* Laboratory of Cell Adhesion and Signaling, Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer
Center, New York 10021; and Laboratory of Molecular Oncology, The Rockefeller University, New York 10021
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Abstract |
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The extracellular matrix exerts a stringent control on the proliferation of normal cells, suggesting the existence of a mitogenic signaling pathway activated by integrins, but not significantly by growth factor receptors. Herein, we provide evidence that integrins cause a significant and protracted activation of Jun NH2-terminal kinase (JNK), while several growth factors cause more modest or no activation of this enzyme. Integrin-mediated stimulation of JNK required the association of focal adhesion kinase (FAK) with a Src kinase and p130CAS, the phosphorylation of p130CAS, and subsequently, the recruitment of Crk. Ras and PI-3K were not required. FAK-JNK signaling was necessary for proper progression through the G1 phase of the cell cycle. These findings establish a role for FAK in both the activation of JNK and the control of the cell cycle, and identify a physiological stimulus for JNK signaling that is consistent with the role of Jun in both proliferation and transformation.
Key words: integrins; focal adhesion kinase; Jun NH2-terminal kinase; Jun; cell cycle ![]() |
Introduction |
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NORMAL cells require adhesion to extracellular matrix components to proliferate in vitro, suggesting
that integrins activate signaling pathways that are
necessary for cell cycle progression (reviewed in Giancotti,
1997). In principle, integrins could cooperate with growth
factor receptors to produce a synergistic stimulation of
one or more mitogenic signaling pathways. Indeed, the results of several studies support this model (reviewed in
Ruoslahti and Reed, 1994
; Clark and Brugge, 1995
; Parsons, 1996
; Clark and Hynes, 1997
; Schwartz, 1997
; Yamada and Geiger, 1997
; Howe et al., 1998
; Schlaepfer and
Hunter, 1998
). However, such a mechanism does not readily explain the strict adhesion requirement for growth
displayed by normal cells. In addition, or instead, integrins
could activate a signaling pathway that is not significantly
activated by growth factors, but is necessary for cell proliferation. The identification of such a pathway would provide a more complete understanding of the anchorage-dependent growth than is currently available.
Integrins activate common, as well as subgroup-specific,
signaling pathways. A subset of integrins, which includes
1
1,
5
1,
v
3, and
6
4, is coupled to the Ras-extracellular signal-regulated kinase (ERK)1 signaling pathway
by the adaptor protein Shc (Mainiero et al., 1995
; Wary et al.,
1996
). Shc binds directly to the uniquely large cytoplasmic
domain of
4 when this integrin undergoes tyrosine phosphorylation (Mainiero et al., 1995
; 1997
). In contrast, the recruitment of Shc by
1 and
v integrins is indirect, requiring the interaction of the integrin
subunit with the
membrane adaptor caveolin-1 and associated tyrosine kinase, Fyn (Wary et al., 1998
). Biochemical and genetic evidence suggest that integrins recruit Shc independently of
focal adhesion kinase (FAK), and that this event is necessary and sufficient to activate the Ras-ERK pathway. In
primary cells, inhibition of integrin-mediated Shc signaling results in cell cycle arrest, despite the presence of growth
factors, suggesting that a combined stimulation of Ras by
Shc-linked integrins and growth factor receptors is required for progression through the G1 phase of the cell cycle (Wary et al., 1996
, 1998
).
Certain integrins appear to associate preferentially with
specific growth factor receptors and contribute to their activation (Miyamoto et al., 1996; Schneller et al., 1997
;
Moro et al., 1998
; Soldi et al., 1999
). For example,
v
3
combines with the platelet-derived growth factor (PDGF)
receptor. Hence, fibroblasts show enhanced proliferation
in response to PDGF when attaching to the
v
3 ligand vitronectin than they do on the
1 integrin ligand collagen
(Schneller et al., 1997
). The selective association of integrins with growth factor receptors represents a second potential mechanism of integrin-specific growth control.
Whereas the aforementioned pathways are activated
only by certain integrins, the tyrosine kinase FAK is activated by most integrins (Parsons, 1996). Activated FAK
undergoes autophosphorylation at Tyr 397 and thereby
binds to the SH2 domain of the Src-family kinase Src or
Fyn (Schaller et al., 1994
; Schlaepfer et al., 1994
). The Src-family kinase then phosphorylates a number of FAK-associated proteins, including p130CAS and paxillin, which contain multiple docking sites for the adaptor proteins Crk
and Nck (Schaller and Parsons, 1995
; Richardson and Parsons, 1996
; Vuori et al., 1996
; Schlaepfer et al., 1997
). In addition, Src phosphorylates FAK at a tyrosine residue
able to recruit Grb2 (Schlaepfer et al., 1994
). It is possible
that FAK contributes to the activation of the Ras-ERK
cascade by these (Schlaepfer et al., 1997
) and potentially
other mechanisms (Chen et al., 1996
; King et al., 1997
; Lin
et al., 1997
; Renshaw et al., 1997
). Although previous studies have provided direct evidence for a role of FAK in cell
migration (Ilic et al., 1995
; Fincham and Frame, 1998
; Cary
et al., 1998
; Klemke et al., 1998
) and protection from apoptotic cell death (Frisch et al., 1996b
; Ilic et al., 1998
), it is
unclear whether FAK also regulates cell proliferation, and
if so, by what mechanism.
