From the Departments of Molecular Pharmacology and
¶ Developmental and Molecular Biology and the
Division of
Endocrinology, Albert Einstein College of Medicine,
Bronx, New York 10461
Received for publication, June 21, 2000, and in revised form, November 13, 2000
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ABSTRACT |
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Caveolin-1 serves as the main coat protein of
caveolae membranes, as an intracellular cholesterol shuttle, and
as a regulator of diverse signaling molecules. Of the 12 residues
conserved across all caveolin isoforms from all species examined to
date, only Ser80 and Ser168 could serve
as phosphorylation sites. We show here that mimicking chronic
phosphorylation of Ser80 by mutation to Glu
(i.e. Cav-1(S80E)), blocks phosphate incorporation. However, Cav-1(S168E) is phosphorylated to the same extent as wild-type
caveolin-1. Cav-1(S80E) targets to the endoplasmic reticulum membrane,
remains oligomeric, and maintains normal membrane topology. In
contrast, Cav-1(S80A), which cannot be phosphorylated, targets to
caveolae membranes. Some exocrine cells secrete caveolin-1 in a
regulated manner. Cav-1(S80A) is not secreted by AR42J pancreatic adenocarcinoma cells even in the presence of dexamethasone, an agent
that induces the secretory phenotype. Conversely, Cav-1(S80E) is
secreted to a greater extent than wild-type caveolin-1 following dexamethasone treatment. We conclude that caveolin-1 phosphorylation on
invariant serine residue 80 is required for endoplasmic
reticulum retention and entry into the regulated secretory pathway.
A 22-kDa phosphoprotein first detected in Rous sarcoma
virus-transformed fibroblasts (1), and normal fibroblasts
overexpressing an activated form of the transforming kinase Src (2),
was identified simultaneously as caveolin (now termed caveolin-1), the
principal coat protein of caveolae (3), and as VIP21, an integral
membrane protein component of detergent resistant, trans-Golgi derived vesicles (4). These initial descriptive reports all foreshadowed the
functions of this molecule. First, caveolin-1 is a
transformation-dependent substrate of v-Src, and is
down-regulated in a number of cancer-derived cell lines (reviewed in
Ref. 5). Second, it is a component of the cell's transport machinery,
contributing to the influx and efflux of cellular cholesterol (6-10),
and to the polarized transport of proteins (4, 11, 12). Finally, it is
the driving force for caveolae formation (13-17), actively
contributing to the organization of lipids (18-22) and signaling
proteins (reviewed in Ref. 23) within these vesicular invaginations of
the plasma membrane.
A wealth of biochemical and cell biological knowledge has now been
gathered about the structure of caveolins. Several laboratories have
validated the hypothesis that a central hydrophobic segment anchors the
protein to the membrane, and splits the molecule into two cytoplasmic
domains (24): first, antibodies directed against the extreme N and C
termini fail to stain caveolin-1 in unpermeabilized cells (25, 26).
Likewise, artificial glycosylation sites introduced at the extreme N
and C termini are not glycosylated (25). Third, caveolin-1 is not
labeled when cells are subjected to surface biotinylation, suggesting
that there is no extracellular domain (27). Finally, the N terminus of
caveolin-1 undergoes tyrosine phosphorylation (1, 2, 28), and the C
terminus of caveolin-1 undergoes palmitoylation (26), both cytoplasmic modifications.
Two other mammalian caveolins have been identified and characterized
(29-31). Caveolin-2 is expressed in many cell types that express
caveolin-1 (12, 32); while caveolin-3 is limited to striated muscle
cells (cardiac and skeletal) and neuroglia (33, 34). Caveolin-1 and
caveolin-3 can each form homotypic, high molecular mass oligomers
containing ~14-16 individual molecules (25, 27, 31). In the
ER,1 homo-oligomerization is
mediated by a 40-aminoacyl residue domain (residues 61-101 in
caveolin-1) (27). In the Golgi, adjacent homo-oligomers undergo a
second stage of oligomerization; they interact with one another through
contacts between the C-terminal domains of caveolin, in a side by side
packing scheme (35, 36). These oligomer-oligomer interactions then
produce an interlocking network of caveolin molecules that gives rise
to the striated caveolar coat seen by scanning electron microscopy (3).
Although much has been learned about caveolin structure and function,
the role of phosphorylation has not been defined. Constitutively active
v-Src phosphorylates caveolin-1 on Tyr14 in
vitro (28). At the permissive temperature, expression of temperature-sensitive v-Src results in tyrosine phosphorylation on
Tyr14 in vivo (37). A reversible flattening and
aggregation of caveolae at the cell membrane occurs as a result of
temperature-sensitive v-Src activation (37, 38).
In addition to these changes in caveolae morphology, we recently
uncovered a novel interaction between caveolin-1 and the SH2 domain
containing adapter molecule GRB7. Caveolin-1 binds GRB7 following
growth factor-stimulated (and Src-catalyzed) phosphorylation of
caveolin-1 tyrosine residue 14. Mutation of this residue to prevent
phosphorylation undermines GRB7 association. When coexpressed, Src,
Cav-1, and GRB7 dramatically increase anchorage-independent growth and
cell migration (39). Downstream effector molecules remain to be
identified, but this series of protein-protein interactions defines the
first biological function for caveolin-1 tyrosine phosphorylation.
Here, we used a mutational approach to characterize the possible
functional significance of phosphorylation at two serine residues
conserved across all caveolin isotypes from all species examined to
date. Mutation of Ser80 to glutamate, a substitution that
mimics chronic phosphorylation, leads to loss of caveolin-1 from
caveolae membranes and tight association with endoplasmic reticulum
(ER) membranes of fibroblasts. Mutation of Ser80 to alanine
neither hinders efficient targeting of caveolin-1 to caveolae, nor
disrupts interaction with wild-type caveolin-1. However, in cells of
the exocrine pancreas, where caveolin-1 undergoes regulated secretion,
we find that mutation of Ser80 to glutamate results in
enhanced secretion, while mutation of Ser80 to alanine
blocks exocytosis completely. These findings suggest that
phosphorylation of caveolin-1 on Ser80 regulates caveolin-1
entry into the regulated secretory pathway by binding to ER membranes.
Materials--
All cell culture materials and LipofectAMINE PLUS
were from Life Technologies, except for donor calf serum (JRH
Biosciences). The following materials were purchased from the indicated
suppliers: [9,10-3H]palmitic acid, American
Radiochemicals; [32P]orthophosphate, PerkinElmer Life
Sciences; calyculin A, okadaic acid, Calbiochem; Fugene 6 transfection
reagent, Complete MiniTM tablets, and pepstatin A, Roche Molecular
Biochemicals; benzamidine, leupeptin, and soybean trypsin inhibitor,
bovine serum albumin (Fraction V), methyl- Plasmids--
The canine caveolin-1 cDNA was as we described
previously (41). Mutagenesis was performed using standard methods and
verified by dideoxynucleotide sequencing.
