Previously, we localized PKCα 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 PKCα in the caveolin-rich (caveolin
) light fractions (Fig. A
, lanes 1–7
). Little PKCα was detected in the heavier fractions (lanes 8–14
) that had the bulk of the plasma membrane protein (Fig. D
), although these fractions contained all of the detectable integrin β3 (lanes 8–14
Figure 1 Effects of EGTA (A), calcium (B), and PMA pretreatment (C) on the presence of PKCα in caveolae membrane fractions. Rat-1 cells grown 24 h in the absence of serum were incubated in the presence (C) or absence (A and B) of 100 nM PMA for 20 min (more ...)
Association of PKC with Caveolae
To determine if the EGTA had stripped away PKCα from caveolae during the isolation, we prepared cell fractions using the same buffer with 1 mM Ca++ added (Fig. B). Under these conditions, the caveolin-rich fractions contained a much higher concentration of PKCα. Since all the protein in each fraction was loaded on the gel, the majority of the PKCα we detected was in these fractions (compare lanes 1–7 with lanes 8–14). The protein profile (Fig. D, diamonds) as well as the distribution of caveolin-1 and integrin 1β3 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 PKCα, even though calcium was not in the isolation buffer (fractions 1–7, compare Fig. , B and C, PKCα). PKCα was not detected in the bulk membrane fractions under either condition (Fig. , B and C, lanes 10–14). These results suggest PKCα 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. ). PKCλ was concentrated in caveolae, but unlike PKCα, 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). PKCε 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 PKCε 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.
Figure 2 Effects of EGTA (lane 1) or calcium (lane 2) in the isolation buffer or pretreatment of cells with PMA (lane 3) on the presence of PKC isoenzymes in caveolae membrane fractions. Rat-1 cells grown 24 h in the absence of serum were incubated in the (more ...)
We used immunoblotting to measure the relative amount of PKCα in the cytosol, noncaveolae membrane (NCM), and caveolae membrane (CM) fractions after various isolation conditions (Fig. ). When Ca++ was in the isolation buffer, PKCα was enriched in caveolae (compare lane 12 with 11) but not noncaveolae fractions (compare lane 7 with 6). The slight increase in PKCα 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 PKCα when Mg++ was substituted for Ca++. Exposing cells to PMA for 20 min caused an apparent increase in the amount of PKCα 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 PKCα (compare lane 5 with 1) and completely eliminated the protein from the caveolae fractions (compare lane 15 with 14).
Figure 3 Comparative blotting activity of PKCα in cytosol, noncaveolae membrane (NCM), and caveolae membrane (CM) fractions. Rat-1 cells were grown in the absence of serum for 24 h and either incubated in the presence of PMA for 20 min (lanes 4, 9 (more ...)
Binding of PKCα to Caveolae
The lack of detectable PKCα in the bulk plasma membrane fractions rich in integrin β3 (Fig. 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 PKCα has a specific affinity for caveolae. We used a solid phase assay to determine if caveolae were able to bind PKCα (Fig. ). Caveolae and noncaveolae membranes were isolated in the absence of Ca++ so that PKCα was not present (see Fig. ). Equal amounts of caveolae (Fig. A, bars 1–6) and noncaveolae (bar 7) membrane protein were air dried on the bottom of 96-well plates and assayed for PKCα binding. When caveolae membranes were incubated in the presence of the complete binding mixture (1.5 nM PKCα, 1 mM Ca++, 30 μM PS, 100 μM ATP) at 37°C for 30 min (bar 1), significant amounts of PKCα bound to caveolae membranes. By contrast, very little PKCα bound to noncaveolae membranes (bar 7). Binding to caveolae was prevented by removing either PKCα (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, PKCα did not bind to caveolae when the incubation was carried out at 4°C (bar 6).
Figure 4 PKCα binding to isolated caveolae and noncaveolae membrane fractions using either a solid phase (A and B) or solution (C) assay. The indicated membrane fractions were prepared form Rat-1 cells grown 24 h in the absence of serum using standard (more ...)
PKCα binding to caveolae membranes in the solid phase assay was saturable (Fig. B, squares). Half-maximal binding occurred at ~0.5 nM PKCα, suggesting a high- affinity interaction with the membrane. Binding of PKCα to noncaveolae membranes (circles) was no greater than binding to dishes coated with albumin (diamonds).
