* Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9039;
and Adirondack Biomedical Research Institute, Lake Placid, New York 12946
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Abstract |
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Previously, we showed caveolae contain a
population of protein kinase C (PKC
) that appears
to regulate membrane invagination. We now report
that multiple PKC isoenzymes are enriched in caveolae of unstimulated fibroblasts. To understand the mechanism of PKC targeting, we prepared caveolae lacking
PKC
and measured the interaction of recombinant
PKC
with these membranes. PKC
bound with high
affinity and specificity to caveolae membranes. Binding was calcium dependent, did not require the addition of
factors that activate the enzyme, and involved the regulatory domain of the molecule. A 68-kD PKC
-binding
protein identified as sdr (serum deprivation response)
was isolated by interaction cloning and localized to caveolae. Antibodies against sdr inhibited PKC
binding. A 100-amino acid sequence from the middle of sdr
competitively blocked PKC
binding while flanking sequences were inactive. Caveolae appear to be a membrane site where PKC enzymes are organized to carry
out essential regulatory functions as well as to modulate signal transduction at the cell surface.
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Introduction |
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THE protein kinase C (PKC)1 family of phospholipid-dependent kinases are important regulators of
growth, differentiation, and gene expression (8, 22).
Based on the requirements for activation, the 12 mammalian PKC isoenzymes can be grouped into three categories (10): PKC,
I,
II, and
require calcium, phosphatidylserine (PS), and diacylglycerol (DAG) for activity;
PKC
,
,
,
, and µ require PS and DAG; and PKC
,
,
and
need only PS. All isoenzymes have similar catalytic
domains but differ in the structure of their regulatory domains. The intramolecular interaction between a 17-
amino acid-long "pseudosubstrate" and the catalytic site may be a critical step in controlling the activity of many of these enzymes (5).
Most cells express multiple isoforms of PKC, and each has a specific set of functions (5). These isoenzymes, however, display little substrate specificity in in vitro assays. Therefore, other mechanisms must govern the specific function of each isoenzyme in the cell. One way to achieve specificity is by targeting individual isoenzymes to select locations in the cell (18), using high-affinity interactions between the enzyme and a subcellular compartment. The isoenzyme could be constitutively present in the target compartment or recruited there after the cell receives a stimulus. A variety of PKC-binding proteins (10) and lipids (22) have been identified that might function to compartmentalize PKC isoenzymes.
One place on the plasma membrane where PKC appears to be a resident protein is caveolae (24, 25). Both
cell fractionation and immunogold labeling of whole
plasma membranes show that PKC
is highly concentrated in caveolae of unstimulated cells (25). Despite the
presence of many different resident and migratory proteins in this domain (14), a 90-kD protein is the major
PKC
substrate detected in intact cells as well as isolated
caveolae (25). Phosphorylation in vitro occurs in the absence of activators such as DAG or PS (25), suggesting the
enzyme is constitutively active when located in this compartment. The uptake of molecules by caveolae is linked
to PKC
kinase activity (25), so the enzyme may play a
key role in regulating the internalization of caveolae. Therefore, a mechanism must exist for directing PKC
to
caveolae and regulating substrate specificity at this site.
We now report that caveolae isolated from Rat-1 cells display a Ca++-dependent, high-affinity PKC
binding activity that may be involved in targeting the enzyme to this
domain. Using interaction cloning together with immunolocalization and a competitive binding assay, we have
identified a protein component of this binding site as serum deprivation response protein (Sdr) (7).
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Materials and Methods |
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Materials
Fetal calf serum was from Hazleton Research Products, Inc. (Lenexa,
KS). DME, trypsin-EDTA, penicillin/streptomycin, and OptiPrep were
from GIBCO BRL (Gaithersburg, MD). Percoll was from Pharmacia Biotech (Piscataway, NJ). EGF was from CalBiochem (San Diego, CA). Human recombinant PKC and PKC
were from PanVera Corporation (Madison, WI). 125I-radiolabeled streptavidin with specific activity of
20-40 µCi/µg and ECL reagent were obtained from Amersham Corp.
