Journal: Frontiers in Cell and Developmental Biology

Article Title: PP2A Regulates Phosphorylation-Dependent Isomerization of Cytoplasmic and Mitochondrial-Associated ATR by Pin1 in DNA Damage Responses

doi: 10.3389/fcell.2020.00813

Figure Lengend Snippet: Protein phosphatase 2A (PP2A) specifically dephosphorylates cytoplasmic ATR at S428 and its action involves direct binding to ATR. (A) A549 cells were treated with siRNAs against three protein phosphatases (PP2Ac, PP4c, and PP5c) followed by UV treatment at 40 J/m 2 with a 2 h recovery. Analysis of cytoplasmic extracts reveals that PP2A is required to dephosphorylate cytoplasmic pATR (S428). (B) siRNA knockdown of PP2Ac in A549 cells was followed by UV treatment at 40 J/m 2 with a 2 h recovery. pATR (S428) levels are increased in cytoplasm with PP2Ac knockdown while the phosphorylation status of ATR in the nucleus remains unchanged. WB of whole cell extracts shows PP2A siRNA knockdown efficiency in A549 cells (C) HCT 116 ATR flox/– cells were transfected with N-terminal Flag-tagged wild-type (WT) or S428A mutant ATR expression constructs and UV treated at 40 J/m 2 with a 2 h recovery. Analysis of the cytoplasmic extracts reveals that PP2A specifically targets the pATR (S428) residue. The pATR (S428) observed in the S428A cells reflects some phosphorylation of the residual endogenous ATR remaining in the ATR flox/– cells (right panel). (D) PLA revealing that in A549 cells there is a direct interaction between ATR and PP2A, especially after UV irradiation at 40 J/m 2 . A DAPI-staining overlay is used to show location of the nuclei. The bar graph represents a statistical analysis of the PLA images. (E) A549 cells treated with UV at 40 J/m 2 were lysed, followed by co-immunoprecipitation of PP2A using anti-ATR antibody. *Stands for p value < 0.01.

Article Snippet: Antibodies and their dilutions for western blots include pATR S428 (Cell Signaling 2853) 1:1000, PP2Ac (Cell Signaling 2038) 1:1000, GAPDH (Cell Signaling 5174) 1:1000, PARP 1 (Cell Signaling 9532) 1:1000, Bid (Cell Signaling 2002) 1:1000, or (Santa Cruz Biotechnology sc6538) 1: 500, Beta Actin (Invitrogen MA1-140) 1:5000, mtHSP70 (Invitrogen MA3-028) 1:1000, ATR (Bethyl Laboratories, Inc., A300-137A, A300-138A) 1:8000, ATRIP (ABclonal A7139) 1:1000, PP2A-Cα/β (Santa Cruz Biotechnology sc-80665) 1:500, Cleaved Caspase-3 (Cell Signaling 9664) 1:1000, PP4c (Bethyl Laboratories, Inc., A300-835A) 1:8000, PP5c (Bethyl Laboratories, Inc., A300-909A) 1:8000.

Techniques: Binding Assay, Transfection, Mutagenesis, Expressing, Construct, Irradiation, Staining, Immunoprecipitation

Journal: Frontiers in Cell and Developmental Biology

Article Title: PP2A Regulates Phosphorylation-Dependent Isomerization of Cytoplasmic and Mitochondrial-Associated ATR by Pin1 in DNA Damage Responses

doi: 10.3389/fcell.2020.00813

Figure Lengend Snippet: Protein phosphatase 2A (PP2A) specifically dephosphorylates cytoplasmic ATR at S428 and its action involves direct binding to ATR. (A) A549 cells were treated with siRNAs against three protein phosphatases (PP2Ac, PP4c, and PP5c) followed by UV treatment at 40 J/m 2 with a 2 h recovery. Analysis of cytoplasmic extracts reveals that PP2A is required to dephosphorylate cytoplasmic pATR (S428). (B) siRNA knockdown of PP2Ac in A549 cells was followed by UV treatment at 40 J/m 2 with a 2 h recovery. pATR (S428) levels are increased in cytoplasm with PP2Ac knockdown while the phosphorylation status of ATR in the nucleus remains unchanged. WB of whole cell extracts shows PP2A siRNA knockdown efficiency in A549 cells (C) HCT 116 ATR flox/– cells were transfected with N-terminal Flag-tagged wild-type (WT) or S428A mutant ATR expression constructs and UV treated at 40 J/m 2 with a 2 h recovery. Analysis of the cytoplasmic extracts reveals that PP2A specifically targets the pATR (S428) residue. The pATR (S428) observed in the S428A cells reflects some phosphorylation of the residual endogenous ATR remaining in the ATR flox/– cells (right panel). (D) PLA revealing that in A549 cells there is a direct interaction between ATR and PP2A, especially after UV irradiation at 40 J/m 2 . A DAPI-staining overlay is used to show location of the nuclei. The bar graph represents a statistical analysis of the PLA images. (E) A549 cells treated with UV at 40 J/m 2 were lysed, followed by co-immunoprecipitation of PP2A using anti-ATR antibody. *Stands for p value < 0.01.

