ATR Plays a Direct Antiapoptotic Role at Mitochondria Which Is Regulated by Prolyl Isomerase Pin1

Isomerization of ATR by Prolyl Isomerase Pin1 Is Inhibited by UV

To explore possible cytoplasmic activities of ATR, cells were treated with DNA damaging agents, followed by subcellular fractionation and Western blot analysis (WB). As shown in Figure 1A-top panel, DNA damage (particularly UV damage) induced an electrophoretic mobility shift of cytoplasmic but not nuclear ATR. The induced-cytoplasmic ATR (cytoATR) migrated slower in 3–8% gradient SDS-PAGE than the nuclear or non-induced cytoATR, and is designated as ATR-H relative to the latter (ATR-L). UV-induced ATR-H formation also was observed in keratinocytes (Figure 1A-bottom panel). ATR-H formation was dose- and post-UV recovery time-dependent (Figure 1B). The UV-induced ATR-H was observed in other human cell lines including primary fibroblasts (Figure S1A). The identity of ATR-H was confirmed via complementation of ATR deficient (ATR^flox/−) cells and mass spectrometry analysis of isolated ATR-H from UV-treated cells (Figures S2A and S2B, respectively). Also, cytoplasmic localization of ATR increased from ~26% to ~47% following UV treatment.

These observations implied that a post-translational modification to ATR protein may occur. Although ATR (301 kD) is known to undergo phosphorylation upon DNA damage (Liu et al., 2011; Nam et al., 2011), phosphorylation did not cause the ATR shift (Figure S2C). The same was true for modifications by glycosylation, ubiquitinylation, sumoylation, palmitoylation, prenylation and N-myristoylation (Figure S2C or data not shown). Also, ATR-H is not a biosynthetic precursor to ATR-L as it occurred with translation inhibitors (data not shown). Alternatively, this mobility shift may reflect a backbone conformational change which persists in SDS-denaturing gel electrophoresis. Since isomerization of peptidyl-propyl bonds may confer a backbone conformational change, possible isomerization of ATR was examined. Prolyl isomerases cyclophilin-40 (CyP40), cyclophilin-A (CyPA) and Pin1 are known to be active in the cytoplasm. As shown in Figure 1C, Pin1 depletion resulted in formation of ATR-H even in the absence of UV (comparing ratios of ATR-H to ATR-L in lanes 1 vs. 7), while depletion of Cyp40 or CyPA had little effect (lanes 1 vs. 3 or 5). ATR-H formation in untreated cells transfected with Pin1 siRNA was also tested and confirmed in a keratinocyte cell line (Figure S1D). As expected, the siRNA effects on ATR-H production in the UV-induced cells were minimal as ATR-H already predominated (lane 2). These results suggest that Pin1was necessary for ATR-L formation and that lack of Pin1 led to ATR-H accumulation. Likewise, ATR-H was overwhelmingly found in the cytoplasm of Pin1^−/− mouse embryonic fibroblasts (MEF) even without UV as compared with the predominant ATR-L in Pin1^+/+ MEF (Figure 1D, upper, lanes 3 vs. 1). Interestingly, for nuclear ATR lack of Pin1 resulted in only a very small amount of ATR-H with/without UV (lanes 7 and 8). Moreover, Pin1^−/− MEF were transfected with Pin1-WT, and Pin1-S71D expression vectors (Figure 1D, lower). Since phosphorylation of Pin1 at Ser71 inhibits Pin1 isomerase activity (Lee et al., 2011), the phospho-mimic S71D mutation should make Pin1 catalytically inactive. As expected, little ATR-L was found in the cytoplasm of Pin1^−/− cells expressing Pin1-S71D even without UV, while there was a much higher level of cytoplasmic ATR-L in Pin1^−/− cells expressing Pin1-WT. Together, these data support an essential role of Pin1 in maintaining ATR-L.