(Arlington, IL). Antibodies were obtained from the following sources:
anti-caveolin-1 mAb IgG, anti-caveolin-1 polyclonal antibody IgG, anti-PKC
, -PKC
, -PKC
IgGs (mAb), anti-RACK1 IgG (mAb), and anti-
integrin
3 IgG (mAb) were from Transduction Laboratories (Lexington,
KY); peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG were from Organon Teknika (West Chester, PA); biotinylated goat anti-mouse IgG was from Vector Laboratories (Burlingame, CA); and TRITC-goat anti-mouse IgG [H+L] and FITC-goat anti-rabbit IgG [H+L] were from
Zymed Laboratories Inc. (South San Francisco, CA). Polyclonal anti-sdr
peptides were produced by standard methods. The PKC
pseudosubstrate
peptide (RFARKGALRQKNVHENKN) was synthesized by University
of Texas Southwestern Medical Center Polymer Core Facility. Immulon I
Removawell 96-well plates were purchased from Dynatech Laboratories
(Chantilly, VA). Immobilon transfer nylon was from Millipore (Bedford,
MA). Crystalline bovine serum albumin and phorbol-12-myristate-13-acetate (PMA) were from Sigma Chemical Co. (St. Louis, MO). 1,1,1-trichloroethane was from Aldrich Chemical Co., Inc. (Milwaukee, WI).
Methods
Cell Culture. Rat-1 cells (6 × 105) were seeded in 100-mm-diam dishes and grown in 10 ml of DME supplemented with 10% (vol/vol) fetal calf serum for 4 d. Cells were then incubated for 24-48 h in DME without serum before each experiment. Normal human fibroblasts were cultured on coverslips as previously described (6).
Isolation of Caveolae Fractions. Detergent-free caveolae fractions were prepared by the method of Smart et al. (26). All steps were carried out at 4°C. Cells were collected by scraping in 5 ml of ice-cold buffer A (0.25 M sucrose, 1 mM EDTA, 20 mM tricine, pH 7.8, with or without 1 mM CaCl2) and pelleting at 1,400 g for 5 min. After douncing, the postnuclear supernatant fraction was obtained, layered on top of 23 ml of 30% Percoll solution prepared in buffer A, and centrifuged at 84,000 g for 30 min (model Ti60 rotor; Beckman Instruments, Fullerton, CA). The plasma membrane band was collected and sonicated. The sonicate was mixed with 2 ml of 50% OptiPrep prepared in buffer B (0.04 M sucrose, 1 mM EDTA, 20 mM tricine, pH 7.8, with or without 1 mM CaCl2) to make a 23% OptiPrep solution. The mixture was placed on the bottom of a centrifuge tube (model SW 41; Beckman Instruments) and overlaid with a linear 20 to 10% OptiPrep gradient (designated OptiPrep 1). After centrifugation at 52,000 g for 90 min in a swinging bucket rotor (model SW 41; Beckman Instruments), fractions were either analyzed directly (700 µl/ fraction), or the top 5 ml of the gradient (fractions 1-7) was collected, mixed with 4 ml of 50% OptiPrep, overlaid with 2 ml of 5% OptiPrep in buffer A, and centrifuged at 52,000 g for 90 min (designated OptiPrep 2). An opaque band located just above the 5% interface was collected and designated the caveolae membrane fraction. Pooled fractions 8-14 from OptiPrep 1 were designated as the noncaveolae membrane fraction.
Electrophoresis and Immunoblots. Each sample was concentrated by TCA precipitation and washed in acetone. Pellets were suspended in Laemmli sample buffer (12), heated at 95°C for 3 min, and loaded onto 12.5% SDS polyacrylamide gel using the method of Laemmli (12). The separated proteins were transferred to nylon supports. The nylon was blocked in buffer C (20 mM Tris, pH 7.5, 137 mM NaCl, 0.5% Tween-20) containing 5% dry milk for 1 h at room temperature. Primary antibodies were diluted in buffer C containing 1% dry milk and incubated with the nylon samples for 1 h at room temperature. The nylon was washed four times for 10 min each in buffer C plus 1% dry milk and incubated with the appropriate HRP-labeled anti-IgG for 1 h at room temperature. The nylon was then washed, and the bands were visualized by enhanced chemiluminescence.
PKC Binding to Caveolae.
PKC binding to caveolae was carried out
using either a solid phase or a solution assay. The solid phase radioimmune assay was modified from the method of Zhang et al. (28). Immulon I Removawell strips were washed twice with distilled water. Either caveolae
or noncaveolae membranes isolated in the absence of calcium (3 µg) or
BSA (3 µg) in 50 µl of buffer A were air dried to the bottom of each well.
