The actinobacterial transcription factor RbpA binds to the principal sigma subunit of RNA polymerase

RbpA is a small non-DNA-binding transcription factor that associates with RNA polymerase holoenzyme and stimulates transcription in actinobacteria, including Streptomyces coelicolor and Mycobacterium tuberculosis. RbpA seems to show specificity for the vegetative form of RNA polymerase as opposed to alternative forms of the enzyme. Here, we explain the basis of this specificity by showing that RbpA binds directly to the principal σ subunit in these organisms, but not to more diverged alternative σ factors. Nuclear magnetic resonance spectroscopy revealed that, although differing in their requirement for structural zinc, the RbpA orthologues from S. coelicolor and M. tuberculosis share a common structural core domain, with extensive, apparently disordered, N- and C-terminal regions. The RbpA-σ interaction is mediated by the C-terminal region of RbpA and σ domain 2, and S. coelicolor RbpA mutants that are defective in binding σ are unable to stimulate transcription in vitro and are inactive in vivo. Given that RbpA is essential in M. tuberculosis and critical for growth in S. coelicolor, these data support a model in which RbpA plays a key role in the σ cycle in actinobacteria

A signalling function of phosphatidylinositol 3,4-bisphosphate in cell migration of breast cancer cells

SHIP2 is a phosphatase that belongs to the family of the phosphoinositide 5-phosphatases. It is known to dephosphorylate PI(3,4,5)P3 to PI(3,4)P2 imparting a tight control of the PI 3-kinase pathway. Over the last decade, SHIP2 has been described as a tumor promotor or tumor suppressor in several cancer types such as glioblastoma, colorectal cancer or breast cancer cells. Several studies have proposed a role of SHIP2 in breast cancer cells, but its tumor promoting function was unclear at the beginning of this thesis especially in terms of its mode of regulation. In 2013, the INPPL1 gene that encodes SHIP2 has been found to be mutated in opsismodysplasia (OPS), a rare autosomal recessive disease characterized by delayed bone maturation but no molecular mechanism was provided to explain the mechanism. In this thesis, we first contributed to establish a negative regulation of SHIP2 on cell migration in 1321 N1 glioblastoma (GBM) cells. Our studies revealed a dephosphorylation activity of SHIP2 on PI(4,5)P2 at the plasma membrane to control cell migration. This study was done in collaboration with Dr. Elong Edimo in the lab. We have also shown that the regulation of cell motility cannot be generalized to all the GBM cells. In LN229 and U-251 GBM cells we observed a positive regulation of cell migration by SHIP2. We next took advantage of a unique model comparing fibroblasts derived from non-affected and OPS patients (in collaboration with Dr. Valérie Cormier-Daire). We have shown that the fibroblasts from the OPS patients are SHIP2 deficient and migrate slower as compared to fibroblasts from non-affected individuals. Finally, the major part of the thesis was the study of breast cancer cells: in the model MDA-MB-231 cells, we established a positive regulation of SHIP2 on cell migration. We extended this regulation on cell migration to different breast cancer cell models using a SHIP2 inhibitor AS1949490. We confirmed that this inhibitor blocks the phosphatase activity of SHIP2 and showed its selectivity towards SHIP2 in cell migration assay. In MDA-MB-231 cells we deciphered a second messenger role of PI(3,4)P2 to control cell migration. Our data in this model rely on the use of SHIP2 depleted cells obtained by lentiviral infection and shRNA. We confirmed the positive role of SHIP2 on cell migration in the model of rat chondrosarcoma SHIP2CRISPR cells (in collaboration with Dr. Pavel Krejci).A major goal of this thesis was achieved thanks to in-vivo studies: using MDA-MB-231 cells injected in SCID mice, we found a tumor promoting role of SHIP2 by determining the tumor weight. We also observed less lung metastasis of SHIP2 depleted injected cells as compared to control cells suggesting SHIP2 to be important for invasiveness of triple negative breast cancers.Doctorat en Sciences biomédicales et pharmaceutiques (Médecine)info:eu-repo/semantics/nonPublishe

The impact of PI 5-phosphatases on phosphoinositides in cell function and human disease.

