![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Molecular and Cellular Biology, September 2003, p. 6385-6395, Vol. 23, No. 18
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.18.6385-6395.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224,1 Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 021152
Received 7 February 2003/ Returned for modification 2 April 2003/ Accepted 13 June 2003
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
5' exonuclease activity. These findings suggest a novel signaling pathway by which c-Abl mediates WRN nuclear localization and catalytic activities in response to DNA damage. |
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
5' helicase, 3'5' exonuclease, and DNA-dependent ATPase activities (17, 18). The human syndromes defective in the RecQ helicase genes, including Werner, Bloom, and Rothmund-Thomson syndromes, are autosomal recessive disorders that share a common feature of genomic instability associated with segmental progeroid and cancer predisposition (27). However, Werner syndrome (WS) displays more symptoms of normal aging than others and is considered a model system for segmental progeroid (26). WS cells are hypersensitive to certain DNA cross-linking therapeutic drugs and, to a lesser extent, ionizing radiation (33, 34, 52). WS cells also show defects in resolving recombinational intermediates and in maintaining the stability of broken DNA ends (31, 35, 37). It has been shown that WRN relocalizes from the nucleolus to the nucleoplasm in response to DNA damage (16, 38); however, the mechanism of this WRN trafficking is not understood. A number of protein partners of WRN have been identified. WRN interacts physically and functionally with replication protein A, Ku70, Ku80, the catalytic subunit of DNA-PK (DNA-PKCS), p53, DNA polymerase 
, Bloom syndrome protein (BLM), and FEN-1 (reviewed in reference 5). The known functions of these proteins suggest that WRN is likely involved in pathways of DNA replication, recombination, and DNA repair. The nuclear form of the ubiquitously expressed c-Abl tyrosine kinase is activated by genotoxic stress, including DNA double strand breaks and cross-links (23). In response to ionizing radiation, the ataxia telangiectasia mutated (ATM) kinase activates nuclear c-Abl (3, 43). Another pathway of c-Abl activation is mediated via interaction with and phosphorylation by DNA-PK, a complex containing DNA, Ku70, Ku80, and DNA-PKCS (20, 22). Activation of the nuclear c-Abl by DNA damage contributes to apoptosis by mechanisms that partly depend on p53, p73, and Rad9 (1, 15, 53, 55, 57). In addition, cellular responses to DNA damage involve interaction of c-Abl with DNA repair proteins, including Rad51, Rad52, BRCA1, and a UV-damaged DNA binding protein (9, 14, 24, 56). Tyrosine phosphorylation by c-Abl plays important regulatory roles in the DNA damage response. Phosphorylation of Rad51 by c-Abl inhibits its strand exchange activity (56), and phosphorylation of DNA-PKCS by c-Abl dissociates the Ku heterodimer from DNA-PK (20, 22). Recently, an ATM-dependent BRCA1 phosphorylation by c-Abl has been identified (14). Thus, c-Abl appears to function in mediating pathways involved in homologous recombination and/or nonhomologous end-joining (NHEJ).
Almost all patients with chronic myeloid leukemia (CML) and a subset of those with acute lymphocytic leukemia carry the constitutively activated BCR-ABL tyrosine kinase, a fusion product of a reciprocal chromosome translocation (40). Structurally, c-Abl tyrosine kinase activity is self-suppressed by its N-terminal sequence (32). For BCR-ABL, this inhibitory sequence is removed, resulting in an uncontrolled, constitutive activation of its tyrosine kinase activity that contributes to CML. BCR-ABL is antiapoptotic when it resides in the cytoplasm but becomes proapoptotic when it binds to the compound STI-571 (Gleevec or Imatinib) and, in turn, promotes the BCR-ABL relocalization to the nucleus (48). The 2-phenylaminopyrimidine derivative STI-571 binds to the kinase domain of the Abl kinase with an exceptionally high affinity, resulting in an inhibition of its tyrosine kinase activity (42). Cells expressing BCR-ABL show increased chromosomal aberrations and decreased levels of DNA-PKCS (12). In contrast, expression of BCR-ABL in mouse myeloid cells stimulates Rad51 expression and DNA recombinational repair (46). Therefore, understanding the molecular mechanisms of DNA metabolism in CML may provide insight into strategies for therapeutic intervention.
