From the Craniofacial Developmental Biology and
Regeneration Branch, NIDCR, National Institutes of Health, Bethesda,
Maryland 20892-4370 and the
Laboratory of Cell Signaling, NHLBI,
National Institutes of Health, Bethesda, Maryland 20892-0320
Received for publication, January 27, 2003, and in revised form, March 4, 2003
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ABSTRACT |
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Integrin transmembrane receptors generate
multiple signals, but how they mediate specific signaling is not clear.
Here we test the hypothesis that particular sequences along the
Integrin transmembrane receptors sense the extracellular
environment and convey bidirectional signals regulating cell behavior and fate (for recent reviews, see Refs. 1-6). Signals transmitted by
integrins regulate cell migration, growth, differentiation, and
decisions for survival or death. Cell survival can be regulated through
integrin-mediated modulation of cell shape and adhesion in cooperation
with growth factors or integrin control of the activity of tyrosine and
serine/threonine kinases such as
FAK,1 Src, and
integrin-linked kinase (see Refs. 4, 5, 7, 8, and 9-12). A variety of
integrin heterodimers and mechanisms have been implicated in
integrin-mediated survival of various cell types (13-24), indicating
the complexity of this signaling.
An important effector of cell survival is protein kinase B (PKB), also
called Akt (25-28). Akt is a Ser/Thr protein kinase that becomes fully
active after dual phosphorylation of Thr308 and
Ser473 (29-31). Integrins can act as positive modulators
of Akt by stimulating its activation pathway involving upstream
phosphoinositide 3-kinase (PI3K) (18, 19, 32).
Recently, it was shown that an integrin can also regulate the
inactivation of Akt by affecting protein phosphatase 2A (PP2A) (33).
PP2A is an abundant and ubiquitous Ser/Thr phosphatase that is involved
in the regulation of a wide variety of cellular activities. Its
substrate specificity is tightly regulated by subunit composition,
post-translational modifications, and association with different
intracellular components (see Refs. 34-36). PP2A can dephosphorylate
and inactivate Akt (37-40); it can also associate with the
Although Previous mutational studies of To test our hypothesis of site-specific integrin regulation of
signaling, we tried to identify an amino acid residue within the
Reagents and Antibodies--
Antibodies to human
Sheep anti-mouse magnetic microbeads (4.5 µm) were purchased from
Dynal. Okadaic acid, AG1433, PD98059, and UO126 were from Calbiochem.
Human plasma fibronectin was purified as previously described (52). The
pHA262pur puromycin resistance plasmid was generously provided by Dr.
Hein te Riele (Netherlands Cancer Institute) (53).
Generation of Mutant Cell Culture, Transfection, and Establishment of Stable Cell
Lines--
The GD25 Integrin Activation by Clustering and Isolation of Integrin-based
Complexes--
Sheep anti-mouse IgG magnetic beads (4.5 µm; Dynal)
were coated with TS2/16 or K20 antibodies according to the
manufacturer's instructions. GD25 cells expressing
For the isolation of integrin-based complexes, serum-starved cells were
mixed with beads (five beads/cell) coated with K20 antibody and
processed as described (51). Briefly, the complexes of beads and bound
cells were isolated with a Dynal magnetic concentrator and were washed
with 1 ml of cold CSK buffer (50 mM NaCl, 300 mM sucrose, 3 mM MgCl2, protease
inhibitor mixture (Roche Applied Science), 1 mM sodium
vanadate, 50 mM NaF, and 1 mM
phenylmethylsulfonyl fluoride in 10 mM PIPES, pH 6.8)
without detergent. The pellet containing cell-bead complexes was
extracted with 0.5 ml of cold CSK buffer containing 0.5% Triton X-100
and sonicated for 10 s with a 50-watt ultrasonic processor (model
GE 50 set at amplitude 20; Aldrich). The bead-protein complexes were
washed five times with 1 ml of cold CSK buffer containing detergent and
prepared for Western blot analysis or PP2A assay. Phosphatase
inhibitors were omitted from the CSK buffer when integrin-based
complexes were prepared for assaying PP2A.
Co-immunoprecipitation and Immunoblotting--
GD25 cells
expressing Flow Cytometry, Immunofluorescence, and Apoptosis Assay--
For
flow cytometry, cells were detached with trypsin-EDTA, washed with
culture medium, and incubated with the indicated antibodies for 30 min
on ice in PBA buffer (phosphate-buffered saline, 0.5% bovine serum
albumin, 0.02% NaN3). Stained cells were washed, incubated
with fluorescein isothiocyanate-conjugated secondary antibodies in the
same buffer for an additional 30 min, washed again, and analyzed using
a FACSCalibur flow cytometer (BD Biosciences). For cell sorting,
trypsinized cells were washed and stained in complete medium with
fluorescein isothiocyanate-conjugated anti-human
Cells for immunofluorescence analysis were plated on fibronectin (5 µg/ml) precoated glass coverslips (12 mm; Carolina Biological Supply
Co.) and cultured overnight in the absence of serum. Samples were fixed
with 4% paraformaldehyde in phosphate-buffered saline containing 5%
sucrose for 20 min and permeabilized with 0.5% Triton X-100 in
phosphate-buffered saline for 3 min. Primary antibodies were used at 10 µg/ml and visualized with secondary CY3- or fluorescein isothiocyanate-conjugated antibody.
For assaying apoptosis induced by serum starvation, cells were cultured
in the absence of serum for 72 h. After this period, the cells
were fixed and stained without permeabilization with 2 µM
Hoechst 33342 fluorochrome for 5 min. Stained samples were mounted in
GEL/MOUNTTM (Biomeda Corp.) containing 1 mg/ml
1,4-phenylenediamine (Fluka) to reduce photobleaching.
Immunofluorescent images were obtained with a Zeiss Axiophot microscope
equipped with a Photometrics CH350 cooled CCD camera. Digital images
were obtained using MetaMorph 3.5 software (Universal Imaging). The
same software was used to score the number of apoptotic (bright) and
nonapoptotic (dark) nuclei from each of two randomly chosen fields from
samples obtained from three separate experiments.
Akt Dephosphorylation, Akt Kinase, MAPK, and PP2A Phosphatase
Assays--
Cells were cultured overnight on fibronectin-coated dishes
(5 µg/ml) in DMEM, 1% bovine serum albumin and then stimulated with
20 ng/ml PDGF BB for 15 min, washed, and cultured in the same medium
containing 30 µM PDGF kinase inhibitor AG 1433. Samples were collected at the indicated time points after PDGF stimulation and
were prepared for Western blot analysis with anti-phospho-Akt (Ser473) antibody. In some experiments, the cells were
pretreated for 15 min with 1 µM okadaic acid before
stimulation with PDGF, and the same concentration of this PP2A
inhibitor was present throughout the entire experiment.
Akt kinase activity was evaluated using an Akt kinase assay
kit from Cell Signaling according to the manufacturer's protocol. Briefly, cells cultured as described above were lysed, endogenous Akt
was immunoprecipitated, and its ability to phosphorylate a recombinant
GSK-3
For assaying MAPK activity and Akt phosphorylation after spreading on
fibronectin, cells were trypsinized, washed with trypsin inhibitor, and
rotated for 30 min in DMEM, 1% bovine serum albumin at 37 °C. Equal
numbers of cells were plated on dishes precoated with 5 µg/ml
fibronectin and blocked with 1% heat-denatured bovine serum albumin.
