MTG16 interacts with PHD2 and induces ubiquitination and proteasomal degradation of HIF1α.

<p><b>A.</b> Raji-MTG16 cells were co-transfected with HA-HIF1α and FLAG-Ubiquitinin and incubated 48 h at 4% O₂ with 20 ng/ml of doxycycline inducing production of MTG16. Cell lysate was subjected to immunoprecipitation with anti α-HA antibody followed by Western blotting using α-FLAG antibody to detect ubiquitinated HIF1α, which was increased in doxycycline induced cells. Middle panel shows co-precipitation of MTG16 by α-HA but not by α-β-actin, serving as a negative control. The lower panel shows the input of HIF1α in whole cell lysate. <b>B.</b> The intensity of the HIF1α-ubiquitin bands in A (top) were quantified by densitometry and expressed as HIF1α-ubiquitin/β-actin ratio. The level of ubiquitinated HIF1α was increased in cells incubated with doxycycline; n = 3, ***P<0.001. <b>C</b>. Cells were incubated 12 h at 4% O₂ with 20 ng/ml of doxycycline to induce production of MTG16. Both MTG16 and HIF1α co-precipitated with prolylhydroxylase D2 (PHD2) in whole cell lysates. <b>D.</b> By use of Western blotting with an anti-hydroxyproline antibody to hydroxyproline-564 in HIF1α (Hyp-564) hydroxylation of HIF1α was detected within 4 h of incubation with 20 ng/ml doxycycline at 20% O₂. Hours on abscissa refer to beginning of incubation with doxycycline. The expected downregulation of HIF-1α with time after induction of MTG16 is seen. <b>E.</b> Experiments were performed to detect hydroxylation of HIF1α also at 4% O₂ during incubation with 20 ng/ml doxycycline. Hours on abscissa refer to beginning of hypoxic conditions. The results confirm hydroxylation of Pro-564 in HIF1α by MTG16 at 4% O₂. <b>F</b>. Cells incubated at 20% O₂ with and without 20 ng/ml of doxycycline to induce production of MTG16 were co-incubated with the proteasomal inhibitor MG132 for 24 h. The inhibitor strongly protected against degradation of HIF1α. <b>G</b>. Similar experiments as in F were performed at 4% O₂. The results confirmed strong protection against MTG16–induced degradation by MG132. The exposure of photographic film was 20 sec for A, E and G and 90 sec for D and F.