| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
(Received for publication, December 22, 1994) From the
We have previously shown that overexpression of the SH2- and
SH3-containing Nck adaptor protein causes transformation of mammalian
fibroblasts. To elucidate the mechanism by which it deregulates growth,
we have sought to identify potential effectors for Nck. We report that
a serine/threonine kinase, which we term NAK (for Nck-associated kinase), associates with Nck in vivo and in vitro. Using glutathione S-transferase fusion proteins generated with isolated domains
of Nck, we demonstrate that NAK binds specifically to the second of
Nck's three SH3 domains. NAK is complexed with Nck in a wide
variety of cell types, including NIH3T3, A431, PC12, and HeLa cells. SH2 ( The effects of receptor binding have been more
difficult to address for a distinct class of SH2-containing proteins,
the so called adaptor proteins, which include
p47 The signaling pathways that are
regulated by other adaptor proteins remain undefined. We have
previously demonstrated that overexpression of Nck in mammalian
fibroblasts results in transformation(17) . To better
understand the precise role of Nck in regulating cell growth, we sought
to identify molecules that interact with Nck. We and others (8, 17, 18, 19) have demonstrated
that Nck binds to activated tyrosine kinases of both the receptor and
cytoplasmic subtypes via its SH2 domain. However, little is known about
the downstream effector pathways regulated by Nck. We previously
described the association of Nck with a serine/threonine kinase in
vitro(17) . In the current study, we demonstrate that Nck
binds to a serine/threonine kinase in vivo and that this
interaction is mediated by the second of Nck's three SH3 domains.
Furthermore, they demonstrate that Nck binds to multiple kinases via
distinct domains, potentially linking tyrosine kinases to a
serine/threonine kinase pathway in the cell.
For the filter binding assays, we prepared an additional fusion
protein construct in a pGEX-2T-derived plasmid, which was modified to
encode a cAMP-dependent protein kinase phosphorylation site after the
GST-encoding sequence(20) . The entire nck sequence,
generated by polymerase chain reaction, was inserted into the BamHI site of this vector; this construct is denoted
pGEX-2TK-Nck.
Phosphoamino acid
analysis was conducted as previously described(17) .
Figure 1:
Nck co-immunoprecipitates specifically
with a kinase that phosphorylates MBP on threonine. A, in
vitro kinase assay. Pre-immune (pi) or anti-Nck
immunocomplexes were prepared from 250 µg of 3Y1 cell lysate as
described under ``Experimental Procedures,'' washed, and
subjected to an in vitro kinase assay by the addition of 10
µCi of [
Figure 2:
GST fusion constructs containing various
domains of Nck. A, schematic diagram of GST fusion proteins.
Portions of the Nck protein contained in each fusion protein are drawn,
with SH3 domains stippled and SH2 darkstippled. The name of each construct is denoted at the right. The GST-Nck fusion protein represents the entire Nck
molecule. B, Coomassie stain of purified GST fusion proteins.
Bacteria expressing the various fusion proteins were lysed and
incubated with glutathione-Sepharose beads. Proteins were eluted and
dialyzed as previously described, fractionated by SDS-PAGE, and
Coomassie stained. Fusion proteins are labeled at the top of
each lane. The 27-kDa protein present in some of the samples
(GST-3, GST-2, and GST-32) represents cleavage of the chimera to yield
GST. Molecular masses in all figures are indicated in
kDa.
Figure 3:
The
second SH3 domain of Nck mediates interaction with NAK. A, MBP
kinase assay. Leftpanels, the indicated fusion
proteins were incubated with SR3Y1 RIPA lysates, precipitated with
glutathione-Sepharose, washed, and subjected to an in vitro kinase assay, SDS-PAGE, and autoradiography. The toppanel represents the top portion of the gel, in which the
fusion proteins run; the bottompanel represents the
low molecular weight portion of the gel where MBP runs. Amounts of
fusion protein used vary (from 1-5 µg) as do exposure times
for the various lanes. Rightpanel, GST-3 fusion
protein was incubated with SR3Y1 lysate, precipitated, and subjected to
a kinase assay. The MBP band is indicated with arrow; the upperband at 40 kDa represents the GST-3 protein. B, phosphoamino acid analysis. Phosphoamino acid content of
the MBP bands are as in A, with the fusion protein used as the
precipitating agent indicated at the top of each lane.