Homogenates of cells in suspension were analyzed by Western blotting
with anti-phospho-MAPK and anti-phospho-Akt antibodies after the
indicated plating times. Relative phosphorylation levels for each time
point were calculated as a ratio between densitometry readings obtained
using anti-phospho-MAPK and anti-total MAPK antibodies or
anti-phospho-Akt and anti-actin antibodies.
PP2A activity was measured with a Ser/Thr phosphatase assay kit
(Upstate Biotechnology) according to the manufacturer's protocol. In
some experiments, a specific phospho-Akt1/PKB peptide (Upstate Biotechnology) was used as a substrate. Total PP2A activity was measured after immunoprecipitation of the PP2Ac catalytic subunit with
anti-PP2A antibody clone 1D6. PP2A activity in integrin-based complexes
was measured after their isolation with anti- PI3K Assay and Measurement of Phosphoinositide Content--
Cell
lysates were obtained from serum-starved cells after overnight plating
on fibronectin as described above. The lysates were immunoprecipitated
with agarose-conjugated 4G10 anti-phosphotyrosine antibody or with a
mixture of isoform-specific anti-p110 PI3K or anti-p85 PI3K antibodies.
PI 3-kinase was assayed as described (54). After thin layer
chromatography on LK6D plates (Whatman), 32P-labeled
phosphoinositides were detected by autoradiography.
For phosphoinositide measurement, serum-starved cells plated on
fibronectin were washed with phosphate-free DMEM and labeled with 0.5 mCi/ml ortho[32P]phosphate (Amersham Biosciences) for
1 h. Phospholipids were extracted from the cells as previously
described (55), and the chloroform phase was vacuum-dried and subjected
to deacylation. Samples were dissolved in methylamine solution (33%
methylamine in ethanol (Fluka)/water/n-butanol; 5/2/1
(v/v/v)), incubated at 53 °C for 1 h, and vacuum-dried. The
dried material was dissolved in water, and the acyl moieties were
removed by two chloroform extractions. The aqueous phases containing
the glycerophosphoinositol phosphates were analyzed by HPLC with a
Partisil SAX 10 column using a 70-min nonlinear gradient of 0-1
M NH4H2PO4 at a flow rate of 1.2 ml/min. Radioactivity in the eluate was monitored with an
on-line Flow-One detector (Packard). Retention times of individual
glycerophosphoinositol phosphates were determined using radiolabeled
standards. Results are expressed as the percentage of total
phospholipid radioactivity attributed to each deacylated phosphoinositide species.
Integrin-mediated Regulation of Akt Activity and Cell Survival
Depends on a Single Conserved Tryptophan within the Integrin
Cytoplasmic Domain--
To test our hypothesis that a particular
sequence or site might be involved in selective integrin signaling, we
focused on integrin-regulated Akt (PKB) signaling, which is relevant to
cell survival but is poorly understood. We first identified the amino acid residues that are conserved between six human
Each of the point-mutated human
The first screen was for effects on the activity of the
serine/threonine protein kinase Akt/PKB, which plays a central role in
the survival of a wide range of cell types (25-28). The activity of
Akt, which is regulated by phosphorylation, was probed with phosphospecific antibodies. The level of phosphorylation on
Ser473 in each of the sorted cell lines was determined on
whole cell lysates in comparison with Akt phosphorylation in cells
expressing wild type
Full activation of Akt occurs via phosphorylation of both
Thr308 in the kinase domain and of Ser473 in
the carboxyl-terminal regulatory domain of the molecule (29-31). In
W775A-expressing cells, both residues showed a 5-fold decrease in
phosphorylation when the cells were grown
in the absence of serum (Figs.
2A and 3A) and a
2-fold decrease in the presence of 10% fetal bovine serum (not shown)
when compared with parallel Tryptophan Mutation Specifically Targets the Akt Signaling
Pathway--
Integrins participate in a number of signaling pathways
that are often complex and interdependent. To test whether mutation of
the tryptophan residue of the Downstream Effects of the W775A Mutation on the Akt Signaling
Pathway--
The decrease of Akt phosphorylation in W775A
integrin-expressing cells was reflected in a similar 5-fold decrease in
Akt kinase enzymatic activity when compared with
The strong suppression of the Akt pathway in cells expressing the
integrin tryptophan point mutant suggested that these cells would be
more vulnerable to apoptosis. Indeed, when GD cell lines were subjected
to serum starvation for 3 days, more than 35% of the W775A cells
showed apoptotic nuclei, whereas only 5% of the cells expressing the
wild type Mutation of the Tryptophan Does Not Interfere with Major
Cytoplasmic Interactions and with Targeting of the Mutant Integrin to
Focal Adhesions--
Introduction of amino acid substitutions might
lead to conformational changes in the integrin cytoplasmic domain that
could affect protein-protein interactions even distal to the mutation. Although the mutated tryptophan is not immediately adjacent to the
membrane-proximal sequence KLLXXXXD, which is important for integrin heterodimer formation (60, 61), we confirmed the continued
ability of the W775A integrin to dimerize with the
Several cytoplasmic proteins have been shown to bind directly or
indirectly to the
Functional Tryptophan Mutation Partially Reduces the
Ligand-binding/Activation State of
Integrin ligation and clustering have been linked to Akt activation
(17, 20, 73). Our findings raised the possibility that the moderately
lower activation state of the W775A integrin mutant might contribute to
the observed Akt inhibition and suggested that reactivation with
antibodies might compensate for this deficiency. To test this
possibility directly, wild type or mutant integrins were clustered with
beads coated with activating antibody TS2/16 (Fig. 5B) or
activated by plating the cells on fibronectin-coated surfaces (Fig.
5C). Although the clustering and activation of Tryptophan Mutation Does Not Affect Signaling Pathways Responsible
for Akt Activation--
Current models for Akt regulation postulate
that Akt phosphorylation depends on the activity of the upstream enzyme
PI3K, and inhibitors of this kinase such as wortmannin prevent Akt
activation (29, 75, 76). We tested the activity of PI3K in
The protein kinase that phosphorylates Akt on Thr308 is
3-phosphoinositide-dependent protein kinase 1 (PDK1) (30,
77). Although PDK1 is considered constitutively active, recent studies
have demonstrated that autophosphorylation at Ser421 is
essential for the activity of PDK1 (78) and that its plasma membrane
localization is important for activation of Akt (79). PDK1 in both
The activation of Akt is mediated by the PI3K products
phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol
3,4-bisphosphate. Akt binds via its pleckstrin homology domain to one
or both of these phosphoinositides at the plasma membrane, which alters
Akt conformation and renders it accessible to phosphorylation (75, 77).
