A comparison of BRCA1 nuclear localization with 14 DNA damage response proteins and domains: Identification of specific differences between BRCA1 and 53BP1 at DNA damage-induced foci

BRCA1 is an important mediator of the DNA damage response pathway. Previous studies have identified a number of proteins that associate with BRCA1 at nuclear foci after ionizing radiation (IR)-induced DNA damage. However, the co-localization patterns of BRCA1 and various DNA damage response proteins have not yet been systematically quantified and compared within the same experimental system. In this study, a new inducible human cell line was established to allow unambiguous detection of YFP–BRCA1 at nuclear foci. Quantitative 2-D microscopic analysis was performed to compare the intranuclear co-localization of YFP–BRCA1 with 10 cellular proteins and 4 cellular domains before and after IR. Intriguingly, YFP–BRCA1 displayed significantly better focal co-localization with BARD1, RAP80 and Abraxas than with the upstream foci-initiating proteins γH2AX and MDC1. In contrast to previous reports, we found that the co-localization between YFP–BRCA1 and 53BP1 foci was surprisingly weak. Quantitative analyses of 3-D confocal images showed that ~ 60% of 53BP1 foci were unrelated to YFP–BRCA1 foci, ~ 35% of foci were abutting and only ~ 5% of foci co-localized. The YFP–BRCA1 and 53BP1 nuclear foci were distinctively separated within the first 3 h after IR. In addition, in situ nuclear retention analysis revealed YFP–BRCA1 and BARD1 are less mobile than 53BP1 at IR-induced nuclear foci. Our findings indicate that BRCA1–BARD1 and 53BP1 are proximal but not overlapping at DNA break sites and are consistent with recent evidence for distinct roles of these proteins in the DNA damage response pathway.     

Mediator of DNA damage checkpoint protein 1 regulates BRCA1 localization and phosphorylation in DNA damage checkpoint control

BRCA1 is a tumor suppressor involved in DNA repair and damage-induced checkpoint controls. In response to DNA damage, BRCA1 relocalizes to nuclear foci at the sites of DNA lesions. However, little is known about the regulation of BRCA1 relocalization following DNA damage. Here we show that mediator of DNA damage checkpoint protein 1 (MDC1), previously named NFBD1 or Kiaa0170, is a proximate mediator of DNA damage responses that regulates BRCA1 function. MDC1 regulates ataxia-telangiectasia-mutated (ATM)-dependent phosphorylation events at the site of DNA damage. Importantly down-regulation of MDC1 abolishes the relocalization and hyperphosphorylation of BRCA1 following DNA damage, which coincides with defective G(2)/M checkpoint control in response to DNA damage. Taken together these data suggest that MDC1 regulates BRCA1 function in DNA damage checkpoint control.     

DNA damage-induced cell cycle checkpoint control requires CtIP, a phosphorylation-dependent binding partner of BRCA1 C-terminal domains

The BRCA1 C-terminal (BRCT) domain has recently been implicated as a phospho-protein binding domain. We demonstrate here that a CTBP-interacting protein CtIP interacts with BRCA1 BRCT domains in a phosphorylation-dependent manner. The CtIP/BRCA1 complex only exists in G(2) phase and is required for DNA damage-induced Chk1 phosphorylation and the G(2)/M transition checkpoint. However, the CtIP/BRCA1 complex is not required for the damage-induced G(2) accumulation checkpoint, which is controlled by a separate BRCA1/BACH1 complex. Taken together, these data not only implicate CtIP as a critical player in cell cycle checkpoint control but also provide molecular mechanisms by which BRCA1 controls multiple cell cycle transitions after DNA damage.     

