Tyrosine-phosphorylated caveolin-1: immunolocalization and molecular characterization - PubMed (original) (raw)

Tyrosine-phosphorylated caveolin-1: immunolocalization and molecular characterization

R Nomura et al. Mol Biol Cell. 1999 Apr.

Free PMC article

Abstract

Caveolin-1 was discovered as a major substrate for v-Src, but the effect of its tyrosine phosphorylation has not been known. We generated a specific antibody (PY14) to caveolin-1 phosphorylated at tyrosine 14 and studied the significance of the modification. By Western blotting of lysates of v-Src-expressing cells, PY14 recognized not only a 22-kDa band (the position of nonphosphorylated caveolin-1) but bands at 23-24 and 25 kDa. Bands of slower mobility were diminished by dephosphorylation and were also observed for mutant caveolin-1 lacking tyrosine 14. By immunofluorescence microscopy, PY14 did not label normal cells but detected large dots in v-Src-expressing cells. Immunoelectron microscopy revealed that the dots corresponded to aggregated caveolae and/or vesicles of various sizes; besides, the label was observed in intramembrane particle-free areas in the plasma membrane, which appeared to have been formed by fusion of flattened caveolae. A positive reaction with PY14 was found in normal cells after vanadate or pervanadate treatment; it occurred mainly at 22 kDa by Western blotting and was not seen as large dots by immunofluorescence microscopy. Detergent solubility, oligomerization, and association with caveolin-2 were observed similarly for caveolin-1 in normal and v-Src-expressing cells. The results indicate that phosphorylation of caveolin-1 in v-Src-expressing cells occurs at multiple residues and induces flattening, aggregation, and fusion of caveolae and/or caveolae-derived vesicles.

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Figures

Figure 1

Figure 1

Western blots using anti–caveolin-1 antibodies and/or PY14. (A) Comparison of 3Y1 and SR-3Y1. Anti–α-caveolin-1 antibody (sc-894) reacted with both samples, but PY14 labeled the latter sample alone. The reaction for SR-3Y1 was broad but with prominent bands at 22, 23–24, and 25 kDa. (B) _src_ts NRK cells. Reaction with PY14 became detectable only after the cell had been transferred to the permissive temperature. (C) Effect of alkaline phosphatase treatment of nitrocellulose blots of SR-3Y1 lysate. After dephosphorylation, the reactivity with PY14 and anti-phosphotyrosine was lost, whereas the reaction with sc-894 remained. (D) Effect of alkaline phosphatase treatment on immunoprecipitates from SR-3Y1 lysate. Immunoprecipitates obtained with sc-894 were dephosphorylated before being subjected to SDS-PAGE and reacted with sc-894 on blots. Bands above 22 kDa were lost after dephosphorylation. (E) Reactivity of monoclonal anti–caveolin-1 antibodies (clones C060 and 2297) with 3Y1 and SR-3Y1. The antibodies recognize both α and β isoforms in 3Y1 samples, but in SR-3Y1 ones the reaction with the α isoform is much weaker than that with the β isoform.

Figure 2

Figure 2

Immunofluorescence microscopy. (A and B) sc-894 intensely labeled both 3Y1 (A) and SR-3Y1 (B) cells. Note that the labeling occurs as peripheral patches in 3Y1 but as randomly distributed dots in SR-3Y1 cells. (C and D) PY14 does not bind to 3Y1 (C) but labels SR-3Y1 cells in a similar pattern as sc-894 (D); note that some dots are apparently larger than others. (E–G) Most _src_ts NRK cells were negative with PY14 at 39°C (nonpermissive temperature) (E), but after the temperature was lowered to 33°C (permissive temperature), they showed intense labeling in peripheral patches at 2 h (F) and randomly distributed large dots at 8 h (G). Bar, 10 μm.

