From the Department of Biochemistry and Molocular Biology and Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202
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ABSTRACT |
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Adapter proteins made up of Src homology (SH)
domains mediate multiple cellular signaling events initiated by
receptor protein tyrosine kinases. Here we report that Grb4 is an
adapter protein closely related to but distinct from Nck that is made
up of three SH3 domains and one SH2 domain. Northern analysis indicated
that both genes are expressed in multiple tissues. Both Nck and Grb4 proteins could associate with receptor tyrosine kinases and the SH3-binding proteins PAK, Sos1, and PRK2, and they synergized with
v-Abl and Sos to induce gene expression via the transcription factor
Elk-1. Although neither protein was transforming on its own, both Nck
and Grb4 cooperated with v-Abl to transform NIH 3T3 cells and
influenced the morphology and anchorage-dependent growth of
wild type Ras-transformed cells. Nck and Grb4 therefore appear to be
functionally redundant.
Growth factor binding to receptor protein tyrosine kinases
(R-PTKs)1 induces their
dimerization and trans-phosphorylation, creating docking sites for
proteins containing SH2 and PTB protein interaction domains (1). Many
of these phosphotyrosine-binding proteins are effector enzymes,
e.g. phospholipase C Another abundantly expressed adapter protein, Nck, consists of three
juxtaposed SH3 domains and a C-terminal SH2 domain (4). The SH2 domain
of Nck has been reported to bind a variety of growth factor receptors,
including those for EGF (5), PDGF (6), vascular endothelial growth
factor (7), and hepatocyte growth factor (8), as well as the insulin
receptor substrate, IRS-1 (9), Eph (10, 11), p62dok (12), and
focal adhesion kinase (13). The SH3 domains of Nck can interact with
proline-rich motifs in multiple binding partners, including the Ser/Thr
protein kinases PAK (14-17), Prk2 (18), casein kinase I In 1992, a partial mouse cDNA that encoded for an SH2 domain and
part of an SH3 domain was isolated from a bacterial expression library
and designated Grb4 (34). The predicted amino acid sequence shared 74%
identity with human Nck. It was not clear from this original study
whether the partial grb4 cDNA encoded the mouse ortholog
of Nck or a separate protein. Here we demonstrate that grb4
is, indeed, a unique human gene. Isolation of the full-length human
grb4 cDNA showed that Grb4 shares the same SH domain
composition and is 69% identical to Nck. Like Nck, Grb4 bound to Sos,
PAK, and Prk2 and to activated growth factor receptors. Both Nck and Grb4 cooperated with v-Abl, Ha-Ras(WT), and Sos1 to modulate cell morphology and transformation and to induce gene expression via the
Elk-1 transcription factor. These findings suggest that Nck and Grb4
may be functionally redundant.
Library Screen--
A human brain Marathon-ready cDNA
library (CLONTECH) was used to isolate full-length
grb4. The 5'-end of the gene was amplified using polymerase
chain reaction with anchor primers provided with the library and nested
grb4 primers located within the middle SH3 domain. Due to
difficulty obtaining the complete 3'-end of the clone by rapid
amplification of cDNA ends, we used further nested grb4
primers to isolate a central portion and specific primers to polymerase
chain reaction amplify the 3' coding region. The grb4
fragments were subcloned into pCR2.1 (Invitrogen) and sequenced.
Full-length grb4 was generated by ligation using existing restriction sites within the three overlapping fragments.
Northern Analysis--
Full-length nck cDNA (4)
or a 266-base pair fragment of grb4 spanning base pairs Mammalian Cell Culture--
Cos, NIH 3T3, and SV40-immortalized
mouse embryo fibroblast cells were grown in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum (Hyclone) or,
for NIH 3T3 cells, in 10% calf serum (Colorado Serum Company or Life
Technologies, Inc.). Transforming focus and soft agar assays were
performed as described (35).