Integrin-mediated adhesion activates not only ERK, but
also Jun NH2-terminal kinase (JNK; Miyamoto et al., 1995;
Mainiero et al., 1997
; MacKenna et al., 1998
). JNK is the
final element of a mitogen-activated protein kinase (MAPK)
cascade known to be activated by stress stimuli, such as
UV radiation, hyperosmolar conditions, and inflammatory
cytokines (Ip and Davis, 1998
). Upon activation, JNK enters the nucleus, and phosphorylates and activates the
transcription factors c-Jun and activating transcription factor 2 (ATF2), thereby regulating AP-1-dependent transcription (Karin et al., 1997
). Because there is evidence
that c-Jun is required for cell proliferation (Riabowol et
al., 1992
; Johnson et al., 1993
), we sought to examine the
mechanism by which integrins activate JNK and test the
hypothesis that activation of this signaling pathway contributes to the control of cell cycle progression.
Our results indicate that integrins cause a significant and protracted activation of JNK, while growth factors appear to be unable to do so. By using various dominant-negative signaling molecules, we provide evidence that the activation of JNK by integrins is mediated by FAK and is necessary for cell cycle progression.
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Materials and Methods |
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Antibodies and Extracellular Matrix Proteins
The mAb M2 to FLAG tag was purchased from Eastman-Kodak and the
anti-CD2 mAb RPA-2.10 from PharMingen. The anti-H-Ras mAb R02120 (clone 18) and anti-p130CAS mAb P27820 (clone 21) were from
Transduction Laboratories. The origin and specificity of the affinity-purified rabbit antibodies to ERK2, phospho-ERK, Src, and GST and of the
anti-5-bromodeoxyuridine (anti-BrdU) mAb were described previously
(Wary et al., 1996; 1998
). The mAb 3C2 reacting with the gag portion of
v-Crk was also described previously (Potts et al., 1987
). Human fibronectin was from GIBCO BRL and poly-L-lysine from Sigma Chemical Co.
Cell Lines, Constructs, and Transfections
293 human embryonic kidney cells were cultured in DME 10% FCS on
gelatin-coated plates. NIH-3T3 mouse fibroblasts were cultured in DME
10% calf serum (CS). Fibroblasts from Src/
and Fyn
/
embryos were
obtained from Philippe Soriano (Fred Hutchinson Cancer Research Center, Seattle, WA) and cultured in DME 10% CS. Human umbilical vein
endothelial cells (HUVECs) were purchased from Clonetics and cultured
on gelatin-coated dishes in human endothelial serum-free medium (SFM;
GIBCO BRL) supplemented with 20% FCS (GIBCO BRL), 10 ng/ml
EGF, 20 ng/ml bFGF, and 1 µg/ml heparin (all from Intergen).
The reporter plasmid pcoll TRE-tk-Luc, in which the expression of luciferase is driven by a single copy of the collagen gene 12-O-tetradecanoylphorbol-13-aretate (TPA)-responsive element (TRE) linked to the
Hepes simplex virus thymidine kinase minimal promoter, was described
previously (Galien et al., 1994). Vectors encoding the FLAG-tagged version of JNK 1, glutatione S-transferase (GST)-Jun, dominant-negative
Ras (N17), and HA-tagged
-galactosidase were described previously
(Mainiero et al., 1997
). The CMV promotor-based pCDM8 vectors encoding CD2-FAK (wild-type), CD2-FAK K454R (kinase dead), and CD2-FAK Y397F were described previously (Chan et al., 1994
). A kinase dead
version of chicken c-Src was obtained from Sara Courtneidge (EMBL,
Heidelberg, Germany) and subcloned in the cytomegalovirus (CMV) promotor-based vector pRK5. The pEBG vectors expressing GST-tagged
MKK4 (wild-type) and MKK4 K129R (kinase dead) from the human
elongation factor 1-
promoter were described previously (Su et al.,
1997
). The Moloney Leukemia Virus (MLV)-LTR based pMEXneo vectors encoding v-Crk (wild-type), v-Crk R273N (SH2 mutant), and v-Crk
D386DRHAD (SH3 insertional mutant) were described previously (Altun-Gultekin et al., 1998
). The pEBG vectors encoding GST-tagged rat
p130CAS (short form) and its substrate region deleted form (SD,
213-
514) were also described (Mayer et al., 1995
). The TAM-67 transactivation domain mutant form of c-Jun (Jun
3-122) was expressed from
pCMV and previously characterized (Brown et al., 1993
). The dominant-negative version of paxillin used in this study carries three phenylalanine
permutations at tyrosine 31, 118, and 187 and is unable to bind to Crk. The
MLV-LTR-based expression vector p
raf-22w encodes an activated version of c-Raf 1 lacking an NH2-terminal segment of 305 amino acids (Stanton et al., 1989
). The vector encoding activated Ras, pDCR-Ha-ras (G12V), was kindly provided by John Westwick (Signal Pharmaceuticals).