CK2 phosphorylates TNFAIP1 to affect its subcellular localization and interaction with PCNA

TNFAIP1 is a protein which can be induced by tumor necrosis factorα (TNFα) and interleukin-6 (IL-6), it may play roles in DNA synthesis, DNA repair, cell apoptosis and human diseases. However, very little has been known about how TNFAIP1 acts in these physiological processes. In this paper, CK2β was identified as a partner of TNFAIP1 by screening the HeLa cDNA library in yeast two-hybrid system with TNFAIP1 as a bait. Furthermore, it was demonstrated that CK2 could phosphorylate TNFAIP1 in vitro and in vivo, which facilitated the distribution of TNFAIP1 in nucleus and enhanced its interaction with PCNA. It is suggested that the phosphorylation of TNFAIP1 may be required for its functions.     

Multiple phosphorylation events regulate the subcellular localization of GGA1

GGAs comprise a family of Arf-dependent coat proteins or adaptors that regulate vesicle traffic from the trans -Golgi network (TGN). GGAs bind activated Arf, cargo, and additional components necessary for vesicle budding through interactions with their four functional domains: VHS, GAT, hinge, and GAE. We identified three sites of phosphorylation in GGA1 by tandem mass spectrometry: S268 and T270 in the GAT domain and S480 in the hinge. Expression of HA-GGA1 in mammalian cells and comparison to endogenous GGA1 confirmed their localization to late Golgi compartments. In contrast, mutations that mimic the phosphoprotein (HA-GGA1[S268D] or HA-GGA1[T270D]) at either of the sites in the GAT domain caused a decrease in the colocalization with markers of the Golgi and TGN and an increase in puncta in cytoplasm. Quantitative comparisons of the extent of colocalization of GGA1 proteins with the known components of GGA1 vesicles revealed that the composition of those markers tested in HA-GGA1[S268D] and HA-GGA1[T270D] vesicles were indistinguishable from those of HA-GGA1 vesicles. We conclude that phosphorylation of the GAT domain can stabilize the coat proteins bound and thus regulate the rate of coat protein dissociation.     

Dishevelled phosphorylation, subcellular localization and multimerization regulate its role in early embryogenesis

Dishevelled (Dsh) induces a secondary axis and can translocate to the membrane when activated by Frizzleds; however, dominant-negative approaches have not supported a role for Dsh in primary axis formation. We demonstrate that the Dsh protein is post-translationally modified at the dorsal side of the embryo: timing and position of this regulation suggests a role of Dsh in dorsal-ventral patterning in Xenopus. To create functional links between these properties of Dsh we analyzed the influence of endogenous Frizzleds and the Dsh domain dependency for these characteristics. Xenopus Frizzleds phosphorylate and translocate Xdsh to the membrane irrespective of their differential ectopic axes inducing abilities, showing that translocation is insufficient for axis induction. Dsh deletion analysis revealed that axis inducing abilities did not segregate with Xdsh membrane association. The DIX region and a short stretch at the N-terminus of the DEP domain are necessary for axis induction while the DEP region is required for Dsh membrane association and its phosphorylation. In addition, Dsh forms homomeric complexes in embryos suggesting that multimerization is important for its proper function.     

Nuclear localization of the cell cycle regulator CDH1 and its regulation by phosphorylation

The anaphase-promoting complex activated by CDC20 and CDH1 is a major ubiquitination system that controls the destruction of cell cycle regulators. Exactly how ubiquitination is regulated in time and space is incompletely understood. Here we report on the cell cycle-dependent localization of CDH1 and its regulation by phosphorylation. CDH1 localizes dynamically to the nucleus during interphase and to the centrosome during metaphase and anaphase. The nuclear accumulation of CDH1 correlates with a reduction in the steady-state amount of cyclin A, but not of cyclin E. A nuclear localization signal conserved in various species was identified in CDH1, and it sufficiently targets green fluorescent protein to the nucleus. Interestingly, a CDH1-4D mutant mimicking the hyperphosphorylated form was constitutively found in the cytoplasm. In further support of the notion that phosphorylation inhibits nuclear import, the nuclear localization signal of CDH1 with two phospho-accepting serine/threonine residues changed into aspartates was unable to drive heterologous protein into the nucleus. On the other hand, abolition of the cyclin-binding ability of CDH1 has no influence on its nuclear localization. Taken together, our findings document the phosphorylation-dependent localization of CDH1 in vertebrate cells.     