While WRN can be serine/threonine phosphorylated by DNA-PK (21, 52), there has been no report of tyrosine phosphorylation of WRN. Recent observations show that, in K562 lymphoblasts from CML patients, WRN distributes throughout the nucleus instead of the nucleolus, where WRN primarily resides in normal cells (38). Therefore, Abl tyrosine kinase may play a specific role in modulating the biological function of WRN. Increasing evidence suggests that WRN plays a role in recombinational repair (34, 37). In addition, nuclear c-Abl is also likely to be involved in this process because it acts as a negative regulator of Rad51, Rad52, and BRCA1, three proteins that promote recombinational repair (14, 24, 56). Both WS and CML cells show increased chromosomal aberrations (5, 12). However, in contrast to WS cells, CML cells are resistant to the DNA cross-linker mitomycin C and to gamma irradiation (46). We thus hypothesized that WRN may interact with the Abl tyrosine kinase in the DNA damage response. Here, we provide evidence for a functional and physical interaction between WRN and c-Abl, including WRN relocalization in response to DNA damage, suggesting that this protein-protein interaction participates in a shared pathway of genome surveillance.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Immunofluorescence. Exponentially growing HeLa cells on glass coverslips were treated with bleomycin (40 µg/ml) for 0 to 30 min. Some cells were pretreated with STI-571 (1 µM) for 24 h as indicated. Cells were washed with phosphate-buffered saline, fixed with ice-cold 3.7% paraformaldehyde for 10 min, permeabilized with 0.3% Triton X-100 at room temperature for 10 min, and incubated with rabbit antiserum against WRN (ab200; Novus, Littleton, Colo.) and mouse anti-nucleolin antibody (MS-3; Santa Cruz). 4',6'-Diamidino-2-phenylindole (DAPI) was used to stain the nuclei. The immunostaining was visualized by a fluorescence microscope (Zeiss Axiovert 200 M), and images were processed by using deconvolution with the software AxioVision, version 3.1.
GST pull-down experiments and ELISA. The GST-WRN and GST-c-Abl fragments and GST were incubated with purified c-Abl and WRN, respectively, and the adsorbents were analyzed by SDS-PAGE as described previously (6). The membrane was analyzed by immunoblotting with the monoclonal anti-Abl or anti-WRN antibody, followed by amido black staining. Enzyme-linked immunosorbent assays (ELISA) were performed as previously described (50). Briefly, either bovine serum albumin (BSA) or full-length c-Abl (0.6 pmol) was coated onto 96-well plates and incubated with full-length WRN (1.2 pmol) in the presence or absence of ethidium bromide (20 ng/µl) for 2 h at 37°C. After extensive washing, the bound WRN was detected with anti-WRN antibodies (Novus) and followed by horseradish peroxidase-conjugated secondary antibody detection.
In vitro kinase assay and in vivo phosphorylation.
For in vitro kinase assays, the affinity-purified WRN and GST-Crk (aa 120 to 225 or 120 to 212) were incubated with the c-Abl fragment in the presence of [
-32P]ATP in a kinase buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM EGTA, 2 mM dithiothreitol) for 15 min at 28°C. Either the tyrosine phosphatase LAR or the c-Abl kinase inhibitor STI-571 (Novartis Pharma AG) (13) was used as a control. For immunocomplex kinase assays, the anti-c-Abl immunoprecipitates from MEFs were added with WRN and incubated in the presence of the phosphatidylinositol-3 kinase inhibitor wortmannin (100 nM) and [-32P]ATP in a kinase buffer (53). Alternatively, the wortmannin (100 nM)-treated anti-WRN immunoprecipitates were incubated with [-32P]ATP in the kinase buffer. Phosphorylated proteins were separated by SDS-PAGE and analyzed by autoradiography and immunoblotting. For in vivo phosphorylation, 293T cells (2.4 x 106 cells/10-cm-diameter culture dish) were cotransfected with 1.5 µg of WRN (16) and 6 µg of wild-type c-Abl or the dominant-negative c-Abl (K-R) mutant vectors (41) for 30 h with the PolyFect reagent (Qiagen, Valencia, Calif.). The cells were lysed in the lysis buffer and followed by SDS-PAGE and immunoblotting to detect phosphotyrosine, c-Abl, and WRN.
WRN activities. Helicase and exonuclease activity assays were conducted as previously described (29). For assays with purified proteins, the c-Abl fragment was preincubated with WRN in a reaction buffer (40 mM Tris-HCl [pH 8.0], 4 mM MgCl2, 5 mM dithiothreitol, 2 mM ATP, 0.1 mg of BSA/ml) for 5 min at 28°C prior to the addition of the 22- or 34-bp forked duplex (5' end-labeled with 32P) as a substrate for measuring WRN helicase or exonuclease, respectively. For assays with immunocomplexes, the anti-immunoglobulin G (IgG) or anti-WRN (Santa Cruz) immunoprecipitates (200 µg of protein) were washed three times in lysis buffer and two times in the WRN activity reaction buffer, followed by the addition of the exonuclease substrate. Helicase and exonuclease products were run on 12% native and 14% denaturing polyacrylamide gels, respectively, visualized with a PhosphorImager, and quantitated by using ImageQuant 5.2 software. For exonuclease assays, the percent digestion was calculated based on the following formula: (intensity of the digested bands/intensity of the digested plus undigested bands) x 100%. Statistics were performed by using the t test.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To determine whether WRN physically associates with c-Abl, we performed ELISA with His-tagged purified full-length proteins (Fig. 1B). The results demonstrated that WRN bound c-Abl directly. Because the interaction was unaffected by the presence of ethidium bromide, this binding was not dependent on DNA. As a control, WRN did not bind to BSA. Therefore, WRN binds to c-Abl directly in vitro.
