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INTRODUCTION |
The integrins have received a great deal of attention in the
literature over the last few years, not only for their ability to bind
and model the extracellular matrix, but also due to their ability to activate a number of cell signaling cascades that influence a range of biological processes including cell growth, differentiation, migration, and apoptosis (1). The distal stretches of many of these
signaling pathways are reasonably well defined. For instance, ligation
of integrins is well established to both activate the MEK/ERK1 signaling axis and
to prolong the activation of the pathway in response to growth factors
and thereby confer anchorage-dependent growth (2). However,
precise descriptions of the upstream events in integrin signaling
remain elusive.
Much of the evidence that kinases associate physically with integrins
is controversial. The integrin-linked kinase was identified as a ligand
for the cytodomain of
1 integrin by yeast two-hybrid analysis, and engagement of
1 integrins has been shown
to activate integrin-linked kinase (3). However, recent work in
Drosophila suggests that the main function of
integrin-linked kinase is that of an adaptor rather than a kinase (4).
On the other hand pp125FAK is well established to be
activated by ligation of integrins and to be a key activator of
numerous downstream signaling cascades (5), but it is unclear whether
pp125FAK associates physically with integrins. Recently,
however, good data have been obtained indicating that the tyrosine
kinase, Syk, associates with
3 integrin cytodomains, and
activation of Syk by clustering of
IIb
3
integrin is likely to be a key upstream event in the activation of
platelets and locomotion of hemopoietic cells (6).
The transmembrane and extracellular domains of integrins also form
so-called "lateral associations," and these are proposed to couple
integrins to key signaling kinases. The
5
1,
v
3, and
1
1 heterodimers associate with caveolin
(7), most probably via their transmembrane stretches. There is no
evidence that this interaction is direct, but it is likely to be
important in linking integrins to the tyrosine kinase, Fyn, and the
adaptor protein, Shc. The recruitment of Shc may be a critical link
between integrins and the Ras/Raf/MEK/ERK signaling cascade and the
regulation of anchorage-dependent growth (7). Integrins are
now well established to form stable complexes with proteins of the
transmembrane 4 superfamily (8). These tetraspannins have been shown to
bind tyrosine kinases (9), phosphotidylinositol 4-kinase (10), and
conventional protein kinase C (11), and the observed modulatory effects
of integrin-tetraspannin complexes on adhesion-dependent signaling may involve these associations.
Prior to engagement with the extracellular matrix, integrins must be
rendered competent to bind ligand via a process termed "inside-out"
signaling (12). There are several ways in which the availability of
ligand-competent integrin may be increased. Growth factors have
recently been shown to regulate delivery of
v
3 from endosomal compartments to the
plasma membrane, and this process is necessary for efficient integrin
function (13). Once upon the cell surface, conformational changes in an
individual heterodimer may increase its affinity for monovalent ligand
(12). This is well documented to be a key event in the activation of
IIb
3 following treatment of platelets
with thrombin. In addition to affinity modulation, the avidity of an
integrin for a multivalent matrix ligand may be increased by regulating
the clustering of active heterodimers (14). For example, clusters of
v
3 integrin form at early stages during
cell spreading and clearly prior to the assembly of focal complexes
(15). Several growth factor-activated signaling pathways have been
implicated in the activation of integrins prior to focal complex
assembly. For instance, the small GTPase, Rab4 regulates delivery of
v
3 to the plasma membrane (13); phosphatidylinositol 3-kinase (16) and H-Ras (17) are clearly involved
in integrin affinity modulation; and the calcium-dependent protease, calpain, has recently been shown to mediate integrin clustering (15). However, the overall picture is far from complete.
Clearly, any kinase or other signaling protein found to associate with
an integrin shortly following cell activation but prior to its
incorporation into focal complexes would be potentially of interest as
a possible regulator of integrin function. To identify these proteins,
we immunoprecipitated integrins from fibroblasts shortly following
activation with platelet-derived growth factor (PDGF), and screened
them for associated proteins that were rich in phosphotyrosine and
phosphothreonine. We report that following PDGF addition, active ERK1
is an abundant component of
v
3 integrin immunoprecipitates. The association of ERK with
v
3 forms in plasma membrane complexes
prior to delivery of integrin to focal complexes. Moreover, we find
that association of active ERK1 with
v
3
integrin is necessary for cells to spread effectively on vitronectin
and thus may define a mechanism whereby a growth factor-activated signaling pathway can directly influence integrin function.
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EXPERIMENTAL PROCEDURES |
Materials--
Monoclonal rat anti-mouse
5
integrin (clone 5H10-27 (MFR5)), hamster anti-mouse
3
integrin (clone 2C9.G2), and mouse anti-human
3 integrin
(clone VI-PL2) were purchased from Pharmingen (San Diego, CA).
Monoclonal mouse anti-ERK1/2 (s13-6200) from Nymed (San Francisco, CA)
was used for immunofluorescence, and polyclonal rabbit anti-ERK1/2
(sc-93) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and
monoclonal anti-Thr202/Tyr204 phospho-p44/42
ERK (clone E10) from New England Biolabs (Beverly, MA) were used for
Western blotting. Peroxidase-conjugated anti-phosphotyrosine (clone
PY-20H) was from BD Transduction Laboratories (San Diego, CA).
Monoclonal mouse anti-phosphothreonine (clone 14B3) and PD98059 (513000) were from Calbiochem. Fluorescein isothiocyanate-conjugated goat anti-mouse and Texas Red-conjugated anti-rabbit immunoglobulins were from Southern Biotechnology (Birmingham, AL). Texas Red-conjugated phalloidin was purchased from Molecular Probes (Leiden, The
Netherlands). Magnetic sheep anti-mouse IgG Dynabeads (Dynal, Oslo,
Norway) and bovine serum albumin (BSA) were from First Link Ltd.
NHS-SS-biotin (21331) and enhanced chemiluminescence reagents (ECL)
were from Pierce and Warriner Ltd. (Chester, Cheshire, UK). Cell
culture medium and Maxisorb 96 well plates were from Invitrogen,
and fetal calf serum (FCS) was from Sera-Q (Tunbridge Wells, Kent, UK). The Fugene 6 transfection reagent was from Roche Diagnostics GmbH (Mannheim, Germany). PDGF-BB (100-14B) was from PreproTech Inc. (Rocky
Hill, NJ). Streptavidin-conjugated horseradish peroxidase was from
Amersham Biosciences (Little Chalfont, Buckinghamshire, UK). All
other reagents including a monoclonal antibody against the hexa-His
epitope tag (anti-polyHISTIDINE, clone HIS-1) were purchased
from Sigma.
