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Sci Rep
2018 Feb 08;81:2683. doi: 10.1038/s41598-018-21040-6.
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Protein interactomes of protein phosphatase 2A B55 regulatory subunits reveal B55-mediated regulation of replication protein A under replication stress.
Wang F
,
Zhu S
,
Fisher LA
,
Wang W
,
Oakley GG
,
Li C
,
Peng A
.
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The specific function of PP2A, a major serine/threonine phosphatase, is mediated by regulatory targeting subunits, such as members of the B55 family. Although implicated in cell division and other pathways, the specific substrates and functions of B55 targeting subunits are largely undefined. In this study we identified over 100 binding proteins of B55α and B55β in Xenopus egg extracts that are involved in metabolism, mitochondria function, molecular trafficking, cell division, cytoskeleton, DNA replication, DNA repair, and cell signaling. Among the B55α and B55β-associated proteins were numerous mitotic regulators, including many substrates of CDK1. Consistently, upregulation of B55α accelerated M-phase exit and inhibited M-phase entry. Moreover, specific substrates of CDK2, including factors of DNA replication and chromatin remodeling were identified within the interactomes of B55α and B55β, suggesting a role for these phosphatase subunits in DNA replication. In particular, we confirmed in human cells that B55α binds RPA and mediates the dephosphorylation of RPA2. The B55-RPA association is disrupted after replication stress, consistent with the induction of RPA2 phosphorylation. Thus, we report here a new mechanism that accounts for both how RPA phosphorylation is modulated by PP2A and how the phosphorylation of RPA2 is abruptly induced after replication stress.
Figure 1. The protein interaction network of B55α. (A) The top 30 most abundant proteins identified as B55α-associated proteins. Recombinant B55α was purified, and incubated in interphase Xenopus egg extracts for 30âmin. B55α-associated protein complexes were then purified and analyzed by mass spectrometry, as described in Materials and Methods. The identified proteins and numbers of peptides are shown. The proteins previously known to bind B55α are shown in red. (B) B55α-associated proteins were classified into various functional groups. (C) Representative B55α-associated proteins involved in distinct cellular pathways are shown.
Figure 2. The protein interaction network of B55β. (A) The top 30 most abundant proteins identified as B55β-associated proteins. B55β-associated protein complexes were purified and analyzed by mass spectrometry, as described in Materials and Methods. The identified proteins and numbers of peptides are shown. The proteins previously known to bind B55β are shown in red. (B) B55β-associated proteins were classified into various functional groups. (C) Representative B55β-associated proteins involved in distinct cellular pathways are shown. (D) The numbers of common and unique binding proteins of B55α and B55β are shown.
Figure 3. Validation of B55α and B55β-associated proteins. B55α or B55β pull-down was performed in Xenopus egg extracts. A control pull-down was performed using the same volume of amylose beads that were not conjugated with proteins. The extract input (approximately 20%), control (ctr) pull-down and B55α or B55β pull-down samples were analyzed by immunoblotting using PP2A, Mcm2, Ube3a, Mastl, Plk1, Incenp, Smc2, and MBP antibodies.
Figure 4. Regulation of the cell cycle by B55α and B55β. (A) Metaphase-arrested Xenopus egg extracts were supplemented with recombinant B55α or control buffer for 10âmin. These extracts were then released into interphase by the addition of Calcium at time 0. The extract samples were collected at the indicated time points and analyzed by immunoblotting. Phosphorylation (band-shift) of Cdc27 and Cdc25, and the global phosphorylation of Cdk substrates are markers of mitosis. (B) Interphase Xenopus egg extracts were supplemented with recombinant B55α or control buffer. The extract samples were collected at the indicated time points and analyzed by immunoblotting. Mitosis is indicated by Cdc27 phosphorylation. (C) MBP-B55α and B55β were purified and examined by Coomassie staining. (D) Interphase Xenopus egg extracts were supplemented with recombinant B55α, B55β, or control buffer. The extract samples were collected at the indicated time points and analyzed by immunoblotting. Mitosis is indicated by Cdc27 phosphorylation.
Figure 5. RPA and B55α association. (A) RPA2 IP was performed in HeLa cell lysates as described in Material and Methods. The input at 10%, control IP (with blank beads), and RPA2 IP were analyzed by immunoblotting for B55α and RPA2. (B) B55α IP was performed in HeLa cell lysates. The input at 10%, control IP (with blank beads), and B55α IP were analyzed by immunoblotting for B55α and RPA2. (C) HA-B55α was expressed in HeLa cells, which were treated with or without HU (1âmM, 24âh). HA IP was performed in the cell lysates. The input at 10%, control IP (with blank beads), and B55α IP were analyzed by immunoblotting for B55α and RPA2. (D) RPA2 IP was performed in the lysates of HeLa cells that were pre-treated with HU (1âmM, 24âh) or mock-treated. The input at 10%, control IP (with blank beads), and B55α IP were analyzed by immunoblotting for B55α and RPA2. (E) The level of B55α in RPA2 IP was examined by immunoblotting and quantified using ImageJ. The mean values and standard deviations were calculated from three independent experiments. Statistical significance was analyzed using an unpaired 2-tailed Studentâs t-test. A p-valueâ<â0.05 was considered statistically significant (*). (F) The expression level of B55α was examined by immunoblotting and quantified using ImageJ. The mean values and standard deviations were calculated from three independent experiments. Statistical significance was analyzed using an unpaired 2-tailed Studentâs t-test. A p-valueâ<â0.05 was considered statistically significant (*).
Figure 6. B55α-mediates RPA2 dephosphorylation. (A) HeLa cells with or without expression of HA-B55α, were treated with HU (1âmM, 24âh) as indicated. The cell lysates were analyzed by immunoblotting for RPA2, B55α, phospho-RPA2 Ser-4/Ser-8, Ser-33, phospho-Chk1 Ser-317, and β-actin. (B) The level of RPA2 S4/8 phosphorylation was examined by immunoblotting, as in panel A, and quantified using ImageJ. The mean values and standard deviations were calculated from three independent experiments. Statistical significance was analyzed using an unpaired 2-tailed Studentâs t-test. A p-valueâ<â0.001 was considered statistically highly significant (**). (C) HeLa cells were treated with B55α expression and HU, as in panel A. Immunofluorescence was performed using phospho-RPA2 Ser-4/Ser-8 antibody. (D) The percentage of cells exhibited positive RPA2 S4/8 phosphorylation was examined by immunofluorescence, as in panel C, and quantified. The mean values and standard deviations were calculated from three independent experiments. Statistical significance was analyzed using an unpaired 2-tailed Studentâs t-test. A p-valueâ<â0.05 was considered statistically significant (*).
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