ATR Isomerization in Cellular Responses to UV Damage of DNA

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Summary DNA damage is a major cause of human cancers and many other human diseases. In response to DNA damage, cells activate DNA damage response (DDR) pathways such as DNA damage checkpoints, DNA repair, and apoptosis. ATR (ataxia telangiectasia and Rad3-related), a member of the phosphoinositide 3-kinase-related protein kinases (PIKK) family, is a major DNA damage checkpoint protein kinase which plays a critical role in DDR by signaling DNA damage, activating checkpoints, arresting cell cycle progression and facilitating DNA repair to restore DNA integrity. Interestingly, a body of evidence from mouse model and human epidemiologic studies shows that unlike ATM, a closely related ATR-like PIKK family member whose deficiency promotes carcinogenesis, ATR inhibition suppresses carcinogenesis. Moreover, ATR knockout is embryonically lethal. These suggest an involvement of ATR in regulating cell death. Although ATR has been extensively studied as a checkpoint kinase in DDR in the nucleus, little is known about its functions in the cytoplasm or mitochondria, the cellular organelle for activating DNA damage-induced apoptosis. A recent finding from the P.I.’s lab reveals that (a) besides its hallmark nuclear checkpoint functions, ATR is a pro-survival protein functioning directly at mitochondria against UV damage; (b) ATR contains a BH3-like domain that allows ATR to act like a Bcl-2 family protein; (c) importantly, mitochondrial ATR is a prolyl cis-isomeric form of ATR regulated by Pin1 while in contrast, nuclear ATR is a trans-isomeric form of ATR; and finally (d) mitochondria activity of ATR is independent of its checkpoint kinase activity and ATRIP. In this project, we will test the hypotheses that (1) Prolyl isomerization alters the structure of ATR, transforming ATR functions for mitochondria-specific activities to promote cell survival or nuclear functions as a DNA damage checkpoint regulator; (2) Antiapoptotic activity of ATR at mitochondria plays an important role in mediating carcinogenesis in vivo, and thus, suppressing such activity may reduce carcinogenesis/tumorigenesis and provide a strategy for cancer prevention and treatment; and (3) ATR’s trans-isomeric form is required for its DNA damage checkpoint activity, and post- translational modifications of ATR and/or ATR-ATRIP complex formation may play a role in stabilizing ATR in the trans-isomeric form in the nucleus. These hypotheses will be tested in the following specific aims. Aim 1: To define the structure-function relationships of ATR prolyl isomers, and of ATRH-tBid and ATRH- Pin1 interactions; Aim 2: To determine the role of prolyl isomerization in the nuclear functions of ATR; and Aim 3: To determine the in vivo effects of ATR isomers on carcinogenesis and tumorigenesis. The proposed studies represent an innovative effort highly relevant to cancer biology and also having implications in other human diseases such as neurodegenerative and cardiovascular diseases.