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J. Biol. Chem., Vol. 276, Issue 47, 43987-43993, November 23, 2001
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From the Department of Molecular Medicine, Cornell University,
Ithaca, New York 14853
Received for publication, May 25, 2001, and in revised form, August 22, 2001
ACK2 (activated Cdc42-associated tyrosine
kinase-2) is a nonreceptor tyrosine kinase that is a specific
target/effector for the GTP-binding protein Cdc42. Thus far the
biological function of this tyrosine kinase has not been determined.
Using an inducible eukaryotic expression system in fibroblasts, we
demonstrate that ACK2 can strongly influence cell shape and growth as
well as focal complex formation. ACK2 was found to associate with the
focal adhesion complex components talin and vinculin, but not with the focal adhesion kinase (FAK), in a kinase-independent manner. The tyrosine kinase activity of FAK was also inhibited in cells
overexpressing both wild-type and kinase-defective ACK2. This may be
due to a competition between ACK2 and FAK for Src, which is an
essential cofactor for FAK activation, as we have found that ACK2
specifically binds Src in cells. The ACK2-Src interaction appears to be
mediated by the SH3 domain of Src, and the phosphorylation of ACK2 is
enhanced in cells overexpressing the hyperactivated Src(Y527F) mutant. Overexpression of both wild-type and kinase-defective ACK2 also results
in a severe inhibition of cell growth. In addition, ACK2 dissolves
actin stress fibers and disassembles focal complexes but in a
kinase-dependent manner. These results, taken together with
previous studies demonstrating an association of ACK2 with integrin ACKs1 (activated
Cdc42-associated tyrosine kinases) are a family of nonreceptor tyrosine
kinases that specifically interact with the GTP-bound, active form of
Cdc42, a Rho family GTPase (1, 2). We have cloned and characterized one
member of this tyrosine kinase family, ACK2 (1, 3). While Cdc42 has
been shown to play important roles in a variety of cellular activities including actin cytoskeletal organization, cell adhesion, intracellular trafficking, DNA biosynthesis, and cell growth regulation (4-10), little is known about the function of the ACKs in cells.
Our initial studies indicated that ACK2 was involved in cell adhesion
signaling (3). Cell attachment strongly enhanced the tyrosine
phosphorylation of ACK2. Unlike the focal adhesion kinase (FAK), which
has a tyrosine kinase activity that is specifically increased by cell
adhesion on fibronectin-coated plates, the tyrosine kinase activity of
ACK2 is stimulated when attaching cells to either fibronectin- or
polylysine-coated plates. Interestingly the stimulation of the tyrosine
kinase activity of ACK2 by adhesion is, at least partially, mediated by
integrin To further address the cellular function of the Cdc42-specific target
ACK2, we used a Tet-off inducible eukaryotic cell expression system.
Here we show that the overexpression of ACK2 in NIH3T3 cells strikingly
affected cell morphology as well as inhibited the tyrosine kinase
activity of FAK, perhaps as an outcome of the binding of ACK2 to c-Src.
These ACK2-mediated effects were all largely independent of ACK2
tyrosine kinase activity as was a marked inhibition of cell growth that
accompanied the inducible expression of ACK2. The expression of ACK2
also resulted in the disruption of actin stress fibers and focal
complexes, although these effects were dependent on ACK2 tyrosine
kinase activity. Overall the data indicate that the Cdc42 target ACK2
participates in the down-regulation of focal adhesion complex
organization and acts as a negative regulator of mitogenic signaling activity.
Materials--
Puromycin was purchased from Calbiochem,
tetracycline and anti-vinculin antibody were obtained from Sigma, and
Texas red-conjugated phalloidin was from Molecular Probes. The Tet-off
inducible expression system was described previously (11).
Selection of Tet-off Inducible Cell Lines--
The
HindIII/EcoRV-digested Myc-tagged ACK2
cDNA from pcDNA3 Myc-ACK2 and the corresponding Myc-tagged
ACK2(K158R) cDNA were cloned into the pTet-splice vector to obtain
pTet Myc-ACK2 or pTet Myc-ACK2(K158R). We then co-transfected ptTAK (3 µg/60-mm dish) with either the pTet-splice (control) vector, the pTet
Myc-ACK2, or the pTet Myc-ACK2(K158R) (3 µg/60-mm dish) together with
the puromycin-resistant gene plasmid (0.3 µg/60-mm dish) into NIH3T3 cells (3 × 105 cells/60-mm dish) in the presence of
tetracycline (1 µg/ml). After 48 h, the cells were transferred
to 100-mm dishes and cultured overnight in DMEM plus 10% calf serum
(CS) and 1 µg/ml tetracycline. The colony selection was performed by
adding puromycin (5 µg/ml) into the culture medium. The positive
colonies were identified following removal of tetracycline for 24 h by immunoblotting the protein with an anti-Myc antibody.
