates in the activation of PI3K and Akt stimulation of EDG3 receptor.
Subject(s)
DNA-Binding Proteins/genetics , I-kappa B Proteins , Lysophospholipids , Phosphatidylinositol 3-Kinases/metabolism , Phospholipase D/metabolism , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/metabolism , Sphingosine/pharmacology , 1-Butanol/pharmacology , Animals , CHO Cells , Cell Membrane/drug effects , Cricetinae , Cricetulus , DNA-Binding Proteins/metabolism , Enzyme Activation , NF-KappaB Inhibitor alpha , Proto-Oncogene Proteins c-akt , Receptors, Lysophospholipid , Sphingosine/analogs & derivatives , Streptomyces/enzymologyABSTRACT
5'-upstream region of the phospholipase C-beta2 gene, 810 bp, was cloned and characterized. S1 nuclease mapping and primer extension analyses revealed that a single transcriptional start site locates at 284 nucleotides upstream from the beginning of translation. The 5-upstream region lacks both TATA motif and typical initiator sequence, but retains GC-rich segment. Two putative regulatory regions, a negative region (-636/-588) and a positive region (-98/ -13) were identified in the upstream region of PLC-beta2 gene. We suggest that the transcription of PLC-beta2 may be regulated by binding of regulatory proteins to the negative and/or positive regulatory regions located in the upstream of the gene.
Subject(s)
Isoenzymes/chemistry , Isoenzymes/genetics , Type C Phospholipases/chemistry , Type C Phospholipases/genetics , Base Sequence , Cells, Cultured , Chloramphenicol O-Acetyltransferase/metabolism , Cloning, Molecular , Conserved Sequence , Gene Deletion , Molecular Sequence Data , Mutagenesis, Site-Directed , Phospholipase C beta , Promoter Regions, Genetic , Protein Binding , Single-Strand Specific DNA and RNA Endonucleases/metabolism , Transcription, Genetic , TransfectionABSTRACT
Insulin-like growth factor-I (IGF-I) regulates muscle differentiation through phosphatidylinositol 3-kinase (PI 3-kinase). Also it was recently reported that PI 3-kinase is involved in the activation of phospholipase C-gamma1 (PLC-gamma1). We investigated whether PLC-gamma1 therefore plays a role in IGF-I-induced muscle differentiation using H9c2 rat cardiac myoblasts as a model. IGF-I was able to activate PLC-gamma1 via both PI 3-kinase-dependent and tyrosine phosphorylation-dependent mechanisms in this model. However, PI 3-kinase appeared to play a more important role than tyrosine phosphorylation in IGF-I activation of PLC-gamma1. In addition, PLC-gamma1 activation was independent of Akt/protein kinase B (Akt/PKB). Importantly, PLC-gamma1 was involved in IGF-I-induced muscle differentiation in parallel with Akt/PKB. Taken together, these results suggest that IGF-I regulation of muscle differentiation is dependent on the activation of PLC-gamma1 and Akt/PKB, both of which are downstream mediators of PI 3-kinase.
Subject(s)
Insulin-Like Growth Factor I/pharmacology , Isoenzymes/metabolism , Myocardium/cytology , Myocardium/metabolism , Protein Serine-Threonine Kinases , Type C Phospholipases/metabolism , Animals , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Line , Enzyme Activation/drug effects , Kinetics , Phosphatidylinositol 3-Kinases/metabolism , Phospholipase C gamma , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Rats , Signal TransductionABSTRACT
Mammalian phospholipase D (PLD) plays a key role in several signal transduction pathways and is involved in many diverse functions. To elucidate the complex molecular regulation of PLD, we investigated PLD-binding proteins obtained from rat brain extract. Here we report that a 43-kDa protein in the rat brain, beta-actin, acts as a major PLD2 direct-binding protein as revealed by peptide mass fingerprinting in combination with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. We also determined that the region between amino acids 613 and 723 of PLD2 is required for the direct binding of beta-actin, using bacterially expressed glutathione S-transferase fusion proteins of PLD2 fragments. Intriguingly, purified beta-actin potently inhibited both phosphatidylinositol-4,5-bisphosphate- and oleate-dependent PLD2 activities in a concentration-dependent manner (IC50 = 5 nm). In a previous paper, we reported that alpha-actinin inhibited PLD2 activity in an interaction-dependent and an ADP-ribosylation factor 1 (ARF1)-reversible manner (Park, J. B., Kim, J. H., Kim, Y., Ha, S. H., Kim, J. H., Yoo, J.-S., Du, G., Frohman, M. A., Suh, P.-G., and Ryu, S. H. (2000) J. Biol. Chem. 275, 21295-21301). In vitro binding analyses showed that beta-actin could displace alpha-actinin binding to PLD2, demonstrating independent interaction between cytoskeletal proteins and PLD2. Furthermore, ARF1 could steer the PLD2 activity in a positive direction regardless of the inhibitory effect of beta-actin on PLD2. We also observed that beta-actin regulates PLD1 and PLD2 with similar binding and inhibitory potencies. Immunocytochemical and co-immunoprecipitation studies demonstrated the in vivo interaction between the two PLD isozymes and actin in cells. Taken together, these results suggest that the regulation of PLD by cytoskeletal proteins, beta-actin and alpha-actinin, and ARF1 may play an important role in cytoskeleton-related PLD functions.
