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La phosphoinositide phosphatase SHIP2 est une protéine capable de moduler le PI(3,4,5)P3, le PI(4,5)P2 et le PI(3,4)P2 qui sont des phosphoinositides importants lors des remaniements membranaires de la cellule. La régulation de la composition en phosphoinositides des membranes permettant l’assemblage de complexes protéiques est primordiale pour la génération de protrusions essentielles pour la migration et l’invasion cellulaire. D’autre part, SHIP2 possède des propriétés d’échafaudage permettant sa participation à plusieurs complexes multi-protéiques et à son ancrage à des localisations subcellulaires spécifiques. Plusieurs études ont montré que SHIP2 pouvait jouer un rôle dans le développement de certains cancers dont le cancer du sein. Au cours de ce travail, nous avons mis en évidence deux nouveaux partenaires de SHIP2 :IRSp53 et CIN85. Ces deux protéines participent respectivement à la formation d’invadopode et à l’endocytose. IRSp53 et CIN85 lient la même région C-terminale riche en prolines (PRR-II) de SHIP2, mais toutefois pas par les mêmes séquences. Cette région contient plusieurs motifs consensus permettant l’interaction de SHIP2 avec plusieurs autres partenaires connus comme l’intersectine, une protéine impliquée dans l’endocytose et Mena, un partenaire commun de SHIP2 et d’IRSp53 impliqué dans l’invasion cellulaire. Nous avons également montré que la mutation de la F1053 située dans la PRR-II de SHIP2 est, essentielle pour la liaison à l’intersectine, impliquée dans la stabilité de la liaison avec Mena et IRSp53 mais pas impliquée dans la liaison de CIN85. De plus, nous montrons qu’à la différence d’IRSp53 qui n’en possède qu’un, CIN85 possède trois domaines SH3 mais ne doit lier que deux motifs dans la PRR-II de SHIP2 afin d’assurer sa complète interaction.Dans les cellules dérivées du cancer du sein MDA-MB-231, nous avons montré que Mena n’est pas nécessaire pour l’interaction entre SHIP2 et IRSp53. Cependant, nous avons observé que l’absence de Mena d, Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished

Proliferating cells increase glucose metabolism to generate ATP and to supply precursors for macromolecular biosynthesis. Despite the fact that aerobic respiration produces more ATP, cancer cells convert most of their glucose to lactate under aerobic conditions, a phenomenon known as the “Warburg effect”, but which is not restricted to cancer cells. Fructose-2,6-bisphosphate (Fru-2,6-BP) is the most potent positive allosteric effector of 6-phosphofructo-1-kinase (PFK-1), and hence of glycolysis. Fru-2,6-BP is synthesized and degraded by a bifunctional, homodimeric enzyme, called 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBPase-2). Four PFK-2/FBPase-2 isoenzymes, designated PFKFB1-4, have been identified in mammals encoded by four genes and each gene generates several isoforms by alternative splicing. Previous studies showed that Fru-2,6-BP concentrations in cancer cells can reach levels 10 – 100 times higher than required to stimulate PFK-1. Therefore, we investigated whether Fru-2,6-BP might play a role in coupling glycolysis to cell proliferation and protein synthesis. Freshly isolated rat thymocytes were chosen as a non-cancerous model to study the metabolic changes that occur during the transition from the resting to the proliferating state. Mitogen stimulation of rat thymocytes with concanavalin A (ConA) induced time-dependent increases in medium lactate accumulation (6-fold), Fru-2,6-BP content (4-fold), expression of PFKFB3 and PFKFB4 isoenzymes (~2-fold and ~15-fold, respectively) and rates of cell proliferation (~40-fold) and protein synthesis (~10-fold) after 68 h of incubation compared with resting cells. In parallel, increased expression and phosphorylation of translation factors, such as eukaryotic initiation factor-4E-binding protein-1 (4E-BP1) and ribosomal protein S6 (rpS6), were observed in ConA-stimulated thymocytes. In a parallel in vitro study, protein kinase B (PKB) was shown to phosphorylate PFKFB3 at Ser461, leading to PFK, (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2017

Metabolic enzymes are very specific. However, most of them show weak side activities toward compounds that are structurally related to their physiological substrates, thereby producing side products that may be toxic. In some cases, ‘metabolite repair enzymes’ eliminating side products have been identified. We show that mammalian glyceraldehyde 3-phosphate dehydrogenase and pyruvate kinase, two core glycolytic enzymes, produce 4-phosphoerythronate and 2-phospho-L-lactate, respectively. 4-Phosphoerythronate strongly inhibits an enzyme of the pentose phosphate pathway, whereas 2-phospho-L-lactate inhibits the enzyme producing the glycolytic activator fructose 2,6-bisphosphate. We discovered that a single, widely conserved enzyme, known as phosphoglycolate phosphatase (PGP) in mammals, dephosphorylates both 4-phosphoerythronate and 2-phospho-L-lactate, thereby preventing a block in the pentose phosphate pathway and glycolysis. Its yeast ortholog, Pho13, similarly dephosphorylates 4-phosphoerythronate and 2-phosphoglycolate, a side product of pyruvate kinase. Our work illustrates how metabolite repair enzymes can make up for the limited specificity of metabolic enzymes and permit high flux in central metabolic pathways.

