Blood, Vol. 93 No. 4 (February 15), 1999:
pp. 1430-1432
CORRESPONDENCE
Regulated Binding of the Fanconi Anemia Proteins, FANCA and FANCC
 |
LETTER |
To the Editor:
In a recent article in BLOOD, Kruyt and
Youssoufian1 examined the cellular localization and
possible interaction of the Fanconi anemia (FA) proteins, FANCA and
FANCC. There are several inconsistencies between their data and our
published work.2 We would like to clarify our results and
offer an explanation for the new discordant data.
Several studies support the existence of a physical interaction between
FANCA and FANCC in the nucleus. First, for lymphoblasts, primary
fibroblasts, and primary bone marrow cells expressing normal
(endogenous) levels of the FANCA and FANCC proteins, we detected a
physical complex of FANCA and FANCC.2 The FANCA/FANCC protein complex was detected by reciprocal immunoprecipitation/Western blotting protocols, with either anti-FANCA or anti-FANCC antisera. Second, the FANCA/FANCC complex was detected in protein fractions from
the cytoplasm and the nucleus of primary cells. The
coimmunoprecipitation was more efficient from nuclear
extracts.3 Other studies have also used confocal microscopy
to localize FANCC to the nucleus.4 Third, the interaction
of FANCA and FANCC in a complex is critical to the function of the
proteins. For lymphoblast lines derived from FA patients, mutant FANCC
proteins fail to bind to FANCA,2 and mutant FANCA proteins
fail to bind to FANCC.5 Functional complementation of these
cells rescued FANCA/FANCC binding. Fourth, new experimental evidence,
independent of the use of anti-FANCA antisera, demonstrated a
FANCA/FANCC complex (I. Garcia-Higuera, unpublished
observation). For these studies, we generated an amino terminal Flag-tagged FANCA protein and expressed this protein in an
FA-A cell line, GM6914. The Flag-tagged FANCA protein corrected the MMC
sensitivity of the transfected cells and cofractionated with FANCC from
an anti-Flag column. Fifth, we have shown that the FANCA protein is a
phosphoprotein and that its phosphorylation correlates with FANCC
binding.5 FANCA is not phosphorylated and the FANCA/FANCC
complex is not detected in FA cells derived from other FA
complementation groups (groups B, E, F, G, and H), suggesting that
products of other FA genes regulate the assembly of the nuclear
complex.5 According to this model (Fig
1), other FA proteins may act
as the kinase or adaptor proteins of the complex.
Despite this overwhelming evidence supporting an FANCA/FANCC complex,
Kruyt and Youssoufian1 have concluded that no such complex
exists. Their failure to detect the complex can be readily explained by
their use of different anti-FANCA and anti-FANCC antisera, different
immunoprecipitation protocols, and different cell lines for their
analysis. Whereas the anti-FANCA antibody used in their study detected
endogenous FANCA protein on Western blot of lymphoblasts, it did not
appear to immunoprecipitate endogenous FANCA protein from cell lines.
The antibody is therefore not suitable for immunoprecipitating
endogenous cellular complexes of FANCA and FANCC. Also, the
investigators performed their immunoprecipitation in RIPA buffer, which
in our hands disrupts FANCA/FANCC complexes. Finally, the investigators
used 293 cells overexpressing FANCA and FANCC. In our experience,
transfected cells overexpressing FANCA and FANCC paradoxically yield
less coimmunoprecipitation of FANCA and FANCC, because overexpressed
free monomeric FANCA and FANCC compete with the endogenous FANCA/FANCC
complex for antibody binding. Also, when FANCA and FANCC were
overexpressed, a corresponding increase in the amount of FANCA/FANCC
complex was not observed, because complex formation appears to be
limited by the expression of other FA gene products.5 In
short, the FANCA/FANCC complex is most readily detected in nuclear
extracts of primary cells, expressing normal levels of the FANCA and
FANCC protein, and with the use of high-affinity antisera capable of immunoprecipitating endogenous (low) levels of FANCA or FANCC protein.
We maintain our conclusion that the FANCA and FANCC proteins physically
and functionally interact, in accordance with our published work.2,3,5 It will be interesting to determine whether other FA proteins, such as the recently cloned FANCG
protein,6 are also components of the nuclear FANCA/FANCC
protein complex.
