Etude du rôle de la phosphatase SHIP2 dans un modèle d'astrocytomes humains

Le metabolisme des phosphoinositides est constitue dfun reseau complexe dfenzymes et de seconds messagers participant a la regulation de nombreux processus cellulaires :la proliferation, la croissance, la survie et la migration. Le phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), est un second messager intracellulaire tres important dont le taux est controle a la fois au niveau de sa synthese par des kinases et au niveau de sa degradation par des phosphatases. La phosphatase PTEN (phosphatase and tensin homolog deleted on chromosomes 10), gene suppresseur de tumeurs frequemment mutee dans des cancers (comme dans des glioblastomes), le dephosphoryle en position 3. Il existe egalement une activite á inositol 5-phosphatase â pour de nombreux derives du myo-inositol. Cfest la proteine SHIP2 (SH2-containing Inositol 5-phosphatase 2), une phosphatase membre de la famille des phosphatidylinositol polyphosphate 5-phosphatases qui est, entre autre, responsable de cette activite.Le but principal de ce travail de these a ete de mettre en evidence le role pro ou anti-oncogenique de SHIP2 dans un modele cellulaire humain de glioblastomes :les cellules 1321 N1. Ce modele cellulaire a comme particularite lfabsence dfexpression de PTEN au niveau proteique.La caracterisation des clones cellulaires deficients pour SHIP2 nous a permis de mettre en evidence une augmentation du taux du PtdIns(3,4,5)P3 par rapport a des cellules qui expriment un taux normal de SHIP2. Par un simple comptage, nous avons observe une augmentation du nombre de cellules en culture par rapport aux cellules controles. Lfimmunomarquage de la F-actine nous a permis de montrer une difference de morphologie entre les cellules N1 shSHIP2 et les cellules controles. Au travers de Western blots, nous avons observe que la phosphorylation de la PKB ainsi que celle de Erk1/2 etait augmentee dans les cellules deficientes pour SHIP2 en comparaison aux cellules qui expriment SHIP2.Dans les cellules COS-7 transfectees par SHIP2 sauvage, nous avons identifie par spectrometrie de masse 8 sites de phosphorylation sur Tyr, Ser et Thr. En systeme endogene dans les cellules N1, notre etude a demontre la presence de 3 sites de phosphorylations sur Ser (position 132 et 1258) et Thr (position 1254). Nous avons genere deux anticorps specifiques contre la proteine SHIP2 phosphorylee sur la Ser132 et Thr1254. Lfanticorps contre la Ser 132 nous a permis de mettre en evidence une colocalisation entre pSHIP2 Ser132 et le facteur dfepissage SC35 present dans les speckles nucleaires. Cette donnee nous suggere un role nucleaire nouveau pour SHIP2.De maniere originale nous avons mis en evidence dans les cellules dfastrocytomes N1 trois localisations subcellulaires de SHIP2 :i) a la membrane plasmique au niveau des adherences focales ou SHIP2 serait impliquee dans le controle du taux de PtdIns(3,4,5)P3/ PtdIns(4,3)P2, et de la migration ii) au niveau perinucleaire ou SHIP2 ainsi que la forme phosphorylee sur Ser132 seraient impliquees dans des interactions proteiques via ses proprietes de á docking protein â et enfin iii) dans le noyau specifiquement dans les speckles ou la forme phosphorylee de SHIP2 sur Ser132 serait impliquee dans le controle du taux de PtdIns(4,5)P2, mais egalement dans la signalisation nucleaire impliquant les phosphoinositides.La presence de SHIP2 dans les adherences focales nous a amene a rechercher par quel mecanisme SHIP2 pouvait se retrouver a cette localisation. Nous avons par immunoprecipitation suivie dfune analyse par spectrometrie de masse identifie la myosine 1c (Myo1c) comme nouveau partenaire de SHIP2. Les cellules deficientes pour la Myo1c presentent une modification de la morphologie cellulaire, ainsi qufune modification de la localisation intracellulaire de SHIP2. Les cellules N1 deficientes pour la Myo1c montrent aussi une diminution de lfexpression de la sous unite regulatrice de la PI 3-kinase (p85ƒ¿) ainsi qufune absence de la phosphorylation de la PKB sur la Ser473 et la Thr308. En outre, ces cellules N1 shMyo1c presentent un taux eleve dfapoptose compare aux cellules controles.En conclusion, ces travaux ont permis de montrer que dans un modele humain de glioblastomes N1, SHIP2 etait phosphorylee sur la Ser 132 et que cette phosphorylation semblait etre requise pour son activite phosphatase. SHIP2 possedait trois localisations subcellulaires probablement associees a des fonctions differentes, qufelle interagissait au minimum avec les proteines partenaires nouvelles lamin A/C et Myo1c. Enfin SHIP2 exercait un controle negatif sur la proliferation et la migration de ces cellules. Ces resultats mettent en evidence plusieurs fonctions originales de SHIP2 comme son role dans la migration et la proliferation qui ouvrent des perspectives nouvelles dans lfetude de cette phosphatase dans le contexte general et surtout physiopathogique des fonctions attribuees aux phosphoinositides.Doctorat en Sciences biomédicales et pharmaceutiquesinfo:eu-repo/semantics/nonPublishe