The NIH3T3 cell lines stably transfected with pTet, pTet-Myc-ACK2, or
pTet-Myc-ACK2(K158R) were sustained in DMEM containing 10% CS, 5 µg/ml puromycin, and 1 µg/ml tetracycline. To determine the
expression of the recombinant proteins, the cells in each cell line
were seeded in four dishes (60 mm) (two dishes for pTet cells) at a
density of 3 × 105 cells/dish and cultured overnight
in DMEM plus 10% CS and 1 µg/ml tetracycline. The induction was
initiated by replacement of tetracycline-containing medium with regular
medium (DMEM plus 10% CS) for various periods of time (ranging from 0 to 72 h) prior to harvesting the cells. The cells in each dish
were lysed in 300 µl of lysis buffer (40 mM Hepes, pH
7.4, 100 mM NaCl, 1% Triton X-100, 25 mM
Apoptosis Assay--
Apoptosis assays were performed using an
apoptosis detection kit with annexin V-FITC staining purchased from
Calbiochem (Oncogene). Cells that were stably transfected with Tet
(vector) or pTet-Myc-ACK2 were cultured on coverslips in DMEM plus 10%
calf serum and 1 µg/ml tetracycline overnight. Tetracycline then was
removed from the culture medium for 28 h to overexpress ACK2. As a
positive control, cells containing the pTet vector were treated at the same time with 200 ng/ml Fas ligand for 12 h to induce apoptosis. Untreated cells containing the pTet vector served as negative controls.
The annexin V-FITC staining procedures were modified based on the
instructions of the manufacturer (Calbiochem). Briefly, the cells were
washed with DMEM serum-free medium twice and incubated with annexin
V-FITC (0.5 µg/ml) in the binding buffer (10 mM Hepes, pH
7.4, 150 mM NaCl, 2.5 mM CaCl2, 1 mM MgCl2, 4% bovine serum albumin) at 22 °C
for 15 min. The cells were then washed with DMEM plus 5 mM
CaCl2 three times and incubated in propidium iodide (30 µg/ml in PBS) in the binding buffer at 22 °C for 5 min to stain
dead cells. After washing (three times) with DMEM plus 5 mM
CaCl2, the cells were fixed with 3.7% formaldehyde plus 5 mM CaCl2 at 22 °C for 10 min and followed
subsequently by three washes with DMEM plus 5 mM
CaCl2. The staining was observed under a fluorescent microscope.
Immunofluorescence--
Cells that were preplated on glass slips
with or without induction were rinsed with PBS twice, fixed in 3.5%
formaldehyde for 10 min, and then permeabilized with 0.2% Triton X-100
for 10 min. The cells were blocked with 0.2% bovine serum albumin in
PBS for 5-10 min and then incubated with primary antibodies for 30 min
at 37 °C. After washing (three times) with PBS, the cells were
incubated with secondary antibodies and phalloidin (conjugated to
fluorescent dyes) for 30 min at 37 °C. The cells were then washed
with PBS (three times), mounted on a glass slide, and observed under a
fluorescence microscope.
Immunoprecipitations--
The cells in 60-mm dishes were lysed
in 500 µl of lysis buffer (40 mM Hepes, pH 7.4, 100 mM NaCl, 1% Triton X-100, 25 mM
BrdUrd Incorporation--
The BrdUrd incorporation
experiments were performed as described previously (11). Briefly, the
cells were synchronized at G0 phase by serum starvation,
and the expression of ACK2 and the kinase-dead mutant was induced for
24 h before labeling the cells with 100 µM BrdUrd in
regular medium for 18 h. The cells were then fixed and stained
with anti-BrdUrd and Hoechst as described in the preceding section. The
percentage of incorporation of BrdUrd was determined by the ratio of
the number of cells showing anti-BrdUrd staining to cells showing
Hoechst staining from five to eight microscopic fields (about 110-200 cells).