Cells, Media, and Transfection Methods--
Madin-Darby canine
kidney epithelial cells were cultured in Dulbecco's modified Eagle's
medium (DMEM) supplemented with 10% donor calf serum, 2 mM
glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin. Human
embryonic kidney 293T and COS-7 cells were cultured in DMEM
supplemented with 10% fetal bovine serum (FBS), 2 mM
glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin. AR42J
cells were obtained from the American Type Cell Collection (CRL-1492)
and were propagated in Kaighn's modified Ham's F-12 nutrient mixture
(F-12K) supplemented with 20% FBS, 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin. 293T cells were transfected by calcium phosphate precipitation. COS-7 cells (grown to
40% confluence in a 3.5-mm diameter dish) were transfected with Fugene
6 using 2 µg of DNA and 3 µl of transfection agent (diluted in 97 µl of serum-free medium) as directed by the manufacturer. AR42J cells
were transfected with LipofectAMINE PLUS as described below.
Immunoblotting--
Samples were subjected to SDS-PAGE under
reducing conditions. Proteins were transferred to nitrocellulose
membranes, and stained with Ponceau S (Sigma). Membranes were then
washed in 10 mM Tris, pH 8.0, 150 mM NaCl,
0.05% Tween 20 (wash buffer), blocked in wash buffer supplemented with
2% nonfat milk and 1% bovine serum albumin (blocking buffer),
incubated with primary antibody, washed again, and incubated with a
secondary antibody conjugated with horseradish peroxidase. Bound IgGs
were detected using a chemiluminescent substrate according to the
manufacturer's instructions (Pierce). Monoclonal mouse IgG and
donkey-anti mouse IgG were diluted in wash buffer supplemented with 1%
bovine serum albumin. Polyclonal rabbit IgG and donkey anti-rabbit IgG
were diluted in blocking buffer.
Protease Protection Assay--
The topology of caveolin-1 was
monitored as described (42, 43). 293T cells were transfected with
caveolin-1 cDNA. Thirty-six hours after transfection, cells were
scraped into the culture medium, pelleted by centrifugation,
resuspended in PBS, and pelleted again. All subsequent steps were
performed at 4 °C. Cells were then resuspended in 0.4 ml of
iso-osmotic buffer (20 mM Tricine, pH 7.8, 250 mM sucrose, 0.1 mM EDTA), and homogenized by 30 passes through a 22-gauge needle. Nuclei were removed by centrifugation at 1000 × g for 5 min. Membranes were then pelleted by
centrifugation at 20,000 × g for 10 min and
resuspended in 0.4 ml of digestion buffer (100 mM sodium
phosphate, pH 7.4, 150 mM NaCl, 4 mM KCl, 2 mM MgCl2, and 0.02% (w/v) NaN3.
One-hundred microliter aliquots were then incubated with or without
trypsin for 30 min. Where indicated, samples were adjusted to 60 mM n-octylglucoside prior to addition of
trypsin. Proteins were precipitated with trichloroacetic acid, pelleted
by centrifugation, resuspended in 1% SDS, separated by SDS-PAGE, and
transferred to nitrocellulose sheets.
Immunoprecipitation--
Thirty-six hours post-transfection,
293T cells (grown to confluence in a 100-mm diameter dish) were washed
twice with ice-cold PBS and scraped into 1 ml of IP buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100,
60 mM n-octylglucoside, 0.1 mM
Na3VO4, 50 mM NaF, 30 mM Na4P2O7, 0.1 µg/ml
okadaic acid supplemented with protease inhibitors). Debris was removed
by centrifugation at 21,000 × g for 10 min. Lysates
were pre-cleared by incubation with 30 µl of Protein-A Sepharose for
45 min at 4 °C and then transferred to fresh tubes containing 30 µl of a 1:1 slurry of Protein A-Sepharose and IP buffer. Two
micrograms of IgG were added to the mixture. For coimmunoprecipitation
studies, rabbit anti-GFP IgG or mouse anti-caveolin-3 IgG were used as
an irrelevant control. Following a 4-h incubation at 4 °C, immune
complexes were collected by centrifugation, washed six times in 1 ml of IP buffer and four times with 1 ml of 10 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA; and
were disrupted by boiling in 1% SDS.
Triton X-100 Extraction, Purification of Caveolae-enriched
Membranes, Hypotonic Lysis and Fractionation, and Alkaline Carbonate
Extraction--
Triton X-100 extraction, purification of caveolin-rich
membranes, hypotonic lysis, and fractionation of proteins into soluble and particulate fractions, and extraction with alkaline sodium carbonate were all performed exactly as we described in detail previously (36).
Metabolic Labeling--
[3H]Palmitate
incorporation was monitored as we described previously (44). Briefly,
16 h post-transfection, cells were washed twice with PBS and
cultured for 1 h in labeling medium (DMEM, 5% dialyzed FBS, 5 mM sodium pyruvate). Cells were labeled for 4 h with
300 µCi of [3H]palmitic acid in 2 ml of labeling
medium, washed twice with PBS, and subjected to lysis in
immunoprecipitation buffer. Proteins were immunoprecipitated, and
separated by SDS-PAGE under nonreducing conditions. The gel was
impregranted with sodium salicilate, dried, and processed for
autoradiography, as we described previously (45). An aliquot of the
immunoprecipitate was subjected to SDS-PAGE, transferred to
nitrocellulose and detected by immunoblot analysis.
Steady state incorporation of [32P]orthophosphate was
monitored as follows: Madin-Darby canine kidney cells, grown to
confluence in two 150-mm diameter dishes, were serum-starved for
12 h, and then cultured in phosphate-free DMEM, supplemented with
5% dialyzed FBS, 1 mCi/ml of [32P]orthophosphate for
4 h. Cells were then scraped into 2 ml of 25 mM Mes,
pH 6.5, 150 mM NaCl, 1% Triton X-100, 0.1 mM
Na3VO4, 50 mM NaF, 30 mM Na4P2O7, 0.1 µg/ml
okadaic acid, 100 nM calyculin A, supplemented with
protease inhibitors. The lysate was homogenized and caveolae were
purified by sucrose density centrifugation as described (17, 36).
Twelve 1-ml fractions were collected from the gradient. Proteins were
precipitated with trichloroacetic acid (10% final concentration) and
resuspended by boiling in RIPA buffer (50 mM Tris pH, 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1%
SDS) for 1 min. Caveolin-1 was immunoprecipitated as described above
using anti-caveolin-1 rabbit IgG N-20. Following SDS-PAGE and transfer
to nitrocellulose, proteins were detected by autoradiography and
immunoblot analysis.