Transcriptional Activation Assays--
Activation of Elk-1 was
determined by co-transfection of cells with both Gal4-Elk-1 and
Gal4-Luc constructs. Gal4-Elk-1 encodes a fusion protein containing the
Gal4 DNA binding domain together with the transactivation domain of
Elk-1 (containing ERK phosphorylation sites). Gal4-Luc encodes the
luciferase gene driven by a minimal promoter containing tandem Gal4 DNA
binding sites (36). NIH 3T3 cells were cotransfected with 125 ng of
Gal4-Elk-1, 2.5 µg of Gal4-Luc, and 1 µg of pSR Mammalian Protein Expression and GST Fusion Protein
Interaction--
For transient overexpression of Prk2 and PAK
proteins, 100-mm plates of 50% confluent Cos cells were transfected
with 6 µg of either pFLAG-CMV2(prk2) (18) or
pCMV6(pak(WT)) (16) using LipofectAMINE (Life Technologies,
Inc.) and harvested after 48 h. Sos1 was stably overexpressed from
pRC-bac(sosF*) (39) in NIH 3T3 cells. Confluent plates of
cells were lysed in 1 ml lysis buffer consisting of 20 mM
Tris, pH 7.4, 50 mM NaCl, 0.5% IGEPAL CA-630, 5 mM EDTA, 10% glycerol, 19 µg/ml aprotinin, 1 mM Na3VO4, and 1 mM
phenylmethylsulfonyl fluoride. Cell lysates were cleared by
microcentrifugation for 10 min at 4 °C. GST fusion proteins of Nck,
Grb4, or a control GST (containing the 40 C-terminal amino acids of
Prk2) were expressed in BL21-DE3(lysE) bacteria as described (18).
2-10 µg of glutathione-agarose bead-bound protein was tumbled with
0.5 ml of each individual cell lysate for 3 h at 4 °C. Beads
were then washed twice with the above lysis buffer and once with
phosphate-buffered saline. Protein was solubilized by boiling in 2×
Laemmli buffer, and 50% of the resultant sample from Prk2- or
Sos-incubated GST fusion proteins was separated by SDS-PAGE (7% gel).
Because PAK co-migrated with the GST-Nck and GST-Grb4 fusion proteins
during SDS-PAGE separation, bead-bound fusion proteins incubated with
lysate from cells overexpressing PAK were further incubated for 30 min
at room temperature with a peptide representing residues 6-21 of PAK1
to specifically release bound PAK into the solution. 50% of the
supernatant from this competition was separated by SDS-PAGE (8% gel).
GST-fused SH2 Domain Interaction with Tyrosine-phosphorylated
Proteins--
Using human cDNA as a template, individual SH2
domains of Grb2 (codons 57-154), Nck (codons 275-377), and Grb4
(codons 278-388) were generated by polymerase chain reaction and
subcloned into pGEX-2T. Glutathione-agarose bead-bound GST fusion
proteins of the SH2 domains were prepared as described above. Mouse
embryo fibroblasts (40) were serum-starved for 48 h prior to
stimulation with EGF (100 ng/ml, Upstate Biologicals Inc.) or PDGF-AA
(50 ng/ml, Collaborative Biomedical Products) for the indicated times and lysed in 50 mM HEPES, 150 mM NaCl, 10%
glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM sodium
pyrophosphate. 25% of the lysate from a confluent 100-mm plate was
then incubated with 5 µg of GST-fused SH2 domain proteins or
full-length GST adapter proteins, as indicated, for 2 h at
4 °C. The bead-bound proteins were washed as described above. The
proteins were separated by SDS-PAGE (10% gel) and detected using the
anti-phosphotyrosine antibody, PY99.
Co-immunoprecipitation of Sos1--
Cos cells were transfected
with pFLAG-CMV2(nck) or pFLAG-CMV2(grb4) together
with pRC-bac(sosF*) using LipofectAMINE. After 48 h,
cells were lysed as above, and tagged Nck and Grb4 were immunoprecipitated with 3 µg of M2 anti-FLAG antibody. Proteins were
separated by SDS-PAGE and co-precipitated Sos1 detected by Western
blotting using anti-Sos antibody.
Western Blotting--
Proteins separated by SDS-PAGE were
transferred onto Immobilon-P polyvinylidene fluoride membrane
(Millipore) and blocked for 1 h at room temperature with 5% milk
powder in TBS-Tween 20 (Tris-buffered saline, 0.05%
polyoxyethylene-sorbitan monolaurate) or, in the case of phosphorylated
tyrosine detection, with 3% bovine serum albumin in TBS-Tween 20. All
antibody dilutions and wash steps were performed in TBS-Tween 20. Primary antibody incubations were for 1 h at room temperature with
indicated antibodies. Secondary incubations were done for 30 min at
room temperature with antibody conjugated to horseradish peroxidase
(Amersham Pharmacia Biotech) and detected by enhanced chemiluminescence
(Amersham Pharmacia Biotech). Anti-Nck antibody was from Pharmingen,
anti-HA tag antibody was from Berkeley Antibody Co., anti-FLAG (M2) was
from Sigma, anti-Sos was from Transduction Laboratories, and PY99 was
from Santa Cruz Biotech. Other anti-Nck sources included Transduction Laboratories and Upstate Biotechnology.
