From the Herman B Wells Center for Pediatric
Research, Departments of § Microbiology/Immunology and
¶ Pediatrics, Indiana University School of Medicine,
Indianapolis, Indiana 46202 and
Onyx Pharmaceuticals,
Richmond, California 94806
Received for publication, October 9, 2000
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ABSTRACT |
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Members of the Ras superfamily of signaling
proteins modulate fundamental cellular processes by cycling between an
active GTP-bound conformation and an inactive GDP-bound form.
Neurofibromin, the protein product of the NF1 tumor
suppressor gene, and p120GAP are GTPase-activating proteins (GAPs) for
p21Ras (Ras) and negatively regulate output by accelerating
GTP hydrolysis on Ras. Neurofibromin and p120GAP differ markedly
outside of their conserved GAP-related domains (GRDs), and it is
therefore unknown if the respective GRDs contribute functional
specificity. To address this question, we expressed the GRDs of
neurofibromin and p120GAP in primary cells from Nf1 mutant
mice in vitro and in vivo. Here we show that
expression of neurofibromin GRD, but not the p120GAP GRD, restores
normal growth and cytokine signaling in three lineages of primary
Nf1-deficient cells that have been implicated in the pathogenesis of neurofibromatosis type 1 (NF1). Furthermore, utilizing a GAP-inactive mutant NF1 GRD identified in a family with
NF1, we demonstrate that growth restoration is a function of
NF1 GRD GAP activity on p21Ras. Thus, the GRDs
of neurofibromin and p120GAP specify nonoverlapping functions in
multiple primary cell types.
Mutations in NF1 cause neurofibromatosis type 1 (NF1),1 a common disorder
characterized by increased risk of specific benign and malignant tumors
that primarily arise from neural crest-derived tissues. Children with
NF1 are predisposed to juvenile myelomonocytic leukemia (JMML) (1, 2),
and ~10% of heterozygous Nf1 mutant mice also develop a
JMML-like myeloproliferative disorder. JMML bone marrows and other
NF1-associated tumors frequently show loss of constitutional
heterozygosity at NF1, consistent with its tumor suppressor
function. Although neurofibromin is a large protein, the GRD is the
only segment known to function in growth control. Like p120GAP, the GRD
of neurofibromin binds Ras with high affinity and induces a
105-fold increase in the rate of GTP hydrolysis. The high
degree of sequence homology within the catalytic domains of p120GAP and neurofibromin led to the hypothesis that these proteins may be functionally interchangeable (3). However, Nf1 and
Gap mutant mice have distinct phenotypes, and mutations in
the human p120GAP catalytic domains are not associated with
any disease state. Moreover, neurofibromin and p120GAP differ markedly
in their non-GAP domains. p120GAP is a smaller protein that contains a
number of modules common to signaling proteins including Src homology-2
and Src homology-3 and pleckstrin homology domains. None of these
domains are present in neurofibromin. In fact, neurofibromin is most
closely related to products of the yeast IRA1 and
IRA2 genes, and its GRD can complement the heat shock
phenotype of IRA mutant strains resulting from hyperactive
Ras (4).
Nf1-deficient hematopoietic cells and fibroblasts provide an
excellent system for dissecting the role of neurofibromin in regulating
cell growth, because efficient vectors for transducing genes into these
cells are available. Furthermore, Ras signaling and cell growth can be
assayed in these primary cell populations. A hallmark of both human
JMML cells and of murine Nf1-deficient myeloid progenitor
cells is a selective hypersensitivity of cultured CFU-GM progenitors to
granulocyte macrophage-colony stimulating factor (GM-CSF) (5). Whereas
homozygous Nf1 mutant (Nf1 Isolation of Fetal Hematopoietic Cells--
Nf1+/ Genotyping Fetal Tissues--
Genomic DNA was isolated from
fetal tissues as described previously (7). The targeting vector used to
disrupt the murine Nf1 gene truncates exon 31 and inserts a
neomycin resistance gene (neo) (11). Previously described primers were
employed to distinguish disrupted and wild type genes in an assay
based on the polymerase chain reaction (11).
