(Received for publication, April 21, 1997, and in revised form, June 4, 1997)
From the Department of Cell Biology, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Co., Ann Arbor, Michigan 48105
Previous work from this laboratory has shown that insulin stimulates the tyrosine phosphorylation of caveolin in 3T3-L1 adipocytes (Mastick, C. C., Brady, M. J., and Saltiel, A. R. (1995) J. Cell Biol. 129, 1523-1531). This phosphorylation is specific for insulin and involves the activation of a tyrosine kinase downstream of the insulin receptor. We report here that tyrosine phosphorylation of caveolin is detected only in fully differentiated adipocytes, not in fibroblasts (preadipocytes), despite the fact that both cell types express caveolin-1 and active insulin receptor. Caveolin copurifies with caveolin tyrosine kinase activity in both preadipocytes and adipocytes. Accumulating evidence indicates that this kinase is the Src family kinase Fyn. Fyn is expressed in the preadipocytes and the adipocytes and is colocalized with caveolin in low density Triton-insoluble complexes in both cell types. In adipocytes, overexpression of wild type Fyn leads to increased basal phosphorylation of caveolin and hyperphosphorylation of caveolin in response to insulin. In vitro kinase assays suggest that Fyn may be activated in response to insulin through the binding of a tyrosine-phosphorylated insulin receptor substrate protein. Previous work suggested that this protein may be c-Cbl (Ribon, V., and Saltiel, A. R. (1997) Biochem. J. 324, 839-846). In 3T3-L1 adipocytes, Cbl binds to Fyn in an insulin-dependent manner, and Cbl phosphorylation is adipocyte-specific. Here we show that phosphorylated Cbl is translocated into caveolin-enriched Triton-insoluble complexes after insulin stimulation. This may lead to the cell type-specific, compartmentalized activation of Fyn and the specific phosphorylation of proteins in the caveolae in response to insulin in adipocytes.
Like receptors for many growth factors, the insulin receptor is a tyrosine kinase that undergoes activation upon insulin binding, leading to the tyrosine phosphorylation of a specific set of substrate proteins (1, 2). Like other growth factors, insulin acts as a mitogen in many cell types and therefore shares a number of signaling pathways with these growth factors (3). However, in adipocytes insulin increases glucose uptake and metabolism 10 to hundreds of fold, whereas growth factors such as PDGF1 and EGF are without effect on these processes (4-7). This indicates that key regulators of metabolic pathways exist in adipocytes that respond uniquely to insulin signaling. Despite intensive investigation, the basis for this specificity remains largely unknown.
One proposed mechanism for specificity in signal transduction involves
the spatial compartmentalization of signaling proteins (8). The
specialized regions of the plasma membrane termed caveolae have been
implicated in the segregation of signal initiation events (9, 10).
Caveolae are small invaginations of the plasma membrane with unique
protein and lipid compositions (9-12). These structures have a
characteristic striated coat, which is made up largely of the caveolins
(10, 13). Caveolin was originally identified as a major phosphoprotein
in v-Src-transformed cells (14-16), and caveolin expression is
down-regulated in cell lines transformed by a number of different
oncogenes (17). In many different cell types, caveolin copurifies with
or binds to signaling molecules, including
glycosylphosphatidylinositol-anchored proteins, receptors, and
effectors (18-26). Caveolins have also been reported to have intrinsic
GDP dissociation inhibitor (GDI) and GTPase-activating protein (GAP)
activities for G-protein -subunits (27, 28). Despite these reports,
the exact role and composition of these caveolin-containing structures
remain controversial (29-31; reviewed in Ref. 8).
Caveolae are abundant in adipocytes, covering a significant fraction of the inner surface of the plasma membrane (32). Caveolins-1 and -2 are highly expressed in adipocytes (33-35), and caveolin-1 expression increases upon adipocyte differentiation (34). Recently, we reported that caveolin is tyrosine-phosphorylated in response to insulin in 3T3-L1 adipocytes (36). Consistent with an important role for caveolae in insulin action, phosphorylation of caveolin is specific for insulin and does not occur in response to other growth factors such as PDGF or EGF, suggesting that caveolae may be involved in an insulin-specific signaling pathway.
Here we show that the tyrosine phosphorylation of caveolin in response to insulin is specific for the differentiated adipocyte phenotype. Although preadipocytes express active insulin receptors, caveolin-1, and the caveolin-kinase Fyn, caveolin is not tyrosine-phosphorylated in response to insulin in these cells. The data indicate that a key molecule(s) in the signaling pathway leading from the activated insulin receptor to caveolin phosphorylation is regulated by adipocyte differentiation.