These results have several important implications. First,
they identify a novel class of ligands for SH3 domains, namely
serine/threonine kinases. Second, they confirm that Nck is an adaptor
protein, binding to tyrosine kinases via its SH2 domain, potentially
linking them to a serine/threonine kinase bound to its second SH3
domain. And third, they show that the SH3 domains of Nck are
non-redundant, since neither the first nor third SH3 domains of Nck are
necessary or sufficient for NAK association.
Figure 4:
NAK substrates and cation requirements.
Anti-Nck immunocomplexes were prepared from 3Y1 cells, split in five
samples, and then subjected to kinase assays using various conditions.
All lanes except lane2 utilize
Mg
Many serine/threonine
kinases require the presence of Mg
Figure 5:
Full-length Nck and its second SH3 domain
coprecipitate with a kinase of 65 and 69 kDa in in-gel kinase assays.
Whole cell lysates or immunoprecipitates were fractionated on Laemmli
gels containing MBP (0.5 mg/ml) in the resolving phase, subjected to a
kinase assay in situ, and visualized by autoradiography. WC, whole cell lysate (20 µg); lane1 is
from 3Y1 cells, and lane2 is from the Y4 cell line.
The remaining lanes are immunoprecipitates from 3Y1 cells and
are labeled with the immunoprecipitating agent. Nck, anti-Nck
immunoprecipitation; pi, pre-immune; GST, GST
protein; and GST-SH3, fusion protein expressing the second SH3
domain of Nck alone. Arrow indicates migration of the 69-kDa
kinase that coprecipitates with Nck, as well as its isolated SH3
domain.
Whole cell lysates of 3Y1 and a nck-overexpressing cell line (Y4) were also subjected to
in-gel kinase assays but failed to reveal any differences in kinase
activities between the two (Fig. 5, lanes1 and 2). This observation suggests that NAK activity may
not be deregulated in nck-transformed cells. Such an
interpretation is supported by experiments described below.
Figure 6:
Nck
binds to 65- and 69-kDa proteins by filter binding assays. Whole cell
lysates (approximately 50 µg) were fractionated by SDS-PAGE and
transferred to membranes. Membranes were then incubated with
GST-2TK-Nck fusion protein, which was
Interaction of Nck with this
65- and 69-kDa doublet of proteins was conserved in a number of other
cell types, including human HeLa and A431 cells (Fig. 6). In
A431 cells, binding occurred in an EGF-independent manner. Similarly,
these proteins were detected in murine NIH3T3 fibroblasts as well as in
lysates of 16-day-old murine whole embryos (Fig. 6). Again,
binding of Nck to this doublet was growth factor-independent in NIH3T3
cells.
Figure 7:
NAK is not activated in nck- or
v-src-transformed cells. Anti-Nck immunoprecipitates were
prepared from 250 µg of lysate of various cell lines, indicated at
the top of each lane, and subjected to in vitro MBP kinase assays. Y1, Y2, and Y4 are three nck-transformed cell lines, which overexpress low, high, and
intermediate levels of nck,
respectively.
Figure 8:
NAK localizes to P100 (membrane) fraction. A, the indicated cell lines were lysed hypotonically,
subjected to ultracentrifugation, and the supernatant collected as S100
fraction (S); the pellet was resuspended and collected as P100 (P). GST-Nck fusion protein was added to each fraction,
precipitated with glutathione-Sepharose, and subjected to a kinase
assay. Migrations of GST-Nck and MBP are indicated. B,
phosphoamino acid analysis of the MBP bands shown in A.
In this study, we demonstrate that Nck binds to a
serine/threonine kinase via its second SH3 domain; we have termed this
kinase NAK. These results have several important implications. First,
they identify a novel class of ligand for SH3 domains, namely serine
kinases. Second, they confirm that NCK acts as an adaptor protein,
binding to tyrosine kinases via its SH2 domain, and potentially linking
them to a serine/threonine kinase bound to its second SH3 domain.
Third, they demonstrate that the SH3 domains of Nck are not redundant,
since neither the first or third SH3 domains can bind NAK. To elucidate
the mechanism by which nck transforms cells, we explored the
role of NAK as a potential effector for Nck. Our data show that nck overexpression does not result in deregulation of NAK activity.