To map this direct binding, we incubated purified c-Abl with bacterially expressed WRN fragments fused to GST. Analyses of the adsorbates demonstrated that c-Abl bound to the N-terminal GST-WRN (aa 1 to 120 and 114 to 240) and the central GST-WRN (aa 500 to 946) fragments but not to the acidic GST-WRN (aa 239 to 499), the C-terminal GST-WRN (aa 949 to 1432) fragment, or GST alone (Fig. 1C). To map the region of c-Abl that binds to WRN, we incubated purified WRN with two c-Abl signaling domains, SH2 and SH3, fused to GST. Analyses of the adsorbates demonstrated that WRN bound to the GST-c-Abl-SH3 domain but not to the GST-c-Abl-SH2 domain or GST alone (Fig. 1D). Equal amounts of GST and the GST fragments for incubation were confirmed by amido black staining (data not shown). Taken together, our data indicate that c-Abl binds to two regions of WRN containing the exonuclease and helicase domains, and WRN binds to the SH3 domain of c-Abl.
WRN is tyrosine phosphorylated and dissociates from c-Abl after bleomycin treatment followed by WRN relocalization. Next, we were interested in exploring whether the cellular association between WRN and c-Abl differs during the process of a cellular DNA damage response pathway. The radiomimetic therapeutic agent bleomycin leads to DNA strand breaks, WRN serine/threonine phosphorylation, and WRN relocalization (21, 22, 38). We therefore tested whether the cellular association between WRN and c-Abl is affected by this compound. Treatment of HeLa cells with bleomycin at 10 µg/ml resulted in a time-dependent decrease of the amount of WRN in the anti-c-Abl immunoprecipitates (Fig. 2A, left panels). When HeLa cells were treated with a higher dose of bleomycin (40 µg/ml), we found a complete disruption of the association between WRN and c-Abl 10 min after the treatment (Fig. 2A, right panels). c-Abl protein levels in the immunoprecipitates remained unchanged, except for those from cells at 60 min after the high-dose bleomycin treatment (Fig. 2A). Thus, bleomycin treatment disrupts the in vivo association between WRN and c-Abl. In comparison with the input control, it is estimated that about 10% of WRN is c-Abl associated in the unstressed cells.
|
Next, we tested for the location of the endogenous WRN by immunostaining in a time course study. While nucleolin remains in the nucleolus regardless of the bleomycin treatment (40 µg/ml), a proportion of WRN was found in the nucleoplasm at 10 min, followed by a significant WRN translocation at 30 min (Fig. 2D). Twenty-four hours after removing bleomycin, a significant proportion of the nucleoplasmic WRN returned to the nucleolus (Fig. 2D), and there was no detectable WRN tyrosine phosphorylation in either the soluble or the insoluble fraction (data not shown). Strikingly, pretreatment of cells with STI-571 (1 µM, 24 h) attenuated the bleomycin-induced WRN relocalization, suggesting a functional link between the DNA damage-induced nuclear c-Abl kinase activity and WRN relocalization (Fig. 2D). We observed that, at 10 min after bleomycin treatment, the majority of WRN was found in the insoluble fraction and in the nucleolus by immunoblotting and immunofluorescence methods, respectively. It is likely that the tyrosine-phosphorylated WRN is released during the process of soluble protein extraction. Collectively, these results suggest the following sequence of events in response to DNA damage: (i) WRN is tyrosine phosphorylated, (ii) it dissociates from c-Abl, and (iii) it relocalizes from the nucleolus to the nucleoplasm.
WRN is tyrosine phosphorylated by c-Abl in vitro and in vivo.
To determine whether WRN is a substrate for c-Abl, we incubated purified WRN with a c-Abl fragment containing the SH2 and tyrosine kinase domains (Fig. 1D, map) in the presence of [-32P]ATP. Analysis of the products by autoradiography showed that c-Abl tyrosine kinase phosphorylated WRN in vitro, whereas there was no detectable phosphorylation of WRN when the c-Abl was heat inactivated or when the phosphorylation reactions were reversed by incubation with LAR (Fig. 3A, upper left panel). Immunoblotting with the same membrane demonstrated that WRN was tyrosine phosphorylated and that total WRN protein levels were not affected by the treatment (Fig. 3A, middle and bottom panels). The c-Abl fragment was autophosphorylated and was resistant to the tyrosine phosphatase treatment (data not shown). As positive and negative controls, the c-Abl fragment phosphorylated GST-Crk (aa 120 to 225) but not the GST-Crk (aa 120 to 212), which does not contain tyrosine phosphorylation sites (Fig. 3A, right panel). The WRN phosphorylation by c-Abl was also attenuated in the presence of STI-571 (Fig. 3B).
|
To test whether WRN is phosphorylated by c-Abl in vivo, we cotransfected 293T cells with expression vectors for WRN and wild-type c-Abl or the kinase-inactive c-Abl(K-R) mutant. Results from immunoblotting showed similar amounts of c-Abl protein and of WRN protein in the different cotransfected cells (Fig. 4A). Analysis of anti-WRN immunoprecipitates with an anti-phosphotyrosine antibody demonstrated WRN tyrosine phosphorylation by c-Abl, but this phosphorylation was dramatically reduced in cells expressing the kinase-inactive c-Abl (K-R) (Fig. 4B). Some bands are nonspecific, as they appear at comparable amounts in all three lanes. Therefore, WRN is an in vivo substrate of c-Abl.
