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INTRODUCTION |
Rho family small GTP-binding proteins have been implicated in a
diversity of biological activities including cell morphology, cytoskeletal organization, cell adhesion, and gene transcription (1-7). We have focused on one member of the Rho family, Cdc42, which
has been shown to induce cell filopodia formation, activate the c-Jun
kinase (JNK1),1 and cause
cell anchorage-independent growth (2, 5, 6, 8-11). A number of
downstream targets of Cdc42 have been characterized and can be
classified into two groups. One class represents the CRIB (for
Cdc42/Rac-interactive binding) motif-containing proteins that includes
the PAKs (1), ACKs, Wiscott-Aldrich syndrome proteins, and mixed
lineage kinases (12-19). The second class of targets lack CRIB motifs
and include the IQGAPs, p70 S6 kinase, and the 85-kDa regulatory
subunits (p85) of phosphatidylinositol 3-kinase (20-25). Functional
analyses of these targets indicate that some are involved in
cytoskeletal organization, such as the Wiscott-Aldrich syndrome
proteins, IQGAPs and PAKs, whereas others regulate JNK activation or
mitogenesis, including the PAKs, mixed lineage kinase-3, p70 S6 kinase,
and phosphatidylinositol 3-kinase. The specific cellular activities of
Cdc42 that are mediated by these targets have been variable and
sometimes contradictory. Thus, activation of Cdc42 has been suggested
both to stimulate (17, 26-28) and inhibit (29, 30) F-actin
polymerization, as well as to enhance (24, 25) and inhibit (31) cell
growth. The complexity of the effects mediated by Cdc42 may reflect a subtle balance or coordination between cytoskeletal organization and
mitogenesis that is necessary for cells to process signals that
regulate both cell morphology and cell growth.
It is known that cell adhesion in adherent cells directly regulates
both cytoskeletal organization and cell mitogenesis (32-34). Anchorage-independent growth becomes a criterion for cell
transformation, indicating that cell adhesion has a key function for
normal cell growth. In fact, overexpression of integrin molecules or
their associated proteins prevents abnormal cell growth and recovers anchorage-dependent growth (35, 36), suggesting that
integrin signaling mediates a balance between cytoskeletal organization and mitogenic progression. However, the connection between these two
cellular events is not clear. Previous studies indicate that Rho
proteins play important roles in cell adhesion (1, 2, 34, 37, 38). Rho
has been shown to regulate focal contact complex assembly and actin
stress fiber formation (38). Overexpression of a GTPase-defective
mutant, Cdc42(G12V), or a constitutively active mutant, Cdc42(F28L),
resulted in anchorage-independent growth (10, 11), suggesting that
Cdc42 may mediate cell adhesion signals that regulate both cytoskeletal
organization and mitogenesis. Recent studies have shown that Cdc42
mediates integrin
1 signaling and promotes cell
migration (39). The Cdc42 targets that mediate these effects are not
known, although a number of Cdc42 targets have been reported to
regulate F-actin polymerization and depolymerization (17, 26-30).
The ACKs are members of a family of non-receptor tyrosine kinases that
specifically interact with Cdc42 (14, 15). Here we demonstrate that
ACK-2 is activated by cell adhesion on a substratum in a
Cdc42-dependent manner. The activation does not require
cell spreading. The RGD peptide and an anti-integrin
1
antibody inhibit the activation of ACK-2 by cell adhesion, and ACK-2
was co-immunoprecipitated with integrin
1, indicating a
role for integrins in the regulation of this Cdc42 target.
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EXPERIMENTAL PROCEDURES |
Materials--
Fibronectin, RGD peptides, anti-integrin
1 monoclonal antibody, and
anti-
5
1 polyclonal antibody were
purchased from Life Technologies, Inc. Polylysine, trypsin inhibitor,
cycloheximide, and latex beads (6 µM) were purchased from
Sigma. Anti-flag antibody (M5) was purchased from Eastman Kodak Co.
Anti-FAK antibody was prepared and used as described previously (40).
Anti-phosphotyrosine (4G10) was purchased from Upstate Biotechnology
Inc., and horseradish peroxidase-conjugated anti-phosphotyrosine (PY20)
was purchased from Oncogene; anti-Erk was obtained from Santa Cruz
Biotechnology, and cytochalasin D was from Calbiochem.
Cell Culture and Transfection--
COS-7 cells were grown in
DMEM plus 10% fetal bovine serum at 37 °C, 5% CO2.