Phosphorylation--
COS cells were transiently transfected with
pFLAG-CMV2(nck) or (grb4) or pCGN HA-tagged
grb2. After 24 h, cells were incubated overnight in
phosphate-free Dulbecco's modified Eagle's medium, supplemented with
0.5% dialyzed fetal bovine serum and 1.25 mCi of
32PO4 (NEN). Cells were stimulated for 10 min
with 50 µM forskolin/0.2 mM isobutyl
methylxanthine, Me2SO, 1 µM phorbol myristate
acetate, or 10% FBS, washed two times with ice-cold phosphate-buffered saline, and lysed in the above lysis buffer supplemented with 10 mM NaF, 0.1 mM ZnCl2, 10 mM sodium pyrophosphate, 10 mM [35S]Methionine
Labeling--
Ha-Ras(Q61L)-transformed NIH 3T3 cells were
metabolically labeled overnight with 250 µCi of
[35S]cysteine/methionine (Amersham Pharmacia Biotech) per
60-mm plate in cysteine/methionine-free Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum that had been dialyzed against 20 mM HEPES, pH 7.4. Cell monolayers were washed
with phosphate-buffered saline and lysed in 150 mM NaCl,
1% IGEPAL CA-630, 0.25% deoxycholate, 20 mM Tris, pH 7.4, 5 mM EDTA, 10% glycerol, 19 µg/ml aprotinin, 1 mM Na3VO4, and 1 mM
phenylmethylsulfonyl fluoride. The lysate was cleared by
microcentrifugation and then tumbled with 20 µg of the indicated GST
fusion proteins on glutathione-agarose beads for 3 h at 4 °C.
Beads were then washed three times with lysis buffer and once with
phosphate-buffered saline. 0.5 ml of each protein sample was separated
by SDS-PAGE (7% gel) and then fixed and stained with 0.25% Coomassie
Blue to confirm equal loading of GST fusion proteins. The gel was
soaked in Amplify (Amersham Pharmacia Biotech), dried, and exposed to
film for 48 h at Isolation of Full-length Grb4 cDNA--
Sequence
homology between human nck and a partial mouse
grb4 cDNA isolate indicated that grb4 could
either be the mouse ortholog of nck or a separate, related
gene (34). Human expressed sequence tags, as well as an additional
partial cDNA clone,2
supported the notion that grb4 was a separate gene.
Therefore, to obtain the full-length Grb4 cDNA, we screened a human
brain library using polymerase chain reaction/rapid amplification of cDNA ends (see under "Materials and Methods"). Like Nck, Grb4 consisted of three N-terminal SH3 domains and a C-terminal SH2 domain
with short linker regions. The two proteins share 69% identity, with
most divergence occurring in the regions between the SH domains (Fig.
1). An in-frame stop codon was located
just 5' of the presented sequence, indicating that the Grb4 N terminus
does not extend beyond that of Nck. Indeed, significant homology
between the nck and grb4 5' untranslated regions
supports a common ancestry. Overall Nck has slightly more identity with
the Xenopus Nck homolog than does Grb4, whereas the
Drosophila protein is somewhat more divergent (Fig. 1).
Nck and Grb4 mRNAs Are Ubiquitously
Expressed--
Because Nck and Grb4 shared such a high degree of
homology within their functional SH domains, we anticipated that they
might serve similar functions in different tissues. We looked for
differential expression of nck/grb4 mRNAs in a panel of
16 human tissues using commercially available Northern blots.
Surprisingly, both nck and grb4 messages were
almost ubiquitously expressed (Fig. 2). This apparent ubiquitous expression pattern was not due to
cross-hybridization of the probes because their messages were of
distinct sizes (the nck mRNA was slightly smaller).
Furthermore, there were slight variations in abundance of the major
transcripts in different tissues, most notably pancreas and thymus. The
broad tissue distribution of nck was consistent with
previously findings (5).
Nck and Grb4 Bind to Common Target Proteins--
We next
tested the two adapter proteins to see whether the minor deviations in
SH domain sequence might impart differential binding properties.
Incubation of GST fusions of SH domain adapter proteins with lysates
from [35S]methionine-labeled NIH 3T3 cells revealed that,
although distinct from that of Grb2, the binding profiles of Nck and
Grb4 were similar to each other (Fig.
3A).