|
|
80% of the substrate (Fig. 6B, lane 3), and incubation with increasing amounts of the c-Abl fragment resulted in a complete inhibition of the WRN exonuclease activity (Fig. 6B, lanes 4 to 7). c-Abl alone did not digest the WRN exonuclease substrate (Fig. 6B, lane 2). These results demonstrate that WRN phosphorylation by Abl kinase inhibits its exonuclease activity on the 34-bp forked duplex DNA. Next, we used a 22-bp forked duplex substrate (Fig. 6C) (29) to determine whether tyrosine phosphorylation of WRN affects its helicase activity. WRN (1.0 nM) alone almost completely unwound the substrate (Fig. 6C, lane 2), whereas c-Abl alone displayed no unwinding activity (Fig. 6C, lane 1). Preincubation of WRN with c-Abl resulted in a c-Abl dose-dependent inhibition (up to 87% inhibition) (Fig. 6, lane 5) of WRN helicase activity (Fig. 6C, lanes 3 to 6). It should be noted that, similar to BCR-ABL, lack of the N-terminal inhibitory sequence makes this c-Abl fragment highly active, so the amounts of the c-Abl fragment used are much smaller than those of WRN. Gel shift experiments demonstrated that the c-Abl fragment did not form a stable complex with either of the two substrates (data not shown), excluding the possibility that the inhibition of WRN activities was due to blockage of the substrate by c-Abl. Therefore, tyrosine phosphorylation of WRN by c-Abl inhibits both its exonuclease and helicase activities.
|
5' direction, and the digestion was enhanced (80% increase, P < 0.05) when the cells were pretreated with STI-571. Similar results were obtained by using a 32-bp, 3'-recessed substrate with one blunt end (10) (data not shown), indicating that this effect is not substrate specific. It should be emphasized that, although the majority of proteins in the immunocomplexes are WRN, we cannot exclude the possibility that other proteins with similar roles in the WRN complex also act on the substrate. These results suggest that treatment of the K562 CML cells with STI-571 increases the exonuclease activity in the WRN complex in vivo. |
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
De novo synthesized WRN is destined to the nucleolus by its nucleolar targeting sequence (49). Some DNA damage agents induce WRN relocalization to foci in the nucleoplasm via a poorly understood mechanism (16, 38). DNA strand breaks induce an ATM- or DNA-PK-dependent c-Abl activation (3, 22), which is likely to tyrosine-phosphorylate WRN in the nucleolus and to release it into the nucleoplasm. Consistent with this notion, in CML cells where Abl kinase is constitutively activated, WRN is distributed throughout the nucleus (38). This observation is in line with the association between DNA damage-induced WRN acetylation and its relocalization (4), as c-Abl is required for some DNA damage-induced acetylation events (11). As both c-Abl and BLM interact with WRN and are part of the BRCA1-associated genome surveillance complex (BASC) (50, 51), WRN may dissociate from BASC after DNA damage, as does c-Abl (14). It is likely that WRN exists in a complex with c-Abl in the nucleolus together with other BASC components prior to DNA damage (2). It is unclear why WRN relocalization induced by 4-nitroquinoline-1-oxide or serum depletion can be prevented by a tyrosine phosphatase inhibitor, Na3VO4, in COS cells (16). This c-Abl/WRN pathway may depend on the stage of cell cycle because DNA damage does not activate c-Abl kinase until cells are committed to enter the S phase (25), and WRN does not localize to the nucleolus in S-phase-arrested cells (16). Collectively, we propose that there is an Abl kinase-mediated cellular response to DNA damage that phosphorylates and translocates the nucleolar WRN to the sites of DNA damage.
What is the biological significance of the inhibition of WRN catalytic activities by c-Abl? Similar to DNA-PK (21, 52), c-Abl also inhibits WRN exonuclease and helicase activities. The ATM kinase is considered an early responder to DNA damage, especially of DNA strand breaks. The bleomycin-induced WRN serine/threonine phosphorylation by DNA-PK appears to be independent of ATM (21, 52). In contrast, ionizing radiation-induced c-Abl activation requires the ATM kinase (3), suggesting that WRN phosphorylation by c-Abl is a separate pathway. NHEJ and homologous recombination are the two major pathways that repair DNA double strand breaks. As DNA-PK is required for NHEJ, the inhibition of WRN catalytic activities due to its phosphorylation by DNA-PK may prevent cells from shuttling damage to this error-prone pathway. In contrast, WRN phosphorylation by activated Abl kinase may facilitate WRN export from the nucleoli while temporarily holding WRN in a catalytically inactive state during the process of relocalization. Dissociation of WRN from c-Abl, and possibly dephosphorylation of WRN by the c-Abl-mediated tyrosine phosphatase (8), may attenuate the tyrosine phosphorylation of WRN such that the inhibition of WRN catalytic activities is reversed at repair foci. As the c-Abl fragment does not bind WRN stably but is sufficient to phosphorylate WRN, the inhibition of WRN catalytic activities appears to be independent of c-Abl binding to WRN. However, there may exist another pathway of WRN in ribosomal DNA metabolism (44), as a small portion of WRN is usually retained in the nucleolus after DNA damage. Therefore, WRN may act as a central coordinating protein of different DNA repair pathways, and its role may be regulated by phosphorylation and other forms of posttranscriptional modification.
