ABSTRACT
CS proteins may have additional functions beyond their role in D N A repair, possibly an involvem ent in transcription following genotoxic attack.8CSA encodes a W D repeat protein that apparently does not interact or cofractionate with CSB protein.9 C SB is a 168-kDa protein that belongs to the SW I2/SN F2 family o f chromatin remodelling proteins,10 exhibits ATPase activity11 and has conserved helicase motifs.3 CSB can interact with the X P B /E R C C 311 and p62 components o f T F IIH ,12 RNA polymerase II (pol II),9,13 the N E R protein X P G 14 and p53.15 X PB /ER C C 3 and p62 are two o f the six core subunits o f the dual function transcription/repair factor T F IIH that is shared between the N E R and pol II basal transcription apparatus.16 This protein has been shown to play a role in both remodelling the chromatin structure and disrupting protein-D N A interactions.17,18 CSB is also part o f and able to stimulate the enzymatic activity o f complexes containing pol I and pol II. These complexes also contain T F IIH , a basal transcription factor.11,19 Additionally, CSB cells were shown to have a defect in transcription, both in vivo and in vitro, even in the absence o f stress such as U V exposure.20 Significandy, an adequate model, describing at which stage o f transcription CSB is operational beyond its involvement in T C R , is missing.It is clear that an impasse provided by an arrested pol II would severely affect transcription. In that sense, CS could, indeed, be considered the Transcription syndrome*. But the arrest o f transcription also provides a strong signal for a cell-death (apoptosis) pathway.21 In this context, CS could also be characterized as a disease o f excessive cell death by apoptosis; a disease that adversely affect rapidly metabolizing cells, such as neurons, which generate high levels o f reactive oxygen species. The apoptosis model could also, in principle, explain the problems o f stunted growth and neurological deterioration. It might also explain why CS patients are no t prone to skin cancer, even with severe sunlight sensitivity; after all, dead cells do no t form tumors. Interaction o f CSB with p53The p53 tum or suppressor gene is the most commonly altered gene in cancer22 and germline transmission o f a single m utant p53 allele is frequendy associated with Li-Fraumeni syndrome, a disorder characterized by a predisposition to a variety o f cancers.23 The anti-neoplastic effect o f p53 occurs, at least in part, via inhibiting propagation o f cells w ith unrepaired D N A damage by enhancing D N A repair, prom oting cell cycle arrest and /or facilitating apoptosis.24 p53 accumulates in a dose-dependent manner in cells following exposure to genotoxic agents, including U V light, through transcriptional and posttranscriptional mechanisms.25 The induction o f p53 is thought to lead to the transactivation o f p53-responsive genes. In addition, p53 may have transactivation-independent functions.24,26CSB protein can interact with p53,15 suggesting that p53 may play a role in transcription-coupled NER. The carboxy-terminal dom ain o f p53 (C T D ) is sufficient for interaction with CSB and deletion o f the p53 C T D prevents association o f p53 with CSB.27 Interestingly, p53 mutants from hum an cancer cells can bind to CSB proteins to a similar or greater extent than wild type p53, in contrast to the p53 m utants that have generally lost the sequence-specific D N A binding activity needed for transcriptional activation. In this regard, it is conceivable that m utant p53 could bind to im portant cellular targets that are normally occupied by wild type p53, resulting in a dom inant negative phenotype in the presence o f wild type p 53 or a dom inant oncogenic phenotype in the absence o f wild type p53.28 In agreement with this concept, cells homozygous for m utant p53 are genetically more unstable than heterozygous and wild-type cells.29,30 However, it is not clear how the binding o f p53 to CSB proteins and hence the potential m odulation o f N ER activity, relates to the phenotype o f genomic stability. A t least two scenarios can be postulated: CSB m ight recruit p53 to sites o f transcription-coupled repair, or p53 m ight recruit CSB to sites o f D N A damage, perhaps using the ability o f the p53 C T D to recognize a D N A heteroduplex31 or single-stranded D N A .32In view o f the role o f CSB on p53-responsive gene expression, the inability to transcribe genes in CSB cells, following genotoxic attack, raises questions concerning the role o f the CSB protein in general and its relationship with p53. The recovery o f RNA synthesis after genotoxic attack