The SH3 domains of Nck have been shown to bind to a number of target
proteins, such as Sos, PAK, and PRK2, all of which contain proline-rich
peptide sequences (14-18, 22, 41). We therefore addressed whether
GST-Grb4 would also interact with these molecules. Recombinant Grb4 was
found to precipitate FLAG-tagged PRK2 and Myc-tagged PAK1 from cell
lysates with similar efficacy to Nck (Fig. 3B). Sos1 could
also be co-immunoprecipitated by FLAG-tagged Grb4 or Nck from Cos cell
lysates. The SH2 domain of Nck binds to a number of activated growth
factor receptors and their substrates (5-13). Grb4 and Nck interacted
with a similar pattern of phosphotyrosine-containing proteins.3 To look more
specifically at interaction with these proteins, serum-starved mouse
embryo fibroblasts were challenged with EGF or PDGF prior to incubation
of lysates with glutathione bead-immobilized full-length adapter
proteins or isolated SH2 domains. As shown in Fig. 3C, the
SH2 domains of Grb2, Nck, and Grb4 all precipitated activated EGF and
PDGF receptors with similar efficiency.
Nck and Grb4 Serve as Substrates for Protein Kinases--
Nck has
been reported to be phosphorylated on multiple sites (primarily Ser/Thr
residues) following agonist stimulation (5, 16, 33, 42-44).
Examination of the Nck sequence revealed that most consensus
phosphorylation sites are located between the SH domains, where Grb4
and Nck are most divergent. Following 32PO4
labeling of Cos cells transiently overexpressing FLAG-tagged Nck or
Grb4, both molecules were heavily phosphorylated, even in the absence
of challenge with forskolin/IBMX (Fig.
4), serum, or phorbol myristate
acetate.3 Therefore, although the regions between the SH3
domains share less homology, both proteins are still susceptible to
phosphorylation. Indeed, three consensus PKA phosphorylation sites are
conserved in the region between the first and second SH3 domains of Nck and Grb4 (see Fig. 1). No such sites were present in the epitope tag.
Adapter Proteins Cooperate with v-Abl and Sos to Induce
Transcriptional Activation--
Nck has been reported to activate Ras
and or Ras-induced phenomena by binding the Ras guanine nucleotide
exchange factor, Sos, and replacing the role of Grb2 in mediating
growth factor-induced recruitment of Sos to the site of Ras activation
(22). Because Sos could co-precipitate with Nck and Grb4, we looked at
the ability of these adapters to stimulate transcriptional activation
via Elk-1 that is located downstream of the Ras/ERK cascade. In these assays, we co-transfected cells with a plasmid encoding the tyrosine kinase v-Abl to provide an upstream initiating signal (45). As shown in
Fig. 5, co-transfection of NIH 3T3 cells
with Grb2 augmented the transcriptional activity induced by v-Abl. Nck
and Grb4 also enhanced the ability of Abl to induce luciferase
expression, and although the effect of these adapter proteins was less
than that seen with Grb2, they had similar potency to each other,
suggesting a common mode of action. None of the adapter plasmids
enhanced luciferase activity when transfected in the absence of
v-Abl.
Because PAK has been implicated in Ras-independent ERK activation (14,
46), we next tested whether Nck and Grb4 might signal through PAK to
induce gene expression. In our assay system, PAK did not induce
transcriptional activation via Elk-1, either alone or in combination
with Nck or Grb4.3 As shown for v-Abl, however, we did find
that both Nck and Grb4 could weakly synergize with Sos to induce
transcriptional activation (Fig. 5).
Nck and Grb4 Cooperate with Ras and v-Abl to Induce Morphologic
Transformation of NIH 3T3 Cells--
Although it has previously been
reported that Nck can induce transformation of rodent (NIH 3T3 and 3Y1)
fibroblast cell lines (5, 33), in our hands Nck did not affect the
morphology, growth rate, or saturation density of either cell
type.3 When NIH 3T3 cells were cotransfected with pZIP
Ha-ras(WT) and pZIP(nck) or
pZIP(grb4), however, we found that a number (1-10%) of the
Ras-induced foci took on a unique morphology of highly rounded cells
(Fig. 6). In contrast, Grb2 had no effect
on Ras-induced focus forming activity (47).3 When cells
coexpressing Ras/Nck or Ras/Grb4 were isolated from the round
transforming foci and plated in semi-solid medium, they developed
anchorage-independent colonies that were distinct from those induced by
oncogenic Ras(Q61L). Ras(Q61L) expression caused the formation of
spherical colonies in approximately 50% of the transfected cells,
whereas nearly 100% of Nck/Ras(WT)- and Grb4/Ras(WT)-expressing cells
grew into diffuse colonies with poorly defined edges (Fig. 6C).
