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INTRODUCTION |
Cell contact with growth factors and extracellular matrix
activates one or more of the
MAPK1 pathways, and these
responses appear to be critical for the appropriate regulation of cell
division and differentiation (1, 2). Although many of the molecules
that participate in these processes have been defined, there are still
significant gaps in our understanding of how multiple signals are
integrated into an appropriate outcome.
The MAPK kinase family of proteins (ERK, JNK, and p38) can
phosphorylate and activate one or more transcription factors and are
activated by a cascade of upstream kinases (for reviews, see Refs. 3
and 4). The JNK series of kinases that are part of the process that
leads to the activation of AP-1-containing promoters (6) appear to be
especially critical for transducing an immediate response to a changing
environment and for regulating cell proliferation (5). JNK is itself
activated following phosphorylation by a JNK kinase (7-10), which in
turn is also activated by phosphorylation. Although several kinases
have been described that can phosphorylate the JNK kinases, the most
important appear to be the MEKK proteins (11). Activation of the MEKK
proteins is dependent on one or more of the small GTP-binding proteins
such as Cdc42, Rac, and Ras (12-14). Although the activation of the
MAPK kinases can be described in terms of a linear series of sequential
activations, it has become clear that many of the relevant components
are assembled into complexes through scaffolding molecules such as MP1
(15) and JIP-1 (16).
Growth factors such as EGF can stimulate all of the MAPK kinases (11),
and for the ERK kinases, many of the components of the pathway are well
established (17). In contrast, the signaling from EGF to the JNK
pathway has received relatively less attention. The ability of EGF to
activate guanylate exchange factors such as Sos and the known
involvement of the GTP-binding proteins in JNK activation suggest that
EGF may also be linked to JNK via guanylate exchange factors (18). A
role for phosphatidylinositol 3-kinase in linking EGF to JNK has also
been reported (19).
The immediate outcome of integrin receptor activation is the formation
of a large complex of kinases and adaptor proteins. This complex
contains at least the kinases FAK, c-Crk, and c-Src and the adaptor
molecule Cas (Crk-associated
substrate) (20-22). FAK is primarily responsible for
phosphorylating a number of proteins involved in cytoskeletal assembly
(23, 24), whereas c-Src (or Src-like kinases) can phosphorylate Cas
(25) and can activate the MAPK pathway (26, 27). Cas, originally
identified as a hyperphosphorylated protein following induced
expression of the viral oncogene Crk (v-Crk) (28), is phosphorylated in
response to growth factors acting through receptor tyrosine kinases
(29) and integrin-mediated signaling (30). As Cas has an SH3-domain as
well as multiple SH2-binding motifs, Cas may well assemble a number of
proteins such as FAK (20) and the phosphatases PTP-PEST (31) and PTP1B
(32) into a single complex. Cas may be a critical component by
which extracellular events influence cell morphology and
survival (33-35).
A theme running through the mechanism by which transcription factors
such as AP-1 are activated by receptor tyrosine kinases and integrin
receptors is the importance of adaptor proteins. These adaptor proteins
contain one or multiple domains that mediate protein-protein or
protein-lipid interactions (for review, see Ref. 36). In addition to
integrating independent extracellular signals, cell type-specific
expression of the adaptor molecules may be critical in determining the
cell type-specific response to extracellular stimuli. Although most of
the more extensively characterized signal transduction molecules are
ubiquitously expressed (15, 37, 38), a few adaptor molecules with a
limited pattern of expression have been identified, such as N-Shc
(37) and Grb-IR (39).
To identify additional adaptor proteins that are involved in growth
factor and integrin signaling, we have searched an expressed sequence
tag (EST) data base for sequences that share homology with
SH2-containing adaptor proteins. By this method, we isolated a cDNA
encoding a family of novel SH2-containing proteins. The structure and
characteristics of at least NSP1 (novel
SH2-containing protein 1) suggest
that this protein may play an important role in cell proliferation and
fetal development.