All tissue culture reagents were purchased from
Life Technologies, Inc. Insulin (porcine) was purchased from Sigma;
IGF-I (human recombinant, receptor grade) was purchased from
Mallinckrodt (Chesterfield, MO). Monoclonal and polyclonal
anti-caveolin antibodies were purchased from Transduction Laboratories
(Lexington, KY). Monoclonal anti-phosphotyrosine antibodies were
purchased from Upstate Biotechnology Inc. (Lake Placid, NY) and
Transduction Laboratories. Monoclonal anti-insulin receptor antiserum
was purchased from Oncogene Sciences (Cambridge, MA), and polyclonal
anti-IRS-1 antisera was prepared as described (37). Polyclonal anti-Fyn antiserum (-Fyn-268-389) was prepared as described (36) or was
purchased from Santa Cruz Biotechnology (Santa Cruz, CA)
(
-Fyn-29-48); monoclonal anti-Fyn antiserum was purchased from
Transduction Laboratories. Polyclonal anti-Src antiserum was purchased
from Santa Cruz; this antibody cross-reacts with Fyn and Yes.
Polyclonal anti-c-Cbl antiserum was purchased from Santa Cruz.
Prestained molecular weight markers (high range), protein A-agarose,
and horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse IgG antibodies were purchased from Life Technologies, Inc.
Octylthioglucoside and Triton X-100 were purchased from Boehringer
Mannheim. All other reagents were purchased from Sigma. Protein
concentrations were determined using the BCA protein determination kit
from Pierce.
3T3-L1 fibroblast cells (CCL 92.1; American Type Culture Collection, Rockville, MD) were grown in DME (high glucose) supplemented with 10% calf serum, 2 mM L-glutamine, 50 units/ml penicillin, and 50 mg/ml streptomycin. They were differentiated into adipocytes on 150-mm culture plates as described (38). Cells were used for experiments 7-10 days after initiation of differentiation. At this point in the differentiation protocol, most of the cells (>90%) were filled with fat droplets. These cells showed large increases in glucose uptake and metabolism in response to nanomolar concentrations of insulin when serum-deprived as described below. Preadipocytes were grown to confluence and maintained in DME supplemented with calf serum in parallel with the differentiated cells.
Generation of Fyn Expressing Cell LinesFyn kinase constructs (a kind gift from Dr. S. A. Courtneidge, EMBL, Heidelberg, Germany) (39) were subcloned into the BamHI site of pUHD 10.3 (a kind gift from H. Bujard, ZMDH, Heidelberg, Germany) (40). 3T3-L1 cells were co-transfected using LipofectAMINE (Life Technologies, Inc.) with Fyn constructs (linearized with Pvu I) and an empty pOPI3CAT vector containing neomycin resistance (Stratagene Cloning Systems, La Jolla, CA; linearized with NotI and BstXI) at a 1:10 ratio. After 2 days, resistant cells were selected in DME supplemented with 750 µg/ml G418 (Life Technologies, Inc.). Cell lines were isolated from single cells by limiting dilution and were screened for Fyn expression by Western blotting.
Preparation of Triton-insoluble ComplexesCaveolin-enriched Triton-insoluble complexes were prepared essentially as described (36). Adipocytes were incubated overnight in DME supplemented with 0.5% calf serum. Insulin or IGF-1 (100 nM) were added, and incubation was continued at 37 °C for the indicated times. Cells for basal samples were treated identically, without addition of growth factors. Cells (1 or 2 150-mm plates per condition) were washed 3 times in ice-cold phosphate-buffered saline, harvested by scraping into 25 mM MES, pH 6.0, 150 mM NaCl, 1% Triton X-100, 1 mM NaVO4, 10 mg/ml aprotinin, 1 mM benzamidine, and 0.1 mM PMSF (MES/NaCl/Triton X-100), and homogenized (10 strokes in glass homogenizer with a Teflon pestle). To prepare low density, Triton-insoluble complexes, 2 ml of cell lysate were diluted 1:1 with 80% sucrose (40% sucrose final concentration), 25 mM MES, pH 6.0, 150 mM NaCl. A gradient was formed by overlaying the extract with 2 ml each of 35, 25, 15, and 5% sucrose in 25 mM MES, pH 6.0, 150 mM NaCl. The gradients were centrifuged for 20 h at 39,000 rpm in an SW40Ti rotor (Beckman Instruments, Palo Alto, CA). Triton-insoluble complexes enriched in caveolin were collected as a flocculent band of material just below the 15-25% interface using a 19-gauge needle and a syringe (approximately 1-2 ml). This fraction was diluted 2- to 3-fold with MES/NaCl/Triton X-100, and the complexes were collected by centrifugation at 13,500 rpm in a microcentrifuge for 20 min at 4 °C. Alternatively, 1-ml fractions were collected from the top of the gradients using a 19-gauge needle and syringe. Fractions were assayed directly for caveolin and Fyn and then low density, Triton-insoluble complexes prepared from pooled fractions.
To prepare caveolin-enriched Triton-insoluble pellets, cells were lysed as described above and then centrifuged for 5 min at 1500 rpm in a microcentrifuge (to remove nuclei). The supernatant was then centrifuged at 13,500 rpm at 4 °C to prepare Triton-insoluble pellets (pellet) and post-nuclear supernatants (supernatant). Triton-insoluble pellets were washed 1 × with 1 ml of MES/NaCl/Triton X-100. To detect IRS-1 phosphorylation, post-nuclear supernatants were prepared from cells solubilized with MES/NaCl/Triton X-100 supplemented with 1 mM EGTA, 10 mM Na4P207, and 100 mM NaF.
