Nemir M, DeVouge M W, Mukherjee B B
Department of Biology, McGill University, Montreal, Quebec, Canada.
J Biol Chem. 1989 Oct 25;264(30):18202-8.
We have reported previously that the 69-kDa major phosphoprotein, secreted by normal rat kidney (NRK) cells, is osteopontin, a glycosylated bone matrix protein. Here we show that this 69-kDa osteopontin is secreted by NRK cells in both phosphorylated (pp69) and nonphosphorylated (np69) forms, with estimated isoelectric points of 3.8 and 4.5, respectively. Electrophoretic analysis of radioiodinated cell surface proteins immunoprecipitated with an anti-69-kDa osteopontin serum, demonstrates that the 69-kDa osteopontin is also present on the cell surface, but only its phosphorylated form (pp69) shows such cell surface association. Because osteopontin mediates cell adhesion and spreading, and contains an Arg-Gly-Asp-Ser cell-binding sequence, our observations strongly suggest that the cell surface localization of pp69 osteopontin is receptor-mediated, and the modification by phosphorylation may be crucial for its receptor binding activity. We also report that antisera directed against either fibronectin or 69-kDa osteopontin co-immunoprecipitate both np69 osteopontin and fibronectin as a heat-dissociable complex. In contrast, pp69 osteopontin does not co-precipitate with fibronectin. These observations demonstrate an interactive relationship between np69 and soluble fibronectin. Furthermore, compared to NRK cells, vanadyl sulfate-treated NRK cells which acquire a reversible transformed phenotype, including anchorage-independent growth, show increased levels of pp69 on the cell surface, concomitant with significantly decreased levels of pp69 and elevated levels of np69 in the conditioned media. The data presented here establish transformation sensitivity of NRK cell-secreted osteopontin with respect to its secretion and cell surface localization, and demonstrate that phosphorylated and nonphosphorylated forms of osteopontin have different physiological properties, which may regulate the functional roles of this extracellular matrix protein.
我们之前报道过，正常大鼠肾（NRK）细胞分泌的69-kDa主要磷蛋白是骨桥蛋白，一种糖基化的骨基质蛋白。在此我们表明，这种69-kDa骨桥蛋白以磷酸化（pp69）和非磷酸化（np69）两种形式由NRK细胞分泌，估计其等电点分别为3.8和4.5。用抗69-kDa骨桥蛋白血清免疫沉淀放射性碘化的细胞表面蛋白进行电泳分析，结果表明69-kDa骨桥蛋白也存在于细胞表面，但只有其磷酸化形式（pp69）显示出这种细胞表面结合。由于骨桥蛋白介导细胞黏附和铺展，且含有Arg-Gly-Asp-Ser细胞结合序列，我们的观察结果强烈表明，pp69骨桥蛋白的细胞表面定位是受体介导的，磷酸化修饰可能对其受体结合活性至关重要。我们还报道，针对纤连蛋白或69-kDa骨桥蛋白的抗血清可共同免疫沉淀np69骨桥蛋白和纤连蛋白，形成一种热解离复合物。相比之下，pp69骨桥蛋白不与纤连蛋白共沉淀。这些观察结果证明了np69与可溶性纤连蛋白之间的相互作用关系。此外，与NRK细胞相比，经硫酸氧钒处理的NRK细胞获得了可逆的转化表型，包括不依赖贴壁生长，其细胞表面pp69水平升高，同时条件培养基中pp69水平显著降低，np69水平升高。此处呈现的数据确定了NRK细胞分泌的骨桥蛋白在其分泌和细胞表面定位方面的转化敏感性，并表明骨桥蛋白的磷酸化和非磷酸化形式具有不同的生理特性，这可能调节这种细胞外基质蛋白的功能作用。