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EXPERIMENTAL PROCEDURES |
Cloning of NSP1, NSP2, and NSP3 cDNAs--
A proprietary EST
data base (Incyte) was screened for sequences that encode homologues of
proteins known to be involved in intracellular signaling. One EST from
a fetal pancreas library encoded an SH2 domain related to human SHC. A
full-length cDNA encoding NSP1 was cloned from a human
fetal kidney cDNA plasmid library in pRK vector using standard
polymerase chain reaction and hybridization protocols. The full-length
NSP1 cDNA sequence was then used to rescreen the EST
data base. Two related cDNAs, NSP2 and NSP3,
were subsequently cloned. The Flag epitope (DYKDDDDK) was added in
frame to the N terminus of the NSP1 cDNA construct using
in vitro mutagenesis (Stratagene) to create pRK.NSP1.Flag. All three tyrosine residues in NSP1 were independently mutated to
phenylalanine (Y16F, Y95F, and Y231F) using in vitro mutagenesis.
Northern Hybridization Analysis--
The human
multiple-tissue Northern I, fetal II, and human immune system II
blots (CLONTECH) were hybridized with a
32P-random primer-labeled NSP1 probe
representing the SH2 domain and a 32P-end-labeled
oligonucleotide NSP2 probe corresponding to amino acids
270-284 and NSP3 probe corresponding to amino acids
475-491 according to the manufacturer's instructions.
Cell Culture and Transfection--
COS and 293 cells were
maintained in Dulbecco's modified Eagle's medium with 10% fetal
bovine serum, 2 mM L-glutamine, 100 units/ml
penicillin, and 100 mg/ml streptomycin at 37 °C. CHO-IR cells (CHO
cells overexpressing insulin receptor, provided by Craig Crowley,
Genentech, Inc.) and CHO cells were cultured in nutrient mixture
F-12/Dulbecco's modified Eagle's medium supplemented as described
above. Liposome-mediated transfections using DOSPER (Boehringer
Mannheim) or Superfect (QIAGEN Inc.) were carried out on CHO, COS, or
293 cells in accordance with the manufacturers' instructions.
Immunoprecipitation and Western Blot Analysis--
Transiently
transfected cells were lysed on ice for 1 h in 1 ml of
immunoprecipitation assay buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 1% Triton X-100, 2 mM EDTA 10 mM sodium pyrophosphate, 10 mM sodium fluoride,
and 2 mM orthovanadate) containing freshly added protease
inhibitors (1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride,
10 mM leupeptin, 2 mg/ml aprotinin, and 1 mM
pepstatin). Samples were immunoprecipitated with anti-Flag affinity gel
(Kodak Scientific Imaging Systems), anti-EGF receptor antibody
(Calbiochem), or anti-c-Myc antibody (Boehringer Mannheim). The
immunoprecipitates were subjected to either Western blotting or an
in vitro kinase assay. For Western blotting, following
SDS-polyacrylamide gel electrophoresis, proteins were transferred onto
nitrocellulose membrane (Novex) and analyzed using the
anti-phosphotyrosine antibody PY20 (Transduction Laboratories), the
anti-p130Cas antibody (Transduction Lab), or the anti-Flag
antibody and detected with the ECL system (Pierce).
JNK Kinase and Luciferase Assays--
293 cells were
cotransfected with 2.5 µg of the pJNK-Myc or pRK.ERK2.Flag plasmid
and the various NSP1 expression plasmids by Superfect. Anti-c-Myc or
anti-Flag immunoprecipitates were generated 36 h after
transfection and subjected to JNK immune complex kinase assays using
activating transcription factor-2 (Santa Cruz Biotechnology) or myelin
basic protein (Upstate Biotechnology, Inc.) as a substrate. One-half of
the reactions were separated by SDS-polyacrylamide gel electrophoresis
and transferred to nitrocellulose membranes. The membranes were
Western-blotted with anti-JNK1 (Santa Cruz Biotechnology) or anti-Flag
antibody to show the amount of JNK or ERK2 in each lane.
CHO cells were grown in six-well culture plates and cotransfected with
0.5 µg of the firefly luciferase reporter plasmids, 2 µg of the
various NSP1 expression plasmids, and 50 ng of the Renilla
luciferase reporter plasmids (Promega) by Superfect. Firefly and
Renilla luciferase activities were determined 36 h
after transfection. The Renilla luciferase activity was used
to normalize the firefly luciferase activity according to the
manufacturer's recommendations.
