From the Department of Medicine (GI Division), Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215
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ABSTRACT |
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We examined the possibility that the
6A and
6B cytoplasmic domain
variants of the
6
1 integrin
differentially activate p42 and p44 mitogen-activated protein (MAP)
kinases. P388D1 macrophages that express equivalent surface levels of
either the
6A
1 or
6B
1 integrin were used to examine this
issue. Adhesion to laminin-1 mediated by the
6A
1 integrin triggered activation of a
substantial fraction of total p42 and p44 MAP kinases as assessed using
a mobility shift assay, immunoblot analysis with a phosphospecific MAP
kinase antibody, and an immune complex kinase assay. In contrast, ligation of the
6B
1 integrin did not
trigger significant MAP kinase activation. These data were confirmed by
antibody clustering of the
6
1 integrins.
Both the
6A
1 and
6B
1 integrins were capable of activating
the p70 ribosomal S6 kinase and this activation, unlike MAP kinase
activation, is dependent on phosphoinositide 3-OH kinase. Activation of
MAP kinase by
6
1 requires both Ras and
protein kinase C activity. A functional correlate for differential activation of MAP kinase was provided by the findings that the
6A
1 transfectants migrated significantly
better on laminin than the
6B
1
transfectants and this migration was dependent on MAP kinase activity
based on the use of the MAP kinase kinase (MEK1) inhibitor PD98059. Our
findings demonstrate that the
6
1 integrin can activate MAP kinase, that this activation is regulated by the
cytoplasmic domain of the
6 subunit, and that it relates to
6
1-mediated migration.
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INTRODUCTION |
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The mechanisms by which integrins modulate the activity of the
ERK11 and ERK2 members of the
MAP kinase family are of interest because these kinases regulate cell
growth (1-4) and have been implicated in other important functions
such as cell migration (5). Initial studies demonstrated that MAP
kinase could be activated by integrin-mediated attachment to the
extracellular matrix (6-8). More recently, the possible involvement of
other signaling molecules including Ras and focal adhesion kinase (FAK)
in MAP kinase activation by integrins has been explored, an area that
remains controversial (9-13). These studies, however, did not address
the possibility that specificity exists among integrins for their
ability to modulate MAP kinase activity. Recently, however, this
possibility was substantiated by the finding that only a subset of
integrins (1
1,
5
1, and
v
3)
can activate MAP kinase (14). The ability of these integrins to
activate MAP kinase was found to be mediated by the recruitment and
phosphorylation of Shc and to be specified by the membrane-proximal portion of the extracellular domain of the integrin
subunit, its
transmembrane segment, or both. Other integrins
(
2
1,
3
1, and
6
1) were unable to induce MAP kinase
activation in these assays (14).
An issue that has not been resolved with respect to integrin-mediated
activation of MAP kinase is the possible role of the cytoplasmic domain
of the integrin subunit in regulating this activation. This
possibility is supported by studies that have established a specific
role for this cytoplasmic domain in regulating integrin-mediated
functions (see, e.g., Ref. 15). In this direction, we are
interested in understanding how the
6A and
6B isoforms of the
6 integrin subunit
regulate the signaling properties of the
6
1 integrin. Previously, we reported that
ligation of the
6A
1 integrin in P388D1
macrophages induces a much more marked induction of protein tyrosine
phosphorylation than does ligation of the
6B
1 integrin in the same cells (16). An
important issue that arises from these data is whether the cytoplasmic
domain sequence of the
6 subunit can influence MAP
kinase activation. This issue is also timely because of the report
discussed above concluding that the
6
1
integrin cannot activate MAP kinase (14). In this study, we demonstrate
that the
6 cytoplasmic domain sequence regulates MAP
kinase activation and that the differential activation of MAP kinase by
the
6A
1 and
6B
1 integrins is linked to the ability of
these cells to migrate on laminin-1.