Phosphoinositides (PIs) are recognized as major signaling molecules in many different functions of eukaryotic cells. PIs can be dephosphorylated by multiple phosphatase activities at the 5-, 4- and 3- positions. Human PI 5-phosphatases belong to a family of 10 members. Except for INPP5A, they all catalyze the dephosphorylation of PI(4,5)P2 and/or PI(3,4,5)P3 at the 5- position. PI 5-phosphatases thus directly control the levels of PI(3,4,5)P3 and also participate in fine-tuning regulatory mechanisms of PI(3,4)P2 and PI(4,5)P2. Second messenger functions have been demonstrated for PI(3,4)P2 in invadopodium maturation and in lamellipodia formation. PI 5-phosphatases can use several substrates on isolated enzymes, and it has been challenging to establish their real substrate in vivo. PI(4,5)P2 has multiple functions in signaling, including interaction with scaffold proteins, ion channels, and cytoskeleton proteins. PI 5-phosphatase isoenzymes have been individually implicated in human diseases, such as the oculocerebrorenal syndrome of Lowe, through mechanisms which include lipid-controlling. Oncogenic and tumor suppressive functions of PI 5-phosphatases have also been reported in different cell contexts. The mechanisms responsible for genetic diseases and for tumor suppressor or oncogenic functions are not fully understood. The regulation of PI 5-phosphatases is thus crucial in understanding cell functions.SCOPUS: re.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

IRSp53 is a novel interactor of SHIP2: A role of the actin binding protein Mena in their cellular localization in breast cancer cells.

A tight control of the machineries regulating membrane bending and actin dynamics is very important for the generation of membrane protrusions, which are crucial for cell migration and invasion. Protein/protein and protein/phosphoinositides complexes assemble and disassemble to coordinate these mechanisms, the scaffold properties of the involved proteins playing a prominent role in this organization. The PI 5-phosphatase SHIP2 is a critical enzyme modulating PI(3,4,5)P3, PI(4,5)P2 and PI(3,4)P2 content in the cell. The scaffold properties of SHIP2 contribute to the specific targeting or retention of the protein in particular subcellular domains. Here, we identified IRSp53 as a new binding interactor of SHIP2 proline-rich domain. Both proteins are costained in HEK293T cells protrusions, upon transfection. We showed that the SH3-binding polyproline motif recognized by IRSp53 in SHIP2 is different from the regions targeted by other PRR binding partners i.e. CIN85, ITSN or even Mena a common interactor of both SHIP2 and IRSp53. We presented evidence that IRSp53 phosphorylation on S366 did not influence its interaction with SHIP2 and that Mena is not necessary for the association of SHIP2 with IRSp53 in MDA-MB-231 cells. The absence of Mena in MDA-MB-231 cells decreased the intracellular content in F-actin and modified the subcellular localization of SHIP2 and IRSp53 by increasing their relative content at the plasma membrane. Together our data suggest that SHIP2, through interaction with the cell protrusion regulators IRSp53 and Mena, participate to the formation of multi-protein complexes. This ensures the appropriate modulations of PIs which is important for regulation of membrane dynamics.info:eu-repo/semantics/publishe

The role of phosphoinositide 5-phosphatases activities in glioblastoma: control of cell motility via phosphoinositide PI(4,5)P2 hydrolysis

info:eu-repo/semantics/nonPublishe

Lipid phosphatases SKIP and SHIP2 regulate fibronectin-dependent cell migration in glioblastoma