In untreated cells, c-Abl localizes to both the nucleus and the cytoplasm while BCR-ABL is exclusively cytoplasmic. Inhibition of tyrosine kinase activity and nuclear export by using STI-571 and leptomycin B, respectively, cause a portion (20 to 25%) of BCR-ABL to reside in the nucleus (48). This raises intriguing questions. Which Abl protein phosphorylates the nuclear WRN, and how does this phosphorylation respond to STI-571 treatment in CML cells? Evidence suggests that cellular localization of c-Abl and BCR-ABL is quite dynamic between the nucleus and the cytoplasm (19, 48), partially due to the coexistence of the nuclear localization and export signals in their C termini (Fig. 1D). It is conceivable that the cytoplasmic BCR-ABL phosphorylates and activates c-Abl when it is in the cytoplasm. After cycling back to the nucleus, c-Abl may phosphorylate WRN. Alternatively, the newly synthesized WRN could be tyrosine phosphorylated prior to translocation to the nucleus. A search for the consensus sequence of the c-Abl phosphorylation motif revealed two putative WRN phosphorylation sites: Tyr640 (VYVTP) and Tyr839 (VIHYGAP). Future experiments should be conducted to verify tyrosine phosphorylation sites on WRN.
As a caretaker of the human genome (28), inactivation of WRN catalytic activities may contribute to increased genomic instability in CML cells. BCR-ABL-expressing cells accumulate more DNA damage than normal cells, and they exhibit an induction of homologous recombination and a reduction of NHEJ (12, 46). Rad51 plays a central role in homologous recombination (47). Prince et al. and Saintigny et al. (35, 37) have demonstrated a coordinated regulation of WRN and Rad51 in the homologous recombination pathway. It has been shown that Rad51 colocalizes with WRN in the nuclear foci in response to DNA damage (38). Interestingly, this Rad51-WRN colocalization is observed in the K562 CML cells where the Abl kinase activity is constitutively activated. Whether this interaction holds true in non-CML cells remains to be determined; however, Abl kinase is likely to mediate this repair pathway (7, 56). As multiple mutations in the kinase domain of BCR-ABL are found in patients who develop Imatinib resistance (39), new therapeutic strategies should also aim at optimizing the DNA repair response in CML.
Importantly, c-Abl tyrosine kinase activity is increased in cells from individuals with BRCA1 mutations (14). As WRN and BRCA1 follow similar kinetics of their interaction with c-Abl, this suggests that an increase in oncogenic tyrosine kinase activity may be associated with the increased cancer predisposition found in WS and other genome instability syndromes. Indeed, genomic instability is common in both normal aging and many types of cancers characterized by activated tyrosine kinase (45). By cDNA microarray analysis, we have recently found that genes transcriptionally affected by normal aging or WRN mutations are very similar (K. J. Kyng and V. A. Bohr, unpublished data). Since WS reflects segmental progeroid in many ways, this type of uncontrolled tyrosine phosphorylation may contribute to the mechanisms of aging and its associated increase in cancer incidence.
|
ACKNOWLEDGMENTS |
|---|
This work was supported in part by grant CA-29431 (to D.K.).
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and their collaboration to induce apoptosis. Nature 399:809-813.[CrossRef][Medline]2. Andersen, J. S., C. E. Lyon, A. H. Fox, A. K. Leung, Y. W. Lam, H. Steen, M. Mann, and A. I. Lamond. 2002. Directed proteomic analysis of the human nucleolus. Curr. Biol. 12:1-11.[CrossRef][Medline]
3. Baskaran, R., L. D. Wood, L. L. Whitaker, C. E. Canman, S. E. Morgan, Y. Xu, C. Barlow, D. Baltimore, A. Wynshaw-Boris, M. B. Kastan, and J. Y. Wang. 1997. Ataxia telangiectasia mutant protein activates c-Abl tyrosine kinase in response to ionizing radiation. Nature 387:516-519.[CrossRef][Medline]
4. Blander, G., N. Zalle, Y. Daniely, J. Taplick, M. D. Gray, and M. Oren. 2002. DNA damage-induced translocation of the Werner helicase is regulated by acetylation. J. Biol. Chem. 277:50934-50940.