ImmunoprecipitationPost-nuclear supernatants and
caveolin-enriched Triton-insoluble complexes were prepared as described
above. The supernatants were retained (post-nuclear supernatants), and
the pellets were solubilized in 10 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, 60 mM
octylthioglucoside, 1 mM NaVO4, 10 mg/ml
aprotinin, and 10 mg/ml leupeptin (solubilization buffer) 1-2 h on
ice. The samples were precleared with protein A-Sepharose and
immunoprecipitated with 4 µg of polyclonal rabbit anti-caveolin
antibody, 8 µl of monoclonal anti-IR antibody, 8 µl of polyclonal
anti-IRS-1 antiserum, 2 µg of monoclonal anti-Cbl antibody, and
protein A-Sepharose (25 µl of packed beads). After washing 5 times
with solubilization buffer or MES/NaCl/Triton X-100, proteins were
eluted using Laemmli sample buffer (4% SDS, 115 mM
Tris-Cl, pH 6.8, 1 mM EDTA, 10% glycerol, 4 mg/ml
bromphenol blue) with 0.7% -mercaptoethanol. Anti-caveolin
immunoprecipitates were eluted with Laemmli sample buffer without
reducing agents.
Assays were performed essentially as described (36). Briefly, Triton-insoluble pellets or low density Triton-insoluble complexes were resuspended in 5 µl of MES/NaCl/Triton X-100 and 20 µl of kinase assay buffer (22.5 mM Hepes, pH 7.5, 12.5 mM MgCl2, 1.25 mM MnCl2, 1.25 mM EGTA). The reaction was initiated with the addition of 1 mM ATP (or buffer); the samples were incubated for 10 min at room temperature, and the reaction was terminated with 4 × concentrated Laemmli sample buffer. Protein tyrosine phosphorylation was determined by Western blotting. In some experiments, reactions were terminated with 0.5 ml of solubilization buffer supplemented with 5 mM EDTA, and caveolin was immunoprecipitated prior to analysis.
To determine the effects of tyrosine-phosphorylated peptides on
caveolin-kinase activity, the complexes were preincubated for 1 h
at 4 °C with 100 µM peptide or Me2SO (2%)
prior to assay. The peptides used were pY-MT (EPQ-pY-EEIPIYL) and MT
(EPQYEEIPIYL) derived from the middle T antigen, and pY-VPM derived
from tyrosine 751 of the human PDGF receptor. The peptides were
synthesized, purified, and characterized as described previously (41).
In vitro competition binding assays using glutathione
S-transferase fusion proteins of SH2 domains indicate that
the pY-MT peptide binds with high affinity to the SH2 domains of Fyn
and Src, but not p85, whereas the pY-VPM peptide binds with high
affinity to both SH2 domains of PI 3
-kinase but not to the SH2 domains
of Fyn or Src (data not shown).
Samples in Laemmli sample buffer were boiled for 5 min; proteins were separated by SDS-polyacrylamide gel electrophoresis, and the proteins were transferred to nitrocellulose (0.2-m pore size; Schleicher and Schuell) for 2 h at 400 mA in 20% methanol, 192 mM glycine, 25 mM Tris, 0.005% SDS. The membranes were blocked by incubation for 1 h in TBST (20 mM Tris, pH 7.6, 150 mM NaCl, 0.05% Tween 20, 0.1% Brij) with 1% ovalbumin and 1% bovine serum albumin (anti-phosphotyrosine) or with 5% non-fat dried milk (anti-caveolin, anti-Fyn, anti-Cbl). The membranes were incubated overnight with both monoclonal anti-phosphotyrosine antibodies diluted in TBST with 1% ovalbumin and 1% bovine serum albumin (1/4000) or with monoclonal anti-caveolin antibody (1/2000), monoclonal, or polyclonal anti-Fyn antibodies (1 µg/ml), or polyclonal anti-c-Cbl (1/2500) antibody diluted in TBST with 1% milk. After washing 4 times in TBST, the membranes were incubated for 30 min with horseradish peroxidase-conjugated goat anti-mouse IgG antibody or goat anti-rabbit IgG antibody diluted in the appropriate blocking buffer (1/3000 or 1/5000), washed 2 times in TBST, 2 times in TBS (20 mM Tris, pH 7.6, 150 mM NaCl), and the labeled proteins visualized by the enhanced chemiluminescence method (Amersham Corp.).
Image ProcessingAutoradiographs were quantified by computer-assisted video densitometry using the BioImage system (Imaging Systems, Millipore Corp., Ann Arbor, MI). Figures of autoradiographs were constructed using the images collected above and Adobe Photoshop software (Adobe Systems Incorporated, Mountain View, CA).