SHIP2 signalling at the plasma membrane, in the nucleus and at focal contacts.

Phosphoinositide 5-phosphatases are critical enzymes in modulating the concentrations of PI(3,4,5)P(3), PI(4,5)P(2) and PI(3,5)P(2). The SH2 domain containing inositol 5-phosphatases SHIP1 and SHIP2 belong to this family of enzymes very much involved in physiopathology and development. Therefore activity and localization of the enzymes are particularly important taking into account both catalytic and non-catalytic mechanisms of the SHIP phosphatases. Several different mechanisms have been reported for SHIP2 targeting that often result from specific protein:protein interactions. In unstimulated astrocytoma cells, SHIP2 has a perinuclear and cytoplasmic localization. In serum-stimulated cells, SHIP2 can be localized at the plasma membrane and at focal contacts in polarized cells. A phosphorylated form of SHIP2 on S132 can be found in the nucleus and nuclear speckles. When present at the plasma membrane, SHIP2 may control the intracellular level of PI(3,4,5)P(3) thereby producing PI(3,4)P(2). When present in the nucleus, SHIP2 probably associates to other nuclear proteins such as lamin A/C and could potentially control nuclear PI(4,5)P(2). Finally, its presence at focal adhesions and lamellipodia could suggest a role in cell adhesion and migration. It is proposed that the complex phenotype observed in SHIP2 mutant mice in tissue development and growth could result from the addition of plasma membrane and nuclear effects consecutive to SHIP2 alteration.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Reversible Ser/Thr SHIP phosphorylation: a new paradigm in phosphoinositide signalling? Targeting of SHIP1/2 phosphatases may be controlled by phosphorylation on Ser and Thr residues.

Phosphoinositide (PI) phosphatases such as the SH2 domain-containing inositol 5-phosphatases 1/2 (SHIP1 and 2) are important signalling enzymes in human physiopathology. SHIP1/2 interact with a large number of immune and growth factor receptors. Tyrosine phosphorylation of SHIP1/2 has been considered to be the determining regulatory modification. However, here we present a hypothesis, based on recent key publications, highlighting the determining role of Ser/Thr phosphorylation in regulating several key properties of SHIP1/2. Since a subunit of the Ser/Thr phosphatase PP2A has been shown to interact with SHIP2, a putative mechanism for reversing SHIP2 Ser/Thr phosphorylation can be anticipated. PI phosphatases are potential target molecules in human diseases, particularly, but not exclusively, in cancer and diabetes. Therefore, this novel regulatory mechanism deserves further attention in the hunt for discovering novel or complementary therapeutic strategies. This mechanism may be more broadly involved in regulating PI signalling in the case of synaptojanin1 or the phosphatase, tensin homolog, deleted on chromosome TEN.Journal ArticleResearch Support, Non-U.S. Gov'tFLWINSCOPUS: ar.jinfo:eu-repo/semantics/publishe

New functions of the inositol polyphosphate 5-phosphatases in cancer.

