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Checkpoint-dependent phosphorylation of Med1/TRAP220 in response to DNA damage
Hyun-Ju Kim1,2, Jeanho Yun1,2
1Peripheral Neuropathy Research Center, College of Medicine, Dong-A University, Busan 49201, Korea.
Abstract:
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.
Insights
Mediator complex subunit 1 (Med1) is phosphorylated by Chk2 during DNA damage. Med1 knockdown increases UV sensitivity and impairs DNA repair gene transcription, revealing its role in the DNA damage response.
Area of Science:
- Molecular Biology
- Cellular Biology
- Biochemistry
Background:
- Mediator complex subunit 1 (Med1), also known as Thyroid hormone receptor-associated protein 220 (TRAP220), is crucial for hormone responses and tumorigenesis.
- The involvement of Med1 in the DNA damage response (DDR) remains largely uncharacterized.
Purpose of the Study:
- To investigate the role of Med1 in the DNA damage response.
- To identify the specific kinases involved in Med1 phosphorylation upon DNA damage.
- To elucidate the functional consequences of Med1 phosphorylation in cellular response to DNA damage.
Main Methods:
- Induction of DNA damage using gamma-irradiation, UV-irradiation, and hydroxyurea.
- Analysis of Med1 phosphorylation using in vivo and in vitro assays.
- Inhibition of checkpoint kinases (Chk1/Chk2) using specific inhibitors and caffeine/wortmannin.
- Identification of phosphorylation sites using sequence analysis.
- Assessment of cellular sensitivity to UV irradiation following Med1 knockdown.
- Microarray analysis to evaluate gene expression changes in response to Med1 knockdown.
Main Results:
- DNA damage induced Med1 phosphorylation in vivo, which was dependent on the DNA damage checkpoint pathway.
- Checkpoint kinase 2 (Chk2) was identified as the primary kinase responsible for phosphorylating Med1 at Serine 671.
- Physical interaction between Chk2 and Med1 was confirmed.
- Med1 knockdown led to increased sensitivity to UV irradiation.
- Med1 knockdown impaired the UV-induced transcription of key DNA repair and cell cycle regulators (e.g., p21, Gadd45).
Conclusions:
- Med1 is a novel target of Chk2-mediated phosphorylation within the DNA damage response pathway.
- Med1 plays a significant role in cellular responses to DNA damage, potentially by regulating the transcription of repair and cell cycle genes.
- These findings highlight Med1 as a potential therapeutic target in cancer treatment related to DNA damage repair mechanisms.
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