NIH3T3 cells were grown in DMEM plus 10% calf serum at 37 °C, 5%
CO2. The cells were split at 3 × 105/60-mm dish within 24 h before transfection. DNA
transfections were performed using LipofectAMINE according to the
manufacturer's standard protocols (Life Technologies, Inc.). For
transient transfections in COS-7 cells, the cDNAs for ACK-2 and
Cdc42 were expressed using the pcDNA3 vector. For stable
transfections, the expression vector was pLTR. To select stable cell
lines for ACK-2, the pLTRHA-ACK-2 (HA-tagged) was co-transfected with a
plasmid carrying the neomycin-resistant gene into NIH3T3 cells. G418
(500 µg/ml)-resistant cell colonies were selected. The expression of
HA-tagged ACK-2 in each colony was determined by immunoblotting the
cell lysates with anti-HA antibody.
Tet-off Inducible Cell
Lines--
HindIII/EcoRV digested Myc-tagged
ACK-2 cDNA from pcDNA3 Myc-ACK-2 was cloned into the
pTet-splice vector (Life Technologies, Inc.) to obtain pTet Myc-ACK-2.
We then co-transfected ptTAK (3 µg/60-mm dish) and pTet-splice
(vector alone) or pTet Myc-ACK-2 (3 µg/60-mm dish) with a
puromycin-resistant gene plasmid (0.3 µg/60-mm dish) into NIH3T3
cells (3 × 105/60-mm dish) in the presence of
tetracycline (1 µg/ml). After 48 h, the cells were transferred
to a 100-mm dish and cultured overnight in DMEM plus 10% calf serum
and 1 µg/ml tetracycline. Colony selection was performed by adding
puromycin (5 µg/ml) to the culture medium. Positive colonies were
determined by immunoblotting with anti-Myc antibody.
Immunoprecipitation--
Confluent 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
-glycerophosphate, 1 mM sodium orthovanadate, 10 µg/ml
leupeptin, and 10 µg/ml aprotinin) or RIPA buffer (40 mM
Hepes, pH 7.4, 100 mM HCl, 1% Triton X-100, 0.5% sodium
deoxycholate, 0.1% SDS, 25 mM
-glycerophosphate, 1 mM EDTA, 1 mM sodium orthovanadate, 10 µg/ml
leupeptin, and 10 µg/ml aprotinin) with rocking for 15-30 min at
4 °C. The lysates were cleared by centrifugation at 14,000 rpm for 2 min. Aliquots of the lysates (200-500 µl) were used for
immunoprecipitation. After the primary antibody was incubated with
lysates on ice for 30 min, protein A or protein G beads (Sigma) were
added, and the mixture was rocked at 4 °C for 1 h. The beads were washed twice with 700 µl of lysis buffer and finally resuspended in 20 µl of 2× SDS-PAGE sample buffer. The immunoprecipitated proteins were separated by SDS-PAGE.
Coating Plates or Latex Beads with Polylysine or Fibronectin and
Cell Adhesion--
Polylysine (10 µg/ml) or fibronectin (10 µg/ml)
in PBS was added to plates (2 ml/35-mm plate) and incubated at 4 °C
overnight. The plates were subsequently washed (3 times) with BSA (2 mg/ml) in PBS and then incubated at 37 °C with 2 ml of BSA (2 mg/ml) in PBS for 1.5-2 h. The plates were then washed (3 times) with PBS and
ready for use. For BSA control plates, treatment procedures were the
same as described above. Cells remained in suspension and did not
attach to BSA-coated plates. The coating of latex beads was performed
essentially as described previously (41). Briefly, 20 µl of the latex
beads were incubated with either polylysine (50 µg/ml) or fibronectin
(50 µg/ml) in PBS overnight at 4 °C with rotation. After washing
with PBS (3 times), the beads were incubated with BSA (2 mg/ml) at
37 °C for 2 h, washed with PBS (2 times), resuspended in 200 µl of DMEM, and then were ready for use. For cell adhesion, the cells
were trypsinized, resuspended with trypsin inhibitor solution, and
washed twice with serum-free DMEM medium. The cells were then
resuspended in DMEM. In some cases, the cells were preincubated with
DMEM plus cycloheximide or anti-integrin antibodies or RGD peptides at
37 °C for 30 min. The cells were finally added onto substratum
precoated culture dishes or mixed with latex beads and incubated at
37 °C for the indicated time and directly lysed with lysis buffer.
