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INTRODUCTION |
Caveolin is a small integral membrane protein and the principal
component of caveolae membrane domains in vivo (1). Caveolae are plasma membrane-attached vesicular organelles that have a characteristic diameter in the 50-100-nm range (2, 3). Although caveolae are present in most cells, they are particularly abundant in
terminally differentiated cells such as adipocyte, endothelial, and
skeletal muscle cells. Recent studies have revealed the presence of at
least three mammalian caveolin subtypes, caveolin-1, caveolin-2, and
caveolin-3, with different tissue distribution (see Ref. 4, and
references therein). Different from the role originally proposed, i.e. as a protein that organizes the caveolae structure and
accumulates many signaling molecules, a new role for caveolin as a
regulator of transmembrane signaling has been suggested by recent data. Several research teams have demonstrated that caveolin is able to
recognize and recruit into caveolae, and to regulate the activities of
several proteins involved in cellular signaling cascades, such as the
heterotrimeric G-proteins, Src kinases, nitric-oxide synthase, epidermal growth factor, and platelet-derived growth factor receptors, as well as protein kinase C (see Ref. 4, and references therein). The
interaction between caveolin and other proteins is mediated through a
short stretch of the membrane-proximal region (or caveolin scaffolding
domain), encoded by residues 82-101, which recognizes and binds
proteins containing the sequence motif
X
XXXX
or
XXXX
XX
, where
is an aromatic residue
(5).
Previous studies have demonstrated that caveolin is phosphorylated on
tyrosine by the oncogenic viral Src kinase (v-Src), and that caveolin
is associated with normal cellular Src (c-Src) and other Src family
tyrosine kinases (6, 7).
Low Mr phosphotyrosine-protein phosphatase
(LMW-PTP)1 is expressed as
two molecular isoforms in mammalian species. These isoforms originated
from two different mRNAs produced through an alternative splicing
mechanism (8). In addition to mammals, the enzyme is expressed in a
sole molecular form in several microorganism species (9-12). It
belongs to the large family of protein-tyrosine phosphatases (PTPs)
that are divided into four subfamilies: the tyrosine-specific
phosphatases, the VH1-like dual specificity phosphatases, the cdc25
phosphatases, and the low molecular weight phosphatases. All PTPs have
the active site signature sequence CXXXXXR, and all share
the same catalytic mechanism (13). Arginine is involved in substrate
binding, whereas cysteine performs the nucleophilic attack on the
substrate phosphorus atom, producing a covalent enzyme-phosphate
intermediate, whose hydrolysis is the limiting step of the catalytic
process. An aspartic acid residue assists the nucleophilic reaction by
donating a proton to the leaving group in the transition state.
Although the function of LMW-PTP in microorganisms is still debated
(10, 12), several papers produced in the last few years have indicated
that the enzyme participates in the tyrosine kinase receptor function
(14-18). Specifically, the enzyme is involved in the down-regulation
of PDGF and insulin receptors (14-16). In the case of the PDGF
receptor, the overexpression of the enzyme in NIH-3T3 cells (both as
wild type (wt) and as a dominant-negative (dn) mutant (C12S), a form
able to bind substrates but catalytically inactive) produces opposite
phenotypic effects; the wild type enzyme decreases cell growth rate,
whereas the dn mutant increases it. The analysis of this action
mechanism demonstrated that LMW-PTP binds and dephosphorylates the
activated receptor in vivo. In particular, LMW-PTP is
involved in pathways that regulate the transcription of the early genes
myc and fos in response to growth factor stimulation.
Recent reports have also demonstrated that LMW-PTP is localized
constitutively in cytosol and cytoskeleton, and that upon growth factor
stimulation c-Src is able to bind and phosphorylate only the
cytoskeleton-associated enzyme. As a consequence of its phosphorylation, LMW-PTP increases its activity about 20-fold, and this
strongly influences both cellular adhesion and migration. The target of
cytoskeleton-associated LMW-PTP is p190Rho-GAP, which is phosphorylated
on tyrosine after PDGF-stimulation (19). In the case of insulin
signaling, the specific involvement of the enzyme in the Src pathway
has been demonstrated. Overexpression of the dn form of the enzyme also
influences glucose uptake and glycogen synthesis upon insulin
stimulation (16).