occurs following, at least, two distinct transcriptional pathways.33 The first one, which is directed by p53, concerns only p53-responsive genes and does no t require CSB. The second one is mediated by CSB. In UV-irradiated CSB cells, p53-responsive genes are activated and the recovery o f the transcription o f housekeeping genes is affected. Moreover, in CSB cells, the vitamin-D dependent response is diminished, independently o f U V irradiation. In this context, p53 activator possess, in addition to its D N A binding property, some additional functions exhibited by CSB. CSB was found to play a role in remodelling the chromatin structure,17 which could facilitate the subsequent access o f the general transcriptional machinery to certain genes, such as housekeeping or VDR-targeted genes. Consequently, p53 recruits specific chromatin remodelling factors to allow the initiation o f the transcription for its own targeted genes. This observation would explain why p53-targeted genes are devoid o f CSB, assuming the fact that CSB could function to convert the conform ation o f the prom oter into an accessible structure. JNK Phosphorylation o f CSBC -jun-N H 2 kinases (JN K ) are amongst the UV-activated protein kinases that play an importan t role in cellular stress response via the phosphorylation o f c-jun, ATF2 and p53.34'36 JN K activity in the cell is tightly controlled by both, protein kinases and protein phosphatases. Various types o f stimuli activate JN K through phosphorylation by the dual specificity kinases M KK 4 or M KK 7.3738 In contrast, mitogens and stress stimuli can inactivate JN K through induction o f the expression o f JN K phosphatases, which include serine/threonine phosphatases, tyrosine phosphatases and dual specificity (threonine/tyrosine) phosphatases.39-41 Phosphorylation o f c-jun by JN K was shown to be im portant for its stability and for m ediating its activities in transcription, replication and transformation.42 Activation o f stress-related protein kinases by U V irradiation involves cell surface receptors and membrane components, including src tyrosine kinases as well as Grb2-SOS and Raf-1.43"46 JN K was shown capable o f interacting w ith Grb2 and w ith p21ras, which serve as docking sites for its phosphorylation by upstream kinases.44,47 JN K activation by U V irradiation requires the presence o f nuclear D N A lesion.48,49 In this regard, study has shown a link between improper repair activity o f transcribed genes and expression/activities o f A P I and ATF family members as representatives o f UV-responsive element-binding proteins.50 JN K pathway is thus one o f the critical sensors and downstream effectors o f persistent D N A damage in transcribing genes.51 The interrelation between N E R and transcription has been docum ented by findings in which D N A damage was preferentially repaired transcribing strands o f active genes.52,53 Importantly, analyses o f primary fibroblast cell lines from patients w ith CSB revealed poor JN K activation after U V irradiation when compared w ith repair-proficient, normal hum an fibroblast cell lines.54 Improper JN K activation is expected to affect activities o f its substrates, ATF2, ELK1, c-jun and p53, which were shown to participate in the U V response in various pathways, including changes in the rate o f cell cycle distribution,55,56 growth arrest55 and changes in the rate o f D N A synthesis57 and D N A repair.58The role o f JN K in apoptosis provides an alternative biological end point by which these kinases contribute to the U V response.59"61 However, more recent studies showed that JN K signaling can regulate apoptosis both positively and negatively, depending on the cell type, cellular context and the nature and dose o f treatment.62,63 Strong and sustained JN K activation is predominantly associated w ith induction or enhancement o f apoptosis, whereas transient JN K activation can result in cell survival.63,64 In line w ith the presumed pro-apoptotic function o f sustained JN K activation, the low dose UV-induced apoptotic response in CSB cells was preceded by delayed and long-lasting JN K activation.51 Moreover, this sustained JN K activation was accompanied by prolonged phosphorylation and activation o f the endogenous JN K substrate c-jun, a com ponent o f transcription factor AP-1 that can act as a pro-apoptotic effector o f JN K .65,66 Interestingly, the impaired JN K activation found in CSB cells was limited to U V radiation and was n o t seen after exposure to other forms o f stress, such as induced by H 20 2 or heat shock.54 These finding suggest that there is an impaired upstream signal transduction com ponent which is part o f the pathway required to mediate U V effects. In this regard, it is reported that different forms o f stress use
different cellular pathways to mediate JN K activation for U V versus heat shock in transformed mouse fibroblasts.67 D N A lesions could mediate activation o f protein kinases by recruiting respective repair gene products to the damaged site. Deficient repair activity could result from improper recruitment o f the necessary N ER components (i.e., ERCC6, the m utant repair gene in CSB cells), yielding impaired JN K activation. Indeed, damaged D N A failed to activate JN K in vitro assays performed with CSB proteins.54 This impairment further suggests that a signal that is em itted from damaged D N A to activate JN K is not properly sent (or received) in CSB cells.The role o f the M APK family members p38 and ERK in DNA-damage-induced apoptosis in CSB cells remains unclear. Preliminary experiments using the p38 inhibitor SB203580 indicate that p38 does no t play a significant role in the induction o f apoptosis. Inhibition o f ERK activation via the inhibitor U 0126 also does no t seem to affect UV-induced apoptosis in the hum an CSB cells, suggesting that ERK might play an auxiliary role. Finally, it remains to be established whether JNK-dependent apoptosis in CSB cells involves its transcription factor