Cell Culture and Transfection--
Swiss and NIH 3T3 mouse
fibroblasts were grown in Dulbecco's modified Eagle's medium (DMEM)
with 10% FCS and 100 units/ml penicillin, 100 µg/ml streptomycin,
and 0.25 µg/ml amphotericin B at 37 °C with 10% CO2.
For transient transfection experiments, NIH 3T3 fibroblasts were grown
to 50% confluence, fed with fresh DMEM containing 10% fetal calf
serum, and transfected with integrin, ERK, and Rab constructs (integrin
and Rab cDNAs were ligated into in pcDNA3 and are as described
in Ref. 13; His-ERK1, HisERK1K>R, HisERK2, and His-ERK2K>R were in
pCMV5 and are as described in Ref. 18) using Fugene 6 according to the
manufacturer's instructions. The ratio of Fugene 6 to DNA was
maintained at 3 µl of Fugene 6:1 µg of DNA. Immunoprecipitations,
integrin recycling, and cell spreading assays were carried out 24 h post-transfection.
Expression and Purification of GST-Integrin Cytodomain Fusion
Proteins--
PCR-amplified DNA fragments corresponding to aa
728-762, 728-756, 728-748, and 728-741 of the human sequence of
3 integrin and to aa 764-798 of the human sequence of
1 integrin were subcloned into the
BamHI-EcoRI site of the pGEX-2TK vector. GST
fusion proteins were expressed in Escherichia coli strain
BL-21 and purified as described previously (19).
Immunoprecipitations and Pull-downs--
Cells were grown to
90% confluence, serum-starved for 30 min, and treated with PDGF (10 ng/ml), epidermal growth factor (30 ng/ml), or lysophosphatidic acid (1 µg/ml) where appropriate. Following this, cells were washed twice in
ice-cold PBS and lysed in a buffer containing 200 mM NaCl,
75 mM Tris-HCl pH 7, 15 mM NaF, 1.5 mM Na3VO4, 7.5 mM EDTA,
and 7.5 mM EGTA, 0.5% (v/v) Triton X-100, 0.25% (v/v)
Igepal CA-630, 50 µg/ml leupeptin, 50 µg/ml aprotinin, and 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride (1.14 µl/cm2 culture area; giving a final concentration of
0.3% (v/v) nonionic detergent following dilution of the lysis buffer
in the PBS wetting the cells) and scraped from the dish with a rubber
policeman. Lysates were passed three times through a 27-gauge needle
and clarified by centrifugation at 10,000 × g for 10 min at 4 °C. For immunoprecipitations, magnetic beads conjugated to
sheep anti-mouse IgG were blocked in PBS containing 0.1% (w/v) BSA and
then bound to anti-integrin or anti-His6 monoclonal
antibodies. For pull-downs, anti-mouse magnetic beads were bound to
mouse anti-rabbit IgG, followed by rabbit anti-GST and finally GST or
GST-integrin cytodomain fusion proteins as appropriate. Antibody and
fusion protein-coated beads were incubated with lysates for 2 h at
4 °C with constant rotation. Unbound proteins were removed by
extensive washing in lysis buffer, and specifically associated proteins
were eluted from the beads by boiling for 10 min in Laemmli sample
buffer. Proteins were resolved by SDS-PAGE (8% gels under reducing
conditions for ERKs, the hexa-His epitope, phosphotyrosine, and
phosphothreonine; 6% gels under nonreducing conditions for integrins)
and analyzed by Western blotting as described previously (20).
Integrin Recycling Assay--
This was performed as described
previously in (13). Cells were serum-starved for 30 min, transferred to
ice, washed twice in cold PBS, and surface-labeled at 4 °C with 0.2 mg/ml NHS-SS-biotin in PBS for 30 min. Labeled cells were transferred
to serum-free DMEM at 22 °C for 15 min to allow internalization of
tracer into early endosomes. Cells were returned to ice and washed
twice with ice-cold PBS, and biotin was removed from proteins remaining
at the cell surface by incubation with a solution containing 20 mM sodium 2-mercaptoethanesulfonate (MesNa) in 50 mM Tris, pH 8.6, and 100 mM NaCl for 15 min at
4 °C. The internalized fraction was then chased from the cells by
returning them to 37 °C in serum-free DMEM in the absence or
presence of 10 ng/ml PDGF-BB for 10 min. Cells were returned to ice,
and biotin was removed from recycled proteins by a second reduction
with MesNa. MesNa was quenched by the addition of 20 mM
iodoacetamide for 10 min, and the cells were lysed in 200 mM NaCl, 75 mM Tris, 15 mM NaF, 1.5 mM Na3VO4, 7.5 mM EDTA
and 7.5 mM EGTA, 1.5% (v/v) Triton X-100, 0.75% (v/v) Igepal CA-630, 50 µg/ml leupeptin, 50 µg/ml aprotinin, and 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride. Lysates were
passed three times through a 27-gauge needle and clarified by
centrifugation at 10,000 × g for 10 min. Supernatants
were corrected to equivalent protein concentration, and levels of
biotinylated integrin were determined by capture enzyme-linked
immunosorbent assay. Maxisorb 96 well plates were coated overnight with
5 µg/ml anti-
3 integrin monoclonal antibodies in 0.05 M Na2CO3, pH 9.6, at 4 °C and
blocked in PBS containing 0.05% (v/v) Tween 20 (PBS-T) with 5% (w/v)
BSA for 1 h at room temperature. Integrins were captured by
overnight incubation of 50 µl of cell lysate at 4 °C. Unbound
material was removed by extensive washing with PBS-T, and wells were
incubated with streptavidin-conjugated horseradish peroxidase in PBS-T
containing 1% (w/v) BSA for 1 h at 4 °C. Following further
washing, biotinylated integrins were detected by chromogenic reaction
with ortho-phenylenediamine.
Immunofluorescence--
Cells were plated onto glass coverslips
and grown to 50-70% confluence over 3 days. Cells were serum-starved
for 30 min and treated with 10 ng/ml PDGF-BB for the indicated times
prior to fixation in PBS containing 2% (w/v) paraformaldehyde for 20 min at room temperature. Nonspecific binding sites were blocked with PBS containing 10% (v/v) fetal calf serum (PBS-FCS) for 1 h, and cells were incubated with hamster anti-m
3 monoclonal
antibody at 5 µg/ml in PBS-BSA at room temperature for 1 h.
Following this, cells were permeabilized with 0.2% (v/v) Triton X-100
in PBS for 5 min and then reblocked in PBS-FCS. Integrin was detected
by sequential application of a rabbit anti-hamster secondary antibody, followed by fluorescein isothiocyanate-conjugated goat anti-rabbit tertiary antibody. The cells were counterstained for ERKs using mouse
anti-ERK1/2 monoclonal antibodies followed by a Texas Red-conjugated goat anti-mouse secondary antibody. Where appropriate, the actin cytoskeleton was counterstained with Texas Red-conjugated phalloidin in
PBS for 10 min at room temperature. Cells were viewed on a Leica
confocal laser-scanning microscope, and images were presented either as
an extended focus projection (using the Leica "maxproj" algorithm)
of a number of optical slices encompassing the depth of the cell (as
for Figs. 4 and 6) or a single Z-section optical slice (as for Fig.