Finally, the involvement of LMW-PTP in the regulation of other growth
factor receptors has been reported. Stein et al. (20) have
found that LMW-PTP is involved in the ephrine-B1 receptor function;
Rigacci et al. (18) and Rovida et al. (17),
respectively, have shown that the fibroblast growth factor receptor and
the macrophage colony-stimulating factor receptor functions are
regulated by LMW-PTP.
In this paper we demonstrate that tyrosine-phosphorylated caveolin is a
potential physiological substrate for LMW-PTP.
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EXPERIMENTAL PROCEDURES |
Materials--
Human recombinant LMW-PTP was prepared as
described previously (21). Monoclonal anti-phosphotyrosine (PY-20) and
polyclonal anti-caveolin (sc-894) antibodies were from Santa Cruz
Biotechnology (Santa Cruz, CA). Monoclonal anti-phosphocaveolin (PY14)
antibody was purchased from Transduction Laboratories (Lexington, KY). The sc-894 antibody is raised against a peptide corresponding to amino
acids 2-21 mapping at the amino terminus of caveolin-1, while the
PY-14 monoclonal antibody is raised against a tyrosine-phosphorylated peptide corresponding to amino acids 9-18 mapping at the amino terminus of caveolin-1. Rabbit polyclonal anti-LMW-PTP antibody was
produced in our laboratory. Protein A-Sepharose beads was from Sigma.
Soluble PTP-1B (recombinant glutathione S-transferase fusion
protein corresponding to full-length human PTP-1B) was purchased from
Upstate Biotechnology (Lake Placid, NY). Electrophoresis reagents were
purchased from Bio-Rad. Enhanced chemiluminescence (ECL) reagents were
from Amersham Pharmacia Biotech. All other reagents were the purest
commercially available.
Protein Determination--
Protein concentration was assayed by
the bicinchoninic acid method (BCA kit) purchased from Sigma.
Cell Cultures and Transfections--
NIH-3T3 fibroblasts, stable
transfected NIH-3T3 cell lines overexpressing wild type LMW-PTP or its
C12S dn mutant (prepared as described by Chiarugi et al.
(Ref. 22)), and H-end endothelial cells (kindly provided from F. Bussolino, University of Torino, Italy) were grown at 37 °C in a
humidified atmosphere containing 5% CO2 in Dulbecco's
modified Eagle's medium, supplemented with 10% fetal calf serum and
antibiotics (penicillin, streptomycin). NIH-3T3IR (a cell line
overexpressing insulin receptor; Ref. 23) were kindly provided by AR
Saltier (Parke-Davis Pharmaceutical Research Division, Ann Arbor, MI).
NIH-3T3IR cells transfected with the C12S (dn) mutant gene for LMW-PTP
(dnNIH-3T3IR) were prepared as described previously (16) and routinely
cultured in Dulbecco's modified Eagle's medium containing with 10%
fetal calf serum and 75 units/ml hygromycin, in 5% CO2
humidified atmosphere.