NIH-3T3 cells were transiently transfected with Lipofectamine according to the manufacturer's instructions (GIBCO BRL). 293 cells were plated at 6 × 106 per 15-cm diam dish for 8 h and then transfected overnight with various amounts of plasmid by the calcium phosphate method. All transfections were normalized to the same total amount of DNA with empty vector. Cells were allowed to recover for 12 h before growth factor starvation.
Biochemical Methods
To monitor the activation of JNK and ERK during G1, HUVECs were synchronized in G0 by a 24 h incubation in human endothelial SFM containing 0.2% FCS. They were then detached with 0.02% EDTA, collected in SFM containing 0.2% heat-inactivated BSA, washed in the same medium, and kept in suspension at a density of 106/ml for 15 min at room temperature to recover. Aliquots consisting of 1.5 × 107 cells were plated on 15-cm diam dishes coated with 20 µg/ml fibronectin and postcoated with 0.2% heat-inactivated BSA in SFM supplemented with ITS+1 (Sigma Chemical Co.), EGF (10 ng/ml), bFGF (20 ng/ml), and heparin (1 µg/ml) for the indicated times. Cells from an identical aliquot were pelleted and lysed in suspension as a control. Before biochemical analysis, NIH-3T3 cells were serum starved for 18 h and 293 cells for 24 h in DME containing 0.2% CS or FCS, respectively. After detachment with 0.02% EDTA, cells were collected in DME containing 0.2% heat-inactivated BSA, washed in the same medium, and kept in suspension at a density of 106/ml for 15 min at room temperature to recover. Aliquots consisting of 1.5 × 107 cells were plated on 15-cm diam dishes, coated with 20 µg/ml fibronectin and postcoated with 0.2% heat-inactivated BSA for the indicated times. Cells from an identical aliquot were pelleted and lysed in suspension as a control. NIH-3T3 cells were treated with growth factors as indicated.
To analyze the activation of JNK, cells were extracted for 30 min on
ice with 0.5 ml/dish of modified Triton lysis buffer (25 mM Hepes, pH 7.5, 300 mM NaCl, 0.1% Triton X-100, 0.2 mM EDTA, 20 mM -glycerophosphate, 1.5 mM MgCl2, and 0.5 mM DTT) containing phosphatase and protease inhibitors. Aliquots containing 0.5 mg of total proteins were brought
to 0.8 ml with modified Triton lysis buffer and diluted to 1.2 ml with HBB
buffer (20 mM Hepes, pH 7.7, 50 mM NaCl, 0.05% Triton X-100, 0.1 mM
EDTA, 20 mM
-glycerophosphate, 2.5 mM MgCl2, and 10 mM DTT)
supplemented with phosphatase and protease inhibitors. Endogenous
JNK was precipitated with 5 µg of GST-Jun fusion protein coupled to glutathione agarose beads (Hibi et al., 1993
). The beads were washed four
times in HBB buffer, twice in kinase buffer (20 mM Hepes pH 7.5, 20 mM
-glycerophosphate, 10 mM MgCl2, and 10 mM DTT), and incubated with
35 µl of kinase buffer containing 10 µCi of
[32P]ATP (ICN) and 20 µM
cold ATP. Recombinant FLAG-tagged JNK 1 was immunoprecipitated
with the anti-Flag mAb M2. The beads were washed as above and incubated with 35 µl of kinase buffer containing 5 µg of GST-Jun, 10 µCi of
[32P]ATP, and 20 µM cold ATP. After 30 min of incubation at 30°C, the
samples were boiled in sample buffer and separated by SDS-PAGE.
Immunoprecipitation and immunoblotting were performed essentially
as described previously (Mainiero et al., 1995). Secondary reagents for immunoblotting included peroxidase-conjugated protein A and affinity-purified rabbit anti-goat IgGs.
