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ITPKB phosphorylates inositol 1,4,5-trisphosphate into inositol 1,3,4,5-tetrakisphosphate and controls signal transduction in various hematopoietic cells. Surprisingly, it has been reported that the ITPKB messenger RNA level is significantly increased in the cerebral cortex of patients with Alzheimer's disease, compared with control subjects. As extracellular signal-regulated kinases 1/2 activation is increased in the Alzheimer brain and as ITPKB is a regulator of extracellular signal-regulated kinases 1/2 activation in some hematopoietic cells, we tested whether this increased activation in Alzheimer's disease might be related to an increased activity of ITPKB. We show here that ITPKB protein level was increased 3-fold in the cerebral cortex of most patients with Alzheimer's disease compared with control subjects, and accumulated in dystrophic neurites associated to amyloid plaques. In mouse Neuro-2a neuroblastoma cells, Itpkb overexpression was associated with increased cell apoptosis and increased β-secretase 1 activity leading to overproduction of amyloid-β peptides. In this cellular model, an inhibitor of mitogen-activated kinase kinases 1/2 completely prevented overproduction of amyloid-β peptides. Transgenic overexpression of ITPKB in mouse forebrain neurons was not sufficient to induce amyloid plaque formation or tau hyperphosphorylation. However, in the 5X familial Alzheimer's disease mouse model, neuronal ITPKB overexpression significantly increased extracellular signal-regulated kinases 1/2 activation and β-secretase 1 activity, resulting in exacerbated Alzheimer's disease pathology as shown by increased astrogliosis, amyloid-β40 peptide production and tau hyperphosphorylation. No impact on pathology was observed in the 5X familial Alzheimer's disease mouse model when a catalytically inactive ITPKB protein was overexpressed. Together, our results point to the ITPKB/inositol 1,3,4,5-tetrakisphosphate/extracellular signal-regulated kinases 1/2 signalling pathway as an important regulator of neuronal cell apoptosis, APP processing and tau phosphorylation in Alzheimer's disease, and suggest that ITPKB could represent a new target for reducing pathology in human patients with Alzheimer's disease with ITPKB expression.

Mice genetically-deficient for the B isoform of the inositol 1,4,5-trisphosphate 3-kinase (or Itpkb) have a severe defect in thymocytes differentiation and thus lack peripheral T cells. In order to study the functional role of Itpkb in peripheral T cells, we constructed a new mouse where a transgene encoding mouse Itpkb is specifically and transiently expressed in thymocytes of Itpkb(-)(/)(-) mice. This allows a partial rescue of mature thymocyte/T cell differentiation and thus the functional characterization of peripheral T cells lacking Itpkb. We show here that Itpkb(-)(/)(-) CD4(+) and CD8(+) peripheral T cells present important functional alterations. Indeed, an increased activated/memory phenotype as well as a decreased proliferative capacity and survival were detected in these T cells. These Itpkb-deficient peripheral T cells have also an increased capacity to secrete cytokines upon stimulation. Together, our present results define the important role of Itpkb in peripheral mature T cell fate and function in mouse, suggesting a potential role for Itpkb in autoimmunity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Inositol Inpp5k (or Pps, SKIP) is a member of the inositol polyphosphate 5-phosphatases family with a poorly characterized function in vivo. In this study, we explored the function of this inositol 5-phosphatase in mice and cells overexpressing the 42-kDa mouse Inpp5k protein. Inpp5k transgenic mice present defects in water metabolism characterized by a reduced plasma osmolality at baseline, a delayed urinary water excretion following a water load, and an increased acute response to vasopressin. These defects are associated with the expression of the Inpp5k transgene in renal collecting ducts and with alterations in the arginine vasopressin/aquaporin-2 signalling pathway in this tubular segment. Analysis in a mouse collecting duct mCCD cell line revealed that Inpp5k overexpression leads to increased expression of the arginine vasopressin receptor type 2 and increased cAMP response to arginine vasopressin, providing a basis for increased aquaporin-2 expression and plasma membrane localization with increased osmotically induced water transport. Altogether, our results indicate that Inpp5k 5-phosphatase is important for the control of the arginine vasopressin/aquaporin-2 signalling pathway and water transport in kidney collecting ducts.