[32P]Orthophosphate incorporation into mutated caveolin-1
was monitored as follows: 16 h post-transfection, 293T cells were washed twice with PBS and cultured for 3 h in phosphate-free DMEM supplemented with 1 mCi/ml [32P]orthophosphate.
Caveolin-1 was immunoprecipitated. [32P]orthophosphate
and protein expression were assessed as described above.
Velocity Gradient Centrifugation--
Thirty-six hours
post-transfection, 293T cells grown to confluence in a 10-cm dish were
subjected to lysis in 1 ml of 25 mM Mes, pH 6.5, 150 mM NaCl, 60 mM octylglucoside (MBS-OG),
supplemented with protease inhibitors. Two-hundred microliters of the
lysate was loaded atop a 5-40% linear sucrose gradient (prepared in
MBS-OG) cast in an 11 × 45-mm ultraclear centrifuge tube. Samples
were subjected to centrifugation at 200,000 × g
(44,000 rpm in Sorval rotor TH-660) for 12 h. Twelve 375-µl
fractions were collected and equal volume aliquots of each were
subjected to SDS-PAGE and immunoblot analysis (27).
Immunofluoresence Localization--
Thirty-six hours
post-transfection, COS-7 cells were washed twice in phosphate-buffered
saline supplemented with 1 mM MgCl2 and 0.1 mM CaCl2 (PBS-CM); fixed in ice-cold
methanol/acetone, 1:1 (v/v), for 10 min at Cholesterol Depletion--
COS-7 cells were washed twice with
PBS, and then treated for 5 min at 37 °C with 10 mM
methyl- Caveolin-1 Secretion Assay--
Caveolin-1 secretion by cells of
the exocrine pancreas was monitored as described (47), with minor
modifications. AR42J rat pancreatic adenocarcinoma cells grown to 60%
confluence in a 100-mm diameter plate were transfected with
LipofectAMINE PLUS as follows: caveolin-1 cDNA (6 µg of DNA) was
mixed with 20 µl of PLUS reagent and 30 µl LipofectAMINE in a final
volume of 1.5 ml of Opti-MEM, exactly as suggested by the manufacturer.
Cells were washed twice with PBS; the medium was replaced with 5 ml of
Opti-MEM; and the DNA-lipid complex was added. After 3 h, cells were washed twice in PBS, and cultured for an additional 33 h in
F-12K, 20% FBS. Cells were washed again, cultured in 2.5 ml of F-12K,
20% FBS, 4 µM leupeptin, 10 µg/ml soybean trypsin
inhibitor, 500 µM benzamidine, 5 µg/ml pepstatin A, and
were treated with 100 nM dexamethasone (or vehicle,
ethanol) for 12 h. The medium was collected, adjusted to pH 7.9 with HEPES (50 mM final concentration). Triton X-100 was
added (1% final concentration) and caveolin-1 was immunoprecipitated
as described above using mAb 2234, and detected by immunoblotting with
rabbit anti-c-Myc IgG A14 or mouse anti-c-Myc IgG 9E10.
Phosphorylated Caveolin-1 Targets to Caveolae at Steady
State--
Caveolin-1 is phosphorylated on serine and tyrosine
residues. Phosphoamino acid analysis reveals that the ratio of
phosphoserine to phosphotyrosine is 10:1 (48). The role of caveolin-1
tyrosine phosphorylation in signal transduction and subcellular
localization has been examined recently (39). However, the role of
serine phosphorylation in caveolin-1 function has not been studied.
We performed metabolic labeling of Madin-Darby canine kidney
epithelial cells to assess the steady state distribution of
phosphorylated caveolin-1. Cells were labeled with
[32P]orthophosphoric acid, cultured in complete medium,
and then subjected to lysis in cold Triton X-100 buffer. The lysate was then fractionated in an established sucrose density gradient that separates caveolae membranes from the bulk of cellular proteins (41,
49). Fig. 1 shows that the bulk of the
32P label remained at the bottom of the gradient (loading
zone fractions 9-12). In contrast caveolin-1 immunoreactivity and
[32P]orthophosphate incorporation were detected in the
low density fraction (fraction 5) enriched in purified caveolae. Since
caveolin-1 is phosphorylated on serine and tyrosine residues in a ratio
of 10:1 (48), caveolin-1 phosphate incorporation at steady state can be
attributed largely to serine phosphorylation.
Mutation of Ser80 Blocks Caveolin-1
Phosphorylation--
Inspection of the caveolin family of primary
sequences reveals that only 12 residues are conserved across all
isoforms from every species examined to date (i.e. from
Caenorhabditis elegans to Homo sapiens) (50). Of
these 12 invariant residues, only Ser80 and
Ser168 (in caveolin-1) could serve as potential
phosphorylation sites (Fig.
2A). Thus, we mutated
Ser80 and Ser168 individually to glutamate (a
substitution that mimics chronic phosphorylation), and compared the
amount of [32P]orthophosphate incorporated by the mutants
to the amount incorporated by wild-type caveolin-1. Fig. 2B
shows that Cav-1(S80E) incorporated significantly less
[32P]orthophosphate than either wild-type caveolin-1 or
Cav-1(S168E), suggesting that Ser80 is a major site of
phosphorylation.
Expression and Characterization of Serine Mutant Caveolin-1
Proteins--
To systematically assess the role of caveolin-1
phosphorylation on either of the invariant serine residues, we next
prepared two more mutants that mimic chronic dephosphoryaltion
(i.e. Cav-1(S80A) and Cav-1(S168A)). All four mutants were
expressed in HEK 293T cells (Fig. 3) to
comparable levels indicating that mutation of these conserved residues
did not alter protein stability.
Caveolin-1 forms homotypic oligomers of 14-16 monomers (25, 27).
Homo-oligomerization is mediated by a 40-aminoacyl residue domain in
the N terminus, i.e. residues 61-101 in caveolin-1 (27). Also, the C-terminal domain mediates oligomer-oligomer interaction in
an isoform-specific manner (35). Therefore, we assessed whether mutation of the conserved serine residues would perturb
oligomerization. We were particularly concerned that because
Ser80 resides in the homo-oligomerization domain, mutation
of this residue might disrupt oligomerization, as occurs when the
oligomerization domain is deleted (17, 35), or subjected to
penta-alanine scanning mutagenesis (51). Conversely, formation of
inappropriately large aggregates would indicate that either
Ser80 or Ser168 is critical to the structural
integrity of caveolin-1 and mutation to another residue would grossly
compromise this integrity. This latter concern arises from our recent
characterization of the two limb-girdle muscular dystrophy (type 1C)
caveolin-3 proteins; both mutant molecules reside in the Golgi as high
molecular weight aggregates that undergo rapid degradation (44).