5).
Cell Adhesion and Spreading Assays--
24-well tissue culture
plates were coated overnight at 4 °C with fibronectin (F-1141;
Sigma) or vitronectin (V-8379; Sigma) at concentrations of 20 µg/ml
and then blocked with 2% (w/v) BSA. Cells were transfected with Rab4
or His-ERK constructs in conjunction with a
-galactosidase-expressing marker construct and, 24 h following transfection, were harvested by trypsinization and collected by centrifugation in the presence of 20 µg/ml soyabean trypsin
inhibitor. The cell suspensions were added immediately to ligand coated
wells in serum-free DMEM containing 10 ng/ml PDGF-BB in the presence and absence of 12 µM PD98059. Cells were allowed to
attach for 60 min, and nonadherent cells were removed by washing six
times with PBS. Attached cells were fixed for 1 min in 0.2%
glutaraldehyde containing 5 mM EGTA, and
-galactosidase-expressing cells were visualized by incubation with 5 mM potassium ferricyanide and 1 mg/ml X-gal
(5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside) overnight at 37 °C. To obtain an index of cell spreading, the area
of cells expressing
-galactosidase was determined by delineation of
the cell envelope using the NIH Image software.
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RESULTS |
Active ERK1 Is Associated with
v
3
Integrin--
Swiss 3T3 fibroblasts were serum-starved for 30 min and
then stimulated with 10 ng/ml PDGF for 10 min or allowed to remain quiescent. Cells were immediately cooled to 4 °C and surface-labeled with NHS-SS-Biotin. Labeled cells were lysed in a buffer containing 0.5% (v/v) Triton X-100 and 0.25% (v/v) Igepal, and
5
1 and
v
3 integrin heterodimers were immunoprecipitated from lysates using monoclonal antibodies to either the mouse
5 or mouse
3 integrin chains, respectively. PDGF increased the
surface expression of
v
3 (but not
5
1) by ~2-fold (Fig.
1A), and this is consistent with our
previous work documenting the rapid stimulation of
v
3 integrin recycling following growth
factor addition (13).

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Fig. 1.
Active ERK1 is associated with endogenous
mouse
v 3
integrin in Swiss 3T3 fibroblasts. Swiss 3T3 fibroblasts were
serum-starved for 30 min and incubated in the presence and absence of
10 ng/ml PDGF-BB (A-D, F), 30 ng/ml epidermal
growth factor, or 1 µg/ml lysophosphatidic acid (F) for 10 min or treated with 10 ng/ml PDGF-BB for the times indicated
(E). Cells were surface-labeled with 0.2 mg/ml NHS-SS-biotin
for 30 min at 4 °C and lysed in a buffer containing 0.5% (v/v)
Triton X-100 and 0.25% (v/v) Igepal CA-630. Lysates were
immunoprecipitated (IP) with monoclonal antibodies against
mouse 5 (m 5) and
3 (m 3) integrins. Immobilized material
was then analyzed by Western blotting with peroxidase-conjugated
streptavidin (SA-HRP) (A), anti-phosphotyrosine
(PY) (B), anti-phosphothreonine (PT)
(C), anti-ERK1/2 (D-F), and anti-phospho-ERK1/2
(D). The positions of 5, v,
1, and 3 integrin chains, immunoglobulin
heavy chain (IgG HC), and ERK1/2 are indicated.
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To gain insight into key biochemical events that occur during the early
stages of focal complex assembly, we investigated the complement of
cell-signaling proteins that coimmunoprecipitate with integrins shortly
following treatment of cells with growth factor. Therefore, integrin
immunoprecipitates were analyzed by Western blotting with antibodies
recognizing phosphotyrosine and phosphothreonine. In serum-starved
cells, low levels of phosphotyrosine- or phosphothreonine-containing
proteins coimmunoprecipitated with either
5
1 or
v
3
integrins (Fig. 1, B and C). However, following the addition of PDGF, a 44-kDa protein (p44) that was rich in both
phosphotyrosine and phosphothreonine was particularly abundant in
immunoprecipitates of
v
3 integrin (Fig.
1, B and C). p44 was only present at low levels
in
5
1 immunoprecipitates, and, moreover,
there was no phosphotyrosine signal at 120 or 70 kDa, indicating that
pp125FAK and paxillin were not associated with either
integrin. We were, however, able to coimmunoprecipitate small
quantities of a phosphotyrosine-containing protein of 190 kDa with
v
3, and this is likely to represent association of the PDGF receptor with the integrin (21).
A previous report has documented the presence of the ERKs in newly
forming focal complexes (22). Both p44 ERK1 and p42 ERK2 would be
expected to be rich in phosphotyrosine and phosphothreonine following
cell activation, so we tested for the presence of these kinases in the
integrin immunoprecipitates. Western blotting with an antibody that
recognizes both p44 ERK1 and p42 ERK2 revealed that p44 comprised ERK1,
and this was associated with
v
3 only following PDGF treatment (Fig. 1D). A small quantity of ERK1
was found to be associated with
5
1
integrin; however, this was not increased by the addition of PDGF (Fig.
1D). Interestingly, only relatively small amounts of p42
ERK2 were associated with
v
3, indicating
that recruitment of ERK1 was isoform-specific. ERK1 must be
phosphorylated on both threonine 202 and tyrosine 204 to be active, and
the presence of signals for phosphotyrosine and phosphothreonine in p44
implied that it was indeed active ERK1. This was confirmed by Western
blotting using a phosphospecific antibody that recognized ERKs only
when phosphorylated at both of these positions (Fig. 1D).
The phosphotyrosine-containing protein at 60 kDa (p60; Fig.
1B) is likely to represent a Src family kinase. We are
currently investigating the identity of p60 and the significance of its
association with
v
3 integrin.
We investigated the time course over which the
ERK1·
v
3 complex was established.
