Checkpoint-dependent phosphorylation of Med1/TRAP220 in response to DNA damage

Mediator complex subunit 1 (Med1)/Thyroid hormone receptor-associated protein 220 (TRAP220), an essential component of thyroid hormone receptor-associated proteins (TRAP)/mediator, plays important roles in hormone responses and tumorigenesis. However, the role of Med1 in the DNA damage response has not been studied. In this study, we found that DNA damage, resulted from γ-irradiation, ultraviolet (UV)-irradiation, or hydroxyurea, induced phosphorylation of Med1 in vivo. Phosphorylation of Med1 was abrogated by either caffeine or wortmannin treatment, suggesting that Med1 is phosphorylated through the DNA damage checkpoint pathway. A checkpoint kinase 1 (Chk1)/checkpoint kinase 2 (Chk2) consensus phosphorylation motif was identified at Serine 671 of Med1 and Ser671 motif was primarily phosphorylated by Chk2 in vitro. Moreover, the in vivo phosphorylation of Med1 was abrogated by a Chk2 inhibitor, and physical interaction between Chk2 and Med1 was observed, confirming that Chk2 is responsible for Med1 phosphorylation upon DNA damage. These results suggest that Med1 is a novel target for the DNA damage checkpoint pathway and may participate in the DNA damage response. Consistent with this notion, knockdown of Med1 expression caused a significant increase in cellular sensitivity to UV irradiation. Moreover, microarray analysis revealed that the UV-induced activation of the transcription of important regulators of cell cycle control and DNA repair, including p21, Gadd45, Rad50, DnaJ, and RecQL, was impaired upon Med1 knockdown. Taken together, our data suggest that Med1 is a novel target for Chk2-mediated phosphorylation and may play a role in cellular DNA damage responses by mediating proper induction of gene transcription upon DNA damage.