Differentiation of 3T3-L1 fibroblasts into adipocytes
is accompanied by a significant increase in the responsiveness of
glucose uptake and metabolism to
insulin.2 To further explore
the role of caveolae in insulin action, the tyrosine phosphorylations
of caveolin and the caveolin-associated proteins were compared in
preadipocytes and adipocytes (Fig. 1). Cells were grown to confluency and either maintained in calf serum (pread.) or induced to differentiate (adip.) as
described (see "Experimental Procedures"). 10 days after the
initiation of differentiation, cells were serum-starved over night and
then incubated with insulin (100 nM, 5 min). The cells were
lysed in MES/NaCl/Triton X-100; caveolin was immunoprecipitated, and
tyrosine phosphorylation was determined by anti-phosphotyrosine Western
blotting. As reported previously (36), in response to insulin there was
a significant increase in the tyrosine phosphorylation of two forms of
caveolin (22 and 24 kDa) and a 29-kDa caveolin-associated protein in
adipocytes (Fig. 1A). This was not observed in the
preadipocytes, although basal tyrosine phosphorylation of both proteins
was detected in these cells. In this experiment, the amount of cell
lysate (mg of protein) used for the preadipocyte and adipocyte samples
was adjusted such that comparable amounts of caveolin were
immunoprecipitated from both (Fig. 1B). However, while the
adipocytes express both the 22- () and 24-kDa (
) forms of
caveolin-1, the preadipocytes express only the 24-kDa form (see
below). The preadipocytes and adipocytes express comparable numbers
of active insulin receptors (Fig. 1C, approximately 3-fold
increase upon differentiation). These receptors appear to be fully
functional, as IRS-1 was highly phosphorylated in response to insulin
in both cell types (Fig. 1D). The identity of the 185-kDa
phosphoprotein as IRS-1 was confirmed by immunoprecipitation (data not
shown).
Many signaling pathways stimulated by insulin are shared by IGF-I.
Although adipocytes express predominantly insulin receptors (43),
preadipocytes express similar levels of both IGF-1 and insulin
receptors (Fig. 2). To maximally
stimulate the insulin/IGF-I pathway in the preadipocytes, we used high
concentrations (100 nM) of IGF-1 to stimulate both the
IGF-1 receptors and insulin receptors in these cells. The major
tyrosine-phosphorylated proteins detected in the post-nuclear
supernatants after insulin stimulation were the -subunit of the
insulin receptor (95 kDa) and the
-subunit of the IGF-I receptor
(105 kDa) (Fig. 2A). The identity of the 185-kDa
tyrosine-phosphorylated protein as IRS-1 was verified by
immunoprecipitation (Fig. 2B). Although there was a
rapid phosphorylation of IRS-1 in these cells, there was no
tyrosine phosphorylation of caveolin in response to IGF-I (Fig.
2C), despite immunoprecipitation of a significant amount of
caveolin from these cells (Fig. 2D). Together with Fig. 1,
these data indicate that the block in caveolin phosphorylation in the
preadipocytes occurs downstream of the insulin receptor itself.
Colocalization of Caveolin and Fyn in Low Density Triton-insoluble Complexes
It has previously been reported that caveolin protein
expression increases 20-fold upon differentiation of 3T3-L1 fibroblasts into adipocytes (34). We found that while there is an increase (approximately 3-fold) in total caveolin expression upon
differentiation, there was a significant amount of the 24-kDa or
-isoform of caveolin-1 expressed in the fibroblasts (Fig.
3A, lower panel). Although there was little increase in the expression of caveolin-1
(less than
2-fold), a large increase was detected in the expression of the 22-kDa
or
-isoform of caveolin-1 (approximately 11-fold) upon
differentiation (the caveolin migrated as a doublet after differentiation). The differences between our results and those of
Scherer et al. (36) may be due to differences in antibodies or variations in the cell lines. We have previously reported that different caveolin-1 antibody preparations recognize either one or both
of the caveolin-1 isoforms. Scherer et al. (36) detected only a single band of caveolin, with an apparent molecular mass of
approximately 22 kDa, the molecular mass of caveolin-1
, which is
consistent with our results. Both isoforms of caveolin-1 are phosphorylated in the adipocytes in response to insulin treatment (Fig.
1 and Ref. 36). In addition, it has been shown that caveolin-1
is a
substrate for v-Src in vitro and in vivo (44).
Therefore, the caveolin expressed in the preadipocytes should serve as
a substrate for tyrosine phosphorylation, if the caveolin and the caveolin-kinase are colocalized in caveolae, and the kinase is activated in response to insulin.
The caveolin in both the preadipocytes and adipocytes was enriched in
low density Triton-insoluble complexes (Fig. 3, C and D). However, the efficiency with which the caveolin was
recovered in these fractions increased upon differentiation. There was
a significant increase in the total amount of protein isolated in the
caveolar fractions after differentiation, although it remained only a
small fraction of the total protein in the cell lysates (data not
shown). Although the mechanism is still unclear, the increase in the
recovery of caveolin in the low density complexes in differentiated
cells may result from the increased expression of caveolin-1
(perhaps decreasing the Triton solubility of the complexes) or from an
increase in the abundance of a limiting component that confers low
density or Triton insolubility on the caveolin-containing complexes
(i.e. cholesterol or sphingolipids). The failure to detect
caveolin phosphorylation in the preadipocytes was not simply due to the
reduced efficiency of caveolin localization in caveolae. There was no
increase in the tyrosine phosphorylation of caveolin found in the low
density Triton-insoluble complexes in the preadipocytes in response to
insulin (Fig. 3B), although basal phosphorylation of
caveolin and the 29-kDa caveolin-associated protein could be detected
in these fractions with long exposures (data not shown). In contrast,
there was a clear increase in the phosphorylation of the caveolins and
the 29-kDa caveolin-associated protein in response to insulin in the
same fractions from the adipocytes.
