The Fanconi anemia proteins functionally interact with the protein kinase regulated by RNA (PKR)

Protein kinase regulated by RNA (PKR) plays critical roles in cell growth and apoptosis and is implicated as a potential pathogenic factor of Alzheimer's, Parkinson's, and Huntington's diseases. Here we report that this proapoptotic kinase is also involved in Fanconi anemia (FA), a disease characterized by bone marrow (BM) failure and leukemia. We have used a BM extract to show that three FA proteins, FANCA, FANCC, and FANCG, functionally interact with the PKR kinase, which in turn regulates translational control. By using a combined immunoprecipitation and reconstituted kinase assay, in which an active PKR kinase complex was captured from a normal cell extract, we demonstrated functional interactions between the FA proteins and the PKR kinase. In primary human BM cells, mutations in the FANCA, FANCC, and FANCG genes markedly increase the amount of PKR bound to FANCC, and this PKR accumulation is correlated with elevated PKR activation and hypersensitivity of BM progenitor cells to growth repression mediated by the inhibitory cytokines interferon-gamma and tumor necrosis factor-alpha. Specific inhibition of PKR by 2-aminopurine in these FA BM cells attenuates PKR activation and apoptosis induction. In lymphoblasts derived from an FA-C patient, overexpression of a dominant negative mutant PKR (PKRK296R) suppressed PKR activation and apoptosis induced by interferon-gamma and tumor necrosis factor-alpha. Furthermore, by using genetically matched wild-type and PKR-null cells, we demonstrated that forced expression of a patient-derived FA-C mutant (FANCCL554P) augmented double-stranded RNA-induced PKR activation and cell death. Thus, inappropriate activation of PKR as a consequence of certain FA mutations might play a role in bone marrow failure that frequently occurred in FA.     

FANCC interacts with Hsp70 to protect hematopoietic cells from IFN-gamma/TNF-alpha-mediated cytotoxicity

The Fanconi anemia (FA) complementation group C gene product (FANCC) functions to protect hematopoietic cells from cytotoxicity induced by interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and double-stranded RNA (dsRNA). Because apoptotic responses of mutant FA-C cells involve activation of interferon-inducible, dsRNA-dependent protein kinase PKR, we sought to identify FANCC-binding cofactors that may modulate PKR activation. We identified the molecular chaperone Hsp70 as an interacting partner of FANCC in lymphoblasts and HeLa cells using 'pull-down' and co-immunoprecipitation experiments. In vitro binding assays showed that the association of FANCC and Hsp70 involves the ATPase domain of Hsp70 and the central 320 residues of FANCC, and that both Hsp40 and ATP/ADP are required. In whole cells, Hsp70-FANCC binding and protection from IFN-gamma/TNF-alpha-induced cytotoxicity were blocked by alanine mutations located in a conserved motif within the Hsp70-interacting domain of FANCC. We therefore conclude that FANCC acts in concert with Hsp70 to prevent apoptosis in hematopoietic cells exposed to IFN-gamma and TNF-alpha.     

Fanconi anemia proteins FANCA, FANCC, and FANCG/XRCC9 interact in a functional nuclear complex

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight complementation groups (A to H). Three FA genes, corresponding to complementation groups A, C, and G, have been cloned, but their cellular function remains unknown. We have previously demonstrated that the FANCA and FANCC proteins interact and form a nuclear complex in normal cells, suggesting that the proteins cooperate in a nuclear function. In this report, we demonstrate that the recently cloned FANCG/XRCC9 protein is required for binding of the FANCA and FANCC proteins. Moreover, the FANCG protein is a component of a nuclear protein complex containing FANCA and FANCC. The amino-terminal region of the FANCA protein is required for FANCG binding, FANCC binding, nuclear localization, and functional activity of the complex. Our results demonstrate that the three cloned FA proteins cooperate in a large multisubunit complex. Disruption of this complex results in the specific cellular and clinical phenotype common to most FA complementation groups.     

