(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 ()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) .
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(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) .
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 [-
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.
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 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.
Many serine/threonine
kinases require the presence of Mg 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).
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 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.
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. 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.
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. 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.
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 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.
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- 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.
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. 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.