Objective:Tumor cells rely heavily on glycolysis regardless of oxygen tension,a phenomenon called the Warburg effect.Hexokinase Ⅱ (HKⅡ) catalyzes the first irreversible step of glycolysis and is often overexpressed in tumor cells.Mitochondrial HKⅡ couples glycolysis and oxidative phosphorylation while maintaining mitochondrial membrane integrity.In this study,we investigated the role of HKⅡ in promoting the Warburg effect in cancer cells.Methods:HKⅡ-mediated phosphorylation of the alpha subunit of pyruvate dehydrogenase (PDHA1) was tested in HEK293T cells and clear cell renal cell carcinoma (ccRCC) specimens using gene knockdown,west blotting,immunohistochemistry,and immunofluorescence.Results:It was determined that HKⅡ could not only transform glucose into glucose-6-phosphate,but also transfer the phosphate group of ATP onto PDHA1.In addition,it was found that HKⅡ increased the phosphorylation of Ser293 on PDHA1,decreasing pyruvate dehydrogenase (PDH) complex activity and thus rerouting the metabolic pathway and promoting the Warburg effect.The overexpression of HKⅡ correlated with the phosphorylation of PDHA1 and disease progression in ccRCC.Conclusions:The data presented here suggest that HKⅡ is an important biomarker in the evaluation and treatment of cancer.