By raising a specific antibody to caveolin-1 phosphorylated at tyrosine 14, we could analyze the distribution and molecular characteristics of the modified molecule in detail. The result of Western blotting indicates that phosphorylation of caveolin-1 in v-Src–expressing cells occurs not only at tyrosine 14 but also at other residues; that is, there were several bands reactive with PY14 in both SR-3Y1 and srcts
NRK cells; at least one upper band was detected even for the mutant caveolin-1 lacking tyrosine 14, and after dephosphorylation, caveolin-1 was detected as a single band at 22 kDa. The possibility that the upper bands were modified β isoform was excluded, because all of them were recognized by anti–α-caveolin-1 (sc-894). Phosphorylation sites besides tyrosine 14 are not known at present, but they could be serine (or threonine), as reported in chicken cells (Glenney, 1989
), or tyrosine residues other than tyrosine 14.
In v-Src–expressing cells, caveolin-1 showed a distribution different from that in their normal counterpart: formation of patches along cell edges was lost, and caveolin-1 occurred in large flat areas of the plasma membrane and in aggregated vesicles in the cytoplasm. The freeze fracture immunoelectron microscopy clearly showed that the flat areas labeled by PY14 in the plasma membrane were demarcated by the absence of IMPs and seen in the vicinity of caveolae. In normal cells, most caveolin-1 labeling occurred in patches of deep and shallow caveolae as observed previously (Fujimoto and Fujimoto, 1997
; Nomura et al., 1997
), and labeled flat IMP-free areas were hardly seen. The size of the caveolin-1–positive IMP-free areas in cells transformed by v-Src was significantly larger than shallow caveolae or few flat structures seen in their normal counterpart. Moreover, whereas shallow caveolae were labeled by sc-894 only inefficiently in normal cells (Fujimoto, Kogo, Nomura, Takahashi, and Une, unpublished results), the IMP-free areas in v-Src–expressing cells were densely labeled. Based on these results, we assume that the large caveolin-1–positive IMP-free areas in v-Src–expressing cells are distinct from shallow caveolae and probably formed by mutual fusion of flattened caveolae.
The cytoplasmic structure labeled positively by PY14 looked like racemose caveolae, that is, a group of caveolae sharing the lumen and connected to the cell surface. Racemose caveolae have been described in various cells in vivo (Bundgaard et al., 1983
; Bundgaard, 1991
) and in vitro (Parton et al., 1994
) and were induced in cultured keratinocytes by disorganizing the actin cytoskeleton with cytochalasin D (Fujimoto et al., 1995
). However, the aggregated vesicles observed in the present study may be different from racemose caveolae, because most of them did not appear to share the lumen; moreover, many of them were found in the deep cytoplasm and thus were unlikely to be open to the cell surface; they were also different from racemose caveolae induced by cytochalasin D in that coaggregation of F-actin was not observed. The mechanism responsible for formation of the aggregated vesicles is not known. But considering that membranes of the vesicles are closely apposed in the aggregates and that flattened caveolae in the plasma membrane appear to fuse, as discussed above, the aggregated vesicles might be also formed by adhesion and fusion of caveolae and/or caveolae-derived vesicles. As a result, some of them may become larger vesicles, whereas others remain as caveola-sized vesicles and are closely apposed to other ones.
We looked for changes in the molecular properties of caveolin-1 between normal and v-Src–expressing cells, but detergent solubility, oligomer formation, and association with caveolin-2 were found to be the same in 3Y1 and SR-3Y1 cells. Caveolin-1 is thought to form oligomers in the endoplasmic reticulum (ER) before being transported through the Golgi to the plasma membrane (Monier et al., 1995
). Because tyrosine phosphorylation by myristylated v-Src is likely to occur at the plasma membrane, only some but not all constituent caveolin-1 molecules in an oligomer may be modified. If this assumption is correct, phosphorylated and nonphosphorylated molecules should coexist in an oligomer, and the characteristics of phosphorylated molecules may be masked in the biochemical experiments. Consistent with this, immunolabeling with PY14 occurred in the same manner as that with sc-894 in v-Src–expressing cells. But the negative result in the present biochemical experiments does not exclude the possibility that phosphorylation alters the molecular properties of caveolin-1 in some aspects. In fact, by Western blotting, a battery of monoclonal anti–caveolin-1 antibodies, whose epitopes are in a segment common to the α and β isoforms, showed much lower reactivity to the α isoform than to the β isoform only in v-Src–expressing cells. Because phosphorylation by v-Src was shown to occur in the α isoform–specific segment (Li et al., 1996b
), it is peculiar that reactivity to the antibodies was reduced. The observation might suggest that some conformational change of α-caveolin-1 occurs as a result of multiple phosphorylation. Therefore, although the caveolin-scaffolding domain, which is supposed to exert the regulatory function of caveolin-1 on various proteins (Li et al., 1996a
), is distant from the α-specific segment, its function could be also modified in the phosphorylated molecule.