JNK Assays--
The flag-tagged JNK1 was immunoprecipitated with
anti-flag antibody (M5) from the lysates of cells that were transiently
transfected with pcDNA3 flag-JNK1 or pcDNA3 flag-JNK1 plus
pcDNA3 Myc-ACK-2 and/or pcDNA3 HA-Cdc42(T17N). The
immunocomplex beads were washed twice with lysis buffer and once with
JNK assay buffer (20 mM Hepes, pH 7.4, 10 mM
MgCl2, 1 mM sodium orthovanadate) and then were
mixed with the kinase assay buffer and GST-Jun (5 µg). The phosphorylation was initiated by adding 5 µCi of
[
-32P]ATP (17 mCi/nmol) and performed at 22 °C for
20 min. The reaction was stopped by adding 2× SDS sample buffer, and
the samples were boiled for 7 min and loaded onto a 10%
SDS-polyacrylamide gel. The gel was transferred onto a polyvinylidene
difluoride membrane that was used for autoradiography and immunoblotting.
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RESULTS |
Cell Adhesion Stimulates Tyrosine Phosphorylation of ACK-2 That Is
Independent of Cell Spreading--
Our previous studies have shown
that cell attachment activated ACK-2, whereas cell detachment resulted
in its dephosphorylation (15). To investigate the relationship between
cell adhesion and the activation of ACK-2 further, we precoated plates
with either polylysine or fibronectin and then added cells expressing ACK-2 onto the plates. When the cells were plated onto polylysine or
fibronectin for either 30 or 60 min at 37 °C, the tyrosine phosphorylation of ACK-2 was markedly increased (Fig.
1A). On polylysine-coated
plates, the cells were firmly attached within 5 min and maintained a
round shape for at least 30 min before they began to flatten and spread
(not shown). This suggests that the activation of ACK-2 by cell
adhesion does not require cell spreading or focal adhesion complex
assembly.

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Fig. 1.
ACK-2 is activated by cell adhesion.
A, COS-7 cells were transfected with either pcDNA3
(vector) or pcDNA3 HA-ACK-2 (4 µg/60-mm plate) for 48 h and
serum-starved overnight. The cells were trypsinized at 37 °C for 10 min, washed in PBS plus trypsin-inhibitor (20 µg/ml) once, and then
with PBS, DMEM, and finally resuspended in DMEM. The cell aliquots then
were incubated either in an Eppendorf tube (lane 3) or
replated onto fibronectin- (lanes 2, 5, and 7) or
polylysine (lanes 1, 4, and 6)-coated plates at
37 °C in 5% CO2 for the indicated times. Both the
resuspended and adhered cells were collected and lysed. The HA-tagged
ACK-2 was immunoblotted with either anti-phosphotyrosine antibody
(PY20) (top panel) or anti-HA antibody (12C5) (bottom
panel). B, the experimental procedure was basically the
same as above except the control for cell adhesion was BSA-blocked
plates. Cytochalasin D (2.5 µg/ml) or ethanol (solvent for
cytochalasin D) was added during cell adhesion. Top panel,
blotted with anti-phosphotyrosine (PY); bottom
panel, blotted with anti-HA. CytoD, cytochalasin D;
PL, polylysine; FN, fibronectin. C,
the experimental procedure was basically the same as above except the
cells were transfected with pcDNA3 Myc-tagged ACK-2 instead of
pcDNA3 HA-tagged ACK-2 and pretreated with cycloheximide (20 µg/ml) at 37 °C for 30 min before plating, as well as during
plating. The cells were allowed to plate onto precoated plates at
37 °C for 30 min. CH, cycloheximide. D, the
Tet-off-inducible Myc-tagged ACK-2 cell line was used in this
experiment. NIH3T3 cells that were stably transfected with pTet
Myc-ACK-2/ptTAK were cultured in DMEM plus 10% calf serum and 1 µg/ml tetracycline (non-induced condition) to 90% confluence and
subsequently cultured in DMEM without serum and tetracycline (induced
condition) for 20 h. The cells were trypsinized and collected as
described above and treated with 20 µg/ml cycloheximide at 37 °C
for 30 min before incubation with latex beads. Cells
(~106) were mixed with precoated latex beads (~2 × 108) in 400 µl of DMEM at 37 °C for 30 min with
gentle shaking. The cells were lysed with RIPA buffer. Tyrosine
phosphorylation of ACK-2 and the amount of Myc-tagged ACK-2 were
detected with horseradish peroxidase-conjugated anti-phosphotyrosine
antibody (PY20, Oncogene) (top panel) and
anti-Myc antibody (bottom panel), respectively.