Pervanadate Treatment, Preparation of Lysates, and in Vitro
Dephosphorylation Experiments--
Prior to cell lysis, 80-90%
confluent cultures of cells (NIH-3T3 fibroblasts and H-end endothelial
cells) were treated for 30 min with 0.1 mM pervanadate (20 µl of a fresh solution containing 50 mM sodium
orthovanadate and 50 mM H2O2 was
added to 10 ml of medium). Cells were collected and then lysed with 20 mM Tris-HCl buffer, pH 7.4, containing 1 mM
EDTA, 0.1 M NaCl, 1% Triton X-100, 10% glycerol, 1 mM phenylmethanesulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 5 mM iodoacetate. Iodoacetate was
added in order to inactivate irreversibly all intracellular PTPs (all members of PTP family contain an active site essential cysteine residue; Ref. 13). After incubation at 4 °C for 30 min, 10 mM dithiothreitol was added to inactivate any unreacted
iodoacetic acid, and the insoluble material was removed by
centrifugation at 102,000 × g for 40 min. Lysates of
pervanadate-treated cells (1.5-3.5 mg of protein/ml) containing
tyrosine-phosphorylated proteins were incubated at 25 °C in the
absence or presence of LMW-PTP or PTP-1B (0.05-0.1 µM
final concentration). Aliquots were removed at various times for
analysis by SDS-PAGE and anti-phosphotyrosine immunoblotting.
Detergent-free Purification of Caveolin-rich Membrane
Fractions--
Low density caveolae-enriched domains were isolated as
described by Song et al. (24). Briefly, one confluent 100-mm
dish, washed twice with ice-cold phosphate-buffered saline (10 mM sodium phosphate and 0.15 M NaCl, pH 7.2),
was scraped into 0.5 ml of sodium carbonate buffer (500 mM
sodium carbonate, pH 11, 25 mM MES, 150 mM
NaCl, 1 mM phenylmethanesulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin). Cells were homogenized extensively using a Dounce homogenizer (50 strokes). The homogenate was then adjusted to 45% sucrose by the addition of 0.65 ml of 80% sucrose in
25 mM MES, 150 mM NaCl, 1 mM
phenylmethanesulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml
aprotinin, and placed at the bottom of an ultracentrifuge tube. A
5-35% discontinuous sucrose gradient was formed above the 45% layer,
by adding 2.5 ml of 35% sucrose and 1.3 ml of 5% sucrose, both in 250 mM sodium carbonate, pH 11, 25 mM MES, 150 mM NaCl, 1 mM phenylmethanesulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin. The gradient was centrifuged at
170,000 × g for 20 h using a Beckman SW50.1
rotor. For the analysis of the resulting gradient, 0.35-ml fractions
were collected from the top to the bottom of the gradient. The
insoluble pellet (fraction 15) was dissolved into 50 µl of Laemmli
sample buffer. Western blot analysis was performed using both
anti-caveolin and anti-LMW-PTP antibodies.
Immunoprecipitation Experiments--
Confluent NIH-3T3 cells
overexpressing C12S dnLMW-PTP cultured in 100-mm dishes were washed
with phosphate-buffered saline, lysed in ice-cold lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100,
60 mM n-octylglucoside, 2 mM EDTA, 1 mM orthovanadate, 100 mM NaF, 1 mM
phenylmethanesulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml
aprotinin), and insoluble material was removed by centrifugation at
10,000 × g for 10 min. Lysate (500 µg of protein)
was incubated overnight with 1 µg of anti-caveolin antibody or 1 µg
of anti-LMW-PTP antibody. After a 1-h incubation with protein
A-Sepharose beads at 4 °C, the immunocomplexes were collected and
washed extensively (three times) with lysis buffer. The beads were
suspended in 20 µl of 2-fold concentrated Laemmli electrophoresis
buffer (without 2-mercaptoethanol), separated by SDS-PAGE, and
electroblotted onto polyvinylidene difluoride membranes for detection.
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RESULTS |
LMW-PTP Dephosphorylates Tyrosine-phosphorylated Caveolin and Few
Other Tyrosine-phosphorylated Proteins Contained in Pervanadate-treated
Cell Lysates--
We have treated both NIH-3T3 and H-end cells with
pervanadate and found that protein tyrosine phosphorylation was
dramatically enhanced with respect to untreated cells, which contain
virtually undetectable levels of phosphotyrosine (25). Other authors
have previously reported similar findings using different cell lines (26, 27); the enhancement of tyrosine phosphorylation is due to the
inhibition of all cellular PTPs by pervanadate. We have performed
time-course dephosphorylation experiments by incubating lysates from
pervanadate-treated H-end cells with LMW-PTP. Aliquots of the
incubation mixtures were withdrawn at various times and analyzed by
Western blotting with anti-phosphotyrosine antibodies (PY20). Fig.
