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Other Proteins and Their Interactions with FA Gene Products

and .

Fanconi anemia (FA) is a genetically heterogeneous disorder, consisting of at least nine complementation groups (FA-A, -B, -C, -D1, -D2, -E, -F, -G, —L).1-3 To date, eight FA genes, FANC-A, -C, -D1 (BRCA2), -D2, -E, -F, -G, —L, have been identified, but the function of each gene product remains unclear. One approach to clarifying these actions is the identification of binding partners, which may yield indirect clues if the biologic function of the binding partner is known. In this chapter, we will review the putative interacting partners of FANCC and FANCA, the first two FA gene products identified.

FANCC-Binding Proteins

Proteins Involved in Oxidative Stress Metabolism: GSTP1 and RED

A number of early reports suggested involvement of reactive oxygen species (ROS) in inducing chromosomal damage and cell death in FA cells.4,5 Some investigators have also suggested that the sensitivity of FA cells to mitomycin C (MMC) and diepoxybutane (DEB) may be attributed to aberrant redox cycling and oxidative stress.6 This hypothesis was supported by the observation that glutathione S-transferase P1-1 (GSTP1), which is involved in intracellular detoxification of toxic and carcinogenic substances, binds to FANCC, resulting in an increase in the catalytic activity of GSTP1.7 Loss of this activation in FA-C cells was found to lead to increased oxidative damage. GSTs are involved in the detoxification of DEB.8 In addition, it had been reported that wild-type FANCC interacts with and reduces the catalytic activity of NADPH cytochrome P450 reductase (RED), an integral microsomal enzyme that can transfer electrons from NADPH to cytochrome P450 isozymes and cytochrome C.9 Since MMC is activated by intracellular reduction with RED, FANCC may protect the cell from active MMC by attenuating its activation. Moreover, reduction of RED activity might prevent the generation of ROS, thereby protecting cells from oxidative stress. Taken together, the oxygen-dependent and redox-related sensitivity of FA-C cells to DEB and MMC may be due to the interaction of FANCC with GSTP1 and RED. Recently, FANCG was also reported to bind to cytochrome P450 2E1 (CYP2E1), a member of the P450 superfamily, suggesting a possible role of FANCG in protection against oxidative DNA damage.10

Transcriptional Factor and Signal Transducer: FAZF and STAT1

Using the yeast two-hybrid system, Hoatlin et al (1999) identified a novel 486 amino-acid gene product, which was named Fanconi anemia zinc finger (FAZF), as interacting with FANCC.11 FAZF contains three C-terminal zinc finger domains as well as a conserved N-terminal BTB/POZ domain, which is homologous to the promyelocytic leukemia zinc finger protein, PLZF. PLZF was known to act as a transcriptional repressor when associated with nuclear corepressors,12 and FAZF is thought to act in a similar fashion, binding to the same sequences as PLZF. Since PLZF has an essential role in limb patterning, acting as a growth inhibitory and pro-apoptotic factor in the limb bud,13 it has been proposed that the FANCC-FAZF interaction may somehow be associated with growth inhibition and developmental anomalies, which are common clinical features of FA.

Other investigators have reported that FA-C cells are hypersensitive to interferon gamma (IFN-γ).14,15 IFN-γ stimulates activation of the transcription factor STAT1 through docking to activated IFN-γ receptors, resulting in STAT1 phosphorylation.16 Pang et al (2000) have presented evidence that FANCC binds to STAT1 to aid receptor docking and, in the absence of functional FANCC, STAT1 phosphorylation was not observed. They propose that FANCC plays a role in controlling STAT1-activated transcription, which provides a mitogenic stimulus and activation of apoptotic responses through control of interferon response factor-1. In the absence of FANCC, an imbalance between these pathways might result in apoptosis of hematopoietic progenitor cells and bone marrow failure.17

Cell Cycle Regulator: cdc2

Several studies have suggested that FA cells have a molecular defect in cell cycle progression, which has been ascribed to G2 phase arrest.18,19 For cell cycle progression from G2 to M phase, the cyclin-dependent kinase, cdc2, assembles with cyclin A and cyclin B and phosphorylates multiple substrates in the pathway.20,21 FANCC was reported to coimmunoprecipitate with cdc2, and expression of FANCC protein appeared to be subject to cell cycle-specific regulation, with peak levels observed at the G2/M transition period.22

FANCA-Binding Proteins

From yeast two-hybrid experiments using a C-terminal FANCA fragment as bait, three FANCA-binding proteins, BRG1,23 IKK224 and SNX5,25 have been identified.

A Component of Chromatin-Remodeling Enzyme Complex: BRG1

BRG1, brm-related gene 1, is a key component of the SWI/SNF complex, which remodels chromatin structure through a DNA-dependent ATPase activity.26 FANCA was demonstrated to associate with the endogenous SWI/SNF complex.23 Additionally, a significant increase in the molecular chaperone GRP94 was observed among BRG1-associated factors isolated from FANCA-deficient cells, which was not seen in either normal control cells or FANCA-deficient cells complemented by wild-type FANCA. GRP94 was previously identified as a FANCC-binding protein.27

As discussed elsewhere, an active form of FANCD2 was found to colocalize with the breast cancer susceptibility protein BRCA1 within ionizing radiation-induced nuclear foci.28 BRCA1 was initially identified as mutated in patients with familial breast and ovarian cancer, and its putative functions include DNA damage repair,29 regulation of the G2/M phase checkpoint,30 and regulation of centrosome duplication.31 BRCA1 has also been reported to directly interact with BRG1 and is associated with the human SWI/SNF complex.32 A functional interaction between BRCA1 and BRG1 was suggested by the observation that p53-mediated stimulation of transcription by BRCA1 was completely abrogated by coexpression of a dominant-negative BRG1 mutant.

