Phosphorylation of the 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase/PFKFB3 family of glycolytic regulators in human cancer.

Fructose 2,6-bisphosphate (F2,6BP) is a potent activator of phosphofructokinase, which is a rate-limiting enzyme of glycolysis. The concentration of F2,6BP depends on the activity of the bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase). Four genes encoding PFK-2/FBPase have been identified and termed PFKFB1 to PFKFB4. PFKFB3 protein is expressed in high levels in human tumors in situ. The purpose of this study was to determine the role of functional interactions between the phosphorylation of PFKFB3 and activated glycolysis in human cancer cells. cDNA from several human tumor cell lines and human colon carcinoma were analyzed by reverse transcription-PCR to identify different splicing variants of PFKFB3. The effect of phosphorylation of Ser461 was studied by recombinantly replacing this residue with glutamate (PFKFB3S461E). The phosphorylation of PFKFB3 protein in human cancer was determined by immunostaining using an anti-phospho-PFK-2(PFKFB3) antibody. Two splicing variants of PFKFB3 are expressed in human cancer cell lines: PFKFB3-ACG and PFKFB3-AG. Quantitative, real-time PCR analysis confirmed the overexpression of PFKFB3 mRNA in colon carcinoma, with the dominant variant being the PFKFB3-ACG isoform that contains a phosphorylation site at Ser461. Forced expression of PFKFB3-ACG in COS-7 cells resulted in enhanced glycolysis. Introduction of PFKFB3-ACGS461E into COS-7 cells led to increased the lactate production and cell proliferation. Highly phosphorylated PFKFB3 protein was found in human tumor cells, vascular endothelial cells, and smooth muscle cells, as determined by immunostaining with an anti-phospho-PFK-2(PFKFB3) antibody. These findings support a potential role for the phosphorylation of PFKFB3 protein in the progression of cancer and angiogenesis.