Tyrosine phosphorylation of protein bands corresponding to the PDGF
receptor (190 kDa), pp125FAK (125 kDa), and paxillin (70 kDa) was maximal ~4 min after PDGF addition and subsided over the
following 12 min (Fig. 1E, upper panel). Recruitment of ERK1 to
v
3 integrin was slower than this and was
maximal at 8 min following PDGF addition (Fig. 1E,
lower panel). The association persisted for at
least 16 min following PDGF addition (Fig. 1E) but abated
somewhat over the following hour (data not shown). PDGF,
lysophosphatidic acid, and epidermal growth factor were all able to
induce substantial increases in tyrosine phosphorylation of a number of
cellular proteins (notably of a band corresponding to
pp125FAK) (Fig. 1F, upper
panel). However, of the growth factors tested in the present
study, only PDGF was able to elicit appreciable recruitment of ERK1 to
v
3 (Fig. 1F, lower
panel). Increased tyrosine phosphorylation of a 36-kDa
protein (marked p36 in Fig. 1, E and
F, upper panels) was PDGF-specific
and, moreover, occurred at a rate that paralleled recruitment of ERK1
to
v
3. Preliminary data indicate that p36
is likely to be annexin II (not shown), and the possible role of this
protein in the assembly of integrin-containing complexes is discussed later.
To determine whether ERK1 was also able to associate with human
v
3 and to confirm that its presence in
mouse
v
3 immunoprecipitates was not an
artifactual characteristic of the antibody employed, human integrins
were transfected into NIH 3T3 fibroblasts, and
v
3 was immunoprecipitated with a
monoclonal antibody that was specific for the human
3
integrin chain (h
3). Surface labeling indicated that
this antibody did not precipitate mouse
v
3 (Fig. 2A), and accordingly we were
unable to detect any phosphotyrosine/phosphothreonine-containing proteins or ERKs associated with anti-human
3 monoclonal
antibody-coated beads when they were incubated with lysates prepared
from mock-transfected cells even following PDGF treatment (Fig. 2,
B-D). Following transfection of NIH 3T3 fibroblasts with
the human
v and
3 integrin chains, surface-labeled proteins corresponding to the
v
3 heterodimer were prominent in the
immunoprecipitates. These displayed increased surface expression
following the addition of PDGF (Fig. 2A), although Western
blotting with an antibody recognizing the h
3 chain
revealed that the total quantity of integrin present in the
immunoprecipitates was unaffected (Fig. 2A). Moreover, in
transfected cells, a profile of
phosphotyrosine/phosphothreonine-containing proteins, similar to that
found associated with the mouse integrin, were coimmunoprecipitated with human
v
3 following the addition of
PDGF (Fig. 2, B and C), and Western blotting
revealed that the 44-kDa band (p44) comprised active ERK1 (Fig.
2D).

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Fig. 2.
ERK1 is associated with human
v 3
integrin expressed in NIH 3T3 fibroblasts. NIH 3T3 fibroblasts
were transfected with human v 3 integrin
(h v 3; A-D),
v 3 in combination with His-ERK1 or
His-ERK2 (E-F), or empty vector control (A-D;
mock) and then serum-starved and challenged with PDGF-BB for
10 min. Following surface labeling, cells were lysed and
immunoprecipitated (IP) with monoclonal antibodies against
human 3 integrin (A-E; h 3) or
the hexa-His epitope (6XHis) (F). Immobilized
material was analyzed by Western blotting with
peroxidase-conjugated streptavidin (SA-HRP) (A),
anti-human 3 integrin (h 3) (A
and E-F), anti-phosphotyrosine (PY)
(B), anti-phosphothreonine (PT) (C),
anti-ERK1/2 (D), anti-phospho-ERK1/2 (D), or the
hexa-His epitope (6XHis) (E and F).
The positions of v and 3 integrin chains,
immunoglobulin heavy chain (IgG HC), ERK1/2, and
HisERK1/2 are indicated.
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To confirm the ability of
v
3 integrin to
recruit ERK1 in preference to ERK2 and to further test the specificity
of this interaction, we performed immunoprecipitation studies on NIH
3T3 fibroblasts following transient expression of epitope-tagged ERKs. Following the addition of PDGF, recruitment of His-ERK1 to
v
3 immunoprecipitates was seen using a
monoclonal anti-His6 antibody to detect the epitope-tagged
protein (Fig. 2E). Furthermore, Western blotting with
anti-h
3 indicated that
v
3
integrin was precipitated by immunoisolation of His-ERK1 using magnetic
beads conjugated to anti-His6 (Fig. 2F). In
contrast, following transient expression of His-ERK2, this
epitope-tagged kinase was not detected in integrin immunoprecipitates
(Fig. 2E); nor did
3 integrin co-precipitate with His-ERK2 (Fig. 2F). It is important to note that in
these experiments cellular expression of His-ERK1 was somewhat greater than His-ERK2. This discrepancy was only observed when the ERKs were
coexpressed with
v
3 integrin (compare
Fig. 2, E and F, with the expression levels of
His-ERKs in Fig 9C, where no exogenous
v
3 is expressed) and may indicate that
overexpression of
v
3 integrin can support
increased cellular levels of ERK1. It is unlikely, however, that the
lack of co-immunoprecipitation of His-ERK2 with
v
3 (and vice versa) is due to
this discrepancy, since we have performed experiments in which the
expression of His-ERK2 was raised to exceed that of His-ERK1 (by
increasing the quantity of cDNA employed for the transfection) and
obtained similar results.
Recruitment of ERK to the
3 Integrin
Cytodomain--
A number of studies have indicated that the
cytodomains of integrin
subunits are responsible for recruiting
signaling kinases and cytoskeletal proteins to the heterodimer (6, 23).
To test the involvement of the
3 cytodomain in ERK1
recruitment, we constructed a series of
3 integrin
truncation mutants (Fig. 3A).
These were aimed at sequentially deleting the membrane-distal NITY
motif including Ile757 (
757), which has been
implicated in the assembly of focal adhesions, the sequence intervening
in the membrane-proximal NPXY and the NITY motifs (
749),
and finally the removal of a large proportion of the integrin
cytodomain (
728).

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Fig. 3.
The 3
integrin cytodomain recruits ERK. A and B,
full-length 3 integrin and the 757, 749 and 728
truncation mutants of 3 integrin shown in A
were transiently expressed in NIH 3T3 fibroblasts together with the
v integrin subunit. Transfected cells were serum-starved
and challenged with 10 ng/ml PDGF-BB for 10 min. Following this, the
monolayers were lysed and immunoprecipitated with monoclonal antibodies
against human 3 integrin. Immobilized material was
analyzed by Western blotting with anti-human 3 integrin
(B; upper panel) and anti-ERK1/2
(B; lower panels). C and
D, lysates from PDGF stimulated NIH 3T3 fibroblasts were
incubated with magnetic beads conjugated to GST or the GST-integrin
cytodomain fusion proteins indicated in C. Immobilized
material was analyzed by Western blotting for ERK1/2 (D;
upper panels), and the loading of the GST fusion
proteins was confirmed by Western blotting for GST (D;
lower panels).