FANCC, FANCE, and FANCD2 form a ternary complex essential to the integrity of the Fanconi anemia DNA damage response pathway

Fanconi anemia (FA) is a genetically heterogeneous disorder characterized by bone marrow failure, cancer predisposition, and increased cellular sensitivity to DNA-cross-linking agents. The products of seven of the nine identified FA genes participate in a protein complex required for monoubiquitination of the FANCD2 protein. Direct interaction of the FANCE protein with both fellow FA complex component FANCC and the downstream FANCD2 protein has been observed in the yeast two-hybrid system. Here, we demonstrate the ability of FANCE to mediate the interaction between FANCC and FANCD2 in the yeast three-hybrid system and confirm the FANCE-mediated association of FANCC with FANCD2 in human cells. A yeast two-hybrid system-based screen was devised to identify randomly mutagenized FANCE proteins capable of interaction with FANCC but not with FANCD2. Exogenous expression of these mutants in an FA-E cell line and subsequent evaluation of FANCD2 monoubiquitination and DNA cross-linker sensitivity indicated a critical role for the FANCE/FANCD2 interaction in maintaining FA pathway integrity. Three-hybrid experiments also demonstrated the ability of FANCE to mediate the interaction between FA core complex components FANCC and FANCF, indicating an additional role for FANCE in complex assembly. Thus, FANCE is shown to be a key mediator of protein interactions both in the architecture of the FA protein complex and in the connection of complex components to the putative downstream targets of complex activity.     

Cytoplasmic FANCA-FANCC Complex Interacts and Stabilizes the Cytoplasm-dislocalized Leukemic Nucleophosmin Protein (NPMc)

Eight of the Fanconi anemia (FA) proteins form a core complex that activates the FA pathway. Some core complex components also form subcomplexes for yet-to-be-elucidated functions. Here, we have analyzed the interaction between a cytoplasmic FA subcomplex and the leukemic nucleophosmin (NPMc). Exogenous NPMc was degraded rapidly in FA acute myeloid leukemia bone marrow cells. Knockdown of FANCA or FANCC in leukemic OCI/AML3 cells induced rapid degradation of endogenous NPMc. NPMc degradation was mediated by the ubiquitin-proteasome pathway involving the IBR-type RING-finger E3 ubiquitin ligase IBRDC2, and genetic correction of FA-A or FA-C lymphoblasts prevented NPMc ubiquitination. Moreover, cytoplasmic FANCA and FANCC formed a cytoplasmic complex and interacted with NPMc. Using a patient-derived FANCC mutant and a nuclearized FANCC, we demonstrated that the cytoplasmic FANCA-FANCC complex was essential for NPMc stability. Finally, depletion of FANCA and FANCC in NPMc-positive leukemic cells significantly increased inflammation and chemoresistance through NF-κB activation. Our findings not only reveal the molecular mechanism involving cytoplasmic retention of NPMc but also suggest cytoplasmic function of FANCA and FANCC in NPMc-related leukemogenesis.     

Fanconi anemia protein complex is a novel target of the IKK signalsome

Fanconi anemia (FA), a genetic disorder predisposing to aplastic anemia and cancer, is characterized by hypersensitivity to DNA-damaging agents and oxidative stress. Five of the cloned FA proteins (FANCA, FANCC, FANCE, FANCF, FANCG) appear to be involved in a common functional pathway that is required for the monoubiquitination of a sixth gene product, FANCD2. Here, we report that FANCA associates with the IkappaB kinase (IKK) signalsome via interaction with IKK2. Components of the FANCA complex undergo rapid, stimulus-dependent changes in phosphorylation, which are blocked by kinase-inactive IKK2 (IKK2 K > M). When exposed to mitomycin C, cells expressing IKK2 K > M develop a cell cycle abnormality characteristic of FA. Thus, FANCA may function to recruit IKK2, thus providing the cell a means of rapidly responding to stress.     

The nuclear accumulation of the Fanconi anemia protein FANCE depends on FANCC

The Fanconi anemia (FA) protein FANCE is an essential component of the nuclear FA core complex, which is required for monoubiquitination of the downstream target FANCD2, an important step in the FA pathway of DNA cross-link repair. FANCE is predominantly localized in the nucleus and acts as a molecular bridge between the FA core complex and FANCD2, through direct binding of both FANCC and FANCD2. At present, it is poorly understood how the nuclear accumulation of FANCE is regulated and therefore we investigated the nuclear localization of this FA protein. We found that FANCE has a strong tendency to localize in the nucleus, since the addition of a nuclear export signal does not interfere with the nuclear localization of FANCE. We also demonstrate that the nuclear accumulation of FANCE does not rely solely on its nuclear localization signal motifs, but also on FANCC. The other FA proteins are not involved in the nuclear accumulation of FANCE, indicating a tight relationship between FANCC and FANCE, as suggested from their direct interaction. Finally, we show that the region of FANCE interacting with FANCC appears to be different from the region involved in binding FANCD2. This strengthens the idea that FANCE recruits FANCD2 to the core complex, without interfering with the binding of FANCC.     