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To confirm further that activation of ACK-2 by cell adhesion does not
require cell spreading, we treated the cells with cytochalasin D, a
reagent that disrupts F-actin. This caused the cells to round-up and
lose their ability to spread. However, cytochalasin D did not affect
cell attachment onto either polylysine- or fibronectin-coated plates,
suggesting that such treatment did not disrupt the interaction of
integrins with fibronectin. As shown in Fig. 1B, treatment with cytochalasin D also did not affect the activation of ACK-2 upon
the attachment of cells to fibronectin-coated plates. Thus, ACK-2
activation by cell adhesion is only correlated with cell attachment and
not with cell spreading. In addition, the activation of ACK-2 by
attachment onto polylysine-coated plates is not due to the new
synthesis of extracellular matrix proteins, because pretreatment of
cells with cycloheximide, an inhibitor of protein synthesis, did not
block ACK-2 activity (Fig. 1C).
In order to examine the effects of cell adhesion on the activation of
ACK-2 further, we performed cell adhesion experiments with
extracellular matrix molecule-coated latex beads (diameter 6 µm) as
described by Miyamoto et al. (41). The data presented in
Fig. 1D show that like the case when cells are plated onto polylysine or fibronectin, the adherence of cells to either polylysine- or fibronectin-coated beads strongly activates ACK-2.
The Activation of ACK-2 Tyrosine Phosphorylation by Cell Adhesion
Is Cdc42-dependent--
To determine whether the
activation of ACK-2 by cell adhesion requires Cdc42, we co-transfected
ACK-2 with either the wild type, constitutively active, or dominant
negative forms of Cdc42 in COS-7 cells. After 48 h, the cells were
plated onto BSA- or fibronectin-coated plates, and the tyrosine
phosphorylation of ACK-2 was detected by immunoblotting with
anti-phosphotyrosine antibody. When vector (pcDNA3) was
co-transfected with ACK-2, the tyrosine phosphorylation of ACK-2 was
enhanced by cell adhesion onto fibronectin-coated plates, compared with
the phosphorylation detected in suspended cells (i.e.
BSA-coated plates) (Fig. 2, lanes 3 and 4). When ACK-2 was
co-transfected with the GTPase-defective Cdc42(Q61L) mutant, the
tyrosine phosphorylation of ACK-2 showed a slight enhancement relative
to control cells plated on fibronectin (Fig. 2, compare lanes
4 and 6). Even in suspended cells, the tyrosine
phosphorylation of ACK-2 was enhanced upon the expression of
Cdc42(Q61L) (compare lanes 3 and 5). When
Cdc42(T17N), a dominant negative mutant, was co-transfected with ACK-2,
the tyrosine phosphorylation of ACK-2 was strongly inhibited (Fig. 2,
lanes 7 and 8). These data indicate that the
stimulation of ACK-2 activity upon cell adhesion was dependent on
Cdc42.

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Fig. 2.
Activation of ACK-2 by cell adhesion is
Cdc42-dependent. The experimental procedures were the
same as in Fig. 1. ACK-2 was Myc-tagged, and the amounts of either
pcDNA3 encoding Myc-ACK-2, pcDNA3 encoding HA-Cdc42(T17N), or
pcDNA3 encoding HA-Cdc42(Q61L) for transfection were 2 µg per
60-mm plate. Top panel, anti-phosphotyrosine (PY20);
bottom panel, anti-Myc antibody. FN,
fibronectin.
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The Activation of ACK-2 by Cell Adhesion Is Distinct from the
Activation of FAK--
It is well known that FAK, a non-receptor
tyrosine kinase, is specifically activated upon cell adherence to
fibronectin-coated plates (42-44). Thus, we compared the cell
adhesion-dependent activation of ACK-2 with that of FAK. As
expected, when cells were directly lysed from culture plates (not
detached), both ACK-2 and FAK were highly autophosphorylated (Fig.
3, lane 1), whereas when cells were detached and resuspended in BSA-blocked plates for up to 60 min,
both ACK-2 and FAK were dephosphorylated (Fig. 3, lane 2).
When replated onto polylysine or fibronectin-coated plates for 5 min,
the cells attached to polylysine-coated plates but not to the
fibronectin-coated plates. Under these conditions, the tyrosine
phosphorylation of ACK-2 was stimulated upon plating on polylysine
(Fig. 3, bottom panel, lane 3). Neither ACK-2 nor FAK was
activated on fibronectin-coated plates because there was no cell
attachment at this time point (Fig. 3, lane 4). After replating for 20 min, 90% of the cells adhered to both polylysine- and
fibronectin-coated plates. However, only cells attached to fibronectin
were able to spread. The cells on polylysine remained round even after
having been replated for 60 min (data not shown). ACK-2 was activated
on both polylysine- and fibronectin-coated plates after replating for
20 min (Fig. 3, lanes 5-8, bottom panel), whereas FAK was
only activated on fibronectin-coated plates (Fig. 3, lanes 6 and 8). However, when the cells were treated with
cytochalasin D during their replating onto fibronectin-coated plates,
the cells were not able to spread but rather adhered to the plates with a rounded morphology, and the autophosphorylation of FAK was
significantly decreased (data not shown). Thus, whereas FAK activation
upon cell adhesion requires cell spreading or actin-cytoskeletal
organization, ACK-2 activation appears to only require cell attachment
to a substratum.