The coated wells were washed quickly three times with 250 µl of buffer D
(25 mM Hepes, pH 7.0, 125 mM potassium acetate, 2.5 mM magnesium
acetate, 1 mg/ml glucose, 0.1 mM EDTA, 1 mM DTT, 1 mg/liter leupeptin, 1 mg/liter pepstatin A) containing 1 mg/ml heat-denatured BSA, incubated with buffer D containing 2 mg/ml heat-denatured BSA for 45 min at
room temperature, and washed three times with 250 µl of buffer D plus 1 mg/ml heat-denatured BSA. The indicated PKC mixtures (100 µl) were
added and incubated for 30 min at the indicated temperature. The wells
were washed rapidly seven times at 4°C with 250 µl of buffer D plus 1 mg/ml heat-denatured BSA. Each sample was fixed with 250 µl of 3% paraformaldehyde in buffer D for 30 min at room temperature. The amount of
PKC
bound to EDTA-stripped membrane was determined by radioimmunoassay as previously described (28) using anti-PKC
(1 µg/ml), biotinylated goat anti-mouse IgG (2 µg/ml), and 125I-streptavidin (2 µCi/ml).
Isolation of sdr.
Interaction cloning (4) was used to isolate a 68-kD
PKC-binding protein designated as clone 34. Analysis of the sequence showed that clone 34 was identical to a previously cloned protein known
as sdr (7). Clone 34/sdr cDNA was ligated in frame into the pTrc (InVitrogen, Carlsbad, CA) or pQE (Qiagen, Chatsworth, CA) bacterial expression vector to produce recombinant His-tagged fusion proteins. The expressed sequences corresponding to polypeptides containing amino acids
1-168, 145-250, and 250-417 were purified by nickel-nitrilotriacetic acid
chromatography according to the manufacturer's instructions. The purified peptides were used to raise antisera in rabbits. Antisera were purified
by affinity chromatography using the expressed sequences coupled to
Sepharose.
ELISA Assay.
Fragments of clone 34/sdr containing residues 1-168,
145-250, or 250-417 (2.8 µg/ml in PBS, 100 µl per well) were bound to individual wells of a 96-well dish, and the wells were blocked with BSA (2%
in PBS). PKC (20 ng of recombinant PKC
) or RD
(60 ng of recombinant maltose-binding protein [MBP] fused to RD
) were added to the
wells in buffer E (PBS plus 0.1 mg/ml BSA, 1 mM EGTA, 0.466 mM
CaCl2, and 2.1 mM MgCl2). Reactions were incubated for 2 h at room
temperature. Where indicated, PS (2 µg/ml) was included in the buffer.
Wells were rinsed with buffer E and incubated with either PKC
-specific
mAb M4 or anti-MBP polyclonal IgG for 1 h (New England Biolabs, Boston, MA) followed by the appropriate secondary antibody conjugated to
HRP for 1 h, all in PBS plus 1 mg/ml BSA. Bound antibody was detected
by adding 12 pmol/well of the substrate 2,2'azino-di[3 ethylbenzthiazoline
sulfonate] in PBS and incubating for 15-60 min. Reaction was quantified
by measuring the absorbance at 405 nm. Nonspecific binding of PKC
, and MBP-RD
was determined using BSA-blocked wells that did not contain peptides. PS did not influence nonspecific binding. Total bound
PKC
or MBP-RD
corresponds to the amount of antibody binding to
wells coated with 20 ng PKC
or 60 ng of MBP-RD
alone.
Indirect Immunofluorescence. Normal human fibroblasts and Rat-1 cells grown on glass coverslips were washed quickly with buffer F (100 mM sodium phosphate, pH 7.6, containing 3 mM KCl and 3 mM MgCl2) and then fixed in methanol/acetic acid/1,1,1 trichloroethane (60:10:30) for 20 min. Cells were quickly rinsed three times with 50% methanol followed by three times with buffer F. Cells were incubated with buffer F containing 0.8% bovine serum albumin for 30 min, followed by buffer F containing 20 µg/ml mAb anti-caveolin-1 plus a 1:10 dilution of anti-sdr IgG for 60 min. Finally, cells were incubated for 60 min in the presence of buffer F containing 20 µg/ml goat anti-mouse IgG conjugated to TRITC and 20 µg/ml goat anti-rabbit IgG conjugated to FITC. After incubation, cells were washed and mounted in a 2.5% solution of 1,4-diabicyclo-(2,2,2) octane. All incubations were at room temperature. Samples were photographed using a Zeiss Photomicroscope III (Thornwood, NY).