The availability of these phosphoinositides depends not only on PI3K
activity but also on their rate of degradation by lipid phosphatases
like phosphatase and tensin homologue deleted on chromosome 10 (PTEN) (80). To evaluate the balance between the production and
degradation of these lipid mediators, we quantified the
phosphoinositides of 32P-radiolabeled GD25 The Tryptophan Mutation Causes Local Activation of PP2A in Integrin
Complexes--
The level of Akt phosphorylation at steady state is the
result of an equilibrium between specific kinase and phosphatase
activities that act on the Thr308 and Ser473
residues of Akt (38). To examine Akt-specific phosphatase activity, cells were stimulated with PDGF to phosphorylate and activate Akt, and
samples were taken every 15 min to assess the subsequent decreases in
phosphorylation of Akt. After the initial PDGF stimulation, cells were
maintained in the presence of AG1433, a PDGF receptor kinase inhibitor,
in order to eliminate residual receptor kinase activity and to be able
to identify just the rate of dephosphorylation. Immediately after
stimulation, both cell lines showed strong stimulation of Akt
phosphorylation, confirming that the inhibition in W775A cells is also
fully reversible by a growth factor (Fig.
7A, PDGF). Nevertheless, whereas cells expressing the wild type
Activation of total cellular PP2A by the
We hypothesized that a specific subpopulation of PP2A associated with
To test the activity of the integrin-associated PP2A fraction, integrin
complexes were isolated from In this study, we tested the hypothesis that signaling selectivity
exists at specific sites along integrin cytoplasmic tails. If such a
site-specific function were to exist, interference with a particular
site should selectively inhibit only a single signal transduction
pathway. We focused on the serine/threonine kinase Akt, since it is a
primary regulator of cell survival, and the mechanisms by which
integrins regulate its activity are not clearly established. In support
of our hypothesis, we have identified a key tryptophan residue at
position 775 of the To search for a specific signaling site, amino acid residues that are
conserved between the different human The activation of Akt is achieved by phosphorylation at two sites,
Thr308 (by PDK1) and Ser473 (by an unresolved
kinase), and it is dependent on the availability of
phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate produced by PI 3-kinase (31, 75, 79). Integrins can
function as upstream regulators of PI3K, thus activating Akt and cell
survival (18, 19, 32). Since the W775A integrin mutant inhibited Akt
phosphorylation at both residues and increased apoptosis 7-fold,
we tested for the activity of PI3K and PDK1 and the levels of
Akt-activating phosphoinositides present in cells with this mutant.
Unexpectedly, neither the kinase activities nor relevant
phosphoinositide levels differed when cells expressing wild type and
mutant integrins were compared, implying that the PI3K-dependent Akt-activating pathway was not affected by
the integrin mutation.
Focal adhesion kinase has been identified as an upstream intermediate
in integrin survival signaling. FAK forms a complex with
p130cas that activates the JNK survival pathway (13) and is
capable of suppressing the p53-regulated cell death pathway (14). FAK- and Shc-dependent increases in Bcl-2 protein expression in
response to integrin engagement that protect Chinese hamster ovary
cells from apoptosis have also been described (17). However, the
effects of the The ability of MAPK (extracellular signal-regulated kinase) to protect
against cell death has also been documented (15, 16). Integrins can
activate the MAPK pathway through multiple mechanisms (see Refs. 11,
12, 86), and interference with this activation may influence cell
survival. Regardless of whether the tryptophan was mutated, integrins
were able to elicit similar MAPK responses immediately after plating of
the cells or after prolonged incubation on fibronectin. Moreover,
blocking MAPK activity with the mitogen-activated extracellular
signal-regulated kinase-activating kinase 1/2 inhibitors PD 98059 and
U0126 did not inhibit Akt phosphorylation in GD Our results underscore the importance of inhibition rather than
defective activation of Akt. Indeed, we found that local activation of
protein phosphatase 2A accounted for the W775A integrin effect on Akt.
GD25 cells stably expressing the tryptophan integrin mutation dephosphorylated Akt much faster than cells expressing wild type integrin, and this process was blocked by the specific PP2A inhibitor okadaic acid. Trp775 is a well conserved residue present in five 1 integrin cytoplasmic domain may exist that are
intimately related to specific integrin-mediated signaling pathways.
Using systematic alanine mutagenesis of amino acids conserved between
different
integrin cytoplasmic domains, we identified the
tryptophan residue at position 775 of human
1 integrin
as specific and necessary for integrin-mediated protein kinase B/Akt
survival signaling. Stable expression of a
1 integrin
mutated at this amino acid in GD25
1-null cells resulted
in reduction of Akt phosphorylation at both Ser473 and
Thr308 activation sites. As a consequence, the cells were
substantially more sensitive to serum starvation-induced apoptosis when
compared with cells expressing wild type
1 integrin.
This inactivation of Akt resulted from increased dephosphorylation by a
localized active population of protein phosphatase 2A. Both Akt and
protein phosphatase 2A were present in
1
integrin-organized cytoplasmic complexes, but the activity of this
phosphatase was 2.5 times higher in the complexes organized by the
mutant integrin. The mutation of Trp775 specifically
affected Akt signaling, without effects on other integrin-activated
pathways including phosphoinositide 3-kinase, MAPK, JNK, and p38 nor
did it influence activation of the integrin-responsive kinases focal
adhesion kinase and Src. The identification of Trp775 as a
specific site for integrin-mediated Akt signaling supports the concept
of specificity of signaling along the integrin cytoplasmic domain.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 integrin (41), suggesting the possibility of a close spatial/functional relationship between these molecules.
integrin subunit cytoplasmic domains are relatively short
(except for
4, which is 1000 amino acids, the rest range between 46 and 70 residues), they are saturated with binding sites for
cytoplasmic proteins that participate in integrin-mediated signaling
and cytoskeletal interactions (see Refs. 1, 42, and 43). We
hypothesized that different sequences within the integrin tail are
essential for regulation of distinct pathways of signal transduction.
In fact, a site conserved between different integrin subunits might be
dedicated to a unique signaling pathway, such that a specific integrin
mutation would affect only one out of many integrin-mediated signaling pathways.
integrin cytoplasmic domains have
focused mainly on the possible roles of phosphorylation sites along the
integrin tail (44-48). They have documented the importance of these
residues for basic cellular processes including adhesion, organization
of the cytoskeleton and focal adhesions, and cell migration without
identifying effects on specific signaling pathways.
1 integrin cytoplasmic domain that was relatively
specifically involved in integrin-mediated survival signaling. Here we
report the identification of tryptophan 775 in the
1
integrin cytoplasmic domain as being both necessary and specific for
Akt activation and cell survival. This residue was found to be involved
in interactions that regulate the local activity of PP2A within
integrin-organized complexes. We also found that Akt is a constituent
of these complexes and is a substrate for PP2A. The interactions
mediated by Trp775 appear to be specific for Akt signaling,
since we were unable to identify any other integrin-mediated signaling
pathways affected by mutating this residue.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 integrins included rat monoclonal antibody 9EG7
(Pharmingen), mouse monoclonal antibodies 12G10 (49), TS2/16 (50), and
K20 (Immunotech), and rabbit antibody Rab 4080 (51). Anti-mouse
5 integrin antibody (monoclonal antibody 1928) was from
Chemicon. Antibodies against actin, tubulin, and vinculin were from
Sigma; anti-total Akt, anti-phospho-Akt, anti-phospho-PDK1, and
anti-total PDK1 antibodies and the Akt kinase assay kit were from Cell
Signaling; anti-PP2A and anti-phospho-FKHRL1 were from Upstate
Biotechnology, Inc.; anti-phospho-FAK and anti-phospho-Src were from
BioSource; anti-phospho- and anti-total p38 and anti-phospho- and
anti-total mitogen-activated protein kinase (MAPK) were from New
England Biolabs; anti-Bcl-2 and anti-phospho- and anti-total c-Jun
N-terminal kinase (JNK) were from Santa Cruz; and anti-
-actinin was
from ICN. Anti-talin rabbit polyclonal antibody was generously provided
by Dr. Keizo Takenaga (NIDCR, NIH, Bethesda, MD). Cy3- and fluorescein
isothiocyanate-conjugated secondary antibodies were from Jackson
ImmunoResearch Laboratories.