Inositol 1,4,5-trisphosphate 3-kinase B (or Itpkb) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), its reaction product, play an important role in the control of B lymphocyte fate and function in vivo. In order to investigate the fine mechanisms of Itpkb and Ins(1,3,4,5)P4 action in B cells, we crossed Itpkb(-/-) mice with transgenic mice expressing a 3-83μδ B cell receptor (BCR) specific for membrane-bound MHC-I H2-K(b) and H2-K(k) molecules. On a non-deleting H2-K(d) genetic background, we show that Itpkb is important for the control of Bim protein expression and B cell survival rather than for the control of B cell development from one stage to another. Analyses of cell surface markers expression, proapoptotic Bim protein expression, in vitro survival and in vivo turnover demonstrated that BCR transgenic Itpkb(-/-) B cells exhibit an anergic phenotype with the notable exception of their enhanced antigen-induced calcium signalling. On a deleting H2-K(b) genetic background, we show that Itpkb is not essential for BCR editing or negative selection. These data establish Itpkb as an important regulator of B cell survival and anergy in vivo., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

In mammals, Ins(1,4,5)P3, the well known calcium mobilization messenger, is phosphorylated in the cytosol at the 3-position of the inositol ring to yield Ins(1,3,4,5)P4 by Ins(1,4,5)P3 3-kinases A, B and C isoforms as well as by inositol polyphosphate multikinase (Ipmk). Studies in gene-deficient mice have revealed that these enzymes and Ins(1,3,4,5)P4, their reaction product, play essential role in multiple physiological processes, ranging from synaptic plasticity, hematopoietic cell survival, development and function, to mRNA export, transcriptional regulation and chromatin remodelling. Rather than to provide an unique and “universal” mechanism of Ins(1,3,4,5)P4 action, these studies in genetically-modified mice point for a role of this inositide in the control of calcium mobilization, of the subcellular localisation of PH domain-containing target proteins, and of higher inositol phosphate production. Mice deficient for the B isoform of inositol 1,4,5-trisphosphate 3-kinase (Itpkb) develop profound alterations in T and B cells as well as in neutrophils and mast cells. Our recent studies indicate that the 3-kinase Itpkb and Ins(1,3,4,5)P4 are important for the survival of naïve mature B cells and the control of proapoptotic Bim protein expression, rather than for the control of B cell transition from one developmental stage to another. They also suggest that Itpkb is an important component in the control of B cell anergy.

Many neutrophil functions are regulated by phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) that mediates protein membrane translocation via binding to pleckstrin homolog (PH) domains within target proteins. Here we show that inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), a cytosolic small molecule, bound the same PH domain of target proteins and competed for binding to PtdIns(3,4,5)P3. In neutrophils, chemoattractant stimulation triggered rapid elevation in Ins(1,3,4,5)P4 concentration. Depletion of Ins(1,3,4,5)P4 by deleting the gene encoding InsP3KB, which converts Ins(1,4,5)P3 to Ins(1,3,4,5)P4, enhanced membrane translocation of the PtdIns(3,4,5)P3-specific PH domain. This led to enhanced sensitivity to chemoattractant stimulation, elevated superoxide production, and enhanced neutrophil recruitment to inflamed peritoneal cavity. On the contrary, augmentation of intracellular Ins(1,3,4,5)P4 concentration blocked PH domain-mediated membrane translocation of target proteins and dramatically decreased the sensitivity of neutrophils to chemoattractant stimulation. These findings establish a role for Ins(1,3,4,5)P4 in cellular signal transduction pathways and provide another mechanism for modulating PtdIns(3,4,5)P3 signaling in neutrophils.

The contribution of the B isoform of inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] 3-kinase (or Itpkb) and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P(4)], its reaction product, to B cell function and development remains unknown. Here, we show that mice deficient in Itpkb have defects in B cell survival leading to specific and intrinsic developmental alterations in the B cell lineage and antigen unresponsiveness in vivo. The decreased B cell survival is associated with a decreased phosphorylation of Erk1/2 and increased Bim gene expression. B cell survival, development, and antigen responsiveness are normalized in parallel to reduced expression of Bim in Itpkb(-/-) Bim(+/-) mice. Analysis of the signaling pathway downstream of Itpkb revealed that Ins(1,3,4,5)P(4) regulates subcellular distribution of Rasa3, a Ras GTPase-activating protein acting as an Ins(1,3,4,5)P(4) receptor. Together, our results indicate that Itpkb and Ins(1,3,4,5)P(4) mediate a survival signal in B cells via a Rasa3-Erk signaling pathway controlling proapoptotic Bim gene expression.