Generation of Recombinant Retroviral Plasmids--
Recombinant
retrovirus constructs were developed using the murine stem cell virus
(MSCV) backbone developed by Dr. Robert Hawley (12). The internal
sequences of these constructs are under the transcriptional control of
the myeloproliferative sarcoma retrovirus promoter. The construct also
contains a puromycin resistance gene, pac, which is under
the transcriptional control of the phosphoglycerate kinase promoter. By
using standard cloning techniques, three viruses were developed for use
in these experiments as follows: 1) a virus expressing the full-length
NF1 GRD (13) and pac (MSCV-NF1
GRD-pac); 2) a construct encoding the p120GAP GRD and pac
(13) (MSCV-p120GAP GRD-pac); and 3) a construct encoding the selectable
marker gene alone (MSCV-pac). The NF1 GRD and p120GAP GRD
constructs both contain a KT3 epitope tag at the 3' end of the GRD
sequences. Mutagenesis of NF1 GRD (R1276P) was accomplished
utilizing an in vitro site-directed mutagenesis kit (Stratagene).
Transfection of Retrovirus Plasmid into Packaging Cell Lines and
Evaluation of Retroviral Titer--
Recombinant retrovirus plasmids
were transfected as previously described into a GP +E 86 packaging cell
line previously developed by Dr. Arthur Banks (14). Evaluation of the
retroviral titer of individual clones expressing the NF1 GRD
cDNA or p120 GRD cDNA was determined using serial dilutions of
viral supernatant to infect NIH-3T3 cells. Clones expressing a high
retroviral titer (1 × 106) were evaluated to
determine the expression of recombinant protein by Western blotting of
the packaging cell lines, using an anti-KT3 antibody (Babco) (13).
Expression and Activity of Recombinant Protein in Primary
Cells--
Murine embryonic fibroblasts were transduced with the
respective retroviruses, and expression of NF1 GRD and
p120GAP GRD was analyzed by Western blotting using the anti-KT3
antibody (13). Activity of recombinant protein was then determined by
incubating immunoprecipitated protein with Ras-GTP and measuring
phosphate release in a GAP activity assay as described previously
(15).
Retroviral Infection of Hematopoietic Progenitors--
The
transduction protocol has been previously described (16) and was
employed here with minor modifications. Briefly, low density
mononuclear cells recovered from genotyped livers were prestimulated
for 48 h in liquid cultures of Iscove's modified Dulbecco's
medium containing 20% fetal bovine serum (HyClone) supplemented with
SCF (100 ng/ml) (PeproTech) and interleukin-6 (200 units/ml)
(PeproTech). Cells were transduced on mitomycin C-treated E86 producer
cells in the presence of SCF, interleukin-6, and Polybrene (5 µg/ml)
for 48 h. Transduced cells were then plated in methylcellulose
culture as described below.
Methylcellulose Cultures--
Following culture, fetal liver
cells were recovered and plated at a concentration of 5 × 104 cells/ml in triplicate methylcellulose cultures
containing increasing log doses of GM-CSF (0.01-10 ng/ml) (Peprotech)
and puromycin (1 µg/ml) to select for transduced progenitors.
Peritoneal cells were plated at a concentration of 1 × 105 cells per plate in methylcellulose cultures containing
10 units/ml and 100 ng/ml SCF. Cells cultured were maintained at
37 °C in a humidified incubator containing 5% CO2 and
95% O2, and colony-forming unit-granulocyte macrophage
(CFU-GM) and colony forming unit-mast (CFU-Mast) were scored on day 10 of culture.
Growth Kinetics Assays--
Murine embryonic fibroblasts (MEFs)
from individual Nf1+/+ and Nf1 Kinase Assays--
Activation of ERK and Akt was determined in
primary MEFs and c-Kit+ hematopoietic cells by culturing
cells in media containing 1% fetal calf serum, 2%
penicillin/streptomycin, and 1% L-glutamine for 24 h
to establish quiescence. Cells were then stimulated with 5 ng/ml GM-CSF
(c-Kit+ cells) or 100 ng/ml EGF (MEFs) (PeproTech). Cells
were lysed in nonionic lysis buffer as described previously (9) and
equalized for protein concentration using a Pierce BCA assay (Pierce).
ERK2 immunoprecipitations were carried out with an anti-ERK2 (C-14) antibody (Santa Cruz Biotechnology) and protein A-Sepharose beads (Amersham Pharmacia Biotech) for 2 h at 4 °C. Akt
immunoprecipitations were carried out with an anti-Akt antibody (New
England Biolabs). To determine activity of the respective kinases,
immunobeads were subjected to an in vitro kinase reaction
using an Elk-1 fusion protein (ERK2) (New England Biolabs) or histone
2B (Akt) (Roche Molecular Biochemicals) as substrate. Briefly, the
reactions were carried out in 30 µl containing 20 mM
MgCl2, 0.1 M sodium vanadate, 1 M
dithiothreitol, 30 mM In Vivo Mast Cell Knock-in Model--
1 × 106
transduced and selected BMMCs were injected into the peritoneums of
nine W/Wv mice. 10 weeks following transplantation,
peritoneal mast cells were recovered by peritoneal lavage and
enumerated by trypan blue exclusion as described previously (17).
