Aoki T, Nomura R, Fujimoto T
Department of Anatomy and Cell Biology, Gunma University School of Medicine, 3-39-22 Showa-machi, Maebashi, 371-8511, Japan.
Exp Cell Res. 1999 Dec 15;253(2):629-36. doi: 10.1006/excr.1999.4652.
Caveolin-1, a scaffolding protein of caveolae, is known to be tyrosine-phosphorylated by Src kinases. Recently we generated a specific antibody to caveolin-1 phosphorylated at tyrosine-14 (PY14) (R. Nomura and T. Fujimoto, 1999, Mol. Biol. Cell 10, 975-986). In the present study, by applying PY14 to sections of normal rat tissues, we found that tyrosine phosphorylation of caveolin-1 occurred in limited locations, including the endothelium of the continuous capillaries and small venules. Cultured endothelial cells were not labeled by PY14 under a standard culture condition, but became positively labeled when exposed to oxidative stresses and/or tyrosine phosphatase inhibitors. The reaction was prohibited by pretreating the cells with herbimycin A or genistein. Vasoactive reagents or physical stimuli did not cause the phosphorylation. Concomitant with the tyrosine phosphorylation, the number of invaginated caveolae decreased drastically, and vesicles labeled intensely for caveolin-1 appeared in the cytoplasm; the average diameter of the vesicles was larger than that of caveolae. The result implies that tyrosine phosphorylation of caveolin-1 occurs at tyrosine-14 in the normal rat endothelium in vivo and may induce caveolar vesiculation and/or fusion.
小窝蛋白-1是小窝的一种支架蛋白，已知可被Src激酶酪氨酸磷酸化。最近我们制备了一种针对酪氨酸14位点磷酸化的小窝蛋白-1（PY14）的特异性抗体（R.野村和藤本T.，1999年，《分子生物学与细胞》10，975 - 986）。在本研究中，通过将PY14应用于正常大鼠组织切片，我们发现小窝蛋白-1的酪氨酸磷酸化发生在有限的部位，包括连续毛细血管和小静脉的内皮。在标准培养条件下，培养的内皮细胞未被PY14标记，但在暴露于氧化应激和/或酪氨酸磷酸酶抑制剂时会被阳性标记。用赫伯霉素A或染料木黄酮预处理细胞可抑制该反应。血管活性试剂或物理刺激不会导致磷酸化。伴随着酪氨酸磷酸化，内陷的小窝数量急剧减少，细胞质中出现了小窝蛋白-1标记强烈的囊泡；囊泡的平均直径大于小窝。结果表明，小窝蛋白-1的酪氨酸磷酸化在正常大鼠体内内皮的酪氨酸14位点发生，可能诱导小窝囊泡化和/或融合。