Regulation of the Fanconi anemia group C protein through proteolytic modification

The function of the Fanconi anemia group C protein (FANCC) is still unknown, though many studies point to a role in damage response signaling. Unlike other known FA proteins, FANCC is mainly localized to the cytoplasm and is thought to act as a messenger of cellular damage rather than an effector of repair. FANCC has been shown to interact with several cytoplasmic and nuclear proteins and to delay the onset of apoptosis through redox regulation of GSTP1. We investigated the fate and function of FANCC during apoptosis. Here we show that FANCC undergoes proteolytic modification by a caspase into a predominant 47-kDa ubiquitinated protein fragment. Lack of proteolytic modification at the putative cleavage site delays apoptosis but does not affect MMC complementation. These results suggest that FANCC function is regulated through proteolytic processing.     

The Hsp90 chaperone complex is both a facilitator and a repressor of the dsRNA-dependent kinase PKR.

PKR, a member of the eukaryotic initiation-factor 2alpha (eIF-2alpha) kinase family, mediates the host antiviral response and is implicated in tumor suppression and apoptosis. Here we show that PKR is regulated by the heat shock protein 90 (Hsp90) molecular chaperone complex. Mammalian PKR expressed in budding yeast depends on several components of the Hsp90 complex for accumulation and activity. In mammalian cells, inhibition of Hsp90 function with geldanamycin (GA) during de novo synthesis of PKR also interferes with its accumulation and activity. Hsp90 and its co-chaperone p23 bind to PKR through its N-terminal double-stranded (ds) RNA binding region as well as through its kinase domain. Both dsRNA and GA induce the rapid dissociation of Hsp90 and p23 from mature PKR, activate PKR both in vivo and in vitro and within minutes trigger the phosphorylation of the PKR substrate eIF-2alpha. A short-term exposure of cells to the Hsp90 inhibitors GA or radicicol not only derepresses PKR, but also activates the Raf-MAPK pathway. This suggests that the Hsp90 complex may more generally assist the regulatory domains of kinases and other Hsp90 substrates.     

Loss of the Fanconi anemia group C protein activity results in an inability to activate caspase-3 after ionizing radiation

Fanconi anemia (FA) is a human genetic disease featuring cancer predisposition, genetic instability and DNA damage hypersensitivity. Although abnormalities in DNA repair and cell cycle checkpoint have been proposed as the underlying defect in this syndrome, these hypotheses did not provide full explanations of the complex phenotype. Although not exclusive of such possibilities, alterations in the control of apoptosis might account for the pleiotropic phenotype of this syndrome. We and others have previously reported a deregulation of the apoptotic response to mitomycin C, suggesting that the products of the Fanconi anemia group C protein (FANCC) contribute to the regulation of apoptosis. To explore the functional importance of the apoptotic alterations in FA we analyzed biochemical steps of the execution phase of apoptosis stimulated by another DNA damaging agent, the gamma-ray using FA cell lines derived from complementation group C (FA-C) independent patients. It is shown that the poly(ADP-ribose) polymerase, a target of caspase-3, is not cleaved in FA-C after ionizing radiation (IR). Moreover, caspase-3 is not processed in its active form and, its activity is not increased by IR in FA-C cells compared to normal cells. Altogether, these results demonstrate that loss of the FANCC activity results in a deficiency of the IR-induced apoptosis which is due to an inability to activate caspase-3. Our work suggests that apoptosis signaling induced by mitomycin C and IR is subject to common regulation involving the FANCC protein.     

Nucleophosmin interacts with and inhibits the catalytic function of eukaryotic initiation factor 2 kinase PKR

In normal cells the protein kinase PKR effects apoptosis in response to various extra and intracellular cues and can also function to suppress the neoplastic phenotype. Because most neoplastic cells are resistant to certain apoptotic cues, we reasoned that an early molecular event in carcinogenesis or leukemogenesis might be the inactivation of PKR by expression or activation of intracellular PKR inhibitors. Seeking novel PKR-modulating proteins we report here that nucleophosmin (NPM), a protein frequently overexpressed in a variety of human malignancies, binds to PKR, and inhibits its activation. Co-immunoprecipitation and in vitro binding experiments showed that NPM associated with PKR. Kinase assays demonstrated that recombinant NPM inhibited PKR activation in a dose-dependent manner. In addition, purified recombinant NPM was phosphorylated by activated PKR. Most importantly, overexpression of NPM suppressed PKR activity, enhanced protein synthesis, and inhibited apoptosis. Lymphoblasts from patients with Fanconi anemia (FA) expressed low levels of NPM, which correlated with high ground-state activation of PKR and cellular hypersensitivity to apoptotic cues, but enforced expression of NPM in these mutant cells reduced aberrant apoptotic responses. Inhibition of PKR by NPM may be one mechanism by which neoplastic clones evolve in sporadic malignancies and in neoplastic cells arising in the context of the cancer predisposition syndrome, Fanconi anemia.     