Retroviruses encoding NF1 GRD (MSCV-NF1
GRD-pac), p120GAP GRD (MSCV-p120GAP GRD-pac), and the
control retrovirus (MSCV-pac) are shown in Fig.
1A. Each retrovirus expresses
pac sequences that confer puromycin resistance. Recombinant
GRD peptide expression was analyzed by immunoblotting utilizing an
antibody specific to a KT3 epitope tag incorporated into both GRD
peptides (13). Immunoblot analysis of transduced
Nf1
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
/
) embryos die in utero, adoptive transfer of
Nf1
/
fetal liver cells consistently induces a
JMML-like myeloproliferative disorder in irradiated recipients (6, 7).
GM-CSF plays a central role in establishing and maintaining this
phenotype in vivo (8). Primary
Nf1
/
hematopoietic cells demonstrate
constitutive activation of Ras-ERK signaling with hyperactivation in
response to GM-CSF and other cytokines (7). Similarly, mast cells and
fibroblasts have a hyperproliferative phenotype in response to stem
cell factor (SCF) (9) and epidermal growth factor (EGF) (10), respectively.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
mice were mated to produce Nf1
/
embryos.
Pregnant Nf1+/
females were sacrificed at day 13.5 of
gestation. Individual fetal livers were placed in Iscove's modified
Dulbecco's medium supplemented with 20% fetal calf serum as described
previously (7). A single cell suspension was prepared by passing the
hepatic tissues through progressively smaller needles
(16-27-gauge).
/
embryos were transduced with viral supernatant then selected by adding
puromycin (1 µg/ml) to the cultures. Selected fibroblasts were seeded
in a 6-well tissue culture-treated plate at a concentration of 2 × 105 cells per well and cultured for 48 h. One µCi
of tritiated thymidine was added to all cultures during the last 6 h of culture incubation. Triplicate cultures were collected on glass
filters using a cell harvester, and thymidine incorporation was
determined by scintillation counting. In vitro proliferation
of BMMCs was assayed as previously described (9). BMMCs were deprived
of growth factors for 24 h, and 2 × 105 cells
were plated in triplicate in 24-well dishes in 1 ml of RPMI containing
1% glutamine, 10% fetal bovine serum, and 100 ng/ml SCF in a
37 °C, 5% CO2, humidified incubator. Viable cells were
enumerated by trypan blue exclusion 72 h following culture initiation.
-glycerol phosphate, 5 mM EGTA, 20 mM MOPS, 1 µM ATP,
and 10 µg of substrate protein in the presence of 2.5 µCi of
[
-32P]ATP. Reaction mixtures were incubated at
30 °C for 30 min, and reactions were terminated by the addition of
10 µl of sample buffer. Reaction mixtures were resolved by SDS-10%
polyacrylamide gel electrophoresis. Gels were dried and subjected to
autoradiography. The relative amounts of incorporated radioactivity
were determined by densitometry using NIH free software.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
/
fibroblasts shows that NF1
GRD and p120GAP GRD are expressed at similar levels in primary cells
(Fig. 1B). Furthermore, the immunoprecipitated GRD peptides had similar GAP activity stimulating equivalent phosphate release from
GTP-loaded Ras (Fig. 1C). Together these data demonstrate that recombinant NF1 and p120GAP GRD peptides are expressed
at equivalent levels in fibroblasts and accelerate GTP hydrolysis on
Ras.
View larger version (21K):
[in a new window]
Fig. 1.
Recombinant proteins. A,
constructs developed to express NF1 GRD, p120GAP GRD, or the
selectable marker gene alone are indicated. LTR, long
terminal repeat. B, expression of recombinant retroviral
proteins in three MEF lines analyzed by Western blotting using a KT3
monoclonal antibody. NF1 GRD is ~55-kDa and p120GAP GRD
runs at ~40 kDa. C, GAP activity of recombinant
proteins recovered from lysates of transduced MEFs. Purified p120 GAP
was used as a positive control for Ras GAP activity. Activities of
KT3-immunoprecipitated recombinant proteins and control GAP are
expressed as fold GAP activity over GAP activity observed in
pac-transduced MEFs, which have been ascribed a value of 1. NS, not significant; p < 0.15.
