Tyrosine-phosphorylated caveolin-1: immunolocalization and molecular characterization.
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.
Nomura
R
R
Department of Anatomy and Cell Biology, Gunma University School of Medicine, Maebashi 371-8511, Japan.
Fujimoto
T
T
D000818:Animals; D051242:Caveolin 1; D051243:Caveolin 2; D022461:Caveolins; D002460:Cell Line; D002461:Cell Line, Transformed; D005614:Freeze Fracturing; D006801:Humans; D007150:Immunohistochemistry; D007668:Kidney; D008565:Membrane Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection
C495540:CAV1 protein, human; C495537:Cav1 protein, rat; D051242:Caveolin 1; D051243:Caveolin 2; D022461:Caveolins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D019000:Phosphotyrosine