Choi H S, Bailey S C, Donahue K A, Vanasse G J, Sytkowski A J
Division of Hematology and Oncology, New England Deaconess Hospital, Boston, Massachusetts.
J Biol Chem. 1990 Mar 5;265(7):4143-8.
We have shown previously that purified human erythropoietin rapidly alters the phosphorylation of an integral erythroid membrane protein, pp43 (Choi, H.-S., Wojchowski, D. M., and Sytkowski, A. J. (1987) J. Biol. Chem. 262, 2933-2936). We have now purified pp43 to apparent homogeneity and have prepared antibodies to it. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer of membrane proteins to nitrocellulose, the antibodies identified pp43 and a series of higher molecular weight antigenically related proteins, up to 50 kDa, in erythropoietin-responsive Rauscher murine erythroleukemia cells and in normal murine erythroid cells. Examination of purified subcellular fractions confirmed the localization of pp43 and the related proteins to the plasma membrane. Phosphorylation with [gamma-32P]ATP demonstrated that, in contrast to pp43, these higher molecular weight proteins were not phosphorylated. Marked differences in both the abundance of pp43 and related proteins and the degree of erythropoietin-sensitive pp43 phosphorylation were found between the plasma membranes of Rauscher cells and those of "non-responsive" Friend murine erythroleukemia cells. In addition only trace amounts of a 50-kDa antigenically related protein and no phosphorylated pp43 were detected in the plasma membranes of two erythropoietin-insensitive human erythroid cells lines, K562 and HEL. The results suggest that the abundance and degree of phosphorylation of pp43 and the antigenically related proteins is strongly correlated with the erythropoietin responsiveness of the particular erythroid cell types.
我们之前已经表明，纯化的人促红细胞生成素能迅速改变一种完整的红细胞膜蛋白pp43的磷酸化状态（Choi, H.-S., Wojchowski, D. M., and Sytkowski, A. J. (1987) J. Biol. Chem. 262, 2933 - 2936）。我们现已将pp43纯化至表观均一，并制备了针对它的抗体。在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳以及将膜蛋白电转移至硝酸纤维素膜之后，这些抗体在促红细胞生成素反应性的劳舍尔小鼠红白血病细胞和正常小鼠红细胞中鉴定出了pp43以及一系列分子量更高的抗原相关蛋白，分子量高达50 kDa。对纯化的亚细胞组分的检测证实了pp43和相关蛋白定位于质膜。用[γ - 32P]ATP进行磷酸化表明，与pp43不同，这些分子量更高的蛋白未被磷酸化。在劳舍尔细胞的质膜与“无反应性”的弗瑞德小鼠红白血病细胞的质膜之间，发现了pp43及相关蛋白丰度以及促红细胞生成素敏感的pp43磷酸化程度的显著差异。此外，在两种促红细胞生成素不敏感的人红细胞系K562和HEL的质膜中，仅检测到微量的50 kDa抗原相关蛋白，未检测到磷酸化的pp43。结果表明，pp43及抗原相关蛋白的丰度和磷酸化程度与特定红细胞类型的促红细胞生成素反应性密切相关。