In Vitro Assays of FAK Autophosphorylation and Exogenous
Substrate Phosphorylation--
The FAK was immunoprecipitated as
follows. The lysate (1.2 mg of total protein) from uninduced or induced
wild-type or kinase-defective ACK-expressing cells was incubated with 2 µl of anti-FAK antibody for 1.5 h at 4 °C followed by the
addition of 5 µl of protein A-agarose beads (Sigma) overnight at
4 °C. The beads containing FAK immunocomplexes were washed three
times with ice-cold lysis buffer and divided into three equal aliquots,
which were used for assaying FAK activity (autophosphorylation and
phosphorylation of exogenous substrates) and immunoblotting.
When assaying FAK activity, the immunocomplexes were further washed
twice with ice-cold kinase buffer (50 mM Tris/HCl, pH 7.4, 10 mM MnCl2) and then incubated in 40 µl of
kinase buffer including 10 µCi of [
For immunoblotting, the complexes were further washed twice with
ice-cold lysis buffer, suspended in 20 µl of 1× SDS-PAGE sample
buffer, boiled for 5 min, and resolved on 9% SDS-polyacrylamide gels.
After protein transfer onto nitrocellulose membranes, FAK was blotted
with the anti-FAK antibody (1:5000).
Precipitation of ACK2 by GST-SH3 Domains--
The immobilized
GST-Src-SH3, Grb2-SH3, and Crk-SH3 domains (20 µg each) on
glutathione beads were incubated with COS7 cell lysates (200 µg) that
contained Myc-tagged ACK2 at 4 °C for 2-3 h with rotation. The
beads were washed three times with cell lysis buffer and subjected to
analysis by SDS-PAGE. The SH3 domain-associated ACK2 was detected by
Western blotting with an anti-Myc antibody.
ACK2 Causes Cell Shape Changes--
To begin to
examine the cellular effects of ACK2, we cloned a Myc-tagged cDNA
of ACK2 and its kinase-defective mutant ACK2(K158R) into pTet-splice, a
eukaryotic expression vector that contains a tetracycline
transactivator regulatory sequence upstream from a minimal human
cytomegalovirus promoter. After co-transfection of pTet-ACK2 or
pTet-ACK2(K158R) with pTet-tTAK, the tetracycline transactivator
plasmid, and pMet-puro (a plasmid that contains a puromycin-resistant
gene), positive (NIH3T3 cell) clones were selected for overexpression
of wild-type ACK2 or ACK2(K158R). The expression of wild-type ACK2 or
ACK2(K158R) was induced by removing tetracycline from the medium at
various times (Fig. 1A). While
we have observed some variability for the time required for the maximal
expression of ACK2 following its induction, for the experiments
presented here, ACK2 expression peaked at ~24 h (postinduction).
Cells overexpressing wild-type ACK2 showed dramatic changes in
morphology with a time course mirroring that for ACK2 expression (Fig.
1B). The cells that overexpressed wild-type ACK2 showed a
significant degree of branching (Fig. 1B, see panels
j-l) with some of the branches extending to a length of 200 µm
(compared with the average diameter of ~50 µm for the control
cells). Generally, similar changes in cell morphology were observed
upon overexpression of the kinase-defective mutant ACK2(K158R).
However, whereas after 24 h of induction of wild-type ACK2 we
often observed significant extensions or long spikes (as many as four
to five per cell), under similar conditions of induction the
kinase-defective ACK2 counterpart showed less branching with typically
one or two very long spikes extending from the ends of the cell body.
We noted that the branching structure of those cells expressing
wild-type ACK2 was dramatically inhibited by cell-cell contact, whereas the spikes exhibited by cells expressing ACK2(K158R) were not affected
by cell-cell contact (data not shown). Cells that contained the vector
alone did not show any changes in morphology after removal of
tetracycline (Fig. 1B, panels a-f).
The Interaction of ACK2 with Known Components of Focal Complexes
Occurs in a Kinase-independent Manner--
Previously we showed that
ACK2 could be co-immunoprecipitated in a complex with the integrin
However, the inhibition of FAK activity by ACK2 may also involve c-Src.
While screening for potential binding partners for ACK2, we found that
the SH3 domain of Src was highly effective in its ability to bind to
the proline-rich carboxyl-terminal domain of ACK2. The upper
panel in Fig. 4A compares
the abilities of different GST-SH3 domains to co-precipitate Myc-tagged
ACK2, while the middle panel compares the binding of the
GST-SH3 domain constructs to dynamin, and the lower panel
compares the relative amounts of the GST-SH3 domains that were assayed.
