Christophe Erneux

The lipid phosphatase SHIP2 controls insulin sensitivity.

Insulin is the primary hormone involved in glucose homeostasis, and impairment of insulin action and/or secretion has a critical role in the pathogenesis of diabetes mellitus. Type-II SH2-domain-containing inositol 5-phosphatase, or ‘SHIP2’, is a member of the inositol polyphosphate 5-phosphatase family. In vitro studies have shown that SHIP2, in response to stimulation by numerous growth factors and insulin, is closely linked to signalling events mediated by both phosphoinositide-3-OH kinase and Ras/mitogen-activated protein kinase. Here we report the generation of mice lacking the SHIP2 gene. Loss of SHIP2 leads to increased sensitivity to insulin, which is characterized by severe neonatal hypoglycaemia, deregulated expression of the genes involved in gluconeogenesis, and perinatal death. Adult mice that are heterozygous for the SHIP2 mutation have increased glucose tolerance and insulin sensitivity associated with an increased recruitment of the GLUT4 glucose transporter and increased glycogen synthesis in skeletal muscles. Our results show that SHIP2 is a potent negative regulator of insulin signalling and insulin sensitivity in vivo.

THE DIVERSITY AND POSSIBLE FUNCTIONS OF THE INOSITOL POLYPHOSPHATE 5-PHOSPHATASES

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, i.e., inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), inositol 1,3,4, 5-tetrakisphosphate (Ins(1,3,4,5)P4), phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P2) or phosphatidylinositol 3,4, 5-trisphosphate (PtdIns(3,4,5)P3). In mammalian cells, this family contains a series of distinct genes and splice variants. All inositol polyphosphate 5-phosphatases share a 5-phosphatase domain and various protein modules probably responsible for specific cell localisation or recruitment (SH2 domain, proline-rich sequences, prenylation sites, etc.). Type I Ins(1,4,5)P3 5-phosphatase also uses Ins(1,3,4,5)P4 but not the phosphoinositides as substrates. This enzyme is targeted to specific membranes by means of a prenylation site. Type II 5-phosphatases can use both PtdIns(4,5)P2 and PtdIns(3,4,5)P3 as substrates. Five mammalian enzymes and multiple splice variants are known: INPP5P or inositol polyphosphate 5-phosphatase II, OCRL (a Golgi protein implicated in the Lowe oculocerebrorenal syndrome), synaptojanin (a protein involved in the recycling of synaptic vesicles), SHIP 1 and SHIP 2 (or SH2-containing inositol 5-phosphatases). As discussed in this review, the substrate specificity, regulatory mechanisms, subcellular localisation and tissue specificity indicate that the different 5-phosphatase isoforms may play specific roles. As known in the dephosphorylation of tyrosine containing substrates by the tyrosine protein phosphatases or in the metabolism of cyclic nucleotides by the cyclic nucleotide phosphodiesterases, inositol polyphosphate 5-phosphatases directly participate in the control of second messengers in response to both activation or inhibitory cell signalling.

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.

Tyrosine Phosphorylation and Relocation of SHIP Are Integrin-mediated in Thrombin-stimulated Human Blood Platelets

The SH2 domain-containing inositol 5-phosphatase, SHIP, known to dephosphorylate inositol 1,3,4,5-tetrakisphosphate and phosphatidylinositol 3,4,5-trisphosphate has recently been shown to be expressed in a variety of hemopoietic cells. This 145-kDa protein is induced to associate with Shc by multiple cytokines and may play an important role in the negative regulation of immunocompetent cells mediated by FcγRIIB receptor. We report here that SHIP is present in human blood platelets and may be involved in platelet activation evoked by thrombin. Platelet SHIP was identified by Western blotting as a single 145-kDa protein. Both phosphatidylinositol 3,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate 5-phosphatase activities could be demonstrated in anti-SHIP immunoprecipitates of platelet lysate. Thrombin stimulation induced a tyrosine phosphorylation of SHIP, this effect being prevented if platelets were not shaken or if RGD-containing peptides were present, indicating an aggregation-dependent, integrin-mediated event. Moreover, although the intrinsic phosphatase activity of SHIP did not appear to be significantly increased, tyrosine-phosphorylated SHIP was relocated to the actin cytoskeleton upon activation in an aggregation- and integrin engagement-dependent manner. Finally, the striking correlation observed between phosphatidylinositol 3,4-bisphosphate production and the tyrosine phosphorylation of SHIP, as well as its relocation to the cytoskeleton upon thrombin stimulation, suggest a role for SHIP in the aggregation-dependent and GpIIb-IIIa-mediated accumulation of this important phosphoinositide.

Inositol 1,3,4,5-tetrakisphosphate is essential for T lymphocyte development

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) is phosphorylated by Ins(1,4,5)P3 3-kinase, generating inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). The physiological function of Ins(1,3,4,5)P4 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)P3 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)P3 amounts or calcium mobilization in Itpkb−/− thymocytes. These data indicate that Itpkb and Ins(1,3,4,5)P4 define an essential signaling pathway for T cell precursor responsiveness and development.