5. Brosh, R. M., Jr., and V. A. Bohr. 2002. Roles of the Werner syndrome protein in pathways required for maintenance of genome stability. Exp. Gerontol. 37:491-506.[CrossRef][Medline]
6. Brosh, R. M., Jr., C. von Kobbe, J. A. Sommers, P. Karmakar, P. L. Opresko, J. Piotrowski, I. Dianova, G. L. Dianov, and V. A. Bohr. 2001. Werner syndrome protein interacts with human flap endonuclease 1 and stimulates its cleavage activity. EMBO J. 20:5791-5801.[CrossRef][Medline]
7. Chen, G., S. S. Yuan, W. Liu, Y. Xu, K. Trujillo, B. Song, F. Cong, S. P. Goff, Y. Wu, R. Arlinghaus, D. Baltimore, P. J. Gasser, M. S. Park, P. Sung, and E. Y. Lee. 1999. Radiation-induced assembly of Rad51 and Rad52 recombination complex requires ATM and c-Abl. J. Biol. Chem. 274:12748-12752.
8. Cong, F., S. Spencer, J. F. Cote, Y. Wu, M. L. Tremblay, L. A. Lasky, and S. P. Goff. 2000. Cytoskeletal protein PSTPIP1 directs the PEST-type protein tyrosine phosphatase to the c-Abl kinase to mediate Abl dephosphorylation. Mol. Cell 6:1413-1423.[CrossRef][Medline]
9. Cong, F., J. Tang, B. J. Hwang, B. Q. Vuong, G. Chu, and S. P. Goff. 2002. Interaction between UV-DDB proteins and the c-Abl tyrosine kinase. J. Biol. Chem. 277:34870-34878.
10. Cooper, M. P., A. Machwe, D. K. Orren, R. M. Brosh, D. Ramsden, and V. A. Bohr. 2000. Ku complex interacts with and stimulates the Werner protein. Genes Dev. 14:907-912.
11. Costanzo, A., P. Merlo, N. Pediconi, M. Fulco, V. Sartorelli, P. A. Cole, G. Fontemaggi, M. Fanciulli, L. Schiltz, G. Blandino, C. Balsano, and M. Levrero. 2002. DNA damage-dependent acetylation of p73 dictates the selective activation of apoptotic target genes. Mol. Cell 9:175-186.[CrossRef][Medline]
12. Deutsch, E., A. Dugray, B. AbdulKarim, E. Marangoni, L. Maggiorella, S. Vaganay, R. M'Kacher, S. D. Rasy, F. Eschwege, W. Vainchenker, A. G. Turhan, and J. Bourhis. 2001. BCR-ABL down-regulates the DNA repair protein DNA-PKcs. Blood 97:2084-2090.
13. Druker, B. J., S. Tamura, E. Buchdunger, S. Ohno, G. M. Segal, S. Fanning, J. Zimmermann, and N. B. Lydon. 1996. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat. Med. 2:561-566.[CrossRef][Medline]
14. Foray, N., D. Marot, V. Randrianarison, N. D. Venezia, D. Picard, M. Perricaudet, V. Favaudon, and P. Jeggo. 2002. Constitutive association of BRCA1 and c-Abl and its ATM-dependent disruption after irradiation. Mol. Cell. Biol. 22:4020-4032.
15. Gong, J. G., A. Costanzo, H. Q. Yang, G. Melino, W. G. Kaelin, Jr., M. Levrero, and J. Y. Wang. 1999. The tyrosine kinase c-Abl regulates p73 in apoptotic response to cisplatin-induced DNA damage. Nature 399:806-809.[CrossRef][Medline]
16. Gray, M. D., L. Wang, H. Youssoufian, G. M. Martin, and J. Oshima. 1998. Werner helicase is localized to transcriptionally active nucleoli of cycling cells. Exp. Cell Res. 242:487-494.[CrossRef][Medline]
17. Gray, M. D., J. C. Shen, A. S. Kamath-Loeb, A. Blank, B. L. Sopher, G. M. Martin, J. Oshima, and L. A. Loeb. 1997. The Werner syndrome protein is a DNA helicase. Nat. Genet. 17:100-103.[CrossRef][Medline]
18. Huang, S., B. Li, M. D. Gray, J. Oshima, I. S. Mian, and J. Campisi. 1998. The premature ageing syndrome protein, WRN, is a 3'5' exonuclease. Nat. Genet. 20:114-116.[CrossRef][Medline]
19. Ito, Y., P. Pandey, N. Mishra, S. Kumar, N. Narula, S. Kharbanda, S. Saxena, and D. Kufe. 2001. Targeting of the c-Abl tyrosine kinase to mitochondria in endoplasmic reticulum stress-induced apoptosis. Mol. Cell. Biol. 21:6233-6242.
20. Jin, S., S. Kharbanda, B. Mayer, D. Kufe, and D. T. Weaver. 1997. Binding of Ku and c-Abl at the kinase homology region of DNA-dependent protein kinase catalytic subunit. J. Biol. Chem. 272:24763-24766.
21. Karmakar, P., J. Piotrowski, R. M. Brosh, Jr., J. A. Sommers, S. P. Miller, W. H. Cheng, C. M. Snowden, D. A. Ramsden, and V. A. Bohr. 2002. Werner protein is a target of DNA-dependent protein kinase in vivo and in vitro, and its catalytic activities are regulated by phosphorylation. J. Biol. Chem. 277:18291-18302.