Cell migration is an important process that occurs during development and has also been linked to the motility of cancer cells. Cytoskeleton reorganization takes place in the migration process leading to lamellipodia formation. Understanding the molecular underpinnings of cell migration is particularly important in studies of glioblastoma, a highly invasive and aggressive cancer type. Two members of the phosphoinositide 5-phosphatase family, SKIP and SHIP2, have been associated with cell migration in glioblastoma; however, the precise role these enzymes play in the process—and whether they work in concert—remains unclear. Here, we compared phosphoinositide 5-phosphatases expression in glioblastoma primary cells and cell lines and showed that SHIP2 and SKIP expression greatly varies between different cell types, while OCRL, another phosphoinositide 5-phosphatase, is constitutively expressed. Upon adhesion of U-251 MG cells to fibronectin, SHIP2, SKIP, and PI(4,5)P2 colocalized in membrane ruffles. Upregulation of PI(4,5)P2 was observed in SKIP-depleted U-251 MG cells compared to control cells, but only when cells were adhered to fibronectin. Both PTEN-deficient (U-251) and PTEN-containing (LN229) glioblastoma cells showed a decrease in cell migration velocity in response to SKIP downregulation. Moreover, a SHIP2 catalytic inhibitor lowered cell migration velocity in the U-251 MG cell line. We conclude that integrin activation in U-251 cells leads to colocalization of both SKIP and SHIP2 in ruffles, where they act as potential drivers of cell migration. Depending on their expression levels in glioblastoma, phosphoinositide 5-phosphatases could cooperate and synergize in the regulation of cell migration and adhesion.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Lipid phosphatases SKIP

Cell migration is an important process that occurs during development and has also been linked to the motility of cancer cells. Cytoskeleton reorganization takes place in the migration process leading to lamellipodia formation. Understanding the molecular underpinnings of cell migration is particularly important in studies of glioblastoma, a highly invasive and aggressive cancer type. Two members of the phosphoinositide 5-phosphatase family, SKIP and SHIP2, have been associated with cell migration in glioblastoma; however, the precise role these enzymes play in the process—and whether they work in concert—remains unclear. Here, we compared phosphoinositide 5-phosphatases expression in glioblastoma primary cells and cell lines and showed that SHIP2 and SKIP expression greatly varies between different cell types, while OCRL, another phosphoinositide 5-phosphatase, is constitutively expressed. Upon adhesion of U-251 MG cells to fibronectin, SHIP2, SKIP, and PI(4,5)P2 colocalized in membrane ruffles. Upregulation of PI(4,5)P2 was observed in SKIP-depleted U-251 MG cells compared to control cells, but only when cells were adhered to fibronectin. Both PTEN-deficient (U-251) and PTEN-containing (LN229) glioblastoma cells showed a decrease in cell migration velocity in response to SKIP downregulation. Moreover, a SHIP2 catalytic inhibitor lowered cell migration velocity in the U-251 MG cell line. We conclude that integrin activation in U-251 cells leads to colocalization of both SKIP and SHIP2 in ruffles, where they act as potential drivers of cell migration. Depending on their expression levels in glioblastoma, phosphoinositide 5-phosphatases could cooperate and synergize in the regulation of cell migration and adhesion.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

Lipid phosphatases SKIP and SHIP2 regulate fibronectin-dependent cell migration in glioblastoma

Cell migration is an important process that occurs during development and has also been linked to the motility of cancer cells. Cytoskeleton reorganization takes place in the migration process leading to lamellipodia formation. Understanding the molecular underpinnings of cell migration is particularly important in studies of glioblastoma, a highly invasive and aggressive cancer type. Two members of the phosphoinositide 5-phosphatase family, SKIP and SHIP2, have been associated with cell migration in glioblastoma; however, the precise role these enzymes play in the process—and whether they work in concert—remains unclear. Here, we compared phosphoinositide 5-phosphatases expression in glioblastoma primary cells and cell lines and showed that SHIP2 and SKIP expression greatly varies between different cell types, while OCRL, another phosphoinositide 5-phosphatase, is constitutively expressed. Upon adhesion of U-251 MG cells to fibronectin, SHIP2, SKIP, and PI(4,5)P2 colocalized in membrane ruffles. Upregulation of PI(4,5)P2 was observed in SKIP-depleted U-251 MG cells compared to control cells, but only when cells were adhered to fibronectin. Both PTEN-deficient (U-251) and PTEN-containing (LN229) glioblastoma cells showed a decrease in cell migration velocity in response to SKIP downregulation. Moreover, a SHIP2 catalytic inhibitor lowered cell migration velocity in the U-251 MG cell line. We conclude that integrin activation in U-251 cells leads to colocalization of both SKIP and SHIP2 in ruffles, where they act as potential drivers of cell migration. Depending on their expression levels in glioblastoma, phosphoinositide 5-phosphatases could cooperate and synergize in the regulation of cell migration and adhesion.SCOPUS: ar.jinfo:eu-repo/semantics/publishe