Irene Garcia-Higuera
Alan D. D'Andrea
Department of Pediatric Oncology
Dana-Farber Cancer
Institute
Harvard Medical School
Boston, MA
| |
REFERENCES |
1.
Kruyt FAE, Youssoufian H:
The Fanconi anemia proteins FAA and FAC function in different cellular compartments to protect against cross-linking agent cytotoxicity.
Blood
92:2229, 1998[Abstract/Free Full Text]
2.
Kupfer GM, Naf D, Suliman A, Pulsipher M, D'Andrea AD:
The Fanconi anemia proteins, FAA and FAC, interact to form a nuclear complex.
Nat Genet
17:487, 1997[Medline]
[Order article via Infotrieve]
3.
Naf D, Kupfer GM, Suliman A, Lambert K, D'Andrea AD:
Functional activity of the Fanconi anemia protein, FAA, requires FAC binding and nuclear localization.
Mol Cell Biol
18:5952, 1998[Abstract/Free Full Text]
4.
Hoatlin ME, Christianson TA, Keeble WW, Hammond AT, Zhi Y, Heinrich MC, Tower PA, Bagby GC Jr:
The Fanconi anemia group C gene product is located in both the nucleus and cytoplasm of human cells.
Blood
91:1418, 1998[Abstract/Free Full Text]
5.
Yamashita T, Kupfer GM, Naf D, Suliman A, Joenje H, Asano S, D'Andrea AD:
The Fanconi anemia pathway requires FAA phosphorylation and FAA/FAC nuclear accumulation.
Proc Natl Acad Sci USA
95:13085, 1998[Abstract/Free Full Text]
6.
de Winter JP, Waisfisz Q, Rooimans MA, van Berkel CGM, Bosnoyan-Collins L, Alon N, Carreau M, Bender O, Demuth I, Schindler D, Pronk JC, Arwert F, Hoehn H, Digweed M, Buchwald M, Joenje H:
The Fanconi anaemia group G gene is identical with human XRCC9.
Nat Genet
20:281, 1998[Medline]
[Order article via Infotrieve]
Response
To the Editor:
Garcia-Higuera and D'Andrea present a model in which FANCA and FANCC
form a complex in the cytoplasm that is mediated by unidentified adaptor molecules.1-3 We have been unable to
confirm the central element of this model, namely, an interaction
between FANCA and FANCC.4 A number of their allusions to
our work are also inaccurate.
(1) Differences in methodology. We tried to recapitulate the
experimental conditions of D'Andrea et al by using a variety of
conditions (ionic and nonionic detergents at different concentrations, physical methods of cell disruption, and different salt concentrations) to detect potentially low-affinity interactions. We used the same pair
of mutant and cDNA-complemented FA-A lymphoblasts used by D'Andrea et
al. Likewise, to perform comparative immunoprecipitations with our own
antibody, we obtained their carboxy-terminal antibody that was used
successfully in their laboratory. None of these strategies yielded a
positive interaction. Garcia-Higuera and D'Andrea point out that this
interaction can only be demonstrated by immunoprecipitation of
endogenous FANCA and FANCC complexes. This argument seems to run
counter to their own data, because they were clearly able to
demonstrate interactions between retrovirally overexpressed FANCA and
FANCC.2 Although it is prudent to be cautious about
methodological differences, we do not believe that there is a fatal
flaw in our experimental strategy.
(2) Location and functional compartments of FA proteins.
Although D'Andrea has recently changed his view on this matter
(compare Kupfer et al1 and Yamashita et al5),
our early studies6,7 as well as theirs5 showed
that FANCC is primarily cytoplasmic. We are aware of one other
published study that addresses this issue.8 Using 293 cells
overexpressing the FANCC cDNA, only a minor pool (~10%) of
the transfected cells showed FANCC protein in the nucleus, whereas the
majority showed cytoplasmic staining. Even if there is a minor pool of
FANCC in the nucleus, a more important consideration relates to the
cellular site of action of FANCC. Previously, we had shown that
cytoplasmic localization was essential for the complementation function
of FANCC, whereas targeting to the nucleus abolished this
function.7 More recently, we looked at the expression of
FANCA and FANCC within individual cells and observed their
movement.4 Whereas FANCC remained in the cytoplasm, FANCA
was found in the nucleus, cytoplasm, or both. To the extent that FANCA
is both nuclear and cytoplasmic, our data agree with that of D'Andrea.
