title: Role of Akt/PKB and PFKFB isoenzymes in the control of glycolysis, cell proliferation and protein synthesis in mitogen-stimulated thymocytes.
abstract: Proliferating cells depend on glycolysis mainly to supply precursors for macromolecular synthesis. Fructose 2,6-bisphosphate (Fru-2,6-P2) is the most potent positive allosteric effector of 6-phosphofructo-1-kinase (PFK-1), and hence of glycolysis. Mitogen stimulation of rat thymocytes with concanavalin A (ConA) led to time-dependent increases in lactate accumulation (6-fold), Fru-2,6-P2content (4-fold), 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase isoenzyme 3 and 4 (PFKFB3 and PFKFB4) protein levels (~2-fold and ~15-fold, respectively) and rates of cell proliferation (~40-fold) and protein synthesis (10-fold) after 68h of incubation compared with resting cells. After 54h of ConA stimulation, PFKFB3 mRNA levels were 45-fold higher than those of PFKFB4 mRNA. Although PFKFB3 could be phosphorylated at Ser461 by protein kinase B (PKB) in vitro leading to PFK-2 activation, PFKFB3 Ser461 phosphorylation was barely detectable in resting cells and only increased slightly in ConA-stimulated cells. On the other hand, PFKFB3 and PFKFB4 mRNA levels were decreased (90% and 70%, respectively) by exposure of ConA-stimulated cells to low doses of PKB inhibitor (MK-2206), suggesting control of expression of the two PFKFB isoenzymes by PKB. Incubation of thymocytes with ConA resulted in increased expression and phosphorylation of the translation factors eukaryotic initiation factor-4E-binding protein-1 (4E-BP1) and ribosomal protein S6 (rpS6). Treatment of ConA-stimulated thymocytes with PFK-2 inhibitor (3PO) or MK-2206 led to significant decreases in Fru-2,6-P2content, medium lactate accumulation and rates of cell proliferation and protein synthesis. These data were confirmed by using siRNA knockdown of PFKFB3, PFKFB4 and PKB α/β in the more easily transfectable Jurkat E6-1 cell line. The findings suggest that increased PFKFB3 and PFKFB4 expression, but not increased PFKFB3 Ser461 phosphorylation, plays a role in increasing glycolysis in mitogen-stimulated thymocytes and implicate PKB in the upregulation of PFKFB3 and PFKFB4. The results also support a role for Fru-2,6-P2in coupling glycolysis to cell proliferation and protein synthesis in this model.
authors.affiliation: Université catholique de Louvain and de Duve Institute, Avenue Hippocrate 75, B-1200 Brussels, Belgium.
authors.forename: Amina
authors.initials: A
authors.lastname: Houddane
authors.affiliation: Université catholique de Louvain and de Duve Institute, Avenue Hippocrate 75, B-1200 Brussels, Belgium.
authors.forename: Laurent
authors.initials: L
authors.lastname: Bultot
authors.affiliation: Departament de Ciències Fisiologiques, IDIBELL, Campus de Ciències de la Salut, Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona E-08907, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Novellasdemunt
authors.affiliation: Université catholique de Louvain and de Duve Institute, Avenue Hippocrate 75, B-1200 Brussels, Belgium.
authors.forename: Manuel
authors.initials: M
authors.lastname: Johanns
authors.affiliation: Université catholique de Louvain and de Duve Institute, Avenue Hippocrate 75, B-1200 Brussels, Belgium.
authors.forename: Marie-Agnès
authors.initials: MA
authors.lastname: Gueuning
authors.affiliation: Université catholique de Louvain and de Duve Institute, Avenue Hippocrate 75, B-1200 Brussels, Belgium.
authors.forename: Didier
authors.initials: D
authors.lastname: Vertommen
authors.affiliation: Université catholique de Louvain and de Duve Institute, Avenue Hippocrate 75, B-1200 Brussels, Belgium.
authors.forename: Pierre G
authors.initials: PG
authors.lastname: Coulie
authors.affiliation: Departament de Ciències Fisiologiques, IDIBELL, Campus de Ciències de la Salut, Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona E-08907, Spain.
authors.forename: Ramon
authors.initials: R
authors.lastname: Bartrons
authors.affiliation: Université catholique de Louvain and de Duve Institute, Avenue Hippocrate 75, B-1200 Brussels, Belgium.
authors.forename: Louis
authors.initials: L
authors.lastname: Hue
authors.affiliation: Université catholique de Louvain and de Duve Institute, Avenue Hippocrate 75, B-1200 Brussels, Belgium. Electronic address: mark.rider@uclouvain.be.
authors.forename: Mark H
authors.initials: MH
authors.lastname: Rider
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003208:Concanavalin A / Q000494:pharmacology; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D006019:Glycolysis / Q000187:drug effects; D006575:Heterocyclic Compounds, 3-Ring / Q000494:pharmacology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D019169:Jurkat Cells; D025481:Phosphofructokinase-2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D014176:Protein Biosynthesis / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D038601:Ribosomal Protein S6 / Q000378:metabolism; D060168:Thymocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; C499526:Eif4ebp1 protein, rat; D006575:Heterocyclic Compounds, 3-Ring; D047908:Intracellular Signaling Peptides and Proteins; C548887:MK 2206; C542952:Pfkfb4 protein, rat; D010750:Phosphoproteins; D038601:Ribosomal Protein S6; D003208:Concanavalin A; C526918:Pfkfb3 protein, rat; D025481:Phosphofructokinase-2; D051057:Proto-Oncogene Proteins c-akt
keywords: Fru-2,6-P(2); Glycolysis; PFKFB isoenzymes; PKB; Proliferation; Protein synthesis