Our previous work (36) indicated that phosphorylation of caveolin occurs through the activation of a tyrosine kinase downstream of the insulin receptor, which is a resident of the caveolae. Accumulating evidence indicates that this kinase is the Src family kinase Fyn (36; see below). The expression and subcellular distribution of Fyn were compared in preadipocytes and adipocytes (Fig. 3). Fyn is easily detected in the preadipocytes by Western blotting (Fig. 3A, upper panel). The levels of Fyn kinase per mg of protein (but not per cell) decreased upon differentiation. Fyn was highly enriched in low density caveolin-enriched Triton-insoluble complexes from both preadipocytes and adipocytes (Fig. 3, C and D, fractions 4 and 5).
Copurification of Caveolin and Caveolin-Tyrosine Kinase ActivityWe have previously observed in in vitro
kinase assays that caveolin copurifies with a caveolin-tyrosine kinase
in Triton-insoluble complexes (36). Isolation of these complexes
results in a significant activation of the caveolin kinase activity in
these fractions, as incubation of complexes from either basal or
insulin-stimulated cells with ATP resulted in a significant and
comparable increase in the level of phosphorylation of caveolin and
associated proteins. The small apparent effect of insulin on the
activity of the caveolin kinase in vitro is due to the
elevated (near maximal) activity of the kinase in the complexes
prepared from basal cells. No detectable insulin receptor kinase
activity was detected in these fractions (36). We took advantage of
this activation of the caveolin kinase activity to determine if
caveolin was colocalized with functional caveolin tyrosine kinase in
both preadipocytes and adipocytes. In vitro kinase assays
were performed on either Triton-insoluble pellets (TIP) or highly
purified low density Triton-insoluble complexes from both cell types
(Fig. 4). Intact complexes were incubated
with or without 1 mM ATP in kinase assay buffer for 10 min,
and kinase activity was measured as an increase in tyrosine phosphorylation by Western blotting. Prior to analysis, the
Triton-insoluble pellet samples were solubilized with
octylthioglucoside, and caveolin-associated proteins were isolated by
immunoprecipitation. The entire low density, Triton-insoluble complex
samples were analyzed. Incubation of either of the complexes with ATP
resulted in a significant increase in the tyrosine phosphorylation of
caveolin and the 29-kDa caveolin-associated protein in samples from
both preadipocytes and adipocytes. As previously observed (36), an
additional 27-kDa caveolin-associated protein was also
tyrosine-phosphorylated in vitro, although this protein was
not phosphorylated in response to insulin in the intact cells. These
data indicate that the caveolin kinase is expressed and properly
localized in complexes with caveolin in the preadipocytes; however, it
is not activated in response to insulin in the intact cells.
Tyrosine Phosphorylation in Cell Lines Overexpressing Fyn
To
explore in more detail whether Fyn is the insulin-regulatable
caveolin-tyrosine kinase, 3T3-L1 cells were stably transfected with
plasmids encoding either kinase-inactive Fyn or wild type Fyn kinase.
We were unable to differentiate any cell lines that expressed the
kinase negative Fyn construct, so the effect of this construct on
caveolin phosphorylation could not be determined. However, a cell line
expressing high levels of the wild type Fyn kinase was isolated and
analyzed (Fig. 5). This cell line
differentiated well, as determined by accumulation of fat droplets,
with kinetics that were faster than the parental cells (data not
shown). Unexpectedly, the Fyn-overexpressing cell line grew
significantly more slowly than the parental cells. Fyn levels were
elevated in both the post-nuclear supernatants and the
caveolin-enriched Triton-insoluble pellets in the overexpressing cell
line (Fig. 5, A and B). In cells overexpressing
Fyn, there was a significant increase in the basal level of
phosphorylation in the caveolin-enriched, low density, Triton-insoluble
complexes, suggesting that the high level of expression of Fyn was
sufficient to cause activation of the kinase in this fraction (Fig.
5C). In contrast, there was very little increase in basal
phosphorylation seen in the Triton-insoluble pellet and post-nuclear
supernatant fractions from these cells (Fig.
6B).
Overexpression of Fyn led to increased basal tyrosine phosphorylation of four proteins in the low density, Triton-insoluble complexes with apparent molecular masses of 22, 24, 27, and 29 kDa. Hyperphosphorylation of these proteins was observed in response to insulin (Fig. 5C). These four proteins were the only proteins in the low density, Triton-insoluble fractions to show an increase in phosphorylation after insulin stimulation. Similar levels of caveolin were isolated in the fractions from the two cells types (Fig. 5D).