This can be interpreted in several ways. One possibility is that the
human Nck protein expressed in 3Y1 cells is incapable of interacting
with the endogenous rat NAK. This seems unlikely, since incubation of
these lysates with a GST-Nck fusion protein efficiently precipitates
NAK activity in vitro (see Fig. 3). We favor the
hypothesis that NAK exists at limiting levels in the cell, such that
overexpression of Nck does not lead to increased coprecipitation of NAK
activity. This idea is supported by immunodepletion experiments, where
pre-clearance of NAK from 3Y1 lysates by immunoprecipitation of
endogenous Nck vastly reduces the amount of NAK precipitable by
exogenously added GST-Nck. Our data further demonstrate that total NAK
activity associated with Nck is not increased by a number of mitogenic
stimuli, including v-src transformation, platelet-derived
growth factor, EGF, insulin, nerve growth factor, and serum. In the last several years, numerous groups have
contributed to an increased understanding of how SH3 domains may
function in cell signaling. Early studies (23) identified
proline-rich regions as SH3 binding moieties. Furthermore, both direct
biochemical and circumstantial evidence indicated that they play a role
in G protein signaling. (i) The Grb2 adaptor protein binds to mSOS, an
activator of the p21 More recently,
other targets of SH3 domains have been identified. For example,
phosphatidylinositol 3-kinase has been shown to interact with the SH3
domains of Src family kinases(26, 27) . Other reports
indicate that the SH3 domain of Src also binds a serine/threonine
kinase in vitro(28) , which appears to be distinct
from NAK based on its substrate specificity. Interestingly, the SH3
domain of phospholipase C- Much work remains to elucidate the exact role of NAK in
mediating Nck signaling. Identification of this kinase is a primary
goal. Because the phosphorylation of MBP by NAK is on threonine, we
explored the possibility that this might be a member of the MAP
kinase/Erk family. However, we have excluded this based on
immunoblotting of anti-Nck immunocomplexes with MAP kinase antibodies,
and utilization of more specific substrates.
Volume 270,
Number 13,
Issue of March 31, 1995 pp. 7359-7364
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
THE Nck ADAPTOR PROTEIN BINDS TO A SERINE/THREONINE KINASE VIA AN
SH3 DOMAIN (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)and SH3 domains are peptide motifs found in a
wide variety of molecules that have demonstrated roles in regulating
cell growth(1, 2) . It has been demonstrated that
virtually all SH2-containing proteins bind to activated tyrosine
kinases by a common mechanism(1, 2) . Specifically,
engagement of growth factor receptors by their cognate ligands
stimulates the intrinsic catalytic activity of the receptors, resulting
in the autophosphorylation of multiple sites in their cytoplasmic
domains(3) . These phosphorylation sites are recognized and
bound by the SH2 domains of specific proteins. Binding of
SH2-containing proteins to activated receptors can effect multiple
responses. In most instances, the recruited protein acts as a substrate
for the receptor. In the case of phospholipase C-
, this tyrosine
phosphorylation has been shown to enhance its specific
activity(4) . For other molecules, however, no such
modification of activity has been observed. Rather, it has been
proposed that association with the receptor serves to juxtapose the
protein with its physiological substrate. Such appears to be the case
with the Ras GTPase-activating protein, whose target,
p21
, is localized at the plasma
membrane(5) .
(6) , the p85 regulatory subunit
of phosphatidylinositol 3-kinase(7) , Nck(8) ,
Grb2/Sem-5(9, 10) , and Shc(11) . These
adaptor proteins consist of little more than SH2 and SH3 domains and
are believed to couple activated tyrosine kinases to various effector
pathways. Since adaptor proteins possess no recognizable catalytic
sequences, the molecular nature of their downstream effects has been
more difficult to analyze. Despite this fact, understanding of the
cellular pathways regulated by several adaptor proteins has grown
rapidly in the last several years. For example, the p85 regulatory
subunit of phosphatidylinositol 3-kinase serves to mediate interaction
of the p110 catalytic subunit with activated growth factor receptors,
resulting in its activation(7, 12, 13) . The
second adaptor with a delineated effector is Grb2/Sem-5 (names of the
murine and Cenorrhabditis elegans homologs,
respectively)(9, 10) . Multiple investigators have
recently shown that Grb2, in concert with Shc, functions in
p21 activation by binding to the guanine
nucleotide exchange factor for p21,
mSOS(14, 15, 16) . Moreover, both biochemical
and genetic evidence reveal that the SH3 domains of Grb2 are required
for interaction with mSOS and for biological
function(9, 10) .