Simulations of the effects of inositol 1,4,5-trisphosphate 3-kinase and 5-phosphatase activities on Ca2+ oscillations

Inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) is responsible for Ca2+ mobilization in response to external stimulation in many cell types. The latter phenomenon often occurs as repetitive Ca2+ spikes. In this study, the effect of the two Ins-1,4,5-P3 metabolizing enzymes (Ins-1,4,5-P3 3-kinase and 5-phosphatase) on the temporal pattern of Ca2+ oscillations has been investigated. On the basis of the well-documented Ins-1,4,5-P3 3-kinase stimulation by the Ca2+/calmodulin complex and of the experimentally-determined kinetic characteristics of these enzymes, we predict that 5-phosphatase primarily controls the levels of Ins-1,4,5-P3 and, thereby, the occurrence and frequency of Ca2+ oscillations. Consequently, the model reproduces the experimental observation performed in Chinese hamster ovary cells that 5-phosphatase overexpression has a much more pronounced effect on the pattern of Ca2+ oscillations than 3-kinase overexpression. We also investigated, in more detail, under which conditions a similar effect could be observed in other cell types expressing various Ins-1,4,5-P3 3-kinase activities.

Phosphoinositide phosphatases in a network of signalling reactions

Phosphoinositide phosphatases dephosphorylate the three positions (D-3, 4 and 5) of the inositol ring of the poly-phosphoinositides. They belong to different families of enzymes. The PtdIns(3,4)P2 4-phosphatase family, the tumour suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN), SAC1 domain phosphatases and myotubularins belong to the tyrosine protein phosphatases superfamily. They share the presence of a conserved cysteine residue in the consensus CX5RT/S. Another family consists of the inositol polyphosphate 5-phosphatase isoenzymes. The importance of these phosphoinositide phosphatases in cell regulation is illustrated by multiple examples of their implications in human diseases such as Lowe syndrome, X-linked myotubular myopathy, cancer, diabetes or bacterial infection.

Cloning and expression of human brain type I inositol 1,4,5-trisphosphate 5-phosphatase High levels of mRNA in cerebellar Purkinje cells

In brain and many other tissues, Type I inositol 1,4,5‐trisphosphate (InsP3) 5‐phosphatase is the major isozyme hydrolysing the calcium‐mobilizing second messenger InsP3. We recently reported the cloning and expression of dog thyroid InsP3 5‐phosphatase. During the course of this cloning, screening of a human brain cDNA library allowed us to isolate a cDNA clone D1 with 91% sequence identity with the thyroid sequence. When clone D1 was expressed in Escherichia coli, the fusion protein had InsP3 5‐phosphatase activity. M r estimates of the recombinant enzyme made by unmunodetection, activity assay after SDS/PAGE or silver staining were consistent with the calculated molecular mass. In situ hybridization on human cerebellum sections localised the mRNA for this enzyme to the Purkinje cells.

Isoprenylated Human Brain Type I Inositol 1,4,5-Trisphosphate 5-Phosphatase Controls Ca2+ Oscillations Induced by ATP in Chinese Hamster Ovary Cells

d-myo-Inositol 1,4,5-trisphosphate (InsP3) 5-phosphatase and 3-kinase are thought to be critical regulatory enzymes in the control of InsP3 and Ca2+ signaling. In brain and many other cells, type I InsP3 5-phosphatase is the major phosphatase that dephosphorylates InsP3 andd-myo-inositol 1,3,4,5-tetrakisphosphate. The type I 5-phosphatase appears to be associated with the particulate fraction of cell homogenates. Molecular cloning of the human brain enzyme identifies a C-terminal farnesylation site CVVQ. Post-translational modification of this enzyme promotes membrane interactions and changes in specific activity. We have now compared the cytosolic Ca2+ ([Ca2+] i ) responses induced by ATP, thapsigargin, and ionomycin in Chinese hamster ovary (CHO-K1) cells transfected with the intact InsP35-phosphatase and with a mutant in which the C-terminal cysteine cannot be farnesylated. [Ca2+] i was also measured in cells transfected with an InsP3 3-kinase construct encoding the A isoform. The Ca2+ oscillations detected in the presence of 1 μm ATP in control cells were totally lost in 87.5% of intact (farnesylated) InsP35-phosphatase-transfected cells, while such a loss occurred in only 1.1% of the mutant InsP3 5-phosphatase-transfected cells. All cells overexpressing the InsP3 3-kinase also responded with an oscillatory pattern. However, in contrast to control cells, the [Ca2+] i returned to base-line levels in between a couple of oscillations. The [Ca2+] i responses to thapsigargin and ionomycin were identical for all cells. The four cell clones compared in this study also behaved similarly with respect to capacitative Ca2+ entry. In permeabilized cells, no differences in extent of InsP3-induced Ca2+release nor in the threshold for InsP3 action were observed among the four clones and no differences in the expression levels of the various InsP3 receptor isoforms could be shown between the clones. Our data support the contention that the ATP-induced increase in InsP3 concentration in transfected CHO-K1 cells is essentially restricted to the site of its production near the plasma membrane, where it can be metabolized by the type I InsP35-phosphatase. This enzyme directly controls the [Ca2+] i response and the Ca2+oscillations in intact cells.

The role of calmodulin for inositol 1,4,5-trisphosphate receptor function

Intracellular calcium release is a fundamental signaling mechanism in all eukaryotic cells. The ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate receptor (IP(3)R) are intracellular calcium release channels. Both channels can be regulated by calcium and calmodulin (CaM). In this review we will first discuss the role of calcium as an activator and inactivator of the IP(3)R, concluding that calcium is the most important regulator of the IP(3)R. In the second part we will further focus on the role of CaM as modulator of the IP(3)R, using results of the voltage-dependent Ca(2+) channels and the RyR as reference material. Here we conclude that despite the fact that different CaM-binding sites have been characterized, their function for the IP(3)R remains elusive. In the third part we will discuss the possible functional role of CaM in IP(3)-induced Ca(2+) release (IICR) by direct and indirect mechanisms. Special attention will be given to the Ca(2+)-binding proteins (CaBPs) that were shown to activate the IP(3)R in the absence of IP(3).