The identities of the Fyn substrate proteins in the Triton-insoluble fractions as the caveolins and the caveolin-associated proteins were verified by immunoprecipitation (Fig. 6). Triton-insoluble pellets from basal or insulin-stimulated cells were solubilized with octylthioglucoside, and caveolin-associated proteins were isolated by immunoprecipitation. There was an increase in the basal phosphorylation of the caveolins (22 and 24 kDa) and the 29-kDa caveolin-associated protein and hyperphosphorylation of these three proteins after insulin stimulation (Fig. 6A, upper panel). Interestingly, overexpression of Fyn led to the constitutive phosphorylation of the 27-kDa caveolin-associated protein. The phosphorylation of this protein had previously only been detected in vitro. Surprisingly, there was no change in the phosphorylation of caveolin, or the caveolin-associated proteins in the preadipocytes (Fig. 6C, upper panel), despite a significant increase in the amount of Fyn localized to the Triton-insoluble pellets in these cells (Fig. 6D), indicating that overexpression of Fyn is not sufficient to activate this kinase in the preadipocytes. Similar amounts of caveolin were immunoprecipitated from both preadipocytes and adipocytes from both cell lines (Fig. 6, A and C, lower panel). Taken together, these data strongly suggest that the block in caveolin phosphorylation in the preadipocytes is in the signaling pathway leading to the activation of the caveolin kinase Fyn, rather than in the localization or expression of the kinase itself.
Potential Mechanism for Fyn Activation in AdipocytesHow is the Fyn that is resident in caveolae activated in response to insulin? Src family kinases share two tyrosines that can be phosphorylated in vivo (45). The Src family kinases are normally phosphorylated at the C-terminal regulatory site (tyrosine 527 in Src) by Csk. This phosphorylation inhibits the activity of the kinases, by inducing the binding of this residue to the SH2 domain in the N termini of these kinases in an intramolecular interaction. This allows for an intramolecular SH3 domain interaction within the kinases, folding the kinases into inactive conformations (46, 47). There is an additional autophosphorylation site that is required for full activation of these kinases. The Src family kinases can be activated either through the dephosphorylation of the tyrosine 527 residue (48) or through the displacement of the phosphorylated C-terminal tail by another tyrosine-phosphorylated protein binding to the SH2 and SH3 domains (49, 50).
Fyn was constitutively associated with the caveolinenriched,
low density, Triton-insoluble complexes in 3T3-L1 adipocytes (Fig. 7A,
-Fyn-(29-48) and
-Fyn-(268-389)). The Fyn
in these complexes exactly comigrated with the 60-kDa phosphoprotein
found in these complexes (Fig. 7A,
-ptyr), and
immunoprecipitation with an anti-Fyn antibody completely immunodepleted
the Fyn from the supernatants of octylthioglucoside-solubilized
complexes, indicating that Fyn is the predominant Src family kinase in
these fractions (36). There is no detectable change in the
phosphorylation of Fyn in the low density, Triton-insoluble complexes
in response to insulin (Fig. 7B). Consistent with this, it
has been shown that CD45 is largely excluded from the Triton-insoluble
fractions in lymphocytes (51). Fyn colocalizes with caveolin in
Triton-insoluble complexes from both preadipocytes and adipocytes (Fig.
7C,
-Fyn and
-Src), and insulin has no effect on the
localization or phosphorylation of Fyn in either cell type.
Interestingly, however, differentiation leads to a significant increase
in the constitutive tyrosine phosphorylation of Fyn in the
Triton-insoluble pellets (Fig. 7C,
-ptyr).
Specific tyrosine-phosphorylated peptides
stimulate the caveolin tyrosine kinase activity in vitro.
Low density, caveolin-enriched, Triton-insoluble complexes
(A, B, and E) and Triton-insoluble pellets (C and D) were prepared from basal cells
() or cells stimulated with insulin (+) for increasing amounts of
time (0-30 min). A, anti-phosphotyrosine
(
-ptyr) or anti-Fyn (
-Fyn-(29-48);
-Fyn-(268-389)) Western blots of low density
Triton-insoluble complexes from basal cells or cells stimulated with
insulin for 5 min. B, anti-phosphotyrosine Western blot of
low density Triton-insoluble complexes prepared from adipocytes
stimulated with insulin for increasing amounts of time. C,
anti-Fyn, anti-Src (
-Src; recognizes Src, Fyn, and Yes),
or anti-phosphotyrosine Western blots of Triton-insoluble pellets from
preadipocytes (pread.) or adipocytes (adip.)
under basal or insulin-stimulated conditions (5 min). These samples are
supernatants from an anti-caveolin immunoprecipitation, and the bands
from residual antibodies are also shown (Ab). D,
Triton-insoluble pellets; E, low density Triton-insoluble
complexes from basal cells were incubated with the indicated peptides
(100 µM) for 1 h at 4 °C and then further
incubated for 10 min with or without 1 mM ATP at 25 °C.
The samples were analyzed by anti-phosphotyrosine Western blotting. MT
and pY-MT are peptides derived from the middle T
antigen; pY-MT binds with high affinity to the SH2 domains of Src and
Fyn. pY-VPM is derived from tyrosine 751 of the
human PDGF receptor and binds with high affinity to both SH2 domains of
PI 3
-kinase but not the SH2 domains of Src or Fyn. Filled arrowheads indicate the positions of proteins whose
phosphorylation is increased with addition of pY-MT, and the open
arrowhead indicates the 27-kDa protein whose phosphorylation is
inhibited by pY-MT.