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Fig. 3.
Activation of ACK-2 by cell adhesion is
distinct from FAK activation. ACK-2 was activated by cell adhesion
on both polylysine and fibronectin, and FAK was activated by cell
adhesion on fibronectin. NIH3T3 cells were stably transfected with
pLTRHA-ACK-2 and cultured in DMEM + 10% calf serum + G418 (500 µg/ml). The cell adhesion experiments were performed as described
under "Experimental Procedures." FAK was immunoprecipitated from
cell lysates and blotted with anti-phosphotyrosine (PY)
antibody. The phosphorylation of ACK-2 was detected from the cell
lysates. Top panel, tyrosine phosphorylation of FAK upon
cell adhesion on either polylysine and fibronectin; bottom
panel, tyrosine phosphorylation of ACK-2 upon cell adhesion on
either polylysine and fibronectin. Not detached refers to
cells that were directly lysed from culture plates. PL,
polylysine; FN, fibronectin. Lane 9 represents a
vector control (cells were not detached).
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Previous studies have shown that FAK activation results in a
stimulation of Erk activity (45). Therefore, we examined whether the
overexpression of ACK-2 could also stimulate Erk activity, by using an
anti-Erk antibody and determining whether an
activation-dependent change in the electrophoretic mobility
of the Erks occurred. As shown in Fig.
4A, consistent with previous
studies, adhesion of cells on fibronectin resulted in the stimulation
of Erk activity (compare lanes 2, 5, and 7 with
lane 3). Adhesion of cells on polylysine also stimulated Erk
activity (compare lanes 1, 4 and 6 with
lane 3) but to a lesser extent compared with fibronectin. However, we did not observe a significant effect on Erk activity upon
expression of ACK-2 (compare lanes 1 and 2 with
lanes 6 and 7). This suggests that ACK-2 does not
input into the Ras/Raf/Erk pathway.

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Fig. 4.
Effects of ACK-2 on mitogen-activated protein
kinase activities. A, ACK-2 activation is not
correlated with Erk activation during cell adhesion. The experimental
procedures were exactly as described for Fig. 1. The Erks were
immunoblotted with anti-Erk antibody (Santa Cruz Biotechnology). The
electrophoretic mobility shifts corresponding to Erk activation are
indicated by arrows. B, ACK-2 activates JNK. The
indicated amounts of pcDNA3 HA-ACK-2 were co-transfected with
flag-tagged JNK (0.5 µg). The JNK assay was performed following
immunoprecipitation with the anti-flag antibody (anti-M5, Kodak). The
proteins were fractionated by SDS-PAGE and subsequently transferred
onto Immobilon membranes. Top panel, autoradiography;
bottom panel, immunoblot with anti-flag antibody.
C, pcDNA3 HA-Cdc42(T17N) (2 µg) was co-transfected
along with pcDNA3-HA-ACK-2 and flag-tagged JNK1 (0.5 µg). The JNK
kinase assay, protein fractionation, and blotting were the same as
above. JNK activity was quantitated from the radioactivity with a
PhosphorImager.
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To determine whether ACK-2 influences the activity of the
stress-responsive mitogen-activated protein kinase, the c-Jun kinase (JNK1), we co-transfected the cDNAs encoding ACK-2 and flag-tagged JNK into COS-7 cells and assayed JNK activity after immunoprecipitation with an anti-flag antibody. As shown in Fig. 4B, the
expression of ACK-2 was accompanied by a significant activation of JNK
activity. In order to determine whether Cdc42 was required for this
activation event, we co-transfected the cDNA encoding a
dominant-negative mutant of Cdc42 (Cdc42(T17N)) with the cDNAs
encoding ACK-2 and flag-tagged JNK. As shown in Fig. 4C, the
expression of dominant-negative Cdc42(T17N) inhibited the activation of
JNK by ACK-2.