Importantly, Fig. 4A shows
that all four serine mutant caveolins form oligomers of the appropriate
size. Thus, mutation of Ser80 or Ser168 does
not cause global derangements in caveolin-1 structure, as manifested by
changes in the size of the caveolin-1 oligomer. Thus, any differences
observed in the localization or function of these mutant caveolin-1
molecules may be attributed reasonably to mimicry of chronic
phosphorylation or dephosphorylation.
To assess the effect of mutation of Ser80 to either Ala or
Glu on hetero-oligomerization with wild-type caveolin-1, untagged wild-type caveolin-1 was coexpressed with Cav-1(S80A) or Cav-1(S80E). Cells were subjected to lysis, and proteins were immunoprecipitated with anti-c-Myc rabbit IgGs. After SDS-PAGE and transfer to
nitrocellulose, blots were probed with mouse anti-caveolin-1 IgG 2234 to unambiguously identify tagged and untagged caveolin-1 proteins (Fig.
4B). Fig. 4C shows that both Cav-1(S80A) and
Cav-1(S80E) hetero-oligomerize with wild-type caveolin-1. This result
further supports the contention that mutation of this invariant residue
to either Ala or Glu does not result in gross disruption of the
protein's structure.
Chronic Phosphorylation of Caveolin-1 at Serine 80 Leads to
Exclusion from Caveolae and Targeting to ER Membranes--
Caveolin-1
resists solubilization in nonionic detergents at cold temperatures (4,
11, 41). This physical property reflects the concentration of
glycosphingolipids, acylated signaling molecules and saturated lipids
in caveolae and related lipid domains (52). Fig.
5A shows that Cav-1(S80E) is
partially soluble in Triton X-100, while Cav-1(S80A), Cav-1(S168A), and
Cav-1(S168E) are insoluble. As controls, we verified that wild-type
caveolin-1 is insoluble in Triton X-100. These results suggest that
Cav-1(S80E) is excluded, at least partially, from caveolae
membranes.
We have exploited caveolin-1's detergent insolubility to purify
caveolin-rich membrane domains from a variety of cell types and tissues
(41, 49, 53). Using an established sucrose-density centrifugation
method, we determined if mutation of caveolin-1 at the conserved serine
residues affects targeting to caveolae membranes. Fig. 5B
shows that Cav-1(S80E) targets much less efficiently to these domains
than wild-type caveolin-1, Cav-1(S80A), Cav-1(S168A), or
Cav-1(S168E).
To determine where Cav-1(S80E) resides in cells, we transfected COS-7
cells with wild-type and serine mutant caveolin-1 cDNAs and
performed immunofluorescence microscopy. This cell line, like 293T and
other cancer cell lines, shows little or no expression caveolin-1
endogenously, and was selected for these studies because it adheres
tightly to coverslips, whereas the 293T line is less adherent (36).
Fig. 6 illustrates that, like wild-type
caveolin-1, Cav-1(S80A), Cav-1(S168A), and Cav-1(S168E) have punctate
distribution patterns suggestive of concentration in vesicles at or
near the plasma membrane. Interestingly, Cav-1(S80E) appears to be
distributed in a reticular patter consistent with ER localization.
Double labeling of Cav-1(S80E) transfected cells with antibodies
directed against the c-Myc epitope and calnexin, a membrane bound ER
protein, confirmed that Cav-1(S80E) targets to the ER (Fig.
7).
The subcellular location of caveolin-1 is sensitive to cellular
cholesterol levels (54). Indeed, depletion of cellular cholesterol results in relocation of caveolar caveolin-1 to the lumen of the ER
(42, 55). This pharmacological manipulation reflects the underlying
physiologic role of caveolin-1 as an intracellular shuttle of
cholesterol (7-9). Since Cav-1(S80E) colocalized with an ER marker, we
used an established fractionation scheme (17) to determine whether the
protein is soluble, or membrane-bound. Fig.
8A shows that nearly all of
wild-type caveolin-1 is in the particulate fraction. Likewise,
Cav-1(S80E) partitions in the pellet fraction. As internal controls to
validate our fractionation method, blots were probed with antibodies
against a cytosolic protein (GDP dissociation inhibitor), and a
veritable integral membrane protein (lysosome associate membrane
protein-1). Both control proteins were detected in the appropriate
fractions, with GDP dissociation inhibitor partitioning in the
supernatant (56), and lysosome-associated membrane protein-1
partitioning in the pellet (57).
To determine whether Cav-1(S80E) was tightly associated with membranes,
cells were extracted with alkaline sodium carbonate, an agent that
strips membranes of peripherally associated proteins, but not plasma
membrane-bound caveolin-1 (11, 17, 56). Fig. 8B shows that
like wild-type caveolin-1, Cav-1(S80E) resisted alkaline sodium
carbonate extraction. As a control, we verified that the peripherally
associated membrane protein early endosomal antigen 1 is solubilized
completely by alkaline sodium carbonate (58).
ER Membrane-bound Cav-1(S80E) Has the Same Membrane Topology as
Plasma Membrane-bound Wild-type Caveolin-1, but Undergoes Less
Palmitoylation--
Our fractionation results indicated that
Cav-1(S80E) was not a soluble resident of the ER. Rather it was tightly
associated with the ER membrane. Plasma-membrane bound caveolin-1 is
believed to assume an unusual conformation with cytosolically directed N and C termini separated by a hydrophobic domain that is believed to
anchor the protein to the membrane (11, 24, 26). Both the N- and
C-terminal domains undergo post-translational modifications, with
Tyr14 (N terminus) undergoing phosphorylation (28) and
three C-terminal cysteinyl residues undergoing thiopalmitoylation
(26).
We used an established protease protection assay to determine the
membrane topology of Cav-1(S80E) (42, 43). Following isotonic lysis and
homogenization, caveolin-1 is sensitive to tryptic digestion, as the N
and C termini are directed toward the cytoplasm, and there is no
ecto-domain (Fig. 9). However, the
protein enters the lumen of the ER in response to cholesterol depletion, and is thus protected from tryptic digestion in this setting
(42). Since Cav-1(S80E) was membrane-bound, we predicted it would
remain sensitive to tryptic digestion, provided that it had the same
topology as plasma membrane-bound caveolin-1. Fig. 9 shows that like
wild-type caveolin-1, Cav-1(S80E) is sensitive to tryptic digestion:
neither antibodies directed against the caveolin-1 N terminus (N-20),
nor a C-terminal c-Myc epitope tag (9E10) detected caveolin-1 in
protease-treated samples. The integrity of the membrane preparations
was verified by blotting for a luminal ER protein (protein-disulfide
isomerase) which was resistant to protease protection until membranes
were dissolved with octylglucoside.