It has been shown that ligands for the Src SH2 domain, such as
tyrosine-phosphorylated peptides derived from the PDGF receptor or
middle T antigen, can activate Src enzymatic activity (49, 52).
Therefore, we investigated whether the caveolin kinase in the
Triton-insoluble complexes could be stimulated with
tyrosine-phosphorylated peptides that bind to the SH2 domains of Src
family kinases. Intact Triton-insoluble pellets (Fig. 7D) or
low density Triton-insoluble complexes (Fig. 7E) were
preincubated with peptides and further incubated with or without 1 mM ATP. Kinase activity was measured as an increase in
tyrosine phosphorylation by anti-phosphotyrosine Western blotting.
Incubation with a tyrosine-phosphorylated peptide derived from middle T
antigen (pY-MT) increased the autophosphorylation of Fyn (60 kDa)
2-3-fold. The phosphorylations of a number of other proteins were
significantly increased as well (Fig. 7, D and E,
filled arrowheads), including caveolin and the 29-kDa
caveolin-associated protein (Fig. 7E, and data not shown).
This peptide binds with high affinity to the SH2 domains of Src and Fyn
but not to SH2 domains from other proteins such as SHP-2 or PI
3-kinase (41). No stimulation of the kinase was observed with
non-phosphorylated peptide or with a phosphorylated peptide (pY-VPM)
that binds to the SH2 domain of PI 3
-kinase, but not Src or Fyn.
Interestingly, the pY-MT peptide inhibited the phosphorylation of a
27-kDa protein in the Triton-insoluble pellet (Fig. 7D, open
arrowhead).
The in vitro kinase data suggested that insulin might
activate the caveolin kinase through the tyrosine phosphorylation of a
specific insulin receptor substrate protein that binds to the SH2
domain in Fyn. Previous work suggested that the insulin-stimulated tyrosine phosphorylation of Cbl may be involved in the activation of
Fyn in adipocytes (53). Cbl is rapidly and specifically phosphorylated on tyrosine in response to insulin in adipocytes. Cbl binds to Fyn in
adipocytes, and this association is significantly increased after
insulin stimulation. In addition, tyrosine-phosphorylated Cbl from
adipocyte lysates binds specifically to a fusion protein containing the
SH2 domain of Fyn. To further explore the possible relationship between
Cbl phosphorylation and activation of the caveolin kinase, the effect
of insulin on the association of Cbl with caveolin-enriched fractions
was analyzed (Fig. 8). Cells were
stimulated with insulin for increasing amounts of time, and the cells
were fractionated into Triton-insoluble pellets or post-nuclear supernatants as described under "Experimental Procedures." Cbl was
detected in the caveolin-enriched Triton-insoluble pellets by anti-Cbl
Western blotting. Consistent with a role for Cbl in the activation of
Fyn, Cbl was translocated into the Triton-insoluble fractions after
insulin stimulation (Fig. 8A). Cbl association rapidly
increased after insulin stimulation, reaching a maximum of 4-fold
within 1-2 min, and levels declined thereafter. Cbl was
immunoprecipitated either from octylthioglucoside-solubilized complexes
or from the post-nuclear supernatants and analyzed by anti-phosphotyrosine Western blotting (Fig. 7B). The Cbl
translocated into the Triton-insoluble pellets was
tyrosine-phosphorylated, and the kinetics of the association of
tyrosine-phosphorylated Cbl with the Triton-insoluble pellets was
similar to the kinetics of phosphorylation of Cbl in the cell lysates
and to the phosphorylation of caveolin in the Triton-insoluble pellets
(Fig. 8C). Very little of the tyrosine-phosphorylated
insulin receptor and no tyrosine-phosphorylated IRS-1 were detected in
the Triton-insoluble pellets by antiphosphotyrosine Western blotting
(Fig. 8D).
The ability of insulin to stimulate glucose uptake and storage of glucose as glycogen and lipid is significantly increased after adipocyte differentiation.2 Differentiation leads to an increase in the expression of the enzymes involved in the storage of glucose as lipid and glycogen. However, the increase in the responsiveness of these pathways to insulin cannot be fully accounted for by the combined increases in insulin-stimulated glucose uptake and the increased levels of expression of these enzymes after differentiation.3 This indicates that the metabolic signaling pathways stimulated by insulin may be up-regulated by differentiation as well.
Many of the signaling pathways stimulated by insulin are shared by other growth factors, such as EGF and PDGF. However, insulin stimulates glucose uptake and metabolism in adipocytes, whereas EGF and PDGF have little effect on these processes. Interestingly, caveolin-1 is specifically tyrosine-phosphorylated in response to insulin. Despite the presence of receptors for other growth factors such as PDGF or EGF on adipocytes, caveolin phosphorylation does not occur in response to these other ligands (36). We demonstrate here that the tyrosine phosphorylation of caveolin is also differentiation-dependent in 3T3-L1 cells (Figs. 1 and 2). Despite comparable levels of expression of active insulin receptors and caveolin in both preadipocytes and adipocytes, stimulation of the insulin receptor is uncoupled from the phosphorylation of caveolin in the preadipocytes. This suggests that caveolin is not a direct substrate of the insulin receptor and that other proteins are required for this phosphorylation.
