Showing papers by "David Communi published in 1995"

Tissue and cell specific expression of Ins(1,4,5)P3 3-kinase isoenzymes

Abstract: The phosphorylation of Ins(1,4,5)P3 (InsP3) to Ins(1,3,4,5)P4 (InsP4) is catalysed by InsP3 3-kinase. Molecular-biological data have shown the presence of two human isoenzymes of InsP3 3-kinase, namely InsP3 3-kinases A and B. We have isolated from a rat thymus cDNA library a 2235 bp cDNA (clone B15) encoding rat InsP3 3-kinase B. Northern-blot analysis of mRNA isolated from rat tissues (thymus, testis, brain, spleen, liver, kidney, heart, lung and intestine) revealed that a rat InsP3 3-kinase B probe hybridized to a 6 kb mRNA in lung, thymus, testis, brain and heart. In contrast, Northern-blot analysis of the same tissues probed under stringent conditions with a rat InsP3 3-kinase A probe hybridized to a 2 kb mRNA only in brain and a 1.8-2.0 kb mRNA species in testis. Northern-blot analysis of three human cell lines (HL-60, SH-SY5Y and HTB-138) probed with a human InsP3 3-kinase B probe showed the presence of a 6 kb mRNA in all cell lines, except in the human neuroblastoma cell line (SH-SY5Y), where two mRNA species of 5.7 and 6 kb were detected. Using the same blot, no hybridization signal could be seen with a human InsP3 3-kinase A probe. Altogether, our data are consistent with the notion that the two InsP3 3-kinase isoenzymes, A and B, are specifically expressed in different tissues and cells.

Production of recombinant human brain type I inositol- 1,4,5=trisphosphate 5-phosphatase in Escherichia coli Lack of phosphorylation by protein kinase C

Abstract: The dephosphorylation of inositol 1,4,5-trisphosphate (InsP3) to inositol 1,4-bisphosphate is catalyzed by InsP3 5-phosphatase. The coding region of human brain type I InsP3 5-phosphatase was expressed as a fusion protein with the maltose-binding protein (MBP) in Escherichia coli, using the pMAL-cR1 vector. The relative molecular mass of the purified fusion protein (MBP-InsP3-5-phosphatase) was approximately M(r) 85,000 as analysed by SDS/PAGE. The yield was about 10 mg fusion protein/l lysate. After cleavage from MBP with factor Xa, the specific activity of recombinant 5-phosphatase was 120-250 mumol.mg-1.min-1. The molecular mass of purified protein by SDS/PAGE was M(r) 43,000. The activity was inactivated by p-hydroxymercuribenzoate. The possibility that protein kinase C might phosphorylate InsP3 5-phosphatase was tested on the purified 43,000 M(r) protein. In this study, we show that recombinant 5-phosphatase is not a substrate of protein kinase C.

Active site labelling of inositol 1,4,5-trisphosphate 3-kinase A by phenylglyoxal.

Abstract: Chemical modification by phenylglyoxal, an arginine-specific reagent, of both native and recombinant rat brain inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] 3-kinase A was accompanied by irreversible inhibition of enzyme activity. This effect was prevented in the presence of the substrate ATP but not Ins(1,4,5)P3. The modification reaction obeyed pseudo-first-order rate kinetics. Complete inhibition of activity corresponded to incorporation of 1.2 mol of phenylglyoxal per mol of protein. A single [14C]phenylglyoxal-modified peptide was isolated following alpha-chymotrypsin digestion of the radiolabelled Ins(1,4,5)P3 3-kinase and reverse-phase HPLC. ATP prevented the incorporation of radioactivity to this peptide. The peptide sequence (i.e. QWREGISSSTTL) corresponded to amino acids 315 to 326 of rat brain Ins(1,4,5)P3 3-kinase A. An estimate of the radioactivity of the different phenylthiohydantoin amino acid derivative showed the modified amino acid to be Arg-317. The data directly identify a reactive arginine residue as part of the ATP-binding site. Arg-317 is located within a sequence segment which is conserved among the catalytic domain of Ins(1,4,5)P3 3-kinase isoenzymes A and B in human and rat species.

Implication des phosphatiyl inositols et de leur produits d'hydrolyse dans la signalisation cellulaire

Abstract: Ins(1,4,5)P3 is a well-known inositol lipid-derived intracellular second messenger mobilizing intracellular calcium from internal stores. It is generated through two major signaling pathways: the former involves G proteins-coupled receptors and the latter tyrosine kinase-linked receptors. Ins(1,4,5)P3 mobilizes intracellular calcium by binding to its receptor, showing structural and physiological similarities with the ryanodine receptor (another intracellular calcium channel). It is metabolized by both an Ins(1,4,5)P3 5-phosphatase and 3-kinase reaction. Biochemical and molecular studies indicated at each step of the signal transduction pathway extensive molecular heterogeneity in the synthesis and degradation of inositol lipids derived molecules. Ins(1,4,5)P3 3-kinase consists in a family of calmodulin sensitive isoenzymes producing Ins(1,3,4,5)P4 (three isozymes). Ins(1,4,5)P3 5-phosphatase dephosphorylates both Ins(1,4,5)P3 and Ins(1,3,4,5)P4. It is interesting to note that the Lowe's oculocere-brorenal syndrome may be caused by a defect in a gene that encodes an enzyme that metabolizes Ins(1,4,5)P3, since type III of Ins(1,4,5)P3, 5-phosphatase shows high levels of sequence homology with the protein encoded by the Lowe's syndrome deficient gene. PtdIns(4,5)P2 is the phospholipase C substrate to generate Ins(1,4,5)P3 and diacylglycerol (DAG) but also the substrate of a specific 3-kinase to produce PtdIns(3,4,5)P3, another potential second messenger. Like phospholipase C, PtdIns(4,5)P2 3-kinase may be activated through G protein-coupled receptors or tyrosine kinase-linked receptors. The levels of Ins(3,4,5,6)P4 may also be regulated through the activation of receptors by extracellular signals and this molecule may also play a role of second messenger.