Article Snippet: Antibodies and their dilutions for western blots include pATR S428 (Cell Signaling 2853) 1:1000, PP2Ac (Cell Signaling 2038) 1:1000, GAPDH (Cell Signaling 5174) 1:1000, PARP 1 (Cell Signaling 9532) 1:1000, Bid (Cell Signaling 2002) 1:1000, or (Santa Cruz Biotechnology sc6538) 1: 500, Beta Actin (Invitrogen MA1-140) 1:5000, mtHSP70 (Invitrogen MA3-028) 1:1000, ATR (Bethyl Laboratories, Inc., A300-137A, A300-138A) 1:8000, ATRIP (ABclonal A7139) 1:1000, PP2A-Cα/β (Santa Cruz Biotechnology sc-80665) 1:500, Cleaved Caspase-3 (Cell Signaling 9664) 1:1000, PP4c (Bethyl Laboratories, Inc., A300-835A) 1:8000, PP5c (Bethyl Laboratories, Inc., A300-909A) 1:8000.

Techniques: Binding Assay, Knockdown, Phospho-proteomics, Transfection, Mutagenesis, Expressing, Construct, Residue, Irradiation, Staining, Immunoprecipitation

Journal: Frontiers in Cell and Developmental Biology

Article Title: PP2A Regulates Phosphorylation-Dependent Isomerization of Cytoplasmic and Mitochondrial-Associated ATR by Pin1 in DNA Damage Responses

doi: 10.3389/fcell.2020.00813

Figure Lengend Snippet: Protein phosphatase 2A (PP2A) dephosphorylates cytoplasmic pATR (S428) in a UV dose- and recovery time-dependent manner and is independent of ATRIP. (A) Cytoplasmic pATR (S428) dephosphorylation by PP2A is dependent on post-irradiation recovery time in A549 cells. The WB shows that the level of ATR phosphorylation at S428 depends on the recovery time following UV irradiation at 40 J/m 2 . PP2A inhibitor treatment (PP2Ai) abolishes this dephosphorylation at all recovery times. (B) Cytoplasmic pATR (S428) dephosphorylation by PP2A is UV dose dependent and is attenuated with PP2A inhibitor treatment of A549 cells. Control A549 cells show a reduction in the level of pATR (S428) that is dependent on the dose of UV irradiation used, but retain their level of pATR (S428). (C) A549 cells, depleted of ATRIP by siRNA knockdown, were divided into two groups with one treated with PP2A inhibitor and the other left as a control. WB assay shows that this ATRIP knockdown does not affect PP2A’s dephosphorylation of pATR (S428) in the cytoplasm of A549 cells. The right panel shows the efficiency of the ATRIP knockdown.

Article Snippet: Antibodies and their dilutions for western blots include pATR S428 (Cell Signaling 2853) 1:1000, PP2Ac (Cell Signaling 2038) 1:1000, GAPDH (Cell Signaling 5174) 1:1000, PARP 1 (Cell Signaling 9532) 1:1000, Bid (Cell Signaling 2002) 1:1000, or (Santa Cruz Biotechnology sc6538) 1: 500, Beta Actin (Invitrogen MA1-140) 1:5000, mtHSP70 (Invitrogen MA3-028) 1:1000, ATR (Bethyl Laboratories, Inc., A300-137A, A300-138A) 1:8000, ATRIP (ABclonal A7139) 1:1000, PP2A-Cα/β (Santa Cruz Biotechnology sc-80665) 1:500, Cleaved Caspase-3 (Cell Signaling 9664) 1:1000, PP4c (Bethyl Laboratories, Inc., A300-835A) 1:8000, PP5c (Bethyl Laboratories, Inc., A300-909A) 1:8000.