The in vivo dynamic organization of BRCA1-A complex proteins at DNA damage-induced nuclear foci

The breast cancer associated gene 1 (BRCA1)‐A protein complex assembles at DNA damage‐induced nuclear foci to facilitate repair of double‐stranded breaks. Here, we describe the first systematic comparison of the dynamics, copy number and organization of its core components at foci. We show that the protein pools at individual foci generally comprise a small immobile fraction (～20%) and larger mobile fraction (～80%), which together occupy the same focal space but exist at different densities. In the mobile fraction, Abraxas (CCDC98) and the heterodimer BARD1–BRCA1 share similar rates of dynamic exchange (complete turnover in ～500 seconds). In contrast, RAP80, which is required for initial foci assembly, was more dynamic with 25‐fold faster turnover at mature foci. In addition, Abraxas, BARD1, BRCA1 and Merit40 (NBA1) were stably retained in the immobile fraction of foci under conditions causing loss of BRCC36 and RAP80, suggesting a shift to RAP80‐independent localization after foci formation. These results, combined with our finding that RAP80 (～1200 copies per focus) is twofold more abundant than Abraxas/BARD1/BRCA1 at foci, suggest new models defining the dynamic organization of BRCA1‐A complex at mature foci, wherein the unusually fast turnover of RAP80 may contribute to its regulation of BRCA1‐dependent DNA repair.     

Cell cycle regulation of the BRCA1/acetyl-CoA-carboxylase complex

Germ-line alterations in BRCA1 are associated with an increased susceptibility to breast and ovarian cancer. The BRCA1 protein has been implicated in multiple cellular functions. We have recently demonstrated that BRCA1 reduces acetyl-CoA-carboxylase alpha (ACCA) activity through its phospho-dependent binding to ACCA, and further established that the phosphorylation of the Ser1263 of ACCA is required for this interaction. Here, to gain more insight into the cellular conditions that trigger the BRCA1/ACCA interaction, we designed an anti-pSer1263 antibody and demonstrated that the Ser1263 of ACCA is phosphorylated in vivo, in a cell cycle-dependent manner. We further showed that the interaction between BRCA1 and ACCA is regulated during cell cycle progression. Taken together, our findings reveal a novel mechanism of regulation of ACCA distinct from the previously described phosphorylation of Ser79, and provide new insights into the control of lipogenesis through the cell cycle.     

Fyn-induced phosphorylation of beta-adducin at tyrosine 489 and its role in their subcellular localization

Fyn is a Src-family tyrosine kinase involved in neuronal development, transmission, and plasticity in mammalian central nervous system. We have previously reported that Fyn binds to a cytoskeletal protein, beta-adducin, in a phosphorylation-dependent manner. In the present report, we show that Fyn phosphorylates beta-adducin at tyrosine 489 located in its C-terminal tail domain. Phosphorylation of beta-adducin at Y489 was required for its association with the Fyn-SH2 domain. An antibody specific to the phosphorylated form of beta-adducin was raised in rabbits and showed that Y489 of beta-adducin was phosphorylated in wild type, but not in Fyn(-/-) mice, suggesting that Y489 of beta-adducin is phosphorylated downstream of Fyn in vivo. After phosphorylation at Y489, beta-adducin was translocated to the cell periphery, and colocalized with Fyn. These results suggest that Fyn phosphorylates and binds to beta-adducin at Y489, resulting in translocation of beta-adducin to the Fyn-enriched regions in the plasma membrane.     