For further confirmation, endogenous cytoATR in control and UV-exposed cells was immunoprecipitated (IPed), washed with high-salt buffer, and subjected to an in vitro Pin1 isomerization assay. Since Pin1 recognizes the phosphorylated Ser/Thr-Pro motif of its substrates and Cdk1 was shown to phosphorylate Ser/Thr-Pro motif (Bernis et al., 2007; Holt et al., 2009), Cdk1 was used for in vitro phosphorylation of ATR though it is unknown if this occurs in vivo. In addition, to minimize isomerization of ATR-H back to ATR-L during purification of ATR from cells, no phosphatase inhibitors were used. As shown in Figure 1E, Cdk1 did phosphorylate ATR at Ser428 in vitro and the Cdk1-treated ATR-H was efficiently converted to ATR-L by Pin1 (lane 7). In addition, two lots of recombinant ATR were purified from HEK293 cells transfected with a pcDNA-ATR overexpression construct. As shown in Figure 1F, the purified ATR co-migrated with the ATR-H form, suggesting that ATR-L maintenance requires prolyl isomerase in cells and during protein purification. Also, purified Pin1 binds to the recombinant ATR-H after Cdk1 phosphorylation (Figure 1G). It should be noted that the enzymatic isomerization was carried out at 30°C while the IP at 4°C to facilitate maximum binding while preventing ATR isomerization. Interestingly, UV-irradiation led to phosphorylation of Pin1 at Ser71 in the cytoplasm of cells (Figure 1H), inactivating Pin1 and DAPK1 appears to be the kinase that phosphorylates Pin1 Ser71 upon UV (Figure 1I). Together these data suggest that: (a) UV may dephosphorylate ATR-H at its Pin1-binding site (Figure 1E, ATR-H/ATR-L ratios of lanes 7 vs. 5 and 1); (b) UV inactivation of Pin1 in the cytoplasm due to S71-phosphorylation by DAPK1 led to ATR-H formation (Figure 1H); and (c) active Pin1 and ATR-H phosphorylation at its Pin1-recognition site are required for ATR-L formation (Figure 1E).

ATR Contains a BH3-like Domain and Interacts with Bid at Mitochondria

To investigate the cellular functions of ATR-H, mitochondria were isolated from the cytoplasm of UV-treated cells. Comparison between the two subcellular compartments showed that ATR-H was exclusively associated with mitochondria (Figure 2A) and not free in the cytosol. The ATR-H association with mitochondria also was supported by immunofluorescence microscopic analysis of ATR colocalization with the mitochondria-specific protein MHSP70 (Figure 2B). Furthermore, transmission electron microscopy (TEM) coupled with immunogold labeling was employed to visualize the localization of ATR to the mitochondria in the cytoplasm (Figure 2C), showing that ATR accumulated at mitochondria after UV irradiation. More detailed localization of ATR-H on mitochondria was examined by an alkali extraction assay which separates outer and inner membrane fractions of mitochondria (Bannwarth et al., 2012). ATR-H associated with the outer membrane rather than inner membrane (Figure 2D). This mitochondrial association appears due to a direct interaction between ATR and apoptotic protein Bid as demonstrated in the Duolink in situ proximity ligation assay (PLA) (Figure 2E). Bid plays an important role in facilitating proapoptotic activities (McDonnell et al., 1999; Zinkel et al., 2005). No interaction was found between ATR and the other tested apoptotic proteins. The ATR-H-Bid interaction was further confirmed by the co-immunoprecipitation (co-IP) of ATR-H with Bid (Figure 5D).

Protein sequence alignment of ATR with several Bcl2 family members reveals that ATR contains three potential BH3-like domains (Figure 3A), a hallmark of Bcl2 family proteins (Adams and Cory, 1998; Lessene et al., 2008). Domain-deletion expression constructs were made to examine these potential BH3-like domains in the ATR-Bid interaction. These constructs (ATR-WT, -Δ1 (aa175–187), -Δ2 (aa432–474) or -Δ3 (aa2345–2357)) had similar expression levels in the cytoplasm after transfection into ATR^flox/− cells (Figure 3B). PLA revealed that ATR-Bid interaction (red foci) was normal in ATR-WT, -Δ1, or -Δ3 cells, but was abrogated in ATR-Δ2 cells (Figures 3C and 3D). In support, dramatically much less ATR-H was found in association with mitochondria in ATR-Δ2 cells than in ATR-WT cells (Figure 3E). This indicates that the BH3-like domain (aa462–474) of ATR was required for ATR-Bid interaction. To assess Pin1 effects on the UV-induced ATR-Bid interaction, cells were depleted of Pin1, which significantly enhanced ATR-Bid foci formation even without UV (Figure 3F).

