Phospholipase D (PLD) hydrolyzes phosphocholine into choline and phosphatide acid, and these metabolites play an important role in regulating cell physiology and biochemistry. To study the biological function of phospholipase D3 (PLD3) during the insulin stimulation in C2C12 myoblasts, we constructed PLD3 over-expressed cell lines (C2C12/pPLD3) and investigated the phosphorylation of Akt. The results showed that the level of phosphorylated Akt (P-Akt) was significantly increased in control C2C12 cells when insulin concentration was elevated during cell treatment, whereas the level of P-Akt in C2C12/pPLD3 cells was not changed. When extending the time of insulin treatment, P-Akt level in C2C12/pPLD3 cells was increased around 2 folds, but the total level of P-Akt in C2C12/pPLD3 was still lower than that in control group. 1-Butanol, a PLD inhibitor, could completely block Akt phosphorylation in C2C12 cells that even stimulated by insulin. However, 1-Butanol did not inhibit the Akt phosphorylation in C2C12/pPLD3 cells, but increased the phosphorylation up to 6 folds higher than control cells. The level of Akt phosphorylation in control C2C12 cells was increased significantly when stimulated by phosphatidic acid (PA), while there was no change in C2C12/pPLD3 cells with the similar treatment. When cells simulated by both PA and insulin, P-Akt level in both C2C12/pPLD3 cells and C2C12 cells were down regulated. Our observations indicated that PLD3 over expression may inhibit Akt phosphorylation and further block the transduction of insulin signaling in C2C12 cells.
Cell Line ; Humans ; Insulin ; pharmacology ; Myoblasts ; cytology ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Phospholipase D ; biosynthesis ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; chemistry ; drug effects ; Signal Transduction

Phospholipase C(PLC) plays a central role in signal transduction and it is important in cellular growth, differentiation and transformation. There are currently ten known mammalian isozymes of PLC identified and cloned. However, there are no report of PLC distribution in human lung tissue or their significances in pulmonary diseases. Presence of various PLC isozymes in normal human lung tissue was studied from surgical specimens. PLC isozymes in tissue extracts of the lung were partially purified by successive chromatographic steps on heparin-sepharose CL-6B conventional and TSKgel heparin-5PW HPLC columns and their activities were assayed. PLC activity peaks identified in the chromatography were immunoblotted with specific antibodies against ten known mammalian PLC isozymes(PLC-beta 1-4, -gamma 1-2, and -delta 1-4). In addition, immunohistochemical staining of the lung tissue was performed to determine subcellular and histological localization of PLC isozymes. The results indicate that normal human lungs contain beta 1, beta 3, gamma 1, and delta 1, isozymes of PLC. The order of amount present in the lung tissue was PLC-delta 1 > gamma 1 >beta 1 >> beta 3, in descending order. On immunohistochemistry, PLC-gamma 1 was most widely distributed and was present in bronchiolar epithelium, in type I and type II pneumocytes as well as in fibroblasts of the interstitial tissue. PLC-delta 1 was present in the cytoplasm of the bronchiolar epithelium whereas PLC-beta 1 was localized to the apical membranous portion of the same epithelium. PLC-beta 3 was seen in the nucleus of the respiratory and alveolar lining epithelium as well as in the nucleus of lung fibroblasts.
Adult ; Chromatography, Agarose ; Female ; Heparin/chemistry ; Human ; Immunohistochemistry ; Isoenzymes/isolation & purification/*metabolism ; Lung/*enzymology/pathology ; Male ; Phospholipase C/isolation & purification/*metabolism