substrates c-Jun and ATF-2 and /o r its target genes ATF-3 and c-fos. Interestingly, induction o f ATF-3 can accelerate apoptosis in certain cell types,68 whereas c-fos is anti-apoptotic in UV-treated mouse embryo fibroblasts.69,70 Functional Role for Tyrosine Phosphorylation of CRB by c-AblThe c-Abl tyrosine kinase is a ubiquitously expressed proto-oncogene that contains SH3, SH 2 and catalytic domains in its N-term inal region.71 C ontained w ithin the C-term inus are nuclear localization motifs, a bipartite DNA-binding dom ain and F-and G-actin binding domains. Alternative splicing results in the expression o f two c-Abl isoforms ( la and lb ), both o f which are detectable in the nucleus and the cytoplasm. Recent studies show that c-Abl takes on an auto-inhibitory conformation and its activation requires posttranslational modifications such as phosphorylation and myristoylation.72,73 Physiological functions dependent on c-Abl remain largely elusive. Certain insights have been derived from the findings that c-Abl is activated in response to D N A damage.74 Nuclear c-Abl interacts w ith the DN A-dependent protein kinase (D N A -PK )/ Ku complex.75,76 Phosphorylation o f c-Abl by the catalytic subunit DNA-PKcs induces c-Abl activity.76 O ther works have demonstrated that c-Abl is activated by the product o f the gene ataxia telangiectasia m utated (ATM).77,78 Activation o f nuclear c-Abl by D N A damage contributes to induction o f apoptosis by mechanisms in part dependent on the p53, p73 and Rad9.79'83 Nuclear c-Abl also contributes to D N A damage-induced activation o f the JNK/stress-activated protein kinase and p38 mitogen-activated protein kinase pathways.74,84*86 In addition, the finding that c-Abl interacts w ith the Rad51 protein in response to D N A damage has supported a role for c-Abl in coordinating recombinational D N A repair with the induction o f apoptosis.87 Recent studies have dem onstrated that c-Abl shuttles between the nucleus and the cytoplasm.88,89 In contrast, oncogenic forms o f Abl, including v-Abl and Bcr-Abl, localize exclusively in the cytoplasm and induce cellular transformation by prom oting proliferation and inhibiting apoptotic cell death.89 91 In this regard, recent studies have suggested that cytoplasmic c-Abl confers cell proliferation and survival.92 By marked contrast, activation o f nuclear c-Abl by many sources o f D N A damage is associated w ith inhibition o f cell growth and induction o f apoptosis.89,93 These findings indicate that the intracellular localization o f c-Abl is im portant in dictating either survival or apoptotic responses. However, the molecular mechanism behind the nucleo-cytoplasmic shuttling o f c-Abl was unknown until recently. O ur recent studies revealed that c-Abl translocates into the nucleus in response to D N A damage or oxidative stress.94Structure-function analysis o f c-Abl indicates that it has a protein /protein interaction SH3 domain, which is likely to participate in interactions between c-Abl and its kinase substrates. SH3 domains bind preferentially to proline-rich motifs such as P-X-X-P.95,96 Tyrosine phosphorylation, particularly by c-Abl tyrosine kinase, has been reported to play a role in the regulation o f certain D N A repair proteins. c-Abl-mediated phosphorylation o f DNA-topoisomerase I (Topo I) at Tyr268 in vitro and in cells conferred activation o f the Topo I function.97 Moreover, activation o f c-Abl by treatm ent o f cells w ith ionizing radiation was associated w ith c-Abl-dependent
phosphorylation o f Topo I and induction o f Topo I activity.97 Rad51 is a key element o f recom-binational D N A repair and its activity is regulated by phosphorylation o f the tyrosine residue at position 315 by c-Abl tyrosine kinase.98 Importandy, CSB is a phospho-protein with an N-terminal proline-rich region and as expected, a recent study demonstrated that CSB binds to c-Abl and is a substrate for c-Abl tyrosine kinase.99 Oxidative stress stimulates c-Abl-mediated phosphorylation o f CSB and this event is blocked by STI-571, a specific inhibitor o f c-Abl kinase. Oxidative stress-induced c-Abl auto-phosphorylation and tyrosine phosphorylation o f CSB appear to require CSB ATPase, because it was no t observed in cells expressing an ATPase-deficient m utant o f CSB. CSB and c-Abl colocalize in the nucleus and redistribute in the nucleolus in cells treated with H 20 2. This colocalization is inhibited by pretreatm ent with STI-571. The redistribution o f c-Abl and CSB may facilitate interaction between the two proteins and tyrosine phosphorylation o f CSB. Finally, tyrosine-phosphorylated CSB may serve as a signal for repair proteins to localize to D N A damage and may help m aintain active transcription in the nucleolus. Taken together, tyrosine phosphorylation o f CSB plays an im portant role in regulating and/or coordinating the cellular response to genotoxic stress. Concluding RemarksThe multisystem character o f CS and the complexity o f the genotype-phenotype relationship suggest that the underlying gene products are involved in basal cellular processes. Through the participation o f CSB in several direct and functional protein interactions, the CS phenotype becomes sensitive to genetic variation affecting concentration, activity and structure o f its many interaction partners.