Finally, we determined if the C-terminal cysteinyl residues of
Cav-1(S80E) are thio-acylated. To this end, cells were labeled with
tritiated palmitic acid. After detergent lysis, caveolin-1 was
recovered by immunoprecipitation and palmitate incorporation was
assessed by autoradiography. Fig. 10
shows that Cav-1(S80E) is palmitoylated to significantly lower levels
than wild-type caveolin-1. Cav-1(S80A), Cav-1(S168A), and Cav-1(S168E)
are palmitoylated to the same extent as wild-type caveolin-1. Although
these results are uniformative regarding topology, they suggest that
palmitoylation occurs once caveolin-1 has traversed the ER. Indeed,
both Golgi-retained limb-girdle muscular dystrophy 1C caveolin-3
mutants are palmitoylated to the same extent as wild-type caveolin-3
(44). This suggests that palmitoylation of caveolins occurs after exit
from the ER, at the level of the Golgi.
Although Cav-1(S80E) was bound to ER membranes, and was not in the
lumen of this compartment, we suspected that phosphorylation of
caveolin-1 on this residue might be a regulatory switch for targeting
to the ER in response to cholesterol depletion. However, immunofluorescence microscopy revealed that the overwhelming majority of Cav-1(S80A) colocalized with calnexin when cells were treated with
the cholesterol depleting agent methyl- Caveolin-1 Phosphorylation on Ser80 Regulates Entry
into the Regulated Secretory Pathway of Pancreatic Acinar
Cells--
Recently, Anderson and colleagues (47) reported that
caveolin-1 is secreted by cells of the exocrine pancreas after mice are
treated with the secretagogues dexamethasone, cholecystekinin, or
secretin (47). Furthermore, recombinant expression of caveolin-1 in the
rat pancreatic adenocarcinoma cell line AR42J recapitulates the
regulated secretion seen in whole animals (47). Although the function
of secreted caveolin-1 remains elusive, understanding the regulation of
this process may aid in unraveling its purpose.
We verified that AR42J cells secrete wild-type caveolin-1 when treated
with dexamethasone, but not with vehicle alone (Fig. 12). To assess whether serine
phosphorylation of caveolin-1 influences secretion in this cell system,
we expressed Cav-1(S80A) and Cav-1(S80E) in AR42J cells. Twelve hours
after administration of dexamethasone, the cell culture medium was
collected and subjected to immunoprecipitation with anti-caveolin-1
antibodies. Total cellular proteins were collected as well. Fig.
12B shows that Cav-1(S80A) was not secreted, while wild-type
caveolin-1 and Cav-1(S80E) were, suggesting that serine phosphorylation
of caveolin-1 is required for secretion. Indeed, 2-2.5 times as much
Cav-1(S80E) was recovered from the cell culture supernatant than
wild-type caveolin-1. Conversely, more wild-type caveolin-1 was found
in the intracellular pool than Cav-1(S80E) (Fig. 12B, lower
panel). Cav-1(S168A) and Cav-1(S168E) were secreted to the same
level as wild-type caveolin-1 (Fig. 12D).
To assess whether mimicking chronic phosphorylation of caveolin-1 on
Ser80 is sufficient to drive secretion, we compared the
amount of Cav-1(S80E) secreted in the presence or absence of
dexamethasone. Fig. 12C shows that like wild-type
caveolin-1, Cav-1(S80E) was not detected in the cell culture medium in
the absence of dexamethasone. Because dexamethasone-driven
reorganization of internal membranes is required to induce the
secretory phenotype of AR42J cells (59), this finding does not exclude
the possibility that in normal exocrine pancreatic cells, caveolin-1
phosphorylation on Ser80 is sufficient to target caveolin-1
to the exocytic pathway.
Because caveolin-1 is a multifunctional protein that moves among
membrane compartments, and is even found in complex with cytosolic
chaperone proteins (8), the molecular events associated with caveolin-1
localization have been studied intensely for nearly a decade (2). Cell
density (24, 60, 61) and cholesterol levels (7-9, 42, 55) are the best
characterized external determinants of caveolin-1 subcellular
localization. However, an emerging body of work indicates that
caveolin-1 has localization signals in its primary sequence that govern
its subcellular localization (17, 36, 51, 62). Superimposed on these
determinants are reversible modifications like phosphorylation that
might alter the protein's position and function in the cell (37,
38).
What information does the sequence of caveolin-1 itself contain
regarding the protein's subcellular traffic? We recently demonstrated that the caveolin scaffolding domain (residues 82-101 of caveolin-1) is necessary for caveolin-1 targeting to caveolae (17); and is
sufficient to target a heterologous soluble protein to these membrane
microdomains (36). We also found that caveolin-1 contains a trans-Golgi
localization signal in its C-terminal domain (36). The steady state
localization of caveolin-1 reflects, in part, these two cues. The role
of phosphorylation is less clear, but Src-dependent
tyrosine phosphorylation of caveolin-1 causes a reversible flattening
and aggregation of caveolae at the cell membrane (37, 38).
Here, we have taken a mutational approach to determine the role of
serine phosphorylation in the subcellular traffic of caveolin-1. We
find that mimicking chronic phosphorylation of invariant serine residue
80 by mutating it to glutamate results in exclusion from caveolae
membranes, and tight association with ER membranes in fibroblasts (293T
and COS-7). Importantly, this mutation does not disrupt
homo-oligomerization, or hetero-oligomerization with wild-type
caveolin-1. In contrast, mutation of this invariant serine residue to
alanine, mimicking chronic dephosphorylation, does not alter the
subcellular localization of caveolin-1 in these cell types. Table
I summarizes the properties of the mutant
caveolin-1 proteins examined in this study.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cyclodextrin, Sigma;
trypsin, Worthington Biochemical Corp.; rabbit anti-caveolin-1 IgG
(N-20), rabbit anti-c-Myc IgG (A14), rabbit anti-GFP IgG (FL), and
mouse anti-c-Myc IgG (9E10), Santa Cruz Biotechnology; monoclonal
anti-lysosome-associated membrane protein-1, anti-early endosomal
antigen 1 and anti-protein-disulfide isomerase, BD Transduction
Laboratories; fluorescein isothiocyante and
rhodamine-trisamine-conjugated donkey anti-mouse and donkey anti-rabbit
antibodies, Jackson Immunochemicals. Monoclonal anti-caveolin-1 (clones
2234 and 2297) and anti-caveolin-3 (clone 26) were gifts from Dr.
