Houddane Amina, Bultot Laurent, Novellasdemunt Laura, Johanns Manuel, Gueuning Marie-Agnès, Vertommen Didier, Coulie Pierre G, Bartrons Ramon, Hue Louis, Rider Mark H
Université catholique de Louvain and de Duve Institute, Avenue Hippocrate 75, B-1200 Brussels, Belgium.
Departament de Ciències Fisiologiques, IDIBELL, Campus de Ciències de la Salut, Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona E-08907, Spain.
Cell Signal. 2017 Jun;34:23-37. doi: 10.1016/j.cellsig.2017.02.019. Epub 2017 Feb 22.
Proliferating cells depend on glycolysis mainly to supply precursors for macromolecular synthesis. Fructose 2,6-bisphosphate (Fru-2,6-P) 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-P content (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-P content, 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-P in coupling glycolysis to cell proliferation and protein synthesis in this model.
增殖细胞主要依靠糖酵解来为大分子合成提供前体。果糖2,6 -二磷酸（Fru - 2,6 - P）是6 -磷酸果糖-1 -激酶（PFK - 1）因而也是糖酵解最有效的正构别构效应剂。用伴刀豆球蛋白A（ConA）刺激大鼠胸腺细胞，与静息细胞相比，孵育68小时后，乳酸积累（增加6倍）、Fru - 2,6 - P含量（增加4倍）、6 -磷酸果糖-2 -激酶（PFK - 2）/果糖-2,6 -二磷酸酶同工酶3和4（PFKFB3和PFKFB4）蛋白水平（分别约增加2倍和15倍）以及细胞增殖率（增加约40倍）和蛋白质合成率（增加10倍）随时间呈依赖性增加。ConA刺激54小时后，PFKFB3 mRNA水平比PFKFB4 mRNA高45倍。虽然PFKFB3在体外可被蛋白激酶B（PKB）在Ser461位点磷酸化从而导致PFK - 2激活，但在静息细胞中几乎检测不到PFKFB3 Ser461磷酸化，在ConA刺激的细胞中也仅略有增加。另一方面，将ConA刺激的细胞暴露于低剂量的PKB抑制剂（MK - 2206）后，PFKFB3和PFKFB4 mRNA水平分别降低了90%和70%，提示PKB对两种PFKFB同工酶的表达具有调控作用。用ConA孵育胸腺细胞导致翻译因子真核起始因子-4E结合蛋白-1（4E - BP1）和核糖体蛋白S6（rpS6）的表达及磷酸化增加。用PFK - 2抑制剂（3PO）或MK - 2206处理ConA刺激的胸腺细胞，可导致Fru - 2,6 - P含量、培养基中乳酸积累以及细胞增殖率和蛋白质合成率显著降低。在更易于转染的Jurkat E6 - 1细胞系中，通过小干扰RNA（siRNA）敲低PFKFB3、PFKFB4和PKBα/β证实了这些数据。这些发现表明，PFKFB3和PFKFB4表达增加而非PFKFB3 Ser461磷酸化增加在有丝分裂原刺激的胸腺细胞糖酵解增加中起作用，并提示PKB参与PFKFB3和PFKFB4的上调。结果还支持在该模型中Fru - 2,6 - P在将糖酵解与细胞增殖和蛋白质合成偶联中的作用。
