PHD1 interacts with ATF4 and negatively regulates its transcriptional activity without prolyl hydroxylation

Abstract

Cellular response to hypoxia plays an important role in both circulatory and pulmonary diseases and cancer. Hypoxia-inducible factors (HIFs) are major transcription factors regulating the response to hypoxia. The α-subunits of HIFs are hydroxylated by members of the prolyl-4-hydroxylase domain (PHD) family, PHD1, PHD2, and PHD3, in an oxygen-dependent manner. Here, we report on the identification of ATF4 as a protein interacting with PHD1 as well as PHD3, but not with PHD2. The central region of ATF4 including the Zipper II domain, ODD domain and β-TrCP recognition motif were involved in the interaction with PHD1. Coexistence of PHD1 stabilized ATF4, as opposed to the destabilization of ATF4 by PHD3. Moreover, coexpression of ATF4 destabilized PHD3, whereas PHD1 stability was not affected by the presence of ATF4. Mutations to alanine of proline residues in ATF4 that satisfied hydroxylation consensus by PHDs did not affect binding activity of ATF4 to PHD1 and PHD3. Furthermore, in vitro prolyl hydroxylation assay clearly indicated that ATF4 did not serve as a substrate of both PHD1 and PHD3. Coexpression of PHD1 or PHD3 with ATF4 repressed the transcriptional activity of ATF4. These results suggest that PHD1 and PHD3 control the transactivation activity of ATF4.