Extracellular matrix enhances heregulin-dependent BRCA1 phosphorylation and suppresses BRCA1 expression through its C terminus

Germ line mutations in the breast cancer susceptibility gene BRCA1 account for the increased risk of early onset of familial breast cancer, whereas overexpression of the ErbB family of receptor tyrosine kinases has been linked to the development of nonfamilial or sporadic breast cancer. To analyze whether there is a link between these two regulatory molecules, we studied the effects of ErbB-2 activation by heregulin (HRG) on BRCA1 function. It was previously demonstrated that HRG induced the phosphorylation of BRCA1, which was mediated by the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Since altered interaction between cells and the surrounding extracellular matrix (ECM) is a common feature in a variety of tumors and since ECM modulates intracellular signaling, we hypothesized that ECM may affect the expression and HRG-dependent phosphorylation of BRCA1. Following stimulation by HRG, a strong increase in [(3)H]thymidine incorporation was observed in human T47D breast cancer cells seeded on plastic (PL). When T47D cells were seeded on laminin (LAM) or Matrigel, HRG induced a significantly higher proliferation than it did in cells seeded on PL. T47D cells seeded on poly-L-lysine had an abrogated mitogenic response, indicating the involvement of integrins in this process. HRG treatment induced a transient phosphorylation of BRCA1 that was enhanced in T47D cells grown on LAM. LAM-enhanced BRCA1 phosphorylation was mediated through alpha(6) integrin upon HRG stimulation. Accordingly, T47D cells grown on LAM had the greatest increase in ErbB-2 activation, PI3K activity, and phosphorylation of Akt. A similar pattern of BRCA1 mRNA expression was observed when T47D cells were seeded on PL, LAM, or COL4. There was a significant decrease in the steady state of the BRCA1 mRNA level on both the LAM and COL4 matrices compared to that for cells seeded on PL. In addition, HRG stimulation caused a significant decrease in BRCA1 mRNA expression that was dependent on protein synthesis. Pretreatment with both the calpain inhibitor ALLN (N-acetyl-Leu-Leu-norleucinal) and the proteosome inhibitor lactacystin inhibited the HRG-induced down-regulation of BRCA1 mRNA expression. Likewise, there was a strong decrease in the protein level of BRCA1 in T47D cells 4 h after treatment with HRG compared to its level in control nontreated T47D cells. Pretreatment with the proteosome inhibitors ALLN, lactacystin, and PSI [N-benzyloxycarbonyl-Ile-Glu-(O-t-butyl)-Ala-leucinal] inhibited also the HRG-induced down-regulation of BRCA1 protein in breast cancer cells. Interestingly, BRCA1 mRNA expression in HCC-1937 breast cancer cells, which express C-terminally truncated BRCA1, was not affected by either LAM or CL4. No phosphorylation of BRCA1 from HCC-1937 cells was observed in response to HRG. While Cdk4 phosphorylated wild-type BRCA1 in response to HRG in T47D cells, Cdk4 failed to phosphorylate the truncated form of BRCA1 in HCC-1937 cells. Furthermore, overexpression of wild-type BRCA1 in HCC-1937 cells resulted in the phosphorylation of BRCA1 and decreased BRCA1 expression upon HRG stimulation while overexpression of truncated BRCA1 in T47D cells resulted in a lack of BRCA1 phosphorylation and restoration of BRCA1 expression. These findings suggest that ECM enhances HRG-dependent BRCA1 phosphorylation and that ECM and HRG down-regulate BRCA1 expression in breast cancer cells. Furthermore, ECM suppresses BRCA1 expression through the C terminus of BRCA1.     

