Colette Moreau

The SH2 domain containing inositol 5‐phosphatase SHIP2 displays phosphatidylinositol 3,4,5‐trisphosphate and inositol 1,3,4,5‐tetrakisphosphate 5‐phosphatase activity

Distinct forms of inositol and phosphatidylinositol polyphosphate 5‐phosphatases selectively remove the phosphate from the 5‐position of the inositol ring from both soluble and lipid substrates. SHIP1 is the 145‐kDa SH2 domain‐containing inositol 5‐phosphatase expressed in haematopoietic cells. SHIP2 is a related but distinct gene product. We report here that SHIP2 can be expressed in an active form both in Escherichia coli and in COS‐7 cells. A truncated 103‐kDa recombinant protein could be purified from bacteria that display both inositol 1,3,4,5‐tetrakisphosphate (InsP4) and phosphatidylinositol 3,4,5‐trisphosphate (PtdIns(3,4,5)P3) phosphatase activities. COS‐7 cell lysates transfected with SHIP2 had increased PtdIns(3,4,5)P3 phosphatase activity as compared to the vector alone.

Stimulation by thyrotropin, cholera toxin and dibutyryl cyclic AMP of the multiplication of differentiated thyroid cells in vitro

Primary cultures of dog thyroid cells have been established. The cells originated from follicles and displayed differentiation characteristics of such cells: iodide trapping and organification, responsiveness of iodide organification and cyclic AMP accumulation to thyrotropin (TSH), induction of a two-dimensional follicular structure by TSH. TSH also stimulated the multiplication of these cells. The effect of TSH was detected with concentrations as low as 100 muU/ml and was reproduced with purified TSH. It was reproduced by cholera toxin (10 ng/ml) and dibutyryl cyclic AMP (10(-5) M). The data show that TSH, which stimulates the function of thyroid tissue, in vivo and in vitro, activates the multiplication of differentiated dog-thyroid follicular cells in primary culture, which suggests that this trophic effect is, partly at least, mediated by cyclic AMP.

The three isoenzymes of human inositol-1,4,5-trisphosphate 3-kinase show specific intracellular localization but comparable Ca2+ responses on transfection in COS-7 cells

Inositol 1,4,5-trisphosphate [Ins(1,4,5) P3] 3-kinase catalyses the phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate. cDNAs encoding three human isoenzymes of InsP3 3-kinase (A, B and C) have been reported previously [Choi, Kim, Lee, Moon, Sim, Kim, Chung and Rhee (1990) Science 248, 64-66; Dewaste, Pouillon, Moreau, Shears, Takazawa and Erneux (2000) Biochem. J. 352, 343-351; Dewaste, Roymans, Moreau and Erneux (2002) Biochem. Biophys. Res. Commun. 291, 400-405; Takazawa, Perret, Dumont and Erneux (1991) Biochem. Biophys. Res. Commun. 174, 529-535]. The localization of InsP3 3-kinase isoenzymes fused at their N-terminus to the green fluorescent protein has been studied by confocal microscopy. The A isoform appeared to associate with the cytoskeleton, whereas the C isoform was totally cytoplasmic. The B isoform had a more complex localization: it appeared in the plasma membrane, cytoskeleton and in the endoplasmic reticulum. The three human isoenzymes of InsP3 3-kinase can thus be distinguished by their N-terminal sequence, sensitivity to Ca2+/calmodulin and localization on transfection in COS-7 cells. We have compared the cytosolic Ca2+ responses induced by ATP in COS-7 cells transfected with the three isoenzymes. Cells expressing high levels of any of the three isoforms no longer respond to ATP, whereas cells expressing low levels of each enzyme showed a reduced response consisting of one to three Ca2+ spikes in response to 100 microM ATP. These effects were seen only in wild-type InsP3 3-kinase-transfected cells. 3-Kinase-dead mutant cells behaved as vector-transfected cells. The results highlight the potential role of the three isoforms of InsP3 3-kinase as direct InsP3 metabolizing enzymes and direct regulators of Ca2+ responses to extracellular signals.