We have found that the carboxyl-terminal SH3 domain of Grb2 is capable
of a weak interaction with ACK2, although in some experiments we have
detected no interaction, and in all cases it was at least 50% less
effective than the SH3 domain of c-Src. Note that under the same
conditions the SH3 domains of both Grb2 and Src bind equally well to
dynamin. Thus far we have not found any other SH3 domain-containing
protein to be capable of associating with ACK2. When Src(Y527F), a
constitutively active Src mutant, was co-transfected with Myc-tagged
ACK2 ACK2 Inhibits Cell Growth--
Another striking effect that
accompanied the overexpression of ACK2 was a significant inhibition of
cell growth. We determined the growth rates of the different cell lines
by either (i) inducing ACK2 expression for different periods of time
and then counting cell numbers (where in each case cells were counted
for a total of 3 days) (Fig.
5A) or (ii) by inducing the
cells for various times and then counting the cells at the different
time periods of induction (Fig. 5B). In both cases, the
growth rates for cells overexpressing wild-type ACK2 were dramatically
reduced. The induced expression of ACK2(K158R) also resulted in a
reduction in cell growth, although the growth inhibition was less
severe than that caused by wild-type ACK2. Similar results were
obtained when assaying DNA synthesis by deoxybromouridine incorporation
(Fig. 5C). Expression of wild-type ACK2, and to a slightly
lesser extent ACK2(K158R), resulted in an inhibition of
serum-stimulated DNA synthesis.
The inhibition of cell growth by ACK2 was not the outcome of programmed
cell death (apoptosis). Fig. 6 shows that
unlike the Fas ligand, which gives rise to a strong apoptotic response
as assayed by staining dead cells with annexin V-FITC, the
overexpression of ACK2 does not induce programmed cell death. At the
present time, we also do not believe that the inhibitory effects of
ACK2 on cell growth can be entirely attributed to the ACK2-mediated inhibition of FAK and/or binding of Src. We have generated a number of
ACK2 mutations, and thus far we have only found that perturbing the SH3
domain of ACK2 alters its ability to inhibit cell growth (data not
shown). This perturbation should not affect the ability of ACK2 to bind
Src or inhibit FAK. However, it does strongly inhibit the binding of
ACK2 to clathrin, presumably by preventing an intramolecular
interaction that allows ACK2 to adapt the appropriate conformation for
binding to clathrin (13). This may mean that the participation of ACK2
in the endocytosis of cell surface receptors (e.g. integrins
and/or growth factor receptors) has a strong role in the ACK2-mediated
inhibition of cell growth.
ACK2 Influences the Actin Cytoskeleton and Focal Adhesion Complexes
in a Kinase-dependent Manner--
To further examine the
cellular effects caused by overexpression of ACK2, actin cytoskeletal
staining was performed. As shown in Fig.
7A, in the absence of
induction (i.e. plus tetracycline), the cytoskeletal
structures for all three cell lines were similar such that each cell
line showed intact actin stress fibers (Fig. 7A, see
panels a-c). After 48 h of induction (i.e.
minus tetracycline), cells expressing wild-type ACK2 were either
completely lacking or had markedly reduced amounts of actin stress
fibers (Fig. 7B, panel b). Both control cells and
cells that express kinase-defective ACK2(K158R) showed intact actin
stress fibers (Fig. 7B, panels a and
c).
Focal adhesion complexes are the docking sites for stress fibers (14).
We have shown that ACK2 is activated by cell attachment (1, 3). The
disassembly of stress fibers by ACK2 may be the result of interactions
between ACK2 and focal adhesion complexes. When we stained control
cells with an antibody against vinculin, a known component of focal
adhesion complexes, we observed that the focal adhesion complexes were
located at the ends of stress fibers (Fig. 7A, panels
d-f). In contrast, vinculin staining was diminished in the cells
that overexpressed wild-type ACK2 and had a branching structure (Fig.
7B, panel e). Control cells containing vector
alone or cells expressing the kinase-defective ACK2 mutant had normal
focal adhesion complexes (Fig. 7B, panels d and
f). These results suggest that the disruption of focal
adhesion complex formation may be the direct cause for the disassembly
of actin stress fibers by ACK2.