Phospholipase Cgamma1 (PLCgamma1) plays an important role in controlling cellular proliferation and differentiation. PLCgamma1 is overexpressed in some tumors, and its overexpression induces solid tumors in nude mice. However, the regulatory mechanisms underlying PLCgamma1-induced cell proliferation are not fully understood. Here we show that overexpression of PLCgamma1 highly phosphorylated glycogen synthase kinase-3beta (GSK-3beta) at serine-9 in 3Y1 fibroblasts. Inhibition of protein kinase C (PKC)s with GF109203X abrogated GSK-3beta phosphorylation by PLCgamma1. We also found that steady-state level of cyclin D1 protein, but not cyclin D1 mRNA, was highly elevated in response to serum stimulation in PLCgamma1-transfected cells as compared with vector-transfected cells. Since GSK-3beta is involved in cyclin D1 proteolysis in response to mitogenic stimulation, PLCgamma1-mediated GSK-3beta phosphorylation may function as a regulation of cyclin D1 accumulation in PLCgamma1-overexpressing cells.
Animals ; Cyclin D1/metabolism ; Epidermal Growth Factor/pharmacology ; Fibroblasts ; Gene Expression ; Glycogen Synthase Kinase 3/chemistry/*metabolism ; Mitogens/pharmacology ; Phospholipase C/genetics/*metabolism ; Phosphorylation/drug effects ; Phosphoserine/*metabolism ; Protein Kinase C/antagonists & inhibitors/*metabolism ; Rats ; Signal Transduction

Both Fas and PMA can activate phospholipase D via activation of protein kinase Cbeta in A20 cells. Phospholipase D activity was increased 4 fold in the presence of Fas and 2.5 fold in the presence of PMA. The possible involvement of tyrosine phosphorylation in Fas-induced activation of phospholipase D was investigated. In five minute after Fas cross-linking, there was a prominent increase in tyrosine phosphorylated proteins, and it was completely inhibited by D609, a specific inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC). A tyrosine kinase inhibitor, genistein, can partially inhibit Fas-induced phospholipase D activation. There were no effects of genistein on Fas-induced activation of PC-PLC and protein kinase C. These results strongly indicate that tyrosine phosphorylation may in part account for the increase in phospholipase D activity by Fas cross-linking and D609 can block not only PC-PLC activity but also tyrosine phosphorylation involved in Fas-induced phospholipase D activation.
Animal ; Antibodies, Monoclonal/immunology/*pharmacology ; Antigens, CD95/immunology/*metabolism ; Bridged Compounds/*pharmacology ; Cell Line ; Cross-Linking Reagents ; Dose-Response Relationship, Immunologic ; Enzyme Activation ; Genistein/pharmacology ; Hydrolysis ; Lymphoma/pathology ; Mice ; Phospholipase C/*antagonists & inhibitors ; Phospholipase D/*metabolism ; Phosphorylation ; Phosphorylcholine/metabolism ; Solubility ; Thiones/*pharmacology ; Tumor Cells, Cultured ; Tyrosine/*metabolism ; Water/chemistry

Both Fas and PMA can activate phospholipase D via activation of protein kinase Cbeta in A20 cells. Phospholipase D activity was increased 4 fold in the presence of Fas and 2.5 fold in the presence of PMA. The possible involvement of tyrosine phosphorylation in Fas-induced activation of phospholipase D was investigated. In five minute after Fas cross-linking, there was a prominent increase in tyrosine phosphorylated proteins, and it was completely inhibited by D609, a specific inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC). A tyrosine kinase inhibitor, genistein, can partially inhibit Fas-induced phospholipase D activation. There were no effects of genistein on Fas-induced activation of PC-PLC and protein kinase C. These results strongly indicate that tyrosine phosphorylation may in part account for the increase in phospholipase D activity by Fas cross-linking and D609 can block not only PC-PLC activity but also tyrosine phosphorylation involved in Fas-induced phospholipase D activation.
Animal ; Antibodies, Monoclonal/immunology/*pharmacology ; Antigens, CD95/immunology/*metabolism ; Bridged Compounds/*pharmacology ; Cell Line ; Cross-Linking Reagents ; Dose-Response Relationship, Immunologic ; Enzyme Activation ; Genistein/pharmacology ; Hydrolysis ; Lymphoma/pathology ; Mice ; Phospholipase C/*antagonists & inhibitors ; Phospholipase D/*metabolism ; Phosphorylation ; Phosphorylcholine/metabolism ; Solubility ; Thiones/*pharmacology ; Tumor Cells, Cultured ; Tyrosine/*metabolism ; Water/chemistry

