BACKGROUND INFORMATION: Repair of damaged DNA is essential for maintaining genomic stability. TP53-binding protein 1 (53BP1) plays an important role in repair of the DNA double-strand breaks. Nuclear localisation of 53BP1 depends on importin β and nucleoporin 153, but the type and location of 53BP1 nuclear localisation signal (NLS) have yet to be determined. RESULTS: Here, we show that nuclear import of 53BP1 depends on two basic regions, namely 1667-KRK-1669 and 1681-KRGRK-1685, which are both needed for importin binding. Lysine 1667 is essential for interaction with importin and its substitution to arginine reduced nuclear localisation of 53BP1. Furthermore, we have found that CDK1-dependent phosphorylation of 53BP1 at S1678 impairs importin binding during mitosis. Phosphorylation-mimicking mutant S1678D showed reduced nuclear localisation, suggesting that phosphorylation of the NLS interferes with nuclear import of the 53BP1 CONCLUSIONS: We show that 53BP1 contains a classical bipartite NLS 1666-GKRKLITSEEERSPAKRGRKS-1686, which enables the importin-mediated nuclear transport of 53BP1. Additionally, we found that posttranslational modification within the NLS region can regulate 53BP1 nuclear import. SIGNIFICANCE: Our results indicate that integrity of the NLS is important for 53BP1 nuclear localisation. Precise mapping of the NLS will facilitate further studies on the effect of posttranslational modifications and somatic mutations on the nuclear localisation 53BP1 and DNA repair.

• MeSH
• 53BP1 genetika   metabolismus   MeSH
• aktivní transport - buněčné jádro MeSH
• arginin chemie   genetika   metabolismus   MeSH
• buněčné jádro genetika   metabolismus   MeSH
• fosforylace MeSH
• HEK293 buňky MeSH
• jaderné lokalizační signály * MeSH
• karyoferiny genetika   metabolismus   MeSH
• lidé MeSH
• lysin chemie   genetika   metabolismus   MeSH
• nádorové buňky kultivované MeSH
• nádory kostí genetika   metabolismus   patologie   MeSH
• osteosarkom genetika   metabolismus   patologie   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Splicing is catalyzed by the spliceosome, a complex of five major small nuclear ribonucleoprotein particles (snRNPs). The pre-mRNA splicing factor PRPF8 is a crucial component of the U5 snRNP, and together with EFTUD2 and SNRNP200, it forms a central module of the spliceosome. Using quantitative proteomics, we identified assembly intermediates containing PRPF8, EFTUD2, and SNRNP200 in association with the HSP90/R2TP complex, its ZNHIT2 cofactor, and additional proteins. HSP90 and R2TP bind unassembled U5 proteins in the cytoplasm, stabilize them, and promote the formation of the U5 snRNP. We further found that PRPF8 mutants causing Retinitis pigmentosa assemble less efficiently with the U5 snRNP and bind more strongly to R2TP, with one mutant retained in the cytoplasm in an R2TP-dependent manner. We propose that the HSP90/R2TP chaperone system promotes the assembly of a key module of U5 snRNP while assuring the quality control of PRPF8. The proteomics data further reveal new interactions between R2TP and the tuberous sclerosis complex (TSC), pointing to a potential link between growth signals and the assembly of key cellular machines.

• MeSH
• elongační faktory genetika   metabolismus   MeSH
• HeLa buňky MeSH
• interakční proteinové domény a motivy MeSH
• lidé MeSH
• malý jaderný ribonukleoprotein U1 metabolismus   MeSH
• malý jaderný ribonukleoprotein U4-U6 metabolismus   MeSH
• malý jaderný ribonukleoprotein U5 genetika   metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• multiproteinové komplexy MeSH
• mutace MeSH
• prekurzory RNA genetika   metabolismus   MeSH
• proteiny tepelného šoku HSP90 metabolismus   MeSH
• proteiny vázající RNA genetika   metabolismus   MeSH
• proteiny vázající vápník metabolismus   MeSH
• proteomika metody   MeSH
• retinopathia pigmentosa genetika   metabolismus   MeSH
• RNA interference MeSH
• sestřih RNA * MeSH
• stabilita proteinů MeSH
• transfekce MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The R2TP complex is a HSP90 co-chaperone, which consists of four subunits: PIH1D1, RPAP3, RUVBL1, and RUVBL2. It is involved in the assembly of large protein or protein-RNA complexes such as RNA polymerase, small nucleolar ribonucleoproteins (snoRNPs), phosphatidylinositol 3 kinase-related kinases (PIKKs), and their complexes. While RPAP3 has a HSP90 binding domain and the RUVBLs comprise ATPase activities important for R2TP functions, PIH1D1 contains a PIH-N domain that specifically recognizes phosphorylated substrates of the R2TP complex. In this review we provide an overview of the current knowledge of the R2TP complex with the focus on the recently identified structural and mechanistic features of the R2TP complex functions. We also discuss the way R2TP regulates cellular response to stress caused by low levels of nutrients or by DNA damage and its possible exploitation as a target for anti-cancer therapy.

• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Cellular senescence guards against cancer and modulates aging; however, the underlying mechanisms remain poorly understood. Here, we show that genotoxic drugs capable of inducing premature senescence in normal and cancer cells, such as 5-bromo-2'-deoxyuridine (BrdU), distamycin A (DMA), aphidicolin and hydroxyurea, persistently activate Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling and expression of interferon-stimulated genes (ISGs), such as MX1, OAS, ISG15, STAT1, PML, IRF1 and IRF7, in several human cancer cell lines. JAK1/STAT-activating ligands, interleukin 10 (IL10), IL20, IL24, interferon gamma (IFNgamma), IFNbeta and IL6, were also expressed by senescent cells, supporting autocrine/paracrine activation of JAK1/STAT. Furthermore, cytokine genes, including proinflammatory IL1, tumor necrosis factor and transforming growth factor families, were highly expressed. The strongest inducer of JAK/STAT signaling, cytokine production and senescence was BrdU combined with DMA. RNA interference-mediated knockdown of JAK1 abolished expression of ISGs, but not DNA damage signaling or senescence. Thus, although DNA damage signaling, p53 and RB activation, and the cytokine/chemokine secretory phenotype are apparently shared by all types of senescence, our data reveal so far unprecedented activation of the IFNbeta-STAT1-ISGs axis, and indicate a less prominent causative role of IL6-JAK/STAT signaling in genotoxic drug-induced senescence compared with reports on oncogene-induced or replicative senescence. These results highlight shared and unique features of drug-induced cellular senescence, and implicate induction of cancer secretory phenotype in chemotherapy.

• MeSH
• bromodeoxyuridin farmakologie   MeSH
• cytokiny genetika   metabolismus   MeSH
• distamyciny farmakologie   MeSH
• HeLa buňky MeSH
• interferony genetika   metabolismus   MeSH
• interleukin-10 genetika   metabolismus   MeSH
• interleukin-6 genetika   metabolismus   MeSH
• interleukin-8 genetika   metabolismus   MeSH
• interleukiny genetika   metabolismus   MeSH
• Janus kinasa 1 genetika   metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• RNA interference MeSH
• signální transdukce účinky léků   MeSH
• stárnutí buněk účinky léků   MeSH
• synergismus léků MeSH
• transkripční faktor STAT1 genetika   metabolismus   MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

• Publikační typ
• abstrakty MeSH

MDC1 and 53BP1 are critical components of the DNA damage response (DDR) machinery that protects genome integrity and guards against cancer, yet the tissue expression patterns and involvement of these two DDR adaptors/mediators in human tumours remain largely unknown. Here we optimized immunohistochemical analyses of human 53BP1 and MDC1 proteins in situ and identified their virtually ubiquitous expression, both in proliferating and quiescent, differentiated tissues. Focus formation by 53BP1 and/or MDC1 in human spermatogenesis and subsets of breast and lung carcinomas indicated physiological and 'pathological' activation of the DDR, respectively. Furthermore, aberrant reduction or lack of either protein in significant proportions of carcinomas supported the candidacy of 53BP1 and MDC1 for tumour suppressors. Contrary to carcinomas, almost no activation or loss of MDC1 or 53BP1 were found among testicular germ-cell tumours (TGCTs), a tumour type with unique biology and exceptionally low incidence of p53 mutations. Such concomitant presence (in carcinomas) or absence (in TGCTs) of DDR activation and DDR aberrations supports the roles of MDC1 and 53BP1 within the ATM/ATR-regulated checkpoint network which, when activated, provides an early anti-cancer barrier the pressure of which selects for DDR defects such as p53 mutations or loss of 53BP1/MDC1 during cancer progression.

