ABSTRACT
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.
Subject(s)
Mice , Animals , Antibodies, Monoclonal/immunology , fas Receptor/immunology , Bridged-Ring Compounds/pharmacology , Cell Line , Cross-Linking Reagents , Dose-Response Relationship, Immunologic , Enzyme Activation , Genistein/pharmacology , Hydrolysis , Lymphoma/pathology , Type C Phospholipases/antagonists & inhibitors , Phospholipase D/metabolism , Phosphorylation , Phosphorylcholine/metabolism , Solubility , Thiones/pharmacology , Tumor Cells, Cultured , Tyrosine/metabolism , Water/chemistryABSTRACT
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.
Subject(s)
Mice , Animals , Antibodies, Monoclonal/immunology , fas Receptor/immunology , Bridged-Ring Compounds/pharmacology , Cell Line , Cross-Linking Reagents , Dose-Response Relationship, Immunologic , Enzyme Activation , Genistein/pharmacology , Hydrolysis , Lymphoma/pathology , Type C Phospholipases/antagonists & inhibitors , Phospholipase D/metabolism , Phosphorylation , Phosphorylcholine/metabolism , Solubility , Thiones/pharmacology , Tumor Cells, Cultured , Tyrosine/metabolism , Water/chemistryABSTRACT
Cdc42 is a member of the Rho family of small GTP-ase and plays an important role in intracellular signaling pathways regulating cell morphology, motility and stimulation of DNA synthesis. We have isolated cDNA encoding Cdc42 from a rat brain cDNA library using PCR-cloning strategy. The sequence of isolated gene revealed an open reading frame of 576 nucleotides with the potential to encode a protein of 191 amino acids with a predicted molecular weight of 21 kD. The resulting sequence was incorporated into the GenBank with accession number, AF205635. Sequence analysis revealed that overall cDNA sequence identity is 96% with human G25K and 52% with rat Chp, a homologue of the GTPase human Cdc42Hs, and having one nucleotide difference from the mouse Cdc42. However, putative protein sequence was identical to the mouse and human brain Cdc42Hs. On expression of the cDNA in COS-7 cells, a protein molecular weight of 21 kD was detected in immunoblotting using anti-human Cdc42 antibodies. Therefore, these results suggest that the cDNA we are reporting is most likely the rat homologue of the GTPase human Cdc42.