Pin1 Isomerizes ATR at the Ser428-Pro Motif near the BH3-like Domain

To identify the Pin1-isomerization site in ATR we examined four Ser/Thr-Pro sites of ATR with reported/predicted S/T phosphorylation (T²²P, S⁴²⁸P, S¹⁰⁰⁷P and T¹⁹⁸⁹P). Alanine mutations were introduced at these Ser/Thr sites in ATR expression constructs which were transfected into ATR^flox/− cells. ATR-S428A was predominately ATR-H even in the absence of UV, while all others were predominately ATR-L (Figure 4A upper). The residual amount of ATR-L in the S428A sample was likely due to the remaining endogenous wild-type ATR in ATR^flox/− cells (Figure 3B, right panel). As expected, UV treatment generated a significant amount of ATR-H in all transfected cells (Figure 4A lower). Consistently, phosphorylation of cytoATR at Ser428-Pro was attenuated in cells treated with UV (Figure 4B upper). This also was true for the endogenous ATR (Figure 4B lower). These data suggest that besides the Ser71 phosphorylation of Pin1 to inhibit its isomerase activity, dephosphorylation of ATR-Ser428 may serve as a complementary mechanism to suppress Pin1 activity on ATR-H. Moreover, ATR-S428A expression abolished Pin1 activity on ATR, leading to a dramatic increase of ATR (ATR-H)-Bid interaction at mitochondria even in the absence of UV (Figure 4C). Together these data strongly support that Pin1 isomerizes ATR at p-Ser428-Pro429 which converts ATR from ATR-H to ATR-L in unstressed cells.

An interesting question remaining is whether ATR-H is the cis- or the trans-isomer. Of the 20 canonical amino acids, peptide bonds formed between any pair of the 19 non-proline amino acids overwhelmingly adopt the trans conformation, while proline could be either the cis- or the trans-isomer with an equilibrium ratio depending on multiple protein features (Fischer and Schmid, 1990; Hinderaker and Raines, 2003). Thus, to identify the isoforms of ATR-H and ATR-L, a P429A point mutation was introduced in an ATR construct for expression in ATR^flox/− cells. This mutation ensures a trans-isoform for S428-A429 bond. This mutation abolished UV-induced conversion of cytoplasmic ATR-L to ATR-H so that ATR-P429A remained in ATR-L form (Figure 4D last lane) and indicates that ATR-H is a cis-isomer while ATR-L is a trans-isomer. Also interestingly, although the N-terminally Flag-tagged ATR-WT expressed in cells could be IPed with anti-Flag antibody, the expressed Flag-tagged ATR-P429A mutant could not (Figure 4E). In contrast, both ATR-WT and its P429A mutant were efficiently IPed using ATR C-terminal antibodies (Figure 4E), suggesting that the Pin1-induced isomerization at P429 results in a conformational change in the N-terminal region of ATR where the BH3-like domain is located (Figure 3A). It is likely that the structural change makes the N-terminal Flag inaccessible to the antibody, and, thus, probably the BH3-like domain is sequestered as well.

ATR-Bid Binding Blocks Recruitment of Bax to Mitochondria and Prevents UV-Induced Apoptosis