Cell Culture
3Y1 rat fibroblasts, all
cell lines derived from 3Y1 (i.e. v-src transformed
3Y1 (SR3Y1), and nck-overexpressing Y1, Y2, and Y4 cell
lines), and NIH3T3 murine fibroblasts were maintained in
Dulbecco's minimal essential medium containing 5% bovine calf
serum at 37 °C.Plasmids and Constructs
Fusion proteins
of various domains of Nck with glutathione S-transferase (GST)
were generated. For the full-length Nck fusion protein (denoted
GST-Nck), a polymerase chain reaction product flanked by BamHI
linkers was inserted into the BamHI site of the pGEX-2T vector
(Pharmacia Biotech Inc.). The fusion construct deleted in the first SH3
domain of Nck was obtained by subcloning the blunted, gel-purified AlwNI-XbaI fragment of nck into the blunted EcoRI site of the pGEX-3X plasmid (Pharmacia); this construct
is denoted GST-332. For the fusion of GST with Nck lacking the first
two SH3 motifs (GST-32), the GST-332 vector was partially digested with BstEII and BamHI, filled in, and religated. The
chimeric protein consisting of GST with the SH2 domain of Nck (GST-2)
was derived as follows. The GST-332 DNA was digested to completion with SmaI and StuI, and the 450-base pair fragment
encoding the SH2 domain was gel purified and subcloned into the SmaI site of the pGEX-3X vector. Finally, the fusion protein
encoding the second SH3 domain of Nck with GST (GST-3) was generated by
digestion of the GST-332 plasmid with SpeI, followed by
partial digestion with BstEII, filling in, and religation. Preparation of Fusion Proteins
GST fusion
proteins were prepared as previously described(17) . For the
GST, GST-2, GST-32, and GST-332 constructs,
isopropyl-1-thio-
-D-galactopyranoside induction was
carried out at 37 °C for 3 h; for the GST-Nck and GST-3 chimeras,
induction was at 27-30 °C, as these proteins were trapped in
inclusion bodies when induced at higher temperatures.Preparation of Cell Lysates and
Immunoprecipitation
Cells were washed twice with ice-cold
phosphate-buffered saline and then lysed in NP-Tx buffer (10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, 50 mM NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 5 mM -glycerophosphate, 1 mM sodium orthovanadate, 1%
Trasylol, 1 mM phenylmethylsulfonyl fluoride). Alternatively,
cells were lysed in RIPA buffer where indicated (10 mM Tris,
pH 7.4, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1%
deoxycholate, 10% glycerol, 0.1% SDS, 1 mM vanadate, 1 mM phenylmethylsulfonyl fluoride, 1% Trasylol). Lysates were
incubated on ice for 10 min and then pelleted in a microcentrifuge at
13,000 rpm for 10 min at 4 °C.Immunoprecipitation, in Vitro Kinase Assays, and
Phosphoamino Acid Analysis
For immunoprecipitations,
anti-Nck (generated against GST-Nck fusion protein) antiserum was added
to lysates and incubated for 1-2 h at 4 °C; 60 µl of a
1:3 slurry of protein A-Sepharose was added, and the lysates were
incubated for 1 h more. For GST fusion protein precipitations,
approximately 1-5 µg of fusion protein and 60 µl of
glutathione-Sepharose beads (1:3 slurry) were used per precipitation.
Immunoprecipitates/complexes were washed four times in NP-Tx buffer.
For kinase assays, samples were then washed twice in kinase buffer (30
mM Tris pH 7.5, 10 mM MgCl
, 1 mM dithiothreitol, 1 mM -glycerophosphate, 1 mM sodium vanadate, 1% Trasylol). Kinase reactions were carried out
in a 30-µl volume of kinase buffer containing 5 µg of myelin
basic protein (MBP) (Sigma) as exogenous substrate, 20 µM cold ATP, and 10 µCi of [-
P]ATP
(3000 Ci/mmol) (Amersham Corp.). Reactions were carried out at room
temperature for 20 min and terminated by the addition of sample buffer,
followed by boiling for 5 min. Samples were fractionated on 15% Laemmli
gels and visualized by autoradiography. For assaying other substrates
and conditions, 10 mM MnCl was used rather than
MgCl where indicated, and casein, histone H1, or enolase
was added instead of MBP as exogenous substrates.Substrate-containing
Gels
Immunoprecipitations were carried out as described
above and fractionated on 10% Laemmli gels containing MBP in the
resolving phase at 0.5 mg/ml. The gel was treated as described (21) and equilibrated in kinase buffer (30 mM HEPES,
pH 7.4, 10 mM MgCl, 10 mM MnCl, 2 mM dithiothreitol, 1 mM vanadate, 5 mM -glycerophosphate) for 30 min.
Reactions were carried out for 1 h in 5 ml of the above buffer
containing 100 µCi of [-P]ATP and 10
µM cold ATP. Finally, the gel was washed in copious
amounts of 5% trichloroacetic acid, 1% sodium pyrophosphate for 10 h.Filter Binding Assays
Gel electrophoresis
and transfer of proteins to polyvinylidene fluoride membranes were
carried out as previously described. Filters were blocked for 2 h in
blocking solution (phosphate-buffered saline containing 2% bovine serum
albumin and 0.1% Triton X-100). Filters were incubated with P-labeled GST-Nck, which was phosphorylated by
cAMP-dependent protein kinase in vitro, as described by Kaelin et al.(20) . Blots were then washed four times for 15
min with phosphate-buffered saline, 0.1% Triton X-100 and subjected to
autoradiography.
Nck Associates with a Serine/Threonine Kinase in
Vivo
Work from several laboratories has shown that the Nck
adaptor protein, which consists of three SH3 domains followed by one
SH2 domain, associates with and is phosphorylated by multiple tyrosine
kinases. However, virtually nothing is known about signaling downstream
of Nck. We have previously demonstrated that Nck, expressed as a GST
fusion protein, can bind to both serine/threonine and tyrosine kinases in vitro. Since the associated serine/threonine kinase
represents a potential effector for Nck, we therefore sought to further
characterize this interaction. To test whether this kinase associates
with Nck in vivo, we immunoprecipitated Nck protein from 3Y1
rat fibroblast lysates. Immunocomplexes were washed and subjected to an in vitro kinase assay by incubation with
[-P]ATP and MBP, a commonly used kinase
substrate. Reaction mixtures were then fractionated by SDS-PAGE and
visualized by autoradiography. These in vitro kinase assays
revealed that Nck co-immunoprecipitates with a kinase that
phosphorylates MBP (Fig. 1A). Phosphoamino acid
analysis demonstrated that MBP in the anti-Nck immunoprecipitations was
phosphorylated on both serine and threonine (Fig. 1B).
In control pre-immune immunoprecipitations, a weak background kinase
activity that phosphorylated MBP on serine only was detected. Thus, Nck
appears to specifically associate with a serine/threonine kinase in
vivo. We have termed this kinase NAK, for Nck-associated kinase.
-P]ATP, 20 µM cold
ATP, and 5 µg of MBP as exogenous substrate. Reactions were
fractionated by 15% SDS-PAGE and visualized by autoradiography. Only
the low molecular weight portion of the gel is shown, as MBP migrates
in the 20-kDa range. B, phosphoamino acid analysis. The band
representing MBP was excised and subjected to phosphoamino acid
analysis as described. Lanes are labeled with the
immunoprecipitating antiserum.
NAK Binds to the Second SH3 Domain of
Nck
To determine which regions of the Nck protein are
required for its association with NAK, various GST fusion proteins were
generated with distinct domains of Nck (Fig. 2A). These
fusion proteins were purified by adsorption to glutathione-Sepharose
beads, which yielded essentially a single band by Coomassie staining (Fig. 2B). The purified fusion proteins were incubated
with lysates of SR3Y1 cells, precipitated by the addition of
glutathione-Sepharose, and subjected to in vitro MBP kinase
assays. All of the GST fusion proteins coprecipitated a kinase(s) that
phosphorylated not only MBP (Fig. 3A, bottompanel) but also the fusion protein itself (Fig. 3A, toppanel). Phosphoamino
acid analysis of the MBP bands revealed that the SH2 domain of Nck
alone was sufficient to precipitate a tyrosine kinase activity (Fig. 3B), which we have previously determined to be
pp60
(17) . ()More
interestingly, however, a kinase that phosphorylated MBP on threonine
was coprecipitated with the fusion constructs containing the second SH3
domain, namely the GST-Nck and GST-332. Deletion of the first SH3 motif
had no effect on the association of NAK. Generation of a GST fusion
protein with the second SH3 domain of Nck alone (GST-3) confirmed that
this motif was sufficient for association of the threonine MBP kinase
activity (Fig. 3A (rightpanel) and B). As a negative control, a GST chimera with the third SH3
domain of Nck was also tested; this construct failed to bind NAK (data
not shown). A weak background kinase with no threonine kinase activity
was also coprecipitated by all of the fusion proteins, including GST
alone.
Substrate Specificity and Cation
Requirements
To further characterize the Nck-associated
kinase, we tested its ability to phosphorylate other substrates in
vitro as well as its cation requirements. Anti-Nck immunocomplexes
from 3Y1 cells were able to phosphorylate casein and histone but not
enolase (Fig. 4). Phosphorylation of casein and histone occurred
exclusively on serine (data not shown).
in the reaction buffer. Lane1,
MBP as exogenous substrate; lane2, MBP as substrate
with Mn as cation; lane3, casein
as substrate; lane4, histone H1 as substrate; lane5, enolase as substrate; lane6, kinase assay of anti-pp60
immunocomplex using enolase as substrate as positive
control. Positions of enolase, H1, and casein are indicated with E, H, and C, respectively. Molecular masses
are indicated in kDa.
and are inactive
when Mn is the only divalent cation present. However,
we found that NAK functioned when Mn only was
included in the kinase reactions (Fig. 4).Substrate-containing Gels Reveal That Nck Binds to a
Kinase of 65 kDa
To obtain a molecular size estimate of
NAK, in-gel kinase assays were performed. As shown in Fig. 5,
Nck co-immunoprecipitated with a kinase of approximately 65 kDa that
phosphorylated MBP and that was not present in control
immunoprecipitations. This protein was also detected by association
with the GST-3 fusion protein but not GST alone. We assume that this
kinase activity is directed toward threonine, since in all previous
experiments this was the only amino acid specifically phosphorylated in
anti-Nck immunoprecipitations. However, this could not be confirmed, as
signals obtained by in-gel kinase assays were too weak to perform
phosphoamino acid analysis.
Interaction of Nck with 65- and 69-kDa Proteins by
Filter Binding Analysis Is Conserved in a Wide Variety of Cell
Types
Association of Nck with a 65- and 69-kDa protein
could also be reproduced using filter binding assays. Lysates were
prepared from various cell types, fractionated by SDS-PAGE, and
transferred to filters. Following denaturation/renaturation, the filter
was probed with a GST-Nck fusion protein labeled with P in vitro by phosphorylation by cAMP-dependent protein kinase.
By this assay, Nck was found to bind to proteins of approximately 65
and 69 kDa in 3Y1 fibroblasts, as well as in nck- and
v-src-transformed 3Y1 cells (Fig. 6). Neither of these
proteins was observed to bind to GST alone (data not shown). Although
the intensity of this 65- and 69-kDa doublet is greater in the Y2 nck-overexpressing cell line in this experiment, this
variation is not reproducible and is dependent on the amount of lysate
loaded. Immunoprecipitation of Nck from cells labeled with
[
S]methionine also showed a coprecipitating
protein of approximately 69 kDa, indicating that association also
occurred in solution (data not shown).
P labeled by in
vitro phosphorylation with cAMP-dependent protein kinase. Filters
were washed and visualized by autoradiography. Cell type is indicated
at the top of each lane: A-E and A+E, A431 cells treated without or with EGF,
respectively; ME16, 16-day-old whole murine embryos; 3T3-P and 3T3+P, NIH3T3 cells treated
without or with platelet-derived growth factor, respectively. Arrows indicate 65- and 69-kDa proteins whose binding is
conserved in all cell lines.
