Abstract

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.

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