Because Nck and Grb4 cooperated with v-Abl to induce transcriptional
activation, we also compared the ability of Nck and Grb4 to cooperate
with v-Abl to induce transformation of NIH 3T3 cells. Co-transfection
of v-Abl with plasmids encoding either adapter protein significantly
enhanced focus formation. Whereas v-Abl alone induced the formation of
only 1-5 small foci/µg of transfected DNA, inclusion of the adapter
proteins Grb2, Nck, or Grb4 resulted in the formation of 10-20 larger
foci/µg that contained highly rounded cells (Fig. 6D).
None of the v-Abl-induced foci could be established into transformed
cell lines. Foci co-expressing the adapter proteins could, however, be
maintained. Furthermore, the cells overexpressing Nck or Grb4 were much
rounder than those expressing Grb2 (Fig. 6D).
A number of SH2/SH3 adapter proteins, including Grb2, Crk, and
Nck, have been identified that couple R-PTKs to downstream effectors. A
partial mouse grb4 cDNA sequence was described in 1992 having 66 and 74% identity to human nck at the DNA and
predicted protein levels, respectively (34). Because a number of
spliced variants and paralogs of the adapter proteins Grb2 (Grb3.3 and Grap) and Crk (CrkI, II, and CrkL) exist (48-50), it was not clear whether Grb4 represented mouse Nck or a novel protein. We have now
isolated a full-length human cDNA encoding the Grb4 protein, demonstrating that it is a distinct gene product rather than an ortholog of Nck.
In contrast to Grap, which shared 59% amino acid identity to Grb2 but
had a very limited tissue distribution (49), the expression of the
grb4 transcript appears to be almost ubiquitous.
Furthermore, with a few exceptions, e.g. pancreas and
thymus, the intensity of grb4 mRNA expression closely
mirrored that of nck. Because all commercial anti-Nck
antibodies tested cross-reacted with Grb43 and we failed to
generate Grb4-specific anti-peptide antisera, we were unable to confirm
the tissue distribution of Nck and Grb4 at the protein level. However,
while this study was under review, Chen et al. (51) reported
the independent isolation of Grb4 as Nck Comparison of the amino acid sequences of Grb4 and Nck indicate that
they are most highly conserved within their SH domains. The key SH
domain residues involved in ligand binding have been predicted by x-ray
crystallographic and NMR structures. Because these residues are mostly
conserved between Nck and Grb4, it was not surprising that the
pull-down profile from [35S]Met-labeled cells,
phosphotyrosine-containing proteins, and specific SH3-binding proteins
was similar for the two adapters. In contrast, it was reported that Nck
binds to the PDGF receptor and to the EGF-receptor-associated adapter
protein p62dok less effectively than Nck Phosphorylation of Nck has been reported to be induced by growth factor
stimulation (5, 42-44) or association with the downstream target
kinase, PAK1 (16). In our hands, both Nck and Grb4 were constitutively
phosphorylated and not sensitive to stimuli. This discrepancy may be
due to the use of Cos cells or overexpression of the proteins in this
study. Regardless, both Nck and Grb4 were phosphorylated to similar
levels. Most consensus phosphorylation sites in Nck are located within
the poorly conserved inter-SH domain regions, providing the potential
for differential regulation of adapter protein binding by
phosphorylation. However, a potential protein tyrosine kinase substrate
site and three consensus cyclic AMP-dependent protein
kinase phosphorylation sites (located between the first and second SH3
domains) are conserved between Nck and Grb4. Which sites are
phosphorylated and whether there is any differential regulation of Nck
versus Grb4 by phosphorylation will require further study.
Indeed, no physiological role has so far been ascribed to Nck phosphorylation.
Nck has been shown to bind to the Ras guanine nucleotide exchange
factor, Sos, in vivo and to activate the c-Fos promoter in a
Ras/ERK-dependent fashion (22). In another study, however, Nck dominant inhibitory mutants, unlike Grb2 mutants, did not block the
activation of ERKs induced by oncogenic ( Two previous studies reported weak transformation of rodent fibroblasts
by Nck (5, 33). Although we saw no effect of Nck or Grb4 on their own,
both proteins could cause a rounding up of Ras(WT)-transformed cells. A
similar phenotype was observed by co-expression of Nck with
R-Ras(G38V).4 The rounding of
the cells suggests a loss of stress fibers and/or cell contact with its
substratum. A similar morphological change was reported following
membrane targeting of PAK in 293T kidney cells using a myristoylated
Nck-SH3 domain (14), implicating PAK in Nck-induced cytoskeletal
events. The morphology of cells derived from the Ras plus Nck/Grb4
foci, as well as the diffuse/migratory nature of the soft agar colonies
compared with those induced by Ras(Q61L), is similar to that induced by
co-expression of Ras with activated Rac1 or RhoA (55). This supports
the notion that Nck is responsible for coupling R-PTK to Rho family
effectors, such as PAK or PRK2.