However, there is complete dyssynchrony in their spatial association.
Moreover, when we enriched FANCA either in the nucleus or the
cytoplasm, nuclear localization of FANCA was necessary for its
complementation function.4 The conclusion that FANCA and
FANCC work in different cellular compartments seems inescapable.
(3) Technical and conceptual flaws with the model. D'Andrea
et al relied only on a single strategy (immunoprecipitation followed by
immunoblotting) to demonstrate a putative interaction between FANCA and
FANCC. We believe that additional methods must be used to validate such
interactions, particularly when experiments are being performed in the
context of the cellular milieu rather than with purified proteins.
Ironically, they always choose to split the interacting partners and
present their data on two separate panels, one probed for FANCA and a
second for FANCC. There is no attempt to show both interacting partners
on the same gel. Although this practice may not be unusual, it
frustrates the ability of the reader to assess the integrity of the
data independently, and any claims about the stoichiometry of the
various subunits remains unfounded. There are also conceptual problems
with their model. If complex formation and nuclear translocation "is
limited by the expression of other FA gene products," then
overexpressed FANCA should accumulate in the cytoplasm, a prediction
that is not supported either by their data2 or our
own.4 The model is also unusual in that most nuclear and
nucleolar complexes (eg, the basal transcription machinery,
ribonucleoprotein complexes) reach the nucleus as smaller subunits and
undergo assembly after translocation.
We suggest that any model on the pathogenesis of FA should take into
account the compartmentalization of these proteins based on their
function: FANCC works in the cytoplasm, at least partly in
collaboration with NADPH cytochrome P450 reductase,9
whereas FANCA works in the nucleus4 in collaboration
with currently unknown targets. In a more general sense, although we
cannot exclude collaborations among other FA-related proteins, we do
not believe that direct or indirect interactions between FANCA and
FANCC account for the similarity in the FA phenotype.
Frank
A.E. Kruyt
Hagop Youssoufian
Department of Molecular and
Human Genetics
Baylor College of Medicine
Houston, TX
| |
REFERENCES |
1.
Kupfer GM, Naf D, Suliman A, Pulsipher M, D'Andrea AD:
The Fanconi anemia proteins, FAA and FAC, interact to form a nuclear complex.
Nat Genet
17:487, 1997
2.
Naf D, Kupfer GM, Suliman A, Lambert K, D'Andrea A:
Functional activity of the Fanconi anemia protein FAA requires FAC binding and nuclear localization.
Mol Cell Biol
18:5952, 1998
3.
Yamashita T, Kupfer GM, Naf D, Suliman A, Joenje H, Asano S, D'Andrea A:
The Fanconi anemia pathways requires FAA phosphorylation and FAA/FAC nuclear accumulation.
Proc Natl Acad Sci USA
95:13085, 1998
4.
Kruyt FAE, Youssoufian H:
The Fanconi anemia proteins FAA and FAC function in different cellular compartments to protect against cross-linking agent cytotoxicity.
Blood
92:2229, 1998
5.
Yamashita T, Barber DL, Zhu Y, Wu N, D'Andrea AD:
The Fanconi anemia polypeptide, FACC, is localized to the cytoplasm.
Proc Natl Acad Sci USA
91:6712, 1994[Abstract/Free Full Text]
6.
Youssoufian H:
Localization of Fanconi anemia C protein to the cytoplasm of mammalian cells.
Proc Natl Acad Sci USA
91:7975, 1994[Abstract/Free Full Text]
7.
Youssoufian H:
Cytoplasmic localization of FAC is essential for the correction of a prerepair defect in Fanconi anemia group C cells.
J Clin Invest
97:2003, 1996[Medline]
[Order article via Infotrieve]
8.
Hoatlin ME, Christianson TA, Keeble WW, Hammond AT, Zhi Y, Heinrich MC, Tower PA, Bagby GC:
The Fanconi anemia group C gene product is located in both the nucleus and cytoplasm of human cells.
Blood
91:1418, 1998
9.
Kruyt FAE, Hoshino T, Liu JM, Joseph P, Jaiswal AK, Youssoufian H:
Abnormal microsomal detoxification implicated in Fanconi anemia group C by interaction of the FAC protein with NADPH cytochrome P450 reductase.
Blood
92:3050, 1998[Abstract/Free Full Text]