Subject(s)
Actins/metabolism , Phospholipase D/antagonists & inhibitors , Phospholipase D/metabolism , Animals , Baculoviridae/metabolism , Brain/enzymology , Brain/metabolism , COS Cells , Cell Line , Cytoskeleton/metabolism , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Glutathione Transferase/metabolism , Immunohistochemistry , Inhibitory Concentration 50 , Insecta , PC12 Cells , Phosphatidylinositol 4,5-Diphosphate/metabolism , Precipitin Tests , Protein Binding , Rats , Recombinant Fusion Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , TransfectionABSTRACT
Superoxide is the most important armory on the primary defense line of monocytes against invading pathogens, and the identification of new stimuli and the characterization of the regulatory mechanism of superoxide generation are of paramount importance. In this study, we identified 3 novel peptides by screening a synthetic hexapeptide combinatorial library and modification of 1 of the peptides. The isolated peptides that can induce superoxide generation in human monocytes are His-Phe-Tyr-Leu-Pro-Met-CONH(2) (HFYLPM), Met-Phe-Tyr-Leu-Pro-Met-CONH(2) (MFYLPM), and His-Phe-Tyr-Leu-Pro-D-Met-CONH(2) (HFYLPm). All 3 peptides also caused intracellular calcium ([Ca(++)](i)) rise. We tested the specificities of the peptides on cells of different origin by looking at [Ca(++)](i) rise. All 3 peptides acted specifically on leukocytes and not on nonimmune cells. Among leukocytes, HL60 and Jurkat T cells were stimulated specifically by MFYLPM or HFYLPM, respectively. As a physiologic characteristic of the peptides, we observed that all 3 peptides induced chemotactic migration of monocytes. Studying receptor specificity, we concluded that the 3 peptides might act on some shared and some distinct receptor(s) on leukocytes. Studying intracellular signaling set in motion by the peptides revealed that HFYLPM, but not MFYLPM or HFYLPm, induced chemotaxis via phosphatidylinositol-3 kinase and protein kinase C. Because HFYLPM, MFYLPM, and HFYLPm not only exhibit different specificities depending on cell type and status of differentiation but also stimulate cells via distinct receptors and signaling, the 3 novel peptides might be useful tools to study leukocyte activation.
Subject(s)
Chemotactic Factors , Chemotaxis, Leukocyte , Leukocytes , Humans , Peptide Library , Reactive Oxygen Species , SuperoxidesABSTRACT
It is well established that a phosphoinositide (PI) cycle which is operationally distinct from the classical plasma membrane PI cycle exists within the nucleus, where it is involved in both cell proliferation and differentiation. However, little is known about the regulation of the nuclear PI cycle. Here, we report that nucleus-localized phospholipase C (PLC) beta1, the key enzyme for the initiation of this cycle, is a physiological target of extracellular signal-regulated kinase (ERK). Stimulation of Swiss 3T3 cells with insulin-like growth factor I (IGF-I) caused rapid nuclear translocation of activated ERK and concurrently induced phosphorylation of nuclear PLC beta1, which was completely blocked by the MEK inhibitor PD 98059. Coimmunoprecipitation detected a specific association between the activated ERK and PLC beta1 within the nucleus. In vitro studies revealed that recombinant PLC beta1 could be efficiently phosphorylated by activated mitogen-activated protein kinase but not by PKA. The ERK phosphorylation site was mapped to serine 982, which lies within a PSSP motif located in the characteristic carboxy-terminal tail of PLC beta1. In cells overexpressing a PLC beta1 mutant in which serine 982 is replaced by glycine (S982G), IGF-I failed to activate the nuclear PI cycle, and its mitogenic effect was also markedly attenuated. Expression of S982G was found to inhibit ERK-mediated phosphorylation of endogenous PLC beta1. This result suggests that ERK-evoked phosphorylation of PLC beta1 at serine 982 plays a critical role in the activation of the nuclear PI cycle and is also crucial to the mitogenic action of IGF-I.