The GTP-binding protein Cdc42 has been implicated in a diversity
of cellular activities that ultimately mediate changes in cell shape,
motility, cell cycle progression, differentiation, and cell survival
(15, 16). Among the major questions concerning the underlying
mechanisms for these different functions are the cellular roles played
by the individual targets of Cdc42. Here we have examined the cellular
effects of the highly specific Cdc42 target ACK2 and found that it has
a significant influence on cell shape, cell cycle progression, and the
actin cytoskeleton. Specifically both wild-type and kinase-dead ACK2
induce cell extensions and inhibit cell growth. The effects are
somewhat enhanced when expressing a kinase-competent ACK2 compared with
the kinase-defective version. Nonetheless, the latter causes clear
changes in cell shape and retards cell growth, arguing that it is the
binding ability (rather than the tyrosine kinase activity) of ACK2 that
is mainly responsible for these effects. However, there is a
significant requirement for the kinase activity of ACK2 in its ability
to dissolve actin stress fibers and disassemble focal complexes. Thus,
one can envisage a model where the binding of ACK2 to integrin
All of this makes it attractive to think of ACK2 as playing a key
regulatory role both through its binding partners and tyrosine phosphosubstrate(s) in coordinating changes in cell shape with a
slowing of cell cycle progression. The coordination of these activities becomes important during cellular differentiation. This is
especially the case when considering the shape changes and effects on
actin stress fibers that accompany neuronal differentiation. Previous
studies have shown that the inhibition of actin stress fiber formation
results in cell membrane protrusion or neurite outgrowth (17).
Microinjection of the constitutively active Rho mutant Rho(G14V) or the
catalytic domain of the p160 RhoA-binding kinase ROK It has been well established that a FAK·Src complex plays a
pivotal role in integrin-mediated cell growth (19-21). Thus, it seems
likely that some of the effects of ACK2 on cell growth are through its
interactions with Src, perhaps by blocking the proper activation of FAK
and preventing its transmission of mitogenic signals through the
Grb2/Ras/extracellular signal-regulated kinase pathway (21, 22).
However, thus far we have not been able to draw a direct correlation
between the ACK2-Src interaction and ACK2-mediated growth inhibition.
We have found that an ACK2 SH3 domain mutant that is incapable of
binding clathrin is also unable to inhibit cell growth (data not
shown). This provides us with a clue that the ACK2-clathrin interaction
is somehow linked to the ACK2-mediated effects on cell cycle
progression. Such a possibility is interesting as proteins that
participate in the endocytosis of growth factor or other mitogenic
receptors are often growth inhibitory. The potential regulatory effects
of ACK2 on growth factor receptor and/or integrin endocytosis giving
rise to a down-regulation of these receptors could strongly contribute to the effects of ACK2 on cell shape and the actin cytoskeleton as well
as cell cycle progression and thereby serve a multipurpose function
toward the generation of a differentiation signal. It will be
interesting to see how general a role ACK2 plays in cellular differentiation processes and whether such roles are connected to the
regulation of ACK2 by integrins and other cell adhesion molecules. Such
a connection may be manifested by the involvement of ACK2 in the
disruption of focal adhesion complexes and the organization of new
integrin complexes necessary for clathrin-coated vesicle endocytosis.
Future studies will be directed toward testing these ideas.
We appreciate the help of Cindy Westmiller in
the preparation of this manuscript.
*
This work was supported by National Institutes of Health
Grants GM40654 and GM47458.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.
Published, JBC Papers in Press, September 4, 2001, DOI 10.1074/jbc.M104819200
The abbreviations used are:
ACK, activated
Cdc42-associated tyrosine kinase;
FAK, focal adhesion kinase;
BrdUrd, deoxybromouridine;
SH3, Src homology 3;
DMEM, Dulbecco's modified
Eagle's medium;
CS, calf serum;
FITC, fluorescein isothiocyanate;
PBS, phosphate-buffered saline;
PAGE, polyacrylamide gel electrophoresis;
GST, glutathione S-transferase;
Tet, tetracycline.
The Nonreceptor Tyrosine Kinase ACK2, a Specific Target for Cdc42
and a Negative Regulator of Cell Growth and Focal Adhesion
Complexes*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 (Yang, W., Lin, Q., Guan, J.-L.,
Cerione, R. A. (1999) J. Biol. Chem. 274, 8524-8530) and clathrin (Yang, W., Lo, C. G., Dispenza, T., and
Cerione, R. A. (2001) J. Biol. Chem. 276, 17468-17473), suggest that the binding and protein tyrosine kinase activities of ACK2 coordinate changes in cell morphology and growth with the disassembly of focal adhesion sites, perhaps to organize new
integrin complexes that are required for endocytosis and/or for
cellular differentiation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1. Data from immunoprecipitation studies
indicated that ACK2 associated with an integrin 1
complex (3), thus suggesting that this tyrosine kinase may have a
regulatory role in the assembly or disassembly of focal adhesion complexes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycerophosphate, 1 mM sodium orthovanadate, 1 mM EDTA, 10 µg/ml leupeptin, and 10 µg/ml aprotinin),
and 20 µg of lysate proteins were loaded onto a 10%
SDS-polyacrylamide gel. The proteins were transferred to a
polyvinylidene difluoride membrane and blotted with an anti-Myc antibody.
