Search

Heat shock protein 90 (Hsp90) regulates diverse signaling pathways. Emerging evidence suggests that Hsp90 inhibitors, such as 17-allylamino-17-demethoxygeldanamycin (17-AAG), enhance DNA damage-induced cell death, suggesting that Hsp90 may regulate cellular responses to genotoxic stress. However, the underlying mechanisms are poorly understood. Here, we show that the Fanconi anemia (FA) pathway is involved in the Hsp90-mediated regulation of genotoxic stress response. In the FA pathway, assembly of 8 FA proteins including FANCA into a nuclear multiprotein complex, and the complex-dependent activation of FANCD2 are critical events for cellular tolerance against DNA cross-linkers. Hsp90 associates with FANCA, in vivo and in vitro, in a 17-AAG–sensitive manner. Disruption of the FANCA/Hsp90 association by cellular treatment with 17-AAG induces rapid proteasomal degradation and cytoplasmic relocalization of FANCA, leading to impaired activation of FANCD2. Furthermore, 17-AAG promotes DNA cross-linker–induced cytotoxicity, but this effect is much less pronounced in FA pathway-defective cells. Notably, 17-AAG enhances DNA cross-linker–induced chromosome aberrations. In conclusion, our results identify FANCA as a novel client of Hsp90, suggesting that Hsp90 promotes activation of the FA pathway through regulation of intracellular turnover and trafficking of FANCA, which is critical for cellular tolerance against genotoxic stress.

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, cancer susceptibility and cellular hypersensitivity to DNA crosslinkers such as mitomycin C (MMC). Current evidence indicates that formation of a nuclear multiprotein complex (core complex) including six FA proteins FANCA/C/E/F/G/L is essential for FANCL/PHF9 ubiquitin ligase-mediated activation of FANCD2 into a monoubiquinated form, which participates in BRCA1 and FANCD1/BRCA2-mediated DNA repair (the FA/BRCA pathway). Subcellular distribution of FANCA plays a crucial role in the regulation of the FA/BRCA pathway. However, the underlying molecular mechanisms are not fully understood. To address this issue, we tried to identify FANCA-associated proteins. To this end, Flag-FANCA ectopically expressed in HeLa cells was immunopurified from the cytoplasmic fraction, using anti-Flag antibody-conjugated sepharose beads. Analysis of the immune complex on SDS polyacrylamide gel electrophoresis revealed that several proteins of Mr. 60–70 kD specifically associated with Flag-FANCA. These proteins were identified as FANCG and Hsc (heat shock cognate protein) 70 by LC-MS/MS. Immunoblot analysis showed that FANCA associated with Hsp90 as well as Hsc70. Hsc70 is an ATP-dependent molecular chaperone highly homologous to Hsp70 and often cooperates with Hsp90 to form a chaperone machinery involved in the regulation of diverse protein functions. Patient-derived FANCA mutants failed to bind FANCC but associated with larger amounts of Hsc70 than wt-FANCA, indicating that the interaction between FANCA and Hsc70 is not mediated by FANCC, as suggested by previous observations of the interaction of FANCC with Hsp70. To study the role of Hsc70 and Hsp90 in the regulation of FANCA, we examined effects of a dominant-negative (dn) form of Hsc70 with inactivated ATPase activity, and a specific inhibitor of Hsp90, 17-AAG (a geldanamycin analog). Overexpression of dn-Hsc70 inhibited nuclear localization of FANCA and inhibited its core complex formation, whereas wt-Hsc70 did not. 17-AAG induced cytoplasmic distribution and proteosomal degradation of FANCA and suppressed FANCD2 mono-ubiquitination. Taken together, these results suggest that Hsc70/Hsp90 chaperone machinery interacts with FANCA and regulates its subcellular distribution and stability, thereby controlling activation of the FA/BRCA pathway.