Protein phosphatase 1 and phosphatase 1 nuclear targeting subunit-dependent regulation of DNA-dependent protein kinase and non-homologous end joining.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a key role in mediating non-homologous end joining (NHEJ), a major repair pathway for DNA double-strand breaks (DSBs). The activation, function and dynamics of DNA-PKcs is regulated largely by its reversible phosphorylation at numerous residues, many of which are targeted by DNA-PKcs itself. Interestingly, these DNA-PKcs phosphorylation sites function in a distinct, and sometimes opposing manner, suggesting that they are differentially regulated via complex actions of both kinases and phosphatases. In this study we identified several phosphatase subunits as potential DSB-associated proteins. In particular, protein phosphatase 1 (PP1) is recruited to a DSB-mimicking substrate in Xenopus egg extracts and sites of laser microirradiation in human cells. Depletion of PP1 impairs NHEJ in both Xenopus egg extracts and human cells. PP1 binds multiple motifs of DNA-PKcs, regulates DNA-PKcs phosphorylation, and is required for DNA-PKcs activation after DNA damage. Interestingly, phosphatase 1 nuclear targeting subunit (PNUTS), an inhibitory regulator of PP1, is also recruited to DNA damage sites to promote NHEJ. PNUTS associates with the DNA-PK complex and is required for DNA-PKcs phosphorylation at Ser-2056 and Thr-2609. Thus, PNUTS and PP1 together fine-tune the dynamic phosphorylation of DNA-PKcs after DNA damage to mediate NHEJ.

Cell cycle-dependent regulation of Greatwall kinase by protein phosphatase 1 and regulatory subunit 3B.

Greatwall (Gwl) kinase plays an essential role in the regulation of mitotic entry and progression. Mitotic activation of Gwl requires both cyclin-dependent kinase 1 (CDK1)-dependent phosphorylation and its autophosphorylation at an evolutionarily conserved serine residue near the carboxyl terminus (Ser-883 in Xenopus). In this study we show that Gwl associates with protein phosphatase 1 (PP1), particularly PP1γ, which mediates the dephosphorylation of Gwl Ser-883. Consistent with the mitotic activation of Gwl, its association with PP1 is disrupted in mitotic cells and egg extracts. During mitotic exit, PP1-dependent dephosphorylation of Gwl Ser-883 occurs prior to dephosphorylation of other mitotic substrates; replacing endogenous Gwl with a phosphomimetic S883E mutant blocks mitotic exit. Moreover, we identified PP1 regulatory subunit 3B (PPP1R3B) as a targeting subunit that can direct PP1 activity toward Gwl. PPP1R3B bridges PP1 and Gwl association and promotes Gwl Ser-883 dephosphorylation. Consistent with the cell cycle-dependent association of Gwl and PP1, Gwl and PPP1R3B dissociate in M phase. Interestingly, up-regulation of PPP1R3B facilitates mitotic exit and blocks mitotic entry. Thus, our study suggests PPP1R3B as a new cell cycle regulator that functions by governing Gwl dephosphorylation.

Phosphatase 1 nuclear targeting subunit is an essential regulator of M-phase entry, maintenance, and exit.

Mitotic progression is regulated largely through dynamic and reversible protein phosphorylation that is modulated by opposing actions of protein kinases and phosphatases. In this study, we show that phosphatase 1 nuclear targeting subunit (Pnuts) functions as a master regulator of mitosis by modulating protein phosphatase 1 (PP1). Overexpression of Pnuts in Xenopus egg extracts inhibited both mitotic and meiotic exit. Immunodepletion of Pnuts from egg extracts revealed its essential functions in mitotic entry and maintenance. The level of Pnuts oscillates during the cell cycle and peaks in mitosis. Pnuts destruction during M-phase exit is mediated by the anaphase-promoting complex/cyclosome (APC/C)-targeted ubiquitination and proteolysis, and conserved destruction motifs of Pnuts. Disruption of Pnuts degradation delayed M-phase exit, suggesting it as an important mechanism to permit M-phase exit.

Incidence of fatal airway obstruction in police officers feloniously killed in the line of duty: a 10-year retrospective analysis.