The observation that Nck and Grb4 only affected the morphology of a
subset of NIH 3T3 cells was surprising. Heterogeneity in the NIH 3T3
cell population has, however, been reported. Although v-Abl induces
cell cycle arrest at G1 in the majority of NIH 3T3 cells,
it transforms a subset representing 5-8% (56). The reason for this
heterogeneity is not clear but may involve the absence of a proposed
inhibitory feedback loop in some 3T3 cells or lack of a v-Abl effector
(56). It would be interesting to determine whether the same population
of cells respond to both Nck- and v-Abl-induced transformation.
Furthermore, this heterogeneity may, at least in part, explain the
differences in biological activity of the Nck adapter protein observed
in different studies on NIH 3T3 cells (Refs. 5, 22, 33, and 41 and this report).
Although activating mutations in ras are found in only ~30% of human
malignancies, Ras activation by upstream oncoproteins, e.g.
BCR/Abl in leukemias or ErbB2/Neu in breast cancer, is also believed to
contribute to transformation. Adapter proteins, such as Grb2, are
required to transduce these deregulated signals from PTKs to Ras (57),
and their amplification could contribute to the transformed phenotype.
Indeed, the adapter protein Grb7 has also been shown to be up-regulated
along with ErbB2 in breast cancer cell lines and tumor samples (58).
Here, we have described the characterization of Grb4, an adapter
protein that shares many structural and functional properties with Nck.
Both proteins share the ability to couple R-PTKs to Ras and to Rho
protein effectors and to cooperate with signaling molecules to promote
cellular transformation. It will be interesting to determine whether
Nck or Grb4 overexpression contributes to human malignancies and in what tissues or at what point in development the body relies on Grb4 expression.
INTRODUCTION
Top
Abstract
Introduction
References
, the protein phosphatases SHP-1 and
SHP-2, and p120 RasGAP (1). However, R-PTKs also bind to a number of
adapter proteins that lack enzymatic activity and contain SH3 in
addition to SH2 domains. Through SH3 domains, adapter proteins can bind
to proline-rich motifs in downstream effectors, often recruiting them
into multiprotein complexes at the plasma membrane. The adapter protein
Grb2, for example, has the domain structure SH3-SH2-SH3 and can bind to
proline-rich sequences in the Ras guanine nucleotide exchange factor,
Sos, recruiting it to the plasma membrane, where it triggers the
Ras-ERK pathway (2, 3).
(19), and
Nck interacting kinase (20). Nck has also been found to interact with
the protein tyrosine kinase Abl (21), Sos (22), the Wiskott-Aldrich
syndrome protein (18, 23), a Drosophila protein tyrosine
phosphatase dPTP61F (24), c-Cbl (25), and SAM68 (26). Although the
physiological significance of most of these interactions is
undetermined, several of the above SH3 binding partners (PAK, PRK2, and
the Wiskott-Aldrich syndrome protein) appear to be downstream effectors
of Rho family GTPases (18, 27, 28). Because Rho proteins have been
implicated in cytoskeletal reorganization (29), Nck may be responsible for coupling R-PTK activation to cytoskeletal regulation. In support of
this notion, the Drosophila homolog of Nck, DOCK, is located in photoreceptor growth cones and is involved in axonal guidance (30).
Use of dominant inhibitory SH domain mutants has also implicated Nck in
dorso-ventral axis development during Xenopus embryogenesis
(31). The nck gene is located at a chromosomal break point
associated with a variety of cancers (32), and Nck has been reported to
weakly transform rodent fibroblast cell lines (5, 33), also implicating
it in oncogenesis.
MATERIALS AND METHODS
44
to +222 were radiolabeled with [
-32P]dCTP using a
random-priming DNA labeling kit (Boehringer Mannheim) as outlined by
the manufacturer. Two human multitissue Northern blots
(CLONTECH) were probed overnight with each
individual fragment in hybridization solution (5× SSPE, 10×
Denhardt's solution, 100 mg/ml denatured sheared salmon sperm DNA,
2.0% SDS plus 50% formamide) at 42 °C. Blots were then washed
extensively in 2× SSC, 0.05% SDS at room temperature followed by two
20-min washes in 0.1× SSC, 0.1% SDS at 50 °C. The blots were
exposed at
80 °C with intensifying screens.
MSVtkneo
(v-abl) (provided by A. M. Pendergast, Duke
University), 2 µg of pZIP (37), 2 µg of pZIP(nck), 2 µg of pZIP(grb4), or 0.67 µg of pZIP(sos1) as
indicated. 24 h posttransfection, cells were serum-starved (0.5%)
overnight, lysed, and analyzed for luciferase activity essentially as
described (38).
glycerol phosphate, and 10 mM p-nitrophenyl phosphate.