• MeSH
• DNA vazebné proteiny biosyntéza   genetika   MeSH
• germinální a embryonální nádory genetika   metabolismus   patologie   MeSH
• imunohistochemie MeSH
• intracelulární signální peptidy a proteiny genetika   metabolismus   MeSH
• jaderné proteiny * biosyntéza   genetika   MeSH
• lidé MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory plic genetika   metabolismus   patologie   MeSH
• nádory prsu genetika   metabolismus   patologie   MeSH
• poškození DNA * MeSH
• testikulární nádory genetika   metabolismus   patologie   MeSH
• trans-aktivátory * biosyntéza   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH

During the evolution of cancer, the incipient tumour experiences 'oncogenic stress', which evokes a counter-response to eliminate such hazardous cells. However, the nature of this stress remains elusive, as does the inducible anti-cancer barrier that elicits growth arrest or cell death. Here we show that in clinical specimens from different stages of human tumours of the urinary bladder, breast, lung and colon, the early precursor lesions (but not normal tissues) commonly express markers of an activated DNA damage response. These include phosphorylated kinases ATM and Chk2, and phosphorylated histone H2AX and p53. Similar checkpoint responses were induced in cultured cells upon expression of different oncogenes that deregulate DNA replication. Together with genetic analyses, including a genome-wide assessment of allelic imbalances, our data indicate that early in tumorigenesis (before genomic instability and malignant conversion), human cells activate an ATR/ATM-regulated DNA damage response network that delays or prevents cancer. Mutations compromising this checkpoint, including defects in the ATM-Chk2-p53 pathway, might allow cell proliferation, survival, increased genomic instability and tumour progression.

• MeSH
• aktivace enzymů MeSH
• alely - nerovnováha genetika   MeSH
• buněčný cyklus MeSH
• checkpoint kinasa 2 MeSH
• cyklin E genetika   metabolismus   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• fosfatasy cdc25 genetika   MeSH
• fosforylace MeSH
• geny p53 genetika   MeSH
• jednonukleotidový polymorfismus genetika   MeSH
• lidé MeSH
• mutace genetika   MeSH
• nádorová transformace buněk * MeSH
• nádorové buněčné linie MeSH
• nádory močového měchýře enzymologie   genetika   metabolismus   patologie   MeSH
• nádory * enzymologie   genetika   patologie   prevence a kontrola   MeSH
• nestabilita genomu MeSH
• onkogeny genetika   fyziologie   MeSH
• poškození DNA * MeSH
• protein-serin-threoninkinasy metabolismus   MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• signální transdukce MeSH
• transkripční faktory E2F MeSH
• transkripční faktory genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH

The ATM kinase is a tumour suppressor and a key activator of genome integrity checkpoints in mammalian cells exposed to ionizing radiation (IR) and other insults that elicit DNA double-strand breaks (DSBs). In response to IR, autophosphorylation on serine 1981 causes dissociation of ATM dimers and initiates cellular ATM kinase activity. Here, we show that the kinetics and magnitude of ATM Ser1981 phosphorylation after exposure of human fibroblasts to low doses (2 Gy) of IR are altered in cells deficient in Nbs1, a substrate of ATM and a component of the MRN (Mre11-Rad50-Nbs1) complex involved in processing/repair of DSBs and ATM-dependent cell cycle checkpoints. Timely phosphorylation of both ATM Ser1981 and the ATM substrate Smc1 after IR were rescued via retrovirally mediated reconstitution of Nbs1-deficient cells by wild-type Nbs1 or mutants of Nbs1 defective in the FHA domain or nonphosphorylatable by ATM, but not by Nbs1 lacking the Mre11-interaction domain. Our data indicate that apart from its role downstream of ATM in the DNA damage checkpoint network, the MRN complex serves also as a modulator/amplifier of ATM activity. Although not absolutely required for ATM activation, the MRN nuclease complex may help reach the threshold activity of ATM necessary for optimal genome maintenance and prevention of cancer.

• MeSH
• amplifikace genu MeSH
• ATM protein MeSH
• DNA vazebné proteiny MeSH
• fosforylace MeSH
• fosfoserin metabolismus   MeSH
• ionizující záření MeSH
• jaderné proteiny chemie   metabolismus   nedostatek   MeSH
• kinetika MeSH
• lidé MeSH
• nádorové supresorové proteiny MeSH
• nádory genetika   prevence a kontrola   MeSH
• poškození DNA genetika   MeSH
• protein-serin-threoninkinasy * metabolismus   účinky záření   MeSH
• proteiny buněčného cyklu chemie   metabolismus   MeSH
• teleangiektatická ataxie MeSH
• vazebná místa MeSH
• viabilita buněk MeSH
• Check Tag
• lidé MeSH