BACKGROUND: According to US military data, airway obstruction is the third leading cause of possibly preventable death in combat. In the absence of law enforcement-specific medical training, military experience has been translated to the law enforcement sector. The purpose of this study was to determine whether airway obstruction represents a significant cause of possibly preventable death in police officers, and whether current military combat lifesaver training programs might have prevented these fatalities. METHODS: De-identified, open-source US Federal Bureau of Investigation (FBI) Uniform Crime Report Law Enforcement Officers Killed and Assaulted (LEOKA) data for the years 1998-2007 were reviewed. Cases were included if officers were on duty at the time of fatal injury and died within one hour from time of wounding from penetrating face or neck trauma. After case identification, letters requesting autopsy reports were sent to the departments of victim officers. Reports were abstracted into a Microsoft Excel database. RESULTS: During the study period, 42 of 533 victim officers met inclusion criteria. Departmental response rate was 85.7%. Autopsy reports were provided for 29 officers; 23 (54.8%) cases remained in the final analysis. All officers died from gunshot wounds. No coroner specifically identified airway obstruction as either a direct cause of death or contributing factor. Based upon autopsy findings, three of 341 officers possibly succumbed to airway trauma (0.9%; 95% CI, 0.0%-1.9%). Endotracheal intubation was the most common advanced airway management technique utilized during attempted resuscitation. CONCLUSION: The limited LEOKA data suggests that acute airway obstruction secondary to penetrating trauma appears to be a rare cause of possibly preventable death in police officers. Based upon the nature of airway trauma, nasopharyngeal airways would not be expected to be an effective lifesaving intervention. This study highlights the requirement for a comprehensive mortality and "near miss" database for law enforcement officers.

Greatwall and Polo-like kinase 1 coordinate to promote checkpoint recovery.

Checkpoint recovery upon completion of DNA repair allows the cell to return to normal cell cycle progression and is thus a crucial process that determines cell fate after DNA damage. We previously studied this process in Xenopus egg extracts and established Greatwall (Gwl) as an important regulator. Here we show that preactivated Gwl kinase can promote checkpoint recovery independently of cyclin-dependent kinase 1 (Cdk1) or Plx1 (Xenopus polo-like kinase 1), whereas depletion of Gwl from extracts exhibits no synergy with that of Plx1 in delaying checkpoint recovery, suggesting a distinct but related relationship between Gwl and Plx1. In further revealing their functional relationship, we found mutual dependence for activation of Gwl and Plx1 during checkpoint recovery, as well as their direct association. We characterized the protein association in detail and recapitulated it in vitro with purified proteins, which suggests direct interaction. Interestingly, Gwl interaction with Plx1 and its phosphorylation by Plx1 both increase at the stage of checkpoint recovery. More importantly, Plx1-mediated phosphorylation renders Gwl more efficient in promoting checkpoint recovery, suggesting a functional involvement of such regulation in the recovery process. Finally, we report an indirect regulatory mechanism involving Aurora A that may account for Gwl-dependent regulation of Plx1 during checkpoint recovery. Our results thus reveal novel mechanisms underlying the involvement of Gwl in checkpoint recovery, in particular, its functional relationship with Plx1, a well characterized regulator of checkpoint recovery. Coordinated interplays between Plx1 and Gwl are required for reactivation of these kinases from the G(2)/M DNA damage checkpoint and efficient checkpoint recovery.

Removal of reactive oxygen species-induced 3'-blocked ends by XPF-ERCC1.

XPF-ERCC1 is a structure-specific endonuclease that is essential for nucleotide excision repair and DNA interstrand cross-link repair in mammalian cells. The yeast counterpart of XPF-ERCC1, Rad1-Rad10, plays multiple roles in DNA repair. Rad1-Rad10 is implicated to be involved in the repair of oxidative DNA damage. To explore the role(s) of XPF-ERCC1 in the repair of DNA damage induced by reactive oxygen species (ROS), cellular sensitivity of the XPF-deficient Chinese hamster ovary cell line UV41 to ROS was investigated. The XPF-deficient UV41 showed sensitivity to hydrogen peroxide, bleomycin, and paraquat. Furthermore, XPF-ERCC1 showed an ability to remove 3'-blocked ends such as 3'-phosphoglycolate from the 3'-end of DNA in vitro. These data suggest that XPF-ERCC1 plays a role in the repair of ROS-induced DNA damage by trimming 3'-blocked ends. The accumulation of various types of DNA damage, including ROS-induced DNA damage due to defects in multiple XPF-ERCC1-mediated DNA repair pathways, could contribute to the accelerated aging phenotypes observed in an XPF-ERCC1-deficient patient.

Phosphatase 1 Nuclear Targeting Subunit (PNUTS) Regulates Aurora Kinases and Mitotic Progression.