Roberto Campos González (BD Transduction Laboratories, Lexington,
KY). Rabbit anti-GDP dissociation inhibitor IgG was a gift from Dr.
Perry E. Bickel (Department of Medicine, Washington University School
of Medicine, St. Louis, MO). Anti-calnexin rabbit IgG were described
previously (40).
20 °C; and air-dried at
room temperature. Cells were then rehydrated in PBS-CM. Epitope-tagged
caveolin-1 was detected with anti-c-Myc antibodies (mAb 9E10 or pAbs
A14). Fluorescently conjugated secondary antibodies were used to
visualize bound IgG on an Olympus IX70 inverted microscope. Images were obtained using a Photonics Cooled CCD camera or a Noran Oz laser scanning confocal microscope with a ×60 NA 1.4 objective lens. All
microscopy was performed at the Analytical Imaging Facility of the
Albert Einstein College of Medicine.
-cyclodextrin (46). After cholesterol depletion, cells were
washed, fixed, and processed for immunofluorescence microscopy.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
At steady-state phosphorylated caveolin-1
targets to caveolae. Madin-Darby canine kidney cells were labeled
with [32P]orthophosphoric acid. Cells were then subjected
to lysis in cold 1% Triton X-100 buffer supplemented with phosphatase
and protease inhibitors. The lysate was homogenized, mixed with an
equal volume of 80% sucrose (4 ml total), prepared in buffer lacking
detergent, and placed at the bottom of an ultra-clear centrifuge tube.
The sample was overlaid with a 5-30% discontinuous sucrose gradient
(4 ml each, both solutions prepared in buffer lacking detergent). The
samples were then subjected to ultracentrifugation at 200,000 × g for 18 h. A light scattering band was observed at the
interface of the 5 and 30% sucrose solutions. Twelve 1-ml fractions
were collected from the top of the gradient. Each fraction was mixed
with 110 µl of 100% trichloroacetic acid, and proteins were
precipitated on ice for 30 min. Following centrifugation at 21,000 × g for 10 min, the sucrose solutions were decanted, and
precipitated proteins were resuspended by boiling in RIPA buffer (see
"Experimental Procedures"). Caveolin-1 was immunoprecipitated with
anti-caveolin-1 rabbit IgG N-20. A, equal volume aliquots of
the resuspended proteins from the gradient were subjected to SDS-PAGE
and autoradiography. Note that the majority of the incorporated
[32P] remained at the bottom of the gradient (fractions
9-12). B, the caveolin-1 immunoprecipitate was subjected to
SDS-PAGE, transfer to nitrocellulose, and autoradiography.
C, the blot shown in B was immunoblotted with
anti-caveolin-1 mouse IgG 2297. Note that caveolin-1 immunoreactivity
and 32P incorporation coincide.
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Fig. 2.
Ser80 is a major site of
caveolin-1 phosphorylation. A, sequence alignment of
caveolin proteins. Twelve residues (boxed) are conserved in
all caveolin isoforms across all species from C. elegans to
H. sapiens (human). Of these invariant residues,
Ser80 and Ser168 are the only ones that can
undergo phosphorylation (marked with arrows). The
homo-oligomerization domain (dashed line) spans residues
61-101, and is followed by the transmembrane domain (solid
line, residues 102-134). B, 293T cells were
transfected and labeled with [32P]orthophosphate as
detailed under "Experimental Procedures." After labeling, cells
were washed and proteins were subjected to immunoprecipitation with
anti-caveolin-1 antibodies. Immunoprecipitated proteins were subjected
to SDS-PAGE and were transferred to nitrocellulose. Autoradiography was
performed to detect incorporation of [32P]phosphate
(upper panel). The membrane was then immunoblotted with
anti-caveolin-1 antibodies to assess the level of protein present
(lower panel). Note that Cav-1(S80E) showed significantly
less [32P]phosphate incorporation than either wild-type
caveolin-1 or Cav-1(S168E).
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Fig. 3.
Construction and expression of serine mutant
caveolin-1. A, structure of caveolin-1 and point
mutants used in this study. Cytoplasmically directed N and C termini
are separated by a putative membrane spanning, hydrophobic domain. The
protein undergoes palmitoylation (downward hatches) on three
cysteinyl residues (Cys133, Cys143, and
Cys156) in the C-terminal third of the molecule. The
positions and identities of the four caveolin-1 serine mutants
characterized in this study are also indicated. All constructs were
tagged with a c-Myc epitope at the C terminus. B, expression
and detection of serine mutant caveolin-1. 293T cells were transfected
with the indicated cDNAs. Lysates were prepared 36 h
post-transfection. Proteins were subjected to SDS-PAGE under reducing
conditions, and transferred to nitrocellulose. Immunoblotting with
anti-c-Myc antibody revealed that all four mutants were expressed to
comparable levels as wild-type (WT) caveolin-1.
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Fig. 4.
Mutation of conserved serine residues does
not perturb caveolin-1 homo-oligomerization. A, 293T
cells were transfected with the indicated cDNAs. Thirty-six hours
post-transfection, lysates were prepared and caveolin-1
homo-oligomerization was assessed using velocity gradient
centrifugation, as described under "Experimental Procedures."
Molecular weight standards were subjected to velocity gradient
centrifugation in a parallel experiment, and their migration is
indicated at the top of the figure. Like wild-type
(WT) caveolin-1, all four mutants formed oligomers of
~14-16 subunits. Blots were probed with anti-caveolin-1 rabbit IgGs
(N-20). B, cells were transfected with cDNAs encoding
untagged wild-type caveolin-1 and caveolin-1 cDNAs bearing a
C-terminal c-Myc epitope tag, as indicated. The lysates were then
subjected to immunoprecipitation with anti-c-Myc rabbit IgG A-14.
Immunoprecipitates were then separated by SDS-PAGE (IP),
along with one-tenth of the lysates (input), after
transfer to nitrocellulose and blotted with anti-caveolin-1 mAb 2234 (67). This allowed unambiguous identification of the c-Myc
epitope-tagged caveolin-1 proteins, and untagged wild-type
caveolin-1. Note that untagged caveolin-1 was not precipitated by
anti-c-Myc IgG unless tagged caveolin-1 was coexpressed. C,
c-Myc-tagged Cav-1(S80A) or Cav-1(S80E) cDNAs were co-transfected
with untagged, wild-type caveolin-1 cDNA. Lysates were prepared and
subjected to immunoprecipitation as in B. As additional
controls, equal fractions of the lysates were subjected to
precipitation with Protein-A Sepharose alone (Protein A),
and an irrelevant rabbit IgG (anti-GFP, FL). After recovery,
proteins were subjected to SDS-PAGE. Note that both Cav-1(S80A) and
Cav-1(S80E) correctly hetero-oligomerized with wild-type caveolin-1,
and were not precipitated by Protein A alone, or the irrelevant
IgG.