We could also detect PKCα binding to caveolae using a solution assay (Fig. 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-PKCα IgG (PKCα). The association of PKCα with the pelleted caveolae fraction was dependent on the presence of PKCα (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 PKCα binding to caveolae membranes. We showed in Fig. that the calcium requirement for PKCα 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 PKCα binding to isolated caveolae (Fig. A). The amount of PKCα bound was the same in the presence or absence of PMA (compare bars 1–3). Furthermore, PMA did not promote PKCα 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 PKCα binding to noncaveolae membranes (data not shown).
Figure 5 Neither PMA (A) nor ATP (B) was required for PKCα binding to caveolae. The solid phase binding assay was using the standard buffer with the indicated additions at 37°C as described in Fig. A. Incubations were carried (more ...)
We originally added ATP to the incubation mixture because PKCα contains an ATP-binding domain that might be required for interacting with caveolae. Fig. B shows, however, that ATP was not required for PKCα 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 PKCα 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 PKCα binding but not ATP, the regulatory domain (RDα) of the molecule may mediate binding to caveolae. We compared the binding to caveolae membranes of recombinant forms of PKCα and RDα (amino acids 1–312). Caveolae (Fig. A, bars 1–4) and noncaveolae (bars 5 and 6) membranes were incubated in the presence of 1.3 nM PKCα or 1.3 nM RDα. When Ca++ was in the buffer (compare bars 1 and 3), equal amounts of either PKCα or RDα 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 RDα-mediated binding is that PKCε, which contains a different regulatory domain that appears not to require calcium for association with caveolae (see Fig. ), did not block PKCα binding to caveolae membranes even when present in >100-fold excess (Fig. B, compare bars 1–5).
Figure 6 The regulatory domain of PKCα (A), but not intact PKCε (B), binds caveolae. The solid phase binding assay was using the standard buffer with the indicated additions at 37°C as described in Fig. A. Incubations were (more ...)
Identification of a PKCα-binding Protein in Caveolae
Most likely, the high-affinity binding of PKCα to caveolae involves an interaction with a resident protein of caveolae. A candidate protein should bind PKCα in the presence of calcium, bind the regulatory domain of PKCα, and be concentrated in caveolae. Several PKC-binding proteins have been identified by probing expression libraries with recombinant PKC (called interaction cloning ). 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 PKCα 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 PKCα-binding domain (Fig. ). 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 (PKCα) or the regulatory domain of PKCα (RDα) in the presence or absence of PS before assaying for the amount bound. Both PKCα (left
) and RDα (right
) bound peptide 145–250 in the presence (hatched bars
) but not the absence of PS (solid bar
). Neither PKCα nor RDα bound the other two peptides.
Figure 7 Localization of the PKCα-binding region of clone 34/ sdr. The indicated peptides were bound to individual wells of a 96-well plate and incubated in the presence of either recombinant PKCα or an MBP–PKCα regulatory domain (more ...)
Fig. 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 PKCα, clone 34/sdr, and caveolin-1 quantitatively cofractionated (fractions 1–8). By contrast, another PKC-binding protein, RACK 1 (receptor for activated C kinase ), was primarily in the bulk plasma membrane fraction (fractions 9–14).
Figure 8 Immunofluorescence (A–D) and cell fractionation (E) localization of clone 34/sdr to caveolae. (A and B) The same sample of normal human fibroblasts grown on coverslips was processed for immunofluorescence colocalization of caveolin-1 (A) and (more ...)
We used the solid phase binding assay to see if anti– clone 34/sdr IgG blocked PKCα binding to caveolae (Fig. 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 PKCα 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 PKCα binding (bar 4). PKCα did not bind to noncaveolae membranes (bar 4). These results suggest clone 34/sdr is a protein component of the PKCα-binding site.
Figure 9 Anti–clone 34/sdr IgG (A) and specific clone 34/sdr peptides (B) block binding of PKCα to caveolae. Caveolae and noncaveolae membranes were prepared from Rat-1 cells, and the solid phase binding assay was used at 37°C to detect (more ...)
A peptide competition assay provided additional evidence that clone 34/sdr was involved in PKCα binding to caveolae (Fig. B). We used subsaturating concentrations of PKCα 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 PKCα 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 PKCα 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 PKCα that can interact during PKCα binding to caveolae membranes.