1A shows the SDS-PAGE
analysis, and Fig. 1B reports the densitometric profiles of
lanes 1-4 of panel A.
LMW-PTP rapidly dephosphorylates a group of low molecular mass bands
(<30 kDa), and at least two additional bands in the 38-55-kDa range.
Other phosphotyrosine-containing proteins were also dephosphorylated, but more slowly. A similar pattern was observed using lysates from
pervanadate-treated NIH-3T3 cells (data not shown).

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Fig. 1.
LMW-PTP dephosphorylation of some
phosphotyrosine-containing proteins from pervanadate-treated H-end cell
lysate. A, lysates from pervanadate-treated H-end cells
were incubated at 25 °C and pH 7.4 in the absence ( ) or in the
presence (+) of LMW-PTP. At various times (0, 2, 4, 8, 12, 20, and 30 min), aliquots containing 20 µg of protein were removed and subjected
to SDS-PAGE/Western blot analysis with PY-20 anti-phosphotyrosine
antibody. B), densitometric profiles of the immunoblots
shown in A. Profiles 1, 2,
3, and 4 correspond to 0, 2, 4, and 8 min,
respectively. C, an experiment similar to that shown in
A was performed using PTP-1B instead of LMW-PTP. Similar
results were obtained in at least three separate experiments.
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In order to verify the specificity of LMW-PTP in comparison with other
tyrosine phosphatases, we carried out the same experiment on the cell
lysate using PTP1B instead of LMW-PTP. The result is reported in Fig.
1C; PTP1B has the capacity to dephosphorylate a broad range
of tyrosine-phosphorylated protein substrates.
Taking into account the findings of Vepa et al. (27), who
demonstrated that caveolin is the highest phosphorylated protein with
low molecular weight in pervanadate-treated endothelial cells, we
performed enzymatic dephosphorylation of caveolin immunoprecipitated from the H-end cell lysate. The samples were incubated for different times with LMW-PTP. Western blot analyses performed respectively with
anti-phosphotyrosine (PY-20) and anti-caveolin (sc-894) antibodies reveal that LMW-PTP rapidly dephosphorylates phosphocaveolin (Fig. 2A). We have also tested the
activity of the phosphotyrosine-protein phosphatase PTP-1B on the
anticaveolin immunoprecipitate. As expected, this enzyme
dephosphorylates phosphocaveolin (Fig. 2B) in agreement with
the finding of Fig. 1C, which shows that PTP-1B displays broad substrate specificity.

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Fig. 2.
Dephosphorylation of immunoprecipitated
caveolin by LMW-PTP. Cell lysates from H-end cells were treated
with sc-894 anti-caveolin antibody. The immunocomplex was collected on
protein A-Sepharose, and then washed extensively with lysis buffer.
Beads were suspended in 20 mM Tris-HCl buffer, pH 7.4, containing 2 mM EDTA, and incubated in the absence ( ) or
in the presence (+) of LMW-PTP. The incubation was performed at
25 °C. At various times (0, 1, 3, 5, 10, and 15 min), identical
aliquots were removed and subjected to SDS-PAGE/Western blot analysis
with PY-20 anti-phosphotyrosine and sc-894 anticaveolin antibodies.
B, the same experiment was performed with PTP-1B instead of
LMW-PTP. IP, immunoprecipitation; WB, Western
blotting. Similar results were obtained in at least three separate
experiments.