To measure transcription from the collagen promotor TRE, NIH-3T3
cells were transiently transfected with the reporter plasmid pcoll TRE-tk-Luc. After 24 h of growth factor starvation, the cells were detached, kept
in suspension for 30 min, and then solubilized or plated on dishes coated
with 20 µg/ml fibronectin for the indicated times in the absence or presence of 20 ng/ml PDGF. Luciferase activity in cell lysates was estimated as
described previously (Mainiero et al., 1997).
Analysis of Cell Cycle Progression
NIH-3T3 cells were transiently transfected with vector encoding -galactosidase in combination with various doses of the indicated constructs.
The cells were allowed to recover in complete medium, synchronized in
G0 by growth factor deprivation, and plated at low density on microtiter
wells coated with 10 µg/ml poly-L-lysine or 20 µg/ml fibronectin in defined
medium (DME supplemented with 6.25 µg/ml insulin, 6.25 µg/ml transferrin, 0.625 ng/ml selenous acid, 1.25 mg/ml BSA, and 5.35 µg/ml linoleic
acid) supplemented with 20 ng/ml PDGF and 10 µM BrdU. After 16 h, the
cells were fixed and stained with X-gal followed by anti-BrdU mAb and
AP-conjugated anti-mouse IgGs. The percentage of X-gal positive cells
that had incorporated BrdU was evaluated microscopically after light
counterstaining with hematoxylin.
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Results |
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Integrins Activate JNK and TRE-dependent Transcription
Preliminary experiments were conducted to examine if JNK was physiologically activated in primary cells progressing through the G1 phase of the cell cycle. HUVECs were synchronized in G0 by growth factor deprivation and detached to simulate the cell rounding physiologically occurring at mitosis. They were then plated on fibronectin in the presence of growth factors to allow entry into and progression through G1.
In vitro kinase assays with the NH2-terminal fragment of
c-Jun as a substrate showed that JNK is activated to a significant level during mid-G1, before the activation of the
D-type cyclin-dependent kinase CDK4 (Fig. 1). Peak JNK
activity was observed 4 h after entry into G1. In contrast,
the activation of ERK was biphasic with a first peak 10 to
20 min after entry into G1 and a second minor peak 8 h
later (Fig. 1). Since unstimulated cells are known to contain detectable levels of c-Jun, but not c-Fos (Karin, 1995), the rapid activation of ERK at the onset of G1 may serve
to induce serum response element (SRE)-dependent transcription of the c-Fos gene before JNK-mediated transcriptional activation of c-Jun. The AP-1 transcription factor Fos/Jun may then promote the expression of genes
necessary for G1 progression. The extent and timing of JNK activation during the cell cycle are thus consistent
with a potential role in the control of G1 progression.
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We next compared the ability of integrins and growth
factor receptors to activate JNK in NIH-3T3 fibroblasts.
Preliminary experiments showed that the activity of JNK
was significantly higher in growth factor deprived, stably
adherent cells than in cells that had been detached and immediately lysed (Fig. 2 A). In accordance with the previous observation that several growth factors cause a relatively modest activation of JNK (Kyriakis et al., 1994;
Minden et al., 1994
), exposure to mitogenic concentrations of PDGF, bFGF, and insulin increased the activity of JNK
only to a limited extent in growth factor starved, stably adherent NIH-3T3 cells. In contrast, PDGF, bFGF, and to a
minor extent, insulin, caused a significant activation of
ERK under the same conditions (Fig. 2 A).
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To further examine the relative contribution of integrins
and growth factor receptors to the activation of JNK,
NIH-3T3 cells were either plated on fibronectin in the absence of growth factors or treated with various doses of
PDGF while in suspension. As shown in Fig. 2 B, adhesion
to fibronectin induced a rapid, strong, and protracted activation of JNK. By contrast, JNK activity increased only
slowly and modestly over time in suspension, perhaps in
response to the activation of a stress pathway, as observed
by others (Frisch et al., 1996a; Khwaja and Downward,
1997
). The activation of JNK caused by integrin ligation
was comparable in intensity to that observed in cells
treated with 5 to 10 ng/ml TNF-
(Fig. 2 B), a known activator of JNK (Kyriakis et al., 1994
; Minden et al., 1994
).
Exposure to a wide range of PDGF concentrations caused little or no activation of JNK in suspended cells (Fig. 2
C). Whereas exposure to 1 µg/ml lysophosphatidic acid
(LPA), which is known to activate FAK (Chrzanowska-Wodnicka and Burridge, 1994
), amplified the activation of
JNK caused by integrin ligation by ~70% (data not
shown), treatment with PDGF did not exert this effect.