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Fig. 5.
Cav-1(S80E) is partially soluble in Triton
X-100 and is excluded from purified caveolae. 293T cells were
transfected with the indicated cDNAs. A, 36 h
post-transfection, cells were extracted with an ice-cold solution of
1% Triton X-100. The extractable fraction was collected and the
inextractable fraction was resuspended in the same volume of 1% SDS.
Equal volume aliquots of both fractions were separated by SDS-PAGE.
Proteins were transferred to nitrocellulose, and sheets were
immunoblotted with anti-c-Myc antibodies. Note that Cav-1(S80E) was
partially soluble in Triton X-100, with roughly half the protein in the
soluble fraction (S), while wild-type (WT) caveolin-1,
Cav-1(S80A), Cav-1(S168A), and Cav-1(S168E) were insoluble, with >95%
of each protein in the insoluble fraction (I). B,
36 h post-transfection, caveolin-rich, detergent-resistant
membranes were isolated by floatation in a sucrose gradient.
Equal masses of protein from each fraction were separated by SDS-PAGE,
transferred to nitrocellulsoe, and subjected to immunoblot analysis.
Note that wild-type caveolin-1, Cav-1(S80A), Cav-1(S168A), and
Cav-1(S168E) were enriched in low density fractions (5 and
6, arrows), while Cav-1(S80E) was preferentially
excluded.
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Fig. 6.
Immunolocalization of serine mutant
caveolin-1. COS-7 cells were transfected with the indicated
cDNAs, fixed, and stained with anti-c-Myc antibody; note that
vector alone transfectants showed no staining. Wild-type caveolin-1
Cav-1(S80A), Cav-1(S168A), and Cav-1(S168E) expressing cells had the
expected punctate pattern of staining. However, Cav-1(S80E)
transfectants had a reticular pattern of staining throughout the
cytoplasm and a strong perinuclear staining pattern.
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Fig. 7.
Cav-1(S80E) colocalizes with the ER membrane
marker calnexin. COS-7 cells were transfected as in Fig. 6. Cells
were fixed and stained with anti-c-Myc antibody and anti-calnexin
antiserum. Scanning confocal microscopy revealed dramatic overlap in
Cav-1(S80E) and calnexin localization in the transfected cell seen in
the middle of each frame.
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Fig. 8.
Cav-1(S80E) is tightly associated with
membranes. A, 36 h post-transfection, 293T cells
were subjected to lysis in hypotonic buffer. Nuclei were discarded by
low speed centrifugation and post-nuclear lysates were separated into
soluble (S) and pellet (P) fractions by
high-speed centrifugation. The soluble fraction was collected and the
pellet was resuspended in the same volume of 1% SDS. Equal volume
aliquots of both fractions were separated by SDS-PAGE. Proteins were
transferred to nitrocellulose, and sheets were immunoblotted with
anti-c-Myc antibodies. Like wild-type (WT) caveolin-1, all
four caveolin-1 mutants were found in the pellet. As internal controls,
we verified that a representative cytosolic protein (GDP dissociation
inhibitor, GDI), and a glycosylated membrane spanning
protein (lysosome associate membrane protein-1; LAMP-1) were
found in the expected fractions. B, 36 h
post-transfection, 293T cells were washed twice in PBS, and once in 150 mM NaCl. Cells were then scraped into 100 mM
Na2CO3, pH 11.5. Following Dounce
homogenization, proteins were fractionated into extractable
(E) and inextractable (I) components. The
extractable fraction was collected and the inextractable fraction was
resuspended in the same volume of 1% SDS. Equal volume aliquots of
both fractions were separated by SDS-PAGE. Proteins were transferred to
nitrocellulose, and detected with anti-c-Myc antibodies. Like wild-type
caveolin-1 (WT), all four mutant caveolin-1 proteins
resisted solubilization by Na2CO3, whereas the
peripherally associated membrane protein EEA1 was completely
solubilized.
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Fig. 9.
Cav-1(S80E) has the same membrane topology as
wild-type caveolin-1. Thirty-six hours after transfection, cells
were collected in PBS, and subjected to iso-osmotic homogenization and
fractionation as described under "Experimental Procedures."
A, the membrane fraction was treated with trypsin and
octylglucoside as indicated. Samples were split in two after digestion,
and equal volume aliquots for each were separated by SDS-PAGE,
transferred to nitrocellulose. Blots were probed with antibodies
against the N-terminal 20 residues of caveolin (rabbit IgG N-20) and
against the C-terminal c-Myc epitope tag (mouse IgG 9E10). Note that
both the N- and C-terminal domains of caveolin-1 are sensitive to
protease digestion. Like wild-type caveolin-1, Cav-1(S80E) was
sensitive to tryptic digestion, indicating that both the N and C
termini of Cav-1(S80E) are directed toward the cytoplasm. To confirm
the integrity of the membrane preparation, blots were probed with
antibodies against the ER luminal protein protein-disulfide isomerase.
As expected, protein-disulfide isomerase was only sensitive to protease
digestion when membranes were solubilized with octylglucoside.
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Fig. 10.
Cav-1(S80E) undergoes less palmitoylation
than wild-type caveolin-1. 293T cells were labeled with
[3H]palmitic acid as described under "Experimental
Procedures." Following immunoprecipitation, proteins were subjected
to SDS-PAGE under nonreducing conditions and were detected by
immunoblot (top panel) or autoradiography
(bottom). Note that all four mutant caveolin-1 molecules
were palmitoylated, but that Cav-1(S80E) showed lower levels of
[3H]palmitate incorporation.
-cyclodextrin (Fig. 11), suggesting that phosphorylation of
Ser80 does not govern this aspect of the caveolin-1
intracellular traffic (i.e. ER retrieval is not regulated by
phosphorylation of Ser80).
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Fig. 11.
Phosphorylation of caveolin-1 on
Ser80 is not required for caveolin-1 retrieval to the
endoplasmic reticulum during cholesterol depletion. COS-7 cells
were transfected with the indicated caveolin-1 cDNAs. Thirty-six
hours after transfection, cells were washed twice with PBS and treated
with serum-free medium supplemented with 10 mM
methyl- -cyclodextrin (M
CD). After a 5-min
incubation at 37 °C, cells were processed for immunofluorescence
microscopy. Note that both wild-type caveolin-1 and Cav-1(S80A)
colocalized with the ER marker calnexin after treatment with
M
CD.
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Fig. 12.