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LMW-PTP Associates with Caveolae-enriched Membrane Domains in Vivo
and Coimmunoprecipitates with Caveolin--
The rationale of the
following experiments, performed on NIH-3T3 fibroblasts overexpressing
wild type or dominant negative LMW-PTPs, is to demonstrate both the
localization of LMW-PTP in caveolae and to investigate the mechanism of
its recruitment into these membrane domains. We lysed confluent cells
and then separated the caveolae-enriched membrane domains from other
cellular components, using the density gradient centrifugation
technique described under "Experimental Procedures." Fifteen
fractions were collected from the top of the gradient, and the
localization of LMW-PTP and caveolin was performed by Western blotting.
Fig. 3 (A and B)
show that both wild type and dnLMW-PTP molecular forms are distributed
in different gradient fractions; they colocalize with caveolin
(fractions 4 and 5), the caveolae domain marker protein, but they are
also present in fractions 12-14 (the loading zone), and in the pellet
(fraction 15). In fraction 1 of the density gradient relative to the
dnLMW-PTP-overexpressing cells, we have detected a positive reaction
with anti-LMW-PTP antibody. The localization of both wt and dn LMW-PTPs
in caveolae suggests that a completely functional active site is not
required for the recruitment of the enzyme into caveolae. The LMW-PTP
localization in the pellet and in the loading zone was expected, since
previous papers reported that LMW-PTP is localized in both cytoplasmic
and cytoskeleton-associated fractions of NIH-3T3 cells (see Ref. 19,
and citations therein).

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Fig. 3.
LMW-PTP associates with caveolae
membranes in vivo and coimmunoprecipitates with
caveolin. Detergent-free sodium carbonate-based lysates were
prepared from NIH-3T3 cells overexpressing wt (A) or dn
(B) LMW-PTP, and fractionated by flotation in sucrose
density gradient. Fractions of 0.35 ml were collected from the top to
the bottom of the gradient. Aliquots (20 µl) of the fractions were
subjected to SDS-PAGE/Western blot analysis with anti-LMW-PTP and
sc-894 anti-caveolin antibodies. C, cell lysates from
NIH-3T3 cells overexpressing wt or dn LMW-PTP were treated with
anti-LMW-PTP antibody, and the immunoprecipitated proteins were
analyzed by SDS-PAGE/Western blot analysis using anti-caveolin
(sc-894), anti-phosphocaveolin (PY-14), and anti-LMW-PTP antibodies.
IP, immunoprecipitation; WB, Western blotting.
Similar results were obtained in at least three separate
experiments.
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To further investigate the association of LMW-PTP with caveolin, we
performed immunoprecipitation experiments with anti-LMW-PTP antibody on
lysates from NIH-3T3 cells overexpressing wild type or dnLMW-PTP.
Western blot analyses of the immunoprecipitate using anti-caveolin
(sc-894), anti-phosphocaveolin (PY-14), and anti-LMW-PTP antibodies
(Fig. 3C) demonstrated that cellular LMW-PTP and caveolin co-immunoprecipitate, indicating that they interact in these cells. Furthermore, the formation of LMW-PTP-caveolin complexes does not
depend on caveolin phosphorylation, since we have not observed a
positive reaction with the PY-14 anti-phosphocaveolin antibody. This
suggests that a site different from the active site of LMW-PTP could be
involved in the binding with caveolin.
Tyrosine-phosphorylated Caveolin Is a Cellular Substrate of
LMW-PTP--
The results described above suggest that
tyrosine-phosphorylated caveolin is a potential substrate for LMW-PTP.
In order to demonstrate this hypothesis, we have performed experiments
with cells overexpressing both the insulin receptor and the dominant negative C12S LMW-PTP mutant.
Insulin-stimulated and non-stimulated cells (neoNIH-3T3IR and
dnNIH-3T3IR) were lysed, and Western blots with anti-phosphotyrosine (PY-20), anti-phosphocaveolin (PY-14), and anti-caveolin (sc-894) antibodies were performed. As expected, the main effect of insulin stimulation of neo cells is an increased phosphorylation level of the
90-kDa insulin receptor
-subunit (Fig.