Mitotic progression is regulated largely by reversible phosphorylation events that are mediated by mitotic kinases and phosphatases. Protein phosphatase 1 (PP1) has been shown to play a crucial role in regulation of mitotic entry, progression, and exit. We previously observed, in Xenopus egg extracts, that phosphatase 1 nuclear targeting subunit (PPP1R10/PNUTS) acts as a mitotic regulator by negatively modulating PP1. This study investigates the role of PNUTS in mitotic progression in mammalian cells, and demonstrates that PNUTS expression is elevated in mitosis and depletion partially blocks mitotic entry. Cells that enter mitosis after PNUTS knockdown exhibit frequent chromosome mis-segregation. Aurora A/B kinase complexes and several kinetochore components are identified as PNUTS-associated proteins. PNUTS depletion suppresses the activation of Aurora A/B kinases, and disrupts the spatiotemporal regulation of the chromosomal passenger complex (CPC). PNUTS dynamically localizes to kinetochores, and is required for the activation of the spindle assembly checkpoint. Finally, PNUTS depletion sensitizes the tumor cell response to Aurora inhibition, suggesting that PNUTS is a potential drug target in combination anticancer therapy. IMPLICATIONS: Delineation of how PNUTS governs the mitotic activation and function of Aurora kinases will improve the understanding of the complex phospho-regulation in mitotic progression, and suggest new options to enhance the therapeutic efficacy of Aurora inhibitors.

Phosphatase 1 Nuclear Targeting Subunit Mediates Recruitment and Function of Poly (ADP-Ribose) Polymerase 1 in DNA Repair.

PARP, particularly PARP1, plays an essential role in the detection and repair of DNA single-strand breaks and double-strand breaks. PARP1 accumulates at DNA damage sites within seconds after DNA damage to catalyze the massive induction of substrate protein poly ADP-ribosylation (PARylation). However, the molecular mechanisms underlying the recruitment and activation of PARP1 in DNA repair are not fully understood. Here we show that phosphatase 1 nuclear targeting subunit 1 (PNUTS) is a robust binding partner of PARP1. Inhibition of PNUTS led to strong accumulation of endogenous DNA damage and sensitized the cellular response to a wide range of DNA-damaging agents, implicating PNUTS as an essential and multifaceted regulator of DNA repair. Recruitment of PNUTS to laser-induced DNA damage was similar to that of PARP1, and depletion or inhibition of PARP1 abrogated recruitment of PNUTS to sites of DNA damage. Conversely, PNUTS was required for efficient induction of substrate PARylation after DNA damage. PNUTS bound the BRCA1 C-terminal (BRCT) domain of PARP1 and was required for the recruitment of PARP1 to sites of DNA damage. Finally, depletion of PNUTS rendered cancer cells hypersensitive to PARP inhibition. Taken together, our study characterizes PNUTS as an essential partner of PARP1 in DNA repair and a potential drug target in cancer therapy. SIGNIFICANCE: These findings reveal PNUTS as an essential functional partner of PARP1 in DNA repair and suggest its inhibition as a potential therapeutic strategy in conjunction with DNA-damaging agents or PARP inhibitors.See related commentary by Murai and Pommier, p. 2460.

Protein interactomes of protein phosphatase 2A B55 regulatory subunits reveal B55-mediated regulation of replication protein A under replication stress.

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 B55ß 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 B55ß-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 B55ß, 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.

Regulation of polo-like kinase 1 by DNA damage and PP2A/B55α.

In addition to governing mitotic progression, Plk1 also suppresses the activation of the G2 DNA damage checkpoint and promotes checkpoint recovery. Previous studies have shown that checkpoint activation after DNA damage requires inhibition of Plk1, but the underlying mechanism of Plk1 regulation was unknown. In this study we show that the specific phosphatase activity toward Plk1 Thr-210 in interphase Xenopus egg extracts is predominantly PP2A-dependent, and this phosphatase activity is upregulated by DNA damage. Consistently, PP2A associates with Plk1 and the association increases after DNA damage. We further revealed that B55α, a targeting subunit of PP2A and putative tumor suppressor, mediates PP2A/Plk1 association and Plk1 dephosphorylation. B55α and PP2A association is greatly strengthened after DNA damage in an ATM/ATR and checkpoint kinase-dependent manner. Collectively, we report a phosphatase-dependent mechanism that responds to DNA damage and regulates Plk1 and checkpoint recovery.

Regulation of Greatwall kinase by protein stabilization and nuclear localization.