To examine whether expression of NF1 GRD and/or p120GAP GRD
can correct the hypersensitivity of Nf1/
myeloid progenitors to GM-CSF, CFU-GM colonies were enumerated in
methylcellulose cultures of transduced Nf1
/
and wild type fetal liver cells. Colony growth was assayed over a range
of GM-CSF concentrations in the presence of puromycin.
Nf1
/
progenitors transduced with either the
control virus or with the p120GAP GRD virus demonstrate a
hypersensitive pattern of CFU-GM colony growth (Fig.
2A). Retroviral mediated
expression of full-length p120GAP in Nf1
/
myeloid progenitors also failed to restore normal cytokine
responsiveness (data not shown). In contrast,
Nf1
/
myeloid progenitors expressing
NF1 GRD displayed a normal pattern of CFU-GM colony growth
in response to GM-CSF in six independent experiments. Similar data were
observed when the proliferative rates of transduced
Nf1
/
fetal liver CFU-GM were analyzed
following stimulation of c-Kit+ cells with saturating
concentrations of SCF, a second cytokine known to induce
hyperproliferation of Nf1-deficient progenitors (7, 9) (data
not shown). Thus, expression of NF1 GRD alone is sufficient
to correct the hyper-responsiveness and aberrant proliferation of
Nf1-deficient myeloid cells.
|
To test whether these observations were operative in another lineage,
we expressed recombinant GRD sequences in
Nf1/
fibroblasts and measured proliferation.
MEFs isolated from Nf1
/
embryos exhibit
increased proliferation in culture compared with wild type MEFs (18,
19). Triplicate cultures of transduced fibroblasts from individual
embryos were established, and proliferation was assessed by thymidine
incorporation 48 h later. Nf1-deficient MEFs transduced
with the control virus or with the p120GAP GRD virus demonstrated a
4-fold higher rate of thymidine incorporation compared with wild type
fibroblasts (Fig. 2B). In contrast, expression of
recombinant NF1 GRD restored the rate of DNA synthesis in
Nf1-deficient MEFs to wild type levels. Similar data were
observed when proliferation was evaluated by cell counting (data not
shown). Therefore, these data confirm the specificity of the NF1
GRD to reduce the hyperproliferation of another primary cell type.
Studies of leukemic cells from children with NF1 and murine
Nf1/
hematopoietic cells have shown elevated
levels of GTP-bound Ras and hyperactivation of ERK in response to
hematopoietic cytokines (6, 7, 20). To determine whether expression of
GRD sequences could reduce ERK activity to wild type levels in
Nf1
/
hematopoietic cells,
Nf1
/
c-Kit+ cells were transduced
with the respective retroviruses, selected in puromycin, and assayed
for ERK kinase activity in response to GM-CSF. Nf1-deficient
cells transduced with the control virus or with the p120GAP GRD virus
had markedly elevated ERK activity in response to GM-CSF compared with
wild type c-Kit+ cells (Fig.
3A). In contrast, ERK
activation in GM-CSF-stimulated Nf1
/
cells
transduced with the NF1 GRD-encoding vector was comparable to wild type cells. A similar pattern of ERK activation was observed when MEFs transduced with the respective retroviruses were stimulated with EGF (Fig. 3B). These biochemical data in
Nf1-deficient hematopoietic progenitors and fibroblasts show
that expression of NF1 GRD selectively reduces hyperactive
Ras-ERK signaling previously shown to be important in mitogenesis. In
addition, we have observed that NF1 GRD but not
p120GAP GRD corrects hyperactivation of Akt, a downstream effector of the Ras-phosphatidylinositol 3-kinase pathway (data not
shown). Collectively, these biochemical observations are consistent with the correction of the hyperproliferative phenotype seen in cell
culture assays.
|
Although these data strongly suggest that the NF1 GRD is
sufficient to control cellular proliferation, the most definitive method to test gene function is to introduce recombinant sequences into
primary cells lacking the gene of interest and examine cellular function in vivo. We have recently demonstrated that bone
marrow-derived mast cells (BMMCs) generated from Nf1+/
mice exhibit increased proliferation to SCF (9). Similarly, we have now
observed that Nf1
/
mast cells derived from
fetal livers also have increased proliferation in response to SCF
compared with wild type mast cells or Nf1
/
mast cells expressing NF1 GRD (data not shown). Given these
observations, we next utilized a mast cell "knock-in" model to
evaluate the effects of transgene expression in vivo (21).