Next, we investigated the effects of ATR-Bid interaction on UV-induced apoptosis. Mitochondria were isolated from UV-irradiated ATR^+/+ and ATR^flox/− cells and post-UV accumulation of proapoptotic protein Bax on mitochondria was analyzed. In Figure 5A, UV-induced Bax accumulation at mitochondria significantly increased in a post-UV time-dependent manner in ATR^flox/− cells while remaining unchanged in ATR^+/+ cells. It is known that Bax binding to tBid (truncated/apoptotically activated Bid) leads to activation of Bax at the mitochondria (Tait and Green, 2010). Once activated, Bax is capable of inducing mitochondrial outer membrane permeablization (MOMP) leading to release of cytochrome c and, thus, apoptosis (Billen et al., 2008; Westphal et al., 2014). Although tBid increasingly accumulated at mitochondria post UV in both ATR^+/+ and ATR^flox/− cells, mitochondrial accumulation of Bax remained unchanged or even decreased in ATR^+/+ cells as compared to the significantly increased accumulation of Bax in ATR^flox/− cells (Figure 5A). Bax is predominately cytosolic under unstressed conditions and upon activation is recruited from the cytoplasmic pool to mitochondria. Since the UV-induced mitochondrial Bax-tBid association dramatically increased in ATR^flox/− cells versus ATR^+/+ cells (Figure 5B), the negative effect of ATR on Bax mitochondrial accumulation is likely because ATR-H-tBid binding may block the Bax-tBid interaction.

Furthermore, an in vitro cytochrome c release assay (Luo et al., 1998) was performed. Mitochondria were isolated from UV-irradiated cells and supplied with purified tBid to induce cytochrome c release. As shown in Figure 5C, significantly more cytochrome c was released from ATR^flox/− than ATR^+/+ cell mitochondria, consistent with the presence or absence of mitochondria-associated ATR-H. Also, more tBid was co-IPed with ATR-H than ATR-L from either a cytoplasmic fraction (including mitochondria) or a mitochondrial-only fraction (Figure 5D). Production of tBid by Bid cleavage under the experimental conditions was confirmed (Figure S3). To further verify ATR’s direct involvement, isolated mitochondria from non-irradiated A549 cells were directly supplied with or without purified ATR-H and titrated with tBid in the cytochrome c release assay. WB analysis shows that the presence of ATR-H significantly reduced the cytochrome c release (Figure 5E). The same was true in an inverse titration study (Figure 5F). Cellular evidence for the direct involvement of ATR in the mitochondria-mediated apoptosis was demonstrated in Figure 5G where deletion of the identified ATR BH3-like domain (aa462–474) significantly reduced the survival relative to ATR-WT cells. In contrast, Pin1 depletion led to a substantial increase in survival of UV-treated cells (Figure 5H), fully consistent with the proapoptotic effect of Pin1 on the UV-induced cell death reported previously (Zacchi et al., 2002). It is worth noting that Pin1 can be either proapoptotic or antiapoptotic depending on its target proteins and the biological pathways it regulates (Sorrentino et al., 2014).

ATR Mitochondrial Function is Independent of its Checkpoint Kinase Activity

Since ATR forms a complex with ATRIP in the nucleus, it is of interest to examine whether ATRIP is involved in the mitochondrial activity of ATR. Surprisingly, little to no ATRIP was found in the cytoplasm as compared to ATRIP in the nucleus (Figure 6A). Consistently, ATRIP, but not ATR, depletion had no effect on the UV-induced mitochondrial accumulation of Bax (Figure 6B). Given that ATRIP plays an essential role in ATR checkpoint activation, we examined the checkpoint kinase activity of cytoATR. ATR was IPed from cytoplasmic and nuclear fractions of ±UV-treated cells, and subjected to an in vitro kinase assay for ³²P-labeling of p53 by ATR. While p53 was efficiently phosphorylated by nuclear ATR, cytoATR failed to label p53 with ³²P (Figure 6C), likely due to the lack of ATRIP. Consistently, ATR kinase inhibition by NU6027 (ATR-KI), but not ATR depletion, failed to promote the UV-induced Bax accumulation at mitochondria in ATR^+/+ cells (Figure 6D). Further confirmation came from the UV-induced ATR-H formation in the cytoplasm of ATR^flox/− cells expressing ATR-WT or ATR-KD (kinase-dead) constructs (Figure S4). It is worth mentioning that the proapoptotic effect of ATR depletion has been shown previously to be independent of p53 (Schoppy et al., 2012). Together our data suggest that the mitochondrial role of ATR may not depend on its checkpoint kinase activity.