Phosphoinositide-specific phospholipase C-gamma1 (PLC-gamma1) has two pleckstrin homology (PH) domains: an amino-terminal domain (PH1) and a split PH domain (PH2). Here, we show that overlay assay of bovine brain tubulin pool with glutathione-S-transferase (GST)-PLC-gamma1 PH domain fusion proteins, followed by matrix-assisted laser-desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), identified 68-kDa neurofilament light chain (NF-L) as a binding protein of amino-terminal PH domain of PLC-gamma1. NF-L is known as a component of neuronal intermediate filaments, which are responsible for supporting the structure of myelinated axons in neuron. PLC-gamma1 and NF-L colocalized in the neurite in PC12 cells upon nerve growth factor stimulation. In vitro binding assay and immunoprecipitation analysis also showed a specific interaction of both proteins in differentiated PC12 cells. The phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P2] hydrolyzing activity of PLC-gamma1 was slightly decreased in the presence of purified NF-L in vitro, suggesting that NF-L inhibits PLC-gamma1. Our results suggest that PLC-gamma1-associated NF-L sequesters the phospholipid from the PH domain of PLC-gamma1.
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Rats ; Protein Interaction Mapping ; Protein Biosynthesis/drug effects ; Protein Binding/drug effects ; Phosphoproteins/chemistry/*metabolism ; Phospholipase C gamma/antagonists & inhibitors/chemistry/*metabolism ; Phosphatidylinositol 4,5-Diphosphate/metabolism ; Peptides/chemistry/metabolism ; PC12 Cells ; Neurofilament Proteins/chemistry/*metabolism ; Nerve Growth Factor/pharmacology ; Molecular Weight ; Molecular Sequence Data ; Microtubules/metabolism ; Microscopy, Fluorescence ; Isoenzymes/metabolism/pharmacology/physiology ; Glutathione Transferase/metabolism ; Blotting, Far-Western ; Blood Proteins/chemistry/*metabolism ; Binding Sites ; Animals ; Amino Acid Sequence

Phospholipase C-gamma1, containing two SH2 and one SH3 domains which participate in the interaction between signaling molecules, plays a significant role in the growth factor-induced signal transduction. However, the role of the SH domains in the growth factor-induced PLC-gamma1 regulation is unclear. By peptide-mass fingerprinting analysis, we have identified SHIP1 as the binding protein for the SH3 domain of PLC-gamma1. SHIP1 was co-immunoprecipitated with PLC-gamma1 and potentiated EGF-induced PLC-gamma1 activation. However, inositol 5'-phosphatase activity of SHIP1 was not required for the potentiation of EGF-induced PLC-gamma1 activation. Taken together, these results suggest that SHIP1 may function as an adaptor protein which can potentiate EGF-induced PLC-gamma1 activation without regards to its inositol 5'-phosphatase activity.
Adaptor Proteins, Signal Transducing ; Amino Acid Sequence ; Animals ; COS Cells/enzymology ; Cercopithecus aethiops ; Enzyme Activation ; Epidermal Growth Factor/*pharmacology ; Immunoprecipitation ; Inositol 1,4,5-Trisphosphate/metabolism ; Molecular Sequence Data ; Phospholipase C/chemistry/*metabolism ; Phosphoric Monoester Hydrolases/chemistry/*metabolism ; Protein Binding ; Signal Transduction ; src Homology Domains/*physiology

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.
Aspergillus Nuclease S1/metabolism ; Base Sequence ; Cells, Cultured ; Chloramphenicol O-Acetyltransferase/metabolism ; Cloning, Molecular ; Conserved Sequence ; Gene Deletion ; Isoenzymes/*chemistry/*genetics ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Phospholipase C/*chemistry/*genetics ; Promoter Regions (Genetics) ; Protein Binding ; Support, Non-U.S. Gov't ; Transcription, Genetic ; Transfection

Animals ; Carcinoma, Squamous Cell ; genetics ; Colorectal Neoplasms ; genetics ; metabolism ; Enzyme Activation ; Esophageal Neoplasms ; genetics ; Genome-Wide Association Study ; Head and Neck Neoplasms ; genetics ; Humans ; Neoplasms ; chemically induced ; enzymology ; genetics ; Phosphoinositide Phospholipase C ; chemistry ; genetics ; metabolism ; physiology ; Signal Transduction ; Skin Neoplasms ; chemically induced ; enzymology ; Stomach Neoplasms ; genetics ; Urinary Bladder Neoplasms ; metabolism ; pathology ; ras Proteins ; metabolism