1A cDNAs--
Human
1A integrin cDNA was kindly provided by Erkki
Ruoslahti (Burnham Institute). Point mutations in the integrin cDNA
were introduced by the QuikChangeTM site-directed
mutagenesis kit (Stratagene) according to the manufacturer's protocol
using the primers listed in Table I. The
resulting constructs were sequenced to confirm the presence of the
desired mutations and the absence of any other alterations introduced
during manipulation.
Oligonucleotide primers used to generate 1 integrin mutants
1-null fibroblast cell line was a
generous gift from Reinhard Fässler (Lund University). Cells were
cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10%
fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml fungizone. Plasmid DNAs encoding wild type or mutant human
1A integrins were co-transfected together with the
puromycin selection vector pHA262pur by electroporation as described
(51). Pools of mixed populations of stable transfectant cells
expressing comparable levels of each of the wild type or mutant
integrins were established by selection in medium containing 10 µg/ml
puromycin and by four consecutive fluorescence-activated cell sortings
with anti-human
1 integrin antibody K20 performed over a
3-month period. Cells expressing the W775A mutation were used up to
passage 10 after establishing a pool of positive cells. Due to
increased apoptosis of the mutant transfectant, a
1
integrin-negative subpopulation becomes detectable after this passage.
1
WT or W775A integrins were trypsinized and serum-starved in
suspension in DMEM with 1% bovine calf serum for 3 h. The
serum-deprived cells were mixed with TS2/16-coated beads (5 beads/cell), and clustering and activation of integrins was allowed to
continue for 1 h at 37 °C with rotation. The complexes of beads
and bound cells were collected by a magnetic particle concentrator
(Dynal) and washed once with phosphate-buffered saline, and then the
cells were lysed and prepared for Western blot analysis.
1 WT or W775A integrins were plated on
fibronectin-coated dishes (5 µg/ml) in DMEM with 1% bovine serum albumin. After overnight incubation, the cells were solubilized on ice
in Nonidet P-40 buffer (137 mM NaCl, 1 mM
CaCl2, 1 mM MgCl2, 1% Nonidet
P-40, 10% glycerol, 20 mM Tris-HCl, pH 8.0, 1 mM sodium vanadate, 50 mM NaF) containing
protease inhibitors and 1 mM phenylmethylsulfonyl fluoride.
Homogenates were clarified by centrifugation at 20,000 × g for 15 min at 4 °C. Immunoprecipitates were obtained
using 4 µg of the indicated antibodies and GammaBindTM
Plus SepharoseTM (Amersham Biosciences). Protein complexes
bound to beads were solubilized in reducing SDS-PAGE sample buffer and
resolved on 4-12% gradient gels (Novex). After electrotransfer to
nitrocellulose membranes (Novex), the filters were blocked (5% nonfat
dry milk in 150 mM NaCl, 50 mM Tris HCl, 0.1%
Tween 20, pH 7.4) and probed with the indicated phosphospecific or
general antibodies, followed by the appropriate secondary horseradish
peroxidase-conjugated antibodies. Immunoblots were visualized using the
ECL system and Hyperfilm x-ray film (Amersham Biosciences).
1
integrin antibody K20 (Immunotech) for 30 min on ice, washed with
complete medium without phenol red, and sorted using a FACStar plus
cell sorter (BD Biosciences).
/
polypeptide containing residues surrounding Ser21 and Ser9 was assessed with
phosphospecific antibodies against these serine residues.
1 integrin antibody K20. After the phosphatase reaction, each sample was analyzed
by Western blotting with anti-PP2A antibodies for determination of PP2A
protein content. PP2A activity was calculated as the ratio between
released free phosphate measured as absorbance at 650 nm with the
phosphatase assay kit and the densitometry signal for PP2A according to
Western blotting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
integrin cytoplasmic tails and substituted an alanine residue sequentially for
each of them in the human
1 integrin sequence (Fig.
1A). We omitted the well
studied conserved NPXY motifs known to be important
for a variety of basic integrin functions including integrin
localization, focal adhesion organization, and cell migration (56, 57),
because mutations in these NPXY regions could affect signal
transduction indirectly in complex ways.
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Fig. 1.
Substitution of the conserved tryptophan 775 residue of the 1 cytoplasmic
domain inhibits integrin-mediated Akt signaling. A,
conserved amino acid residues (boldface type and
underlined) in the human
1A integrin
cytoplasmic domain were identified as residues that are found five or
more times at comparable sites in human
1,
2,
3,
5,
6,
and
7 sequences. They were separately mutated to Ala in
order to generate a set of point mutant human
1
integrins. B, each mutant was stably expressed in
1-null mouse GD25 cells and FACS-sorted with anti-human
integrin antibody K20 to obtain mixed cell populations with similar
levels of integrin expression. The stable transfectant cell lines were
plated overnight on fibronectin in the absence of serum and lysed, and
the levels of Akt phosphorylation were determined by Western blotting
with antibody against phospho-Ser473 Akt. Note that cells
expressing the integrin W775A mutation show a 5-fold decrease in Akt
phosphorylation. C, the expression of comparable levels of
mutant integrins was verified by Western blotting with anti-integrin
1 cytoplasmic domain antibody (Rab 4080). Actin was used
as the internal control for loading. *, p < 0.001.
1 integrins was stably
transfected into
1-null mouse GD25 cells (58). Mixed
populations of positive transfectants with similar levels of integrin
expression (Fig. 1C) were obtained by fluorescence-activated
cell sorting (see "Experimental Procedures") in order to avoid
potential variations associated with the use of single clones or
different expression levels of the mutated integrins.
1 integrin (Fig. 1B).
Only one mutation (W775A) among the nine conserved integrin sites
tested by alanine mutagenesis showed a statistically significant
(p < 0.001) decrease in Akt phosphorylation. This
result suggested that the tryptophan residue at position 775 is
important for integrin-regulated Akt activation. This mutant was used
in all subsequent studies.
1 WT transfectants.
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Fig. 2.
W775A mutation specifically affects Akt
phosphorylation. A, GD25 cells expressing wild type
( 1 wt) or tryptophan mutant (W775A)
integrins were cultured overnight on fibronectin in the absence of
serum in medium supplemented with 0.1 mg/ml GRGDS peptide.
Phosphorylation levels of the indicated kinases were analyzed by
Western blotting with phosphospecific antibodies. Tubulin levels or the
total quantity of each kinase regardless of its phosphorylation state
(total-MAPK; total-JNK; total-p38) were used as internal
controls for loading. Boxed results were obtained from the same
nitrocellulose membrane. B, cells cultured on fibronectin
were serum-starved overnight, trypsinized, and maintained in suspension
in medium without serum for an additional 30 min. The cells were then
allowed to attach to fibronectin (5 µg/ml)-coated dishes for the
indicated times. Lysates from samples taken before plating (0 time) and
at the end of each time point (15-60 min) were analyzed by Western
blotting with antibodies against phosphorylated and total MAPK.
Densitometry obtained with anti-phospho-MAPK antibody was normalized
against anti-total MAPK, and -fold changes in MAPK phosphorylation
after attachment compared with suspension were calculated.
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Fig. 3.
GD25 cells expressing W775A
1 integrin have suppressed Akt
signaling and are more sensitive to apoptosis after serum starvation.
A, GD25 cells expressing wild type (
1
wt) or tryptophan mutant (W775A) integrins were
cultured overnight on fibronectin in the absence of serum, and the
levels of Akt phosphorylation of Ser (p-Akt(Ser473))
and Thr (p-Akt(Thr308)) residues were analyzed by
Western blotting with phosphospecific antibodies. The total amount of
Akt (total-Akt) was used as an internal control for loading.
B, lysates from cells cultured as described for A
were used to test Akt kinase activity in vitro. Endogenous
Akt was immunoprecipitated from 50 µl (lane 1), 100 µl
(lane 2), and 200 µl (lane 3) of lysates from
cells expressing wild type (
1 wt) or tryptophan
mutant (W775A) integrins. The Akt-induced phosphorylation of
a recombinant GSK-3 fusion protein was measured by Western blotting
with antibodies against phospho-GSK-3
serine 21 and phospho-GSK-3
serine 9 (p-GSK-3
/
(Ser21/9)). The
amounts of immunoprecipitated Akt were verified by probing the same
membrane with anti-Akt antibodies (total-Akt). C,
the same cell lysates described for A were probed with
phosphospecific antibodies against the Akt substrate FKHRL1 residues
threonine 32 (p-FKHRL1(Thr32)) and serine 253 (p-FKHRL1(Ser253)). Tubulin was used as an internal
control for the amount of protein loaded from each sample.
D, cells were plated on fibronectin, serum-starved for
78 h, fixed, and then stained with Hoechst 33342 without
permeabilization. Apoptotic (arrows in the
insets) and nonapoptotic (arrowheads in the
insets) nuclei were scored, and the percentage of apoptotic
cells in each sample was determined. Bar, 100 µm.
1 integrin cytoplasmic
domain selectively affects only the Akt survival-related pathway or in addition affects other signaling pathways, we assayed for effects on
important cellular kinases that respond to integrin activation. Activation levels of these kinases were estimated by their level of
site-specific phosphorylation. Activities were measured under steady-state conditions (overnight incubation) in the presence of 0.1 mg/ml RGD peptide. The latter treatment blocks
v
3 integrin-mediated adhesion of GD25
cells, forcing the GD25
1 WT and GD25 W775A cells to
depend on
1 integrins for attachment to fibronectin (47). The major autophosphorylation site of focal adhesion kinase (Tyr397) as well as the Src activation (Tyr418)
and inhibitory (Tyr529) sites were phosphorylated to
similar levels in the two transfectant cell lines (Fig. 2A,
p-FAK (Tyr397),
p-Src(Tyr418), and p-Src
(Tyr529)), suggesting that both kinases are
equally active. Comparable phosphorylation levels were also found for
MAPK, JNK, and p38 MAPK (Fig. 2A, p-MAPK
(Tyr202/Tyr204), p-JNK
(Thr183/Tyr185),
and p-p38
(Thr180/Tyr182)). Moreover,
the dynamic response of MAPK following integrin engagement after
plating on fibronectin was very similar, with maximal activation after
15 min and a nearly complete decline by 1 h after plating (Fig.
2B), implying that the mutant and wild type integrins had
equal abilities for MAPK activation. Similar phosphorylation levels
were also observed for the adapter protein Shc and Crk-associated
substrate p130cas when probed with general
anti-phosphotyrosine antibodies after immunoprecipitation (not shown).
In summary, the phosphorylation levels of all of the
integrin-responding signaling molecules tested were parallel in both
1 WT- and W775A-expressing cell lines, with the sole
exception of Akt (Fig. 2A, p-Akt
(Ser473)). These results demonstrate that the tryptophan
mutation specifically affects Akt signaling, leaving other
integrin-related pathways fully operational.
1 WT
integrin-expressing cells in an in vitro kinase assay (Fig.
3B). This deficiency in Akt activation was associated with
decreased phosphorylation of FKHRL1, which is a member of the Forkhead
family of transcription factors and is one of the downstream effectors
of Akt. Both residues Thr32 and Ser253 of
FKHRL1, which have been shown to be direct targets of Akt kinase (59),
were considerably less phosphorylated in W775A-expressing cells when
compared with FKHRL1 in cells expressing wild type
1
integrin (Fig. 3C).
1 integrin were apoptotic (Fig. 3D).
5 subunit. Co-immunoprecipitation experiments showed that the endogenous mouse
5 integrin subunit binds similar amounts of the
transfected human wild type and mutated
1 integrins
(Fig. 4A). Moreover, the
amounts of
5 subunit detected by FACS analysis on the
surface of
1 WT- and W775A-expressing cells were
similar, whereas the
1-null GD25 cells were negative,
suggesting that the mutant integrin is also equally effective in
stabilizing endogenous
subunit cell surface expression.
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Fig. 4.
The W775A
1 mutation does not hinder pairing with
the
5 subunit, binding to talin,
-actinin, or vinculin, and focal adhesion localization.
A, integrin
5 subunit immunoprecipitates were
prepared from parental GD25 cells (
1
/
) or
cells expressing wild type (
1 wt) or tryptophan
mutant (W775A) integrins after overnight incubation on
fibronectin in the absence of serum and were analyzed by Western
blotting for co-immunoprecipitated
1 integrin. Amounts
of
5 integrin subunit were verified with an
anti-
5 antibody. B, integrin-based complexes
were isolated from wild type (
1 wt) or tryptophan
mutant (W775A) integrin-expressing cells that were
serum-starved in suspension for 3 h. The complexes were purified
with magnetic beads coated with anti-
1 integrin 12G10 as
described under "Experimental Procedures," and the presence of
talin,
-actinin, and vinculin as well as
1 integrins
was analyzed by Western blotting. C, cells were cultured
overnight on fibronectin in the absence of serum, fixed, and
double-stained with anti-integrin (
1 integrin)
and anti-vinculin (vinculin) antibodies. Bar, 10 µm.
1 integrin cytoplasmic domain (see
Ref. 42). Among them are talin (62-64) and
-actinin (65, 66), which
have binding sites that include Trp775. To test the effect
of the tryptophan substitution on these interactions, we isolated
integrin-based complexes using beads coated with anti-integrin antibodies (see "Experimental Procedures" and Ref. 51). The amounts
of talin and vinculin, which may bind to
1 integrin via talin (67, 68), were similar, and
-actinin was even increased in the
complexes formed by the mutant W775A integrin (Fig. 4B). Analogous results were obtained in co-immunoprecipitation experiments (not shown), suggesting that the W775A mutation does not reduce
1 interactions with talin,
-actinin, and vinculin.
1 integrins are capable of localizing to
focal adhesions. We therefore analyzed cellular localization of the
tryptophan mutant by immunofluorescence with two antibodies that
recognize cation- and ligand-induced epitopes on
1
integrin: 12G10 (48) and 9EG7 (69, 70). Both wild type and mutant
integrins reacted with 12G10 (Fig. 4C,
1
integrin) and 9EG7 (not shown) antibodies and co-localized
similarly with the focal adhesion marker vinculin (Fig. 4C,
vinculin) when plated on fibronectin. These results indicate
that tryptophan substitution induces little functional change in the
1 integrin cytoplasmic domain, with the sole exception
of Akt signaling.
1, yet
Restoring Integrin Activation Does Not Compensate for Akt
Inhibition--
Some changes in the cytoplasmic domain of integrins
can affect the conformation of the extracellular portion of the
molecule. This mechanism operates normally in living cells and is known as inside-out signaling (71, 72). It activates ligand binding of
integrin molecules, accompanied by exposure of specific epitopes. To
investigate the ligand binding/activation state of the W775A mutant, we
used the 12G10 and 9EG7 monoclonal antibodies, which recognize cation-
and ligand-induced epitopes on the human
1 integrin.
FACS analysis revealed that cells expressing the tryptophan mutant bind
40% fewer 12G10 or 9EG7 antibodies when compared with cells expressing
the wild type
1 integrin (Fig.
5A, 12G10 and 9EG7). This difference was not the result of different
levels of integrin expression, because binding of the K20 monoclonal antibody, which recognizes total human
1 integrin
regardless of its activation, was similar for the two cell lines (Fig.
5A, K20). Full stimulation of W775A to a level
similar to that of the wild type integrin was obtained with the
activating TS2/16 antibody (50) (Fig. 5A,
TS2/16). These results suggest that the W775A
substitution partially decreases the activation state of the
1 integrin, but this effect is reversible by integrin activators.
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Fig. 5.
W775A integrin is in a lower activation
state, but its reactivation is not sufficient to restore Akt
phosphorylation. A, integrin-null cells
( 1
/
;
) or cells expressing wild type
(
1 wt;
) or mutant (W775A;
)
integrins were stained with anti-
1 integrin antibodies
and analyzed by FACS. The anti-
1 integrin antibodies
used were against ligand- and cation-induced binding sites
(12G10 and 9EG7), activating
(TS2/16), or capable of recognizing total
1 integrins regardless of activation state
(K20). B, cells expressing wild type
(
1 wt) or mutant (W775A) integrins
were serum-starved in suspension and then mixed with magnetic beads
coated with activating TS2/16 antibody for 30 min. Lysates from
treated, or control, nontreated cells were subjected to immunoblotting
analysis with phosphospecific antibodies against Akt (p-Akt
(Ser473)) or focal adhesion kinase
(p-FAK(Tyr473)) or with antibodies recognizing total
Akt (total-Akt) or Bcl-2 (Bcl-2) proteins present
in the lysates. C, cells were serum-starved overnight,
trypsinized, and maintained in suspension in medium without serum for
an additional 30 min. The cells were then plated on dishes precoated
with fibronectin (5 µg/ml) and cultured for the indicated times.
Lysates from samples taken before plating (0 h) and at the end of each
time point (0.5-10 h) were analyzed by Western blotting with
antibodies against phosphorylated Akt (Ser473) and actin.
Phosphorylated Akt (p-Akt) concentrations in arbitrary units
were calculated as the ratio between densitometry obtained with
anti-phospho-Akt antibody and anti-actin antibody.
1 integrins induced 2-fold (after plating) to 3-fold
(with beads) increases in phospho-Akt over the control in each cell
line, the differences in Akt phosphorylation between
1
WT and W775A cells, whether treated or untreated, remained the same
(Fig. 5, B, compare lines
1 WT
(+) with W775A (+), and C, compare
1 WT with W775A at 0-2 h). A stable
increase in the phosphorylation of Akt was observed in
1
WT cells 2-10 h after plating, whereas mutant integrin-expressing
cells were unable to activate Akt (Fig. 5C, compare
1 WT with W775A, 2-10 h). 10 h
after plating, W775A-expressing cells showed similar 5-fold reduction
in Akt phosphorylation that was observed under steady-state conditions (Fig. 3A). The inability of clustering by activating
antibodies to compensate fully for the Akt inhibition in
W775A-expressing cells was also evaluated with respect to their
effectiveness in the converse process of "outside-in" signaling
(74). Bead-bound antibody effectively stimulated FAK
autophosphorylation (Fig. 5B, p-FAK
(Tyr397)) and increased Bcl-2 levels (Fig. 5B,
Bcl-2) similarly in both cell lines; these events have been
associated with integrin outside-in stimulation of survival signaling
pathways (13, 14, 17). These results indicate that restoration of the
active conformation of the extracellular domain of
1
integrin is not able to restore Akt activity in cells expressing the
mutant W775A integrin.
1 WT and W775A integrin-expressing cells by comparing
the amounts of phosphatidylinositol 3-phosphate produced in
vitro by activated PI3K (Fig. 6,
PI3K assay: PI(3)P) or total PI3K (not shown). Activated
PI3K was isolated in an immune complex with anti-phosphotyrosine
antibody 4G10, and total PI3K was isolated using a mixture of
isoform-specific anti-p110 PI3K or anti-p85 PI3K antibodies. In both
wild type and W775A integrin cell lines, the activities of PI 3-kinase
were similar; both were suppressed by wortmannin (Fig. 6A).
This finding suggests that the tryptophan mutation does not interfere
with normal function of this kinase, although the downstream Akt from the same lysates used for PI 3-kinase assays showed marked differences in phosphorylation (Fig. 6A, WB: p-Akt
(Ser473)). Wortmannin completely suppressed Akt
phosphorylation in both cell lines, confirming the role of PI3K as
the central upstream regulator of Akt.
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Fig. 6.
The W775A mutation does not interfere with PI
3-kinase-mediated Akt activation or PDK1 activity. A, GD25
cells expressing either wild type ( 1 wt) or
tryptophan mutant (W775A) integrins were cultured overnight
on fibronectin in complete medium. The cells were treated with (+) or
without (
) 100 nM wortmannin (Wortm.) for 30 min prior to lysis. The lysates were immunoprecipitated with
anti-phosphotyrosine antibody 4G10, and PI3K activity in the immune
complexes was assayed. Autoradiograph of 32P-labeled
phosphatidylinositide 3-phosphate (PI3K assay: PI(3)P) and
Western blot analysis of the same lysates with anti-phosphospecific
(WB: p-Akt (Ser473)) or total (WB:
total-Akt) Akt antibodies is shown. B, GD25 cells
expressing either wild type (
1 wt) or tryptophan
mutant (W775A) integrins were cultured overnight on
fibronectin in the absence of serum, and the levels of PDK1
phosphorylation of Ser (p-PDK1(Ser241)) residue were
analyzed by Western blotting with phosphospecific antibodies. The total
amount of PDK1 (total-PDK1) was used as an internal control
for loading.
1 WT and W775A integrin-expressing cell lines showed comparable levels of Ser421 phosphorylation (Fig.
6B) and membrane localization (not shown), suggesting that
the W775A integrin mutation did not affect PDK1 function.
1
WT and GD25 W775A cells. HPLC analyses of the deacylated lipid products
revealed that both cell lines contained similar amounts of
phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, and
phosphatidylinositol trisphosphate (Table
II), suggesting that the primary
Akt-activating pathway is not affected by this integrin mutation and
thus that the low phosphorylation level of Akt in W775A
integrin-expressing cells is related to a mechanism other than
activation.
Phosphoinositide content of cells expressing wild-type (1
WT) or mutant (W775A) integrins
1
integrin maintained high levels of Akt Ser473
phosphorylation, W775A cells showed a rapid decline of the signal, and
by 75 min after the stimulation, it was barely detectable (Fig.
7A, W775A, 75', No
treatment). Similar results were obtained even without the
inhibition of residual PDGF activity with AG1433 (data not shown).
These dynamics suggested that the expression of the mutant integrin
could be associated with increased PP2A activity, which has been
characterized as a key phosphatase that can dephosphorylate both
Thr308 and Ser407 and thereby inactivate Akt
(33, 37, 39, 40). In fact, treatment with okadaic acid, a specific PP2A
inhibitor, completely blocked the dephosphorylation of Akt
Ser473 (Fig. 7A, OA) and Akt
Thr308 (not shown) in W775A cells, supporting the role of
this phosphatase in the observed effects on Akt.
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Fig. 7.
Akt dephosphorylation in cells expressing
W775A integrin is related to increased integrin-associated PP2A
activity. A, GD25 cells expressing either wild type
( 1 wt) or tryptophan mutant (W775A)
integrins were cultured overnight on fibronectin in the absence of
serum. Cells were then stimulated with 20 ng/ml PDGF-BB for 15 min, the
growth factor was washed away, and the cells were maintained in medium
without serum supplemented with 30 µM PDGF kinase
inhibitor AG1433. Samples were taken immediately after stimulation
(PDGF,
) and at the indicated time points. The experiment
was performed in the absence (No treatment) or presence of 1 µM okadaic acid (OA). Lysates from the samples
were analyzed by Western blotting with anti-phospho-Ser473
Akt (WB: p-Akt (Ser473)) or anti-actin
(WB: actin) antibodies. Actin was used as an internal
control for loading. B, cells were cultured overnight on
fibronectin in the absence of serum and lysed, and then PP2A was
immunoprecipitated with monoclonal antibody clone 1D6. The
immunoprecipitates were assayed for phosphatase activity in the absence
(No treatment,
) or presence of 5 nM okadaic
acid (OA,
) against a phospho-Ser473 Akt
peptide (residues 467-477) using a malachite green Ser/Thr phosphatase
assay kit. After the phosphatase assay, the actual amount of the PP2A
in each immunoprecipitate was determined by Western blotting with
antibodies against PP2A. PP2A activity is presented in arbitrary units
(a.u.) calculated as the ratio between released free
phosphate (absorbance at 650 nm)/min and the densitometry signal for
PP2A from the Western blotting.
2
1 integrin after plating of primary
fibroblasts into collagen gels has been described recently (33).
However, unlike in that study, we observed no general increase in
serine/threonine phosphatase activity in cellular lysates (not shown)
nor differences in the activation of the total PP2A. To assay total
PP2A activity, we immunoprecipitated PP2A with mouse monoclonal
antibody 1D6, which does not interfere with its phosphatase activity.
The immunoprecipitates were tested for activity against a general
threonine phosphopeptide (KRpTIRR, where pT represents
phosphothreonine) (not shown) and against a specific serine
phosphopeptide derived from the sequence of Akt
(KHFPQFpS473YSAS, where pS represents phosphoserine) (Fig.
7B). In both cases, the PP2A activity in
1 WT
and W775A integrin-expressing cells was similar and was inhibited by
more than 90% after treatment with 5 nM okadaic acid.
These results indicate that the observed differences in putative
PP2A-associated Akt dephosphorylation in W775A cells cannot be
explained by changes in total PP2A activity. They instead suggest that
a particular localized subpopulation of this enzyme might be
responsible for the Akt inactivation.
1 integrins was responsible for the Akt inhibition. We
first tested whether this phosphatase is present in the protein complexes organized by the integrin cytoplasmic tail. PP2A was readily
detected in both wild type and mutant integrin complexes isolated with
antibody-coated beads (Fig.
8A, WB: PP2A) or by immunoprecipitation (not shown). Importantly, we also found that Akt is
present in the same complexes (Fig. 8A, WB:
total-Akt), suggesting that Akt dephosphorylation may take place
within the integrin complexes. The amount of PP2A in the complexes
formed by the mutant integrin was slightly increased, but this apparent increase was difficult to reproduce consistently, and the minor increase in quantities alone could not explain the large differences in
Akt phosphorylation, unless there was also altered activity of the
phosphatase recruited into the complex.
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Fig. 8.
The phosphatase activity of PP2A associated
with W775A integrin mutant is increased. A, cells expressing
wild type ( 1 wt) or mutant (W775A)
integrins were serum-starved in suspension and incubated with magnetic
beads coated with anti-
1 integrin antibody K20 for 30 min. The isolated integrin complexes (see "Experimental
Procedures") were tested by Western blotting with the indicated
antibodies. B, the integrin complexes in A were
tested for phosphatase activity against the phospho-Ser473
Akt peptide (residues 467-477) using a malachite green Ser/Thr
phosphatase assay kit either without inhibitors (No
treatment,
) or in the presence of 0.5 µM
inhibitor-2 (I-2,
) or 5 nM okadaic acid (OA,
). PP2A activity is
presented in arbitrary units (a.u.) calculated as in Fig.
6B. Note the increase in PP2A activity present in
W775A-integrin complexes when compared with the
1 WT
complexes (p < 0.001) in the presence or absence of
inhibitor-2.
1 WT and W775A cells and
tested in vitro for phosphatase activity against the
Akt-derived phosphopeptide that includes Ser473 (Fig.
8B). The phosphatase activity in the mutant integrin
complexes was 2.6 times higher than the activity in the complexes
organized by the wild type
1 integrin (Fig.
8B, black bars, p < 0.001). This
phosphatase activity was most probably due to PP2A, because the
addition of 5 nM okadaic acid, a specific PP2A inhibitor at this concentration, inhibited the activity by more than 65% (66.5% for
1 WT and 79% for W775A complexes) (Fig.
8B, open bars). Another phosphatase that can be
inhibited by okadaic acid, but only at a 100-fold higher concentration
than PP2A (IC50 = 0.1 nM), is protein
phosphatase 1 (IC50 = 10 nM). Because of the
composite nature of the integrin complexes, we tested for the presence
of contaminating protein phosphatase 1 activity. When the reactions were carried out in the presence 0.5 µM inhibitor-2, a
specific inhibitor of protein phosphatase 1 with an IC50 of
2 nM, the phosphatase activity was reduced by only 5% for
1 WT and 8% for W775A complexes, and a similar 2.5-fold
higher activity was still measured in the mutant integrin complexes
(Fig. 8B, gray bars, p < 0.001).
Taken together, our results suggest that the protein complexes
organized by
1 integrin contain both a subpopulation of
PP2A and its substrate Akt and that the tryptophan mutation in the
integrin tail leads to a localized increase in activity of PP2A that
inactivates Akt.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 integrin cytoplasmic domain as a
specific site regulating Akt activation. Our results identify a novel
mechanism employed by integrins that controls the level of Akt
phosphorylation via local activity of the phosphatase PP2A.
integrin cytoplasmic domains
were identified, and their potential roles in signaling were tested by
single-residue substitutions using alanine-scanning mutagenesis. Each
full-length human
1 integrin mutant was expressed in
1-null GD25 cells (58). Among these
1
integrin mutants, only the W775A mutant demonstrated defective Akt
signaling. To examine the validity of our "signaling selectivity"
hypothesis, we searched for effects of this mutation on other
integrin-mediated signaling pathways. Previously, the effect of
mutating this residue was studied in the context of a double mutation
within an interleukin-2 receptor
1 tail chimera in a
1 WT background (81). Since no specific signaling
function for this residue has been identified to date, we explored the
mechanism by which its mutation suppresses Akt activation.
1 integrin Trp775 mutation
that we describe on cell survival involve a different mechanism.
Analysis of FAK activation showed similar levels of phosphorylation of
Tyr397 in cells expressing wild type or tryptophan mutant
integrin. This major autophosphorylation is induced by integrin
ligation, and this site binds Src (82). In turn, Src further activates FAK by phosphorylating it at additional sites, including
Tyr861 (83), which was similarly induced in
1 WT and W775A integrin-expressing cells (not shown).
The latter result suggested that Src is equally active in both cell
lines. Indeed, comparable levels were found for phosphorylation of the
activation site within the catalytic loop (Tyr418) (84) and
the inhibitory site (Tyr529) (85) in the carboxyl-terminal
portion of the kinase. We were also unable to find changes in JNK, p38,
or Shc phosphorylation or in Bcl-2 protein levels, indicating that
major kinases activated by integrins and at least several of their
downstream effectors operate similarly in both cell lines and are not
affected by the tryptophan mutation.
1 WT
cells (not shown), suggesting that MAPK is not a mediator of
Akt-regulated survival in this cell type. In summary, consistent with
our hypothesis, the W775A mutation specifically affects Akt-mediated cell survival and does not interfere with other integrin-mediated signaling pathways.
2
1 integrin-induced
activation of PP2A that leads to dephosphorylation of Akt and is
dependent on the
2 cytoplasmic domain was recently
reported (33). In contrast to that study, our results point to the
importance of local activation of PP2A confined within complexes
organized by the mutant integrin, whereas total cellular PP2A activity
was not affected by the W775A mutation. This finding, together with the
fact that GD25 cells do not express
2
1
after reconstitution of
1 integrin (87), suggests that the Trp775 mutation reveals a new relationship between
integrins and PP2A. This phosphatase was present in both
1 WT and W775A integrin complexes together with its
substrate Akt, but the activity of PP2A associated with the mutant
integrin was 2.5 times higher. An attractive mechanism can be envisaged
in which the subpopulation of PP2A associated with integrin cytoplasmic
domains controls Akt phosphorylation locally in integrin molecular
complexes. It is tempting to speculate that "correct" extracellular
matrix interactions retain organization of normal
1
integrin cytoplasmic complexes in which PP2A activity remains
repressed, resulting in continued Akt activation. Inappropriate matrix
conditions or mutation of the tryptophan at position 775 would
interfere with this process by releasing the inhibition of PP2A in
integrin complexes, leading directly to altered cell survival decisions.
integrin subunits (
1,
2,
3,
6, and
7), and to our
knowledge it has not been the subject of single-residue mutational
studies. It remains to be determined whether the effect on Akt
activation that we describe is valid for each of the other
integrins or is restricted to the
1 integrin. The
Trp775 residue resides in an integrin sequence that is
implicated in
-actinin (66) and talin binding (88-90). Interactions
of the
subunit with the PTB-like domain of talin have been shown to be important for integrin activation (63, 64). Alanine substitution of
the Trp775 residue did not interfere with talin binding and
focal adhesion localization, which has been shown to depend primarily
on the integrity of the NPXY motif (56, 57, 63, 64), but it
reduced the activation state of the W775A integrin by 40%, suggesting that it may affect the mechanism of inside-out integrin activation. Moreover, we found that although activation of the mutant integrin with
activating antibodies promoted FAK autophosphorylation and Bcl-2
expression (similar to the wild type), it did not compensate for the
inhibition of Akt. These findings imply again that local interactions
involving Trp775 are more important for this specific
signaling than restoration of the active conformation of the entire
cytoplasmic domain. Double mutation of the tyrosines in the cytoplasmic
domain of the integrin
1 subunit (Tyr783 and
Tyr795) to phenylalanines has also been shown to affect FAK
autophosphorylation but not its focal contact localization or
p130cas phosphorylation (46). Although effects on other
signaling pathways have not been explored, it is possible that this
double mutation could represent another site of specific signaling on
the
integrin cytoplasmic domain. These results, together with our
finding that Trp775 is intimately and selectively involved
in the control of Akt activation, support the concept of specificity of
signaling along the integrin cytoplasmic domain. They open the exciting
possibility of identifying more such distinct sites.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Susan E. LaFlamme for reagents
and discussions in the initial stages of this study, Dr. Reinhard
Fässler for providing GD25 1 integrin-null cells,
Dr. Sue Goo Rhee for valuable discussions, and Dr. Cristina Murga for
assistance with the PI3K assay.
![]() |
FOOTNOTES |
---|
* 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: CDBRB, NIDCR, NIH, Bldg. 30, Rm. 421, 30 Convent Dr. MSC 4370, Bethesda, MD 20892-4370. Tel.: 301-496-4041; Fax: 301-402-0897; E-mail: roumen. pankov{at}nih.gov.
¶ Present address: Division of Basic Science, Fox Chase Cancer Center, Philadelphia, PA 19111.
Published, JBC Papers in Press, March 11, 2003, DOI 10.1074/jbc.M300879200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: FAK, focal adhesion kinase; PKB, protein kinase B; PDK1, 3-phosphoinositide-dependent protein kinase 1; PP2A, protein phosphatase 2A; PDGF, platelet-derived growth factor; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; PI3K, phosphatidylinositol 3-kinase; DMEM, Dulbecco's modified Eagle's medium; PIPES, 1,4-piperazinediethanesulfonic acid; FACS, fluorescence-activated cell sorting; HPLC, high pressure liquid chromatography.
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