After clarification (microcentrifugation for 10 min at 14,000 rpm)
lysates were immunoprecipitated with 3 µg of anti-FLAG or anti-HA
antibody. Proteins were resolved by SDS-PAGE (8%). The gel was
Coomassie-stained, dried, and exposed to Amersham Pharmacia Biotech ECL
film for 4 h.
80 °C.
RESULTS
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Fig. 1.
Alignment of Nck and Grb4 amino acid
sequences. Sequence identity or similarity is indicated by
black or gray boxes, respectively, and individual
SH domains are outlined in black (SH3s) or white
(SH2). X-Nck represents Xenopus laevis Nck, and
Dock is the Drosophila homolog. Sequences were
aligned using ClustalW and colored using Boxshade.
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Fig. 2.
Northern blot analysis of nck
and grb4 expression. The presence of
nck and grb4 mRNA was analyzed in 16 adult
human tissues using 32P-labeled probes. Upper
panels indicate nck expression, and lower
panels indicate grb4 expression.
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Fig. 3.
Nck and Grb4 interact with a common set of
proteins. A, lysates from
[35S]methionine/cysteine-labeled NIH 3T3 cells were
incubated with GST fusion proteins containing the PRK2 C terminus
(negative control), Grb2, Nck, or Grb4. Co-precipitated proteins were
separated by SDS-PAGE and visualized by fluorography. B,
lysates from Cos cells transiently expressing FLAG-tagged PRK2 or
Myc-tagged PAK1 were incubated with 2-10 µg of the indicated GST
fusion proteins. Co-precipitation of PRK2 and PAK1 with GST fusion
proteins was determined by immunoblotting. GST fusion proteins were
detected by Ponceau S staining of polyvinylidene fluoride membranes
prior to blocking. Lower panel, 48 h after
co-transfection of Cos cells with plasmids encoding Sos1 and FLAG-Nck
or FLAG-Grb4, cells were lysed and subjected to immunoprecipitation
with anti-FLAG antibody. Sos co-precipitation was detected by Western
blotting. C, phosphotyrosine-containing proteins
precipitated from EGF-stimulated (upper panel) or
PDGF-stimulated (lower panel) mouse embryo fibroblasts by
the indicated GST fusion proteins were detected using anti-Tyr(P)
antibody, PY99. PDGF stimulation was for 5 min. Similar results were
obtained using full-length adapter proteins or isolated SH2 domains
(see Footnote 3). All data representative of at least three
experiments.
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Fig. 4.
Nck and Grb4 are constitutively
phosphorylated in Cos cells. Cos cells transiently expressing
FLAG-tagged Nck or Grb4 or HA-tagged Grb2 were serum-starved and
labeled overnight with 32PO4. Following a
10-min challenge with 50 µM forskolin/0.2 mM
isobutyl methylxanthine (IBMX) or Me2SO vehicle,
cells were lysed and adapter proteins immunoprecipitated. The
phosphorylation state of Grb2, Nck, and Grb4 was determined by SDS-PAGE
and autoradiography.
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Fig. 5.
Nck and Grb4 cooperate with v-Abl and Sos1 to
induce transcriptional activation via Elk-1. NIH 3T3 cells were
cotransfected with either pSR MSVtkneo(v-abl),
pZIP(sos1) or appropriate empty vector,
pZIP(nck), or pZIP(grb4) as indicated, plus
Gal4-Elk-1 and Gal4-luc reporter plasmids. Luciferase activity was
detected as described (38). Data are representative of five or more
experiments performed in triplicate. The columns and
error bars show mean ± S.D.
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Fig. 6.
Nck and Grb4 can transform NIH 3T3 cells by
cooperating with Ha-Ras(WT) and v-Abl. A, NIH 3T3 cells were
transfected with pZIPH(ras) along with pZIP,
pZIP(nck), or pZIP(grb4), as indicated.
Transforming foci were photographed (magnification, × 10) after
culture in regular growth medium for 14 days. B, individual
transforming foci from A were isolated and selected on G418.
Subconfluent cells were photographed (magnification, × 20).
C, cells from B were grown in 0.6% agar for 14 days. Pooled populations of cells stably expressing Nck and Grb4 in the
absence of Ras did not produce colonies (magnification, × 4).
D, NIH 3T3 cells were transfected with
pSR MSVtkneo(v-abl) along with pZIP,
pZIP(nck), or pZIP(grb4), as indicated.