Inositol polyphosphate 5-phosphatases act on inositol phosphates and phosphoinositides as substrates. They are 10 different isoenzymes and several splice variants in the human genome that are involved in a series of human pathologies such as the Lowe syndrome, the Joubert and MORM syndromes, breast cancer, glioblastoma, gastric cancer and several other type of cancers. Inositol 5-phosphatases can be amplified in human cancer cells, whereas the 3- and 4- phosphatase tumor suppressor PTEN and INPP4B, repectively are often repressed or deleted. The inositol 5-phosphatases are critically involved in a complex network of higly regulated phosphoinositides, affecting the lipid content of PI(3, 4, 5)P3, PI(4, 5)P2 and PI(3, 4)P2. This has an impact on the normal behavior of many intracellular target proteins e.g. protein kinase B (PKB/Akt) or actin binding proteins and final biological responses. The production of PI(3, 4P)2 by dephosphorylation of the substrate PI(3, 4, 5)P3 is particularly important as it produces a new signal messenger in the control of cell migration, invasion and endocytosis. New inhibitors/activators of inositol 5-phosphatases have recently been identified for the possible control of their activity in several human pathologies such as inflamation and cancer.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

The SHIP2 interactor Myo1c is required for cell migration in 1321 N1 glioblastoma cells.

The phosphoinositide 5-phosphatases consist of several enzymes that have been shown to modulate cell migration and invasion. SHIP2, one family member, is known to interact with growth factor receptors and cytoskeletal proteins. In a human model of glioblastoma 1321 N1 cells, we recently identified Myo1c as a new interactor of SHIP2. This was shown in a complex of proteins also containing filamin A. We show here that SHIP2 localization at lamellipodia and ruffles is impaired in Myo1c depleted cells. In the absence of Myo1c, N1 cells tend to associate to form clusters. Cell migration is very much reduced in Myo1c depleted cells, concomitantly with a decrease in FAK Tyr397 phosphorylation, focal adhesion length and PI(4,5)P2 immunostaining. In N1 cells, Myo1c is thus important for lamellipodia formation to assemble a protein complex containing SHIP2 to facilitate cell migration.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

TNFα suppresses IFNγ-induced MHC class II expression on retinal pigmented epithelial cells cultures

Purpose: One major consequence of retinal pigment epithelium (RPE) cell activation during autoimmune uveitis is the induction of MHC II molecules expression at their surface. IFNγ is regarded as the main cytokine involved in this induction. As TNFα plays a central role in autoimmune uveitis, we investigated its effects on IFNγ-mediated MHC II induction on RPE cells. Methods: Retinal pigment epithelium cells (ARPE-19) were stimulated with IFNγ, TNFα and the anti-TNFα antibody infliximab. The expression of MHCII and ICAM-1 was analysed by flow cytometry. The activation and expression of IRF-1 and STAT-1, two proteins involved in IFNγ-signalling pathway, were analysed by WB. Class II transactivator (CIITA) expression was monitored by qRT-PCR and immunoprecipitation. Results: TNFα inhibits IFNγ-induced MHC II expression on ARPE cells in a dose-dependent manner. Infliximab completely reverses the inhibitory effect of TNFα. We did not observe an inhibitory effect of TNFα on the expression of ICAM-1 induced by IFNγ. Similarly, IFNγ-induced STAT1 phosphorylation and IRF1 expression were not affected by TNFα. On the contrary, we found that TNFα suppresses IFNγ-induced CIITA mRNA accumulation and protein expression. Conclusion: TNFα inhibits IFNγ-induced MHC II expression in RPE cells. This inhibitory effect was reversed by infliximab and was not because of a global inhibition of IFNγ -mediated RPE cell activation but rather to a specific down-regulation of CIITA expression. Those findings are consistent with the role of TNFα in the resolution of inflammation and might help to elucidate the complex development of autoimmune uveitis. © 2011 Acta Ophthalmologica Scandinavica Foundation.SCOPUS: ar.jFLWINinfo:eu-repo/semantics/publishe

When worlds collide: inositol pyrophosphates and phosphoinositides intersect at the plasma membrane.