Cell Culture, UV Irradiation, Treatments and Antibodies

UV irradiation employed a 254-nm lamp at a flounce of 0.83 J/m²/sec. All UV treatments unless otherwise noted are 40 J/m² followed by a 2 hr recovery. See Supplemental Experimental Procedures for details.

Cell lysis and Immunoblotting

Cells were harvested by scraping or trypsinization, and suspended in lysis buffer (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1x protease/phosphatase inhibitor cocktail (Thermo, 1861280)). 2x SDS loading buffer was added to the lysates and the mixtures were heated at 95°C for 5 min. Gradient gels of 3–8% Tris-Acetate (TA) SDS-PAGE (Invitrogen, EA0378) were used for maximum resolution of the slower migrating cytoplasmic ATR (ATR-H) band. For display of total ATR proteins standard SDS-PAGE was used. Proteins were transferred from the gels onto PVDF membranes (Millipore, IPVH00010). Chemiluminescent signal was captured using the Fuji Film imager LAS-4000.

RNAi and Plasmid Transfections

The siRNA transfection reagent, Interferin, was purchased from Polyplus and the transfections were performed according to the manufacturer’s instructions. Transfection of plasmids into all cell lines employed jetPEI transfection reagent (Polyplus, 101-10). Site-directed mutagenesis and deletion generation were performed using the In-Fusion HD cloning kit (Clontech, 638909). See the Supplemental Experimental Procedures for details.

Cytoplasmic and Nuclear Protein Extraction

Subcellular fractionation was performed using cytoplasm lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 3 mM CaCl₂, 1.5 mM MgCl₂, 0.34 M Sucrose, 10% Glycerol, 0.1% Triton X-100, protease and phosphotase Inhibitors), and nuclei lysis buffer (50 mM Tris-HCl, pH 7.9, 140 mM NaCl, 3 mM CaCl₂, 1 mM EDTA, 1% NP-40, 10% Glycerol, protease and phosphotase Inhibitors). Briefly, 10 volumes of cell lysis buffer with 1x protease and phosphatase inhibitors cocktail were added to 1 volume of packed cells. After resuspension and incubation on ice for 10 min, cytoplasm was separated from nuclei at 500xg for 7 min at 4°C. Isolated nuclei were washed twice with 500 μL of the nuclear wash buffer, collected by centrifugation. Collected nuclear pellets were suspended in ice-cold nuclear storage buffer, and 1/10 volume of the nuclear lysis reagent was added to lyse nuclei with rotation for 15 min at 4°C. The nuclear lysate was collected by centrifugation at 20,000xg for 15 min at 4°C. In all of the fractionation experiments, WB of β-actin and PARP were assessed to check the quality of fractionation and protein loading.

Alkali Extraction

Isolated mitochondria were treated with 0.1 M Na₂CO₃ (pH 11.5) for 30 min on ice before centrifugation at 16,000xg for 15 min at 4°C. The supernatant containing peripheral membrane proteins was collected. The pellet containing mitochondrial inner membrane proteins was washed once and suspended in 250 mM sucrose, 1 mM EDTA, 20 mM HEPES-NaOH (pH 7.4). Input refers to untreated control mitochondria. Equal proportions were analyzed by WB.

Purification of Recombinant ATR and Pin1 Proteins

Flag-tagged ATR recombinant protein was isolated from HEK 293T cells 48 hrs after transfection with pcDNA3-Flag-ATR plasmid. Briefly, expressed ATR was IPed with monoclonal mouse-anti-FLAG M2 antibody (Sigma) and captured with Protein-G plus agarose beads (Pierce). For purification of Pin1, RK plasmid containing a GST-tagged Pin1 construct was expressed in E.coli (BL21(DE3)). GST-Pin1 protein was captured using glutathione-agarose beads (Sigma).

Mitochondria Isolation and Cytochrome c Release Assay

Mitochondrial isolation was performed using several mitochondrial extraction kits (Qiagen and Thermo) according to the manufacturer’s instructions. Mitochondria also were isolated according to Frezza et al. (Frezza et al., 2007). The cytochrome c assay was performed by subjecting isolated mitochondria to incubation with recombinant tBid (R&D Systems, 883-M8) according to Luo et al. (1998).