4A, lanes
1 and 2). Insulin stimulation of NIH-3T3IR cells
overexpressing dnLMW-PTP causes a dramatic increase of tyrosine
phosphorylation of the insulin receptor with respect to control cells
(Fig. 4A, lanes 3 and 4).
The overexpression of dnLMW-PTP is accompanied by a slight increase in
the phosphorylation level of another band of approximately 60 kDa. The
anti-caveolin Western blot shows that caveolin is present in the
lysates (Fig. 4C), but the PY-20 antibody does not reveal
phosphorylated caveolin (Fig. 4A). Nevertheless, Western
blot analysis performed with the monoclonal antibody PY-14 (which
recognizes Tyr14-phosphorylated caveolin with high
specificity) shows that upon insulin treatment caveolin phosphorylation
is strongly enhanced in neoNIH-3T3IR cells, indicating that a tyrosine
kinase activated upon insulin-signaling is involved (Fig.
4B, lanes 1 and 2). The overexpression of the dominant negative LMW-PTP causes an increase in
caveolin phosphorylation level both in untreated and in
insulin-stimulated cells as compared with the control cells (Fig.
4B). Moreover, we demonstrated that, in these cells,
caveolin coimmunoprecipitates with LMW-PTP (Fig. 4, panels
D-F), and that the amount of immunoprecipitated caveolin is
not dependent on its phosphorylation. In the control cells,
immunoprecipitation with the anti-LMW-PTP antibody does not result in
detectable LMW-PTP levels (Fig. 4F, lanes
1 and 2). Nonetheless, the samples contain
significant amounts of immunoprecipitated caveolin (Fig.
4D). We think that this is due to the low basal level of
LMW-PTP in control cells; in contrast, caveolin is revealed in the
immunoprecipitate because it forms homooligomers (31), which may
interact with other proteins with an unknown stoichiometry.

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Fig. 4.
Effect of insulin stimulation on the
association of endogenous tyrosine-phosphorylated caveolin with
transfected dnLMW-PTP in NIH-3T3IR cells. cDNAs encoding C12S
dominant negative (dn) mutant of LMW-PTP or the vector alone
(neo) were transfected in NIH-3T3IR cells. Cells were
stimulated (+) or not ( ) with insulin (100 nM) for 5 min,
and lysates were analyzed by SDS-PAGE/Western blotting with PY-20
anti-phosphotyrosine antibody (A), with PY-14
anti-phosphocaveolin antibody (B), and with sc-894
anti-caveolin antibody (C). Aliquots of the lysates from the
above cell lines were treated with the anti-LMW-PTP antibody, and the
immunoprecipitated proteins were analyzed by SDS-PAGE/Western blotting
with sc-894 anti-caveolin (D), PY-14 anti-phosphocaveolin
(E), and anti-LMW-PTP (F) antibodies.
IP, immunoprecipitation; WB, Western blotting.
The results are representative of three independent experiments.
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Taken together, the above experiments clearly demonstrate that the
overexpression of the C12S LMW-PTP mutant in NIH-3T3IR cells causes the
enhancement of the tyrosine phosphorylation of some specific proteins
(such as insulin receptor
-subunit and caveolin) because it protects
them against the action of cellular PTPs. Some authors have described
these kinds of mutant as substrate traps, since, although they are
catalytically inactive, they maintain the capacity to bind substrates,
thus protecting them from dephosphorylation (28-30). The insulin
receptor, which is immediately phosphorylated upon insulin stimulation,
is an already known physiological substrate of LMW-PTP (16); our
results suggest that caveolin, which is phosphorylated on tyrosine by a
kinase activated upon insulin signaling, is a potential new cellular
substrate for LMW-PTP.