-glycerophosphate, 1 mM sodium orthovanadate, 10 µg/ml
leupeptin, and 10 µg/ml aprotinin) by rocking for 15-30 min at
4 °C. The lysate was cleared by centrifugation at 14,000 rpm for 2 min, and an aliquot of the lysate (200-500 µl) was used for
immunoprecipitation. After the primary antibody (1 µg/400 µg of
lysate) was incubated with the lysate on ice for 30 min, protein A
beads (Sigma) (1:1) were added, and the mixture was rocked at 4 °C
for 1 h. The beads were washed twice with 700 µl of the lysis
buffer and finally resuspended in 20 µl of 2× SDS-PAGE sample
buffer. The immunoprecipitated proteins were separated by SDS-PAGE.
-32P]ATP with
(for assaying the phosphorylation of exogenous substrates) or without
(for autophosphorylation assays) 5 µg of the exogenous substrate
poly(Glu,Tyr) (E4Y1) for 20 min at 25 °C.
The reactions were stopped by adding 10 µl of 5× SDS-PAGE sample
buffer. The samples were boiled for 5 min, and the proteins were
resolved by SDS-PAGE. The radioactivity of the phosphorylation bands
was visualized and quantitated by autoradiography and phosphorimage analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Overexpression of ACK2 causes morphology
changes in NIH3T3 cells. A, expression of Myc-ACK2 and
Myc-ACK2(K158R) in tetracycline-inducible (Tet-off) cell lines. The
induction of protein expression was performed as described previously
(9). The proteins were blotted with an anti-Myc antibody (9E10). The
numbers at the top of the panel represent the
time of induction. B, ACK2 induces cell branching. The cells
were seeded in a 12-well plate at a density of 103
cells/well and cultured overnight in DMEM plus 10% CS and 1 µg/ml
tetracycline. For induction, the cells were rinsed once with DMEM plus
10% CS and replenished with DMEM plus 10% CS. After the indicated
times of induction (to the left), the cells were
photographed under a microscope. Upper three panels
(a, g, and m) represent no induction.
Panels a-f, pTet (vector control) cell line; panels
g-l, pTet-ACK2 cell line; panels m-r,
pTet-ACK2(K158R) cell line.
1 subunit (3). Here we show that both wild-type and
kinase-defective ACK2 can be co-immunoprecipitated with anti-vinculin
as well as anti-talin antibodies (Fig. 2,
see lanes 8, 9, 14, and
15). We have estimated that ~10% of the total (overexpressed) ACK2 is co-immunoprecipitated with vinculin or talin,
and both of these interactions occur independently of ACK2 tyrosine
kinase activity. We did not find evidence for a stable complex between
ACK2 and FAK based on co-immunoprecipitation experiments. However, we
consistently observed that the tyrosine phosphorylation of FAK was
inhibited in cells that overexpressed ACK2 (see Fig. 2, compare
lanes 2 and 3 with lane 1 or
versus lanes 4-6). This is further examined in Fig.
3. Overexpression of either wild-type ACK2 or the kinase-defective ACK2 mutant caused a clear inhibition of
the ability of FAK to phosphorylate exogenous substrates, as assayed
with the tyrosine-containing polymer E4Y1, with
a corresponding inhibition of the autophosphorylation of FAK. In
different experiments, we have found that the extent of inhibition
ranged from 50-75%. Previous studies have shown that cytochalasin D,
an F-actin depolymerization reagent, also inhibits the tyrosine
phosphorylation of FAK (12), suggesting that ACK2-mediated effects on
the integrity of the actin cytoskeleton may at least in part account
for the inhibition of FAK autophosphorylation.
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Fig. 2.
