Role of Akt/PKB and PFKFB isoenzymes in the control of glycolysis, cell proliferation and protein synthesis in mitogen-stimulated thymocytes

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-2 activation. However, in ConA-stimulated cells, only a slight increase in PFKFB3 Ser461 phosphorylation was detected. Therefore, although PFKFB3 is expressed in many proliferating and cancer cells, the importance of PKB-induced PFKFB3 phosphorylation is unclear. Treatment of ConA-stimulated thymocytes with low doses of PKB inhibitor (MK-2206) resulted in reduced levels of the PFKFB3 and PFKFB4 proteins, suggesting regulation of expression of the two PFKFB isoenzymes by PKB. In parallel, we also showed that MK-2206 induced significant decreases in Fru-2,6-BP content, medium lactate and rates of cell proliferation and protein synthesis in ConA-stimulated thymocytes. As expected, treatment with the PFK-2 inhibitor (3PO) reduced Fru-2,6-BP content and medium lactate accumulation, without affecting PFKFB3 and PFKFB4 expression in ConA-stimulated cells. Surprisingly, rates of cell proliferation and protein synthesis were also significantly decreased by exposure of ConA-stimulated cells to low doses of 3PO. These data were confirmed by 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 stimulating glycolysis in ConA-stimulated thymocytes and implicate PKB in the upregulation of PFKFB3 and PFKFB4 expression. Taken together, the findings support a role of Fru-2,6-BP in the control of glycolysis, cell proliferation and protein synthesis in mitogen-stimulated thymocytes, through a mechanism involving PKB.