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DISCUSSION |
The cellular level of protein phosphotyrosine is generally very
low (25). In order to elevate cellular tyrosine phosphorylation, two
approaches are currently used. First, physiological stimulation of
cells with growth factors, which trigger cascades of tyrosine phosphorylation events, results in tyrosine phosphorylation of only a
limited number of proteins; second, treatment of cells with pervanadate
to inhibit intracellular PTPs results in the phosphorylation of a large
number of proteins. We have used the latter method to obtain cell
lysates and immunoprecipitates useful for revealing the
tyrosine-phosphorylated proteins that are rapidly dephosphorylated by
LMW-PTP in vitro. We performed time-course dephosphorylation
experiments revealing tyrosine-phosphorylated proteins by immunoblot
analysis with the PY-20 antibody. Our findings indicate that the
LMW-PTP dephosphorylates some bands very rapidly, whereas it
dephosphorylates other bands only after longer incubation periods.
Since Vepa et al. (27) reported that caveolin is one of the
most tyrosine-phosphorylated proteins in pervanadate-treated endothelial cells, we performed immunoprecipitation of caveolin species
using anti-caveolin antibody. The incubation of this immunoprecipitate with LMW-PTP and the successive immunoblot analyses using both anti-phosphotyrosine and anti-caveolin antibodies demonstrated that
LMW-PTP very rapidly dephosphorylates phosphocaveolin in vitro. Furthermore, both immunoprecipitation experiments with anti-LMW-PTP or anti-caveolin antibodies and cellular fractionation experiments in wtNIH-3T3 or dnNIH-3T3 cells demonstrated that LMW-PTP
is associated with caveolin in caveolae membrane enriched domains
in vivo. Moreover, we have demonstrated that this
interaction does not require caveolin phosphorylation (Fig.
3C); instead, it is in all probability mediated by a LMW-PTP
sequence motif similar to those found in other caveolin-binding
proteins. In fact, Couet et al. (5), using the caveolin
scaffolding domain to select random peptide ligands from phage display
libraries, proposed the following caveolin-binding motifs:
X
XXXX
or
XXXX
XX
(where
is an aromatic residue
and X is any amino acid). Interestingly, in that study,
nearly 10% of the selected 15-mer peptides contained only two aromatic
residues. The authors also reported that, although a caveolin consensus
motif including four aromatic residues
(
X
XXXX
XX
) has been
identified in several G
subunits, G
q uses valine and leucine as substitutions for two of these aromatic residues. Despite this substitution, G
q has been shown to
coimmunoprecipitate with caveolin. Thus, it seems possible that
caveolin-binding requirements are broader than described previously.
Recently, the caveolin-binding sequences of other caveolin-binding
proteins have been described. Carman et al. (32) have
reported that all known G-protein-coupled receptor kinases contain the
conserved caveolin-binding motif (I/L)XXXXFXXF.
We have observed that this caveolin-binding motif is also present in
LMW-PTP (residues 77-85) (33), suggesting that caveolin interacts with
this LMW-PTP region.
There is increasing evidence that caveolae play a major role in
organizing signal transduction at the cell surface (3, 34). Different
hormone receptors and signal transducers are localized in caveolae, and
specific signaling events originate in caveolae, including the
EGF-dependent activation of Raf-1 (35), interleukin-1
-stimulated production of ceramide (36), and PDGF receptor kinase cascade (37). Furthermore, caveolin-1 is directly involved in the modulation of the activity of heterotrimeric
GTP-binding proteins in vitro (38). Thus, caveolae may be
involved in the organization of these molecules, a necessary step for
the integration of different sources of information during signal transduction.