NAK Is Not Activated in nck- or v-src-transformed
Cells
To ascertain what role NAK might have in mediating nck transformation, we examined the levels of NAK activity in nck-transformed cells. As shown in Fig. 7, NAK activity
was unaltered in nck-overexpressing cell lines (Y1, Y2, and
Y4) relative to parental 3Y1 cells. Experiments were performed to
confirm that anti-Nck serum was not limiting (data not shown). NAK
activity was similarly unaffected in SR3Y1 cells (Fig. 7).
NAK Localizes to P100 (Membrane)
Fractions
Cell fractionation experiments were conducted to
determine where NAK is localized within the cell. Furthermore, since no
changes in total NAK activity were observed in the nck-transformed cell lines, we wished to explore the
possibility that its subcellular location might be altered. Y1, Y2, and
Y4 and parental 3Y1 cells were disrupted by hypotonic lysis and
subjected to ultracentrifugation. Supernatants were collected as the
soluble (S100) fraction and the resolubilized pellet as the membrane
(P100) fraction. Purified GST-Nck fusion protein was added to both
fractions, adsorbed to glutathione-Sepharose, and subjected to MBP
kinase assays (Fig. 8A). Phosphoamino acid analysis
revealed that NAK activity was only present in the P100 fraction in all
cells (Fig. 8B). Appropriate fractionation was
confirmed by probing for the presence of MAP kinase, known to be
present in S100 (and nuclear) fractions(22) , by anti-MAP
kinase immunoblotting (data not shown). NAK was also found to be
exclusively in the P100 fraction in SR3Y1 cells (data not shown). Thus,
membrane localization of NAK is not altered in nck- or
v-src-transformed cells.
While this lack of increased NAK
activity in nck-overexpressing cells may seem confounding, a
similar result has been reported for the p85 regulatory subunit of
phosphatidylinositol 3-kinase(7) . That is,
phosphatidylinositol 3-kinase activity is not increased in cells
overexpressing p85. Thus, neither the NAK specific activity nor its association with
Nck appears to be regulated by these stimuli. This is similar to what
has been reported for Grb2 and mSOS: association of Grb2 with mSOS is
independent of EGF stimulation. Moreover, purified Grb2 has no effect
on the specific activity of mSOS(15) . It has been proposed
that regulation of p21
occurs at the level of mSOS
localization, such that ligand-induced autophosphorylation of the EGF
receptor causes the recruitment of the pre-existing Grb2-mSOS complex
to the membrane, where p21 resides. Regulation of a NAK
substrate may also occur at the level of localization. It is tempting
to speculate that NAK is not the sole effector of Nck but that other
molecules associate with Nck's other SH3 domains and that the
concerted actions of these proteins mediate nck transformation. protooncogene, primarily via its
C-terminal SH3 domain(14, 15, 24) . (ii)
Cicchetti et al.(25) have cloned an Abl-SH3 binding
protein that possesses homology to GTPase-activating proteins for the
Rho family of G proteins. (iii) A novel human Ras GTPase-activating
protein specific for the CDC42 GTPase contains a proline-rich sequence
that binds to the SH3 domains of c-Src and the p85 subunit of
phosphatidylinositol 3-kinase. (iv) Dynamin, a neurally expressed G
protein, binds to and is regulated by the SH3 domains of several
signal-transducing molecules. Together, these observations suggest an
interplay between SH3 motifs and G protein signaling. is very homologous (46%) to the second
SH3 domain of Nck, such that certain monoclonal antibodies against
phospholipase C- cross-react with Nck(18) . Despite this
fact, we do not observe a threonine-directed MBP kinase coprecipitating
with intact phospholipase C- or with a GST fusion of its SH3
motif. NAK therefore appears to be a specific effector for
Nck. The results
from the filter binding assays demonstrate that expression cloning may
be a fruitful and expedient method for identifying Nck binding
proteins. We are currently in the process of cloning such proteins.
Isolation of these proteins will allow us to better understand how Nck
regulates cell growth and how its overexpression mediates
transformation.