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RESULTS |
cDNA Cloning of NSP1, NSP2, and NSP3--
A proprietary data
base was searched for sequences related to signal transduction motifs.
An EST homologous to the SH2 domain of the adaptor protein Shc was
found and used to clone a corresponding full-length cDNA encoding a
576-amino acid protein: NSP1. NSP1 is most closely related to Shc (34%
identity over a 92-amino acid region in the Shc SH2 domain), although
this similarity only covers the SH2 region that appears at the N
terminus of NSP1 (Fig. 1). NSP1 also
contains a proline/serine-rich domain (PS domain) that may
function as an SH3 interaction domain. Rescreening the EST data base
with the NSP1 sequence allowed the cloning of two related cDNAs (NSP2 and NSP3). These three proteins
(calculated molecular masses of 63.1, 92.6, and 77.1 kDa) have an
overall shared identity of between 25 and 39%, and all three proteins
contain SH2 domains and potential SH3 interaction domains. None of the
three NSP proteins contain obvious kinase or phosphatase domains, but
all three do contain an amino acid motif that is consistently found in
proteins with guanylate exchange activity. Whether there is a guanylate exchange activity associated with these proteins is being
investigated.

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Fig. 1.
Structural characteristics and alignment of
NSP1, NSP2, and NSP3. The amino acid sequences and the alignment
of NSP1, NSP2, and NSP3 deduced from the cloned cDNAs are shown.
The approximate delimitation of the SH2 domains and the proline/serine
(PS)-rich regions are indicated and shaded in
gray.
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Expression of NSP1, NSP2, and NSP3 in Human Tissues--
By
Northern hybridization analysis, NSP1 is weakly expressed in
comparison to NSP2 and NSP3, and significant
levels of NSP1 mRNA were only detected in the placenta;
fetal kidney and lung; and adult pancreas, kidney, and lung (Fig.
2A). Whether there is a
biological relevance to the apparent specificity of expression in
organs enriched in secretory epithelial cells is of some interest. In
addition, we have detected expression of NSP1 in some cell lines
including SW480 (data not shown). In contrast, NSP2 and NSP3 are
expressed in a wide variety of fetal and adult tissues. In
hematopoietic tissues, two NSP3 transcripts were detected
(Fig. 2B). The relative level of the two transcripts in
these hematopoietic tissues does vary; some such as the thymus express
only the higher molecular mass form.

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Fig. 2.
Northern blot analysis of
NSP1, NSP2, and NSP3
expression. The human multiple-tissue Northern I and fetal
II (A) and human immune system II (B) blots were
hybridized using 32P-labeled probes complementary to the
appropriate cDNAs (see "Experimental Procedures").
kb, kilobases; PBL, peripheral blood
leukocytes.
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NSP1 Phosphorylation by EGF and Insulin--
As NSP1 contains
three tyrosines, we investigated whether NSP1 could be phosphorylated
in response to extracellular stimuli. COS and CHO-IR cells were used to
investigate responses to EGF and insulin, respectively. Cell extracts
were prepared following transfection with Flag-tagged NSP1 expression
plasmids, and NSP1 was immunoprecipitated with anti-Flag affinity gel
and Western-blotted with an anti-phosphotyrosine antibody. Treatment
with either insulin or EGF for 10 min induced rapid tyrosine
phosphorylation of NSP1 (Fig. 3).

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Fig. 3.
EGF and insulin induce NSP1 tyrosine
phosphorylation. COS (A) and CHO-IR (B)
cells were transfected with pRK.NSP1.Flag. Serum-starved cells were
treated (+) or not ( ) with 25 ng/ml EGF and 100 nM
insulin for 10 min, respectively. The anti-Flag immunoprecipitates were
blotted with anti-phosphotyrosine antibody PY20. The same blots were
stripped and reblotted with anti-Flag antibody. IP,
immunoprecipitation; W, Western blotting. The
encircled P indicates "phospho."