W/Wv mice are mast cell-deficient secondary to a
mutation in the c-kit receptor. Many groups have recently
established that embryonic or adult progenitor mast cells can fully
reconstitute W/Wv mice to wild type levels in all
relevant organs (21-23). In addition, one group has recently proposed
that in vivo reconstitution of mast cell precursors derived
from embryonic stem cells is an approach to evaluate the in
vivo function of a range of embryonic lethal mutations (24).
Therefore, direct injection of cultured mast cells into the peritoneum
of these mice offers a sensitive measure of mast cell function in
vivo. Since these mice contain no peritoneal mast cells and
cultured mast cells can repopulate the peritoneum of these mice,
equivalent numbers of transduced wild type or Nf1-deficient mast cells were injected into the peritoneums of
W/Wv mice, and peritoneal mast cell numbers were
examined 10 weeks following injection. In three independent
experiments, mice transplanted with Nf1
/
mast
cells had a 3-fold increase in peritoneal mast cells compared with mice
transplanted with wild type mast cells or with
Nf1
/
mast cells expressing NF1 GRD
(Fig. 4A). We also assessed
the ability of these reconstituted mast cells of the respective
experimental groups to form clonal mast cell progenitors in
vitro. Mast cell progenitors cultured from the peritoneum of mice
transplanted with Nf1
/
cells expressing only
reporter sequences had a 4-fold increase in mast cell progenitors
compared with wild type cells and Nf1
/
mast
cells expressing NF1 GRD (Fig. 4B). Thus,
introduction of Nf1 GRD sequences is sufficient to restore normal mast cell growth in vitro and in vivo.
|
Given the specificity of the NF1 GRD in correcting the
cellular and biochemical Nf1/
phenotype, we
next tested whether this restoration of normal growth is due to a
direct effect on Ras proteins. We generated a GAP-inactive mutant of
NF1 GRD that harbors a mutation in the arginine finger loop
(R1276P). This mutation, identified in NF1-associated malignancies,
greatly reduces neurofibromin GAP activity (25, 26). The R1276P mutant
recombinant protein had no catalytic activity in a GAP assay (Fig.
5A), and expression failed to
restore normal GM-CSF responsiveness to Nf1
/
hematopoietic progenitors (Fig. 5B). Similarly, expression
of this mutant GRD failed to correct the characteristic ERK2
hyperactivation observed in Nf1
/
MEFs (Fig.
5C). Thus, NF1 GRD exerts a Ras-specific function
as regulation of cellular proliferation is dependent on Ras-GAP
activity.
|
Despite its large size, the GRD encodes the only well established functional domain of neurofibromin. Previous studies in immortalized cell lines indicate that expressing NF1 GRD is sufficient to suppress the transformation capacity of an oncogenic K-Ras in a colon carcinoma cell line (27). However, such data cannot implicate sufficiency of NF1 GRD to restore normal cellular proliferation. The large size of neurofibromin has led investigators to search for other functional domains outside of the GRD. Recently, a missense mutation was described in an NF1 patient that disrupts a protein kinase A (PKA) phospho-acceptor consensus sequence (28), and these investigators hypothesize that these sequences may define a second functional domain of neurofibromin. These data are of additional interest because of recent observations in Drosophila that have suggested a link between cAMP-mediated signaling and NF1 (29). Although our construct does not contain this putative PKA consensus site, expression of recombinant NF1 GRD was sufficient to restore normal growth and ameliorate the biochemical deficits observed in three cell types frequently implicated in NF1-associated malignancies. Thus, the role of the hypothesized PKA sequences in the human NF1 gene and its relationship to Drosophila function remain unclear. It may be possible that PKA can modulate GAP activity by an unidentified mechanism. For example, lipid regulation of neurofibromin GAP activity differs between full-length protein and its isolated GRD (30). Perhaps PKA-dependent phosphorylation of the full-length protein could expose a lipid-binding site and result in modulation of activity. Additionally, despite a retention of structural homology, it is possible that species differences between Drosophila and humans result in a divergence of function.
Importantly, correction of the cellular and biochemical deficits of
Nf1/
cells by NF1 GRD is dependent
on Ras GAP activity as the R1276P arginine-finger mutant, found in
several distinct NF1 malignancies, failed to restore normal
proliferation. This suggests that the primary role of these domains in
these tested cell types is to modulate Ras activity.
