Figure 2. PDHK1 kinase activity can be induced by hypoxia, and it is essential for serine 232 phosphorylation.

(a) Western blots of MIA PaCa-2 control (Hg) and shHIF1α cells treated for 16 h with decreasing oxygen levels showing effect on HIF-regulated PDHK1 and 3 expression and phosphorylation of PDH E1α. (b) WT and HIF1α KO MEFs were treated with hypoxia (0.5% O₂) or 1 mM DMOG and levels of PDHK1 and pSer232-E1α detected by Western blotting. (c) PANC-1 control (Hg (pX335)) and PDHK1 null cells (PDHK1 KO, two clones) treated with hypoxia (0.5% O₂) overnight showing no kinase activity on Ser232 of E1α. Note that phosphorylation of Ser293 and 300 remain unaffected, reflecting activity of the remaining PDHKs. (d) PDH activity measured in extracts of PANC-1 control and PDHK1 null cells described in (c), after treatment with hypoxia (0.5%) or 1 mM DMOG overnight. (mean ± SD, one-way ANOVA, *p < 0.05, **p < 0.01).