Other Assays. Protein concentrations were determined using Bio-Rad Bradford assay (Hercules, CA).
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Results |
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Previously, we localized PKC to caveolae of MA104 cells
using a cell fractionation scheme that depends on the partial insolubility of caveolae in Triton X-100 at 4°C (25). To
avoid potential artifacts associated with the use of detergents, in the current studies we isolated caveolae from purified plasma membranes by flotation on OptiPrep gradients (26). The first (OptiPrep 1) of the two gradients used
in the purification separates light membranes rich in the
caveolae marker caveolin-1 from the bulk plasma membrane protein. The second gradient (OptiPrep 2) further purifies the caveolae from the top seven fractions of the
first gradient. The standard buffer for this cell fractionation procedure contains 1 mM EGTA. Immunoblots of
each fraction (total protein load) from OptiPrep 1 gradients of Rat-1 cell plasma membranes showed low levels of
PKC
in the caveolin-rich (caveolin) light fractions (Fig. 1 A,
PKC
, lanes 1-7). Little PKC
was detected in the
heavier fractions (lanes 8-14) that had the bulk of the plasma membrane protein (Fig. 1 D, squares), although
these fractions contained all of the detectable integrin
3
(lanes 8-14).
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Association of PKC with Caveolae
To determine if the EGTA had stripped away PKC from
caveolae during the isolation, we prepared cell fractions
using the same buffer with 1 mM Ca++ added (Fig. 1 B).
Under these conditions, the caveolin-rich fractions contained a much higher concentration of PKC
. Since all the
protein in each fraction was loaded on the gel, the majority
of the PKC
we detected was in these fractions (compare
lanes 1-7 with lanes 8-14). The protein profile (Fig. 1 D,
diamonds) as well as the distribution of caveolin-1 and integrin 1
3 were unchanged. If the cells were preincubated
in the presence of PMA for 20 min before fractionation,
the light membrane fractions had similar levels of PKC
,
even though calcium was not in the isolation buffer (fractions 1-7, compare Fig. 1, B and C, PKC
). PKC
was not
detected in the bulk membrane fractions under either condition (Fig. 1, B and C, lanes 10-14). These results suggest
PKC
is normally bound to caveolae through a calcium-sensitive interaction with resident molecules.
Other PKC isoforms were also found to be enriched in
caveolae fractions (Fig. 2). PKC was concentrated in caveolae, but unlike PKC
, enrichment was stimulated by a
lack of Ca++ in the isolation buffer (compare lanes 1 and
2). This isoform was also enriched when cells were pretreated with PMA for 20 min (lane 3). PKC
was enriched
in the absence of Ca++ (lane 1), but the presence of Ca++
slightly reduced the concentration (lane 2). Pretreatment
of cells with PMA increased the amount of PKC
in the
caveolae fraction relative to other treatments (lane 3).
Thus, PKC isoenzyme types differ in the amount of calcium required to remain bound to caveolae membrane
during isolation but share the ability to remain bound independently of calcium after cells are pretreated with PMA.
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We used immunoblotting to measure the relative
amount of PKC in the cytosol, noncaveolae membrane
(NCM), and caveolae membrane (CM) fractions after
various isolation conditions (Fig. 3). When Ca++ was in
the isolation buffer, PKC
was enriched in caveolae (compare lane 12 with 11) but not noncaveolae fractions (compare lane 7 with 6). The slight increase in PKC
concentration seen in the cytosol fraction under these conditions
was within experimental variability (compare lane 1 with
2). Both caveolae (lane 13) and noncaveolae (lane 8) fractions had similar low levels of PKC
when Mg++ was substituted for Ca++. Exposing cells to PMA for 20 min
caused an apparent increase in the amount of PKC
in the
caveolae fraction relative to isolation in the absence of Ca++
(compare lane 14 with 11) without changing the amount in
either the cytosol (lane 4) or the noncaveolae (lane 9) fractions. By contrast, extended exposure of cells to PMA
caused a reduction in the cytosolic level of PKC
(compare lane 5 with 1) and completely eliminated the protein
from the caveolae fractions (compare lane 15 with 14).
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Binding of PKC to Caveolae
The lack of detectable PKC in the bulk plasma membrane fractions rich in integrin
3 (Fig. 1 B), even though
we loaded the total protein in each fraction (up to 100 µg/
lane in fractions 11 and 12) on the gel, suggests PKC
has
a specific affinity for caveolae. We used a solid phase assay
to determine if caveolae were able to bind PKC
(Fig. 4).