Although expressing p120GAP GRD or full-length p120GAP in primary cells
induced similar levels of total GAP activity, this did not alter the
cellular or biochemical deficits in Nf1/
cells. This result is consistent with hyperactive Ras signaling in
leukemia cells from patients with NF1 despite near-normal levels of
total cellular GAP activity (20). One potential explanation for our observations regarding the differential functions of the two GAPs is
that neurofibromin and p120GAP may modulate different Ras isoforms in vivo. Alternatively, our experimental observations might
be explained by differences in the catalytic or association kinetics of
NF1 GRD and p120GAP GRD with Ras. The crystal structure of the catalytic regions of the two GRDs has been resolved (26, 31),
analyzed kinetically (32), and described as retaining high structural
homology. Both fragments possess a conserved Ras binding groove, an
arginine finger necessary for catalysis, and stabilization components
including a phenylalanine-leucine-arginine motif important in
positioning of the arginine finger loop. However, differences in amino
acid composition within each of these motifs were hypothesized to alter
interactions with Ras. Specifically, two residues within the Ras
binding groove of the NF1 GRD have been identified that have
electrostatic or hydrogen bonding potential with Glu-37 and Glu-63 of
Ras. The presence of neutral or oppositely charged residues at these
same locations within p120GAP may be responsible, in part, for
differential affinities of the two GAPs for Ras. Additionally, slight
deviations in residues proximal to the arginine finger loop of
NF1 GRD are thought to impart more flexibility to this key
catalytic motif (26). Overall, these differences may translate into a
major alteration in the kinetics of Ras-GTP complex formation with the
respective GAPs. In support of these structural data, kinetic data in
cell-free systems have demonstrated that the affinity of NF1
GRD for Ras is severalfold higher than that of p120GAP at physiological
concentrations of Ras-GTP (33), and the association and dissociation
rate constants of p120GAP-Ras complex are faster than that of the
neurofibromin-Ras complex (33). Our in vitro and in
vivo studies in primary Nf1-deficient cells support
these structural and in vitro biochemical predictions.
Although the studies presented here suggest distinct roles of
NF1 GRD and p120GAP GRD in regulating Ras output in
fibroblasts and hematopoietic cells, the contribution of individual
residues and localization sequences within these domains to GTP
hydrolysis remains unclear. In vitro mutagenesis of p120GAP
and neurofibromin, followed by introduction of these sequences into
Nf1/
cells, may help define these functions.
In addition, such studies may further define how each of these GAPs
coordinates with other signaling proteins and the functional
significance of these interactions. Future studies utilizing these
techniques will provide a better understanding of the physiological
roles of these two GAPs and may further define molecular mechanisms of
disease pathogenesis when NF1-specific GAP function is perturbed.
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ACKNOWLEDGEMENTS |
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We thank Dr. Tyler Jacks for generously providing us with Nf1 heterozygous mice. We also thank Dr. Kevin Shannon for numerous helpful scientific discussions throughout the course of these experiments, and we thank Dr. Jacks, Dr. Karen Cichowski, and Dr. Shannon for helpful discussions during preparation of this manuscript.
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FOOTNOTES |
---|
* This work was supported by March of Dimes Birth Defects Foundation Grant 6FY98-0219, National Institutes of Health Grant R29 CA74177-01, American Cancer Society Grant DB146 (to D. W. C.), and National Institutes of Health Pediatric Scientist Development Program Grant K12-HD0050 (to D. A. I.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Indiana University School of Medicine, Cancer Research Institute, 1044 W. Walnut, Rm. 421, Indianapolis, IN 46202. Tel.: 317-274-4719; Fax: 317-274-8679; E-mail: dclapp@iupui.edu.
Published, JBC Papers in Press, November 15, 2000, DOI 10.1074/jbc.M009202200
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ABBREVIATIONS |
---|
The abbreviations used are: NF1, neurofibromatosis type 1; GAPs, GTPase-activating proteins; GRDs, GAP-related domains; GM-CSF, granulocyte macrophage-colony stimulating factor; SCF, stem cell factor; MSCV, murine stem cell virus; MEFs, murine embryonic fibroblasts; CFU-GM, colony-forming unit-granulocyte macrophage; ERK, extracellular signal-regulated kinase; BMMC, bone marrow-derived mast cells; EGF, epidermal growth factor; MOPS, 4-morpholinepropanesulfonic acid; JMML, juvenile myelomonocytic leukemia; PKA, protein kinase A.
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