FANCE: the link between Fanconi anaemia complex assembly and activity

The Fanconi anaemia (FA) nuclear complex (composed of the FA proteins A, C, G and F) is essential for protection against chromosome breakage. It activates the downstream protein FANCD2 by monoubiquitylation; this then forges an association with the BRCA1 protein at sites of DNA damage. Here we show that the recently identified FANCE protein is part of this nuclear complex, binding both FANCC and FANCD2. Indeed, FANCE is required for the nuclear accumulation of FANCC and provides a critical bridge between the FA complex and FANCD2. Disease-associated FANCC mutants do not bind to FANCE, cannot accumulate in the nucleus and are unable to prevent chromosome breakage.     

A role for the Fanconi anemia C protein in maintaining the DNA damage-induced G2 checkpoint

Fanconi anemia (FA) is a complex, heterogeneous genetic disorder composed of at least 11 complementation groups. The FA proteins have recently been found to functionally interact with the cell cycle regulatory proteins ATM and BRCA1; however, the function of the FA proteins in cell cycle control remains incompletely understood. Here we show that the Fanconi anemia complementation group C protein (Fancc) is necessary for proper function of the DNA damage-induced G2/M checkpoint in vitro and in vivo. Despite apparently normal induction of the G2/M checkpoint after ionizing radiation, murine and human cells lacking functional FANCC did not maintain the G2 checkpoint as compared with wild-type cells. The increased rate of mitotic entry seen in Fancc-/-mouse embryo fibroblasts correlated with decreased inhibitory phosphorylation of cdc2 kinase on tyrosine 15. An increased inability to maintain the DNA damage-induced G2 checkpoint was observed in Fancc -/-; Trp53 -/-cells compared with Fancc -/-cells, indicating that Fancc and p53 cooperated to maintain the G2 checkpoint. In contrast, genetic disruption of both Fancc and Atm did not cooperate in the G2 checkpoint. These data indicate that Fancc and p53 in separate pathways converge to regulate the G2 checkpoint. Finally, fibroblasts lacking FANCD2 were found to have a G2 checkpoint phenotype similar to FANCC-deficient cells, indicating that FANCD2, which is activated by the FA complex, was also required to maintain the G2 checkpoint. Because a proper checkpoint function is critical for the maintenance of genomic stability and is intricately related to the function and integrity of the DNA repair process, these data have implications in understanding both the function of FA proteins and the mechanism of genomic instability in FA.     

Double-stranded RNA-dependent protein kinase (PKR) is a stress-responsive kinase that induces NFkappaB-mediated resistance against mercury cytotoxicity

The interferon-inducible, double-stranded (ds)RNA-dependent protein kinase (PKR) plays a major role in antiviral defense mechanisms where it down-regulates translation via phosphorylation of eukaryotic translation initiation factor 2alpha. PKR is also involved in the activation of nuclear factor kappaB (NFkappaB) through activation of the IkappaB kinase complex. Activation of PKR can occur in the absence of dsRNA and in such case is controlled by intracellular regulators like the PKR-activating protein (PACT), the PKR inhibitor p58(IPK), or heat-shock proteins (Hsp). These regulators are activated by stress stimuli, supporting a role for PKR in response to stress; however the final outcome of PKR activation in stress situations is unclear. We present here evidence that expression and activation of PKR contributes to an increased cellular resistance to mercury cytotoxicity. In two cell lines constitutively expressing PKR (THP-1 and Molt-3), treatment with the PKR inhibitor 2-aminopurine increases their sensitivity to mercury. In contrast, Ramos cells, which do not constitutively express PKR, present an increased resistance to mercury when PKR expression is induced by polyIC or interferon-beta treatment. This protective effect is inhibited by 2-aminopurine. We also show that exposure of Ramos cells to mercury leads to the induction of Hsp70. Treatment of cells with Hsp70 or NFkappaB inhibitors suppresses the PKR-dependent protection. We propose a model where PKR, modulated by Hsp70, activates a NFkappaB-mediated protective pathway. Because the cytotoxicity of mercury is primarily due to the generation of reactive oxygen species, our results suggest a more general function of PKR in the mechanisms of cellular response to oxidative stress.     

Fanconi anemia group C protein prevents apoptosis in hematopoietic cells through redox regulation of GSTP1.

