The Fanconi anemia pathway requires FAA phosphorylation and FAAyFAC nuclear accumulation (mitomycin Cyleukemiaycancer susceptibility)

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight complementation groups (A–H). Two FA genes, corresponding to complementation groups A and C, have been cloned, but the function of the FAA and FAC proteins remains unknown. We have recently shown that the FAA and FAC proteins bind and form a nuclear complex. In the current study, we analyzed the FAA and FAC proteins in normal lymphoblasts and lymphoblasts from multiple FA complementation groups. In contrast to normal controls, FA cells derived from groups A, B, C, E, F, G, and H were defective in the formation of the FAAyFAC protein complex, the phosphorylation of the FAA protein, and the accumulation of the FAAyFAC protein complex in the nucleus. These biochemical events seem to define a signaling pathway required for the maintenance of genomic stability and normal hematopoiesis. Our results support the idea that multiple gene products cooperate in the FA Pathway. Fanconi anemia (FA) is an autosomal recessive disease characterized by genomic instability, cancer susceptibility, and progressive bone marrow failure (refs. 1–3; for ref. 3 see section, “Fanconi anemia”). Somatic cell fusion studies have defined at least eight genetic complementation groups (FA-A through FA-H; refs. 4–6), displaying similar phenotypes, suggesting a functional relationship of the FA genes. The genes corresponding to FA-A and FA-C have been cloned (7–9), and mutations in FAA and FAC account for 80% of patients with FA (6, 10). The FAA and FAC proteins have no sequence similarity to other proteins in GenBank, and their biochemical functions remain unknown. We have recently determined that FAA and FAC bind and form a protein complex in the nucleus (11). The FA proteins may therefore cooperate in some nuclear function, such as DNA repair, DNA replication, or RNA splicing. Cells derived from patients with FA display multiple phenotypic abnormalities. FA cells are hypersensitive to bifunctional alkylating agents, such as diepoxybutane and mitomycin C (MMC), suggesting a defect in DNA repair. FA cells also exhibit abnormal cell-cycle progression (12–14) and reduced cell survival (15–19). Many of these abnormalities are also evident in primary cells derived from mice homozygous for a disrupted fac gene (16, 20). Currently, how the absence of the FAA protein, the FAC protein, or the FAAyFAC protein complex leads to these cellular abnormalities is unknown. Little is known regarding the nature of the binding interaction between the FAA and FAC proteins. The binding may be a direct protein–protein interaction or may be an indirect interaction, mediated by other adaptor proteins. Regulated posttranslational modifications of the FAA or FAC protein, such as phosphorylation, may also be required for interaction of the FA proteins. It is not yet known whether the FAAyFAC binding interaction is constitutive or inducible under various cellular conditions or stresses. Interaction between the FAA and FAC proteins has functional importance. A patient-derived missense mutation in the FAC protein, FACL554P, prevents the formation of the FAAyFAC complex (11), suggesting that protein binding may be required for the normal (non-FA) cellular phenotype. Whether the interaction of FAA and FAC is required for nuclear translocation of the protein complex also is currently unclear. The expression and binding of FAA and FAC in the various FA complementation groups has not yet been examined. To explore the functional importance of the FAAyFAC protein interaction further, we analyzed FAAyFAC binding, FAA phosphorylation, and FAAyFAC nuclear accumulation in lymphoblast lines derived from normal adult controls or patients with FA. Our results show that the FAA protein is phosphorylated in normal cells and FA-D cells but not in FA cells derived from groups A, B, C, E, F, G, and H. Moreover, the phosphorylation of FAA correlated with both the binding of FAA to FAC and the nuclear accumulation of FAA and FAC. Our results suggest that FAA phosphorylation is an important regulatory event controlling the FA signaling pathway. MATERIALS AND METHODS Characterization of Cell Lines. FA lymphoblast lines and PD7 cells (normal control) were cultured as described (21–23). All FA cell lines used in this study were sensitive to MMC as described (6, 7, 11). FA lymphoblast lines were also analyzed by transduction with retroviral vectors containing FAA or FAC cDNAs (24). As expected, FAA and FAC cDNAs failed to complement the MMC sensitivity of FA cells derived from groups B, D, E, F, G, and H. Production of pMMP-FAA and pMMP-FAC Retroviral Supernatants and Infection of Lymphoblast Lines. The FAA cDNA or FAC cDNA was subcloned into the retroviral vector, pMMP (25), as described (11, 24). For production of pMMPFAA or pMMP-FAC retrovirus, 293GPG helper cells (25) were grown to 90% confluence in 10-cm dishes and electroporated by lipofection for 8 h at 37°C with 10 mg of plasmid DNA in 6 ml of Opti-MEM (GIBCO) containing 8.7 mlyml LipoThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y9513085-6$2.00y0 PNAS is available online at www.pnas.org. This paper was submitted directly (Track II) to the Proceedings office. Abbreviations: FA, Fanconi anemia; MMC, mitomycin C; wt, wild type. §To whom reprint requests should be addressed. e-mail: aodandrea@ farber.harvard.edu.