2.

Hexokinase (HK) II phosphorylates Ser293 of the alpha subunit of pyruvate dehydrogenase (PDHA1). (A) Purified HKII was incubated with either wild type PDHA1 or PDHA1 mutant S293A. After incubation, PHDA1 was purified by Flag beads. Phosphorylation level of serine 293 of PDHA1 (P-S293-PDHA1) were determined and quantified; (B) Purified wide type PDHA1 and S293A mutant were treated with recombinant wide type HKII or its catalytic dead mutant HKII^mut, and the levels of P-Ser of PDHA1 in the reaction mixture after treatment were determined; (C) Flag-tagged PDHA1 was co-expressed with either HA-tagged HKII or HA-tagged HKII mutant in HeLa cells. P-Ser levels of Flag bead-purified PDHA1 from each culture were determined and quantified; (D) Flag-tagged PDHA1 was co-expressed with HA-tagged HKII in HeLa cells. P-Ser levels of Flag bead-purified PDHA1 from each cell cultured with and without 3-bromopyruvate (3BP, 5 mmol/L) supplementation were determined and quantified; (E) PDK1 was knocked down in HeLa cells and HA-tagged HKII was transfected into PDK1 knockdown cells. Then the cells were treated with serial diluted 3BP and P-S293-PDHA1 was tested; (F) HA-tagged HKII was co-expressed with either Flag-tagged PDHA1, Flag-tagged PDHA1 mutants S293A or S293E in HeLa cells. The P-Ser levels of Flag bead-purified PDHA1 and mutants from each culture were determined and quantified; (G) PDK1 was knocked down in HeLa cells and HKII and PDHA1, S293A, S300A and S232A mutants were transfected into knockdown cells, then Pan-P-Serine was tested; (H) HA-tagged WT HKII or HA-tagged HKII mutant was overexpressed in HeLa cells. P-S293 levels of endogenous PDHA1 of each culture were determined; (I) HA-tagged HKII was overexpressed in HeLa cells. P-S293 levels of endogenous PDHA1 of each culture with and without 3BP (5 mmol/L) supplementation were determined; (J) P-S293 levels of endogenous PDHA1 in HeLa cells before and after HKII knockdown by independent shRNAs were compared; PDK1 (K); PDK2 (L); PDK3 (M) and PDK4 (N) were knocked down, respectively in HeLa cells and HKII was transfected into the cells, then P-S293-PHDA1 was tested.; (O) HA-tagged HKII overexpressed HeLa cells were treated with or without 2-DG (10 mmol/L) or 3BP (5 mmol/L) and P-S293 levels of endogenous PDHA1 were determined; (P) PDK1 was knocked down in HeLa cells and HA-tagged HKII was overexpressed in the cells. The endogenous P-S293 levels of PDHA1 of each culture with and without 3BP (5 mmol/L) or 2-DG (10 mmol/L) supplementation were determined.