Characterization of D-myo-inositol 1,4,5-trisphosphate phosphatase in rat brain.

Rat brain homogenates contain significant amounts of inositol 1,4,5-trisphosphate phosphatase in both 180,000xg (60 min) particulate and supernatant fractions. As other membrane-bound enzymes (e.g. guanylate cyclase), particulate inositol 1,4,5-trisphosphate phosphatase activity is highly sensitive to low concentrations of Triton X-100 (0.03%). Higher concentrations of detergent (1%) partially solubilized the enzyme. Thiol blocking agents (e.g. p-hydroxymercuribenzoate) inactivate inositol 1,4,5-trisphosphate phosphatase activity (an effect reversed with 2-mercaptoethanol). It is thus suggested that enzymatic activity requires the presence of -SH groups.

The two SH2-domain-containing inositol 5-phosphatases SHIP1 and SHIP2 are coexpressed in human T lymphocytes.

Abstract The activation of many hematopoietic cells via cytokine receptors, as well as B and T cell receptors, leads to the tyrosine phosphorylation of Shc and its association with both Grb2-Sos1 complexes and with a 145 kDa protein referred to as the SH2 containing inositol 5-phosphatase (SHIP1). In a search of putative 5-phosphatase isoenzymes, we have isolated a second SH2 domain containing inositol 5-phosphatase, referred to as (SHIP2). Both SHIP1 and SHIP2 are coexpressed in human T lymphocytes. This was shown at the protein level by Western blot analysis in transformed T cell lines and in peripheral blood T lymphocytes either unstimulated or after in vitro activation through TCR-CD3 complex. SHIP1 protein level was not modulated after activation of T lymphocytes, in contrast to SHIP2, which was increased after longterm stimulation. SHIP1 was tyrosine phosphorylated in resting naive T cells. This was not observed in the transformed T cell lines. T lymphocyte is therefore a model of coexpression of the two SH2-containing inositol 5-phosphatases SHIP1 and SHIP2.

Interaction of the catalytic domain of inositol 1,4,5-trisphosphate 3-kinase A with inositol phosphate analogues.

The levels of inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] in the cytoplasm are tightly regulated by two enzymes, Ins(1,4,5)P3 3‐kinase and type I Ins(1,4,5)P3 5‐phosphatase. The catalytic domain of Ins(1,4,5)P3 3‐kinase (isoenzymes A, B and C) is restricted to approximately 275 amino acids at the C‐terminal end. We were interested in understanding the catalytic mechanism of this key family of enzymes in order to exploit this in inhibitor design. We expressed the catalytic domain of rat Ins(1,4,5)P3 3‐kinase A in Escherichia coli as a His‐ and S‐tagged fusion protein. The purified enzyme was used in an Ins(1,4,5)P3 kinase assay to phosphorylate a series of inositol phosphate analogues with three or four phosphate groups. A synthetic route to D‐2‐deoxy‐Ins(1,4,5)P3 was devised. D‐2‐Deoxy‐Ins(1,4,5)P3 and D‐3‐deoxy‐Ins(1,4,6)P3 were potent inhibitors of the enzyme, with IC50 values in the micromolar range. Amongst all analogues tested, only D‐2‐deoxy‐Ins(1,4,5)P3 appears to be a good substrate of the Ins(1,4,5)P3 3‐kinase. Therefore, the axial 2‐hydroxy group of Ins(1,4,5)P3 is not involved in recognition of the substrate nor does it participate in the phosphorylation mechanism of Ins(1,4,5)P3. In contrast, the equatorial 3‐hydroxy function must be present in that configuration for phosphorylation to occur. Our data indicate the importance of the 3‐hydroxy function in the mechanism of inositol trisphosphate phosphorylation rather than in substrate binding.