Caveolae and noncaveolae membranes were isolated in
the absence of Ca++ so that PKC
was not present (see
Fig. 2). Equal amounts of caveolae (Fig. 4 A, bars 1-6) and
noncaveolae (bar 7) membrane protein were air dried on
the bottom of 96-well plates and assayed for PKC
binding. When caveolae membranes were incubated in the
presence of the complete binding mixture (1.5 nM PKC
,
1 mM Ca++, 30 µM PS, 100 µM ATP) at 37°C for 30 min
(bar 1), significant amounts of PKC
bound to caveolae
membranes. By contrast, very little PKC
bound to noncaveolae membranes (bar 7). Binding to caveolae was prevented by removing either PKC
(bar 2) or Ca++ (bar 3)
from the mixture. Mg++ could not substitute for Ca++ (bar
4), and PS was not required (bar 5). Finally, PKC
did not bind to caveolae when the incubation was carried out at
4°C (bar 6).
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PKC binding to caveolae membranes in the solid phase
assay was saturable (Fig. 4 B, squares). Half-maximal
binding occurred at ~0.5 nM PKC
, suggesting a high-
affinity interaction with the membrane. Binding of PKC
to noncaveolae membranes (circles) was no greater than
binding to dishes coated with albumin (diamonds).
We could also detect PKC binding to caveolae using a
solution assay (Fig. 4 C). Caveolae and noncaveolae membranes were prepared and incubated in solution with the
indicated mixtures. At the end of each incubation, the
membranes were recovered by centrifugation, processed
for gel electrophoresis, and immunoblotted with either anti-caveolin-1 IgG (caveolin) or anti-PKC
IgG (PKC
).
The association of PKC
with the pelleted caveolae fraction was dependent on the presence of PKC
(compare
lanes 1 and 2), Ca++ (compare lanes 2 and 3), and temperature (compare lanes 2 and 6), but not PS (compare lanes
2 and 5). Binding was not detected if noncaveolae membrane was substituted for caveolae (compare lanes 2 and
7) or if Ca++ was replaced with Mg++ (compare lanes 2 and 4).
The solid phase assay was used to define further the requirements for PKC binding to caveolae membranes. We
showed in Fig. 1 that the calcium requirement for PKC
association with isolated caveolae was lost when cells were
incubated in the presence of PMA before caveolae isolation. By contrast, the addition of PMA to the in vitro binding assay mixture had no effect on PKC
binding to isolated caveolae (Fig. 5 A). The amount of PKC
bound was the same in the presence or absence of PMA (compare
bars 1-3). Furthermore, PMA did not promote PKC
binding to caveolae when calcium was removed from the
incubation mixture (compare bars 4 and 5 with 2 and 3).
No binding was detected when noncaveolae membranes
(bar 6) or albumin (bar 7) were substituted for caveolae. In other experiments, we found that PMA did not stimulate PKC
binding to noncaveolae membranes (data not
shown).
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We originally added ATP to the incubation mixture because PKC contains an ATP-binding domain that might
be required for interacting with caveolae. Fig. 5 B shows,
however, that ATP was not required for PKC
binding
(compare bars 1 and 2). GTP also had no effect on binding
(data not shown). We still did not detect binding to caveolae at 4°C (compare bars 3 and 4) or to noncaveolae membranes (compare bars 5 and 6) when ATP was removed
from the incubation buffer. Also, the lack of PKC
binding to caveolae at 4°C did not change if PS was removed
from the incubation mixture (data not shown).
Since Ca++ is required for PKC binding but not ATP,
the regulatory domain (RD
) of the molecule may mediate binding to caveolae. We compared the binding to caveolae membranes of recombinant forms of PKC
and RD
(amino acids 1-312). Caveolae (Fig. 6 A, bars 1-4) and
noncaveolae (bars 5 and 6) membranes were incubated in the presence of 1.3 nM PKC
or 1.3 nM RD
. When Ca++
was in the buffer (compare bars 1 and 3), equal amounts of
either PKC
or RD
bound to caveolae membranes. Removal of Ca++ from the buffer (compare bars 2 and 4) reduced binding to the level seen when noncaveolae membranes were substituted for caveolae (compare bars 2 and
4 with 5 and 6). Further evidence for RD
-mediated binding is that PKC
, which contains a different regulatory domain that appears not to require calcium for association
with caveolae (see Fig. 2), did not block PKC
binding to
caveolae membranes even when present in >100-fold excess (Fig. 6 B, compare bars 1-5).