The activation of phospholipase C leads to the formation of both I(1,4,5)P(3) and diacylglycerol (DAG). I(1,4,5)P(3) can be metabolized by dephosphorylation catalyzed by Type I I(1,4,5)P(3) 5-phosphatase and by enzymatic phosphorylation to various inositol phosphates. This last step is catalyzed by three mammalian isoenzymes that specifically phosphorylate the 3-phosphate position of the inositol ring Itpka, Itpkb and Itpkc and a less specific enzyme Ipmk (or inositol multikinase) that phosphorylates I(1,4,5)P(3) at the D-3 and D-6 positions. This study was performed in mice cells in order to understand the synthetic pathway of IP5 and IP6 following PLC stimulation and possible link with Itpk activity. Mouse embryonic fibroblasts (MEF) were prepared from Itpkb(-/-) Itpkc(-/-) mice. Western blot and RT-PCR analysis show that the cells do not express Itpka. In contrast, they do express Ipmk. The cells still produce IP5 and IP6. Our data show that the absence of expression of the three isoenzymes of Itpk does not prevent the formation of IP5 and IP6, at least in mouse embryonic fibroblasts. The nuclear Ipmk plays therefore a critical role in the metabolism of I(1,4,5)P(3) and production of highly phosphorylated IP5 and IP6.

Inositol 1,4,5-trisphosphate 3-kinase (IP(3)-3K) catalyses the phosphorylation of inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate. cDNAs encoding three mammalian isoforms have been reported and referred to as IP(3)-3KA, IP(3)-3KB, and IP(3)-3KC. IP(3)-3KB is particularly sensitive to proteolysis at the N-terminus, a mechanism known to generate active fragments of lower molecular mass. Endogenous IP(3)-3KB has therefore not been formally identified in tissues. We have probed a series of murine tissues with an antibody directed against the C-terminus of IP(3)-3KB and used IP(3)-3KB deficient mouse tissues as negative controls. IP(3)-3KB was shown to be particularly well expressed in brain, lung, and thymus with molecular masses of 110-120kDa. The identification of the native IP(3)-3KB by Western blotting for the first time will facilitate further studies of regulation of its activity by specific proteases and/or phosphorylation.

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) is phosphorylated by Ins(1,4,5)P(3) 3-kinase, generating inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)). The physiological function of Ins(1,3,4,5)P(4) is still unclear, but it has been reported to be a potential modulator of calcium mobilization. Disruption of the gene encoding the ubiquitously expressed Ins(1,4,5)P(3) 3-kinase isoform B (Itpkb) in mice caused a severe T cell deficiency due to major alterations in thymocyte responsiveness and selection. However, we were unable to detect substantial defects in Ins(1,4,5)P(3) amounts or calcium mobilization in Itpkb(-/-) thymocytes. These data indicate that Itpkb and Ins(1,3,4,5)P(4) define an essential signaling pathway for T cell precursor responsiveness and development.

Genetic susceptibility to type 2 diabetes involves many genes, most of which are still unknown. The lipid phosphatase SHIP2 is a potent negative regulator of insulin signaling and sensitivity in vivo and is thus a good candidate gene. Here we report the presence of SHIP2 gene mutations associated with type 2 diabetes in rats and humans. The R1142C mutation specifically identified in Goto-Kakizaki (GK) and spontaneously hypertensive rat strains disrupts a potential class II ligand for Src homology (SH)-3 domain and slightly impairs insulin signaling in cell culture. In humans, a deletion identified in the SHIP2 3' untranslated region (UTR) of type 2 diabetic subjects includes a motif implicated in the control of protein synthesis. In cell culture, the deletion results in reporter messenger RNA and protein overexpression. Finally, genotyping of a cohort of type 2 diabetic and control subjects showed a significant association between the deletion and type 2 diabetes. Altogether, our results show that mutations in the SHIP2 gene contribute to the genetic susceptibility to type 2 diabetes in rats and humans.