However, PDGF changed the time course of JNK activation in cells adhering to fibronectin. Specifically, while adhesion to fibronectin caused maximal stimulation of JNK
in ~10 min, simultaneous exposure to PDGF significantly
delayed the peak of activation of the kinase (Fig. 2 D).
This effect of PDGF may be related to its ability, when
used at mitogenic concentrations as were used here, to
transiently disrupt the cytoskeleton and thus delay integrin-mediated activation of FAK (Rankin and Rozengurt,
1994
). Taken together, these observations indicate that
JNK is activated by integrins, but only to a limited extent
by PDGF, bFGF, and insulin. Exposure to growth factors
may, however, contribute to sustain the activation of JNK
caused by integrin ligation.
Phosphorylation of c-Jun by JNK is required for transcriptional activation of the dimeric transcription factor
AP-1 and for the oncogenic cooperation between c-Jun
and Ha-Ras (Smeal et al., 1991). To examine if the activation of JNK caused by integrin ligation could contribute to
immediate early gene expression by promoting AP-1 dependent transcription from TRE, NIH-3T3 cells were
transiently transfected with a vector encoding the luciferase gene under the transcriptional control of a TRE
and plated on fibronectin in the presence or absence of
PDGF. As shown in Fig. 2, D and E, adhesion to fibronectin promoted TRE-dependent transcription with kinetics
that closely followed that of JNK activation. Simultaneous exposure to PDGF caused a delay in the transcriptional
response to fibronectin, as observed for the activation of
JNK. The induction of TRE-dependent transcription by
integrins required the transcriptional activity of c-Jun because it was suppressed by the TAM-67 dominant-negative form of the transcription factor (93.3% inhibition). Integrin-mediated activation of ERK and SRE-dependent
transcription of Fos (Wary et al., 1996
; Mainiero et al.,
1997
) can increase the levels of AP-1 available for phosphorylation by JNK. However, TRE-dependent transcription could not have occurred in the absence of JNK-mediated phosphorylation of c-Jun.
Integrin-mediated Activation of JNK Requires FAK, Src, p130CAS, Crk, and MKK
The mechanism by which integrins activate JNK was examined by introducing dominant-negative versions of various signaling components into human embryonic kidney
293 cells. Since preliminary experiments suggested that the
ability to activate JNK is shared by all integrins, irrespective of whether they are able or not to recruit Shc (data
not shown), we decided to examine the role of FAK in this
process. Inactivating mutations were introduced at either
the Src SH2-binding site or the ATP-binding site of CD2-FAK, a chimeric, membrane-anchored form of FAK that
localizes efficiently to focal adhesions (Chan et al., 1994;
Frisch et al., 1996b
). We reasoned that membrane attachment would promote the interaction of these FAK mutants with focal adhesion components and thereby facilitate a dominant-negative effect.
As shown in Fig. 3, both CD2-FAK mutants exerted a
dominant-negative effect on fibronectin-mediated activation of JNK, indicating that this process requires the kinase activity of FAK and its association with an Src family
kinase. Accordingly, integrin-mediated activation of JNK
was also suppressed by a kinase dead version of Src. In addition, we observed that the activation of JNK by integrins
is partially defective in Src/
and in Fyn
/
fibroblasts, in
accordance with the notion that FAK can combine with
both kinases (data not shown). Integrin-mediated activation of JNK was also blocked by a kinase inactive version
of MKK4, one of the major enzymes that binds to and
phosphorylates JNK (Fig. 4). In contrast, it was not inhibited by expression of dominant-negative Ras (Fig. 3) or by
exposure to the PI-3K inhibitors Wortmannin (100 nM)
and LY294002 (50 µM) (data not shown). These results indicate that the FAK/Src complex links integrins to MKK4
or a related enzyme, and thereby JNK. In addition, they
suggest that Ras and its substrate, PI-3K, which can activate Rac and thus JNK (Rodriguez-Viciana et al., 1994
;
Nobes et al., 1995
; Klippel et al., 1996
), do not contribute
to this process.
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To examine the mechanism by which the FAK/Src complex activates JNK, we focused on the role of the docking/
adaptor proteins p130CAS and paxillin, which bind to the
FAK/Src complex and become heavily phosphorylated on
tyrosine residues upon integrin engagement (Schaller and
Parson, 1995; Richardson and Parsons, 1996; Vuori et al., 1996
; Schlaepfer et al., 1997
). The activation of JNK by integrins was inhibited to a significant extent by a mutant
form of p130CAS carrying a deletion of the entire substrate
region (SD-CAS; Fig. 4). In contrast, a mutant form of
paxillin carrying phenylalanine substitutions at three tyrosine phosphorylation sites, including all the Crk binding
sites, inhibited this event modestly and only when expressed at relatively high levels (data not shown). These results indicate that p130CAS plays a major role, and paxillin perhaps a minor one, in integrin-mediated JNK activation.
9 out of 15 tyrosine phosphorylation sites within the substrate region of p130CAS conform to the consensus motif
for binding to the SH2 domain of Crk (Sakai et al., 1994).
In addition to the SH2 domain, Crk contains either one or
two SH3 domains able to interact with downstream target-effectors (Mayer et al., 1988
; Matsuda et al., 1992
; ten Hoeve et al., 1993
). Previous studies have indicated that
the adaptor function of Crk is regulated positively by recruitment to the plasma membrane and negatively when
tyrosine 222 becomes phosphorylated and associates intramolecularly with the SH2 domain (Mayer and Hanafusa, 1990
; Feller et al., 1994
).
We reasoned that an SH3 mutant form of the viral version of Crk, which is anchored to the membrane via its gag
sequences and truncated before tyrosine 222, would have
interacted efficiently, via its intact SH2 domain, with
p130CAS, but not with downstream target effectors. As
shown in Fig. 4, expression of this mutant form of Crk
effectively suppressed JNK activation in cells plated on
fibronectin. Conversely, the introduction of a control
construct with a mutated SH2 domain stimulated the activation of JNK, suggesting that the recruitment of Crk to
the plasma membrane and its interaction with downstream
target(s) via the SH3 domain are sufficient to activate JNK
(Fig. 4). These observations indicate that the FAK/Src/
p130CAS complex activates JNK by recruiting Crk to the
plasma membrane. They are also in agreement with previous studies implicating Crk in the activation of JNK
(Tanaka et al., 1997).
Integrin-mediated JNK Signaling Controls Cell Cycle Progression
To examine whether FAK signaling to JNK was required
for cell proliferation, NIH-3T3 fibroblasts were transiently
transfected with various amounts of vectors encoding
wild-type and mutant versions of the signaling components of this pathway, in combination with the marker
-galactosidase. The cells were synchronized in G0 and
plated on fibronectin in the presence of PDGF. Entry of the transfected cells into S phase was evaluated by double
staining with X-gal and anti-BrdU antibodies.
While CD2-FAK, which is constitutively active (Chan
et al., 1994), promoted entry into the S phase to a limited
extent, its kinase dead version suppressed it (Fig. 5). In
both cases, the effects observed were dose dependent. In
addition, whereas wild-type p130CAS did not affect progression through G1, a mutant version carrying a deletion
of the substrate region, which includes all the Crk binding
sites, partially inhibited transit through G1 (Fig. 5). The incomplete effect of this mutant may be due to residual, integrin-induced binding of Crk to paxillin (Schaller and
Parsons, 1995
). In accordance with this hypothesis, dominant-negative Crk suppressed entry into S phase as effectively as the kinase dead version of CD2-FAK. Finally, cell
cycle progression was also suppressed by dominant-negative versions of MKK4 and Jun (Fig. 5). These findings
suggest that integrin-mediated activation of the FAK-
JNK pathway is necessary for progression through the G1
phase of the cell cycle.
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Discussion |
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Although the details of FAK's interaction with a number of cytoskeletal and signaling components are known, the biological function of this kinase is incompletely understood. Our results suggest that FAK mediates activation of JNK and c-Jun in response to integrin ligation, and by doing so, regulates progression through the G1 phase of the cell cycle.
What is the mechanism by which FAK activates JNK?
Upon activation, FAK undergoes autophosphorylation at
tyrosine 397 and combines with the SH2 domain of Src or
Fyn (Parsons, 1996). The most prominent substrates of
the FAK/Src complex are the docking adaptor proteins p130CAS and paxillin (Schaller and Parsons, 1995
; Richardson and Parsons, 1996
; Vuori et al., 1996
). Both contain tyrosine phosphorylation sites conforming to the consensus
for binding to the adaptor protein Crk. However, while
paxillin has only two such sites and does not appear to associate efficiently with Crk in response to integrin ligation
(Schaller and Parsons, 1995
), p130CAS contains nine Crk-binding motifs and associates well with Crk in cells adhering to fibronectin (Vuori et al., 1996
). Our results indicate
that the expression of dominant-negative versions of FAK,
Src, p130CAS, and Crk suppress the activation of JNK by
integrins. Together with complementary results of a recent
study (Dolfi et al., 1998
), these findings provide evidence
that integrin-mediated activation of JNK requires the association of FAK with Src (or Fyn) and p130CAS, and the
recruitment of Crk. It is unlikely that the coupling of Ras
to Rac mediated by PI-3K (Rodriguez-Viciana et al., 1994
; Nobes et al., 1995
; Klippel et al., 1996
) contributes in a significant manner to the activation of JNK by integrins because a dominant-negative form of Ras and specific inhibitors of PI-3K did not interfere with activation of this
pathway. Thus, it appears that the
1 and
v integrins activate JNK and ERK via two separate pathways (see Fig. 6
for a model). By contrast, the
6
4 integrin, which is presumably unable to activate FAK because it does not contain the sequences required for its recruitment, is coupled
to JNK signaling via the Ras-PI-3K-Rac pathway (Mainiero et al., 1997
).
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The mechanism by which Crk activates JNK in response
to integrin ligation remains to be examined. Crk is known
to interact via one of its SH3 domains with the exchange
factor C3G (Tanaka et al., 1994) and previous studies have
indicated that C3G is required for activation of JNK by
the viral oncoprotein v-Crk (Tanaka et al., 1997
). Interestingly, the activation of JNK by v-Crk and C3G appears to
require the sequential action of the mixed lineage kinases
MLK3 and DLK, but not the activity of Rho family GTPases or PAK proteins (Tanaka and Hanafusa, 1998
). In addition, or instead, Crk may activate Rac, and thereby JNK,
by promoting the association of C3G with DOCK 180 or
mSOS (Dolfi et al., 1998
).
The identity of genes regulated by JNK is largely unknown, but they must include genes important for cell proliferation. The evidence for this is several fold: first, deregulated expression of c-Jun or its mutated viral version v-Jun
is sufficient to cause neoplastic transformation of primary
avian and mammalian fibroblasts (Vogt, 1994); second,
primary fibroblasts derived from c-Jun
/
mice display a
severe proliferation defect (Johnson et al., 1993
); and
third, several oncoproteins, including v-Src, activated Ras, v-Crk, Bcr-Abl, and Met, potently activate JNK and there
is evidence to suggest that this activation is required to
cause neoplastic transformation (Derijard et al., 1994
;
Minden et al., 1995
; Raitano et al., 1995
; Johnson et al.,
1996
; Rodrigues et al., 1997
; Tanaka et al., 1997
).
Despite the clear requirement for c-Jun transcriptional
activity in cell proliferation, it has been difficult to identify a physiological, nonstress stimulus for JNK consistent with
its role in the regulation of AP-1 transcription. With the
notable exception of EGF, mitogenic neuropeptides, and
muscarinic receptor ligands, which indeed activate FAK or
the related kinase PYK-2 (Zachary et al., 1992; Coso et al.,
1995
; Tokiwa et al., 1996
; Yu et al., 1996
; Cadwallader et al.,
1997
; Higashita et al., 1997
; Logan et al., 1997
; Slack,
1998
), most growth factors cause a relatively modest activation of JNK (Kyriakis et al., 1994
; Minden et al., 1994
).
Our results indicating that integrin ligation causes a significant activation of JNK and TRE-dependent transcription provide a physiological stimulus for JNK signaling that is
consistent with its role in the control of cell proliferation.
Our present results imply that FAK, which appears to
be activated by all 1 and
v integrins, is required during
G1 progression because of its ability to activate JNK. Recently, it has been shown that a mutant form of FAK,
which is truncated at the COOH terminus and thus unable
to localize to focal adhesions, interferes with both fibronectin-induced activation of ERK and progression through the cell cycle (Zhao et al., 1998
). On the basis of
these results, it has been argued that FAK regulates cell
proliferation by stimulating ERK. However, an alternative
explanation is that this truncated form of FAK acts as a
cytoplasmic sink for all Src-family kinases and thus disrupts not only FAK, but also Shc signaling. In accordance
with this hypothesis, two more specific dominant-negative
forms of FAK, FRNK and FAK-Y397F, inhibit ERK activation to a much lesser degree, but interfere with cell proliferation nonetheless (Zhao et al., 1998
; see also Gilmore
and Romer, 1996
). Although additional mechanisms cannot be excluded, our observation that dominant-negative
forms of FAK, p130CAS, Crk, MKK4, and Jun all inhibit
entry into the S phase provides evidence that FAK regulates cell proliferation by activating JNK.
If FAK is required for cell cycle progression, why then
do the FAK/
cells not display an obvious proliferation
defect (Ilic et al., 1995
)? There are two potential explanations. First, it is now apparent that the FAK
/
cells originally examined by Ilic and colleagues also lack a functional form of the cell cycle regulator p53 (Furuta et al., 1995
). Some of the cell lines generated more recently do
have wild-type p53, but are transformed by the polyoma
middle T antigen (Ilic et al., 1998
). It is possible that the
lack of p53 or presence of middle T antigen bypasses the
requirement for FAK during cell proliferation. In addition, it recently has been shown that FAK
/
cells have elevated levels of PYK-2, which may compensate, at least in
part, for the lack of FAK (Sieg et al., 1998
).
The mitogenic signaling pathway linking integrins to
JNK is likely to be deregulated in, and to contribute to, the
transformation of at least some neoplastic cells. Previous
studies have provided evidence that FAK is overexpressed
in invasive carcinomas (Owens et al., 1995) and that the
constitutively active CD2-FAK induces anchorage-independent growth in MDCK cells (Frisch et al., 1996b
). In
addition, the viral version of Src is a potent oncogene capable of transforming a variety of cells types, and there is
strong genetic evidence that p130CAS is a necessary substrate of v-Src-induced transformation (Honda et al.,
1998
). In accordance with these findings, we have observed that CD2-FAK and p130CAS cooperate with activated Raf to induce anchorage-independent growth in
NIH-3T3 cells (F. Liu and F.G. Giancotti, unpublished results). Finally, v-Crk and v-Jun are potent oncogenes
(Mayer et al., 1988
; Vogt, 1994
). Taken together, these observations suggest that the FAK-JNK pathway can contribute to neoplastic transformation.
Previous studies have provided evidence that 6
4 and
a subset of
1 and
v integrins activate the Ras-ERK signaling cascade by recruiting Shc (Mainiero et al., 1995
;
1997
; Wary et al., 1996
; 1998
). Mitogens and Shc-linked integrins synergize to promote transcription from the Fos
SRE. Accordingly, ligation of integrins linked to Shc enables these cells to progress through the G1 phase of the
cell cycle in response to mitogens, whereas adhesion mediated by other integrins results in growth arrest, despite the
presence of mitogens. These mechanisms also appear to
operate in vivo, as mice lacking the integrin
1 subunit or
the cytoplasmic domain of
4 display cell cycle defects
consistent with the lack of Shc signaling (Murgia et al.,
1998
; Pozzi et al., 1998
). These data suggest that integrin-mediated Shc signaling is necessary for cell cycle progression.
The results of this and previous studies support the
model that integrins control cell cycle progression primarily by regulating immediate early gene expression (Fig.
6). While Shc-linked integrins and growth factor receptors
cooperate to activate the Ras-ERK cascade and promote
SRE-dependent transcription of c-Fos, all 1 and
v integrins appear to be able to stimulate the FAK-JNK pathway in the absence of a significant contribution from
growth factor receptors. It is likely that, in response to integrin ligation, JNK not only acts on preexisting Jun/ATF2
and ATF2/ATF2 dimers, thereby promoting the CRE-dependent transcription of c-Jun, but also activates the
Fos/Jun dimers formed in response to the coordinated action of both integrins and growth factor receptors. The existence of a signaling pathway activated by all integrins
within a signaling network coordinately regulated by a
subset of integrins and growth factor receptors ensures
that the control of cell proliferation exerted by the extracellular matrix is both stringent and integrin-specific.
![]() |
Footnotes |
---|
Address correspondence to Filippo G. Giancotti, Memorial Sloan-Kettering Cancer Center, Box 216, 1275 York Avenue, New York, NY 10021. Tel.: (212) 639-7333. Fax: (212) 794-6236. E-mail: f-giancotti{at}ski.mskcc.org
Received for publication 17 February 1999 and in revised form 25 May 1999.
Maja Oktay's present address is Department of Pathology, Yale University School of Medicine, New Haven, CT 06520.
We are indebted to B. Binetruy, S. Courtneidge, E. Skolnik, K. Vuori, and
J. Westwick for constructs. We thank P. Soriano for the Src/
and Fyn
/
3T3 fibroblasts. We are grateful to members of the Giancotti laboratory
for discussions and comments on the manuscript.
This work was supported by National Institutes of Health grants R01 CA78901 (to F.G. Giancotti) and P30 CA08748 (to the Memorial Sloan-Kettering Cancer Center). F.G. Giancotti is an Established Investigator of the American Heart Association.
![]() |
Abbreviations used in this paper |
---|
ATF2, activating transcrition factor 2; BrdU, 5-bromodeoxyuridine; CS, calf serum; CMV, cytomegalovirus; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; GST, glutathione S-transferase; HUVECs, human umbilical vein endothelial cells; JNK, Jun NH2-terminal kinase; PDGF, platelet-derived growth factor; SD, substrate region deleted; SFM, serum-free medium; SRE, serum response element; TPA, 12-O-tetradecanoylphorbol-13-acetate; TRE, TPA-responsive element.
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