Figure 6. Prolyl hydroxylation of ATF4 on aa 60 and 235 by PHD1/3 limits ATF4 availability.

(A–B) Specificity of shRNA used to KD PHD1/3. Siah1a^−/−::Siah2^−/− MEFs were infected with scrambled control or PHD1 shRNA and PHD3 shRNA alone or in combination, and treated with TM (1 µg/ml) for 6 h. The relative mRNA levels of PHD1 (A) and PHD3 (B) were determined by qPCR. The results are shown as the mean values ± S.E. of three independent experiments. Three independent shRNA were used to confirm the changes shown. (C) PHD1 and PHD3 cooperation is required and mediate the effect of Siah1a/2 on TM-induced CHOP transcription. Siah1a^−/−::Siah2^−/− MEFs were infected with different PHD1 shRNA or PHD3 shRNA, or their combination, or scrambled shRNA. Cells were treated with TM (1 µg/ml) and collected 6 h later. The relative transcription levels of CHOP were determined by qPCR. (D) PHD1 and PHD3 mediate the effect of Siah1a/2 on hypoxia-induced CHOP transcription. Siah1a^−/−::Siah2^−/− MEFs were infected with PHD1 shRNA and PHD3 shRNA alone or in combination, or control vector and exposed to 1% O₂ for 6 h. The relative mRNA level of CHOP was determined by qPCR. (E) PHD3 protein is induced in Siah1a^−/−::Siah2^−/− cells. WT and Siah1a^−/−::Siah2^−/− MEFs were exposed to 1% O₂ for 24 h prior to the analysis for the expression of HIF-1α, PHD3 and β-actin by Western blotting. The arrow points to the position of the endogenous PHD3 protein. (F) Annotated MS/MS spectra resulting in the identification of proline hydroxylation sites at P60 and P235. Identified fragment ions are shown, as are the detected sites of peptide backbone cleavage; m/z, mass to charge ratio. Note that site determining fragment ions resulted in localization of both sites of proline hydroxylation. (G) Mutations of the two identified proline hydroxylation sites at P235 and P60 to alanine stabilize ATF4 protein. 293T cells were transfected either with Flag-ATF4 or Flag-ATF4 presenting either a mutation at P60, P235, or both. After 24 h from transfection cells were treated overnight with vehicle, DMOG (0.5 mM; upper panel), or MG132 (5 µM; lower panel) followed by cell harvest and immunoblot analysis of ATF4 and β-actin. *** p<0.0005, ** p<0.005, * p<0.05 compared to ad shRNA scr. (A–D) in the same condition (student's t-test). The Western blot experiments were repeated three times and the qPCR results are shown as the mean values ± S.E. of three independent experiments.