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The three truncation mutants,
757,
749, and
728, were all
expressed at levels similar to that observed for full-length
3, were immunoprecipitated efficiently with the
anti-h
3 monoclonal antibody (Fig. 3B), and
formed heterodimers with the
v subunit (not shown). ERK1
coimmunoprecipitated equally well with both full-length
3 integrin and
757, indicating that the NITY motif and Ile757 are not necessary for association with the
kinase. However, removal of the 749EATSTFTN759
sequence that intervenes in the NPXY and NITY motifs
resulted in a substantial reduction in ERK1 recruitment (Fig.
3B).
To determine whether the
3 cytodomain was sufficient on
its own to recruit active ERK, lysates from PDGF-stimulated NIH 3T3 fibroblasts were incubated with GST fusion proteins corresponding to
the
3 integrin cytodomain and truncation mutants thereof
(Fig. 3C). Interestingly GST-
3 cytodomain was
able to associate with both ERK1 and ERK2, whereas GST-
1
was less effective in this regard (Fig. 3D) and, consistent
with the immunoprecipitation studies shown in Fig. 3B,
association of ERKs was lost upon removal of the
749EATSTFTN759 sequence between the
NPXY and NITY motifs (Fig. 3D). These results indicate that the cytodomain of the
3 integrin subunit
is both necessary and sufficient to recruit an ERK-containing complex to
v
3 integrin and that the central
region of the cytodomain is involved in establishment of this association.
Recruitment of
v
3 and ERK to Plasma
Membrane Complexes--
We have previously shown that
v
3 integrin is incorporated into punctate
plasma membrane complexes immediately following Rab4-dependent recycling, and these are subsequently
redistributed into peripheral focal complexes (13). Given that ERK1
association with
v
3 was established
within 10 min of PDGF addition, we wished to determine whether the
kinase was also recruited to plasma membrane complexes. Cells were
serum-starved for 30 min and treated with PDGF for 10 min, and surface
v
3 and ERKs visualized by confocal immunofluorescence microscopy. In serum-starved cells, immunoreactive ERKs were seen to focus in the perinuclear region, perhaps suggesting sequestration of the kinase upon an endomembrane compartment or even at
the microtubule organizing center (Fig.
4C). Upon the addition of
PDGF, ERKs rapidly redistributed such that they could now be seen in
the nucleus and also dispersed into a punctate array across the cell
surface (Fig. 4G). This resembled the distribution assumed
by
v
3 (Fig. 4E), and
examination of the higher magnification confocal micrograph shown in
Fig. 5 revealed a close colocalization of
ERKs and
v
3 integrin in these small
punctate structures, which were particularly enriched toward the cell
periphery (Fig. 5E). These complexes did not contain
caveolin 1, so they are distinct from caveolin-containing membrane
islands; nor did they contain paxillin or other markers of focal
adhesions and complexes (data not shown).
5
1 integrin was present in large deposits
and a fibrillar distribution reminiscent of the fibronectin
network and was not seen to be distributed as a punctate array of
plasma membrane complexes (Fig. 4M).

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Fig. 4.
Recruitment of
v 3
integrin and ERK to focal complexes. Swiss 3T3 fibroblasts were
serum-starved for 30 min and then challenged with 10 ng/ml PDGF-BB for
10 min (E-H, M, and N), or 30 min
(I-L) or allowed to remain quiescent (A-D).
Following fixation in 2% paraformaldehyde, surface
v 3 (A, E, and
I; green), 5 1
(M; green) or cellular ERK1/2 (C,
G, and K; green) were visualized by
indirect immunofluorescence, and cells were counterstained with
phalloidin (B, D, F, H,
J, L, and N; red). Surface-only
integrin staining was obtained by the addition of primary antibody
prior to detergent permeabilization. Images are presented as an
extended focus projection through a stack of 5 × 1-µm Z-section
optical slices. Bar, 10 µm.
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Fig. 5.
v 3
integrin and ERK localize to punctate plasma membrane complexes.
Swiss 3T3 fibroblasts were serum-starved for 30 min, challenged with 10 ng/ml PDGF-BB for 10 min, and then fixed in 2% paraformaldehyde.
Surface v 3 integrin was visualized by
indirect immunofluorescence (A and B;
green). Following this, cells were detergent-permeabilized
and counterstained for cellular ERK1/2 (C and D;
red). Colocalization of the two fluorophores is shown in
yellow (E and F). Images are presented
as a single confocal optical slice centered ~0.5 µm above the plane
of the substratum. Bars, 10 µm (A,
C, and E; left panels) and 2.5 µm
(B, D, and F; right
panels).
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Following a longer (30-min) exposure to PDGF, ERKs were still visible
in the nucleus, but the surface complexes were no longer prominent, and
the kinase was incorporated into a fine array of focal complexes in the
peripheral lamellae that paralleled the distribution of
v
3 integrin (Fig 4, I-L).
Taken together, these immunofluorescence and biochemical data suggest
that shortly following growth factor addition, ERK1 and
v
3 form a physical association within
small punctate complexes in the plasma membrane. These then
subsequently redistribute to form focal complexes in the peripheral lamellae.
Association of ERK1 with
v
3 Integrin
Requires the Activity of MEK--
ERK1 is activated by phosphorylation
on threonine 202 and tyrosine 204 by the dual specificity kinase,
MEK1/2. We investigated whether treatment of cells with the MEK
inhibitor, PD98059 (24), affected the association of
v
3 with ERK1 and the recruitment of ERK1
to
v
3-containing complexes. Serum-starved
cells were treated with 12 µM PD98059 for 10 min,
following which they were challenged with PDGF and lysed for
immunoprecipitation or fixed for immunofluorescence. This concentration
of PD98059 completely ablated association of ERK1 with
immunoprecipitates of
v
3 (Fig. 6A), and, in accordance with
this, markedly reduced the appearance of ERK in integrin-rich plasma
membrane complexes (Fig 6, B-G). These data show that ERK1
must be phosphorylated by MEK in order to be recruited to
v
3 integrin.

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Fig. 6.
PD98059 inhibits association of ERK with
v 3
integrin. A, serum-starved Swiss 3T3 fibroblasts were
incubated for 10 min in the absence or presence of 12 µM
PD98059 and then challenged with 10 ng/ml PDGF-BB or allowed to remain
quiescent. Cells were lysed, and v 3
integrin was immunoprecipitated (IP) from the lysates with
monoclonal antibodies against the mouse 3 integrin
chain. Immobilized material was then analyzed by Western blotting with
an antibody against ERK1/2. The migration positions of the ERKs1/2 are
indicated. B-G, serum-starved Swiss 3T3 fibroblasts were
incubated for 10 min in the absence (B-D) or presence
(E-F) of 12 µM PD98059, challenged with 10 ng/ml PDGF-BB, and then fixed in 2% paraformaldehyde. Surface
v 3 integrin was visualized by indirect
immunofluorescence (B and E; green).
Following this, cells were detergent-permeabilized and counterstained
for cellular ERK (C and F; red).
Colocalization of the two fluorophores is shown in yellow
(D and G). Bar, 10 µm.
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It is important to note that the concentration of PD98059 employed for
these experiments (12 µM), albeit sufficient to negate association of ERK1 with integrin, had only partial effects on the
PDGF-induced recruitment of ERKs to the nucleus (Fig.
6F).
Association of ERK1 with
v
3 Is Not
Necessary for Integrin Recycling--
We have previously shown that
PDGF increases recycling of
v
3 from early
endosomes to the plasma membrane via a Rab4-dependent mechanism (13). To investigate the possibility that recruitment of ERK1
to
v
3 is necessary for recycling, we
studied the effect of PD98059 on
v
3
recycling. Recycling of
v
3 from early
endosomes to the plasma membrane was assayed using the enzyme-linked
immunosorbent assay-based method we have described previously (13), and
PD98059 had no effect on the ability of PDGF to drive
v
3 recycling from early endosomes (Fig.
7). These data are consistent with the
images presented in Fig. 6, E-G, where PD98059 suppressed
the colocalization of
v
3 with ERK but did
not affect surface expression of the integrin. It is interesting to
note, however, that the integrin-containing complexes were smaller and
more numerous in the presence of PD98059 (Fig. 6E),
indicating that ERK may act to cluster
v
3.

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Fig. 7.
PDGF-stimulated recycling of
v 3
does not require the activity of MEK. Serum-starved Swiss 3T3
fibroblasts were surface-labeled with 0.2 mg/ml NHS-SS-Biotin for 30 min at 4 °C, and internalization was allowed to proceed for 15 min
at 22 °C in the presence and absence of 12 µM PD98059.
Biotin was removed from receptors remaining at the cell surface by
treatment with MesNa at 4 °C, and cells were rewarmed to 37 °C
for 10 min in the absence or presence of 10 ng/ml PDGF-BB to allow
recycling to the plasma membrane, followed by a second reduction with
MesNa. Cells were lysed, and integrin biotinylation was determined by
capture enzyme-linked immunosorbent assay using microtiter wells coated
with anti-mouse 3 integrin monoclonal antibodies. The
proportion of integrin recycled to the plasma membrane is expressed as
a percentage of the pool of integrin labeled during the internalization
period (values are mean ± S.E. from three separate
experiments).
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Recycling of
v
3 Is Not a Prerequisite
for Association of ERK1 with
v
3--
Having shown that delivery of
v
3 to the plasma membrane was independent
of ERK1 recruitment to the integrin, we wished to determine whether the
recycling of
v
3 integrin was a
prerequisite for its association with ERK1. Recycling of
v
3 from early endosomes to the plasma
membrane was powerfully stimulated by PDGF, and, consistent with our
previous studies (13), this component of integrin vesicular transport
was completely ablated by expression of the dominant negative Rab4
construct, N121Irab4 (Fig.
8A). However, association of
ERK1 with
v
3 was unaffected by expression of N121Irab4 (Fig. 8B), despite the blockade of integrin
recycling effected by this dominant negative construct.

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Fig. 8.
Dominant negative Rab4 does not block
association of ERK1 with
v 3
integrin. NIH 3T3 fibroblasts were transfected with human
v 3 integrin in combination with wild-type
Rab4 (wtrab4) or N121Irab4 as indicated. A,
transfected cells were serum-starved and surface-labeled, and integrin
recycling was performed in the presence and absence of 10 ng/ml PDGF-BB
as for Fig. 7. Biotinylated integrin was determined by capture
enzyme-linked immunosorbent assay using microtiter wells coated with
anti-human 3 monoclonal antibodies. Values are mean ± S.E. from three separate experiments. B, transfected
cells were serum-starved and then challenged with 10 ng/ml PDGF-BB or
allowed to remain quiescent. Cells were lysed, and
v 3 integrin was immunoprecipitated from
the lysates with monoclonal antibodies against the human
3 integrin chain as for Fig. 2. Immobilized material was
then analyzed by Western blotting with an antibody against ERK1/2. The
migration positions of ERK1/2 are indicated.
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Recycling of
v
3 and Active ERK1 Are
Required for Cell Spreading on Vitronectin--
To investigate the
role of ERK1 in
v
3 integrin function,
cells were allowed to spread on vitronectin, a good ligand for
v
3 but not
5
1, in the presence and absence of 12 µM PD98059. PD98059 inhibited cell spreading on
vitronectin by ~40%, indicating a requirement for active ERK in this
process (Fig. 9A). In
contrast, PD98059 did not inhibit spreading on fibronectin. This matrix dependence of PD98059 action indicates that ERK activity is required for the function of a vitronectin-binding integrin and is consistent with our observation that ERK1 is found associated with
v
3 and not
5
1. Inhibition of
v
3 recycling by expression of dominant negative S22Nrab4, also inhibited cell spreading on vitronectin to a
similar extent as PD98059 (Fig. 9A), indicating that the activities of Rab4 and ERK are both required for the function of
v
3 but not
5
1 integrin.

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Fig. 9.
ERK1 regulates cell spreading on
vitronectin. NIH 3T3 cells were transfected with wild-type Rab4,
S22Nrab4, HisERK1, HisERK1K>R, HisERK2, or HisERK2K>R in combination
with a -galactosidase transfection marker. Following trypsinization,
cells were allowed to adhere to either vitronectin (VN) or
fibronectin (FN) in the presence of 10 ng/ml PDGF-BB for
1 h, with or without 12 µM PD98059. The attached
cells were fixed and stained for -galactosidase expression and
photographed with a digital camera, and the area of transfected cells
was determined by delineation of the cell envelope using the NIH Image
software. The data are expressed as a percentage of the cell area of
wild-type Rab4-expressing cells (A) or HisERK1-expressing
cells (B) following spreading on vitronectin. (Values are
mean ± S.E.) The cellular expression levels of the His-tagged ERK
proteins are shown in C.
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Having demonstrated that recruitment of active ERKl to
v
3 and the Rab4-dependent
recycling of the integrin can be evoked independently from one another,
we were interested in determining the effect of inhibiting both of
these events simultaneously. The inhibitory effects of PD98059 and
S22Nrab4 were not additive, indicating that they were affecting the
function of the same integrin and that both efficient recycling
and recruitment of active ERK1 to the integrin are necessary
for
v
3 function.
PD98059 opposes the activation of both ERK1 and ERK2. However, the
observation that cellular
v
3 integrin
associates specifically with ERK1 in immunoprecipitates suggests the
possibility of a special role for this kinase in regulating the
assembly and remodeling of cell contacts with the vitronectin matrix.
To test this, we employed dominant negative mutants of ERK1 and ERK2
(ERK1K>R and ERK2K>R, respectively), which have previously been shown
to oppose ERK-induced c-fos expression and cell
transformation in an isoform-specific fashion (18). Both of these
dominant negative ERK mutants were expressed at similar levels in NIH
3T3 fibroblasts (Fig. 9C), but ERK1K>R inhibited spreading
onto vitronectin by ~60%, whereas ERK2K>R was ineffective in this
regard (Fig. 9B). Additionally, ERK1K>R did not compromise
spreading on fibronectin, indicating that ERK1 activity is particularly
focused toward the function of
v
3 and
does not impinge on the function of other fibronectin-binding integrins
such as
5
1.
 |
DISCUSSION |
Here we show that following addition of PDGF to serum-starved
fibroblasts, a 44-kDa protein that is rich in phosphotyrosine and
phosphothreonine coimmunoprecipitates with
v
3 integrin. Western blotting with
phosphospecific antibodies revealed that this protein was active ERK1.
Experiments in which the C-terminal region of the
3
integrin subunit was truncated, and pull-downs with
GST-
3 fusion proteins show that the cytodomain of the
integrin is both necessary and sufficient to recruit ERK and moreover
that the 749EATSTFTN759 sequence interposing
the NPXY and NITY motifs may be critical to this.
Immediately following PDGF addition, ERK was seen to colocalize with
v
3 in numerous small complexes at the
plasma membrane, and only later did these redistribute to focal
complexes in the peripheral lamellae. The association of ERK1 with
v
3 was particularly sensitive to
treatment of the cells with the MEK inhibitor, PD98059; however, this
compound had no effect on the Rab4-dependent flux of
integrin from early endosomes to the plasma membrane. Correspondingly,
inhibition of Rab4 had no effect on recruitment of ERK1 to
v
3 integrin, indicating that integrin recycling and the recruitment of active ERK1 are not interdependent. Expression of a dominant negative mutant of ERK1 (but not ERK2) significantly reduced the spreading of cells onto vitronectin, whereas
cell spreading on fibronectin was unaffected by inhibition of ERK1,
consistent with a special role for this isoform of ERK in the
regulation of
v
3 (but not
5
1) integrin function. PD98059 also
reduced cell spreading on vitronectin, to the same extent as did
dominant negative Rab4, and the effects of Rab4 and MEK inhibition were
not additive. Taken together, these data indicate that
v
3 must recycle to the plasma membrane
via the Rab4 pathway and recruit ERK1 in order to function efficiently.
Role of Integrins in ERK Translocation--
In resting cells, ERK
is retained in the cytoplasm in tight association with the microtubular
cytoskeleton (25), and it is likely, therefore, that the perinuclear
accumulation of ERK that we observe in serum-starved fibroblasts
indicates association with the microtubule organizing center. Upon
stimulation, ERK translocates from the cytoplasm to the nucleus, where
it influences gene expression by phosphorylating transcription factors.
This enhances expression of a number of early response genes, such as
c-fos (26), and ultimately leads to the induction of cyclin D1 and progression through the G1 phase of the cell cycle
(27). The engagement of integrin is known to profoundly enhance ERK activation in response to growth factor addition, and this provides a
rationale for the much studied phenomenon of
anchorage-dependent growth (28). Enhancement of ERK
signaling is thought to be mediated by a diverse array of
integrin-activated signaling pathways, most of which also lead to
reorganization of the actin cytoskeleton. Indeed, a recent study has
shown that integrin-mediated adhesion is necessary for efficient
nuclear translocation of ERK via a mechanism that clearly requires an
intact actin cytoskeleton (29). It is possible that association of ERK
with the focal adhesion machinery may facilitate delivery of the kinase
to the nucleus. Two aspects of our data, however, argue against this.
First, ERK1 recruitment to
v
3 is only
fully established ~8 min following PDGF addition. However, the
translocation of ERK to the nucleus is, if anything, faster than this,
arguing against a sequence of events whereby ERK is obliged to
associate with
v
3 and passage through
focal complexes in order to reach the nucleus. Second, the
concentration of PD98059 employed in the present study was found to
completely ablate association of ERK1 with
v
3 but had no effect on nuclear
accumulation of ERK. This implies that different pools of cytoplasmic
ERK are destined for transport to the nucleus and the plasma membrane
following growth factor addition, the activation of the former being
less sensitive to treatment of cells with PD98059 than the latter.
Our data indicate that overexpression of
h
v
3 integrin favors increased cellular
expression levels of His-ERK1. This suggests that
v
3 may play a role in the stabilization
of the ERK1 protein, most likely by incorporation of the kinase into an
integrin complex. The coordinated synthesis and degradation of many
signaling proteins is likely to be integral to the establishment of
anchorage-dependent growth, and it is possible that the
ability of
v
3 integrin to support
increased cellular levels of ERK1 may contribute to this.
A Role for ERK at the Plasma Membrane--
A number of recent
studies have shown that ERK has an important role in the cytoplasm and
that this is likely to be distinct from its activity in the nucleus.
The sea star oocyte homologue of ERK1 directly phosphorylates myosin
light chain kinase (30), and more recently activation of ERKs with a
constitutively active MEK has been shown to enhance cell migration via
phosphorylation of myosin light chain kinase (31). A more recent study
has demonstrated that active ERK is recruited to focal adhesions and
controls their assembly by virtue of its ability to phosphorylate and
activate myosin light chain kinase (22). Thus, if phospho-ERK levels are lowered using U0126 (a more potent MEK inhibitor than PD98059), the
assembly of focal complexes is inhibited, and consequently the ability
of cells to spread on the extracellular matrix is compromised. We are
able to confirm that ERK is indeed targeted to focal complexes and
furthermore show that this is likely to be achieved by its association
with an extracellular matrix receptor,
v
3 integrin.
Recruitment of ERK is clearly mediated by the cytodomain of the
3 integrin subunit. The extreme C-terminal region of
3 is known to associate with both endonexin (32) and Syk
(6), and a previous study has shown that Ile757 is critical
for targeting
IIb
3 to focal adhesions
(33). However, removal of the C-terminal NITY motif, including
Ile757, has no effect on ERK recruitment to
v
3 or to GST-
3 cytodomain fusion proteins. On the other hand, our data indicate that the 749EATSTFTN759 sequence (immediately N-terminal
to the NITY motif) is required for association with ERK. It has been
known for some time that this portion of the
3
cytodomain is critical for integrin function. A serine to proline
substitution in this region has been found in a patient with
Glanzmann's thrombasthenia and renders the integrin refractory to
inside-out activation (34), and, furthermore, substitution of the TST
motif for AAA reduces
3 integrin function in cell
attachment, spreading, and the initiation of tyrosine phosphorylation
of pp125FAK and paxillin (35). A recent report has
documented a direct association of ERK with the cytodomain of
6 integrin (36). Synthetic peptides containing the
central region of the
6 cytodomain were shown by these
workers to bind directly to ERK. It is interesting to note that a TSTF
motif in this region is conserved between
3 and
6 and is not present in other
-integrin cytodomains. Further experiments will be aimed at testing the ability of the TSTF
motif in
3 to associate directly with ERKs.
Our data indicate a special relationship between ERK1 and
v
3, which is not shared by ERK2. First,
ERK1 (and not ERK2) is recruited to immunoprecipitates of
v
3, and second His-ERK1K>R opposes
v
3-mediated cell spreading, whereas
His-ERK2K>R is ineffective in this regard. From this, it would be
tempting to speculate that this is due to an integrin-binding site that
is present in ERK1 but not ERK2. However, it is clear that
GST-
3 cytodomain fusion proteins have the capacity to
bind equally well to both ERK1 and ERK2. This may indicate that
selectivity for ERK1 requires the presence of the
v
3 heterodimer or alternatively may be
influenced by accessory factors that are unable to function in the
pull-down assays.
Our data indicate that the association of
v
3 with ERK1 occurs rapidly following the
addition of PDGF and that the resulting complex localizes to punctate
clusters in the plasma membrane prior to its incorporation into focal
complexes. Hitherto, many studies have focused on the role of integrins
in focal adhesions and complexes, and it is generally accepted that
integrins are brought into close proximity with the various signaling
molecules that mediate focal adhesion signaling, as a consequence of
the activity of the Rho subfamily GTPases, such as RhoA and Rac (37). However, it is now becoming clear that certain pathways promote the
association of integrins with other signaling components upstream of
focal complex assembly. A recent study has highlighted a novel integrin
complex, referred to as an integrin cluster, that forms upstream of Rac
activation and focal complex assembly (15). These workers reported that
integrin clusters differ from focal complexes in both their
distribution and molecular composition. The
v
3·ERK-containing complexes described
in the present study also differ in their composition from focal
complexes. For instance, they do not stain for established focal
adhesion markers, such as vinculin and paxillin; nor do any of these
proteins coimmunoprecipitate with
v
3
following the addition of PDGF. It is interesting to speculate what
kind of cellular structure these
v
3·ERK-rich complexes may be. Labeling
experiments with [32P]orthophosphate have indicated that,
even following extensive washing with nonionic detergent (0.5% (v/v)
Triton X-100 and 0.25% (v/v) Igepal), labeled phospholipids are
tightly associated with
v
3
immunoprecipitates. Moreover, the quantity of coimmunoprecipitating phospholipid increased dramatically in response to the addition of
PDGF, and this was opposed by PD98059 (data not shown). Reorganization of lipid rafts and other plasma membrane lipid subdomains has been
reported to occur following activation of a number of signaling pathways (41). It is possible, therefore, that active ERK1 can act at
the plasma membrane to induce clustering of integrin into large
detergent-resistant raftlike membrane microdomains. Preliminary data
indicate that p36 (Fig. 1, E and F) is likely to
be annexin II (not shown). This Ca2+-, phospholipid-, and
actin-binding protein is known to be a substrate for the PDGF receptor
and Src tyrosine kinases (38), and more recently it has been shown to
localize to plasma membrane lipid rafts (39) and participate in the
reorganization of these domains during cell attachment (40). It will be
interesting to determine whether annexin II is recruited to
v
3-rich membrane complexes and to
investigate the possibility that it has a role in recruiting ERK1 to
the plasma membrane.
Integrins have been reported to associate with many other types of
transmembrane and other proteins, which in principle may coalesce to
form an extensive network (42). Indeed,
v
3 integrin is known to associate with
CD47, or integrin-associated protein, in a plasma membrane lipid raft
(43). Also, the integrin-associated tetraspannin, CD81, has been shown
to localize to such a membrane domain (42), and it is interesting in
this regard that we observe CD81 to colocalize with
v
3 in puncta following PDGF treatment (data not shown).
Following expression of dominant negative Rab4,
v
3 would be expected to accumulate in
early endosomes. This construct, however, does not reduce the
recruitment of ERK1 to
v
3
immunoprecipitates and indicates that the association of active kinase
to the integrin may be established either on the surface of endosomes
or at the plasma membrane. Indeed, large integrin-tetraspannin
complexes have been detected on intracellular vesicles (10), and this implies that associations made between integrins and other signaling molecules and membrane proteins may persist while the integrin engages
in endoexocytic cycling. There is mounting evidence that the
incorporation of membrane proteins into raftlike domains is a key
sorting event in the secretory pathway (44). However, it is unlikely
that recruitment of active ERK to
v
3 at
the endosome is necessary to direct its recycling, since the rate of
delivery of early endosomal
v
3 to the
plasma membrane was clearly unaffected by PD98059.
The observation that dominant negative ERK1 compromises cell spreading
on vitronectin, but not on fibronectin, implicates the activity of this
ERK in the activation of
v
3 integrin.
Many integrins, including
v
3, can assume
different states with respect to ligand binding and engagement, and
there are many examples of the transition between these states being
controlled by signaling pathways within the cell, a phenomenon termed
inside-out signaling (12). The affinity of an individual integrin
heterodimer for its ligand may be increased, and changes in the lateral
mobility and clustering of integrins can also affect the avidity of
integrin binding to multivalent ligands. Regulation of either the
affinity or avidity of an integrin for its ligand will have profound
influence on the ability of cells to spread on the extracellular
matrix. The ability of the platelet integrin
IIb
3 to bind fibrinogen has been shown to
be inhibited by dominant negative mutants of Raf-1 or MEK1 (45). This
implicates the MEK/ERK signaling axis in inside-out signaling to
3 integrins, and this could by achieved by influencing
either the affinity state or the clustering of integrin. We favor the
explanation that the recruitment of ERK1 leads to integrin clustering,
since we found that integrin-containing puncta were clearly smaller and
more numerous in the presence of PD98059.
In summary, our studies have identified a novel association of active
ERK1 kinase with
v
3 integrin that is
established in advance of the incorporation of the integrin into focal
complexes. Formation of this complex was not necessary for the
trafficking of
v
3 through the
Rab4-dependent recycling pathway; nor was the activity of
Rab4 required for association of
v
3 with
ERK. However, the activity of ERK1 and Rab4 are clearly required for cells to spread on vitronectin. We suggest that recruitment of ERK1 to
v
3 and the Rab4-dependent
recycling pathway are parallel growth factor-activated events that are
necessary for integrin function.