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EXPERIMENTAL PROCEDURES |
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Cells and Cell Culture--
Details on the generation and
characterization of the P388D1/6 transfectants have been
described previously (17). Cells were maintained in RPMI containing 25 mM HEPES, 15% certified fetal bovine serum, and 300 µg/ml G418. The subclones used in this study had equivalent surface
expression of either the
6A
1 or
6B
1 integrins as assessed by
fluorescence-activated cell sorting using the
6 specific
monoclonal antibody, 2B7 (17).
MAP Kinase Assays--
Tissue culture dishes (60 mm, Costar)
were coated at 4 °C overnight with 30 µg/ml laminin purified from
the Englebreth-Holm-Swarm sarcoma as described (18). The dishes
were rinsed twice with phosphate-buffered saline and once with Puck's
Saline A prior to use. Confluent P388D1 cells were removed from tissue
culture dishes by scraping and maintained in suspension for 30 min at room temperature in Puck's Saline A solution containing 0.5 mM MnCl2. Subsequently, the cells were either
kept in suspension or plated on matrix-coated dishes and incubated at
37 °C for the time periods noted in the Fig. legends. Following this
incubation, the cells were washed once with ice-cold phosphate-buffered
saline and then extracted in 0.2 ml of a buffer containing 20 mM HEPES, pH 7.4, 150 mM NaCl, 0.5% Triton
X-100, 0.1% sodium deoxycholate, 1 mM
Na3VO4, 1 mM
phenymethylsulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml
pepstatin, and 5 µg/ml leupeptin. Protein concentration was
determined using the Bradford dye binding assay (Bio-Rad). Total cell
extracts were electrophoresed on SDS-polyacrylamide gels,
transferred to nitrocellulose, and blotted with one of the the
following antibodies as indicated in the figure legends: ERK1 mAbs
(Santa Cruz Biotechnology, Inc. and Transduction Laboratories, Inc.), a
polyclonal ERK antibody (from John Blenis, Harvard Medical School), or
a phosphospecific ERK polyclonal antibody (New England BioLabs, Inc.).
MAP kinase activity was examined in the P388D1 extracts using an immune
complex kinase assay following the manufacturer's protocol (New
England BioLabs). In some experiments, MAP kinase activation was
assessed after mAb clustering of the 6
1
integrins on the surface of the P388D1 transfectants following the
method of Kornberg et al. (19), except that 20 µg/ml mouse
Fc fragment was incubated with the cells prior to the addition of the
integrin antibody. To examine the effects of calphostin C (Sigma),
wortmannin (Calbiochem, La Jolla, CA), and PD98059 (Calbiochem) on MAP
kinase activation, the cells were treated with these pharmacological reagents for 30 min in suspension prior to plating on laminin-1. Samples containing calphostin C were activated by fluorescent light.
p70 Ribosomal S6 Kinase (S6K) Assays-- The same extracts of the P388D1 transfectants described above for the MAP kinase assays were also used to assay the activation of S6K by a mobility shift assay. Briefly, 20 µg of total cell protein obtained from cells either maintained in suspension or adherent to laminin were electrophoresed on an 8% SDS-polyacrylamide gel. Both the phosphorylated and non-phosphorylated forms of S6K were detected by immunoblotting using an S6K-specific polyclonal antibody (provided by John Blenis).
Dominant Negative Ras Transfections--
Transient transfection
experiments were performed with LipofectAMINE (Life Technologies, Inc.)
according to the manufacturer's instructions. Each 100-mm dish was
co-transfected with 2 µg of pCDNA3-HA-ERK1 (John Blenis) and 6 µg of either pMT3-RasN17 (Larry Feig, Tufts School of Medicine) or
empty vector. After 40 h, the cells were removed from the dishes
and either maintained in suspension or plated on laminin-1 for 30 min
as described above. Cell extracts (200 µg of protein) were
immunprecipitated with an HA antibody (12CA5; Boehringer Mannheim).
After washing three times with lysis buffer, the immune complexes were
resuspended in 50 µl of kinase buffer (25 mM HEPES, pH
7.4, 10 mM MgCl2, 1 mM
dithiothreitol) containing 50 µM ATP, 4 µg of myelin
basic protein (MBP), and 5 µCi of [-32P]ATP. The
samples were incubated at 30 °C for 30 min and the reactions were
terminated by the addition of sample buffer and boiling for 5 min.
Subsequently, the samples were resolved by SDS-PAGE and the
phosphorylated MBP was detected by autoradiography.
Migration Assays-- Cell migration assays were performed using 6.5-mm Transwell chambers (8-mm pore size) (CoStar, Cambridge, MA) as described previously (20). Briefly, RPMI-H containing 15 µg/ml laminin was added to the bottom well, and the filters were coated for 1 h at 37 °C. Cells were resuspended at 106 cells/ml in RPMI-H, and 105 cells were added to the top well of the Transwell chambers. After a 24-h incubation in the presence of PD98059 (25 µM) in Me2SO or Me2SO alone, the cells that had migrated to the lower surface of the filters were fixed, stained, and counted as described (20).
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RESULTS AND DISCUSSION |
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The 6A
1 and
6B
1 Integrins Differentially Induce MAP
Kinase Activation in P388D1 Cells--
To determine whether the
6
1 integrin can induce MAP kinase
activation and whether the cytoplasmic domain of the
subunit plays
a role in this event, we used stable subclones of P388D1 macrophages
that expressed either the
6A
1 or
6B
1 integrins at equivalent levels of
surface expression (16, 20). P388D1 cells do not express the
6 integrin subunit, and expression of either the
6A
1 or
6B
1
integrin enables them to adhere to laminin-1 (20). Activation of ERK1
and ERK2 was assessed initially by a mobility shift assay using cells
that were maintained in suspension or plated on laminin-1-coated dishes
for 40 min. In
6A
1-expressing cells
plated on laminin-1, the phosphorylated forms of both ERK1 and ERK2
increased markedly in comparison to cells maintained in suspension
(Fig. 1A, upper
panel). Under identical conditions, significantly less ERK1 and
ERK2 were shifted to the slower mobility species in
6B
1-expressing cells. Densitometric
analysis of data obtained from three separate experiments revealed that
the ratio of the increase in ERK2 phosphorylation induced by laminin-1
in the
6A
1-expressing cells compared
with the increase in the
6B
1-expressing cells was 6.0 ± 1.5.
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Both the 6A
1 and
6B
1 Integrins Induce p70 S6 Kinase
Activation in P388D1 Macrophages--
The issue of whether the
6B
1 integrin is capable of activating
other kinases in P388D1 cells was addressed by examining the
phosphorylation of the p70 S6K, a Ser/Thr kinase that phosphorylates the 40 S ribosomal subunit protein S6 (22, 23). We chose S6K because
its activation has been shown to be independent of the MAP kinase
pathway (24) and because it can be activated by integrin signaling
(25). The ability of the
6A
1- and
6B
1-expressing cells to activate S6K in
response to laminin adhesion was examined by a mobility shift assay.
Extracts from cells that were maintained in suspension or adherent to
laminin-1 were immunoblotted with a polyclonal antibody that recognizes
both the phosphorylated and non-phosphorylated forms of S6K. Adhesion
to laminin-1 induced a dramatic shift to the more slowly migrating,
phosphorylated forms of S6K in both the
6A
1- and
6B
1-expressing cells (Fig. 2A), indicating that both the
6A
1 and
6B
1
integrins can activate S6K in these cells. Interestingly, S6K
activation was completely inhibited by 100 nM wortmannin
(Fig. 2A), but MAP kinase activation was affected minimally
by this inhibitor (Fig. 2B). These findings demonstrate that
the
6B
1 integrin is capable of
signaling.
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6A
1 Integrin-mediated MAP Kinase
Activation Depends on the Activity of Ras and PKC--
To investigate
the role of the small G protein Ras in the
6
1-mediated MAP kinase activation,
6A
1-expressing P388D1 cells were
co-transfected with cDNAs that encode a hemagglutinin
epitope-tagged ERK1 (HA-ERK1) and a dominant negative form of Ras
(RasN17) that blocks guanine nucleotide exchange factors involved in
the activation of endogenous Ras (26). Cells that expressed these
constructs were either maintained in suspension or plated on laminin-1
for 30 min and HA-immune complexes obtained from extracts of these cells were assayed for their ability to phosphorylate MBP. As shown in
Fig. 3A, MBP phosphorylation
in response to laminin-1 attachment was significantly inhibited in
cells that expressed RasN17. A RasN17-dependent reduction
in the basal level of MBP phosphorylation of cells in suspension was
also observed (Fig. 3A). Although these findings suggest
that
6
1-stimulated MAP kinase activation
is through the canonical Ras pathway in these cells, it should be noted
that the involvement of Ras, as well as FAK, in integrin-mediated MAP
kinase activation remains controversial in other cells (9-13). At the
very least, we can conclude that FAK is not important for
6
1-mediated activation of MAP kinase in
P388D1 cells because these cells, similar to other macrophages, do not
express this kinase.2
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MAP Kinase Activation Is Required for P388D1 Migration on
Laminin--
We reported previously that
6A
1-expressing P388D1 cells migrate
markedly better on laminin-1 than the
6B
1
cells (20). Indeed, this finding has been substantiated by other
studies that have linked the
6A
1 integrin
to cell migration (30, 31). For this reason, we examined the
possibility that the differential activation of MAP kinase by the
6A
1 and
6B
1
integrins is linked to the ability of these integrins to mediate cell
migration on laminin-1. For this purpose, migration assays were
performed in the presence of the MEK1 inhibitor PD98059. This inhibitor
blocked the
6A
1-induced phosphorylation
of both ERK1 and ERK2 (Fig. 4A) but had no effect on S6K
activation (data not shown). The
6A
1-expressing cells were approximately
2.5-fold more migratory on laminin-1 than the
6B
1 cells (Fig. 4B) consistent
with our previous results (20). PD98059 inhibited the migration of
6A
1 cells by 60% at a concentration
range of 12.5-25 µM. The slower migration of the
6B
1-expressing cells was also inhibited
slightly by PD98059, suggesting that the haptotactic migration of these cells is dependent on MAP kinase and that the differential activation of MAP kinase by the
6A
1 and
6B
1 integrins regulates this migration.
The enhanced ability of the
6A
1-expressing cells to migrate on
laminin-1 was not observed on other matrix proteins (20) indicating
that a specific, ligand-dependent activation of MAP kinase
is required. These results are interesting in view of the recent report
that MAP kinase can regulate cell migration by its ability to
phosphorylate myosin light chain kinase (5). For this reason, it will
be informative to determine if the
6A
1 and
6B
1 integrins differ in their ability
to induce the phosphorylation of this kinase.
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ACKNOWLEDGEMENTS |
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We thank Dr. Wengui Yu for helpful discussions on the MAP kinase experiments. We also thank John Blenis and Larry Feig for providing reagents.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant AI39264 (to A. M. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Beth Israel Deaconess
Medical Center, Dana 601, Harvard Medical School, 330 Brookline Ave.,
Boston, MA 02215. Tel.: 617-667-7714; Fax: 617-975-5071; E-mail:
amercuri{at}bidmc.harvard.edu.
1 The abbreviations used are: ERK, extracellular-signal regulated kinase; MAP, mitogen-activated protein; S6K, p70 ribosomal S6 kinase; MBP, myelin basic protein; FAK, focal adhesion kinase; PKC, protein kinase C; mAb, monoclonal antibody; Ab, antibody; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin.
2 J. Wei and L. M. Shaw, unpublished results.
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REFERENCES |
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