Desensitization of carbamylcholine-mediated cyclic GMP accumulation in dog thyroid slices

Previous studies have shown that carbamylcholine through a muscarinic receptor increases Ca2+ influx and consequently cGMP accumulation in dog thyroid slices. A first exposure to carbamylcholine induced desensitization of the cGMP response to a further exposure to this agent. The aim of this work was to investigate the role of carbamylcholine, extracellular calcium or intracellular cGMP in this desensitization. The effect of various combinations of these factors has been studied. Carbamylcholine in the absence of calcium, with and without cGMP accumulation, did not desensitize. On the other hand, an increase in intracellular calcium and its consequent cGMP accumulation did not desensitize carbamylcholine-induced cGMP accumulation either. Previous exposure of dog thyroid cells to carbamylcholine in the presence of calcium slightly decreased ionophore A23187-induced cGMP accumulation. Carbamylcholine desensitization is thus mainly homologous and bears on a step prior to cGMP synthesis. These data suggest that both carbamylcholine interaction with its receptor and extracellular calcium are necessary for desensitization. cGMP is not sufficient for this effect but its possible role is not excluded.

Soluble and particulate inositol 1,4,5-trisphosphate 5-phosphatases show common antigenic determinants.

Inositol 1,4,5-trisphosphate 5-phosphatase catalyses the dephosphorylation of the phosphate in the 5-position from inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate. One particulate and two soluble enzymes were previously described in bovine brain. In this study, we have obtained a precipitating antiserum against soluble type I inositol 1,4,5-trisphosphate 5-phosphatase. The particulate, but not the soluble type II enzyme, was immunoprecipitated by the serum. Inositol 1,4,5-trisphosphate 5-phosphatase activity from crude extracts of rat brain, human platelets and rat liver were immunoprecipitated by the same antibodies, suggesting the existence of common antigenic determinant among inositol 1,4,5-trisphosphate 5-phosphatases of diverse sources.

Phosphorylated SHIP2 on Y1135 localizes at focal adhesions and at the mitotic spindle in cancer cell lines

The SH2 containing inositol 5-phosphatase SHIP2 is a member of the mammalian phosphoinositide polyphosphate 5-phosphatase family. It is a multi-domain protein comprising a central catalytic domain, an SH2 domain at its N-terminus, proline rich sequences and SAM domain at its C-terminus. It can dephosphorylate both phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and can participate in multiple signaling events in response to growth factors such as EGF, FGF or PDGF. Human SHIP2 can be phosphorylated at two major tyrosine residues Tyr986 and Tyr1135. Here, we report two intracellular localizations of pSHIP2(Y1135): pSHIP2(Y1135)-ir localizes at focal adhesions in EGF-stimulated HeLa cells and at the mitotic spindle in HeLa, in GIST882 cells, a human model of gastrointestinal stromal tumors derived cells, and in human astrocytoma 1321N1 cells. pSHIP2(Y1135)-ir is maximal at metaphase. In N1 cells, evidence is provided that the SHIP2 pathway impacts the distribution of mitotic centrosomes, particularly ү-tubulin. Our data reinforce the concept that SHIP2 localization in intact cells is dependent on phosphorylation mechanisms on both Ser/Thr and Tyr residues, i.e. Y1135, in three cancer cell lines.

Regeneration of enzymatic activity after sodium dodecyl sulfate/polyacrylamide gel electrophoresis and zinc acetate staining: The example of inositol 1,4,5-trisphosphate 5-phosphatase

Regeneration of enzyme activity after sodium dodecyl sulfate-gel electrophoresis was investigated with a purified inositol 1,4,5-trisphosphate 5-phosphatase. In order to avoid silver or Coomassie blue staining, we have used zinc acetate. This staining procedure was sensitive, rapid, and reversible provided that zinc cations are chelated and activity is extracted after diffusion out of the gel. The method allows some gel lane staining and identification of the enzyme based on catalytic activity.