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Identification of a PKC-binding Protein in Caveolae
Most likely, the high-affinity binding of PKC to caveolae
involves an interaction with a resident protein of caveolae.
A candidate protein should bind PKC
in the presence of
calcium, bind the regulatory domain of PKC
, and be concentrated in caveolae. Several PKC-binding proteins have
been identified by probing expression libraries with recombinant PKC (called interaction cloning [10]). A protein isolated from such a screen with the required characteristics is clone 34. Clone 34 is a 68-kD protein identical in
sequence to sdr, which was isolated from serum starved
cells (7). In an overlay assay, clone 34/sdr bound the regulatory domain of PKC
only when calcium and PS were
present (data not shown). We used a quantitative binding
assay to localize the region of clone 34/sdr that contains
the PKC
-binding domain (Fig. 7). Samples of histidine-tagged fusion protein containing either amino acids 1-168,
145-250, or 250-417 of clone 34/sdr were bound to individual wells of a 96-well plate. Wells were then incubated in
the presence of either the full-length (PKC
) or the regulatory domain of PKC
(RD
) in the presence or absence
of PS before assaying for the amount bound. Both PKC
(left) and RD
(right) bound peptide 145-250 in the presence (hatched bars) but not the absence of PS (solid bar).
Neither PKC
nor RD
bound the other two peptides.
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Fig. 8 shows the immunofluorescence colocalization of
clone 34/sdr (B) and caveolin-1 (A) in a human fibroblast.
Some anti-clone 34/sdr IgG staining had a perinuclear (N,
nucleus) distribution characteristic of the Golgi apparatus.
Staining was also prominent along the edges of the cell
and in linear patches on the surface (arrowheads). The
edge and surface patches colocalized with caveolin-1 (compare arrowheads between A and B). The mAb anti-
caveolin-1 used to do the colocalization reacted poorly
with Rat-1 cells. Nevertheless, when we used polyclonal
anti-caveolin-1 IgG (C) and anti-clone 34/sdr IgG (D) on
separate sets of cells, a similar edge staining (arrowheads)
was evident in both sets. Immunoblots of total protein
loads from Rat-1 cell OptiPrep 1 gradient fractions (E)
showed that PKC, clone 34/sdr, and caveolin-1 quantitatively cofractionated (fractions 1-8). By contrast, another
PKC-binding protein, RACK 1 (receptor for activated C
kinase [19]), was primarily in the bulk plasma membrane
fraction (fractions 9-14).
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We used the solid phase binding assay to see if anti-
clone 34/sdr IgG blocked PKC binding to caveolae (Fig.
9 A). Good binding was observed when caveolae fractions
were incubated with the complete binding mixture (bar 1).
Addition of 15 µg of the affinity-purified anti-clone 34/sdr
IgG to the incubation mixture reduced PKC
binding by
~50% (bar 2). Increasing the concentration of the antibody did not further reduce binding. The same concentration of polyclonal anti-caveolin-1 IgG, by contrast, had
no effect on PKC
binding (bar 4). PKC
did not bind to
noncaveolae membranes (bar 4). These results suggest
clone 34/sdr is a protein component of the PKC
-binding site.
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A peptide competition assay provided additional evidence that clone 34/sdr was involved in PKC binding to
caveolae (Fig. 9 B). We used subsaturating concentrations
of PKC
in a standard binding assay where each tested
peptide was present in 100-fold excess. Compared with no
additions (bar 1), peptide 1-168 had no effect on PKC
binding (bar 2). Peptide 145-250, by contrast, reduced
binding to the level seen when noncaveolae membranes
were substituted for caveolae (compare bars 3 and 6). Peptide 250-417 did not inhibit binding (bar 4). We also tested
the effect of the PKC
pseudosubstrate peptide on binding (bar 5). This peptide completely blocked binding (bar
5). Therefore, we have localized peptide domains within
both clone 34/sdr and PKC
that can interact during
PKC
binding to caveolae membranes.