The Fanconi anemia group C protein (FANCC) plays an important role in hematopoiesis by ensuring the survival of hematopoietic progenitor cells through an unknown mechanism. We investigated the function of FANCC by identifying FANCC-binding proteins in hematopoietic cells. Here we show that glutathione S-transferase P1-1 (GSTP1) interacts with FANCC, and that overexpression of both proteins in a myeloid progenitor cell line prevents apoptosis following factor deprivation. FANCC increases GSTP1 activity after the induction of apoptosis. GSTP1 is an enzyme that catalyzes the detoxification of xenobiotics and by-products of oxidative stress, and it is frequently upregulated in neoplastic cells. Although FANCC lacks homology with conventional disulfide reductases, it functions by preventing the formation of inactivating disulfide bonds within GSTP1 during apoptosis. The prevention of protein oxidation by FANCC reveals a novel mechanism of enzyme regulation during apoptosis and has implications for the treatment of degenerative diseases with thiol reducing agents.     

The Fanconi anemia core complex forms four complexes of different sizes in different subcellular compartments

Fanconi anemia (FA) is an autosomal recessive disease marked by congenital defects, bone marrow failure, and cancer susceptibility. FA cells exhibit a characteristic hypersensitivity to DNA crosslinking agents such as mitomycin C. The molecular mechanism for the disease remains elusive, but at least 6 FA proteins are known to be part of what is termed the FA core complex. We used affinity pulldown of FLAG-FANCA to pull down the FA complex from whole-cell extracts. Mass spectroscopy detected previously reported FA-binding proteins, including FANCA, FANCC, FANCG, cdc2, and GRP94, thus validating the approach. We further describe a method of purification of the FA core complex in an effort to find novel complex components and biochemical activity to define the function of the complex. By using conventional chromatographic fractionation of subcellular preparations, we report: (i) the FA core complex exists in a cytoplasmic form at 500-600 kDa; (ii) a larger, 750-kDa cytoplasmic form is seen only at mitosis; (iii) a nuclear form achieves a size of 2 megaDaltons; and (iv) a distinct 1-megaDalton FA core complex exists bound to chromatin that contains phosphorylated FANCA after undergoing DNA damage. We are continuing our analysis using mass spectroscopy in an effort to characterize novel binding proteins. These data will help define the biochemical role of the FA core complex in normal cell physiology as well as in the development of the FA disease state.     

Phosphorylation of fanconi anemia (FA) complementation group G protein, FANCG, at serine 7 is important for function of the FA pathway

Fanconi anemia (FA) is an autosomal recessive disease of cancer susceptibility. FA cells exhibit a characteristic hypersensitivity to DNA cross-linking agents. The molecular mechanism for the disease is unknown as few of the FA proteins have functional motifs. Several post-translational modifications of the proteins have been described. We and others have reported that the FANCG protein (Fanconi complementation group G) is phosphorylated. We show that in an in vitro kinase reaction FANCG is radioactively labeled. Mass spectrometry analysis detected a peptide containing phosphorylation of serine 7. Using PCR-mediated site-directed mutagenesis we mutated serine 7 to alanine. Only wild-type FANCG cDNA fully corrected FA-G mutant cells. We also tested the effect of human wild-type FANCG in Chinese hamster ovary cells in which the FANCG homologue is mutant. Human FANCG complemented these cells, whereas human FANCG(S7A) did not. Unexpectedly, FANCG(S7A) bound to and stabilized the endogenous forms of the FANCA and FANCC proteins in the FA-G cells. FANCG(S7A) aberrantly localized to globules in chromatin and did not abrogate the internuclear bridges seen in the FA-G mutant cells. Phosphorylation of serine 7 in FANCG is functionally important in the FA pathway.     

The Fanconi anaemia gene FANCC promotes homologous recombination and error-prone DNA repair

The Fanconi anemia (FA) protein FANCC is essential for chromosome stability in vertebrate cells, a feature underscored by the extreme sensitivity of FANCC-deficient cells to agents that crosslink DNA. However, it is not known how this FA protein facilitates the repair of both endogenously acquired and mutagen-induced DNA damage. Here, we use the model vertebrate cell line DT40 to address this question. We discover that apart from functioning in homologous recombination, FANCC also promotes the mutational repair of endogenously generated abasic sites. Moreover in these vertebrate cells, the efficient repair of crosslinks requires the combined functions of FANCC, translesion synthesis, and homologous recombination. These studies reveal that the FA proteins cooperate with key mutagenesis and repair processes that enable replication of damaged DNA.