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Complex Formation of NSP1 with p130Cas and the EGF
Receptor--
Co-immunoprecipitation experiments were performed to
identify protein partners of NSP1. Under basal conditions
(serum-starved cells attached to tissue culture plates or cells held in
suspension for 30 min), immunoprecipitated, unphosphorylated NSP1 was
found to interact with a 130-kDa protein subsequently identified as p130Cas (Cas) using an anti-Cas antibody (Fig.
4A). The
tyrosine-phosphorylated protein with a molecular mass of ~120 kDa has
not yet been identified. EGF increased NSP1 phosphorylation, but led to
a dephosphorylation of the Cas that was associated with NSP1 (Fig.
4A, upper panel). The apparent decrease in Cas
phosphorylation becomes more significant when the EGF-stimulated
increase in the amount of Cas that is immunoprecipitated is taken into
account (Fig. 4A, center panel). Insulin
treatment of CHO-IR cells induced comparable changes (data not shown).
In contrast to the response to receptor tyrosine kinases, replating of
detached cells on fibronectin stimulated only weak NSP1 phosphorylation
(Fig. 4B), but led to a significant increase in the
phosphorylation of the Cas that was associated with NSP1 and, at the
same time, a transient dissociation in the amount of Cas that was
associated with NSP1. The amount of the NSP1·Cas complex reached a
nadir at ~30 min following contact with the fibronectin and then
returned toward base-line conditions at ~4 h. The band below NSP1
appears to be nonspecific.

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Fig. 4.
NSP1 interacts with p130Cas.
Transfected and serum-starved COS cells were either treated with 25 ng/ml EGF for the times indicated or left untreated (A).
Transfected and serum-starved 293 cells were left attached to plastic
(On dish), held in suspension (Off dish), or
replated onto 10 mg/ml fibronectin (FN)-coated dishes for
the times indicated (B). Cells were lysed, and the anti-Flag
immunoprecipitates were visualized by anti-(P)Tyr antibody PY20
(upper panels). The same blots were stripped and reblotted
with anti-p130Cas (middle panels) or anti-Flag
(lower panels) antibody. IP, immunoprecipitation;
W, Western blotting; EGFR, EGF receptor. The
encircled P indicates "phospho."
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In COS cells treated with EGF, a 170-kDa protein was
co-immunoprecipitated with NSP1 (Fig. 4A). This protein was
rapidly tyrosine-phosphorylated in response to EGF and could be
detected with an antibody against the EGF receptor (data not shown). In
a reverse experiment, we showed that NSP1 could be detected following
immunoprecipitation of the EGF receptor and that the extent of the
interaction significantly increased following exposure to EGF (Fig.
5). Under these conditions, we could
detect EGF receptor-associated NSP1 using the anti-Flag antibody (Fig.
5, upper panel), but could not detect phosphorylated NSP1
using the anti-phosphotyrosine antibodies. It is unclear whether this
is a technical issue (e.g. relative affinities of the two
antibodies) or reflects the state of the NSP1 that remains associated
with the receptor.

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Fig. 5.
NSP1 interacts with the EGF receptor.
Transfected and serum-starved COS cells were either treated with 25 ng/ml EGF for the times indicated or left untreated, and the cell
lysates were immunoprecipitated with anti-EGF receptor
(EGFR) antibody. The associated NSP1 protein was detected
with anti-Flag antibody (upper panel); the same blot was
reblotted with anti-(P)Tyr antibody to show the phosphorylated EGF
receptor (lower panel). IP, immunoprecipitation;
W, Western blotting. The encircled P
indicates "phospho."
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Identification of Phosphorylated Tyrosine Residues in
NSP1--
To determine which tyrosine residues in NSP1 are
phosphorylated, we changed each of the three tyrosines in NSP1 to
phenylalanine. Transfected cells were then stimulated with EGF for 10 min, and NSP1 was immunoprecipitated (Fig.
6). Mutant NSP1Y16F was
phosphorylated normally in response to EGF, suggesting that Tyr-16 may
not be phosphorylated. However, there was no detectable phosphorylation of NSP1Y95F, and NSP1Y231F was weakly
phosphorylated. Note that each of these variants can still interact
with the EGF receptor whether or not NSP1 is phosphorylated,
demonstrating that the amino acid substitution does not lead to gross
protein misfolding and that the lack of phosphorylation is not simply
the result of failure to interact with the EGF receptor. Although other
possibilities exist, these data suggest a sequential phosphorylation
model in which there is an obligatory first phosphorylation of Tyr-95, followed by the phosphorylation of Tyr-231.

