The proto-oncogene vav is expressed solely in hematopoietic cells and plays an important role in cell signaling, although little is known about the proteins involved in these pathways. To gain further information, the Src homology 2 (SH2) and 3 (SH3) domains of Vav were used to screen a lymphoid cell cDNA library by the yeast two-hybrid system. Among the positive clones, we detected a nuclear protein, Ku-70, which is the DNA-binding element of the DNA-dependent protein kinase. In Jurkat and UT7 cells, Vav is partially localized in the nuclei, as judged from immunofluorescence and confocal microscopy studies. By using glutathione S-transferase fusion proteins derived from Ku-70 and coimmunoprecipitation experiments with lysates prepared from human thymocytes and vav is a complicated and interesting molecule because of a number of structural features, several of which have suggested a role for Vav in cell signaling (51). Vav is expressed solely in hematopoietic cells, and its temporal pattern of expression during development precedes and coincides with the onset of hematopoiesis (67). Multiple types of evidence show that Vav is involved in signal transduction. We and others have shown that in T cells, Vav is tyrosine phosphorylated upon activation through the CD2 or the CD3 receptor (8, 44, 52), and it has been suggested that Zap-70 could be responsible for Vav tyrosine phosphorylation (38). Vav is also tyrosine phosphorylated upon activation of B and mast cells through the immunoglobulin M (IgM) antigen receptor (7) and the IgE high-affinity FceRI receptor (44), respectively. Furthermore, Vav is tyrosine phosphorylated upon activation of c-Kit by the steel factor (1) and Flk-2 (16) tyrosine protein kinase receptors, by erythropoietin (Epo) stimulation of the Epo receptor (46), and by interleukin-2, interleukin-3, and alpha interferon treatment of hematopoietic cells (16,20,50). Recently, the generation of mice deficient in vav expression in their lymphoid cells has pointed to the essential role of Vav in antigen receptor-induced proliferation of T and B cells. However, these studies also showed that there are Vav-independent signaling pathways involved in the proliferation of both kinds of cells (24,61,66).Vav has two Src homology 3 (SH3) domains flanking an SH2 domain and a proline-rich region in the amino-terminal SH3 (N-SH3) domain (8, 44). It also contains a cysteine-rich region that displays strong similarity to the zinc butterfly domains of protein kinase C isoforms, a pleckstrin homology region, and a Dbl-like domain with homology to a guanine nucleotide release factor. Therefore, it was postulated that Vav may participate in the activation of small GTP-binding proteins. Vav also has an acidic domain, a leucine zipper motif, a helix-loop-helix domain, and nuclear localization signals, suggesting that Vav could play a role as a nuclear factor (5,14,36,47).The function of Vav in cell signaling is not clear, and little is known about the proteins implicated in these pathways. The proline-rich region of Vav, ...
DNA double-strand break (DSB) repair is not only key to genome stability but is also an important anticancer target. Through an shRNA library-based screening, we identified ubiquitin-conjugating enzyme H7 (UbcH7, also known as Ube2L3), a ubiquitin E2 enzyme, as a critical player in DSB repair. UbcH7 regulates both the steady-state and replicative stress-induced ubiquitination and proteasome-dependent degradation of the tumor suppressor p53-binding protein 1 (53BP1). Phosphorylation of 53BP1 at the N terminus is involved in the replicative stress-induced 53BP1 degradation. Depletion of UbcH7 stabilizes 53BP1, leading to inhibition of DSB end resection. Therefore, UbcH7-depleted cells display increased nonhomologous end-joining and reduced homologous recombination for DSB repair. Accordingly, UbcH7-depleted cells are sensitive to DNA damage likely because they mainly used the errorprone nonhomologous end-joining pathway to repair DSBs. Our studies reveal a novel layer of regulation of the DSB repair choice and propose an innovative approach to enhance the effect of radiotherapy or chemotherapy through stabilizing 53BP1.DNA damage response | UbcH7 | 53BP1 | protein degradation | DSB repair P rompt response to double-strand breaks (DSBs) caused by, for example, ionization radiation (IR), requires sequential and coordinated assembly of DNA damage response (DDR) proteins at damage sites (1). Recent research findings reveal key roles of the tumor suppressor p53-binding protein 1 (53BP1) and BRCA1 in the decision making of DSB repair. 53BP1, together with Rif1, suppress BRCA1-dependent homologous recombination (HR), thereby promoting nonhomologous end-joining (NHEJ) in G1 phase (2-6). Conversely, BRCA1 antagonizes 53BP1/Rif1, favoring HR in S and G2 phases (7,8). In the absence of BRCA1 or with enhanced retention of 53BP1 at DSB sites, cells primarily use the error-prone NHEJ to repair DSBs throughout the cell cycle, which leads to gene rearrangement, cell death, and increased sensitivity to anticancer therapies (9-11). Consistently, BRCA1-null mice are early embryonic lethal (12, 13) and codepletion of TP53BP1 rescued the lethality phenotype of BRCA1-null mice (12)(13)(14).Low expression level of 53BP1 was found to be associated with poor clinical outcome in triple negative breast cancer patients with BRCA1 mutation (12, 15), as well as resistance to genotoxins and poly(ADP-ribose) polymerase inhibitors (12,16,17). This finding is probably because loss of 53BP1 restored HR and promoted cell survival (12-14). Reduced expression of 53BP1 was also observed in tumors from the brain (18), lymph node (19), and pancreas (20). These data indicate that loss of 53BP1 might be a common mechanism for advanced tumors to evade from radiotherapy or chemotherapy. However, molecular mechanisms controlling the protein level of 53BP1 remain less well understood.Here we show that UbcH7, an E2 enzyme involved in the ubiquitin (Ub) pathway, controls the protein stability of 53BP1, thereby determining the DSB repair choice. Loss of UbcH7 sta...
DNA polymerase eta (Pol eta) is a member of the mammalian Y family polymerases and performs error-free translesion synthesis across UV-damaged DNA. For this function, Pol eta accumulates in nuclear foci at replication stalling sites via its interaction with monoubiquitinated PCNA. However, little is known about the posttranslational control mechanisms of Pol eta, which regulate its accumulation in replication foci. Here, we report that the molecular chaperone Hsp90 promotes UV irradiation-induced nuclear focus formation of Pol eta through control of its stability and binding to monoubiquitinated PCNA. Our data indicate that Hsp90 facilitates the folding of Pol eta into an active form in which PCNA- and ubiquitin-binding regions are functional. Furthermore, Hsp90 inhibition potentiates UV-induced cytotoxicity and mutagenesis in a Pol eta-dependent manner. Our studies identify Hsp90 as an essential regulator of Pol eta-mediated translesion synthesis.
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