Caveolin-1 exists in vivo as two molecular species (
and
) that differ in their NH2-terminal sequences. These two
species derive from a single gene through alternate initiation during translation;
-caveolin contains residues 1-178, whereas
-caveolin contains residues 32-178. Li et al. (6) have
demonstrated that only
-caveolin is phosphorylated by v-Src in
vitro and in vivo, and Ko et al. (39)
detected tyrosine phosphorylation only in the
-isoform of caveolin
in the Rat-1 cell line expressing a temperature-sensitive
pp60v-src kinase. Tyrosine phosphorylation of
caveolin has also been detected in normal cells, but appears to occur
in a strictly regulated fashion. For example, insulin stimulates
tyrosine phosphorylation of caveolin-1 only in fully differentiated
3T3-L1 adipocytes but not in fibroblasts (preadipocytes), despite the
fact that both cell types express caveolin-1 and active insulin
receptors (7, 40). The functional consequences of tyrosine
phosphorylation of caveolin-1 are not known. However, several reports
suggest some cellular implications of caveolin phosphorylation. For
example, tyrosine phosphorylation of caveolin-1 has been linked to cell transformation by v-Src (41, 42). Koleske et al. (43)
reported that cells transformed by oncogenes show reduced levels of
caveolin-1 along with attenuated number of caveolae. Lee et
al. (44) found that overexpression of caveolin-1 in oncogenically
transformed cells and in breast cancer cells, where the expression of
caveolin-1 is largely reduced, resulted in substantial growth
inhibition and attenuation of anchorage-independent growth in soft
agar. Recently, Kim et al. (45) reported that COOH-terminal
truncation and/or overexpression of the EGF receptor in B82L
fibroblasts can induce an enhanced tyrosine phosphorylation of
caveolin-1 in response to EGF, suggesting that caveolin-1 plays an
important role in the EGF signaling events that are mediated by
aberrant forms or levels of EGF receptor. An enhanced level of wild
type EGF receptor was detected in various human cancers (46, 47) and
was linked with cell transformation (48-50). Furthermore, truncated EGF receptors that lack COOH-terminal autophosphorylation sites can
also induce mitogenesis and cell transformation (51-54).
Among other possible effects, tyrosine phosphorylation of caveolin-1
may alter cholesterol binding to the caveolae and membrane structure
(39). In fact, several studies have demonstrated that caveolin-1 is a
cholesterol-binding protein (55) and that cholesterol is an important
component of caveolae. Furthermore, the observed traffic of caveolin-1
between the endoplasmic reticulum and the Golgi apparatus has been
implicated in the transport of cholesterol to caveolae (56). Taking
into account that cholesterol level of the caveolae membrane may be
involved in the regulation of mitogenesis, tyrosine phosphorylation of
caveolin-1 may improve the transport of cholesterol, determining a loss
of this important component in the caveolae, and thereby altering their
structure/function, with consequent effects on cell growth control
(45).
In the present study, using a monoclonal antibody highly specific for
tyrosine-phosphorylated caveolin (PY-14) (57, 58), we have shown that
the overexpression of the dn mutant of LMW-PTP (an inactive form able
to bind its substrates) in NIH-3T3IR cells causes the enhancement of
tyrosine-phosphorylated caveolin levels. This effect is produced by the
protection exerted by dnLMW-PTP against the dephosphorylating action of
endogenous LMW-PTP and other cellular PTPs on tyrosine-phosphorylated
caveolin. Insulin stimulation causes a dramatic increase of the amount
of phosphocaveolin level in the dnNIH-3T3IR cells with respect to neo
NIH-3T3IR cells, demonstrating that phosphorylated caveolin is a
cellular target for LMW-PTP.
Eukaryotic cells contain a very large number of different PTPs that
probably act specifically on different cellular substrates and in
different cellular sites. The identification of the physiological substrates of PTPs is a key element in understanding the biological function of this family of enzymes. Taken together, our results on the
localization of LMW-PTP in caveolae, on the in vivo
interaction between LMW-PTP and caveolin, and on the capacity of this
enzyme to rapidly dephosphorylate phosphocaveolin indicate that
tyrosine-phosphorylated caveolin is a relevant substrate for
LMW-PTP. We suggest that this enzyme is a good candidate for the
cellular regulation of phosphorylated caveolin level, with consequent
implications in all the processes that are affected by caveolin
phosphorylation on tyrosine.