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Fig. 6.
Identification of phosphorylated tyrosine
residues in NSP1. Each of the three tyrosine residues in NSP1 was
individually changed to phenylalanine. Variants (Y16F, Y95F, and Y231F)
and wild-type (WT) NSP1 were transfected into COS cells and
treated with 25 ng/ml EGF for 10 min or left untreated. Cell lysates
were immunoprecipitated with anti-Flag antibody and Western-blotted
with anti-phosphotyrosine, anti-p130Cas or anti-Flag antibody. The
encircled P indicates "phospho."
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NSP1 Activates the JNK1 Kinase and AP-1--
To identify
downstream effects of NSP1, 293 cells were cotransfected with NSP1 and
the expression plasmids for JNK1 or ERK2. The kinases were
immunoprecipitated, and their activities were determined with
recombinant ATF-2 or myelin basic protein as a substrate in in
vitro kinase assays. Although there was no apparent increase in
ERK2 kinase activity (Fig.
7A), increased expression of
NSP1 led to a dose-dependent increase in JNK1 activity
(Fig. 7B). Activation of JNK should lead to phosphorylation
of c-Jun and the subsequent increase in expression from reporter genes under the control of AP-1 recognition sequences. Conversely, the failure to activate ERK should be revealed by a lack of activation of
promoters containing a serum response element (SRE). Thus, NSP1 was
cotransfected with the luciferase cDNA controlled by a promoter
containing either AP-1 or SRE interaction sites. Consistent with the
results of the in vitro kinase assays, we have found that
NSP1 expression led to a 7-fold increase in the level of expression of
an AP-1-luciferase gene. Under the same conditions, there was no
increase in expression of the luciferase gene containing SRE
recognition sequences (Fig. 8). In both
experiments, transfection with a plasmid expressing the MAPK kinase
MEKK led to the expected increase in luciferase expression.

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Fig. 7.
Increased expression of NSP1 results in JNK
(but not ERK) activation. 293 cells were transfected with 2.5 µg
of pJNK-Myc (A) or pRK.ERK2.Flag (B) and
increasing amounts of NSP1 expression plasmids as indicated. The
anti-Flag or anti-c-Myc immunoprecipitates were evaluated for their
ability to phosphorylate myelin basic protein (MBP;
A) or ATF-2 (B) in an in vitro kinase
assay (INK). The same blots were used in Western blotting
(W) to show the amount of JNK1 and ERK2 proteins in each
lane. IP, immunoprecipitation.
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Fig. 8.
Increased expression of NSP1 leads to the
activation of the AP-1 (but not SRE) promoter. CHO cells were
cotransfected with 0.5 µg of the pAP-1-Luc or pSRE-Luc reporter
plasmid and 2 µg of the indicated NSP1 expression plasmids. The
relative luciferase activities were generated using Renilla
luciferase (see "Experimental Procedures"). Each point is the
average of triplicates. The results represent one of three
replicates.
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DISCUSSION |
We have isolated a family of novel adaptor proteins (NSP1, NSP2,
and NSP3) that contain an N-terminal SH2 domain, a central proline/serine-rich region, and a C-terminal sequence that is distinctive for the NSP proteins. While this manuscript was in preparation, the sequence of NSP2 was published as BCAR3;
this protein (BCAR3) appears to be implicated in the development of resistance to the cytostatic agent Taxol (40). NSP1
expression may be restricted to tissues with secretory epithelial
cells, whereas NSP2 and NSP3 are expressed in
variety of tissues. In hematopoietic tissues, two
NSP3-related transcripts are detected. The coding potential
of these two NSP3 transcripts is being explored.