Subject(s)
Insulin-Like Growth Factor I/metabolism , Isoenzymes/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinases/metabolism , Mitogens/metabolism , Type C Phospholipases/metabolism , 3T3 Cells , Amino Acid Sequence , Animals , Cell Line , Cell Nucleus/enzymology , Enzyme Activation , Insulin-Like Growth Factor I/pharmacology , Isoenzymes/genetics , Mice , Mitogen-Activated Protein Kinase 3 , Mitogens/pharmacology , Molecular Sequence Data , Mutagenesis, Site-Directed , Phospholipase C beta , Phosphorylation , Serine/genetics , Serine/metabolism , Spodoptera/cytology , Type C Phospholipases/geneticsABSTRACT
The tumor necrosis factor receptor (TNFR)-associated factor (TRAF) proteins play a central role in the early steps of signal transduction by TNFR superfamily proteins, which induce various cellular responses, including apoptosis. Influences of TRAF proteins on the regulation of cell death and physical interactions between TRAFs and caspases have been reported. In this study, we demonstrate that TRAF3 is proteolyzed during cell death in a caspase-dependent manner. TRAF3 was found to be cleaved by incubation with caspase3 in vitro and by Fas- or CD3-triggering in Jurkat-T cells. The Fas- or CD3-induced cleavage of TRAF3 was blocked by caspase inhibitors and by introduction of alanine substitutions for D347 and D367 residues. Furthermore, the amino-terminal fragment of TRAF3 showed a different intracellular localization from the full-length TRAF3 with preferential distribution to particulate fractions and the nucleus. These findings suggest that TRAF3 may be regulated by caspases during apoptosis of T cells.
Subject(s)
Caspases/metabolism , Jurkat Cells/metabolism , Membrane Glycoproteins/pharmacology , Proteins/metabolism , Amino Acid Chloromethyl Ketones/pharmacology , Animals , Apoptosis/drug effects , Apoptosis/physiology , COS Cells/enzymology , COS Cells/metabolism , Caspase 3 , Caspase Inhibitors , Cysteine Proteinase Inhibitors , Fas Ligand Protein , HeLa Cells , Humans , Jurkat Cells/drug effects , Jurkat Cells/enzymology , Muromonab-CD3/pharmacology , Oligopeptides/pharmacology , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , TNF Receptor-Associated Factor 3ABSTRACT
Upon epidermal growth factor treatment, phospholipase C-gamma1 (PLC-gamma1) translocates from cytosol to membrane where it is phosphorylated at tyrosine residues. Caveolae are small plasma membrane invaginations whose structural protein is caveolin. In this study, we show that the translocation of PLC-gamma1 and its tyrosine phosphorylation are localized in caveolae by caveolin-enriched low-density membrane (CM) preparation and immunostaining of cells. Pretreatment of cells with methyl-beta-cyclodextrin (MbetaCD), a chemical disrupting caveolae structure, inhibits the translocation of PLC-gamma1 to CM as well as phosphatidylinositol (PtdIns) turnover. However, MbetaCD shows no effect on tyrosine phosphorylation level of PLC-gamma1. Our findings suggest that, for proper signaling, PLC-gamma1 phosphorylation has to occur at PtdInsP(2)-enriched sites.
Subject(s)
Caveolae/metabolism , Epidermal Growth Factor/metabolism , Isoenzymes/metabolism , Phosphatidylinositols/metabolism , Signal Transduction , Type C Phospholipases/metabolism , Tyrosine/metabolism , beta-Cyclodextrins , Animals , Blotting, Western , COS Cells , Caveolae/enzymology , Caveolae/ultrastructure , Caveolin 1 , Caveolins/metabolism , Cell Compartmentation , Cell Line , Cell Membrane/enzymology , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Cyclodextrins/pharmacology , Hydrolysis , Phospholipase C gamma , Phosphorylation , Precipitin TestsABSTRACT
Apoptotic proteases cleave and inactivate survival signaling molecules such as Akt/PKB, phospholipase C (PLC)-gamma1, and Bcl-2. We have found that treatment of A431 cells with tumor necrosis factor-alpha in the presence of cycloheximide resulted in the cleavage of epidermal growth factor receptor (EGFR) as well as the activation of caspase-3. Among various caspases, caspase-1, caspase-3 and caspase-7 were most potent in the cleavage of EGFR in vitro. Proteolytic cleavage of EGFR was inhibited by both YVAD-cmk and DEVD-fmk in vitro. We also investigated the effect of caspase-dependent cleavage of EGFR upon the mediation of signals to downstream signaling molecules such as PLC-gamma1. Cleavage of EGFR by caspase-3 significantly impaired the tyrosine phosphorylation of PLC-gamma1 in vitro. Given these results, we suggest that apoptotic protease specifically cleaves and inactivates EGFR, which plays crucial roles in anti-apoptotic signaling, to abrogate the activation of EGFR-dependent downstream survival signaling molecules.