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Discussion |
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PKC Binding to Caveolae
Cell fractionation and immunocytochemistry have previously shown that PKC is constitutively present in caveolae and that this is a major cell surface location for the enzyme (25). We used a solid phase binding assay that has
successfully identified other membrane binding sites for
cytosolic proteins (28) to determine if PKC
would bind
to caveolae. PKC
bound with high affinity (binding was
dependent on calcium) did not require the addition of either PMA, PS, or ATP, and only occurred at 37°C. PKC
did not bind to noncaveolae membranes, which contain
>90% of the plasma membrane protein starting material.
The same specific interaction with caveolae was also detected in a solution binding assay. Caveolae, therefore, exhibit a PKC
binding activity that may be responsible for
targeting the enzyme to this compartment.
We found that caveolae contained other members of the
PKC enzyme family. Fractions from untreated cells contained PKC only when calcium was present and PKC
only when calcium was absent from the isolation buffer.
The presence of PKC
was not dependent on calcium, although this cation did appear to reduce the amount of enzyme in the fraction. The calcium concentration needed to retain the enzyme during isolation is a reflection of the
cation requirement for PKC binding to caveolae. These results raise the possibility that local fluctuations in the concentration of calcium can regulate the amount of a PKC
isoenzyme in caveolae. Calcium could function, therefore,
as a regulatory switch that controls the isoenzyme composition of caveolae. This may be especially important at
times when calcium entry occurs at caveolae (2).
PMA did not significantly increase the level of PKC in
caveolae above that normally present when isolation was
carried out under the correct calcium conditions for retention of the isoenzyme (Fig. 2, lane 3). This suggests that
PMA does not stimulate recruitment of cytosolic PKCs to
caveolae but instead stabilizes the resident population of
isoenzyme so it remains bound regardless of the concentration of calcium in the isolation buffer. This conclusion is
supported by the finding that PMA did not induce binding
of PKC to either caveolae or noncaveolae membranes in
vitro (Fig. 5 A).
Recombinant PKC was used in all of the in vitro assays, so binding to isolated caveolae was not dependent on
phosphorylation of the enzyme. Furthermore, the regulatory domain alone bound as well as the whole protein, and
this region does not contain any of the phosphorylation
sites thought to modulate the interaction of PKC
with
the cytoskeleton (20). PMA was also not required for
binding, nor did it block binding (Fig. 4 A), and calcium was required for retention during caveolae isolation.
These are the characteristics of a binding site designed to
recognize inactive, native PKC
within the cell and concentrate the enzyme at caveolae independently of the activation state of the cell. There may be binding sites specific
for each of the major isoenzyme families. The specificity
required to distinguish between isoenzyme families may
be conferred by other PKC-binding proteins together with
cofactors concentrated in caveolae. The PKC isoenzymes
in caveolae are probably engaged in regulating essential
cellular activities.
One activity that PKC appears to regulate at this location is the internalization of caveolae (25). The phosphorylation of a 90-kD caveolae substrate occurs during invagination and sequestration of molecules by caveolae. Cells
lacking PKC
do not have detectable enzyme in caveolae,
and both caveolae invagination and ligand internalization
are blocked. Like many resident proteins of caveolae, the
PKC
in this domain is normally resistant to solubilization by Triton X-100 at 4°C. After stimulation of histamine H1
receptors, membrane-bound PKC
becomes detergent
soluble, suggesting a change in its linkage to the caveolae
membrane. Under these conditions, phosphorylation of
the 90-kD substrate does not occur, and internalization of
caveolae is inhibited. The binding activity we have detected may be essential for positioning PKC
to optimize
the phosphorylation of this protein. Another outcome of
binding is to localize PKC isoenzymes at a site where they
can interact with multiple signaling pathways (2).
Localization of a PKC-binding Protein to Caveolae
A number of PKC-binding proteins have been identified
that could participate in targeting PKC to caveolae (10,
18, 21), including caveolin (23). We focused our attention
on clone 34/sdr because initial immunofluorescence examination suggested it was present in caveolae. Immunofluorescence and cell fractionation of Rat-1 cells clearly show
that the majority of the plasma membrane clone 34/sdr is
concentrated in caveolae. Clone 34/sdr was in caveolae
fractions isolated without calcium even after PMA pretreatment of cells (data not shown). The binding of PKC
to both caveolae and purified clone 34/sdr requires calcium and the regulatory domain of PKC
. In addition, neither activity requires ATP or an activator such as PMA.