Transforming foci were photographed (magnification, × 4) after culture
in regular growth medium for 14-20 days. Individual transforming foci
from D were isolated and selected on G418. Subconfluent
cells were photographed (magnification, × 10). Focus assays are
representative of three or more experiments done in quadruplicate. Soft
agar data are representative of two experiments performed in
triplicate.
DISCUSSION
and confirmed that it was
ubiquitously expressed at the protein level.
(51). This may have
been due to differences between the Grb4 and Nck
sequences. A valine
at codon 26 of Nck
(51) was not present in the Grb4 sequence or that of the human, Drosophila, or Xenopus Nck (Fig.
1). Nck
also has a unique E199Q substitution (51). Another possible
explanation for the different SH2 binding affinities may be the
instability of recombinant GST-Nck or Nck-SH2 domain proteins. We did
observe a significant difference in the ability of Nck or Grb4 SH2
domains to precipitate R-PTKs when not using freshly prepared fusion
proteins.3 Both Nck and Grb4 were expressed at similar
levels in mammalian cells, suggesting that this differential stability
may not be significant in vivo. However, Grb4 but not Nck
was reported to inhibit growth factor-induced DNA synthesis, suggesting
that the adapter proteins play unique roles (51). Because we did not find any difference between the ability of Nck and Grb4 to induce Elk
activation, the effect on DNA synthesis appears not to be mediated by
the Ras/ERK pathway. An explanation for the apparent redundancy in the
function of Nck and Grb4 could be that they are differentially
expressed or regulated during development. For example, differential
regulation has been reported for the three Ras proteins, in which Ki-
but not Ha- or N-Ras was required for embryonic development (52, 53).
It will be interesting to determine whether Grb4 and Nck are
functionally redundant following gene-targeted knockout. Grb4 has also
recently been described as Nck2 (59).
SH3) c-Abl when transiently
overexpressed in 293-T cells (54). Furthermore, overexpression of Nck
did not rescue NIH 3T3 cells from the inhibitory effects of the Sos C
terminus, implying that Nck did not couple Sos to MAP kinase activation
(41). One possible explanation for this discrepancy is that the binding
efficiency of Nck and Grb4 to Sos is weaker than that of Grb2, such
that competition assays are less effective. Although we found no effect
of Nck, Grb4, or Grb2 on basal transcriptional activity, each of the
adapters could co-immunoprecipitate Sos and weakly cooperated with
v-Abl and Sos1 to induce luciferase expression via Elk-1. The
differences in cell types or culture conditions employed in these
studies likely influenced the biological observations. Indeed, we have observed that growth of NIH 3T3 cells in bovine calf serum from various
sources can result in significant differences in the magnitude of
adapter protein-induced transcriptional responses.3
Typically, we have seen less luciferase activity induced by Nck/Grb4 than by Grb2, suggesting that Grb2 is more effective at recruiting Sos
to the site of Ras activation. Transcriptional activation differences
among the adapters could be due to the presence of R-PTKs that
preferentially interact with Grb2. Alternatively, the receptors that
associate with Grb2 may be more effectively localized to the vicinity
of Ras. It has been reported that activation of PAK can result in
phosphorylation of ERKs independently of Ras (14, 46). Because Nck can
bind to PAK and influence both its location and activation (14, 16,
17), it was possible that Nck and Grb4 were circumventing Ras and
activating Elk via PAK. However, in our hands, transfection of
pCMV6(pak1) had no effect on luciferase activity in the
presence or absence of Nck.
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ACKNOWLEDGEMENTS |
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We are grateful to Leigh Mickelson-Young and Chen Bi for technical support and to Zhong-Qing Shi for mouse embryo fibroblasts.
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FOOTNOTES |
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* This work was supported by American Cancer Society Grant RPG 97-007-01-BE, United States Public Health Service Grants CA63139 and P60 DK20542-16, and funds from the Grace M. Showalter Trust (to L. A. Q.).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: Dept. of Biochemistry
and Molecular Biology, 635 Barnhill Drive, MS-4053, Indiana University
School of Medicine, Indianapolis, IN 46202. Tel.: 317-274-8550; Fax:
317-274-4686; E-mail: lquillia{at}iupui.edu.
2 B. K. Kay, personal communication.
3 L. E. Braverman and L. A. Quilliam, unpublished observations.
4 A. D. Cox and L. A. Quilliam, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: R-PTK, receptor protein tyrosine kinase; SH, Src homology domain; GST, glutathione S-transferase; ERK, extracellular signal-regulated kinase; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; PAGE, polyacrylamide gel electrophoresis; WT, wild type.
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REFERENCES |
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