Techniques: De-Phosphorylation Assay, Irradiation, Phospho-proteomics, Control, Knockdown

Journal: Frontiers in Cell and Developmental Biology

Article Title: PP2A Regulates Phosphorylation-Dependent Isomerization of Cytoplasmic and Mitochondrial-Associated ATR by Pin1 in DNA Damage Responses

doi: 10.3389/fcell.2020.00813

Figure Lengend Snippet: Protein phosphatase 2A (PP2A) dephosphorylation of pATR changes ATR to the higher form (ATR-H). (A) UV irradiation of A549 cells at 40 J/m 2 increases the formation of ATR-H in the cytoplasm. Also, the ATR-H is not phosphorylated while the ATR-L band is phosphorylated in both the nucleus and the cytoplasm (cytoplasm to nucleus loading ratio = 3:1). The samples were analyzed on a 3–8% gradient gel. (B) A549 cells were pretreated with DMSO (Ctrl) or transfected with PP2A siRNAs, then UV-irradiated at 40 J/m 2 (–/+UV) followed by a 2 h recovery. The cytoplasmic fraction was analyzed by electrophoresis through a 3–8% gradient gel and WB to show the form of ATR in the cytoplasm. PP2A depletion reduces the levels of cytoplasmic ATR and the amount of ATR-H formed following UV treatment. (C) WB showing PP2A siRNA knockdown efficiency in A549 cells.

Article Snippet: Antibodies and their dilutions for western blots include pATR S428 (Cell Signaling 2853) 1:1000, PP2Ac (Cell Signaling 2038) 1:1000, GAPDH (Cell Signaling 5174) 1:1000, PARP 1 (Cell Signaling 9532) 1:1000, Bid (Cell Signaling 2002) 1:1000, or (Santa Cruz Biotechnology sc6538) 1: 500, Beta Actin (Invitrogen MA1-140) 1:5000, mtHSP70 (Invitrogen MA3-028) 1:1000, ATR (Bethyl Laboratories, Inc., A300-137A, A300-138A) 1:8000, ATRIP (ABclonal A7139) 1:1000, PP2A-Cα/β (Santa Cruz Biotechnology sc-80665) 1:500, Cleaved Caspase-3 (Cell Signaling 9664) 1:1000, PP4c (Bethyl Laboratories, Inc., A300-835A) 1:8000, PP5c (Bethyl Laboratories, Inc., A300-909A) 1:8000.

Techniques: De-Phosphorylation Assay, Irradiation, Transfection, Electrophoresis, Knockdown

Journal: Frontiers in Cell and Developmental Biology

Article Title: PP2A Regulates Phosphorylation-Dependent Isomerization of Cytoplasmic and Mitochondrial-Associated ATR by Pin1 in DNA Damage Responses

doi: 10.3389/fcell.2020.00813

Figure Lengend Snippet: Inhibition of PP2A’s dephosphorylation of pATR and ATR-L accumulation in the cytoplasm leads to reduced association of ATR with tBid on mitochondria. (A) PLA shows that cytoplasmic ATR directly associates with Bid and that this association increases with UV treatment, but the UV-induced association is attenuated by PP2A inhibitor treatment. The nuclei are stained with DAPI and mitochondria are indicated by mtHSP70 immunofluorescence. A549 cells were UV irradiated at 40 J/m 2 with a 2 h recovery. (B) A graphic display of the PLA data shown in (A) . The p < 0.05 refers to the sample (+UV, −PP2Ai) versus any of other samples. (C) ATR-H accumulation at the mitochondria increases with UV treatment at 40 J/m 2 in A549 cells, but is reduced in cells treated with PP2A inhibitor. (D) Biochemical confirmation of the reduction in the cytoplasmic ATR:tBid association observed by PLA (A,B) . A549 cells were pretreated with PP2Ai followed by UV irradiation with a 2 h recovery. After cellular fractionation ATR was immunoprecipitated from the cytoplasmic fraction using C-terminal-specific ATR antibody. The association of tBid with ATR was confirmed by WB.

Article Snippet: Antibodies and their dilutions for western blots include pATR S428 (Cell Signaling 2853) 1:1000, PP2Ac (Cell Signaling 2038) 1:1000, GAPDH (Cell Signaling 5174) 1:1000, PARP 1 (Cell Signaling 9532) 1:1000, Bid (Cell Signaling 2002) 1:1000, or (Santa Cruz Biotechnology sc6538) 1: 500, Beta Actin (Invitrogen MA1-140) 1:5000, mtHSP70 (Invitrogen MA3-028) 1:1000, ATR (Bethyl Laboratories, Inc., A300-137A, A300-138A) 1:8000, ATRIP (ABclonal A7139) 1:1000, PP2A-Cα/β (Santa Cruz Biotechnology sc-80665) 1:500, Cleaved Caspase-3 (Cell Signaling 9664) 1:1000, PP4c (Bethyl Laboratories, Inc., A300-835A) 1:8000, PP5c (Bethyl Laboratories, Inc., A300-909A) 1:8000.

Techniques: Inhibition, De-Phosphorylation Assay, Staining, Immunofluorescence, Irradiation, Cell Fractionation, Immunoprecipitation