Figure 2: HK1 is phosphorylated by c-Src at Tyr732.

(a) HEK 293T cells were co-transfected with HA-c-Src and Flag-HK1, followed by IP for HK1 and western blot (WB) with a pan anti-phospho-tyrosine antibody (Anti-pTyr) for detection of HK1 phosphorylation. CIAP, calf-intestinal alkaline phosphatase. (b) c-Src, but not c-Src-KD (a kinase dead form of c-Src), is able to phosphorylate HK1 in HEK 293T cells. (c) Both PP2 and SU6656, inhibitors of Src family kinases, efficiently antagonize c-Src-mediated phosphorylation of HK1. Transfected HEK 293T cells were treated with or without 10 μM PP2 or 10 μM SU6656 for 4 h before harvest. (d) GST-c-Src, but not GST-c-Src-KD, directly phosphorylated His-HK1 in an in vitro kinase assay. (e) Knockdown of c-Src in HeLa cells impairs tyrosine phosphorylation of HK1. Cells were transfected with pLKO.1-based shRNAs against c-Src or green fluorescence protein (GFP), followed by selection with puromycin (2 μg ml⁻¹). Tyrosine phosphorylation of endogenous HK1 was detected. Relative quantification of blots were analysed by Image J software. (f) HK1 phosphorylation is abolished by PP2 and SU6656 treatment in HeLa cells. c-Src activity was indicated by detecting Y419 phosphorylation level with anti-p-c-Src. (g) Y732F mutation dramatically attenuated c-Src-mediated tyrosine phosphorylation of HK1 in HEK 293T cells. (h) HK1-Y732F mutant failed to be phosphorylated by GST-c-Src. In vitro kinase assay was performed as in d. (i) HK1 was phosphorylated by c-Src at Y732 in HEK 293T cells as detected by anti-p-Y732, a home-made rabbit antibody that can specifically recognize phosphorylated Y732 residue. (j) His-HK1 is directly phosphorylated by GST-c-Src in vitro. (k) Inhibition of c-Src activity by PP2 and SU6656 abolishes HK1 phosphorylation at Y732 in HeLa cells. (l) HeLa cells were treated with 0.1 mM pervanadate (a protein tyrosine phosphatase inhibitor) for 10 min, followed by detection of HK1-Y732 and c-Src-Y419 phosphorylation. (m) Dominant-negative form of c-Src depleted HK1-Y732 phosphorylation stimulated by EGF (100 ng ml⁻¹), PDGF (20 ng ml⁻¹) or insulin-like growth factor (IGF) (100 ng ml⁻¹) in HeLa cells. (n) PP2 blocked the phosphorylation of HK1-Y732 stimulated by PDGF in HeLa cells.