AMP-activated protein kinase is involved in the activation of the Fanconi anemia/BRCA pathway in response to DNA interstrand crosslinks

// Min Jeong Chun 1, * , Sunshin Kim 2, * , Soo Kyung Hwang 1 , Bong Sub Kim 1 , Hyoun Geun Kim 1 , Hae In Choi 1 , Jong Heon Kim 1 , Sung Ho Goh 2 , Chang-Hun Lee 1 1 Cancer Cell and Molecular Biology Branch, Research Institute, National Cancer Center, Ilsandong-gu, Goyang, Gyeonggi, 10408, Korea 2 Precision Medicine Branch, Research Institute, National Cancer Center, Ilsandong-gu, Goyang, Gyeonggi, 10408, Korea * These authors have contributed equally to this work and considered as co-first authors Correspondence to: Chang-Hun Lee, email: chlee@ncc.re.kr Keywords: Fanconi anemia (FA)/BRCA pathway, AMP-activated protein kinase, DNA interstrand crosslinks, phosphorylation, FANCA Received: March 10, 2016      Accepted: July 7, 2016      Published: July 18, 2016 ABSTRACT Fanconi anemia complementation group (FANC) proteins constitute the Fanconi Anemia (FA)/BRCA pathway that is activated in response to DNA interstrand crosslinks (ICLs). We previously performed yeast two-hybrid screening to identify novel FANC-interacting proteins and discovered that the alpha subunit of AMP-activated protein kinase (AMPKα1) was a candidate binding partner of the FANCG protein, which is a component of the FA nuclear core complex. We confirmed the interaction between AMPKα and both FANCG using co-immunoprecipitation experiments. Additionally, we showed that AMPKα interacted with FANCA, another component of the FA nuclear core complex. AMPKα knockdown in U2OS cells decreased FANCD2 monoubiquitination and nuclear foci formation upon mitomycin C-induced ICLs. Furthermore, AMPKα knockdown enhanced cellular sensitivity to MMC. MMC treatment resulted in an increase in AMPKα phosphorylation/activation, indicating AMPK is involved in the cellular response to ICLs. FANCA was phosphorylated by AMPK at S347 and phosphorylation increased with MMC treatment. MMC-induced FANCD2 monoubiquitination and nuclear foci formation were compromised in a U2OS cell line that stably overexpressed the S347A mutant form of FANCA compared to wild-type FANCA-overexpressing cells, indicating a requirement for FANCA phosphorylation at S347 for proper activation of the FA/BRCA pathway. Our data suggest AMPK is involved in the activation of the FA/BRCA pathway.