Chk2 phosphorylation of BRCA1 regulates DNA double-strand break repair

The pathway determining malignant cellular transformation, which depends upon mutation of the BRCA1 tumor suppressor gene, is poorly defined. A growing body of evidence suggests that promotion of DNA double-strand break repair by homologous recombination (HR) may be the means by which BRCA1 maintains genomic stability, while a role of BRCA1 in error-prone nonhomologous recombination (NHR) processes has just begun to be elucidated. The BRCA1 protein becomes phosphorylated in response to DNA damage, but the effects of phosphorylation on recombinational repair are unknown. In this study, we tested the hypothesis that the BRCA1-mediated regulation of recombination requires the Chk2- and ATM-dependent phosphorylation sites. We studied Rad51-dependent HR and random chromosomal integration of linearized plasmid DNA, a subtype of NHR, which we demonstrate to be dependent on the Mre11-Rad50-Nbs1 complex. Prevention of Chk2-mediated phosphorylation via mutation of the serine 988 residue of BRCA1 disrupted both the BRCA1-dependent promotion of HR and the suppression of NHR. Similar results were obtained when endogenous Chk2 kinase activity was inhibited by expression of a dominant-negative Chk2 mutant. Surprisingly, the opposing regulation of HR and NHR did not require the ATM phosphorylation sites on serines 1423 and 1524. Together, these data suggest a functional link between recombination control and breast cancer predisposition in carriers of Chk2 and BRCA1 germ line mutations. We propose a dual regulatory role for BRCA1 in maintaining genome integrity, whereby BRCA1 phosphorylation status controls the selectivity of repair events dictated by HR and error-prone NHR.     

ATM、ATR和DNA损伤介导的细胞周期阻滞

ATM和ATR属于PIKK家族,是DNA损伤检查点的主要成员。它们被不同类型的DNA损伤所激活,通过磷酸化相应的下游蛋白Chk1和Chk2等,调节细胞周期各个检查点,引起细胞周期阻滞,使DNA损伤得以修复。ATM和ATR在维持基因组的稳定性中起到至关重要的作用。本文着重综述有关ATM和ATR在DNA损伤介导的细胞周期阻滞中发挥的作用以及相互关系的最新研究进展。     

Cell growth-dependent subcellular localization of p8

p8 is a stress-induced protein, biochemically related to the architectural factor HMG-I/Y, overexpressed in many cancers and required for tumor expansion. The molecular mechanisms by which p8 may exert its effect in aspects of growth is unknown. Using immunocytochemistry, we found that p8 presents nuclear localization in sub-confluent cells, but it localizes throughout the whole cell in high density grown cells. Cells arrested in Go/G1, either by serum deprivation or by hydroxyurea treatment, show a nucleo-cytoplasmic localization of p8, whether in the rest of the cell cycle stages of actively dividing cells the localization is nuclear. A comparison of p8 sequences from human to fly predicts a conserved bipartite nuclear localization sequence (NLS). The putative NLS has been demonstrated to be functional, since nuclear import is energy dependent (inhibited by sodium azide plus 2-deoxyglucose), and fusion proteins GFP-p8 and GFP-NLSp8 localize to the nucleus, whereas GFP-p8NLSmut in which with Lys 65, 69, 76, and 77 mutated to Ala localized to the whole cell. p8 localization does not involve the CRM1 transporter, since it is insensitive to leptomycin B. Inhibitors of MAPK pathways did not affect p8 subcellular localization. The inhibition of deacetylation with Trichostatin A promotes cytoplasmic accumulation of p8. The results suggest that p8 growth stage-dependent localization is regulated by acetylation, that p8 is not free within the cell but forming part of a complex and that it may exert a role in both subcellular localizations.     

A nuclear export signal and phosphorylation regulate Dok1 subcellular localization and functions