Highly phosphorylated inositol pyrophosphates are present in the cells of many organisms such as yeast, Dictyostelium and mammals. They can act as signal molecules in growth factor and insulin signalling both in cultured cells and in intact mice. Their action involves protein pyrophosphorylation or binding to multiple protein interactors such as PH (pleckstrin homology)-domain-containing proteins. One key enzyme in their synthesis, PPIP5K (diphosphoinositol pentakisphosphate kinase) 1/2, can phosphorylate InsP6 and 5-InsP7 to 1-InsP7 and InsP8 respectively. Stephen Shears's laboratory reported in this issue of the Biochemical Journal that PPIP5K1's unexpectedly high affinity for PtdIns(3,4,5)P3, which is synthesized at the plasma membrane, provides a recruitment mechanism for this enzyme in response to growth factor receptor activation. In competition experiments, they observed that PtdIns(3,4,5)P3 binding to PPIP5K1 could be displaced by inositol pyrophosphates and that PPIP5K1 substrates were more potent inhibitors than PPIP5K1 products. Those findings reveal a mechanism for localized depletion of InsP6 and 5-InsP7 at the plasma membrane and further translocation of PtdIns(3,4,5)P3-binding PH-domain-containing proteins.Journal ArticleSCOPUS: no.jinfo:eu-repo/semantics/publishe

SHIP2 controls plasma membrane PI(4,5)P2 thereby participating in the control of cell migration in 1321 N1 glioblastoma cells

Phosphoinositides, particularly PI(3,4,5)P3, and PI(4,5)P2, are recognized by SHIP2 a member of the inositol polyphosphate 5-phosphatase family. SHIP2 dephosphorylates PI(3,4,5)P3 to form PI(3,4)P2; the latter interacts with specific target proteins (e.g. lamellipodin). Although the SHIP2 preferred substrate is PI(3,4,5)P3, PI(4,5)P2 could also be dephosphorylated to PI4P. Through depletion of SHIP2 in a glioblastoma cell line 1321 N1 cells, we show that SHIP2 inhibits cell migration. In different glioblastoma cell lines and primary cultures, SHIP2 staining at the plasma membrane partly overlaps with PI(4,5)P2 immunoreactivity. PI(4,5)P2 was upregulated in SHIP2-deficient N1 cells as compared to control cells; in contrast, PI4P was very much decreased in SHIP2-deficient cells. Therefore, SHIP2 controls both PI(3,4,5)P3 and PI(4,5)P2 levels in intact cells. In N1 cells, the PI(4,5)P2 binding protein myosin-1c was identified as a new interactor of SHIP2. Regulation of PI(4,5)P2 and PI4P content by SHIP2 controls N1 cell migration through the organization of focal adhesions. Thus, our results reveal a novel role of SHIP2 in the control of PI(4,5)P2, PI4P and cell migration in PTEN-deficient glioblastoma N1 cells.status: publishe

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)P3) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) 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.JOURNAL ARTICLESCOPUS: ar.jinfo:eu-repo/semantics/publishe

Evidence of SHIP2 Ser132 phosphorylation, its nuclear localization and stability.

PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are major signalling molecules in mammalian cell biology. PtdIns(3,4)P2 can be produced by PI3Ks [PI (phosphoinositide) 3-kinases], but also by PI 5-phosphatases including SHIP2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 2]. Proteomic studies in human cells revealed that SHIP2 can be phosphorylated at more than 20 sites, but their individual function is unknown. In a model of PTEN (phosphatase and tensin homologue deleted on chromosome 10)-null astrocytoma cells, lowering SHIP2 expression leads to increased PtdIns(3,4,5)P3 levels and Akt phosphorylation. MS analysis identified SHIP2 phosphosites on Ser132, Thr1254 and Ser1258; phosphotyrosine-containing sites were undetectable. By immunostaining, total SHIP2 concentrated in the perinuclear area and in the nucleus, whereas SHIP2 phosphorylated on Ser132 was in the cytoplasm, the nucleus and nuclear speckles, depending on the cell cycle stage. SHIP2 phosphorylated on Ser132 demonstrated PtdIns(4,5)P2 phosphatase activity. Endogenous phospho-SHIP2 (Ser132) showed an overlap with PtdIns(4,5)P2 staining in nuclear speckles. SHIP2 S132A was less sensitive to C-terminal degradation and more resistant to calpain as compared with wild-type enzyme. We have identified nuclear lamin A/C as a novel SHIP2 interactor. We suggest that the function of SHIP2 is different at the plasma membrane where it recognizes PtdIns(3,4,5)P3, and in the nucleus where it may interact with PtdIns(4,5)P2, particularly in speckles.Comparative StudyJournal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe