From the
Department of Microbiology and Cancer
Center, University of Virginia Health System, Charlottesville, Virginia
22908-0735 and the
Department of Biochemistry
and Molecular Biology, Indiana University School of Medicine, Indianapolis,
Indiana 46202-5122
Received for publication, April 4, 2003
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ABSTRACT |
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INTRODUCTION |
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One of the major initiating events for cell migration is engagement of integrin receptors by ECM ligands. In this way, integrins directly link ECM components to the actin cytoskeleton (for review see Ref. 10). Integrins also recruit cellular proteins, including kinases and adapter molecules such as Cas, to newly formed focal complexes and mature focal adhesions (for review see Ref. 11). Ultimately, many of the signaling molecules that participate in cell adhesion and migration impact the Rho family of small GTPases, which modify the actin cytoskeleton by regulating actin polymerization and the molecular composition of adhesion sites (12). Elucidating the functions of focal adhesion proteins that participate in the regulation of these small GTPases is critical for understanding mechanisms that govern cell motility.
Several pieces of evidence suggest that Cas plays a critical role in cell migration. Consistent with its presence in focal adhesions, Cas becomes tyrosine phosphorylated in response to integrin ligation by numerous ECM components, including fibronectin (FN), collagen, laminin, and vitronectin (8, 1315). In addition, overexpression of Cas has been shown to enhance migration of FG pancreatic carcinoma cells and COS cells (16). Conversely, Cas/ mouse embryo fibroblasts do not migrate as efficiently as their wild-type counterparts unless they are engineered to express ectopic Cas (17, 18). Recent studies (19) have shown that the carboxyl terminus of Cas is required to fully rescue this defect in cell migration.
The Cas carboxyl terminus contains a bipartite binding site for the nonreceptor protein tyrosine kinase (PTK) c-Src (Src) (20). This PTK is not only a mediator of cell proliferation and survival (for review see Refs. 21 and 22), but it also plays an essential role in cell migration. Fibroblasts derived from mice lacking three Src family members (Src, Yes, and Fyn) are deficient in migration, and this defect can be rescued by re-expression of Src alone (23). In addition, the kinase activity of Src has been shown to be essential for integrin-mediated cell adhesion, spreading, and migration (24, 25). Evidence suggests that Src family kinases mediate integrin-dependent Cas phosphorylation (23, 2628) and that Src is the primary PTK responsible for phosphorylation of Cas (29).
Interestingly, Cas is not only a substrate of Src, but also a potent enhancer of Src kinase activity (30, 31). Src is typically found in a closed or kinase-inactive conformation, held in place by repressive intramolecular interactions involving its Src homology-2 (SH2) and -3 (SH3) domains (for review see Ref. 32). Because Cas contains motifs that interact with the SH2 and SH3 domains, it can prevent the adoption of this autoinhibitory conformation by directly binding to Src. We and others (30, 31, 33) have shown that the formation of Src/Cas complexes leads to a significant increase in PTK activity, as well as serum- and anchorage-independent growth. Interactions between Src and Cas may therefore initiate signaling cascades that lead to a number of cellular processes, including cell migration.
In addition to Src, members of a second group of proteins called NSPs (novel SH2-containing proteins) have also been shown to bind to the carboxyl terminus of Cas (34). These proteins contain an amino-terminal SH2 domain and a carboxyl-terminal domain with sequence homology to the Cdc25 family of guanine nucleotide exchange factors (GEFs) for Ras family GTPases. The association between Cas and one of these proteins, Chat (Nsp3), has been shown recently (35) to be involved in the regulation of cell adhesion. A second family member, AND-34/BCAR3 (Nsp2), hereafter referred to as AND-34, also binds to the carboxyl terminus of Cas (36, 37). Murine AND-34 has been shown to exhibit GEF activity toward the small GTPases RalA, Rap1, and R-Ras (37). Because all three of these GTPases have been reported to function in various aspects of cell adhesion, migration, and/or proliferation (for review see Refs. 3840), it is interesting to speculate that AND-34 may contribute to some of the functions attributed to the carboxyl terminus of Cas- and Src/Cas-dependent signaling pathways.
In this study, we focused on determining whether Cas and AND-34 function cooperatively to regulate cell signaling pathways that lead to Src activation and cell migration. The use of strategies involving protein overexpression is supported for these studies by the fact that both Src and Cas are highly expressed in a number of naturally occurring cellular contexts, including human breast tumors (4143). Moreover, the human homologues of both Cas (BCAR1) and AND-34 (BCAR3) were identified in a screen for genes that promoted resistance to the antiestrogen tamoxifen when overexpressed in breast cancer cells (43, 44). We found that Cas promotes haptotactic and chemotactic migration in a manner that depends upon kinase-active Src and that AND-34 functions synergistically with Cas to enhance both cell migration and Src activation. The carboxyl-terminal GEF domain of AND-34 was found to contribute to these activities, as was one of the reported targets of this GEF activity, Rap1. Immunolocalization studies suggest that AND-34 may participate in these processes by relocalizing Cas to lamellipodia located at the leading edge of migrating cells. Together, these data indicate that Cas and AND-34 can function together to stimulate Src signaling pathways and promote cell migration.
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EXPERIMENTAL PROCEDURES |
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Plasmid Construction and MutagenesispRK5 constructs
encoding the genes for Myc-tagged full-length Cas, deletion of the minimal
carboxyl terminus (mCT; deletion of amino acids 693874), and the
minimal carboxyl terminus (mCT; amino acids 693874) have been described
previously (9,
49). Single point mutations
substituting a proline residue for leucine 791 (L791P) in full-length Cas and
mCT, as well as mutation of leucine 762 (L762P) in mCT, were generated using
QuikChange site-directed mutagenesis (Stratagene). The mCT double mutant
(L762/791P) was generated by subjecting mCT-L791P to a second round of
mutagenesis. All mutations were confirmed by automated DNA sequence analysis.
Yellow fluorescent protein (YFP)-tagged Cas was constructed by inserting the
complete Cas cDNA as a BamHI/XbaI fragment into pLP-EYFP-C1
using the CreatorTM cloning and expression system (Clontech). pCDNA-c-Src
was generously supplied by S. J. Parsons (University of Virginia)
(50), and
pCDNA3FLAG2AB-paxillin has been described previously
(30). FLAG-tagged AND-34 was
constructed in pcDNA3 by replacing the polylinker of pcDNA3 with that of
pFLAG-CMV2 (pFLAG3). The expressed sequence tag AI115884
[GenBank]
was found to contain
the entire coding sequence of the mouse BCAR3 homologue, AND-34. The first 24
amino acids of AND-34 were deleted to allow use of an NcoI site to
blunt into the newly created pFLAG3 vector, resulting in pFLAG3-AND-34. AND-34
deleted for the GEF-containing carboxyl terminus (
GEF) was created from
wild-type cDNA by synthesizing a PCR product using a 5' primer that
contained a unique EcoRI site just upstream of codon 25 and a
3' primer containing a unique XbaI site just downstream of
codon 593. The PCR product was digested with EcoRI and XbaI
and ligated in-frame into pCDNA3FLAG2AB in place of the analogous restriction
fragment. A constitutively active RalA construct (pCAG-Ral72L) was generously
provided by L. Feig (Tufts University)
(51), and constitutively
active R-Ras (pCGN-R-Ras87L) was a gift from A. Cox (University of North
Carolina) (52).
Dominant-negative and constitutively active Rap1B constructs (pCMV-Rap1N17 and
pCMV-Rap1V12) were kindly provided by P. Stork (Oregon Health Sciences Center)
(53).
Transfection and Protein ExpressionTransient transfections
in COS-1 and REF-52 cells were performed according to manufacturer
specifications using SuperfectTM purchased from Qiagen, and
C3H10T cell lines were transfected using LipofectAMINE PLUS
(Invitrogen). In those cases where cells were co-transfected with two or more
constructs, immunofluorescence was performed to confirm that >90% of
transfected cells expressed both proteins. Cells were lysed 24 h
post-transfection in modified radioimmune precipitation assay buffer (150
mM NaCl, 50 mM Tris, pH 7.5, 1% Igepal CA-630, 0.5%
deoxycholate) supplemented with protease and phosphatase inhibitors (100
µM leupeptin, 1 mM phenylmethylsulfonyl fluoride,
0.15 unit/ml aprotinin, 1 mM sodium orthovanadate) as described
previously (54). Protein
concentrations were determined using the BCA assay from Pierce.
Immunoprecipitation and ImmunoblottingFor Myc immunoprecipitates, cell lysate was incubated with mAb 9E10 overnight at 4 °C with rotation, and the immune complexes were recovered by a 1-h incubation with protein A-Sepharose beads that had been preincubated with rabbit anti-mouse immunoglobulin. For FLAG immunoprecipitates, cell lysates were incubated with M2 resin (25 µl/mg protein) for 1 h at 4 °C with rotation. Complexes were washed twice in modified radioimmune precipitation assay buffer and twice in Tris-buffered saline, resuspended in 2x Laemmli sample buffer, and boiled for 5 min. Proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and incubated with antibodies as indicated. Proteins were detected by horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin followed by enhanced chemiluminescence (PerkinElmer Life Sciences).
Cell MigrationC3H10T cell lines were transfected
with the indicated constructs and cultured overnight in low-serum migration
medium (DMEM containing 0.05% FCS). The following day, cells were trypsinized,
counted, and a total of 105 cells in 500 µl of migration medium
were loaded into the top of a modified Boyden chamber (24-well, 8.0-µm
BioCoat insert; BD Biosciences). For migration toward FN (Sigma), the
underside of the insert membrane was preincubated with FN overnight at 4
°C (20 µg/ml in phosphate-buffered saline), and the bottom chamber was
filled with migration medium. For migration toward serum, uncoated inserts
were placed in wells filled with DMEM containing 10% FCS. Cells were allowed
to migrate for2hat37 °C, and the nonmigratory cells were then removed from
the top of the membrane with cotton swabs. The underside of the membrane was
fixed in 3% paraformaldehyde for 20 min at room temperature and mounted on
glass slides using VectaShield (Vector Laboratories Inc., Burlingame, CA).
Migrated cells were counted using a Nikon fluorescence microscope, and the
ratio of fluorescent cells to total cells (visualized by phase microscopy) was
used to determine percent migration. This was divided by the percent
transfection for each condition, resulting in the migratory index
(55). The log values for each
group of data were analyzed by single factor analysis of variance. When
significant differences were found between groups at the 5% level, they were
compared using the Student's t test assuming unequal variance. The
data are shown as the mean relative migratory index (normalized to
vector-transfected cells) ± S.D.
ImmunofluorescenceFor immunolocalization of AND-34 wild-type and mutant constructs, REF-52 cells were transfected as described above. The following day, cells were replated on FN-coated coverslips (20 µg/ml) for 45 h. The cells were then fixed in 3% paraformaldehyde for 20 min at room temperature and permeabilized using 0.4% Triton X-100 in phosphate-buffered saline for 5 min at room temperature. Cells were incubated with FLAG M5 mAb in 2% bovine serum albumin/phosphate-buffered saline for 1 h, followed by fluorescein isothiocyanate-conjugated goat anti-mouse and TR-conjugated phalloidin for 1 h. For Cas/AND-34 colocalization, transfected REF-52 cells were plated on FN-coated coverslips for 45 h and then fixed, permeabilized, incubated with FLAG M5 and Cas B polyclonal antisera, and subsequently incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit and TR-conjugated goat anti-mouse antibodies. Cells were visualized through a Nikon fluorescence microscope and photographed using a cooled CCD camera controlled by Inovision Isee software.
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RESULTS |
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AND-34 Synergizes with Cas to Promote Src Activation and Cell MigrationCoincident with their effect on cell migration, interactions between Src and Cas result in the activation of Src, leading to tyrosine phosphorylation of several Src substrates and the promotion of adhesion- and serum-independent growth (30). A recently identified Cas binding partner, AND-34, also demonstrates pro-proliferative effects when overexpressed in breast cancer cell lines grown in the presence of the antiestrogen tamoxifen (44). To determine whether AND-34 participates in Cas-mediated Src activation and substrate phosphorylation, COS-1 cells were transiently co-transfected with plasmids encoding WT Src, Myc-tagged Cas, and/or FLAG-tagged AND-34. PTK activity was determined 24 h post-transfection by measuring the level of pTyr on the Src substrate paxillin, which was expressed as a FLAG-tagged construct, together with the other proteins. Consistent with our previous report (30), co-expression of Src and Myc-tagged Cas resulted in enhanced phosphorylation of paxillin (Fig. 2A, top panel, lane 2). Interestingly, co-expression of AND-34 with Cas and Src dramatically increased paxillin phosphorylation above the level observed when Cas and Src were expressed alone (compare lanes 2 and 3). This effect of AND-34 on Src activity was shown to require Cas, as co-expression of AND-34 and Src in the absence of Cas did not stimulate phosphorylation of paxillin (lane 4). Control immunoblots indicated that roughly equal amounts of FLAG-paxillin were immunoprecipitated from cell extracts (second panel) and that AND-34 and Cas were expressed appropriately (bottom panels). AND-34 also enhanced the phosphorylation of other Src substrates in the presence of Cas, including Shc, cortactin, and Src itself at the autophosphorylation site (Tyr-416) (data not shown).
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One potential mechanism by which AND-34 may enhance Cas-dependent Src activation is through the stabilization of Src/Cas interactions. To address this possibility, Src immunoblots were performed on Myc immune complexes isolated from the same cell extracts used for the experiment shown in Fig. 2A. The amount of Src associated with Myc-Cas was not affected by AND-34 expression (Fig. 2B, middle panel, compare lanes 2 and 3). AND-34 expression also had no effect on the absolute expression level of Cas (top panel) or Src (bottom panel) in these cells. These data indicate that the enhancement of Cas-mediated Src activation by AND-34 does not involve a mechanism that alters Src/Cas interactions per se.
Given that AND-34 appeared to synergize with Cas to promote Src substrate
phosphorylation, we next investigated whether AND-34 expression also
stimulated Cas-mediated migration in cells overexpressing Src.
C3H10T-5H cells were transfected with plasmids encoding YFP, YFP-Cas,
and/or FLAG-AND-34. As was the case in Fig.
1, expression of Cas in these cells increased migration toward
both FN and serum (Fig.
2C, black bars). Expression of AND-34 in the
absence of Cas had no such stimulatory effect (gray bars). When both
Cas and AND-34 were co-expressed, however, a significant increase in migration
toward FN and serum was observed relative to both vector- and Cas-transfected
cells (hatched bars). Taken together, these data indicate that AND-34
is a potent enhancer of both Cas-mediated Src activation and cell
migration.
Cas/AND-34 Interactions Are Not Required for the Promotion of
Src Activation and Cell MigrationAND-34 is a member of the NSP
family of proteins, which contain an amino-terminal SH2 domain and a
carboxyl-terminal region with homology to the Ras GEF Cdc25
(34). To further investigate
the functional synergy between Cas and AND-34, we sought to specifically
define the region of Cas required for AND-34 binding. Data from other
investigators indicated that AND-34 bound to sequences within the extreme
carboxyl terminus of Cas, which contains a divergent helix-loop-helix (dHLH)
domain (37,
56). To determine whether this
region, hereafter referred to as mCT
(9), was required for
Cas/AND-34 binding, COS-1 cells were transiently co-transfected with plasmids
encoding FLAG-tagged AND-34 and Myc-tagged WT Cas, a Myc-tagged Cas molecule
deleted for amino acids 693874 (mCT), or the independently
expressed Myc-tagged mCT (Fig.
3A). Myc immune complexes were isolated from cell
extracts and immunoblotted for Myc and FLAG
(Fig. 3B). AND-34 was
observed to associate with WT Cas under these conditions (third panel,
lane 2) but not with
mCT (lane 3). AND-34 was also able
to associate with the independently expressed mCT molecule (lane 4).
The reduced recovery of AND-34 in mCT immune complexes relative to full-length
Cas was likely caused by less AND-34 expression in this experiment (bottom
panel, compare lanes 2 and 4). However, the fact that
equal amounts of AND-34 were present in cells expressing
mCT, which did
not bind to AND-34, and mCT, which was found to associate with AND-34
(bottom panel, lanes 3 and 4), is consistent with the
carboxyl-terminal mCT region of Cas being both necessary and sufficient for
association with AND-34.
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To address whether the helices of the dHLH motif were themselves critical for AND-34 binding to Cas, single amino acid substitutions were engineered into the mCT construct, replacing conserved leucine residues at positions 762 of the first helix and 791 of the second helix with prolines. Mutation of the analogous leucines in the Cas family member HEF1 has been shown to disrupt dHLH functions associated with protein-protein interactions and subcellular localization (56, 57). WT and mutant Myc-tagged mCT molecules were expressed together with FLAG-tagged AND-34 in COS-1 cells, and Myc immune complexes were then examined for the presence of FLAG-AND-34 (Fig. 3C). Mutation of leucines 762 and 791, either singly or in combination, completely abrogated AND-34 binding to mCT (middle panel, lanes 24). These data suggest either that leucines 762 and 791 are responsible for directly binding to AND-34 or that structures governed by the dHLH motif play an essential role in the association of Cas and AND-34.
Having delineated specific residues of Cas required for AND-34 binding, we next addressed whether the ability of AND-34 to enhance Cas-dependent Src activation required the association of Cas and AND-34. COS-1 cells were transfected with plasmids encoding Src, FLAG-paxillin, FLAG-AND-34, and either WT Myc-Cas or a full-length Myc-Cas molecule containing the L791P mutation. PTK activity, as measured by phosphorylation of paxillin, was found to be identical in the presence of WT Cas or Cas-L791P (Fig. 4A, top panel, compare lanes 1 and 2), despite the fact that AND-34 binding to Cas-L791P was severely compromised (Fig. 4B, middle panel, lane 2). Importantly, Src/Cas interactions were maintained in the presence of the L791P mutation (bottom panel). These data indicate that synergistic enhancement of Cas-dependent Src activity by AND-34 does not require a stable association between Cas and AND-34.
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Function of the AND-34 Carboxyl TerminusThe finding that
AND-34 does not need to stably associate with Cas to augment Cas-dependent Src
activation suggested that its role in this process may be related to functions
of the molecule that are independent of Cas binding. Among its putative
functional domains, AND-34 contains sequences within its carboxyl terminus
that have homology to GEF domains, and expression of this molecule has been
reported to coincide with activation of the small GTPases R-Ras, RalA, and
Rap1 (36,
37). To address whether this
domain was required for promotion of Src activity by Cas, the carboxyl
terminus of AND-34 was deleted to produce a mutant lacking putative GEF
activity (GEF). WT and mutant FLAG-tagged AND-34 molecules were
expressed together with myc-Cas and Src in COS-1 cells, and PTK activity was
measured. Endogenous cortactin was used to measure Src activity in this case
instead of paxillin, because FLAG-paxillin and the AND-34
GEF molecule
co-migrated on SDS-PAGE. As was the case for paxillin, cortactin
phosphorylation was elevated in the presence of overexpressed Cas and Src
(Fig. 5A, top
panel, lane 2), and this was significantly augmented by co-expression of
AND-34 (lane 3). In contrast,
GEF did not demonstrate a
significant synergy with Cas in promoting activation of Src (compare lane
4 with lane 3).
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Because Src activation by Cas and AND-34 correlated with promotion of cell
migration (see Fig. 2), we
investigated whether the inability of the AND-34 GEF mutant to augment
Cas-dependent Src activity was coincident with a similar inability to promote
cell migration. Whereas AND-34 expression was again found to significantly
enhance the effect of Cas on cell migration toward FN
(Fig. 5B) and serum
(data not shown), expression of
GEF had no such effect. Together, these
data indicate that the carboxyl terminus of AND-34 is functionally important
for the promotion of both Src activity and cell migration by Cas.
Functions Associated with the GEF Domain of AND-34 Because the GEF domain located at the carboxyl terminus of AND-34 has been reported to have activity toward RalA, Rap1, and R-Ras (37), we hypothesized that enhancement of Src activation and Src/Cas-dependent cell migration by AND-34 may occur through a mechanism that involves activation of one or more of these GTPases. If this is the case, then activated forms of these GTPases would be expected to function like AND-34 to augment the positive effect of Cas on Src activity and cell migration. To test this hypothesis, COS-1 cells were co-transfected with plasmids encoding Src, Cas, paxillin, and activated forms of R-Ras (R-Ras 87L), Rap1 (Rap1 V12), or Ral (Ral 72L). None of these constitutively active GTPases had any effect on paxillin phosphorylation in the absence of Cas (Fig. 6, A (top panel, lanes 2 and 3) and B (top panel, lane 2)). However, in the presence of Cas, Rap1 V12 dramatically enhanced the level of paxillin phosphorylation above the level induced by Cas alone (Fig. 6A, top panel, compare lanes 4 and 6). Active Ral had no such stimulatory effect (Fig. 6B, top panel, compare lanes 3 and 4), and active R-Ras appeared to actually inhibit Cas-induced paxillin phosphorylation (Fig. 6A, top panel, compare lanes 4 and 5). From these data, it appears that constitutively active Rap1, but not Ral or R-Ras, can stimulate Cas-dependent Src activation in the absence of AND-34.
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If Rap1 activation by AND-34 is required for augmentation of Src activity by Cas, then expression of a dominant-inhibitory Rap1 molecule would be expected to inhibit the positive effect of AND-34 on Src activation. To determine whether this was the case, Cas, Src, paxillin, and AND-34 were expressed in COS-1 cells in the absence or presence of dominant-inhibitory Rap1 N17. FLAG-paxillin immunoprecipitated from these cell extracts was heavily tyrosine phosphorylated in the presence of Cas and AND-34 (Fig. 6C, top panel, lane 4). Expression of Rap1 N17 reduced this phosphorylation (lane 5), albeit not to the level observed when Cas was expressed in the absence of AND-34 (lane 2). Rap1 N17 also caused a slight decrease in paxillin phosphorylation in the presence of Cas alone (compare lanes 2 and 3). Based on these data, it appears that Rap1 can function downstream of AND-34 and Cas in the promotion of Cas-mediated Src activation. The incomplete inhibition of Src activation in the presence of dominant-negative Rap1 could be the result of either technical issues that prevented complete inhibition of endogenous Rap1 activity or the contribution of additional signals emanating from Rap1-independent pathways.
We next investigated whether the stimulatory effect of AND-34 on
Cas/Src-dependent cell migration was dependent on Rap1 activity. As before,
expression of YFP-Cas in Src-overexpressing C3H10T-5H cells promoted
cell migration toward serum, and co-expression of AND-34 caused a further
enhancement in cell migration (Fig.
6D, white bars). Rap1 N17 blocked the
up-regulation of Cas-promoted migration by AND-34 but had no statistically
significant effect on migration in the absence of AND-34 (black
bars). These data, together with the data on Src activation shown in
Fig. 6,
AC, implicate Rap1 as a key mediator of
AND-34 signaling through Src/Cas.
Subcellular Localization of Cas and AND-34 The ability of
AND-34 to promote cell migration though Src/Cas signaling pathways raises the
question of whether the function of these molecules is coordinated at specific
sites within the cell. To examine the subcellular localization of AND-34 in
adherent cells, REF-52 cells were transiently transfected with plasmids
encoding FLAG-tagged AND-34 or GEF and immunostained with FLAG
antibodies to visualize AND-34, as well as phalloidin to visualize polymerized
actin (Fig. 7). WT AND-34 was
strongly localized to membrane ruffles and lamellipodia in areas where
significant cortical actin was detected (panels A-C). Interestingly,
the carboxyl terminus of AND-34 did not appear to be required for this
localization, because
GEF was also present at the cell periphery
(panels D-F). To investigate whether Cas localization was altered in
the presence of overexpressed AND-34, REF-52 cells were transfected with
plasmids encoding Cas alone or in combination with WT or mutant AND-34
molecules (Fig. 8). In the
absence of AND-34, Cas was localized predominantly to discrete focal adhesions
and perinuclear regions of the cell (panel A). When co-expressed with
WT AND-34, Cas showed a dramatic relocalization to membrane ruffles and
lamellipodia, coincident with AND-34 localization (panels
BD). Approximately 80% of co-expressing cells also
underwent a change in morphology, marked by the adoption of a phenotype that
exhibited classical leading and trailing edges, with Cas and AND-34 localized
primarily at the leading edge. Cells expressing
GEF did not exhibit
this polarized morphology, although Cas still co-localized with AND-34 at
membrane ruffles (panels E-G). Thus, the enhancement of cell
migration exhibited by cells co-expressing Cas and WT AND-34 coincided with
both the relocalization of Cas to lamellipodia and the acquisition of a
polarized phenotype.
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DISCUSSION |
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Regulation of Src Activity by Cas, AND-34, and Rap1The
ability of AND-34 to enhance Src activation and cell migration was found to be
absolutely dependent on co-expression of Cas. AND-34 expression in the absence
of Cas was unable to stimulate Src-PTK activity or the migration of
Src-overexpressing C3H10T-5H cells, suggesting that AND-34 does not
directly regulate Src PTK activity. This is further supported by our findings
that the relative steady-state level of Src/Cas complexes was not affected by
AND-34 expression and that there was no requirement for direct binding of
AND-34 to Cas. These data thus argue against a direct effect of AND-34 on the
PTK activity of Src/Cas complexes.
One mechanism through which AND-34 may coordinate with Src and Cas in these processes involves phosphorylation of AND-34 by Src. AND-34 is a phosphoprotein that becomes tyrosine phosphorylated upon stimulation by serum and adhesion to FN (36). Interestingly, in the present study, AND-34 was consistently observed to be phosphorylated in a manner similar to other Src substrates when Cas and Src were overexpressed (data not shown). Although the effect of tyrosine phosphorylation on AND-34 function is currently not known, there is precedent for the regulation of GEF activity in other molecules by tyrosine phosphorylation. For example, the GEF activities of RasGRF1, Vav, and C3G are stimulated by tyrosine phosphorylation, and RasGRF1 in particular is activated following tyrosine phosphorylation by Src (5860). These reports raise the important question of whether AND-34 is a substrate of Src whose GEF activity can be regulated by phosphorylation. We are currently testing this possibility.
The data presented in this report suggest that the carboxyl terminus of AND-34, which contains the putative GEF domain, is important for its ability to synergize with Cas and Src. First, AND-34 molecules lacking the carboxyl terminus were not capable of enhancing Cas-mediated Src activation or cell migration. Second, constitutive activation of one of the reported targets of AND-34 GEF activity, Rap1, was shown to mimic the effect of AND-34 on Cas-mediated Src activation. Finally, a dominant-negative form of Rap1 inhibited AND-34-dependent enhancement of Cas-mediated Src activation and cell migration. Thus, the ability of AND-34 to synergize with Cas appeared to be at least partly dependent on the physical presence of the AND-34 carboxyl terminus and the activity of Rap1. Together, these data are consistent with a model in which the initial activation of Src that is caused by Cas binding is sufficient to phosphorylate AND-34, resulting in stimulation of GEF activity and subsequent activation of Rap1. This in turn may result in a further activation of Src PTK activity and more robust promotion of cell migration.
There are several recent reports that document a relationship between Rap1 activities and Src/Cas functions. Rap1 was shown by Xing et al. (31) to function downstream of Src/Cas complexes to activate transcription through serum response elements and AP-1 sites. In this study, we provide evidence that Rap1 can also function upstream of Src/Cas to induce Src substrate phosphorylation. To the best of our knowledge, this is the first time that Rap1 has been shown to regulate what is typically considered to be an upstream kinase. The mechanism by which Rap1 is able to enhance Src activation and substrate phosphorylation is not understood. Rap1 shares several of the same effectors as Ras, including B-Raf, Ral-specific GEFs, and other molecules such as AF-6, phosphatidylinositol 3-kinase, and RasGAP (reviewed in Ref. 39). Whether any of these downstream targets of Rap1 play a role in its enhancement of Src activation is not known and represents an intriguing area for further study.
An alternative process by which Rap1 could affect Src activation is through
modulation of integrin affinity and/or avidity via inside-out signaling. A
number of studies (39,
6164)
in several cell types have shown that Rap1 can control integrin-mediated
adhesion and potentially induce changes in integrin affinity/avidity for ECM
ligands. In turn, Src activity becomes elevated upon integrin activation and
ligand binding (15,
65,
66). Interestingly, in cells
expressing IIb
3 integrins, Rap1 can augment
ligand binding and affinity/avidity. Src has been shown to be constitutively
associated with this receptor and becomes strongly activated in response to
IIb
3-mediated adhesion
(67,
68).
Although Rap1 has not been shown previously to function upstream of Src, a second putative target of AND-34 GEF activity, Ral, has been reported to promote Src-dependent phosphorylation of the substrates Stat3 and cortactin in response to epidermal growth factor stimulation (69). Moreover, dominant-negative Ral was shown to inhibit epidermal growth factor stimulation of Src activity. In contrast to these findings, we did not observe increased phosphorylation of the Src substrate paxillin in the presence of activated Ral. This suggests that the potential function of Ral as an activator of Src is likely to be dependent on cell context, as well as the mechanism of activation.
Regulation of Cell Migration by Cas, AND-34, and Rap1A role for Rap1 in cell migration is somewhat better established. In several cases, Rap1 has been shown to be a positive regulator of cell migration (70, 71), and one of its targets, the extracellular signal-regulated kinase/mitogen-activated protein kinase signaling cascade, is known to be important for cell migration and invasion (18, 72). However, there is also evidence to suggest that Rap1 activation may be inhibitory for cell migration (73). For example, Ohba et al. (74) showed that fibroblasts lacking the Rap1 GEF C3G exhibited reduced cell adhesion and increased cell migration as compared with wild-type mouse embryo fibroblasts and that expression of activated Rap1 in the C3G-null cells resulted in decreased migration and greater cell adhesion. Ultimately, the effects of Rap1 on cell migration are also likely to be dependent on cell context and the nature and concentration of the migratory stimulus.
It is important to point out that there is currently some debate concerning the relevant targets of the AND-34 GEF domain and whether this molecule is in fact capable of functioning as an authentic GEF (39, 75). We have not specifically tested the requirement for direct activation of Rap1 by AND-34 in this study. Consequently, it is formally possible that Rap1 may function downstream of AND-34 through a mechanism that is independent of AND-34 GEF activity per se. Sakakibara et al. (35) showed in a recent report that the AND-34 family member Chat can induce Rap1 activation through a pathway that involves Cas signaling to C3G. Phosphorylation of the YXXP motifs in the substrate binding domain of Cas can result in its association with the adapter protein Crk (5), which is constitutively bound to the Rap1 GEF C3G (reviewed in Ref. 76). The possibility that AND-34 may function in a similar manner could help to reconcile conflicting data regarding the ability of AND-34 to directly activate Rap1 and other GTPases (37, 39, 75).
Finally, AND-34 may contribute to Cas-induced cell migration through its ability to relocalize Cas to membrane ruffles. Localized assembly of Cas/Crk complexes occurs at the cell periphery and can lead to activation of Rac1 through the recruitment of a second Crk-binding protein, DOCK180 (18, 77, 78). In this way, relocalization of Cas by AND-34 may activate Rac1 at the leading edge, resulting in the extension of lamellipodia and the induction of a polarized phenotype marked by discrete leading and trailing edges that are typically exhibited by migrating cells (79). This is consistent with previous data from a number of other investigators who characterize Cas/Crk as a "molecular switch" that promotes cytoskeletal reorganization and cell migration (16, 18, 72).
The mechanisms by which AND-34 recruits Cas to the cell periphery remain to
be determined, although some clues can be gained from the observation that Cas
was efficiently localized to the cell periphery in cells expressing both the
WT and GEF proteins. The putative GEF activity and Cas binding
functions of AND-34 are both missing from the
GEF construct
(36,
37). Therefore, these
activities appear to be dispensable for Cas re-localization by
AND-34. However, the AND-34 carboxyl terminus was found to be important for
adoption of the polarized phenotype exhibited by cells co-expressing Cas and
AND-34, because polarization was not observed in
GEF-expressing cells.
Determination of the specific features of the AND-34 carboxyl terminus that
are required for the induction of this phenotype will greatly enhance our
understanding of the mechanisms governing cell polarity and motility.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Dept. of Microbiology and Cancer Center, University of Virginia Health System, P. O. Box 800734, Charlottesville, VA. Tel.: 434-924-2513; Fax: 434-982-1071; E-mail: ahb8y{at}virginia.edu.
1 The abbreviations used are: pTyr, phosphotyrosine; ECM, extracellular
matrix; FN, fibronectin; PTK, protein tyrosine kinase; SH, Src homology; NSP,
novel SH2-containing protein; GEF, guanine nucleotide exchange factor; DMEM,
Dulbecco's modified Eagle's medium; FCS, fetal calf serum; mAb, monoclonal
antibody; TR, Texas Red; mCT, minimal carboxyl terminus; YFP, yellow
fluorescent protein; WT, wild-type; KD, kinase-dead; dHLH, divergent
helix-loop-helix; IP, immunoprecipitation; IB, immunoblotting.
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ACKNOWLEDGMENTS |
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REFERENCES |
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