We have previously shown (36) that caveolin copurifies with a caveolin-tyrosine kinase in low density Triton-insoluble complexes from 3T3-L1 adipocytes from both basal and insulin-stimulated cells. Although there was no detectable insulin receptor tyrosine kinase activity in these complexes, the Src family kinase Fyn was highly enriched in the caveolar fractions from 3T3-L1 adipocytes (36). Fyn copurifies with caveolin in low density Triton-insoluble complexes from both preadipocytes and adipocytes (Figs. 3 and 4). Moreover, overexpression of wild type Fyn caused an increase in basal caveolin phosphorylation, as well as hyperphosphorylation of caveolin after insulin stimulation in the adipocytes (Figs. 5 and 6). However, there was no effect of Fyn overexpression on caveolin phosphorylation in the preadipocytes.
Preadipocytes and adipocytes both express significant levels of the insulin receptor, caveolin, and the caveolin kinase Fyn. However, only in the adipocytes does activation of the insulin receptor lead to the phosphorylation of caveolin. The data imply the presence of a mediator required for the segregated activation of Fyn in caveolae, the activity of which is critically dependent on adipocyte differentiation. There was no change in the association or tyrosine phosphorylation of Fyn in the caveolin-enriched fractions in response insulin in either adipocytes or preadipocytes (Fig. 7), indicating that direct modifications of Fyn phosphorylation are not involved in the activation of this kinase in response to insulin. The in vitro kinase assays suggest that the mediator for Fyn activation in adipocytes is an insulin-stimulated tyrosine-phosphorylated protein, which activates Fyn through binding to the SH2 and SH3 domains of the kinase. While the identity of this specific insulin-receptor substrate protein has not been definitively shown, evidence suggests that this molecule may be c-Cbl.
The unique conditions under which caveolin phosphorylation is observed
suggest that the insulin receptor substrate responsible for the
activation of caveolar Fyn would have several unique properties as
follows: 1) phosphorylation showing specificity for insulin in
adipocytes; 2) association with Fyn (through an SH2 domain interaction)
in response to tyrosine phosphorylation; 3) translocation of the
phosphorylated protein into caveolae in response to insulin; and 4)
phosphorylation in 3T3-L1 adipocytes but not in the preadipocytes. Although the well characterized insulin receptor substrates IRS-1/-2 and Shc emerged as potential candidates (54, 55), none fulfilled all of
these requirements. In contrast, the proto-oncogene product c-Cbl
shares many properties with this presumed substrate protein. Insulin
stimulates the phosphorylation of c-Cbl in adipocytes, and this
phosphorylation shows specificity for insulin in this cell type (53).
As has been observed in lymphocytes, Cbl constitutively binds to Fyn in
unstimulated adipocytes through an SH3 domain-mediated interaction, and
insulin-stimulated tyrosine phosphorylation of Cbl increases this
association. The tyrosine-phosphorylated Cbl in adipocytes binds
specifically to fusion proteins containing the SH2 domain of Fyn but
not other insulin-stimulated proteins such as SHP-2 or PI 3-kinase.
Cbl is translocated into caveolin-enriched Triton-insoluble complexes
after insulin stimulation, and the Cbl in these complexes is
tyrosine-phosphorylated (Fig. 8). Most interestingly, unlike the other
known substrates of the insulin receptor such as IRS-1/-2 and Shc, Cbl
phosphorylation is specific for the differentiated adipocyte phenotype
(53).
The basis for the cell type specificity of Cbl phosphorylation is currently unknown. Cbl is expressed at comparable levels in both the preadipocytes and adipocytes. Unlike IRS-1/-2 and Shc, however, Cbl does not directly interact with the insulin receptor (53). We hypothesize that tyrosine phosphorylation of Cbl may require a specific adapter protein (56), which allows for the interaction of Cbl with the insulin receptor (53), and that it is the regulation of the expression of this protein that accounts for the coordinate regulation of the phosphorylations of Cbl and caveolin in response to insulin in adipocytes. Although the model linking phosphorylation of Cbl to the phosphorylation of caveolin through the activation of Fyn is compelling, the exact relationship between these three proteins is likely to be complex (56). For example, Cbl is likely to undergo processive phosphorylation after the activation of Fyn (42, 57). However, the insulin-dependent association of Cbl and Fyn in caveolae leads to the intriguing possibility that the Fyn/Cbl pathway may have a unique function in adipocytes (phosphorylation of caveolin) due to the localization of these proteins to specialized domains of the plasma membrane, the caveolae.
The fact that caveolin expression and phosphorylation are regulated during adipocyte differentiation suggests an important role for caveolae in the differentiated adipocyte phenotype. An understanding of this role will lead to further insights into the general function of caveolae in cells and may lead to insights into the mechanisms of insulin-specific signal transduction.
We thank Drs. Vered Ribon, Matthew Brady, and Dan Lazar for valuable discussions, Parul Matani for technical assistance, and Dr. Sarah Courtneidge for the Fyn constructs.