Caveolin-1 phosphorylation on
Ser80 is necessary for caveolin-1 secretion in AR42J
Cells. To assess the possible role of serine phosphorylation in
caveolin-1 secretion, we transfected AR42J cells with the indicated
cDNAs. Thirty-six hours post-transfection, cells were treated with
100 nM dexamethasone. Twelve hours later, the cell culture
medium was collected, and immunoprecipitated with anti-caveolin-1 IgG
2234, which recognizes an epitope in the first 20 aminoacyl residues
(67). Cellular proteins were recovered in 1% SDS. A, blots
were probed with anti-c-Myc epitope rabbit IgGs (A14) to verify that
full-length protein was recovered. Note that wild-type caveolin-1 was
only detected in the extracellular compartment when cells were treated
with dexamethasone. B, cells were transfected with the
indicated caveolin-1 cDNAs and treated with dexamethasone as
described above. Note that wild-type caveolin-1 and Cav-1(S80E) were
detected in both the intra- and extracellular compartments, while
Cav-1(S80A) was completely intracellular. Band densitometry was
performed with Chemimager software and the results are shown below the
secreted caveolin panel. Cellular proteins ("Cell"), and
a trichloroacetic acid precipitate of the PBS used to wash the cells
("Wash"), after collection of the medium, were run in
parallel to demonstrate that the intracellular caveolin-1 was not
contaminated with extracellular protein. Extracellular caveolin-1 was
detected with anti-c-Myc mouse IgG (9E10), and the light chain of the
precipitating anti-caveolin-1 IgG (2234) was therefore detected.
C, as with wild-type caveolin-1 (see panel A),
Cav-1(S80E) was only secreted when cells were treated with
dexamethasone. D, like wild-type caveolin-1, Cav-1(168A) and
Cav-1(S168E) were secreted when AR42J cells were treated with
dexamethasone.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Summary of the properties of the caveolin-1 serine mutants examined in
this study
Finally, the phenotypes of Cav-1(S168A) and Cav-1(S168E) were indistinguishable from that of wild-type caveolin-1. Since Ser168 is an invariant residue, it is unlikely that it is insensitive to mutation. The C-terminal domain of caveolin-1 mediates oligomer-oligomer interaction (35) and we recently demonstrated that the final 10 residues of caveolin-1 are required for oligomer-oligomer interaction. This stretch of amino acid residues includes Ser168, and mutation of this residue to either Ala or Glu hinders GFP-fused Cav-1(135-178) binding to wild-type caveolin-1. Thus, this invariant residue serves an important structural role that is only revealed in the context of the isolated C terminus (36).
Since Cav-1(S80E) did not show complete loss of phosphate incorporation (Fig. 2B), there are likely other sites of serine phosphorylation that contribute to the subcellular localization of caveolin-1. Our finding that phosphorylated caveolin-1 resides in caveolae of epithelial cells at steady state (Fig. 1) supports this hypothesis. Combinations of serine mutants may be required to dissect the hierarchy of signals governing subcellular localization. We also note that mimicry of serine phosphorylation by mutation to glutamate may not be completely equivalent to sustained phosphorylation; protein-protein interactions dependent on phosphoserine may be lost in mutating the serine residue to glutamate. Alternatively, we cannot exclude the possibility that the S80E mutation does not adequately mimic the phosphorylated form, but rather is inducing a novel phenotype.
Serine Phosphorylation Regulates Caveolin-1 Entry into the Secretory Pathway by Targeting to the ER-- In professional secretory cells of the exocrine pancreas, caveolin-1 undergoes regulated exocytosis, with secretion being stimulated by a panel of secretagogues (47). Here, we found that mutation of Ser80 to Glu increases the amount of caveolin-1 protein secreted in response to dexamethasone. Conversely, mutation of Ser80 to Ala blocks secretion completely.
The opposing effects of mutating Ser80 to either Ala or Glu on caveolin-1 secretion are reminiscent of those exerted by similar mutations of the polymeric immunoglobulin receptor (pIgR) at Ser664. pIgR transcytosis in polarized epithelia is regulated by phosphorylation of Ser664; and pIgR(S664A) undergoes transcytosis at a slower rate than wild-type pIgR, while pIgR(S664D) shows an increased transcytotic rate compared with wild-type pIgR (63). These results reflect an increase (S664A) or decrease (S664D) in the rate of pIgR recycling to the basolateral surface. Thus, serine phosphorylation of membrane proximal Ser residues may serve as a general signal for targeting proteins to distinct membrane compartments. Other signals like tyrosine phosphorylation by Src family kinases (37, 64) and homo-oligomerization (44, 51, 65) regulate caveolin-1 and pIgR subcellular traffic as well.
Although studies with pIgR establish a precedent for serine
phosphorylation mediated regulation of membrane protein trafficking, the point of caveolin-1 entry into the regulated secretory pathway contrasts with the route taken by most proteins that undergo regulated exocytosis in endocrine and exocrine cells. Typically, such proteins are packaged into nascent secretory granules derived from the trans-Golgi network where they undergo proteolytic processing and
aggregation (66). Why is caveolin-1 entry into the regulated secretory
pathway different? First, conventional sorting mechanisms like
proteolytic processing in the trans-Golgi network to generate cargo for
immature secretory granules cannot be used to target caveolin-1 to the
regulated secretory pathway: full-length caveolin-1, not a proteolytic
fragment thereof, undergoes regulated exocytosis. Second, caveolin-1 is
usually an oligomeric protein bound to the cytoplasmic face of the
plasma membrane, whereas secreted products are soluble proteins. Thus,
the professional secretory cell can control caveolin-1 entry into the
regulated secretory pathway by gating its traffic across the ER
membrane (Fig. 13).
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ACKNOWLEDGEMENTS |
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We thank Michael Cammer for help with confocal microscopy, Drs. Perry E. Bickel and Roberto Campos González for antibodies, and Dr. Michael E. Zenilman for advice.
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FOOTNOTES |
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* This work was supported in part by grants from the National Institutes of Health, Muscular Dystrophy Association, and the Komen Breast Cancer Foundation (to M. P. L.).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.
§ Supported by National Institutes of Health Medical Scientist Training Grant T32-GM07288.
** To whom correspondence should be addressed: Dept. of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 Tel.: 718-430-8828; Fax: 718-430-8830; E-mail: lisanti@aecom.yu.edu.
Published, JBC Papers in Press, November 14, 2000, DOI 10.1074/jbc.M005448200
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ABBREVIATIONS |
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The abbreviations used are: ER, endoplasmic reticulum; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PAGE, polyacrylamide gel electrophoresis; pIgR, polymeric immunoglobulin receptor; PBS, phosphate-buffered saline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; Mes, 4-morpholineethanesulfonic acid.
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