))
We thank Gerd Blobel, Glen Scholz, and Beatrice
Knudsen for critical reading of this manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  C. S. Lim, S. H. Kim, J. G. Jung, J.-K. Kim, and W. K. Song Regulation of SPIN90 Phosphorylation and Interaction with Nck by ERK and Cell Adhesion J. Biol. Chem., December 26, 2003; 278(52): 52116 - 52123. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Chen, M. A. White, and M. H. Cobb Stimulus-specific Requirements for MAP3 Kinases in Activating the JNK Pathway J. Biol. Chem., December 13, 2002; 277(51): 49105 - 49110. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. B. Goldberg Actin-Based Motility of Intracellular Microbial Pathogens Microbiol. Mol. Biol. Rev., December 1, 2001; 65(4): 595 - 626. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Pearson, F. Robinson, T. Beers Gibson, B.-e Xu, M. Karandikar, K. Berman, and M. H. Cobb Mitogen-Activated Protein (MAP) Kinase Pathways: Regulation and Physiological Functions Endocr. Rev., April 1, 2001; 22(2): 153 - 183. [Abstract] [Full Text]
|
|
|
|
|
|
L. E. Braverman and L. A. Quilliam Identification of Grb4/Nckbeta , a Src Homology 2 and 3 Domain-containing Adapter Protein Having Similar Binding and Biological Properties to Nck J. Biol. Chem., February 26, 1999; 274(9): 5542 - 5549. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Tu, F. Li, and C. Wu Nck-2, a Novel Src Homology2/3-containing Adaptor Protein That Interacts with the LIM-only Protein PINCH and Components of Growth Factor Receptor Kinase-signaling Pathways Mol. Biol. Cell, December 1, 1998; 9(12): 3367 - 3382. [Abstract] [Full Text]
|
|
|
|
|
|
M. Chen, H. She, E. M. Davis, C. M. Spicer, L. Kim, R. Ren, M. M. Le Beau, and W. Li Identification of Nck Family Genes, Chromosomal Localization, Expression, and Signaling Specificity J. Biol. Chem., September 25, 1998; 273(39): 25171 - 25178. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. M. Anton, W. Lu, B. J. Mayer, N. Ramesh, and R. S. Geha The Wiskott-Aldrich Syndrome Protein-interacting Protein (WIP) Binds to the Adaptor Protein Nck J. Biol. Chem., August 14, 1998; 273(33): 20992 - 20995. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Lussier and L. Larose A Casein Kinase I Activity Is Constitutively Associated with Nck J. Biol. Chem., January 31, 1997; 272(5): 2688 - 2694. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. Quilliam, Q. T. Lambert, L. A. Mickelson-Young, J. K. Westwick, A. B. Sparks, B. K. Kay, N. A. Jenkins, D. J. Gilbert, N. G. Copeland, and C. J. Der Isolation of a NCK-associated Kinase, PRK2, an SH3-binding Protein and Potential Effector of Rho Protein Signaling J. Biol. Chem., November 15, 1996; 271(46): 28772 - 28776. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. M. Bokoch, Y. Wang, B. P. Bohl, M. A. Sells, L. A. Quilliam, and U. G. Knaus Interaction of the Nck Adapter Protein with p21-activated Kinase (PAK1) J. Biol. Chem., October 18, 1996; 271(42): 25746 - 25749. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. August and B. Dupont Association between Mitogen-activated Protein Kinase and the [IMAGE] Chain of the T Cell Receptor (TcR) with the SH2,3 Domain of p56[IMAGE] J. Biol. Chem., April 26, 1996; 271(17): 10054 - 10059. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Bagrodia, S. J. Taylor, C. L. Creasy, J. Chernoff, and R. A. Cerione Identification of a Mouse p21[IMAGE] Activated Kinase J. Biol. Chem., September 29, 1995; 270(39): 22731 - 22737. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Hashimoto, A. Tsubouchi, Y. Mazaki, and H. Sabe Interaction of Paxillin with p21-activated Kinase (PAK). ASSOCIATION OF PAXILLIN alpha WITH THE KINASE-INACTIVE AND THE Cdc42-ACTIVATED FORMS OF PAK3 J. Biol. Chem., February 16, 2001; 276(8): 6037 - 6045. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Yan, K. Nehrke, J. Choi, and D. L. Barber The Nck-interacting Kinase (NIK) Phosphorylates the Na+-H+ Exchanger NHE1 and Regulates NHE1 Activation by Platelet-derived Growth Factor J. Biol. Chem., August 10, 2001; 276(33): 31349 - 31356. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |
