From the La Jolla Cancer Research Center, The Burnham Institute, La Jolla, California 92037
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ABSTRACT |
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Integrin ligand binding induces a signaling
complex formation via the direct association of the docking protein
p130Cas (Cas) with diverse molecules. We report here
that the 14-3-3 Integrin-mediated cell-extracellular matrix
(ECM)1
interactions affect many aspects of cell behavior, including cell
proliferation, differentiation, and migration (1, 2). Upon ligand
binding, integrins recruit a number of signaling proteins to sites of
cell contact with the ECM known as focal adhesions (3). As a result, several focal adhesion proteins are rapidly tyrosine-phosphorylated and
engaged in protein-protein interactions with signaling proteins containing Src homology 2 (SH2) domains. One of the
tyrosine-phosphorylated proteins that orchestrates the assembly of
these signaling complexes is p130Cas (Cas), a docking
protein originally identified as the major tyrosine-phosphorylated substrate in v-Crk and v-Src transformed cells (4-7). Cas has a unique
structure composed of several domains capable of mediating protein-protein interactions (see Fig. 1). An amino-terminal SH3 domain
has been shown to associate with proline-rich regions of the focal
adhesion kinase (FAK) (8), and of the protein tyrosine phosphatases
PTP1B (9) and PTP-PEST (10). Furthermore, the binding of Src family
kinases toward the carboxyl terminus of Cas (11) results in the
tyrosine phosphorylation of this docking protein in a region known as
the substrate domain (SD) (see Fig. 1) (12-14). Nine YDV/TP motifs
that conform to the binding motif for the Crk SH2 domain appear in this
region and, indeed, Cas-Crk interaction is known to take place upon
integrin-mediated cell adhesion (12, 13, 15). Cas also contains a
serine-rich domain (designated here as SR) (see Fig. 1); however,
despite the fact that Cas is known to undergo serine phosphorylation in
response to integrin-mediated cell adhesion (Ref. 16, and data not
shown), the function of this region or the significance of serine
phosphorylation of Cas is unknown.
To get insight into the function of the SR region in Cas, we set forth
to identify the proteins that directly bind Cas within this domain. We
report here that among these molecules are the 14-3-3 proteins. Members
of the 14-3-3 family form homo- or heterodimeric complexes that mediate
interactions between diverse components of signaling pathways,
including the serine/threonine kinase c-Raf-1 (17-20), the tyrosine
phosphatase Cdc25 (21), the phosphatidylinositol 3'-kinase (22), the
proto-oncogene product Cbl (23), the tumor suppressor gene p53 (24),
and the Bcl-2 family member Bad (25) (for a review, see Refs. 26 and
27). In many cases, serine phosphorylation of the binding partner is
crucial in regulating the interaction with 14-3-3 (28), and by using
phosphoserine-oriented libraries, two different binding consensus
motifs for 14-3-3 were identified (29). Through the various
interactions, the 14-3-3 proteins have been proposed to regulate
enzymatic activity of their binding partners, to serve as clustering
proteins bringing together enzymes and substrates, and to function as
"chaperone" molecules that stabilize structural conformations of
the associated proteins (26). Our results reported here suggest a role
for 14-3-3 proteins in integrin signaling pathways and provide new insights into their functional significance in intracellular signaling events.
DNA Constructs and Mutagenesis--
The yeast two-hybrid cloning
vectors pVP16 and pBTM116, the DNA construct pBTM116/lamin, and the
yeast strain L40 have been described in (30). The "bait" construct
used for the two-hybrid screening, pBTM116/Cas(SR), contains the SR
domain of Cas encompassing the amino acids 520 to 712 (Fig. 1)
(numbering according to Sakai et al. (7)), and was generated
by cloning the corresponding cDNA fragment in frame with the LexA
coding sequence into the vector pBTM116. The DNA constructs encoding
glutathione S-transferase (GST) fusion proteins of the
14-3-3 isoforms Yeast Two-hybrid Analysis--
The yeast two-hybrid interaction
screen was performed essentially as described by Vojtek et
al. (30). The plasmid pBTM116/Cas(SR) was introduced into the L40
yeast strain, total protein extracts were prepared from individual
colonies (32), and the expression of LexA-Cas(SR) was assessed by
immunoblotting (anti-LexA antibody) (Santa Cruz Biotechnology). A L40
yeast clone expressing LexA-Cas(SR) was transformed with a murine whole
embryo library (embryonic day 9.5-10.5) constructed in the plasmid
pVP16 (33). The resulted transformants (2 × 106) were
selected using histidine prototrophy and analyzed for
Cell Culture, Adhesion, and Transfection--
Mammalian cells
used in the study were grown in Dulbecco's modified Eagle's medium
containing glutamine Pen-Strep (Irvine Scientific) and 10% fetal calf
serum (Tissue Culture Biologicals). For cell adhesion experiments,
Rat-1 cells were grown to 90% confluency as a monolayer, serum-starved
for 18 h, and detached with trypsin-EDTA treatment followed by
treatment with 0.5 mg/ml of trypsin inhibitor. Cell suspensions were
incubated in 0.5% bovine serum albumin, Dulbecco's modified Eagle's
medium at 37 °C with continuous rotation for the indicated time
periods and plated onto dishes precoated with 10 µg/ml of fibronectin
(FN) or with 2 mg/ml of poly-L-lysine (2 mg/ml). Cell
transfections were performed using the LipofectAMINE reagent (Life
Technologies, Inc.) following the manufacturer's protocol.
Preparation of Cell Lysates, Immunoprecipitations, and
Immunoblotting--
Cell cultures were rinsed with ice-cold
phosphate-buffered saline (PBS) and lysed on ice for 5 min (50 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM
EGTA, 10% glycerol, 1% Triton X-100, 1.5 mM
MgCl2, 200 µM Na3VO4,
50 mM NaF, 0.1 units/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Cell lysates were
cleared by centrifugation for 15 min at 4 °C, normalized for the
protein content and subjected to immunoprecipitation or affinity
association experiments (described later). Immunoprecipitations were
carried out with a commercial anti-Cas antibody (C-20, Santa Cruz
Biotechnology) or with a rabbit polyclonal antibody generated in the
laboratory against the SR domain of Cas and protein A-Sepharose,
followed by SDS-polyacrylamide gel electrophoresis (PAGE).
Immunoblotting was performed with an anti-Cas antibody (Transduction
Laboratories), anti-14-3-3 Affinity Association Using GST Fusion Proteins or
Glutathione-Sepharose--
In vitro association experiments
were carried out with GST fusion proteins containing the full-length
14-3-3 molecule. The fusion proteins were expressed in
Escherichia coli and purified as described (13). Cell
lysates prepared as described above were normalized for the protein
content and incubated with ~10 µg of GST alone or GST fusion
protein, which had been immobilized on glutathione-Sepharose beads.
Protein complexes were allowed to associate for 4 h at 4 °C,
recovered by centrifugation, and washed with lysis buffer. Complexes
were analyzed by standard SDS-PAGE and immunoblotting techniques.
Affinity purification of GST-tagged Cas proteins from mammalian cell
lysates with glutathione-Sepharose 4B (Amersham Pharmacia Biotech) was
carried out by using a similar procedure, except that the lysis buffer
contained 400 mM NaCl, and the washing buffer contained 500 mM NaCl.
Alkaline Phosphatase Treatment--
Cas was immunoprecipitated
from Rat-1 cells using an anti-Cas polyclonal antibody, protein
complexes were recovered by centrifugation, equilibrated with 50 µl
of phosphatase reaction buffer (50 mM Hepes, pH 7.9, 2 mM MgCl2, 140 mM NaCl, 0.5 mM dithiothreitol) and treated with 30 units of calf
alkaline phosphatase (New England Biolabs) for 30 min at 30 °C. The
protein complexes were then eluted with 1% SDS (5 min at 95 °C),
diluted 1:10 with lysis buffer, incubated with GST/14-3-3 fusion
proteins immobilized on Sepharose and analyzed with GST/14-3-3 fusion
proteins as above. Parallel control experiments omitted calf alkaline phosphatase.
Confocal Microscopy--
Rat-1 cells were fixed with 4%
paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS for 5 min at room temperature. The cells were incubated for 1 h at room
temperature with commercial antibodies against 14-3-3 The Serine-rich Domain of Cas Interacts with the 14-3-3 Full-length Cas Interacts with 14-3-3 Proteins in Vitro and in Vivo
in Mammalian Cells--
To examine whether 14-3-3
We next investigated whether 14-3-3 proteins interact with Cas in
vivo. To this end, GST-tagged forms of Cas, GST/Cas, or GST/Cas Serine Phosphorylation of Cas Regulates the Cas-14-3-3
Interaction--
A charge-reversal mutation of lysine-49 in the
peptide binding groove within the 14-3-3 molecule is thought to disrupt
the binding of 14-3-3 to the serine-phosphorylated target proteins (see
"Discussion") and indeed, this mutation has been shown to greatly
decrease the association of 14-3-3 with Raf-1 and exoenzyme S (40). We
found that the association between Cas and 14-3-3 Integrin-mediated Cell Adhesion Induces Cas-14-3-3
Interaction--
Cas is known to become not only tyrosine-, but also
serine-phosphorylated in response to integrin-mediated cell adhesion
(16). We therefore investigated the possibility that the Cas-14-3-3 interaction would be regulated by cell adhesion to extracellular matrix
proteins. Pull-down experiments demonstrated that Cas and 14-3-3 readily interacted in Rat-1 cells that had been cultured as a monolayer
in 10% fetal calf serum and Dulbecco's modified Eagle's medium or
serum-starved for 18 h (Fig. 6).
Upon cell detachment, the Cas-14-3-3 interaction was remarkably reduced
in a time-dependent manner. When the Rat-1 fibroblasts were
replated onto FN substratum, to which the cells adhere in an
Co-localization of Cas and 14-3-3 Proteins--
Previous studies
have demonstrated that Cas localizes in membrane ruffles (39) and focal
adhesions (41) during the initial attachment of cells to matrix
proteins, whereas, in stationary cells, the majority of Cas protein
localizes in the cytosol (42). We examined the subcellular distribution
of Cas and 14-3-3 proteins using digital confocal immunofluorescence
(Fig. 7). Rat-1 cells were kept in
suspension for 40 min followed by replating on FN for 20 min or 4 h. During the initial steps of cell adhesion (20 min), lamellipodia
extensions were observed in the spreading cells. Simultaneous two-color
confocal immunofluorescence showed that 14-3-3 proteins (Fig.
7A) accumulate at the edges of the membrane ruffles where
Cas (Fig. 7D) was highly concentrated. Upon merging the
images, a remarkable co-localization of 14-3-3 with Cas (Fig. 7G) was observed in the lamellipodia structures (yellow).
After 4 h of attachment to FN, lamellipodia structures had
disappeared, and focal adhesion contacts were well organized as defined
by immunostaining with anti-vinculin antibodies, a marker for focal adhesions (43) (Fig. 7E). At this time point, the 14-3-3 proteins were localized throughout the cytosol; however,
co-localization analysis with vinculin also demonstrated the presence
of 14-3-3 proteins within some focal adhesion structures (Fig.
7H). Cytosolic 14-3-3 proteins showed a prominent punctuate
distribution (Fig. 7, B and C) that was very
similar to the one observed for Cas (Fig. 7F). Upon
superimposing the two images, significant co-localization of Cas and
14-3-3 was detected in the cytosol (Fig. 7I). These results
further support the notion that the two proteins interact in
vivo and that this association may have a functional
significance.
The docking protein Cas was originally identified as a target for
tyrosine kinase activity in various transformed cells, and consequently
the tyrosine phosphorylation levels of Cas were found to correlate well
with the transforming activity of various oncogenes (6, 7, 44-47).
Significantly, recent results obtained with Cas Although much of the attention has been focused on the role of the
tyrosine-phosphorylated form of Cas in intracellular signaling events,
our data demonstrate that tyrosine phosphorylation levels of Cas do not
correlate with the ability of Cas to bind 14-3-3. Thus, a mutant
construct in which the major tyrosine-phosphorylated region (substrate
domain) of Cas was deleted readily interacted with the 14-3-3 proteins
in vivo. Moreover, we observed no correlation between the
Cas-14-3-3 interaction and the tyrosine phosphorylation levels of Cas
in transformed cells. Although Cas is heavily tyrosine-phosphorylated in both v-Crk and v-Src transformed cells (5, 7), we only observed
Cas-14-3-3 association in cell lysates prepared from v-Crk, and not
from v-Src-expressing cells (data not shown). Significantly, our
results firmly support a model in which serine phosphorylation of Cas
determines its binding to the 14-3-3 proteins. First, the charge-reversal mutation K49E in 14-3-3 completely abolished its ability to associate with Cas. Crystallographic analysis has
demonstrated that this lysine residue is lining the amphipathic
peptide-binding groove of the 14-3-3 proteins (53), which mediate
electrostatic interactions with phosphoserine residues in the 14-3-3 target proteins (40, 54). Second, the Cas-14-3-3 interaction was highly
reduced when Cas immunoprecipitates were treated with alkaline serine
phosphatase before affinity association experiments; a similar
experimental approach has been used to demonstrate the serine-phosphorylation requirement for association between the docking
protein c-Cbl and 14-3-3 (55). In addition, our results further
demonstrate a role for cell adhesion in the regulation of 14-3-3 function, as we found that the Cas-14-3-3 interaction is highly
enhanced upon integrin-mediated cell adhesion. It is also interesting
to note that several focal adhesion proteins, including Cas (16), FAK
(56), and paxillin (57, 58), have been reported to undergo serine
phosphorylation in response to integrin-mediated cell adhesion. Taken
together, we propose a model in which integrin ligand binding connects
to the multifunctional 14-3-3 proteins by regulating the serine
phosphorylation levels of Cas. This interaction in turn may have an
important functional role in integrin signaling pathways.
Our yeast two-hybrid results together with the in vitro
interaction studies in which very stringent conditions were used to disrupt pre-existing Cas-protein complexes (1% SDS elution and heat
denaturation) suggest that the interaction between Cas and 14-3-3 is
likely to be direct and mediated by the SR domain of Cas. Moreover,
deletion of this domain (mutant Cas During the initial steps of cell adhesion to ECM, the cells extend a
leading edge that adheres to the substratum through integrin receptors.
This process of protrusion of the lamellipodium involves actin
polymerization (60) and the accumulation of components of focal
adhesion structures at the edge of the membrane (61). Recent
observations made with Cas protein interacts with Cas in the yeast two-hybrid
assay. We also found that the two proteins associate in mammalian cells
and that this interaction takes place in a
phosphoserine-dependent manner, because treatment of Cas
with a serine phosphatase greatly reduced its ability to bind
14-3-3
. Furthermore, the Cas-14-3-3
interaction was found to be
regulated by integrin-mediated cell adhesion. Thus, when cells are
detached from the extracellular matrix, the binding of Cas to 14-3-3
is greatly diminished, whereas replating the cells onto fibronectin
rapidly induces the association. Consistent with these results, we
found that the subcellular localization of Cas and 14-3-3 is also
regulated by integrin ligand binding and that the two proteins display
a significant co-localization during cell attachment to the
extracellular matrix. In conclusion, our results demonstrate that
14-3-3 proteins participate in integrin-activated signaling pathways
through their interaction with Cas, which, in turn, may contribute to
important biological responses regulated by cell adhesion to the
extracellular matrix.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
,
,
, and
have been described by Yaffe
et al. (29). The mammalian expression plasmids for the
GST-tagged Cas constructs GST/Cas (coding for the full-length wild-type
Cas) and GST/Cas
(SD) (coding for a form of Cas in which the
substrate domain, amino acids 213-514, has been deleted) are described
by Mayer et al. (31) (see Fig. 1). The 14-3-3
/Myc
cDNA contains the c-Myc epitope at the amino-terminal domain of the
14-3-3
protein and was cloned into the mammalian expression vector
pcDNA3 (Invitrogen). Single point mutations and DNA deletions were
performed using polymerase chain reaction techniques and the
QuickChange site-directed mutagenesis kit (Stratagene). The Cas
(SR)
construct was prepared by deleting the DNA sequences encoding for the
residues 520 to 712 of the full-length Cas cDNA (Fig. 1), followed
by cloning into the mammalian expression vector pSSR
.
-galactosidase activity. Positive yeast clones were selected and
expanded in selective medium for the expression of the gene Leu2
(pVP16-vector). The specificity of the protein-protein interactions was
assessed by transforming the selected yeast clones with the plasmids
pBTM116/Lamin or pBTM116/Cas(SR). Plasmid DNA from positive clones was
isolated, sequenced, and compared with the nucleotide data base at the
National Library of Medicine (www3.ncbi.nlm.nih.gov/). The yeast
two-hybrid protein-protein interactions were also evaluated by
quantifying the
-galactosidase activity in liquid cultures using
o-nitrophenyl-
-galactopyranoside (Sigma) (34).
antibody (Santa Cruz), or anti-c-Myc
antibody (Calbiochem). Immunoreactive bands were detected with
horseradish peroxidase-conjugated anti-mouse IgG or protein A and
enhanced chemiluminescence (SuperSignal Chemiluminescent Substrate) (Pierce).
(Santa Cruz),
vinculin (Bio-Rad) and Cas (Transduction Laboratories). Anti-14-3-3 and
anti-vinculin antibodies were diluted in 5% fetal calf serum, PBS, and
applied to cells that had been incubated with 5% fetal calf serum, PBS for 30 min at room temperature. The anti-Cas antibody was diluted in
0.5% bovine serum albumin, PBS and used without preblocking the cells.
The immunocomplexes were detected using fluorescein isothiocyanate- or
tetramethyl rhodamine-conjugated anti-rabbit/anti-mouse IgG (Jackson
ImmunoResearch Laboratories, Inc.), FluoroGuard Antifade mounting
reagent (Bio-Rad), and visualized by digital confocal immunofluorescence (Zeiss LSM-410 confocal microscope). Images were
captured with a Zeiss Axiovert 405 M inverted microscope with an attached CCD camera (100x Plan-Neofluare).
RESULTS
Protein
in Yeast--
The amino acids 520-712 of the docking protein Cas
encompass a region that is characterized by the presence of multiple
serine phosphorylation consensus motifs, the SR domain (Fig.
1). Importantly, this region is
significantly conserved in Cas-homologous proteins, including Efs/Sin
(35, 36) and HEF1/Cas-L (37, 38). This information prompted us to
investigate the possibility that the S domain of Cas might participate
in intracellular signaling by mediating protein-protein interactions.
The cDNA coding for the SR domain of Cas, LexA-Cas(SR), was used as
a bait, pBTM116-LexA-Cas(SR) for two-hybrid screen of a mouse embryonic
library (2 × 106 transformants) subcloned into pVP16.
Six of the total of 67 selected clones were found to interact in a
specific manner with the LexA-Cas(SR) bait. Plasmid DNA from these
clones was isolated and sequenced. DNA data base analysis demonstrated
that clone number 11 contained a fragment corresponding to the amino
acids 1 to 138 of the mouse 14-3-3
protein fused in frame with the
VP16 protein. This clone was renamed as pVP16/14-3-3(11). As shown in
Fig. 2, yeast transformed with the
pBTM116/Cas(SR) and pVP16/14-3-3(11) plasmids expressed significant
-galactosidase activity and grew in conditional media lacking
leucine, tryptophan, and histidine. The clone pVP16/14-3-3(11) contains
DNA sequences (~60 base pairs) that correspond to the 5'-untranslated
region of the mouse 14-3-3
cDNA that were deleted to generate
the construct pVP16/14-3-3(DUT). Expression of this protein together
with the SR domain of Cas, pBTM116/Cas(SR), resulted in a similar
transactivation as compared with that obtained with the original clone
isolated by two-hybrid screen, pVP16/14-3-3(11) (Fig. 2), confirming a
direct interaction between the 14-3-3
peptide and Cas(SR) domain.
The specificity of the interaction between Cas(SR) and 14-3-3
in
yeast was further demonstrated by the lack of interaction of
pVP16/14-3-3 with other domains of Cas or lamin (Fig. 2).
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Fig. 1.
Schematic representation of Cas and the
constructs used in this work (not in scale). The following regions
in Cas are indicated: SH3 domain (SH3), proline-rich
sequence (P1), substrate domain (this region contains most
of the tyrosine phosphorylation sites in Cas), SR domain
(SR), and Src binding site (SBS, this region
contains a proline-rich sequence that associates with the SH3 domain,
and a tyrosine residue, which upon phosphorylation, binds to the SH2
domain). The yeast two-hybrid screen bait construct coding for the SR
domain of Cas and containing amino acids 520-712 is shown
(pBTM116/Cas(SR)). The Cas (SD) construct contains a deletion of the
entire substrate domain of Cas (amino acids 213-514; the deletion is
indicated as Cas
(SD)). The Cas
(SR) construct lacks the SR domain
of Cas (amino acids 520-712; the corresponding deletion is indicated
as Cas
(SR)).
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Fig. 2.
The serine-rich domain of Cas interacts with
14-3-3 in yeast. A, liquid
assays for
-galactosidase activity. The L40 yeast strain was
cotransformed with LexA DNA-binding domain fusion plasmids expressing
either the serine-rich domain of Cas (pBTM116/Cas(SR)), the Cas
proline-rich sequence P1 (pBTM116/Cas(P1)), or lamin (pBTM116/Lamin),
together with either (i) the plasmid pVP16/14-3-3(11) containing the
activation domain of VP16 (pVP16) fused with the first 138 amino acids
of the mouse 14-3-3
protein, (ii) the plasmid pVP16/14-3-3(DUT), in
which the sequences connecting the VP16 peptide to the 14-3-3 protein
were deleted, or (iii) the plasmid pVP16/Cas(SH3) expressing the SH3
domain of Cas. Three separated colonies per sample were analyzed, and
the results are shown as the average units with the error bars denoting
the standard deviation. B, colony-lift
-galactosidase
assay of yeast growth in restrictive medium lacking tryptophan,
leucine, and histidine. The numbers correspond to the L40 yeast
transformants shown in A.
interacts with
the full-length Cas protein in mammalian cells, a GST fusion protein
coding for 14-3-3
was used in affinity association, or pull-down,
experiments. Analysis of lysates prepared from COS-1, HeLa, and 293 human embryonic kidney cell lines demonstrated that Cas interacts
in vitro with 14-3-3
(Fig.
3A). The association was more
prominent in HeLa and 293 cell lysates, suggesting that the interaction
might be regulated by cellular processes, such as post-translational
protein modifications. Pull-down studies using GST fusion proteins of various 14-3-3 isoforms showed that Cas efficiently associates with the
14-3-3
and
isoforms as well (Fig. 3B, left
panel). Interestingly, the interaction observed between Cas and
14-3-3
was consistently lower than that between the other isoforms,
despite the fact that equal amounts of the different GST fusion
proteins were used in the experiments (Fig. 3B, right
panel).
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Fig. 3.
Full-length Cas from different cell lines
associates in vitro with 14-3-3 proteins.
A, protein lysates prepared from the indicated cell lines
were incubated in vitro with Sepharose-immobilized
GST/14-3-3 , or GST alone as a negative control. The associated
protein complexes and samples of the total protein preparations
(total cell lysate) were analyzed by SDS-PAGE and
immunoblotting using an anti-Cas antibody. B, protein
lysates from HeLa cells were subjected to affinity association analysis
using various isoforms (
,
,
,
) of Sepharose-immobilized
GST/14-3-3 proteins and analyzed as indicated in A (left
panel). A Coomassie-stained SDS-PAGE gel analysis of the purified
GST proteins is shown in the right panel.
(SD), or an untagged form of wild-type Cas were expressed alone or in combination with 14-3-3
-Myc in HeLa (Fig.
4A) or COS-1 cells (Fig.
4B). Protein lysates were prepared 48 h after transfection and the GST-tagged Cas proteins were precipitated with
glutathione-Sepharose (Fig. 4, A and B). The
protein complexes were analyzed by immunoblotting with anti-14-3-3
antibodies that cross-react with all 14-3-3 isoforms (Fig.
4A), or with an anti-Myc antibody that recognizes the
expressed Myc-tagged 14-3-3
protein (Fig. 4B). As shown
in Fig. 4, the recombinant 14-3-3
-Myc protein migrates with an
apparent molecular mass of ~35 kDa, which is slightly larger than
that of the endogenous 14-3-3 proteins (~32-33 kDa). Both endogenous
and recombinant 14-3-3 proteins were found to associate with the
wild-type GST/Cas protein in vivo. A mutant form of Cas in
which the substrate domain has been deleted, GST/Cas
(SD), associated
with the 14-3-3 proteins to a similar extent (Fig. 4A).
Because this mutant form does not become phosphorylated on tyrosine at
a detectable level (39), our results suggest that the Cas-14-3-3
interaction is not dependent on or regulated by the tyrosine
phosphorylation levels of Cas. To rule out the possibility that the
association of 14-3-3 with the Cas constructs was mediated by binding
to the GST tag and not to Cas, we performed co-immunoprecipitation experiments with anti-Cas antibodies. Endogenous 14-3-3 proteins readily co-immunoprecipitated with the endogenous Cas proteins from
Rat-1 cell lysates thereby confirming that 14-3-3 is a component of the
Cas protein complex in vivo (Fig. 4C).
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Fig. 4.
Association of 14-3-3 proteins with Cas
in vivo. A, HeLa cells were
transiently transfected with mammalian expression vectors containing
the cDNAs for 14-3-3 /Myc, for the full-length, untagged Cas
(Cas), or for two GST-tagged forms of Cas:
GST/Cas
(SD), in which the substrate domain of Cas had
been deleted, and GST/Cas, in which the GST tag was fused
with the full-length Cas cDNA. Total protein lysates were prepared
and incubated with glutathione-Sepharose 4B (see "Experimental
Procedures"). The protein complexes were resolved on SDS-PAGE gels
and immunoblotted using an anti-14-3-3
antibody that recognizes all
known 14-3-3 isoforms. B, COS-1 cells were transfected with
the indicated cDNAs and assayed as in A but
immunoblotting was carried out with an anti-c-Myc antibody that
recognizes the exogenously expressed 14-3-3
/Myc protein.
C, endogenous Cas immunocomplexes were prepared from Rat-1
cells and analyzed by immunoblotting with an anti-14-3-3 antibody as in
A. The band corresponding to endogenous 14-3-3 proteins is
indicated in the figure. A protein band of ~60-65 kDa was observed
also in control experiments using irrelevant antibodies and therefore
was determined to be nonspecific.
is also critically
dependent on interactions mediated by this lysine residue. Parallel
pull-down experiments using the wild-type GST/14-3-3
or the
GST/14-3-3
K49E mutant demonstrated that Cas associates exclusively
with the wild-type protein, despite the fact that similar amounts of
bacterially expressed fusion proteins were used in the studies (Fig.
5A). These data prompted us to further investigate the requirement of serine phosphorylation in Cas
for the association with 14-3-3
. To this end, Cas was immunoprecipitated from Rat-1 cell lysates, treated with calf intestinal alkaline phosphatase and subjected to an in vitro
association analysis with GST/14-3-3
and control GST fusion proteins
as described under "Experimental Procedures." Fig. 5B
shows that phosphatase treatment of Cas completely abolished the
interaction with the GST/14-3-3
. The decrease in association was not
because of proteolysis of Cas, as equivalent amounts of Cas protein
were present in all the samples (data not shown). Therefore, we
concluded that phosphoserine residue(s) of Cas are an important
determinant for the binding to 14-3-3. These results together with the
yeast two-hybrid interaction of the serine-rich domain of Cas with
14-3-3
directed us to investigate whether the SR domain in Cas is
the major binding site for 14-3-3 proteins in the context of
full-length Cas. Therefore, we generated a Cas mutant with the entire
SR domain deleted, Cas
(SR). Pull-down experiments showed that
deletion of this domain of Cas greatly diminished, but did not
completely eliminate, the interaction with GST/14-3-3
. Despite the
fact that the protein expression levels of the recombinant proteins and
the endogenous Cas were comparable (Fig. 5C), GST/14-3-3
readily interacted with the endogenous full-length Cas and with the
mutant Cas
(SD), but to a lesser degree with the recombinant
Cas
(SR) protein (Fig. 5C). These results demonstrate that
the SR domain of Cas is a major structural determinant mediating the
association of Cas to 14-3-3 proteins and that additional, yet-to-be
identified 14-3-3 binding domains appear to be present in Cas outside
of this region.
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Fig. 5.
14-3-3 proteins and
Cas associate in a phosphoserine-dependent manner.
A, the single point mutation 14-3-3
K49E disrupts the
interaction in vitro with Cas. Protein lysates from 293 cells were incubated with Sepharose-immobilized GST/14-3-3
wild type
(WT) or GST/14-3-3
K49E. The associated complexes and
total cell lysates were analyzed by SDS-PAGE and immunoblotting with an
anti-Cas antibody. The Coomassie-stained SDS-PAGE gel analysis of the
purified GST fusion proteins is shown on the right panel.
B, dephosphorylation of Cas disrupts its interaction with
14-3-3
proteins. Cas immunoprecipitated from Rat-1 cell lysates were
treated (+) or not (
) with calf alkaline phosphatase as described
under "Experimental Procedures." The samples were then subjected to
affinity association experiments using wild-type or K49E mutant
GST/14-3-3 proteins, and the complexes were analyzed as in A. C, 293 cells were transiently transfected with the cDNA
expressing Cas
(SD) or Cas
(SR) and protein lysates were analyzed
as described in A. Note that GST/14-3-3 associates to a
significantly higher extent with endogenous Cas and the mutant
Cas
(SD) rather than Cas
(SR). The apparent SDS-PAGE molecular
masses of Cas
(SR) and Cas
(SD) proteins are ~110 kDa and ~85
kDa, respectively. Immunoblot analysis of total cell lysate protein
samples shows that the expression levels of the endogenous or
recombinant proteins are comparable.
5
1 integrin-dependent manner, the Cas-14-3-3 interaction was restored. A gradual increase in the
association was observed as early as 20 min after replating of the
cells, with a complete recovery to steady-state levels within 4 h
after replating (Fig. 6A). In contrast, cell adhesion to
poly-L-lysine, to which cells adhere in an
integrin-independent manner, failed to stimulate Cas-14-3-3 interaction
(Fig. 6B). Together, these results demonstrate the
requirement for integrin ligand binding in the signaling pathway that
induces the Cas-14-3-3 association.
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Fig. 6.
Cell adhesion to extracellular matrix
proteins regulates the association of Cas with
14-3-3 . A, Rat-1 cells were
either grown to confluency (Normal), serum starved for
18 h (Serum Free), detached, and kept in suspension for
5 or 45 min (Susp.), or plated on FN for 20 min or 4 h
(Attached). Protein lysates were incubated with equal
amounts of Sepharose-immobilized wild-type GST/14-3-3
or
GST/14-3-3
K49E proteins. The protein complexes were resolved by
SDS-PAGE and immunoblotted with anti-Cas monoclonal antibody
(upper panel). Anti-Cas immunoblotting of the total cell
lysate preparations was used to confirm that equal amounts of protein
were used (lower panel). B, the experiment was
performed following conditions similar to A, but the cells
were allowed to adhere either onto poly-L-lysine
(PLL) or fibronectin (FN) for 50 min.
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Fig. 7.
Subcellular co-localization of 14-3-3 proteins with either Cas or vinculin. Shown are single plane
images of digital confocal immunofluorescence analysis to determine the
subcellular localization of 14-3-3 (panels A, B,
and C), vinculin, used as a marker for focal adhesion
contacts (panel E), and Cas (panels D and
F). Rat-1 cells were detached from ECM and subsequently
allowed to adhere to fibronectin-coated glass surfaces for different
periods of time: 20 min (panels A and D) or
4 h (panels B, C, E, and
F). The immunofluorescence analysis was performed as
indicated under "Experimental Procedures." The lower
panels (G, H, and I) depict the
overlay of the corresponding upper images to show the co-localization
of 14-3-3 and vinculin or Cas (yellow color). The images were obtained
with a 100× magnification. Bar = 10 µM.
DISCUSSION
/
fibroblasts
demonstrate that Cas is essential for Src-induced morphological
transformation and anchorage independence, suggesting a causal role for
Cas in oncogene-induced malignant transformation (48). In
nontransformed cells, the tyrosine phosphorylation levels of Cas are
regulated by extracellular stimuli such as integrin-mediated cell
adhesion (12, 13, 15, 49-51). As a result, Cas associates with the SH2
domain containing adapter protein Crk. This interaction has been shown
to lead to the activation of signaling pathways controlling cellular
functions such as actin organization (52), cell migration (39), and
c-Jun N-terminal kinase activation (63). The primary structure of Cas
suggests that it may participate in additional signal transduction
pathways through multiple differentially regulated protein-protein
interactions. Among the putative signaling domains in Cas is a SR
region, which is significantly conserved (56% similar) between Cas and
a close family member, HEF1/Cas-L (37, 38). The SR region of Cas was
found here to interact with the 14-3-3
protein in a yeast two-hybrid
system. Importantly, this interaction was not limited to yeast as our
results indicate that the full-length Cas interacts both in
vitro and in vivo in mammalian cells with various
isoforms of the 14-3-3 family.
(SR)) did greatly reduce the
ability to bind 14-3-3
, further confirming the importance of
structural 14-3-3 binding determinants located within the SR domain of
Cas. Nevertheless, the binding of the Cas
(SR) mutant to 14-3-3 was
still detectable. Detailed analysis of the primary structure of Cas
revealed the presence of several sequences that conform to classical
14-3-3-consensus motifs (29). Only one of these sequences, RPLPSPP
(amino acids 733-739), which overlaps with the Src-SH3 binding site,
is located outside of the SR domain. Further mutation (S737Q mutation)
of this motif in the context of the Cas
(SR) construct did not affect
the interaction with GST/14-3-3
(data not shown). These results
indicate the presence of additional 14-3-3-recognition sites in Cas
that do not conform to the canonical recognition sequences. The
presence of active 14-3-3 binding sites, distinct from the established
consensus motifs, has been described in other proteins, such as the
docking molecule c-Cbl (55) and the intermediate filament protein
keratin 18 (59).
/
fibroblasts suggest that Cas is a key
molecule in mediating actin polymerization events (48). It has also
been reported that Cas is markedly concentrated in membrane ruffles
(39) and has an important role during cell migration (62) via
interaction with signaling molecules such as c-Crk (39). Our
subcellular localization studies demonstrate that 14-3-3 proteins also
accumulate at the leading edge of the lamellipodia and co-localize with
Cas in these structures. Once the cells stabilize the focal adhesion
structures and become stationary, Cas and 14-3-3 redistribute through
the cytosol displaying a punctuated pattern and extensive
co-localization. Thus, the similar dynamic subcellular distribution
shared by 14-3-3 and Cas suggests that these two proteins may cooperate
in cellular functions, such as the orchestration and stabilization of
cellular contact sites with the ECM. Further studies with
e.g. dominant-interfering mutants of Cas or 14-3-3 will be
required to fully explore the signaling roles that the Cas-14-3-3
complex may have in integrin signaling pathways.
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ACKNOWLEDGEMENTS |
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We thank Dr. S. M. Hollenberg for providing the mouse embryonic cDNA library cloned into pVP16, Dr. H. Hirai for the Cas cDNA, Dr. J. Avruch for the plasmids expressing the GST/14-3-3 and 14-3-3/Myc cDNAs, and Dr. M. B. Yaffe for plasmids expressing GST fusion proteins of the other 14-3-3 isoforms and for useful discussions. We thank Dr. E. Monosov for invaluable technical assistance during the digital confocal analysis and Dr. K. Zeh for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA71560 (to K. V.).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.
Recipient of fellowship from the Human Frontier Science Program.
§ Recipient of fellowship from the American-Italian Cancer Foundation.
¶ A Pew scholar in biomedical sciences. To whom correspondence should be addressed: The Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-646-3100; Fax: 619-646-3199; E-mail: kvuori{at}ljcrf.edu.
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ABBREVIATIONS |
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The abbreviations used are: ECM, extracellular matrix; FN, fibronectin; GST, glutathione S-transferase; PBS, phosphate-buffered saline; SD, substrate domain; SR, serine-rich; SH2, Src homology type 2; SH3, Src homology type 3; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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