Co-immunoprecipitation of ACK2 with vinculin
and talin. The Tet-off inducible cells that were stably
transfected with pTet-Myc-ACK2 or pTet-Myc-ACK2(K158R) were cultured in
100-mm dishes with DMEM plus 10% CS plus 1 µg/ml tetracycline
overnight, and the expression of ACK2 proteins was induced by removing
tetracycline from the medium for 24 h. The cells were then
lysed in 1 ml of the radioimmune precipitation lysis buffer for
each 100-mm dish. 300 µg of lysate proteins were used in each sample
for immunoprecipitation. The anti-vinculin (2 µg), anti-talin (2 µg), or anti-FAK (2 µg) antibody was incubated with the lysates,
and the immunoprecipitation was carried out as described under
"Experimental Procedures." The immunoprecipitation complexes were
separated by SDS-PAGE, transferred onto a polyvinylidene difluoride
membrane, and immunoblotted by either anti-phosphotyrosine antibody or
anti-Myc antibody. +Tet, uninduced samples;
Tet, induced samples; IP, immunoprecipitation;
PY, phosphotyrosine; MW, molecular weight
marker.
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Fig. 3.
ACK2 inhibited tyrosine phosphorylation and
kinase activity of FAK. U, uninduced (+tetracycline);
I, induced ( tetracycline). The upper right
panel shows the quantitation of FAK activity by phosphorimage
analysis of the autoradiograph shown in the upper left
panel. The lower right panel shows inhibition of FAK
autophosphorylation by ACK2. The lower left panel compares
the relative amounts of FAK that were assayed for the different
conditions. WT, wild type; IP,
immunoprecipitation.
NT, an amino-terminal truncation mutant of ACK2, in COS7 cells,
the tyrosine phosphorylation of ACK2NT was dramatically increased
(Fig. 4B, lane 3), demonstrating that Src either
directly, or through another tyrosine kinase, phosphorylates ACK2
in vivo. Stable complex formation between Src and ACK2NT
in vivo was also detected following the immunoprecipitation of either Myc-tagged ACK2NT with an anti-Myc antibody (Fig.
4B, lane 1) or endogenous c-Src with an anti-Src
antibody (Fig. 4C, lane 2). In these experiments,
we estimated that ~5% of the total ACK2 was co-immunoprecipitated
with Src. Although the tyrosine phosphorylation of wild-type ACK2 was
also dramatically enhanced by co-transfection with Src(Y527F), we have
not been able to consistently detect a stable association between
wild-type ACK2 and c-Src in cells (data not shown). These results are
reminiscent of the binding interaction between ACK2 and clathrin, which
is significantly stronger when assaying the ACK2NT mutant (13). This
may mean that the amino-terminal end of ACK2 plays some sort of
regulatory role that weakens the affinity of ACK2 for Src, perhaps in a
manner similar to its effects on the interaction of ACK2 with clathrin.
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Fig. 4.
ACK2 interacts with Src and is
phosphorylated by Src(Y527F). A, ACK2 specifically
interacts with the Src-SH3 domain. Immobilized GST-Src-SH3, Grb2
carboxyl-terminal SH3, and Crk amino-terminal SH3 domains (20 µg
each) on glutathione beads were incubated with Myc-tagged
ACK2-transfected COS7 cell lysates (200 µg) at 4 °C for 2-3 h.
The SH3 domain-associated ACK2 protein was detected by immunoblotting
with anti-Myc antibody (upper panel). The middle
panel shows the results of immunoblotting with anti-dynamin
antibody. The lower panel shows the relative amounts of the
different GST fusion proteins used in the experiments. B,
ACK2 NT is co-immunoprecipitated with and phosphorylated by
Src(Y527F). pcDNA3-Myc-ACK2NT (2 µg/60-mm dish) was
co-transfected with pcDNA3-Src(Y527F) (2 µg/60-mm dish) into COS7
cells. The cells were lysed with a Triton X-100-based lysis buffer. The
immunoprecipitations were performed as described under "Experimental
Procedures" with an anti-Myc antibody (9E10). The
immunoprecipitated proteins were immunoblotted with either an
anti-phosphotyrosine, an anti-Src, or an anti-Myc antibody as indicated
in the figure. Lanes 1 and 3, co-transfection of
pcDNA3-Myc-ACK2NT with pcDNA3-Src(Y527F); lanes 2 and 4, pcDNA3-Myc-ACK2NT alone. C,
ACK2NT is associated with endogenous c-Src. pcDNA3-Myc-ACK2NT
(2 µg/60-mm dish) was transfected into COS7 cells. The cells were
lysed with a Triton X-100-based lysis buffer. The immunoprecipitation
was performed with an anti-Src antibody. The immunoprecipitated
proteins were immunoblotted with either an anti-Myc antibody or
anti-Src antibody as indicated in the figure. Lane 1,
control cells (no transfection); lane 2,
pcDNA3-Myc-ACK2NT-transfected cells. PY,
phosphotyrosine; IP, immunoprecipitation.