Dok1 is believed to be a mainly cytoplasmic adaptor protein which down-regulates mitogen-activated protein kinase activation, inhibits cell proliferation and transformation, and promotes cell spreading and cell migration. Here we show that Dok1 shuttles between the nucleus and cytoplasm. Treatment of cells with leptomycin B (LMB), a specific inhibitor of the nuclear export signal (NES)-dependent receptor CRM1, causes nuclear accumulation of Dok1. We have identified a functional NES (348LLKAKLTDPKED359) that plays a major role in the cytoplasmic localization of Dok1. Src-induced tyrosine phosphorylation prevented the LMB-mediated nuclear accumulation of Dok1. Dok1 cytoplasmic localization is also dependent on IKKbeta. Serum starvation or maintaining cells in suspension favor Dok1 nuclear localization, while serum stimulation, exposure to growth factor, or cell adhesion to a substrate induce cytoplasmic localization. Functionally, nuclear NES-mutant Dok1 had impaired ability to inhibit cell proliferation and to promote cell spreading and cell motility. Taken together, our results provide the first evidence that Dok1 transits through the nucleus and is actively exported into the cytoplasm by the CRM1 nuclear export system. Nuclear export modulated by external stimuli and phosphorylation may be a mechanism by which Dok1 is maintained in the cytoplasm and membrane, thus regulating its signaling functions.     

Inflammation-induced DNA damage and damage-induced inflammation: a vicious cycle

Inflammation is the ultimate response to the constant challenges of the immune system by microbes, irritants or injury. The inflammatory cascade initiates with the recognition of microorganism-derived pathogen associated molecular patterns (PAMPs) and host cell-derived damage associated molecular patterns (DAMPs) by the pattern recognition receptors (PRRs). DNA as a molecular PAMP or DAMP is sensed directly or via specific binding proteins to instigate pro-inflammatory response. Some of these DNA binding proteins also participate in canonical DNA repair pathways and recognise damaged DNA to initiate DNA damage response. In this review we aim to capture the essence of the complex interplay between DNA damage response and the pro-inflammatory signalling through representative examples.     

Cell cycle effects by C-FADD depend on its C-terminal phosphorylation site

Expression of a truncated form of the death receptor adaptor FADD (C-FADD) as a transgene in mice blocks T cell proliferation. Here we provide evidence that the C-terminal phosphorylation site Ser194 in C-FADD affects the cell cycle in nonlymphoid cells as well. High expression of wild type C-FADD but not C-FADD with a S194A point mutation arrested the nontumor cell line MCF10A in G2/M but not the tumor cell line MCF7. BJAB as well as MCF10A cells expressing moderate levels of C-FADD with a S194E mutation mimicking phosphorylated C-FADD were more susceptible to a Taxol-induced G2/M arrest than cells expressing C-FADD S194A suggesting that C-FADD S194E lowers the threshold for G2/M arrest. Our data suggest that C-FADD may affect apoptosis sensitivity of cells by interfering with cell cycle progression and not only by binding to death receptors.     

Functional expression of human DNA topoisomerase I and its subcellular localization in HeLa cells

DNA topoisomerase (topo) I plays an important role in DNA metabolism by relieving the torsional restraints of DNA topology through ATP-independent single-strand DNA breakage. In the present study, we expressed human topo I in HeLa cells by fusing it to enhanced green fluorescent protein (EGFP). The EGFP-topo I fusion protein is functionally active in that it relaxes supercoiled plasmid DNA; forms complexes with DNA, as revealed by band depletion assays; and increases the sensitivity of cells to topo I inhibitors such as topotecan, as determined by growth inhibition assays. In contrast, a mutant form of the EGFP-topo I fusion protein, in which the active Tyr has been replaced by Phe (Y723F), has no such activities. Furthermore, the fusion protein localizes to the nucleus at interphase and completely associates with chromatids at every stage of mitosis. Of importance, the mutant fusion protein (Y723F) displays a pattern of subcellular localization identical to that of the wild-type fusion protein, although the mutant fusion protein is catalytically inactive. These results suggest that in addition to its role in DNA metabolism, topo I might also play a structural role in chromosomal organization; moreover, the association of topo I with chromosomal DNA is independent of its catalytic activity. Finally, the fusion constructs may provide a useful tool to study drug action in tumor cells, as demonstrated by nucleolar delocalization of the fusion proteins in response to treatment with the topo I inhibitor topotecan.