22. Kharbanda, S., P. Pandey, S. Jin, S. Inoue, A. Bharti, Z. M. Yuan, R. Weichselbaum, D. Weaver, and D. Kufe. 1997. Functional interaction between DNA-PK and c-Abl in response to DNA damage. Nature 386:732-735.[CrossRef][Medline]
23. Kharbanda, S., R. Ren, P. Pandey, T. D. Shafman, S. M. Feller, R. R. Weichselbaum, and D. W. Kufe. 1995. Activation of the c-Abl tyrosine kinase in the stress response to DNA-damaging agents. Nature 376:785-788.[CrossRef][Medline]
24. Kitao, H., and Z. M. Yuan. 2002. Regulation of ionizing radiation-induced Rad52 nuclear foci formation by c-Abl-mediated phosphorylation. J. Biol. Chem. 277:48944-48948.
25. Liu, Z. G., R. Baskaran, E. T. Lea-Chou, L. D. Wood, Y. Chen, M. Karin, and J. Y. Wang. 1996. Three distinct signalling responses by murine fibroblasts to genotoxic stress. Nature 384:273-276.[CrossRef][Medline]
26. Martin, G. M., S. N. Austad, and T. E. Johnson. 1996. Genetic analysis of aging: role of oxidative damage and environmental stresses. Nat. Genet. 13:25-34.[CrossRef][Medline]
27. Mohaghegh, P., and I. D. Hickson. 2001. DNA helicase deficiencies associated with cancer predisposition and premature ageing disorders. Hum. Mol. Genet. 10:741-746.
28. Nakayama, H. 2002. RecQ family helicases: roles as tumor suppressor proteins. Oncogene 21:9008-9021.[CrossRef][Medline]
29. Opresko, P. L., J. P. Laine, R. M. Brosh, Jr., M. M. Seidman, and V. A. Bohr. 2001. Coordinate action of the helicase and 3' to 5' exonuclease of Werner syndrome protein. J. Biol. Chem. 276:44677-44687.
30. Orren, D. K., R. M. Brosh, Jr., J. O. Nehlin, A. Machwe, M. D. Gray, and V. A. Bohr. 1999. Enzymatic and DNA binding properties of purified WRN protein: high affinity binding to single-stranded DNA but not to DNA damage induced by 4NQO. Nucleic Acids Res. 27:3557-3566.
31. Oshima, J., S. Huang, C. Pae, J. Campisi, and R. H. Schiestl. 2002. Lack of WRN results in extensive deletion at nonhomologous joining ends. Cancer Res. 62:547-551.
32. Pluk, H., K. Dorey, and G. Superti-Furga. 2002. Autoinhibition of c-Abl. Cell 108:247-259.[CrossRef][Medline]
33. Poot, M., K. A. Gollahon, M. J. Emond, J. R. Silber, and P. S. Rabinovitch. 2002. Werner syndrome diploid fibroblasts are sensitive to 4-nitroquinoline-N-oxide and 8-methoxypsoralen: implications for the disease phenotype. FASEB J. 16:757-758.
34. Poot, M., J. S. Yom, S. H. Whang, J. T. Kato, K. A. Gollahon, and P. S. Rabinovitch. 2001. Werner syndrome cells are sensitive to DNA cross-linking drugs. FASEB J. 15:1224-1226.
35. Prince, P. R., M. J. Emond, and R. J. Monnat, Jr. 2001. Loss of Werner syndrome protein function promotes aberrant mitotic recombination. Genes Dev. 15:933-938.
36. Ren, R., Z. S. Ye, and D. Baltimore. 1994. Abl protein-tyrosine kinase selects the Crk adapter as a substrate using SH3-binding sites. Genes Dev. 8:783-795.
37. Saintigny, Y., K. Makienko, C. Swanson, M. J. Emond, and R. J. Monnat, Jr. 2002. Homologous recombination resolution defect in werner syndrome. Mol. Cell. Biol. 22:6971-6978.
38. Sakamoto, S., K. Nishikawa, S. J. Heo, M. Goto, Y. Furuichi, and A. Shimamoto. 2001. Werner helicase relocates into nuclear foci in response to DNA damaging agents and co-localizes with RPA and Rad51. Genes Cells 6:421-430.[Abstract]
39. Sawyers, C. L. 1999. Chronic myeloid leukemia. N. Engl. J. Med. 340:1330-1340.
40. Sawyers, C. L. 2002. Disabling Abl-perspectives on Abl kinase regulation and cancer therapeutics. Cancer Cell 1:13-15.[CrossRef][Medline]
41. Sawyers, C. L., J. McLaughlin, A. Goga, M. Havlik, and O. Witte. 1994. The nuclear tyrosine kinase c-Abl negatively regulates cell growth. Cell 77:121-131.[CrossRef][Medline]
42. Schindler, T., W. Bornmann, P. Pellicena, W. T. Miller, B. Clarkson, and J. Kuriyan. 2000. Structural mechanism for STI-571 inhibition of abelson tyrosine kinase. Science 289:1938-1942.
43. Shafman, T., K. K. Khanna, P. Kedar, K. Spring, S. Kozlov, T. Yen, K. Hobson, M. Gatei, N. Zhang, D. Watters, M. Egerton, Y. Shiloh, S. Kharbanda, D. Kufe, and M. F. Lavin. 1997. Interaction between ATM protein and c-Abl in response to DNA damage. Nature 387:520-523.[CrossRef][Medline]
44. Shiratori, M., T. Suzuki, C. Itoh, M. Goto, Y. Furuichi, and T. Matsumoto. 2002. WRN helicase accelerates the transcription of ribosomal RNA as a component of an RNA polymerase I-associated complex. Oncogene 21:2447-2454.[CrossRef][Medline]
45. Skorski, T. 2002. Oncogenic tyrosine kinases and the DNA-damage response. Nat. Rev. Cancer 2:351-360.[CrossRef][Medline]
46. Slupianek, A., C. Schmutte, G. Tombline, M. Nieborowska-Skorska, G. Hoser, M. O. Nowicki, A. J. Pierce, R. Fishel, and T. Skorski. 2001. BCR/ABL regulates mammalian RecA homologs, resulting in drug resistance. Mol. Cell 8:795-806.[CrossRef][Medline]
47. Sung, P. 1997. Function of yeast Rad52 protein as a mediator between replication protein A and the Rad51 recombinase. J. Biol. Chem. 272:28194-28197.
48. Vigneri, P., and J. Y. Wang. 2001. Induction of apoptosis in chronic myelogenous leukemia cells through nuclear entrapment of BCR-ABL tyrosine kinase. Nat. Med. 7:228-234.[CrossRef][Medline]
49. von Kobbe, C., and V. A. Bohr. 2002. A nucleolar targeting sequence in the Werner syndrome protein resides within residues 949-1092. J. Cell Sci. 115:3901-3907.
50. von Kobbe, C., P. Karmakar, L. Dawut, P. Opresko, X. Zeng, R. M. Brosh, Jr., I. D. Hickson, and V. A. Bohr. 2002. Colocalization, physical and functional interaction between Werner and Bloom syndrome proteins. J. Biol. Chem. 277:22035-22044.
51. Wang, Y., D. Cortez, P. Yazdi, N. Neff, S. J. Elledge, and J. Qin. 2000. BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures. Genes Dev. 14:927-939.
52. Yannone, S. M., S. Roy, D. W. Chan, M. B. Murphy, S. Huang, J. Campisi, and D. J. Chen. 2001. Werner syndrome protein is regulated and phosphorylated by DNA-dependent protein kinase. J. Biol. Chem. 276:38242-38248.
53. Yoshida, K., K. Komatsu, H. G. Wang, and D. Kufe. 2002. c-Abl tyrosine kinase regulates the human Rad9 checkpoint protein in response to DNA damage. Mol. Cell. Biol. 22:3292-3300.
54. Yu, C. E., J. Oshima, Y. H. Fu, E. M. Wijsman, F. Hisama, R. Alisch, S. Matthews, J. Nakura, T. Miki, S. Ouais, G. M. Martin, J. Mulligan, and G. D. Schellenberg. 1996. Positional cloning of the Werner's syndrome gene. Science 272:258-262.[Abstract]
55. Yuan, Z. M., Y. Huang, M. M. Fan, C. Sawyers, S. Kharbanda, and D. Kufe. 1996. Genotoxic drugs induce interaction of the c-Abl tyrosine kinase and the tumor suppressor protein p53. J. Biol. Chem. 271:26457-26460.
56. Yuan, Z. M., Y. Huang, T. Ishiko, S. Nakada, T. Utsugisawa, S. Kharbanda, R. Wang, P. Sung, A. Shinohara, R. Weichselbaum, and D. Kufe. 1998. Regulation of Rad51 function by c-Abl in response to DNA damage. J. Biol. Chem. 273:3799-3802.
57. Yuan, Z. M., H. Shioya, T. Ishiko, X. Sun, J. Gu, Y. Y. Huang, H. Lu, S. Kharbanda, R. Weichselbaum, and D. Kufe. 1999. p73 is regulated by tyrosine kinase c-Abl in the apoptotic response to DNA damage. Nature 399:814-817.[CrossRef][Medline]
This article has been cited by other articles:
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
, heat-inactivated c-Abl; LAR, a tyrosine phosphatase; +, present; -, absent. (B) Kinase assays were conducted with purified WRN and the c-Abl kinase in the presence of STI-571 (50 nM). (C) Kinase assays were conducted with anti-Abl immunoprecipitates (IP) from c-Abl-/- and wild-type MEFs, with the addition of the purified WRN protein, in the absence or presence of wortmannin (100 nM). Autoradiography and immunoblotting were performed by using the respective antibodies against individual proteins. (D) Kinase assays were conducted by using anti-WRN immunoprecipitates from c-Abl-/- and wild-type MEFs in the presence of wortmannin (100 nM) followed by autoradiography and immunoblotting with the respective antibodies.