Figure 3

Figure 3

Freeze fracture immunoelectron microscopy. (A–C) _src_ts NRK cell cultured at the permissive temperature. (A and B) Labeling by PY14 occurred not only in caveolae of normal shape but also in IMP-free flat areas of various sizes and shapes (arrowheads). (C) Similar labeling was observed with sc-894. (D) In normal NRK cells, virtually all the labeling by sc-894 was seen in deep (single large arrows) and shallow (double small arrows) caveolae, and IMP-free flat areas were scarce. Bar, 100 nm (A, C, and D), 85 nm (B).

Figure 4

Figure 4

Quantitative comparison of sc-894–positive areas in freeze fracture immunoelectron microscopy. The diameter of the labeled areas was measured for shallow caveolae and IMP-free flat areas. They were significantly larger in _src_ts NRK cells than in normal NRK cells (Mann–Whitney U test, p < 0.0001).

Figure 5

Figure 5

Immunoelectron microscopy of ultrathin cryosections. Labeling of _src_ts NRK cells cultured at the permissive temperature with PY14. (A) Caveolae (double small arrows) as well as the flat plasma membrane (arrowheads) are decorated. Aggregated caveolae and/or vesicles found relatively near the cell surface (B–E) or in the deeper cytoplasm (F–H) are also labeled heavily. Some of them are as small as caveolae, but others are apparently larger than caveolae (single arrows). Bar, 100 nm.

Figure 6

Figure 6

Quantitative analysis of the distribution and size of sc-894–positive structures in immunoelectron microscopy of ultrathin cryosections. Vesicles and caveolae were classified into “cell surface” and “intracellular,” depending on whether a portion of them existed within 200 nm from the plasma membrane. The proportion of intracellular labeling in _src_ts NRK cells (29.6%) was significantly more frequent than in normal NRK cells (7.9%) (χ2 test for independence, p < 0.0001). PM, labeling in the seemingly flat portion of the plasma membrane most likely corresponds to both shallow caveolae and IMP-free flat areas seen in freeze replicas; its proportion in the cell surface labeling was also significantly larger in _src_ts NRK cells than in normal NRK cells (χ2 test for independence, p = 0.0087). Moreover, the diameter of the labeled structures was significantly larger in _src_ts NRK cells than in normal NRK cells (Mann–Whitney U test: cell surface, p < 0.0001; intracellular, p = 0.0011).

Figure 7

Figure 7

Biochemical characterization of tyrosine-phosphorylated caveolin-1. (A) Solubility in Triton X-100. The reaction of PY14 in SR-3Y1 as well as that of sc-894 in 3Y1 occurred intensely for the Triton X-100–insoluble material (I) but only weakly for the Triton X-100–soluble one (S). (B) Oligomer formation. Fractions of sucrose density gradient ultracentrifugation were subjected to SDS-PAGE, and caveolin-1 was detected by Western blotting using sc-894 and PY14. Both 3Y1 and SR-3Y1 showed a positive reaction between 150 and 400 kDa. Locations of molecular mass marker proteins are indicated by arrows. (C) Association with caveolin-2. Immunoprecipitates obtained with sc-894 from both 3Y1 and SR-3Y1 cells are positive for caveolin-2.

Figure 8

Figure 8

Western blots of mutant caveolin-1 whose tyrosine 14 was replaced with asparagine. (A) The lysates of transfected 3Y1 and SR-3Y1 cells were reacted with anti–c-myc tag antibody. The band of 3Y1 indicates the position of nonphosphorylated mutant caveolin-1α. At least one additional band is seen above the α isoform in the SR-3Y1 sample. (B) The lysate of transfected SR-3Y1 cells was precipitated with sc-894 and dephosphorylated by alkaline phosphatase before electrophoresis and Western blotting by anti–c-myc antibody. By dephosphorylation, the reaction with the band above the α isoform was abolished.

Figure 9

Figure 9

Normal NRK cells treated with vanadate. (A) Western blotting shows that the reaction with PY14 occurs as a single band at 22 kDa (at 2 h) and as three bands at 22, 23–24, and 25 kDa (at 8 h). (B and C) Immunofluorescence micrographs of normal NRK cells before (B) and after (C) the vanadate treatment. Caveolin-1 was localized as peripheral patches in untreated cells, but they disappeared after the treatment. Bar, 10 μm.

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