OVEREXPRESSION AND BIOCHEMICAL CHARACTERIZATION OF PHOSPHOLIPASE C-BLB

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2 FORMS OF PHOSPHOLIPASE C-BETA-1 GENERATED BY ALTERNATIVE SPLICING

Phospholipase C-beta 1 (PLC-beta 1) exists as two immunologically indistinguishable polypeptides of 150 and 140 kDa and is encoded in rat brain by two distinct transcripts of 5.4 and 7.2 kilobases (kb). cDNA corresponding to the entire 5.4-kb transcript as reported previously reveals an open reading frame that is capable of coding a 1216-amino acid polypeptide (Suh, P. G., Ryu, S. H., Moon, K. H., Suh, H. W., and Rhee, S. G. (1988) Cell 54, 161-169). We have now isolated cDNAs corresponding to the entire 7.2-kb transcript from a rat brain cDNA library. The 7.2-kb transcript differs from the previously reported 5.4-kb transcript by possessing both an additional 118 nucleotides located near the end of the coding sequence and a 1738-nucleotide extension of the 3'-flanking region. The presence of the 118-nucleotide insert in the cumulative 7.2-kb sequence gives rise to an open reading frame that is capable of coding a 1173-amino acid polypeptide (PLC-beta 1b), the carboxyl terminal sequence of which differs from that of the 1216-amino acid polypeptide (PLC-beta 1a) derived from the 5.4-kb transcript. Antibodies were raised against synthetic peptides corresponding to the carboxyl-terminal portions of PLC- beta 1a and PLC-beta 1b. Immunoblot analysis with these isozyme specific antibodies revealed that both PLC-beta 1a and PLC-beta 1b are expressed in rat brain and C(6)Bu-1 glioma cells and that PLC-beta 1a and PLC-beta 1b correspond to the previously identified 150- and 140-kDa PLC-beta 1 enzymes, respectively. Analysis of PLC-beta 1 genomic DNA indicates that PLC-beta 1a and PLC-beta 1b are derived from a single gene by alternative RNA splicing.X1171sciescopu

CLONING OF CDNA-ENCODING RAT PHOSPHOLIPASE C-BETA-4, A NEW MEMBER OF THE PHOSPHOLIPASE-C

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CLONING OF CDNA-ENCODING RAT PHOSPHOLIPASE C-BETA-4, A NEW MEMBER OF THE PHOSPHOLIPASE-C

Phospholipase C-??4(PLC-??4), a new member of phospholipase C isozyme, was purified from bovine cerebellum. The cDNA encoding rat PLC-??4 has been cloned from a cDNA library prepared from rat brain. The predicted open reading frame encodes a protein of 1,176 amino acids with a calculated molecular weight of 134,552. The deduced amino acid sequence exhibits 39, 36, and 36% identity with the sequences of rat PLC-??1, human PLC-??2, and rat PLC-??3, respectively. The amino acid sequence of PLC-??4, especially, shows higher identity (50%) with norpA PLC sequence from Drosophila melanogaster than those of other PLC-?? subtypes, suggesting that the PLC-??4 might be a mammalian PLC equivalent of norpA PLC implicated in photosignal transduction in Drosophila.close261

The promoter activity of the phospholipase C-gamma 2 gene is regulated by a cell-type-specific control element

We have cloned and characterized a genomic DNA spanning the 5'-flanking region, the first and second exons, and the first intron of the human PLC-gamma 2 gene. The proximal upstream region is highly CC-rich and lacks a TATA box, whereas the distal region contains several AT-rich tracts. Multiple transcription initiation sites were identified by primer extension analysis, Based on the transient transfection assays, the major transcriptional activation element was identified between -183 and +43 (G2SE) and a transcriptional repressive element was found between -303 and -184 (G2RE). The expression of PLC-gamma 2 in various cell lines was examined using monoclonal anti-PLC-gamma 2 antibody, PLC-gamma 2 was highly expressed in B-cell lines such as Daudi, SP2, and Ramos cells, whereas it existed at very low levels in Jurkat, 3T3-L1, NBL-7, and C6Bu-1 cells. Moderate levels of PLC-gamma 2 were also detected in C2C12, P19, U937, HL60, A431, and PC12 cells. The 4-kb genomic fragment upstream of -1,654 was able to activate transcription from the PLC-gamma 2 promoter in Daudi and C2C12 cells, but not in Jurkat cells, which is consistent with the PLC-gamma 2 protein expression levels in those cell lines. These results suggest that the cell-type-specific expression of PLC-gamma 2 might be attributed to the transcriptional regulation by the upstream cis-element.close4

The Vr-PLC3 gene encodes a putative plasma membrane-localized phosphoinositide-specific phospholipase C whose expression is induced by abiotic stress in mung bean (Vigna radiata L.)

Phosphoinositide-specific phospholipase C (PI-PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate and diacylglycerol, both of which act as secondary messengers in animal cells. In this report, we identified in Vigna radiata L. (mung bean) three distinct partial cDNAs (pVr-PLC1, pVr-PLC2, and pVrPLC3), which encode forms of putative PI-PLC. All three Vr-PLC genes were transcriptionally active and displayed unique patterns of expression. The Vr-PLC1 and Vr-PLC2 transcripts were constitutively expressed to varying degrees in every tissue of mung bean plants examined. In contrast, the Vr-PLC3 mRNA level was very low under normal growth conditions and was rapidly induced in an abscisic acid-independent manner under environmental stress conditions (drought and high salinity). An isolated genomic clone, about 8.2 kb in length, showed that Vr-PLC1 and Vr-PLC3 are in tandem array in the mung bean genome. The predicted primary sequence of Vr-PLC3 (M-r = 67.4 kDa) is reminiscent of the delta-isoform of animal enzymes which contain core sequences found in typical PI-PLCs, such as the catalytic domain comprising X and Y motifs, a lipid-binding C2 domain, and the less conserved EF-hand domain. Results of in vivo targeting experiment using a green fluorescent protein (GFP) showed that the GFP-Vr-PLC3 fusion protein was localized primarily to the plasma membrane of the Arabidopsis protoplast. The C2 domain was essential for Vr-PLC3 to be targeted to the plasma membrane. The possible biological functions of stress-responsive Vr-PLC3 in mung bean plants are discussed. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.X1149sciescopu

Nuclear phospholipid signaling: phosphatidylinositol-specific phospholipase C and phosphoinositide 3-kinase

Over the last 20 years, numerous studies have demonstrated the existence of nuclear phosphoinositide signaling distinct from the one at the plasma membrane. The activation of phosphatidylinositol-specific phospholipase C (PI-PLC) and phosphoinositide 3-kinase (PI3K), the generation of diacylglycerol, and the accumulation of the 3-phosphorylated phosphoinositides have been documented in the nuclei of different cell types. In this review, we summarize some recent studies of the subnuclear localization, mechanisms of activation, and the possible physiological roles of the nuclear PI-PLC and PI-3 kinases in the regulation of cell cycle, survival, and differentiation