Subject(s)
Caspases/metabolism , ErbB Receptors/metabolism , Isoenzymes/metabolism , Type C Phospholipases/metabolism , Amino Acid Chloromethyl Ketones/pharmacology , Amino Acid Sequence , Caspase 1/metabolism , Caspase 2 , Caspase 3 , Caspase 7 , Caspase Inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Humans , Molecular Sequence Data , Oligopeptides/pharmacology , Peptide Hydrolases/metabolism , Phospholipase C gamma , Phosphorylation , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Tyrosine/metabolismABSTRACT
His-Phe-Tyr-Leu-Pro-Met (HFYLPM) is a synthetic peptide that stimulates Jurkat T cells resulting in intracellular calcium ([Ca2+]i) increase in a pertussis toxin (PTX)-sensitive manner. We have examined the physiological role of the peptide in T cell activity by comparative investigation of intracellular signaling pathways accompanied with HFYLPM-induced T cell chemotaxis with a well-known chemokine, stromal cell-derived factor-1 (SDF-1)-induced signalings. Wortmannin and genistein inhibited both of HFYLPM- and SDF-1-induced Jurkat T cell chemotaxis indicating that phosphoinositide-3-kinase and tyrosine kinase activity were required for the processes. However, U-73122 and BAPTA/AM preferentially blocked HFYLPM- but not SDF-1-induced T cell chemotaxis. It indicates that phospholipase C/calcium signaling is necessary for only chemotaxis by HFYLPM. One of the well-known cellular molecules involving chemotaxis, extracellular signal-regulated protein kinase (ERK), was activated by SDF-1 but not by HFYLPM ruling out a possible role of ERK on the peptide-mediated chemotaxis. These results indicate that the synthetic peptide, HFYLPM, stimulates T cell chemotaxis showing unique signaling and provide a useful tool for the study of T cell activation mechanism.
Subject(s)
Chemokines, CXC/pharmacology , Chemotaxis, Leukocyte/physiology , Peptide Fragments/physiology , T-Lymphocytes/drug effects , Androstadienes/pharmacology , Calcium/metabolism , Cell Line , Chemotaxis, Leukocyte/drug effects , Dose-Response Relationship, Drug , Genistein/pharmacology , Humans , Jurkat Cells , Oligopeptides , Peptide Fragments/chemical synthesis , Peptide Fragments/metabolism , Pertussis Toxin , Phosphatidylinositol 3-Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Signal Transduction/drug effects , Type C Phospholipases/metabolism , Virulence Factors, Bordetella/pharmacology , WortmanninABSTRACT
Areca catechu L. extracts I and II, prepared using two different solvent systems, exhibited strong inhibitory activities against pancreatic cholesterol esterase (pCEase) in vitro. To determine their cholesterol-lowering effects, these two extracts were investigated by analyzing plasma lipid levels, intestinal enzyme activities, and the absorption of cholesteryl oleate. For 6 days, male rats were fed a diet containing cholesteryl oleate (0.5 g/100 g of body weight) either with or without the Areca nut extract supplements. The supplementation of the two Areca nut extracts significantly lowered the concentrations of plasma cholesterol by 13. 4 and 11.7% and plasma triglycerides by 35.0 and 36.9%, respectively, compared with the pre-experimental values. However, when the cholesteryl oleate diet was fed without any Areca nut extract in high-cholesterol control, the plasma cholesterol and triglyceride concentrations significantly increased by 13.6 and 15.9%, respectively, compared with the pre-experimental values. After 6 days of treatment, the intestinal pCEase activities were significantly lower in the groups supplemented with the Areca nut extracts (37.8 and 26.5%) than in the group with no extract supplement (83.2%). The supplements also significantly elevated the excretion of [1,2(n)-(3)H]cholesteryl oleate administered orally, when determined by the large intestinal contents, 930.5 Bq/day (Areca I) and 1,766.3 Bq/day (Areca II) vs. 98.1 Bq/day (high-cholesteryl oleate (CO) control). The inhibition of pCEase activity with the supplementation of the Areca nut extracts could account for the decrease in [1,2(n)-(3)H]cholesteryl oleate absorption that resulted in decreased radioactivity in blood.