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Fig. 5.
ACK2 inhibits cell growth. The
cells were seeded in a 12-well plate and cultured in DMEM plus 10% CS
and 1 µg/ml tetracycline overnight. The initial cell density was
5 × 103 in A and 6 × 104
in B. Induction of the expression of ACK2 or ACK2(K158R) was
performed in two ways: either induction at the different indicated time
points and then counting the cell numbers (in all cases, cells were
grown for 3 days) (A) or cells were induced for different
time periods and then counted (B). The cell growth rate was
determined by counting the cell numbers under a microscope with a
manual cell counter. The numbers in the figures are the averages from
two independent sets of culture. C, ACK2 inhibits DNA
synthesis. The pTet, pTet-Myc-ACK2, or pTet Myc-ACK2(K158R) stably
transfected NIH3T3 cells were seeded at 104/35-mm dish and
cultured in DMEM plus 10% CS plus 1 mg/ml tetracycline overnight. The
cells were synchronized at G0 phase by serum starvation for
24 h, and the expression of ACK2 was induced at the same time by
removing tetracycline in the culture medium. The cells were
subsequently cultured in regular culture medium plus BrdUrd
(BrdU) (100 µM) for 18 h, and then fixed
and stained with anti-BrdUrd antibody and Hoechst. The percentage of
BrdUrd incorporation was determined by the ratio of the number of cells
showing BrdUrd staining to cells showing Hoechst staining from five to
eight microscopic fields (110-200 cells). The variation was calculated
from the differences between microscopic fields.
View larger version (80K):
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Fig. 6.
Overexpression of ACK2 did not induce
apoptosis. The cells were cultured, treated, and stained as
described under "Experimental Procedures." The expression of
Myc-tagged ACK2 was induced by removing tetracycline from the culture
medium for 28 h before annexin V-FITC staining. The positive
control was the pTet (vector)-transfected cells that were treated with
200 ng/ml Fas ligand (FasL) for 12 h, and the negative
control was the pTet (vector)-transfected cells without any treatment.
The green fluorescence is annexin V-FITC staining. The phase
contrast image of the cells was captured from the same field for the
annexin V staining. Notice that Fas ligand induced apoptosis in about
30% of total cells, while overexpression of ACK2 did not result in any
apoptosis.
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[in a new window]
Fig. 7.
ACK2 disassembles actin stress fibers and
focal adhesion complexes. Cells were cultured in 100-mm dishes
with DMEM plus 10% CS and 1 µg/ml tetracycline overnight, and then
for induction of ACK2, cells were cultured without tetracycline for
24 h. The cells were added to glass coverslips in a six-well plate
and cultured under the same conditions (as described above) to continue
the induction for another 24 h. The cells were then fixed and
stained as described under "Experimental Procedures." A,
without induction (+Tet). B, with
induction for 48 h ( Tet). In both A and
B, the panels on the left (a,
b, and c) were stained with phalloidin for
F-actin (red) and the panels on the right
(d, e, and f) were stained with
anti-vinculin (green) for focal adhesion complexes.
Panels a and d represent the pTet (vector
control) cell line, panels b and e represent the
pTet-Myc-ACK2 cell line, and panels c and f
represent the pTet-Myc-ACK2(K158R) cell line.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and/or vinculin, talin, and c-Src initially results in
a modulation of FAK activity and cytoskeletal alterations that lead to
cellular extensions. The activation of ACK2 tyrosine kinase activity
apparently then gives rise to the phosphorylation of a focal contact or
cytoskeletal-associated component(s) that directs focal adhesion
disassembly and the dissolution of actin stress fibers.
into rat
pheochromocytoma (PC12) cells rapidly induced neurite retraction (18),
while introduction of the C3 exoenzyme, which is a specific inhibitor
for Rho activation, promoted neurite outgrowth in PC12 cells (17).
However, the microinjection of a dominant-negative mutant of Cdc42
(Cdc42(T17N)) inhibited neurite outgrowth completely, indicating that
active Cdc42 is required for this neuronal activity. Microinjection of
activated Cdc42 into PC12 cells leads to a loss of actin stress fibers
and focal complexes. Thus, ACK2, which is capable of inducing cellular
extensions and disrupting actin stress fibers, may be responsible for
mediating these Cdc42-stimulated events during a differentiation response.
ACKNOWLEDGEMENT
FOOTNOTES
To whom correspondence should be addressed. Tel.: 607-253-3888;
Fax: 607-253-3659; E-mail: rac1@cornell.edu.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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