NSP1 is phosphorylated in response to insulin and EGF (and insulin-like
growth factor I and heregulin (data not shown)), indicating that NSP1
is a common target for a variety of growth factor receptors. In
response to EGF, NSP1 associates with the EGF receptor and is
phosphorylated. The data are consistent with a direct interaction between the phosphotyrosines on the EGF receptor and the NSP1 SH2
domain and a subsequent receptor kinase-dependent
phosphorylation of NSP1. Preliminary data indicate that the SH2 domain
alone is able to interact with the EGF receptor, although other regions of NSP1 may also participate in this interaction (data not shown). It
is also possible that the EGF receptor-NSP1 interaction is indirect.
NSP1 is only modestly phosphorylated in response to integrin signaling.
This weak integrin-mediated phosphorylation could conceivably be
through FAK or Src-related kinases, both of which are known to
associate with Cas (20, 22, 25, 28, 41).
Co-immunoprecipitation experiments revealed that NSP1 interacts with
p130Cas. Preliminary data from a yeast two-hybrid analysis
are consistent with this result and suggest that the interaction is
direct (data not shown). In the co-immunoprecipitation experiments, the
interaction between NSP1 and Cas could be detected under conditions in
which there was no detectable phosphorylation of NSP1 or Cas,
suggesting that the interaction between NSP1 and Cas is
phosphorylation-independent and may occur via the SH3 domain in Cas and
the proline/serine-rich domain in NSP1. Neither of the demonstrated
associations (EGF receptor and Cas) appears to utilize the
phosphotyrosines in NSP1. This conclusion is reinforced by the
experiments using the tyrosine replacement variants in which both EGF
receptor and Cas interactions can occur in the absence of any
detectable NSP1 phosphorylation.
The relative level of the NSP1·Cas complex and the phosphorylation
status of Cas are quite different between receptor tyrosine kinase and
integrin signaling. Thus, EGF treatment leads to NSP1 phosphorylation,
to dephosphorylation of the Cas that is associated with NSP1, and to an
increase in the amount of the NSP·Cas complex. In contrast, in
response to fibronectin, there is little change in NSP1
phosphorylation, but there is a significant increase in the
phosphorylation of the Cas associated with NSP1. Cas dephosphorylation in response to EGF (42) and Cas phosphorylation in response to
fibronectin (30, 43, 44) have been previously reported. There is also
an apparent dissociation of NSP1 from Cas at short time periods and a
subsequent reassociation at longer (4 h) times. These results led to
the hypothesis that the biological outcome in response to extracellular
signals could be quite distinct in the presence or absence of NSP1. For
example, FAK associates with the SH3 region in Cas via a
PXXP region at the C terminus of FAK (P715SRP;
mouse nomenclature (22)). There are six PXXP signatures in
NSP1. Thus, it is possible that NSP1 could compete for the SH3 region
in Cas and decrease the amount of FAK that is bound to Cas and so alter
FAK-dependent events (21). In contrast, EGF treatment leads
to NSP1 phosphorylation, to dephosphorylation of NSP1-associated Cas,
and to an increase in the amount of the NSP1·Cas complex. This
complex is then likely to have a decrease in the number of proteins
associated with the phosphotyrosines in Cas and so may lead to changes
in downstream signaling.
Increased expression of NSP1 results in JNK activation and increased
expression from an AP-1-dependent promoter. Whether this activation of JNK is related to the EGF-stimulated phosphorylation of
NSP1 or the interaction of Cas with NSP1 is currently unknown, although
preliminary data from a luciferase assay indicate that NSP1Y95F, which is not phosphorylated but still interacts
with both Cas and the EGF receptor, has diminished but not abolished activity in this assay. It has been previously shown that activation of
the JNK kinase cascade is dependent on one of several small GTP-binding
proteins (Cdc42, Rac, and Ras) (12, 14) and on phosphatidylinositol
3-kinase (19). How or whether NSP1 can modify any of these components
of the signaling pathway is under investigation.
The activation of the c-Jun kinases in response to receptor tyrosine
kinase and integrin receptor signaling appears to be critical for
regulating cell proliferation (45-47). Furthermore, genetic analysis
of JNK signaling in Drosophila (5) and the abnormal liver
development in mice genetically lacking the JNK kinase activator MKK4
suggest a critical role for JNK in normal development (48). Thus, the
identification of a novel adaptor protein that functions in these
processes may provide a valuable molecular tool for understanding cell
proliferation and fetal development.