Besides the direct alteration of caveolin-1, various caveolar functions may be changed by dissolution of patches, flattening in the plasma membrane, and formation of the aggregated vesicles. First, the intimate structural relationship between caveolae and the ER (Kogo et al., 1997
) may be disrupted. Caveolae have been hypothesized to be related to Ca2+
influx and extrusion (Fujimoto et al., 1992
; Fujimoto, 1993
) and intracellular cholesterol transport (Fielding and Fielding, 1995
; Smart et al., 1996
), whereas the ER is thought to be a site of Ca2+
storage and the site of de novo cholesterol synthesis (for reviews, see Pozzan et al., 1994
; Fielding and Fielding, 1997
). Although speculative, their close apposition might be related to their functional correlation in Ca2+
regulation and cholesterol, and this relationship may be changed in v-Src–expressing cells. Second, because a significant proportion of caveolae-derived vesicles appear to be sequestered from the cell surface in v-Src–expressing cells, receptors and their downstream signaling molecules that reside in caveolae may be separated from the extracellular milieu. The change may insulate cells from various extracellular ligands. In oncogenically transformed cells, expression of caveolin-1 and the number of caveolae were reported to decrease (Koleske et al., 1995
). In contrast, the expression of caveolin-1 was not much different between normal and v-Src–expressing cells as far as examined by sc-894 in Western blotting. The presence of caveolin-1 in the flat IMP-free plasmalemmal areas as well as intracellular vesicles implies that an alteration of caveolar function in transformed cells could occur without a drastic reduction in caveolin-1 expression.
The present study confirmed the result on endothelial cells that vanadate or pervanadate induces tyrosine phosphorylation of caveolin-1 in normal cells (Vepa et al., 1997
). Although the reaction of vanadate-treated NRK cell lysates with PY14 in Western blotting occurred in the same three bands (22, 23–24, and 25 kDa) as in v-Src–expressing cells, it was most intense at 22 kDa, whereas the 25-kDa band was the strongest in v-Src–expressing cells, the difference most likely reflecting the difference in kinases involved. Effects of tyrosine phosphorylation on caveolin-1 distribution in vanadate-treated cells were the same as in v-Src–transformed cells in that peripheral patches disappeared but were different in that the large dots corresponding to the aggregated vesicles were not seen. In cultured endothelial cells, however, we observed extensive vesiculation of caveolae after the vanadate treatment (Aoki, Nomura, and Fujimoto, unpublished observations). Different cells may have different sensitivity and/or different machinery to respond to tyrosine phosphorylation of caveolin-1. Thus, in contrast to v-Src–expressing cells, in which extensive phosphorylation occurs, diverse phenomena might occur when the level of tyrosine phosphorylation is relatively low.
In summary, the present study revealed the following results in v-Src–expressing cells: 1) caveolin-1 is phosphorylated not only in tyrosine 14 but also in other residues; 2) it is not distributed in peripheral patches as seen in normal cells but is found in IMP-free flat plasmalemmal areas and in aggregated caveolae and/or caveolae-derived vesicles; and 3) Triton X-100 insolubility, oligomer formation, and association with caveolin-2 persisted, but the molecular conformation may be altered. Caveolin-1 can be phosphorylated at tyrosine 14 in normal cells, but its consequence is different from that caused by v-Src. Tyrosine phosphorylation of caveolin-1 in v-Src–expressing cells has been considered a critical event for cellular transformation. Further analysis of its significance will be important for a better understanding of the physiology and pathology of caveolae.