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] 3-kinase catalyses the phosphorylation of Ins(1,4,5)P(3) to Ins(1,3,4,5)P(4). cDNAs encoding two isoenzymes of Ins(1,4,5)P(3) 3-kinase (3-kinases A and B) have been described previously. In the present study, we report the cloning of a full-length 2052 bp cDNA encoding a third human isoenzyme of the Ins(1,4,5)P(3) 3-kinase family, referred to as isoform C. This novel enzyme has a calculated molecular mass of 75. 207 kDa and a K(m) for Ins(1,4,5)P(3) of 6 microM. Northern-blot analysis showed the presence of a transcript of approx. 3.9 kb in various human tissues. Inositol trisphosphate 3-kinase C demonstrates enzymic activity when expressed in DH5alphaF' bacteria or COS-7 cells. Calcium alone decreases the Ins(1,4,5)P(3) 3-kinase activity of the 3-kinase C isoenzyme in transfected COS-7 cells. This inhibitory effect is reversed in the presence of calmodulin. The recombinant bacterial 3-kinase C can be adsorbed on calmodulin-Sepharose in the presence of calcium. The present data show that Ins(1,4,5)P(3) 3-kinase C: (i) shares a conserved catalytic domain of about 275 amino acids with the two other mammalian isoforms, (ii) could be purified on a calmodulin-Sepharose column and (iii) could be distinguished from the A and B isoenzymes by the effects of calcium and of calmodulin.

We describe here a selection strategy allowing the cloning of sequences that contain a functional nuclear targeting signal. Our method relies on the use of green fluorescent protein fusion proteins to identify nuclear targeting sequences. Transfected cells expressing nuclear protein fusions were isolated on the basis of their nuclear fluorescence using flow cytometry and the transfected DNAs were recovered after bacterial transformation with total DNA from pools of sorted cells. Starting from a cDNA expression library, in which only 1% of the expressed proteins were nuclear, we obtained a 70-fold enrichment in nuclear protein-encoding clones after a single round of selection. Among the 63 clones that have been partially sequenced to date, 25 (40%) corresponded to known nuclear proteins and 13 (20%) to previously uncharacterized sequences. Despite their ability to target the green fluorescent protein marker to the cell nucleus, about half of the cloned sequences did not encode canonical basic or bipartite nuclear localization signals. The method can thus be applied to the large-scale cloning of functional nuclear targeting sequences, which opens the way to a wide investigation of nuclear import mechanisms and to the identification of previously unknown nuclear proteins., (Copyright 2000 Academic Press.)

SHIP2 is a new member of the inositol polyphosphate 5-phosphatase family showing homology to SHIP1. The structure of both enzymes is characterized by the presence of a 5' SH2 domain, a central catalytic domain, and a 3' proline-rich region. Recent results suggest that SHIP2 and SHIP1 act downstream of various receptors by removing a phosphate from the 5' position of the phosphatidylinositol 3'-kinase phosphatidylinositol 3,4, 5-triphosphate product and of inositol 1,3,4,5-tetrakisphosphate. Human SHIP2 is highly expressed in adult heart, skeletal muscle, and placenta, whereas SHIP1 expression is limited to the hematopoietic system. We report here the molecular analysis of the mouse SHIP2 cDNA and the corresponding protein, the structure of the gene, and the identification of its promoter. SHIP2 mRNA expression was analyzed in embryonic and adult mouse tissues by reverse transcription-polymerase chain reaction and in situ hybridization. In embryonic day 15.5 mice, SHIP2 was strongly expressed in the liver, specific regions of the central nervous system, the thymus, the lung, and the cartilage perichondrium. In adult mice, SHIP2 mRNA was markedly present in the brain and the thymus and at different stages of spermatozoa maturation in the seminiferous tubules. The subtle differences in the protein structure of SHIP2 and SHIP1 as well as their different patterns of expression are discussed., (Copyright 1999 Academic Press.)

TTF-2 is a thyroid-specific winged-helix transcription factor which has been proposed to play a key role in the hormonal control of thyroglobulin and thyroperoxidase genes transcription in FRTL-5 cells. We have analyzed TTF-2 DNA-binding activity in primary cultures of dog thyrocytes maintained in control condition or in the presence of the cAMP agonist forskolin. Binding of 35S-labelled nuclear proteins to the TTF-2 recognition sequence identified the presence of two molecular species of 41.5 and 42.5 kDa. TTF-2 DNA-binding activity was clearly detectable in nuclear extracts from unstimulated cells and appeared increased in forskolin-treated cells. Thus, the presence of TTF-2 DNA-binding activity does not correlate with the cAMP-dependent activity of thyroglobulin and thyroperoxidase genes in this cell system. In addition, the mutation of the TTF-2 binding site in the thyroglobulin promoter resulted in a very reduced but still clearly cAMP-dependent promoter activity when assayed by transient expression in the same cells. These results do not support a dominant role for TTF-2 in the cAMP-dependent control of thyroglobulin gene transcription in primary cultured thyrocytes.