Anti-clone 34/sdr IgG reduces PKC
binding by 50%, and
a specific peptide (amino acids 145-250) within sdr competitively inhibits binding. These results suggest clone 34/ sdr has a role in targeting PKC
to caveolae.
sdr was originally isolated from NIH 3T3 cells in a
screen for RNA messages that are upregulated during serum deprivation (7). sdr contains a leucine zipper-like motif between amino acids 50 and 100 and two consensus
sites for PKC phosphorylation. One of the phosphorylation sites (amino acids 229-250) is at the amino terminus
of the sdr peptide that binds the regulatory domain of
PKC and blocks its binding to caveolae. SRBC (sdr-
related gene product that binds C-kinase) (9) shares several similarities with sdr, including binding PS as well as
the regulatory domain of PKC and phosphorylation by
PKC. These two proteins belong to a class of molecules
called STICKs (substrates that interact with C-kinase
[10]). Each STICK may have a primary function in targeting a distinct set of PKC isoenzymes to specific locations in
the cell. Interestingly, a fusion protein with cell transforming activity was isolated from colon cancer cells that consists of the first 184 amino acids of SRBC linked to c-Raf
(27). Since activation of c-Raf takes place in caveolae (16),
and a c-Raf containing the COOH-terminal consensus sequence for prenylation is constitutively active (13) in caveolae (17), the SRBC-Raf fusion protein may alter cell
behavior by inappropriately targeting c-Raf to this membrane domain. If this is the case, then the first 184 amino
acids of SRBC are predicted to contain a caveolae binding motif.
The targeting of PKC to caveolae is probably more
complex than a simple one-to-one interaction between the
enzyme and sdr. Unlike caveolae, PKC
binding to purified sdr can occur at 4°C, requires PS, and is not blocked
by the PKC
pseudosubstrate peptide. Caveolae could
provide the needed PS, but it is hard to reconcile the other
two differences if sdr acts alone. Caveolae membrane lipids, unlike surrounding regions of membrane, are in a liquid order phase owing to the high concentration of cholesterol and sphingomyelin (3). The phase properties of
membrane lipids are temperature sensitive, raising the
possibility that a higher lateral mobility of membrane proteins and lipids at 37°C is required for PKC
binding to caveolae. There also must be molecules in caveolae that concentrate the sdr itself because it does not contain any
obvious membrane anchor. Whatever these interactions
turn out to be, they probably influence the amount of
PKC
in caveolae. Finally, the PKC
in caveolae is active
(25), so DAG, a lipid species that is enriched in caveolae
(15), is probably bound to this population of enzyme (22).
The pseudosubstrate of the enzyme, therefore, may be
free to interact with nearby molecules, which could account for why the pseudosubstrate peptide interfered with
PKC
binding to caveolae. We conclude that a protein, or group of proteins, act coordinately in the proper lipid environment to attract PKC
to caveolae.
Compartmentalization of PKC Function by Caveolae
The finding that multiple PKC isoenzymes along with at least one known PKC-binding protein are concentrated in caveolae suggests this is a location where the signaling function of these molecules is compartmentalized. The combination of a unique membrane environment and a close physical association should enable caveolae PKC isoenzymes to perform unique functions that do not occur anywhere else in the cell. Some of these functions may be housekeeping in nature, such as controlling the invagination of caveolae. The proximity of these PKCs to other signaling molecules in this domain (1, 2), however, will naturally facilitate interactions that influence many different signaling events. The immediate goals are to identify caveolae-specific PKC functions and to determine the mechanism(s) used to organize these enzymes at this location on the cell surface. There may be a protein scaffold (11) that holds several isoenzymes in a PKC module, linking receptors with multiple targets through a kinase cascade (22). Molecules like sdr might function as linkers, adaptors, or switches that control interactions among the elements of this module.
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Footnotes |
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Received for publication 20 November 1997 and in revised form 4 March 1998.
Address all correspondence to Richard G.W. Anderson, Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX 75235-9039. Tel.: (214) 648-2346. Fax: (214) 648-7577. E-mail: anders06{at}utsw.swmed.eduWe would like to thank William Donzell and Ann Horton for their valuable technical assistance and Stephanie Baldock for administrative assistance.
This work was supported by grants from the National Institutes of Health, CA53841, HL 20948, and GM 43169, and the Perot Family Foundation.
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Abbreviations used in this paper |
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DAG, diacylglycerol; MBP, maltose-binding protein; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate; PS, phosphatidylserine; sdr, serum deprivation response; RD, regulatory domain.
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