FIGURE 4. Degradation of PHD3 by phospho-mutant forms of Siah2.

A, in vitro ubiquitination (Ub) of WT and mutant Siah2. In vitro translated (IVT) ³⁵S-labeled Siah2-HA was subjected to in vitro kinase assay (or control mock reaction) using recombinant p38 MAPK and either cold or [γ³²P]ATP once (lane 5) or three times (lane 6). The reaction mixture was washed extensively and subjected to an in vitro ubiquitination reaction in the presence of E1, E2 (UbcH5c), and Ub-HA for 25 min at 37 °C. The reaction mixture was then separated on SDS-PAGE, and ubiquitination of GST-Siah2 was monitored by immunoblots with anti-HA antibody. The degree of phosphorylation was monitored by autoradiograph, and total levels of GST-Siah2 are shown (middle panel). Levels of Siah2-HA was monitored by immunoblot analysis with anti-HA antibody (lower panel). B, NIH3T3 cells were transfected with Siah2-HA, Siah2 T24A-HA, Siah2S29A-HA, and PHD3-FLAG. Cell lysates were subjected to immunoblot analysis and probed with the antibodies indicated in the figure. C, 293T cells were co-transfected with 1 or 2 µg of Siah2-HA, Siah2 S29A-HA, Siah2 S29D-HA, and PHD3-FLAG. Cell lysates were subjected to immunoblot analysis and probed with anti-FLAG, anti-HA, and β-actin antibodies. D, HeLa cells were transfected with Siah2-HA, Siah2S29A-HA, and Siah2S29D-HA. Cells were either left untreated or treated with 1% hypoxia. Cell lysates were subjected to immunoblot analysis and probed with anti-PHD3, anti-HA, and anti-β-actin antibodies. E, WT MEFs and MKK3/6 knock-out MEFs were transfected with PHD3-FLAG and various amounts (µg) of Siah2-HA as indicated. 48 h later cells were either untreated or treated with 1% hypoxia for 4 h. Cell lysates were subjected to immunoblot analysis using anti-FLAG antibody and anti-β-actin antibody.