1. Volume 60, Issue 1, Pages 35-46 (October 2015)
ATR Plays a Direct Antiapoptotic Role at Mitochondria, which Is Regulated by Prolyl Isomerase Pin1 
Benjamin A. Hilton, Zhengke Li, Phillip R. Musich, Hui Wang, Brian M. Cartwright, Moises Serrano, Xiao Zhen Zhou, Kun Ping Lu, Yue Zou 
Molecular Cell 
Volume 60, Issue 1, Pages (October 2015)
DOI: /j.molcel
Copyright © 2015 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2015 60, 35-46DOI: (10.1016/j.molcel.2015.08.008)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1
Cytoplasmic ATR Is Prolyl-Isomerized by Pin1, which Is Inhibited upon UV Irradiation
(A) Treatment with DNA-damaging agents reveals the formation of cytoplasmic ATR-H, which exhibited a slower electrophoretic mobility (top). Analysis of the HaCaT keratinocyte cells shows formation of ATR-H (lower).
(B) ATR-H formation is UV dose and recovery time dependent.
(C) siRNA knockdown of indicated peptidylprolyl isomerases shows that ATR-H formation increases in the cytoplasm of non-irradiated A549 cells only after Pin1 knockdown (lane 7).
(D) Pin1 deficient (Pin1−/−) MEF cytoplasm contains only ATR-H, while the proficient cells (Pin1wt) contain ATR-L under normal conditions and ATR-H following UV exposure (top). MEF Pin1−/− cells transfected with RK5 vector, Pin1WT or inactive Pin1S71D plasmids show significant accumulation of ATR-L only in the Pin1WT (bottom).
(E) In vitro isomerization of purified ATR-H to ATR-L by purified recombinant Pin1. ATR-H phosphorylated by Cdk1 prior to Pin1 addition showed maximum conversion to ATR-L (lane 7).
(F) Two lots (1 and 2) of recombinant ATR, purified from HEK293T cells (right), have the same electrophoretic mobility as endogenous ATR-H from UV-treated HCT116 cells.
(G) Recombinant ATR-H phosphorylated by CDK1 directly interacts with recombinant Pin1.
(H) Pin1 is phosphorylated at S71 in response to UV treatment.
(I) Pin1 phosphorylation in response to UV is inhibited by knockdown of DAPK1 resulting in accumulation of ATR-L.
See also Figures S1 and S2.
Molecular Cell  , 35-46DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions

4. Figure 2 ATR Localizes to Mitochondria in Response to UV Irradiation
(A) Cytosolic ATR accumulates at mitochondria (Mito) as ATR-H in a time-dependent manner.
(B) ATR localizes to the mitochondria by immunofluorescence microscopy following UV irradiation.
(C) Immuno-gold labeling followed by transmission electron microscopy (TEM) examination shows that ATR is dispersed in the cytoplasm (-UV) but localizes to the mitochondria following UV irradiation. Areas in rectangles were magnified to highlight mitochondrial localization of ATR.
(D) Alkali extraction reveals that ATR-H interacts with the outer mitochondrial membrane. Intact mitochondria are represented by “Input.” “Pellet” represents the integral membrane proteins, and “Supnt” represents the soluble protein fraction.
(E) Duolink PLA demonstrates that ATR interacts with proapoptotic protein Bid. Focus stacking reveals that the UV-induced ATR-Bid interaction predominately occurs outside of the nucleus.
Molecular Cell  , 35-46DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions

5. Figure 3
ATR Contains a BH3-like Domain that Is Required for Its Interaction with Mitochondria and Bid
(A) Sequence alignment of BH3 domains in Bcl2 family proteins with ATR protein. In the consensus BH3 domain sequence, Φ represents a hydrophobic residue, Z a hydrophilic residue, D/E an acidic residue, A/G a small residue, K/R a basic residue, and X any residue. Three candidate BH3 domains were observed: ATR(175–187), ATR(462–474), and ATR(2345–2357). Previously identified domains of ATR are shown in the lower bar diagram in relation to the ATR BH3-like domain and the Pin1 isomerization site (Ser428Pro).
(B) Recombinant WT-ATR and ATR with deletion of one of the three BH3-like domains (Δ1, Δ2, Δ3, representing ATR lacking aa175–187, aa462–474, or aa2345–2357, respectively) were expressed in the ATR-H form upon UV irradiation (left). ATRflox/− cells exhibit greatly reduced expression of endogenous ATR (right).
(C) ATR-Bid interaction (PLA) requires BH3-like domain ATR(462–474).
(D) Quantification of cells displaying ATR-Bid interaction in (C); three independent PLA experiments were performed.
(E) The BH3-like domain (aa462–474) is necessary for ATR-H localization to mitochondria.
(F) Pin1 isomerization of ATR reduces the ATR-Bid interaction (PLA), as shown by knockdown with indicated siRNA prior to UV treatment. The graph displays the average ATR-Bid interactions observed per cell from three independent experiments; data are represented as mean ± SD.
See also Figure S6.
Molecular Cell  , 35-46DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions

6. Figure 4
Pin1 Isomerizes ATR at the Phospho-Ser428-Pro Motif Near Its BH3-like Domain for ATR-Bid Interaction
(A) Mutation of ATR at S428 resulted in ATR-H formation in non-UV-treated cells.
(B) Phosphorylation of cytoATR at Ser428 in ATRflox/− cells (top). ATR+/+ isolated from cytoplasm was analyzed to confirm phosphorylation status of Ser428 in UV-treated cells (bottom; note that this non-gradient SDS-PAGE does not separate ATR-H from ATR-L). rATR-H, purified recombinant ATR-H.
(C) Absence of phosphorylation of ATR at S428 is needed for ATR-Bid interaction. Data are represented as mean ± SD.
(D) CytoATR mutated at P429 has the same electrophoretic mobility as the ATR-L following UV treatment.
(E) ATRflox/− cells were transfected with constructs for expression of N-terminal Flag-tagged ATR-WT or mutated ATR-P429A followed by IP using anti-Flag antibodies or C-terminal-specific ATR antibodies.
See also Figures S1 and S5.
Molecular Cell  , 35-46DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions

7. Figure 5
ATR Binding to Bid Inhibits Bax and Bak Recruitment to Mitochondria and Prevents UV-Induced Apoptosis
(A) Lack of ATR in ATRflox/− allows recruitment of Bax and tBid to mitochondria following UV treatment.
(B) ATR inhibits Bax-Bid complex formation. Confocal analysis of PLA foci was performed, and the average interactions between Bax-tBid observed per cell were quantified from three independent experiments.
(C) The presence of ATR-H at mitochondria prevents cytochrome c release. Mitochondria isolated from UV-treated ATR+/+ or ATRflox/− cells were supplemented with tBid to initiate cytochrome c release. Marker is the cytosolic fraction of ATR+/+ cells ± UV.
(D) tBid interacts with ATR-H upon UV exposure of cells. tBid co-IPed with ATR form the cytoplasmic and the mitochondrial fractions of UV-treated A549 cells.
(E and F) Recombinant ATR-H can prevent cytochrome c release. Mitochondria isolated from untreated A549 cells were incubated with recombinant ATR-H and then with increasing amounts of tBid (E). The amount of recombinant ATR-H required to inhibit cytochrome c release was assessed (F). Purified RFC1 was used as a negative control for mitochondria binding.
(G) The BH3-like domain Δ2 (aa462–474) of ATR is required for cell survival against UV. ATRflox/− cells were transfected with plasmid constructs as in Figure 3B before UV exposure and tetramethylrhodamine ethyl ester (TMRE) staining.
(H) Silencing Pin1 in cells increases cell survival after UV exposure likely due to ATR-H accumulation.
Data in (B), (G), and (H) are represented as mean ± SD. See also Figure S3.
Molecular Cell  , 35-46DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions

8. Figure 6
Mitochondrial-Specific ATR Functions Independently of Its Checkpoint Kinase Activity and ATRIP
(A) A lack of ATRIP in the cytoplasmic fraction suggests that ATR kinase is not required for ATR-H formation.
(B) ATRIP is not required for inhibition of Bax translocation by ATR-H. Bax association with isolated mitochondria (left) and the efficiency of the ATR and ATRIP knockdowns in whole cell extracts (WCE) are shown (right).
(C) Confirmation of ATR-H’s lack of kinase activity. In vitro phosphorylation of purified GST-p53 was carried out to assess the checkpoint kinase activity of IPed ATR (entire cytoplasmic ATR-H fraction versus one third of nuclear ATR-L fraction) in the presence of [γ-32P] ATP.
(D) ATR kinase activity is not required to prevent Bax recruitment to mitochondria. Association of Bax with the indicated fractions under given conditions was analyzed. Phosphorylation of p53 on Ser15 (pp53(S15)) and Chk1 on Ser345 (pChk1(S345)) were monitored to confirm the loss of checkpoint kinase activity of ATR (below).
See also Figure S4.
Molecular Cell  , 35-46DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions

9. Figure 7
Proposed Mechanisms of Pin1 Regulation of ATR Isomeric Forms and the Function of ATR at Mitochondria in Response to UV Damage
ATR-H (cis-ATR) formation occurs in the cytoplasm due to Pin1 inactivation by UV-induced phosphorylation at Ser71 by DAPK1 or by genetic deficiency; ATR-H can either localize to the mitochondria, where it interacts with tBid inserted into the outer mitochondrial membrane via its now accessible BH3-like domain, and/or bind to cytosolic tBid before mitochondria localization. The common result is that ATR-H functions as an antiapoptotic protein, preventing further recruitment of Bax to mitochondria and subsequent Bax/Bak activation and thus deterring cytochrome c release and apoptosis.
Molecular Cell  , 35-46DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions