From the Department of Molecular Medicine, University
of Massachusetts Medical School, Worcester, Massachusetts 01605 and the
§ Department of Biotechnology, Worcester Polytechnical
Institute, Worcester, Massachusetts 01609
Received for publication, October 18, 2002, and in revised form, February 5, 2003
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ABSTRACT |
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Endothelial cells approaching confluence exhibit
marked decreases in tyrosine phosphorylation of receptor tyrosine
kinases and adherens junctions proteins, required for cell cycle arrest and adherens junctions stability. Recently, we demonstrated a close
correlation in endothelial cells between membrane cholesterol and
tyrosine phosphorylation of adherens junctions proteins. Here, we probe
the mechanistic basis for this correlation. We find that as endothelial
cells reach confluence, the tyrosine phosphatase SHP-2 is recruited to
a low-density membrane fraction in a cholesterol-dependent manner. Binding of SHP-2 to this fraction was not abolished by phenyl
phosphate, strongly suggesting that this binding was mediated by other
regions of SHP-2 beside its SH2 domains. Annexin II, previously
implicated in cholesterol trafficking, was associated in a complex with
SHP-2, and both proteins localized to adhesion bands in confluent
endothelial monolayers. These studies reveal a novel,
cholesterol-dependent mechanism for the recruitment of signaling proteins to specific plasma membrane domains via their interactions with annexin II.
Endothelial cells approaching confluence in culture undergo
profound morphological and functional alterations, including the formation of intercellular junctions and the cessation of cell growth.
These changes are associated with reductions in tyrosine phosphorylation levels of both receptor tyrosine kinases (1, 2) and
cadherins and catenins (3), the major components of adherens junctions.
The dephosphorylation of adherens junctions proteins appears to be
necessary for the formation of stable, confluent endothelial
monolayers, since increased tyrosine phosphorylation of adherens
junctions proteins leads to the disruption of adherens junctions
(4-6). The mechanisms by which cell density decreases tyrosine
phosphorylation of endothelial membrane proteins are unknown.
One possible mechanism is that increased cell confluence modulates the
local environment of signaling proteins in the plasma membrane, thereby
altering their response to extracellular ligands. Recently (7), we
identified membrane cholesterol as a potential determinant of tyrosine
phosphorylation levels in growing endothelial monolayers. Membrane
cholesterol increased markedly with cell density in cultures of growing
endothelial cells, exhibiting levels 3-4-fold higher upon reaching
confluence. Depletion of cholesterol from confluent monolayers of
CPAE1 cells induced the
tyrosine phosphorylation of multiple membrane proteins, including the
adherens junctions proteins Cholesterol might regulate tyrosine phosphorylation levels through the
localization of tyrosine kinases or phosphatases to specific plasma
membrane loci. To begin to identify such kinases or phosphatases, we
have used very low (0.01%) levels of the sterol-containing detergent
digitonin to identify proteins bound to membranes in a
cholesterol-dependent manner (8). At these concentrations, digitonin complexes specifically with unesterified 3- Antibodies and Reagents--
Monoclonal antibodies to Ax II,
SHP-2, and Cell Culture, Homogenization, and
Fractionation--
Subconfluent or confluent cultures of CPAE cells
(ATCC, Manassas, VA) were grown, harvested, and homogenized in cytosol
buffer (25 mM HEPES, pH 7.0, 125 mM potassium
acetate, 2.5 mM magnesium acetate, 0.2 M
sucrose, 1 mM dithiothreitol, 1 mM ATP, 0.01 mg/ml tosylarginine methyl ester, 4 µg/ml leupeptin, 1 mM
benzamidine, 1 mM 1,10-phenanthrene, 0.2 mM phenylmethylsulfonyl fluoride) as previously described
(7) unless otherwise indicated. To prepare unfractionated membranes,
cell homogenates were first centrifuged at 1000 × g
for 10 min to remove nuclei and unbroken cells. The supernatant was
centrifuged at 50,000 rpm (124,000 × g) for 15 min in
a Beckman TL-100 centrifuge, and the membrane pellet was washed three
times by resuspension in cytosol buffer, homogenization with a
tuberculin syringe, and recentrifugation as described above. Washed
membranes were resuspended in cytosol buffer and syringe-homogenized.
When multiple samples were prepared in parallel, they were made equal
in protein concentration. Identical volumes of membranes were extracted
with or without digitonin (0.01%) on ice for 10 min and centrifuged as
before for 15 min. Supernatants and pellets were analyzed by gel
electrophoresis and immunoblotting as previously described (7).
To separate membranes into low- and high-density fractions, 0.5 ml of
unfractionated membranes in cytosol buffer were layered on to 0.4 ml of
a 35% sucrose cushion in cytosol buffer and centrifuged at 97,000 × g for 20 min. Low-density or high-density membranes were
collected from the 0/35% sucrose interface or the bottom of the 35%
cushion, respectively. The membrane fractions were diluted 5-fold with
cytosol buffer and harvested by centrifugation at 135,000 × g for 1 h. The fractions were resuspended in cytosol buffer and made equal in protein concentration, and identical volumes
were extracted with or without digitonin, as described above.
Assay of PTPase Activity--
PTPase activities of membranes or
membrane extracts were determined using a kit (catalogue number V2471)
from Promega. Briefly, samples in cytosol buffer were incubated with
5 nmol of each of two peptide substrates (ENDpYINASL and
DADEpYLIPQQG) in a total volume of 50 µl for 30-60 min in a 96-well
plate. The PTPase reaction was stopped with the addition of 50 µl of
a proprietary acid/molybdate-based dye reagent, and free phosphate was
determined from the absorbance at 600 nm using an inorganic phosphate
standard. The PTPase reaction was linear with respect to both time and
protein concentration in the ranges employed.
Immunoprecipitations--
Unfractionated membranes in cytosol
buffer were mixed with 0.11 volumes of a 10× stock of Nonidet P-40 to
a final concentration of 0.2%. Equal volumes of the supernatant (~25
µl) were immediately diluted 20-fold with cytosol buffer and mixed
with agarose beads conjugated to anti-mouse IgG (Sigma), which had
previously been incubated either with or without a monoclonal antibody
to SHP-2. The mixtures were incubated overnight at 4 °C with
agitation, and the beads were washed six to seven times by
sedimentation at 1 g and resuspension in cytosol buffer plus
0.01% Nonidet P-40. The washed beads were solubilized in SDS gel
sample buffer and analyzed by SDS-polyacrylamide gradient gel
electrophoresis and immunoblotting with antibodies to SHP-2 and Ax II.
Immunofluorescence--
Cells were plated on glass coverslips at
the indicated densities and grown for 2 days. Where indicated,
confluent cells were treated with 4% methyl- Other Biochemical Methods--
Protein mixtures were analyzed on
7.5-17.5% polyacrylamide gradient SDS gels. Polypeptide bands were
stained using undiluted Sypro Ruby protein reagent (Molecular Probes)
overnight, rinsed in water, and imaged using a 300-nm UV
transilluminator. Antibodies were diluted 1:2500-1:5000 for
immunoblotting. horseradish peroxidase-conjugated antibodies were
detected on immunoblots using a chemiluminescence reagent from
PerkinElmer Life Sciences. Protein concentrations were determined using
a Coomassie Blue-based reagent (number 500-0006) from Bio-Rad.
Polyacrylamide gels and immunoblots are representative of at least
three separate experiments.
Dependence of Tyrosine Phosphorylation of Membrane Proteins on
Cholesterol--
Earlier studies (3) had indicated that tyrosine
phosphorylation of adherens junctions proteins decreased as endothelial cells formed confluent monolayers in culture. Recently (7) we reported
that depletion of cholesterol by methyl- Cholesterol-dependent Binding of PTPase Activity to
Membranes--
We sought to identify mechanisms by which cholesterol
could regulate or localize tyrosine kinase or phosphatase activity. As
noted earlier, digitonin, at very low (<0.02%) concentrations, binds
specifically to membrane cholesterol (9) without disrupting other
membrane lipids. Therefore, to determine whether any PTPases bound to
membranes in a cholesterol-dependent manner, we measured the amount of PTPase activity that could be extracted by 0.01% digitonin from membranes prepared from confluent CPAE cells (Fig. 2). Extraction with buffer alone
solubilized 0.2 ± 0.1 units (1 unit = 1 nmol of phosphate
hydrolyzed/min/mg of membrane protein) of activity, whereas buffer
containing digitonin solubilized 1.5 ± 0.2 units of activity.
Thus, digitonin specifically extracted 1.3 ± 0.2 units of
activity, representing 15% of the total membrane-bound activity
(1.3/8.8; see legend to Fig. 2). In contrast, only 0.5 ± 0.2 units of activity could be specifically extracted with
digitonin from membranes from subconfluent cells,
representing just 7% (0.5/7.3; see legend to Fig. 2) of the total
membrane-associated activity. Thus, 2.6-fold more PTPase activity was
specifically extractable with digitonin from membranes prepared from
confluent rather than from subconfluent cells. Treatment of intact
cells with methyl- Cholesterol-dependent Binding of SHP-2 to Membranes
from Confluent Cells--
To determine the identity of the
digitonin-extractable PTPase, we probed extracts from subconfluent or
confluent cells for several known PTPases by immunoblotting. A small
but reproducible amount of SHP-2 was specifically extracted by
digitonin from membranes from confluent, but not subconfluent cells,
although membranes from subconfluent and confluent cells contained
almost equal amounts of the protein (Fig. 2). As reported previously
(7), substantial amounts of Ax II could also be extracted from
membranes from confluent cells with digitonin, and virtually no Ax II
was detected in membranes from subconfluent cells, consistent with
their low levels of cholesterol. Prior treatment of confluent cells
with methyl-
SHP-2 contains two N-terminal Src homology (SH)2 domains, which bind to
numerous tyrosine phosphorylated proteins (5, 14-19). In addition,
SHP-2 contains two tyrosine phosphorylation sites, one of which
binds to the SH2 domain of GRB2 (20). To begin to evaluate the role of
these sites in the cholesterol-dependent binding of SHP-2
to membranes, we extracted membranes from confluent cells with phenyl
phosphate (40 mM), a competitive inhibitor of phosphotyrosine/SH2 interactions (Fig. 3C). In contrast to
digitonin, phenyl phosphate extracted only trace levels of SHP-2 and Ax
II, although SDS gels stained for total protein indicated that several other polypeptides were specifically extracted by phenyl phosphate. Thus, the cholesterol-dependent binding of SHP-2 to
membranes appeared to occur by a mechanism involving other regions of
SHP-2 besides its SH2 domains.
Identification of a Distinct Subcellular Pool of SHP-2, Dependent
on Cell Confluence and Cholesterol--
We reasoned that the
digitonin-extractable SHP-2 in confluent cells might represent a
separate pool of the protein. Since cholesterol-rich plasma membrane
domains often migrate as low-density vesicles when centrifuged through
sucrose density gradients, we separated membranes from confluent CPAE
cells into low-density (LDM) and high-density (HDM) fractions and
extracted them either with or without digitonin (Fig.
4A). SHP-2 was distributed
approximately equally between the LDM and HDM fractions. However, while
over half of the SHP-2 in the LDM could be extracted using digitonin, virtually none of the SHP-2 in the HDM fraction was solubilized by
digitonin. Thus, the digitonin-extractable SHP-2 in confluent cells
represented a distinct pool of the protein residing in a separate
compartment. In contrast to SHP-2,
Since the digitonin-extractable SHP-2 in confluent cells was localized
almost exclusively to the LDM, and subconfluent cells contained no
digitonin-extractable SHP-2, it seemed plausible that SHP-2 was
recruited to the LDM as cells reached confluence. In fact, in contrast
to confluent cells, the LDM fraction from subconfluent cells contained
virtually no SHP-2, while the HDM fraction contained substantial
amounts of the protein. Thus, SHP-2 and Ax II were both recruited to
membranes in the LDM fraction by a cholesterol-dependent
mechanism as endothelial cells reached confluence.
Localization of SHP-2 and Ax II to Adhesion Bands and Stress
Fibers--
To identify the intracellular structures to which Ax II
and SHP-2 were recruited, we visualized SHP-2 and Ax II in CPAE cells by confocal immunofluorescence microscopy (Fig.
5, A and B). In confluent cells, both SHP-2 and Ax II were visualized at sites of
cell-cell adhesion, although diffuse, granular staining was also
observed. Brief (3 min) treatments with methyl-
At the lowest cell density, Ax II immunofluorescence was
generally faint, consistent with the low levels of particulate Ax II in
these cells (Fig. 3A), although some co-linear staining with
actin could be detected, and SHP-2 exhibited clear co-localization with
actin. At somewhat higher densities, both Ax II and SHP-2 were
visualized along actin bundles at the cell periphery, as well as along
stress fibers, although some diffuse granular staining was also
observed. Generally, confluent cells showed a somewhat lower level of
stress fiber staining, suggesting that actin filaments were
redistributed from the cell interior to the periphery as intercellular
junctions formed.
Identification of a Membrane-bound Complex Containing SHP-2 and Ax
II--
Our studies indicated that, as endothelial cells reached
confluence, SHP-2 and Ax II are coordinately recruited to a low-density membrane fraction, apparently derived from cell-cell adhesion sites. We
reasoned, therefore, that SHP-2 and Ax II might be physically associated in confluent CPAE cells. To test this hypothesis, we prepared immunoprecipitates from membrane extracts using a monoclonal antibody to SHP-2 and probed them for Ax II (Fig.
6). A crude membrane fraction from
confluent CPAE cells was extracted with 0.2% Nonidet P-40 and
centrifuged, and the supernatant diluted 20-fold with buffer and
incubated overnight with anti-mouse IgG-agarose beads either with or
without a monoclonal antibody to SHP-2. Under these conditions,
virtually all of the SHP-2 bound to the beads containing the antibody
to SHP-2, while almost no SHP-2 bound to the beads alone. Ax II was
also present in the beads containing the antibody to SHP-2, but not in
the beads without the antibody. Thus, membrane-bound SHP-2 in confluent
CPAE cells can form a complex with Ax II.
We have found that the tyrosine phosphorylation levels of membrane
proteins in endothelial cells appear to be regulated by cholesterol
levels: as endothelial cells reached confluence, tyrosine phosphorylation levels decreased as cholesterol levels increased (7),
and partial depletion of cholesterol with methyl- Our observations indicate that SHP-2 is recruited to sites of cell-cell
contact as endothelial cells reach confluence, apparently as a function
of increasing cholesterol. While SHP-2 has been found to associate with
numerous signaling proteins via its SH2-domains, this recruitment
appears to be at least in part independent of phosphotyrosine/SH2
interactions, since almost none of the membrane-bound SHP-2 in our
preparations was extractable with phenyl phosphate (Fig. 3C)
and the extraction of SHP-2 with digitonin was only seen at high cell
densities, where membrane proteins were tyrosine phosphorylated at very
low levels (Fig. 1; cf. Refs. 3 and 7). In endothelial
cells, the SH2 domains of SHP-2 have been reported to interact with
phosphotyrosines in both platelet endothelial cell adhesion molecule-1
(18) and The finding that Ax II co-immunoprecipitates with SHP-2 from membrane
extracts from confluent endothelial cells suggests that the two
proteins are recruited as a complex to the cell periphery with
increasing cell density and membrane cholesterol. Ax II has also been
localized to adhesions between confluent Madin-Darby canine kidney
cells (25). Ax II has been found to bind directly to anionic liposomes
(11, 12), and cholesterol has been reported to enhance this binding
(26), suggesting that an Ax II/SHP-2 complex could bind directly to
membrane lipid domains enriched in cholesterol. Membrane anchoring
could also occur through an intrinsic membrane protein such as CD44,
which was recently found to anchor Ax II to detergent-resistant,
cholesterol-rich membranes (rafts) in mammary epithelial EpH4 cells
(27). Both Ax II (27-30) and SHP-2 (31) have been identified in rafts
in cultured cells, and cholesterol-sequestering reagents have been
found to dissociate Ax II from both chromaffin granules (26) and
endosomes (32). Alternatively, it is conceivable that the localization
of either Ax II or SHP-2 to sites of cell-cell attachment could occur
through direct binding to actin, since both Ax II (33, 34) and SHP-2 (35) have been reported to bind to actin filaments in vitro, and both proteins were found along stress fibers in subconfluent endothelial cells (Fig. 5).
The nature of the plasma membrane domains to which SHP-2 and Ax II are
recruited with increasing cell confluence is not clear. Ax II has been
found not to co-purify with caveolae isolated from rat lung blood
vessels (36), and caveolin did not localize to cell junctions in
endothelial cells,2 in
contrast to Ax II (Fig. 6). Our findings that much of the membrane-bound Ax II in confluent cells is extractable with low levels
of digitonin and that Ax II is predominantly localized to intercellular
junctions argue that these domains are also found at intercellular
junctions. Nevertheless, we have found that confluent 3T3L1
fibroblasts, which are contact-inhibited, but do not elaborate defined
junctional structures, also contain both elevated levels of cholesterol
and Ax II and SHP-2, which can be extracted with low levels of
digitonin.3 In addition, we
have found that, in growing endothelial monolayers, cholesterol and
membrane-bound Ax II increased prior to the formation of cell junctions
(7). These observations strongly suggest that the domains to which Ax
II and SHP-2 are recruited are not uniquely targeted to intercellular
junctions but might arise more generally in contexts where the
attenuation of tyrosine kinase-based signaling is required. Further
studies will be needed to clarify the precise nature of these domains
and the range of signaling processes which they regulate.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-catenin and pp120, and disrupted
adherens junctions.
-hydroxysterols (principally free cholesterol, in animal cells), forming in
situ precipitates without disrupting other lipid interactions (9, 10). Here, we report that extracts prepared with digitonin from confluent, but not subconfluent endothelial cells, contained the tyrosine phosphatase SHP-2. SHP-2 was found in a complex with Ax II, a
phospholipid-binding protein (11, 12) implicated in cholesterol
trafficking (13), and both SHP-2 and Ax II were visualized at
intercellular junctions in confluent endothelial monolayers. Our
observations indicate that, as endothelial cells reach confluence, a
complex containing SHP-2 and Ax II is recruited to intercellular
junctions by a novel, cholesterol-dependent, mechanism.
These studies suggest that cell density regulates the tyrosine
phosphorylation levels of membrane proteins through the cholesterol-dependent redistribution of tyrosine kinases
and/or phosphatases.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-catenin were purchased from Transduction
Laboratories (Lexington, KY). A monoclonal antibody to
phosphotyrosine (clone 4G10) was purchased from Upstate Biotechnology
(Lake Placid, NY). A polyclonal antibody to actin was purchased from
Sigma. Horseradish peroxidase-conjugated secondary antibodies
were purchased from Promega (Madison, WI). Alexa fluor-conjugated secondary antibodies were purchased from Molecular Probes (Eugene, OR).
Digitonin was purchased from Calbiochem. All other chemicals were
purchased from Sigma.
-cyclodextrin for 3 min
at 37 °C immediately prior to fixation. Cells were then fixed in
ice-cold methanol for 15 min at
20 °C, washed three times with
phosphate-buffered saline (PBS), incubated in blocking solution (fetal
bovine serum (1.0%) plus Triton X-100 (0.5%) in PBS) for 15 min at
room temperature, and incubated overnight at 4 °C with a monoclonal
antibody to either SHP-2 or Ax II and a polyclonal antibody to actin,
both diluted 100-fold in blocking buffer. Coverslips were washed three times for 5 min each in blocking buffer, incubated for 1 h at room
temperature in Alexa 488-conjugated goat anti-mouse IgG and Alexa
594-conjugated goat anti-rabbit IgG, both diluted 1:400 in blocking
buffer, washed as before, and mounted using Anti-Fade Mounting Medium
(Molecular Probes). Cells were imaged using a Leica TCS-SP laser
scanning confocal microscope (Leica, Heidelberg, Germany), using the
argon and krypton lasers to excite the anti-mouse and
anti-rabbit antibodies respectively. Bleed-through was routinely eliminated by adjusting laser intensities and signal gain so that the
cross-channel signal in the scanned images was zero.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cyclodextrin induced
tyrosine phosphorylation of several membrane proteins, including pp120
and
-catenin. To determine the extent to which methyl-
-cyclodextrin reversed tyrosine dephosphorylations, which occur as endothelial cells reach confluence in culture, we directly compared tyrosine phosphorylation levels of membrane proteins from
subconfluent cells, confluent cells, and confluent cells treated with
methyl-
-cyclodextrin (Fig. 1).
Subconfluent cells contained prominent tyrosine phosphorylated proteins
migrating as 60-, 70-, 75-, 80- (
-catenin), 120- (pp120), 135- and
175-kDa species. Tyrosine phosphorylation of these proteins was
virtually undetectable in membranes from confluent cells. Incubation of confluent cells with methyl-
-cyclodextrin for 20 min restored tyrosine phosphorylation levels of most of these proteins, including pp120 and
-catenin, to approximately the same levels as those seen
in subconfluent cells, while the 135-kDa polypeptide generally exhibited somewhat higher levels of phosphorylation. Thus, partial depletion of cholesterol from confluent cells restored tyrosine phosphorylation levels of membrane proteins to the same or higher levels as those in subconfluent cells.
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Fig. 1.
Dependence of tyrosine phosphorylation of
CPAE membrane proteins on cell confluence and cholesterol.
Subconfluent or confluent CPAE cells were incubated with or without 6%
cyclodextrin for 20 min at 37 °C, harvested, and homogenized in
cytosol buffer containing 1 mM ATP and 1 mM
Na3VO4. Postnuclear supernatants were
normalized for total protein, and equal volumes were centrifuged to
yield membranes, which were analyzed on SDS gels, immunoblotted, and
probed with an antibody to phosphotyrosine. Bands labeled as
-catenin and pp120 were identified previously in antiphosphotyrosine
immunoprecipitates (7).
-cyclodextrin reduced the activity specifically
extracted with digitonin from 1.4 ± 0.2 to 0.8 ± 0.2 units
(Fig. 2B), which represented, respectively, 18 and 9% of
the total membrane-associated activity (1.4/7.7 and 0.8/9.0; see legend
to Fig. 2), indicating that this activity did, in fact, associate with
membranes in a cholesterol-dependent manner.
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Fig. 2.
Extraction of PTPase activity from CPAE
membranes with digitonin. Subconfluent or confluent CPAE cells
were incubated with or without 6% cyclodextrin for 20 min at 37 °C,
harvested, and homogenized in cytosol buffer. Membranes were prepared
from postnuclear supernatants, which were then normalized for total
protein, extracted with cytosol buffer, with or without digitonin
(0.01%), and centrifuged. Equal volumes of extracts were assayed for
PTPase activity. Columns marked ` ' represent the difference in
activities of the extracts prepared with or without digitonin. 1.0 unit
of PTPase activity equals 1.0 nmol of phosphate hydrolyzed/min/mg of
protein in membranes prior to extraction. In A, the PTPase
activity of the unextracted membranes from confluent or subconfluent
cells was 8.8 ± 1.0 or 7.3 ± 2.1 units, respectively
(n = 3). In B, the PTPase activity of the
unextracted membranes from untreated or cyclodextrin-treated cells was
7.7 ± 2.6 or 9.0 ± 3.5 units, respectively
(n = 3).
-cyclodextrin substantially reduced the amounts of both
SHP-2 and Ax II extracted by digitonin (Fig.
3B), confirming that both
proteins were at least partially bound to membranes in a
cholesterol-dependent manner.
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Fig. 3.
Extraction of SHP-2 and Ax II from CPAE
membranes by digitonin or phenyl phosphate. A,
membranes were prepared from subconfluent or confluent CPAE cells and
extracted with or without digitonin (0.1%). Supernatants and pellets
were analyzed for Ax II and SHP-2 by immunoblotting. B,
confluent CPAE cells were incubated with or without 6% cyclodextrin
for 20 min at 37 °C, harvested, and homogenized in cytosol buffer.
Membranes were prepared and extracted with or without digitonin
(0.1%). Supernatants were analyzed for SHP-2 and Ax II by
immunoblotting. C, subconfluent or confluent CPAE cells were
incubated with or without 6% cyclodextrin for 20 min, harvested, and
homogenized in cytosol buffer. Membranes were prepared, extracted with
either digitonin (0.01%) or phenyl phosphate (40 mM), and
centrifuged. Supernatants were analyzed on 7.5-17.5% polyacrylamide
gradient SDS gels. Proteins were either immunoblotted and probed for
SHP2 and Ax II or visualized using Sypro Ruby protein stain (Molecular
Probes). The band at the position of 66 kDa in the lane at the
far left is an artifact.
-catenin could not be extracted
from either the LDM or HDM with digitonin, although SHP-2 has been
reported to bind to
-catenin in in vitro assays (5). As
noted previously, virtually all the Ax II was found in the LDM, and a
substantial amount of Ax II could be extracted with digitonin (7).
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Fig. 4.
Extraction of low- and high-density CPAE
membrane fractions with digitonin. A, LDM and HDM
fractions were prepared from confluent CPAE cells by centrifugation of
unfractionated membranes through a 35% sucrose cushion in cytosol
buffer. The LDM and HDM fractions were isolated from the 0/35% sucrose
interface and the bottom of the sucrose cushion, respectively. The
membrane fractions were diluted with cytosol buffer, concentrated by
centrifugation, normalized for total protein, and extracted with buffer
with or without digitonin (0.01%). Supernatants and pellets were
immunoblotted for Ax II, SHP-2, and -catenin. B, LDM and
HDM fractions from subconfluent or confluent cells were prepared by
centrifugation through sucrose, as described for A, and
immunoblotted for SHP-2.
-cyclodextrin almost
entirely dissociated Ax II and SHP-2 from the cell periphery. Generally, actin fibers were more readily visualized after treatment with methyl-
-cyclodextrin, suggesting that stress fibers were released from the cell periphery to the interior, although in some
cells peripheral actin bundles were still seen. In many cells, retraction of the plasma membrane was evident, as noted previously (7).
No bleed-through was detected in the SHP-2 or Ax II channels when cells
were illuminated at the excitation frequency of the actin-bound
fluorophore only (see Supplementary Fig. 1 at http://www.jbc.org).
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Fig. 5.
Localization of SHP-2 and
Ax II to the actin cytoskeleton. CPAE cells were plated at the
indicated cell densities on glass coverslips. After 2 days of growth,
only cells plated at the highest density were confluent. One set of
confluent cultures was treated with 6% methyl- -cyclodextrin for 3 min immediately prior to fixation. Cells were fixed with methanol at
20 °C for 15 min and processed for immunofluorescence microscopy
using a monoclonal antibody to either SHP-2 (A) or Ax II
(B) and a polyclonal antibody to actin. Images shown are
confocal sections in the plane of adherens junctions, i.e.
approximately one-third the distance from the basolateral to the apical
surface. Scale bars equal 40 µm.
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Fig. 6.
Co-immunoprecipitation of Ax II with
SHP-2. Membranes were prepared from confluent CPAE cells,
extracted with 0.2% Nonidet P-40, and centrifuged. The supernatant was
immediately diluted 20-fold with cytosol buffer and incubated overnight
at 4 °C with anti-mouse IgG-agarose beads, which had previously been
incubated with or without a monoclonal antibody to SHP-2. The beads
were then washed extensively and immunoblotted for SHP-2 and Ax
II.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cyclodextrin restored tyrosine phosphorylation levels to those seen in subconfluent cells (Fig. 1). While other studies have also indicated inverse relationships between tyrosine phosphorylation and cholesterol (21-24), our observations represent the first instance in which physiological changes in cholesterol are linked both to physiologically significant tyrosine phosphorylation events and to the subcellular relocalization of a tyrosine kinase or phosphatase. Our observations also provide a mechanism for the attenuation of signaling pathways at
higher cell densities in the presence of high concentrations of
activating ligands.
-catenin (5), both of which have been localized to sites of
cell-cell contact. However, these interactions were observed when
intact cells were treated with orthovanadate, which induces tyrosine
phosphorylation of membrane proteins, and destabilizes adherens
junctions. Thus, it seems plausible that the binding of SHP-2 to
platelet endothelial cell adhesion molecule-1 and
-catenin occurs in
response to signals initiated by growth factors, e.g.
vascular endothelial growth factor, which leads to the breakdown of
intercellular junctions and the resumption of the cell cycle. In
contrast, the cholesterol-dependent binding of SHP-2 to
membranes that we report here was observed in the absence of
orthovanadate and may represent a mechanism for maintaining junctional stability.
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ACKNOWLEDGEMENT |
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We thank Dr. Jeffrey Nickerson for expert instruction and guidance in laser confocal microscopy.
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FOOTNOTES |
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* This work was supported in part by Grant IRG 93-033 from the American Cancer Society (to H. S. S.).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.
The on-line version of this article (available at
http://www.jbc.org) contains a supplementary figure.
¶ Both authors contributed equally to this work.
To whom correspondence should be addressed: Dept. of Molecular
Medicine, 373 Plantation St., Suite 107, University of
Massachusetts Medical Center, Worcester, MA 01605. Tel.: 508-856-6866;
Fax: 508-856-4289; E-mail: howard.shpetner@umassmed.edu.
Published, JBC Papers in Press, February 27, 2003, DOI 10.1074/jbc.M210701200
2 H. S. Shpetner, unpublished observations.
3 A. Burkart, S. Corvera, and H. S. Shpetner, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: CPAE, cow pulmonary aortic endothelial; Ax II, annexin II; PTP, protein tyrosine phosphatase; PBS, phosphate-buffered saline; SH, Src homology; LDM, low-density membrane; HDM, high-density membrane.
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