Four Key Steps Control Glycolytic Flux in Mammalian Cells

Altered glycolysis is a hallmark of diseases including diabetes and cancer. Despite intensive study of the contributions of individual glycolytic enzymes, systems-level analyses of flux control through glycolysis remain limited. Here, we overexpress in two mammalian cell lines the individual enzymes catalyzing each of the 12 steps linking extracellular glucose to excreted lactate, and find substantial flux control at four steps: glucose import, hexokinase, phosphofructokinase, and lactate export (and not at any steps of lower glycolysis). The four flux-controlling steps are specifically upregulated by the Ras oncogene: optogenetic Ras activation rapidly induces the transcription of isozymes catalyzing these four steps and enhances glycolysis. At least one isozyme catalyzing each of these four steps is consistently elevated in human tumors. Thus, in the studied contexts, flux control in glycolysis is concentrated in four key enzymatic steps. Upregulation of these steps in tumors likely underlies the Warburg effect. Here, we perform a systematic analysis of glycolytic flux control in mammalian cells. We identify four key flux-controlling steps: glucose import and phosphorylation, fructose-1,6-bisphosphate production, and lactate export. In contrast, enzyme steps in lower glycolysis do not control pathway flux. Activation of glycolysis in cancer and immune cells is associated with enhanced expression of enzymes catalyzing these four key flux-controlling steps.