Biguanides such as metformin have previously been shown to antagonize hepatic glucagon-stimulated cyclic AMP (cAMP) signalling independently of AMP-activated protein kinase (AMPK) via direct inhibition of adenylate cyclase by AMP. Here we show that incubation of hepatocytes with the small-molecule AMPK activator 991 decreases glucagon-stimulated cAMP accumulation, cAMP-dependent protein kinase (PKA) activity and downstream PKA target phosphorylation. Moreover, incubation of hepatocytes with 991 increases the Vmax of cyclic nucleotide phosphodiesterase 4B (PDE4B) without affecting intracellular adenine nucleotide concentrations. The effects of 991 to decrease glucagon-stimulated cAMP concentrations and activate PDE4B are lost in hepatocytes deleted for both catalytic subunits of AMPK. PDE4B is phosphorylated by AMPK at three sites, and by site-directed mutagenesis, Ser304 phosphorylation is important for activation. In conclusion, we provide a new mechanism by which AMPK antagonizes hepatic glucagon signalling via phosphorylation-induced PDE4B activation., SCOPUS: ar.j, info:eu-repo/semantics/published

PIKfyve (FYVE domain-containing phosphatidylinositol 3-phosphate 5-kinase), the lipid kinase that phosphorylates PtdIns3P to PtdIns(3,5)P2, has been implicated in insulin-stimulated glucose uptake. We investigated whether PIKfyve could also be involved in contraction/AMPK (AMP-activated protein kinase)-stimulated glucose uptake in skeletal muscle. Incubation of rat epitrochlearis muscles with YM201636, a selective PIKfyve inhibitor, reduced contraction- and AICAriboside (5-amino-4-imidazolecarboxamide riboside)-stimulated glucose uptake. Consistently, PIKfyve knockdown in C2C12 myotubes reduced AICAriboside-stimulated glucose transport. Furthermore, muscle contraction increased PtdIns(3,5)P2 levels and PIKfyve phosphorylation. AMPK phosphorylated PIKfyve at Ser307 both in vitro and in intact cells. Following subcellular fractionation, PIKfyve recovery in a crude intracellular membrane fraction was increased in contracting versus resting muscles. Also in opossum kidney cells, wild-type, but not S307A mutant, PIKfyve was recruited to endosomal vesicles in response to AMPK activation. We propose that PIKfyve activity is required for the stimulation of skeletal muscle glucose uptake by contraction/AMPK activation. PIKfyve is a new AMPK substrate whose phosphorylation at Ser307 could promote PIKfyve translocation to endosomes for PtdIns(3,5)P2 synthesis to facilitate GLUT4 (glucose transporter 4) translocation., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

Ezrin, radixin, and moesin (ERM) proteins are known to be substrates of Rho kinase (ROCK), a key player in vascular smooth muscle regulation. Their function in arteries remains to be elucidated. The objective of the present study was to investigate ERM phosphorylation and function in rat aorta and mesenteric artery and the influence of ERM-binding phosphoprotein 50 (EBP50), a scaffold partner of ERM proteins in several cell types. In isolated arteries, ERM proteins are phosphorylated by PKC and ROCK with different kinetics after either agonist stimulation or KCl-induced depolarization. Immunoprecipitation of EBP50 in noradrenaline-stimulated arteries allowed identification of its interaction with moesin and several other proteins involved in cytoskeleton regulation. This interaction was inhibited by Y27632, a ROCK inhibitor. Moesin or EBP50 depletion after small interfering RNA transfection by reverse permeabilization in intact mesenteric arteries both potentiated the contractility in response to agonist stimulation without any effect on contractile response induced by high KCl. This effect was preserved in ionomycin-permeabilized arteries. These results indicate that, in agonist-stimulated arteries, the activation of ROCK leads to the binding of moesin to EBP50, which interacts with several components of the cytoskeleton, resulting in a decrease in the contractile response., info:eu-repo/semantics/published