Activation of ACK-2 by Cell Adhesion Is Mediated by Integrin
1--
We next examined the molecular basis by which
cell adhesion activates ACK-2. The fact that the adhesion of cells onto
fibronectin activates ACK-2 suggested that integrins may be involved in
the activation process. To examine this possibility, we determined whether treatment with an antibody against integrin
1 or
RGD peptides, which block the interaction of fibronectin with
integrins, affected the tyrosine phosphorylation of ACK-2 when plating
the cells on either polylysine or fibronectin (Fig.
5). When cells were treated with the
anti-integrin
1 antibody or the RGD peptides, about
80-90% of the cells were no longer able to adhere onto
fibronectin-coated plates, indicating that the ability of the cells to
adhere to fibronectin was integrin
1-dependent (data not shown). However, treatment with either the anti-integrin
1 antibody or
the RGD peptides did not significantly affect attachment of the cells onto polylysine-coated plates (data not shown), suggesting that the
cell adhesion to polylysine can occur via integrin-independent events.

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Fig. 5.
Inhibitory anti-integrin
1 antibody and RGD peptide block
activation of ACK-2 by cell adhesion on both polylysine and
fibronectin. The cell adhesion procedures were the same as
described for Fig. 1B except that the antibody (1:50
dilution) or RDG peptide (1 mM) was preincubated with cells
in an Eppendorf tube at 37 °C and 5% CO2 for 30 min
before replating. A and B, effects of
anti-integrin 1 on tyrosine phosphorylation of ACK-2
upon cell adhesion on fibronectin and polylysine, respectively;
C and D, effects of RGD peptide on tyrosine
phosphorylation of ACK-2 upon cell adhesion on fibronectin and
polylysine, respectively. ESP represents the GRGESP control
peptide; DNP represents the GRGDNP peptide. In all figures,
the top panel is blotted with anti-phosphotyrosine
(PY); the bottom panel is blotted with anti-Myc
or anti-HA.
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As shown in Fig. 5A, the anti-integrin
1
antibody completely reversed fibronectin-stimulated tyrosine
phosphorylation of ACK-2, and at least partially inhibited the
polylysine-stimulated tyrosine phosphorylation (Fig. 5B). A
similar set of experiments were performed using the RGD peptides (Fig.
5, C and D). We used GRGESP as a control peptide
(labeled ESP in the figures) and GRGDNP as an inhibitory
peptide (labeled DNP). We found that the control peptide,
GRGESP, affected neither cell adhesion nor the tyrosine phosphorylation
of ACK-2 upon cell adhesion (Fig. 5, C and D), whereas GRGDNP inhibited the activation of ACK-2 by cell adhesion onto
either fibronectin- or polylysine-coated plates (Fig. 5C, 4th and 5th lanes; Fig. 5D, 4th lane). These
data strongly suggest that ACK-2 is activated upon cell adhesion onto a
substratum via integrin
1. Although cell attachment to
polylysine was not dependent on integrins, the ability of polylysine to
stimulate ACK-2 activity is integrin-dependent. This
indicates some type of functional coupling between cell-surface
receptors that bind polylysine and integrin
1 which can
in turn influence ACK-2 activity.
ACK-2 Is Constitutively Associated with the Integrin
1 Complex and the Association Is Independent of ACK-2
Tyrosine Kinase Activity--
The implication that integrin
1 mediates the activation of ACK-2 then raises the
question of whether ACK-2 directly associates with the integrin
complex. To address this question, we transfected the cDNA encoding
Myc-tagged ACK-2 or a kinase-defective mutant of ACK-2 (ACK-2(K158R))
into COS-7 cells and then replated the cells onto BSA- or
fibronectin-coated plates. We then immunoprecipitated endogenous
integrin
1 with an anti-integrin
1
antibody and Western-blotted the immunoprecipitated complex with an
anti-Myc antibody to detect integrin-associated Myc-tagged ACK-2.
Unexpectedly, we found that a similar amount of Myc-tagged ACK-2 was
co-immunoprecipitated with integrin
1 (Fig.
6A, right panel, 1st and
2nd lanes) from cells in suspension (BSA-coated plates) and
when cells are attached to fibronectin, suggesting that ACK-2 was
constitutively associated with the integrin
1. Control
experiments with non-immune IgG or using anti-FAK antibody did not
immunoprecipitate ACK-2 (data not shown). The association of ACK-2 with
the integrin
1 complex was totally independent of its
kinase activity or tyrosine phosphorylation (Fig. 6A, left
panel, all lanes, and right panel, 3rd and 4th lanes).

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Fig. 6.
ACK-2 is directly associated with the
integrin 1 complex.
A, co-immunoprecipitation of ACK-2 with integrin
1. COS-7 cells transfected with Myc-tagged
ACK-2 or Myc-tagged ACK-2(K158R), a kinase-defective
mutant, were lysed with RIPA buffer after replating onto BSA or
fibronectin-coated plates. Immunoprecipitation of integrin
1 was performed by adding anti-integrin 1
antibody (10 µl) and 10 µl of protein G beads into 500-1000 µg
of lysate protein and incubated at 4 °C for 3 h with rocking.
The immunoprecipitated sample was divided into 2 parts and blotted with
either anti-phosphotyrosine (PY) (left panel) or
with anti-Myc antibody (right panel). B,
self-association of ACK-2 is inhibited by cell adhesion on fibronectin
or by co-transfection with Cdc42. COS-7 cells were transfected with
pcDNA3 encoding Myc-ACK-2 or pcDNA3 encoding Myc-ACK-2 plus
pcDNA3 encoding HA-Cdc42 and lysed with lysis buffer after
replating onto BSA or fibronectin-coated plates. In the left
panel, immobilized GST-ACK-2 SH3/CRIB domain (20 µg/sample),
which contains both the SH3 and Cdc42-binding motifs, was incubated
with cell lysates, and the precipitated proteins were separated by
SDS-PAGE and blotted with anti-Myc (upper part, >58 kDa) or
anti-HA (lower part, < 58 kDa). In the right
panel, to determine the expression levels of ACK-2 or Cdc42, the
cell lysates were blotted with anti-Myc (upper part, >58
kDa) or anti-HA (lower part, <58 kDa).
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These findings raise the question of how ACK-2 transduces signals upon
cell adherence to a substratum if it is constitutively associated with
integrin
1. The data presented in Fig. 6B
begin to point toward a possible explanation. In these experiments, a
GST fusion protein encoding the SH3 and CRIB domains of ACK-2 was
immobilized on glutathione beads and incubated with lysates from COS-7
cells expressing Myc-tagged full-length ACK-2 or Myc-tagged ACK-2 and
HA-tagged Cdc42. These cells had been replated onto BSA- or
fibronectin-coated plates. As shown in Fig. 6B (left
panel, 1st and 2nd lanes), the GST-SH3/CRIB domain construct bound
more effectively to full-length ACK-2 in lysates from suspended cells (BSA) compared with lysates from adherent cells (fibronectin). However,
the co-transfection of Cdc42 with ACK-2 totally blocked the binding of
the GST-SH3/CRIB domain construct to full-length ACK-2 (Fig. 6B,
left panel, 3rd and 4th lanes). The right
panel of Fig. 6B shows that equal amounts of ACK-2 and
Cdc42 were expressed in each lysate sample. These data suggest that the
interaction between the SH3 domain and a proline-rich sequence of ACK-2
is tightly regulated by Cdc42. One possibility is that in the basal state, ACK-2 undergoes an intramolecular interaction between its SH3
domain and a proline-rich sequence which prevents the binding of
cellular targets and/or phospho-substrates. However, upon the addition
of an excess of the GST-SH3/CRIB domain, one of the proline-rich sequences of ACK-2 may be able to undergo an intermolecular interaction with the GST-SH3/CRIB domain fusion protein, due to an equilibrium between a "closed" state where full-length ACK-2 is engaged in an
intramolecular interaction and an "open" state where the
proline-rich sequences are accessible to intermolecular interactions.
The binding of activated Cdc42 to the CRIB motif, which lies between
the SH3 and proline-rich sequences of ACK-2, may then prevent the
intramolecular interaction between these domains and thereby allow the
binding of cellular target proteins. This in turn would reduce the
amount of cellular ACK-2 that is available to bind the GST-SH3/CRIB
domain construct. We would further propose that upon cell adhesion and the formation of integrin clusters (Cdc42-activated), ACK-2 molecules are brought into sufficient proximity to one another to undergo trans-phosphorylation, thus accounting for the marked increase in the
tyrosine phosphorylation of ACK-2 that occurs under these conditions.
 |
DISCUSSION |
It is well known that Rho-related small GTP-binding proteins
regulate cytoskeletal organization and cell morphology. Given that cell
adhesion induces marked changes in the actin cytoskeleton, it seems
likely that the Rho-related proteins will also play roles in bridging
adhesion-dependent signaling with effects on the
cytoskeletal architecture. Along these lines, Cdc42 and Rac have
recently been shown to mediate integrin
1 signaling in
cell migration (39), suggesting that cell adhesion or integrins induce
the activation of Cdc42 and Rac (see also Ref. 46). Tiam-1, a
guanine-nucleotide exchange factor for Rac, is involved in cell
invasion (47), and Rac has been shown to participate in cadherin
signaling in epithelial cells and to inhibit Ras-induced cell invasion
(48). An obviously important question will be to identify the target molecules for Cdc42 and Rac that mediate the effects of cell adhesion. Based on our initial studies with the non-receptor tyrosine kinase ACK-2 (15), we felt that it was an attractive candidate for such a
role. Specifically, we had earlier shown that ACK-2 was activated upon
cell adhesion (15). Here we show that ACK-2 can be activated by cell
adhesion via the
1 integrin in a
Cdc42-dependent manner and that ACK-2 appears to associate
with an integrin complex.
It is interesting to note that the activation of ACK-2 by cell adhesion
clearly differs from that of FAK. Activation of ACK-2 by cell adhesion
does not require cell spreading nor an intact F-actin structure,
whereas the activation of FAK requires both. There are two possible
explanations for this difference. 1) ACK-2 and FAK participate in
distinct integrin signaling pathways, or 2) ACK-2 is upstream from FAK
during cell adhesion signaling. However, the latter possibility seems
unlikely given that we have not observed that overexpression of ACK-2
enhances FAK tyrosine phosphorylation.
A particularly interesting distinction between ACK-2 and FAK concerns
the ability of polylysine to activate ACK-2. Based on the inhibitory
effects of anti-integrin
1 and GRD peptides on polylysine-induced activation of ACK-2, at least part of the activation of ACK-2 by cell adhesion onto polylysine-coated plates appears to be
mediated through
1 integrin. However, when cells adhere onto polylysine-coated plates, they can only attach to the plates but
are not able to spread, indicating that cells plated on polylysine can
not form stress fibers (45). The inability of the cells to spread when
plated on polylysine probably explains why FAK is not activated under
these conditions. It has been reported that cells adhered onto
polylysine form filopodia, suggesting that Cdc42 may be activated upon
cell adhesion on polylysine (49) and thus providing a link to ACK-2
activation. We have also observed that some protrusions appear from the
bottom of cells that are attached to polylysine-coated plates (data not
shown). These protrusions have been described as point contacts that
are distinct from focal contacts (49, 50). It has been further proposed
that in fibroblasts,
1
1 and
5
1 integrin heterodimers first accumulate in point contacts followed by their redistribution into focal contacts (in
astrocytes, the accumulation of
1
1
heterodimers in point contacts was shown to occur when cells were
plated on either polylysine, fibronectin, or laminin (49)). Thus, cell
attachment on either polylysine or fibronectin, leading to an
accumulation of integrins in point contacts, may represent an early
signal for the activation of Cdc42 and then ACK-2. Met, a receptor
tyrosine kinase that is a proto-oncogene and involved in cell invasion
and tumor cell metastasis, shows a similar activation behavior as ACK-2
upon cell adhesion, i.e. it is activated upon plating cells
on polylysine (51). However, thus far we have not found any signaling
connection between ACK-2 and Met.
Overall, the findings reported here now provide a possible molecular
basis for the signaling connections between integrins/cell adhesion and
Cdc42. The mechanisms underlying the apparent activation of Cdc42 by
cell adhesion, which lead to the recruitment and/or activation of
ACK-2, remain to be delineated. However, it appears that upon
activation, Cdc42 may reverse an intramolecular interaction within
ACK-2 which then makes the kinase accessible to interact with other
binding partners or possibly substrates. At present, we know relatively
little about the downstream signaling pathways that are engaged
following ACK-2 activation, although the Raf-Mek-Erk pathway does not
appear to be involved. Although we have found that overexpression of
ACK-2 stimulates JNK activity, it is difficult to assess the importance
of this activation in vivo given that a number of tyrosine
kinases including Src, Pyk2, Abl, and Btk have also been shown to
stimulate JNK activity (52-55). Moreover, as yet, we have not been
able to show a significant activation of JNK activity upon cell
attachment. Thus, either one or more of the signaling participants that
may connect cell adhesion to JNK activation was limiting in our
experiments, or the observation that overexpression of ACK-2 resulted
in JNK activation reflected an aberrant signaling pathway triggered by
the higher than normal levels of ACK-2. We suspect that ACK-2 may play
some specialized roles in cell differentiation, since it is highly
enriched in brain and skeletal muscle (15). One interesting possibility is that ACK-2 may be activated by extracellular matrix proteins that
guide neurite outgrowth of neuronal cells or the differentiation of
muscle cells. Future efforts will be directed toward determining the
in vivo function of ACK-2 in specific tissues and
identifying its downstream targets.