Interaction between the FA protein complex (FANCA/FANCC/FANCG) and chromatin has been described.33 According to the report, the FA protein complex was excluded from chromatin of cells in mitosis (M phase), precisely analogous to the behavior of BRG1. Taken together, cell cycle-specific interactions between the chromatin-FA protein complex and between FANCA-BRG1-BRCA1 may allow the repair machinery access to sites of damaged DNA or specific gene targets (fig. 1).

Figure 1. FANCA and BRG1.

Figure 1

FANCA and BRG1.

A Component of IKK Signalsome: IKK2

FA cells have been shown to be hypersensitive to apoptosis induced by tumor necrosis factor-alpha (TNF-α).34,35 TNF-α mediates this signal through activation of the caspase cascade, whereas an anti-apoptotic signal is concurrently generated by activation of the NF-κB transcription factor.36,37 NF-κB activation is in turn regulated by proteasomal degradation of inhibitor κB (IκB) by the IκB kinase complex (IKK complex) (fig. 2). FANCA was recently found to associate with the IKK complex through a direct interaction between FANCA and IKK2, a core component of the IKK complex.24 In vitro kinase assays suggested that components of the FANCA protein complex are substrates of IKK2. These studies suggest a functional role for the IKK complex in the biological pathway mediated by FANCA. The IKK complex is responsive to both ROS signaling and to DNA damage and exerts its protective effect by activation of NF-κB, resulting in up-regulation of genes involved in redox regulation, DNA repair and resistance to apoptosis.37 Interaction between FANCA and the IKK complex may therefore explain aberrant apoptosis signaling in FA cells.

Figure 2. The IKK complex pathway.

Figure 2

The IKK complex pathway.

Intracellular Trafficking Molecule: SNX5

Among the family of sorting nexins (SNXs), SNX1 was first identified as a protein that bound to the cytoplasmic domain of the epidermal growth factor (EGF) receptor.38 Based on its homology to a yeast protein (Mvp1p), known to be involved in targeting hydrolases to the vacuole, it was hypothesized that SNX1 was involved in EGF receptor degradation in lysosomes. 39 Over-expression of SNX1 resulted in accelerated degradation of EGF receptor.38 Subsequently, several proteins were cloned and identified as members of the SNX family on the basis of containing an approximately 100 amino-acid region, termed the phox homology (pX) domain,40 which is also present in the yeast proteins Mvp1p, Vps5p and Grd19p. All three yeast proteins are involved in intracellular trafficking of target proteins. The pX domain has since been recognized as an interaction domain with phosphoinositide (PI) lipids.40

A new member of the SNX family, SNX5 was identified as a binding partner for FANCA,25 although the functional significance of this association remains unclear. One possibility is that SNX5 is involved in subcellular trafficking of FANCA. FANCA is known to undergo specific phosphorylation, which may be important for its function.41 Recently, the Akt kinase was reported to regulate FANCA phosphorylation.42 Remarkably, Akt is a member of a family of protein kinases that contain the pleckstrin homology (pH) domain, which also binds a broad range of PI lipids.43 These observations suggest that FANCA may be subject to regulation by PI signaling or metabolism.

As an example, phosphoinositide 3-kinase (PI-3K) plays an essential role in Akt activation through the production of phosphatidylinositol-3,4,5-trisphosphate, a lipid second messenger that somehow signals translocation of Akt to the plasma membrane, where it is phosphorylated and activated by phosphoinositide-dependent kinase-1 (PDK-1) and possibly other kinases. It is possible that the interaction between FANCA and SNX5 may localize FANCA to particular subcellular domains, thus subjecting FANCA to regulation by Akt (fig. 3).

Figure 3. FANCA and SNX5.

Figure 3

FANCA and SNX5.

FA Protein Complex and Human α Spectrin II

With regards to assembly of the FA protein complex, human nonerythroid αII spectrin (αSpIIΣ*) has been shown to play an important role.44 Levels of this protein were reduced in the nuclei of FA cells, and αSpIIΣ* was postulated to act as a scaffold to align or enhance interactions between FA proteins and DNA repair proteins such as ERCC1 and XPF (fig. 4).

Figure 4. FA protein complex and human α spectrin II.

Figure 4

FA protein complex and human α spectrin II.

Conclusion

The FA proteins appear to interact with a variety of proteins, and these associations suggest involvement in a number of functions. However, protein components of two major pathways appear more than once in connection with the FA gene products: chromatin remodeling and stress-mediated kinase complexes. There may be cross-talk, for example, between the FA protein complex, the SWI/SNF complex, and the IKK signalsome (fig. 5). According to this model, the FA protein complex may act to process or repair DNA damage, as a result of interaction with the SWI/SNF complex, BRCA1 and FANCD2, and also modulate apoptosis, through associations with the IKK complex and Akt kinase. The relationship between stress-mediated transcriptional regulation (IKK and NF-κB pathways) and proteins involved in homologous recombination repair (such as BRCA1, BRCA2, etc.) has been well documented45 and serves to underscore how FA proteins may serve pleiotropic functions. Further investigations will be needed to clarify these various functions and the molecular pathophysiology of FA.

Figure 5. Pleiotropic Functions.

Figure 5

Pleiotropic Functions.

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