In vitro Isomerization and Immunoprecipitation Assays

The cytoplasm of A549 cells was isolated after UV treatment. ATR antibody (Bethyl Labs) was added for pull-down overnight. IPed ATR protein was subjected to a 0.6 M NaCl wash to remove associated proteins and suspended in kinase buffer (New England Biolabs). Cdk1-Cyclin B (New England Biolabs, P6020S) was added and incubated for 1 hr at 30°C to phosphorylate the isolated IPed ATR. After centrifugation and washing of the bead-bound ATR purified Pin1 was added to the phosphorylated ATR and incubated for 1 hr at 30 °C. SDS-loading buffer was added and ATR isomerization assayed by 3–8% TA SDS-PAGE and WB. Alternatively, to measure Pin1 binding to phosphorylated ATR the IPed ATR was incubated first with Cdk1 for 1 hr at 30°C, washed thrice, and purified Pin1 added for a 2 hr incubation at 4°C. The ATR-beads were washed three times and suspended in SDS loading buffer for WB analysis. To assess tBid binding to ATR-H following UV treatment of A549 cells, the cytoplasmic fraction or isolated mitochondria was collected. ATR antibody (Bethyl Labs) was added for pull-down overnight. IPed ATR-beads were washed three washes in Co-IP wash buffer (50 mM Tris-HCl, pH 7.6, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.2% Tween-20). The beads were suspended in 1x SDS loading buffer, boiled at 95°C for 5 min and analyzed by WB.

Duolink – in situ Proximity Ligation Assays

The Duolink assay was carried out according to the manufacturer (Sigma, DUO92101). Images were taken using a Life Technologies Evos microscope or a Leica confocal microscope.

Immunogold Labeling and TEM

A549 cells were treated with 40 J/m² UV with a 2 hr recovery. Cells scraped off the surface were collected by centrifugation and rinsed in PBS and fixed in 2% paraformaldehyde/2.5% glutaraldehyde in 0.2 M cacodylate. Fixed samples were dehydrated through alcohol and agar-enrobed before embedding in Lowicryl resin (Lowicryl K4M Embedding Kit, Electron Microscopy Sciences EMS). The resin was polymerized by multiday illumination with ultraviolet light before cutting ultrathin (60 nm) sections which were placed on gold glider grids (EMS, #G200-Au). Sections were blocked in 5% normal donkey serum to prevent nonspecific labeling, rinsed with 0.1 M Sorenson’s buffer before incubation with primary ATR antibody (Bethyl, A300-138A) for two hours at room temperature. Incubation with anti-rabbit secondary antibody conjugated to 20 nm gold particles (Cytodiagnostics) was carried out for two hours at room temperature. Sections were rinsed and imaged by a Philips Tecra 10 transmission electron microscope (FEI Company).

Mitochondrial Membrane Potential Assays

TMRE staining (ab113852) was carried out according to the manufacturer. Samples were analyzed in a BD Accuri C6 flow cytometer.

In vitro Kinase Activity Assay

The in vitro kinase assay was performed on endogenous ATR that was IPed from cytoplasmic or nuclear extracts of A549 cells after UV irradiation. The IPed ATR was washed three times with PBS containing 0.05% NP40, followed by a kinase buffer wash [50 mM HEPES (pH 7.5), 150 mM NaCl, 10 mM MgCl2, 10 mM MnCl2, 2 mM DTT, 10% glycerol, 1x protease and phosphatase inhibitors, and 0.5 mM ATP]. The IPed ATR was suspended in 20 μL of kinase buffer containing 10 μCi of [γ-32P] ATP and 0.5 μg of GST-p53 protein (Signal Chem, P05-30BG). The reaction was incubated at 30°C for 30 minutes and stopped by the addition of SDS loading buffer. Proteins were separated by SDS-PAGE and the radiolabeled proteins were visualized by gel scanning using a PhosphorImage scanner (Fuji, Stamford, CT). IPed endogenous ATR and the amount of GST-p53 in each sample were confirmed by WB.

Statistical Analysis

The statistical analysis of samples was performed with a two- tailed student’s t-test, and a p-value of less than 0.05 was considered as significant.