Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least 11 complementation groups (A, B, C, D1, D2, E, F, G, I, J, and L), and eight FA genes have been cloned. The FANCD1 gene is identical to the breast cancer susceptibility gene, BRCA2. The FA proteins cooperate in a common pathway, but the function of BRCA2/FANCD1 in this pathway remains unknown. Here we show that monoubiquitination of FANCD2, which is activated by DNA damage, is required for targeting of FANCD2 to chromatin, where it interacts with BRCA2. FANCD2-Ub then promotes BRCA2 loading into a chromatin complex. FANCD2 ؊/؊ cells are deficient in the assembly of DNA damage-inducible BRCA2 foci and in chromatin loading of BRCA2. Functional complementation with the FANCD2 cDNA restores BRCA2 foci and its chromatin loading following DNA damage. BRCA2؊/؊ cells expressing a carboxy-terminal truncated BRCA2 protein form IR-inducible BRCA2 and FANCD2 foci, but these foci fail to colocalize. Functional complementation of these cells with wild-type BRCA2 restores the interaction of BRCA2 and FANCD2. The C terminus of BRCA2 is therefore required for the functional interaction of BRCA2 and FANCD2 in chromatin. Taken together, our results demonstrate that monoubiquitination of FANCD2, which is regulated by the FA pathway, promotes BRCA2 loading into chromatin complexes. These complexes appear to be required for normal homology-directed DNA repair.Fanconi anemia (FA) is an autosomal recessive disease characterized by cancer susceptibility and cellular hypersensitivity to DNA cross-linking agents, such as mitomycin C (MMC) and cisplatin. Eight FA genes have been cloned, corresponding to FA subtypes A, C, BRCA2/D1, D2, E, F, G, and L. The encoded FA proteins cooperate in a common pathway-the FA/BRCA pathway (6, 15). Six of the FA proteins (A, C, E, F, G, and L) assemble in a multisubunit nuclear complex (8,18,19), required for the activation (monoubiquitination) of the downstream FANCD2 protein (9). Whether the E3 ubiquitin ligase, BRCA1, also participates in the monoubiquitination of FANCD2 remains unclear (28). The activated FANCD2 protein is subsequently targeted to nuclear foci (9).Whether BRCA2 participates with other FA proteins in this pathway has remained uncertain. On the one hand, BRCA2Ϫ/Ϫ patients share most of the clinical and cellular phenotypic features of other FA subtypes, suggesting a common pathway (13). On the other hand, BRCA2-deficient cells have a more severe defect in homologous recombination repair (21, 30), suggesting that BRCA2 may have functions independent of the FA pathway. Also, BRCA2Ϫ/Ϫ patients generally have a more severe clinical phenotype, with earlier onset of cancer (11). Although two-hybrid studies suggest that BRCA2 may interact with other FA proteins, such as FANCG (14), direct biochemical evidence linking BRCA2 to other FA proteins is lacking.Unlike other FA proteins, BRCA2 has a well-defined role in homology-directed DNA repair (HDR). BRCA2 binds to single-and double-stranded DNA (31), interac...

Summary Following genotoxic stress, cells activate a complex kinase-based signaling network to arrest the cell cycle and initiate DNA repair. p53-defective tumor cells rewire their checkpoint response and become dependent on the p38/MK2 pathway for survival after DNA damage, despite a functional ATR-Chk1 pathway. We used functional genetics to dissect the contributions of Chk1 and MK2 to checkpoint control. We show that nuclear Chk1 activity is essential to establish a G2/M checkpoint, while cytoplasmic MK2 activity is critical for prolonged checkpoint maintenance through a process of post-transcriptional mRNA stabilization. Following DNA damage, the p38/MK2 complex relocalizes from nucleus to cytoplasm where MK2, phosphorylates hnRNPA0, to stabilize Gadd45α mRNA, while p38 phosphorylates and releases the translational inhibitor TIAR. In addition, MK2 phosphorylates PARN, blocking Gadd45α mRNA degradation. Gadd45α functions within a positive feedback loop, sustaining the MK2-dependent cytoplasmic sequestration of Cdc25B/C to block mitotic entry in the presence of unrepaired DNA damage. Our findings demonstrate a critical role for the MK2 pathway in the post-transcriptional regulation of gene expression as part of the DNA damage response in cancer cells.

Heat shock factor 1 (HSF1) regulates the transcription of molecular chaperone hsp genes. However, the cellular control mechanisms that regulate HSF1 activity are not well understood. In this study, we have demonstrated for the first time that human HSF1 binds to the essential cell signaling protein 14-3-3. Binding of HSF1 to 14-3-3 occurs in cells in which extracellular signal regulated kinase (ERK) is activated and blockade of the ERK pathway by treatment with the specific ERK pathway inhibitor PD98059 in vivo strongly suppresses the binding. We previously showed that ERK1 phosphorylates HSF1 on serine 307 and leads to secondary phosphorylation by glycogen synthase kinase 3 (GSK3) on serine 303 within the regulatory domain and that these phosphorylation events repress HSF1. We show here that HSF1 binding to 14-3-3 requires HSF1 phosphorylation on serines 303 and 307. Furthermore, the serine phosphorylation-dependent binding of HSF1 to 14-3-3 results in the transcriptional repression of HSF1 and its sequestration in the cytoplasm. Leptomycin B, a specific inhibitor of nuclear export receptor CRM1, was found to reverse the cytoplasmic sequestration of HSF1 mediated by 14-3-3, suggesting that CRM1/14-3-3 directed nuclear export plays a major role in repression of HSF1 by the ERK/GSK3/14-3-3 pathway. Our experiments indicate a novel pathway for HSF1 regulation and suggest a mechanism for suppression of its activity during cellular proliferation.Heat shock transcription factor 1 (HSF1) is the mammalian regulator of the heat shock response and activates the transcription of heat shock protein (Hsp) molecular chaperone genes (43,47,49). Inactivation of the murine hsf1gene has been shown to confer a complex phenotype, indicating an essential function for hsf1 in growth, in development, and in acute response to stress (35). Disruption of hsf1 (i.e., hsf1 Ϫ/Ϫ ) in mouse embryonic fibroblasts leads to a profound loss of thermotolerance and markedly increased susceptibility to heatinduced apoptosis (11,35). hsf1 is required as a maternal factor during the early cleavage stage of development in the Ϫ/Ϫ mouse embryo (11). hsf1-deficient mice can survive to adulthood but display severe defects in the chorioallantoic placenta that result in increased prenatal lethality (56). In addition, the aging process is associated with degeneration of the heat shock response, and transcriptional activity of HSF1 protein was significantly reduced with age in a cell-free system, as well as in isolated hepatocytes (26). Understanding the processes involved in HSF1 regulation may therefore aid in delineating its role in resistance to stress, development, and aging.Under normal conditions, cellular HSF1 exists in a predominantly transcriptionally repressed state (44, 59). Such HSF1 is monomeric, is constitutively phosphorylated, and lacks the ability to bind the cis-acting heat shock elements (HSEs) located in the promoters of Hsp genes (50, 55). Induction of transcriptional activity by heat shock then results in the conversion of HSF1 from in...