Greatwall (Gwl) functions as an essential mitotic kinase by antagonizing protein phosphatase 2A. In this study we identified Hsp90, Cdc37 and members of the importin α and ß families as the major binding partners of Gwl. Both Hsp90/Cdc37 chaperone and importin complexes associated with the N-terminal kinase domain of Gwl, whereas an intact glycine-rich loop at the N-terminus of Gwl was essential for binding of Hsp90/Cdc37 but not importins. We found that Hsp90 inhibition led to destabilization of Gwl, a mechanism that may partially contribute to the emerging role of Hsp90 in cell cycle progression and the anti-proliferative potential of Hsp90 inhibition. Moreover, in agreement with its importin association, Gwl exhibited nuclear localization in interphase Xenopus S3 cells, and dynamic nucleocytoplasmic distribution during mitosis. We identified KR456/457 as the locus of importin binding and the functional NLS of Gwl. Mutation of this site resulted in exclusion of Gwl from the nucleus. Finally, we showed that the Gwl nuclear localization is indispensable for the biochemical function of Gwl in promoting mitotic entry.

Role of interaction of XPF with RPA in nucleotide excision repair.

Nucleotide excision repair (NER) is a very important defense system against various types of DNA damage, and it is necessary for maintaining genomic stability. The molecular mechanism of NER has been studied in considerable detail, and it has been shown that proper protein-protein interactions among NER factors are critical for efficient repair. A structure-specific endonuclease, XPF-ERCC1, which makes the 5' incision in NER, was shown to interact with a single-stranded DNA binding protein, RPA. However, the biological significance of this interaction was not studied in detail. We used the yeast two-hybrid assay to determine that XPF interacts with the p70 subunit of RPA. To further examine the role of this XPF-p70 interaction, we isolated a p70-interaction-deficient mutant form of XPF that contains a single amino acid substitution in the N-terminus of XPF by the reverse yeast two-hybrid assay using randomly mutagenized XPF. The biochemical properties of this RPA-interaction-deficient mutant XPF-ERCC1 are very similar to those of wild-type XPF-ERCC1 in vitro. Interestingly, expression of this mutated form of XPF in the XPF-deficient Chinese hamster ovary cell line, UV41, only partially restores NER activity and UV resistance in vivo compared to wild-type XPF. We discovered that the RPA-interaction-deficient XPF is not localized in nuclei and the mislocalization of XPF-ERCC1 prevents the complex from functioning in NER.

Monoclonal antibodies against Xenopus greatwall kinase.

Mitosis is known to be regulated by protein kinases, including MPF, Plk1, Aurora kinases, and so on, which become active in M-phase and phosphorylate a wide range of substrates to control multiple aspects of mitotic entry, progression, and exit. Mechanistic investigations of these kinases not only provide key insights into cell cycle regulation, but also hold great promise for cancer therapy. Recent studies, largely in Xenopus, characterized a new mitotic kinase named Greatwall (Gwl) that plays essential roles in both mitotic entry and maintenance. In this study, we generated a panel of mouse monoclonal antibodies (MAbs) specific for Xenopus Gwl and characterized these antibodies for their utility in immunoblotting, immunoprecipitation, and immunodepletion in Xenopus egg extracts. Importantly, we generated an MAb that is capable of neutralizing endogenous Gwl. The addition of this antibody into M-phase extracts results in loss of mitotic phosphorylation of Gwl, Plk1, and Cdk1 substrates. These results illustrate a new tool to study loss-of-function of Gwl, and support its essential role in mitosis. Finally, we demonstrated the usefulness of the MAb against human Gwl/MASTL.

Processing of a psoralen DNA interstrand cross-link by XPF-ERCC1 complex in vitro.

The processing of stalled forks caused by DNA interstrand cross-links (ICLs) has been proposed to be an important step in initiating mammalian ICL repair. To investigate a role of the XPF-ERCC1 complex in this process, we designed a model substrate DNA with a single psoralen ICL at a three-way junction (Y-shaped DNA), which mimics a stalled fork structure. We found that the XPF-ERCC1 complex makes an incision 5' to a psoralen lesion on Y-shaped DNA in a damage-dependent manner. Furthermore, the XPF-ERCC1 complex generates an ICL-specific incision on the 3'-side of an ICL. The ICL-specific 3'-incision, along with the 5'-incision, on the cross-linked Y-shaped DNA resulted in the separation of the two cross-linked strands (the unhooking of the ICL) and the induction of a double strand break near the cross-linked site. These results implicate the XPF-ERCC1 complex in initiating ICL repair by unhooking the ICL, which simultaneously induces a double strand break at a stalled fork.