Courtneidge S, Ralston R, Alitalo K, Bishop J M
Mol Cell Biol. 1983 Mar;3(3):340-50. doi: 10.1128/mcb.3.3.340-350.1983.
A 36,000-dalton cellular protein (p36) has been identified previously as an abundant substrate for phosphorylation by tyrosine-specific protein kinases. Since several of the responsible kinases are associated with the plasma membrane, we explored the subcellular distribution of p36. Biochemical fractionations located p36 on the plasma membrane of both normal and retrovirus-transformed cells. Approximately half of the p36 was bound to the membrane with the affinity of a peripheral membrane protein; the remainder was even more tightly bound. The distribution of p36 among subcellular fractions and its affinity for the plasma membrane were not affected by tyrosine phosphorylation. We determined that p36 is synthesized in the soluble compartment of the cell and then moves rapidly to the membranous compartment. Immunofluorescence microscopy with antibodies directed against p36 revealed two distinct distributions of the antigen: (i) a sharply demarcated crenelated pattern within or immediately beneath the plasma membrane, which we presume to be a correlary of the distribution of p36 in biochemical fractionations; and (ii) diffuse staining in a cytoplasmic location that could not be attributed to a specific feature of cytoarchitecture and could not be easily reconciled with the results of biochemical fractionations. Efforts to detect the secretion of p36 were unsuccessful. No evidence was obtained for exposure of p36 on the cell surface, and no changes in localization were observed as a consequence of neoplastic transformation. During the course of this study, we had the opportunity to pursue a previous report that p36 is a component of the enzyme malate dehydrogenase (Rubsamen et al., Proc. Natl. Acad. Sci. U.S.A. 79:228-232, 1982). We were unable to substantiate this claim. We conclude that at least a substantial fraction of p36 is located on the cytoplasmic aspect of the plasma membrane, where it could be well situated to serve as a substrate for several identified tyrosine-specific kinases. But the function of p36 and its role, if any, in neoplastic transformation of cells by retroviruses possessing tyrosine-specific kinases remain enigmatic.
一种36000道尔顿的细胞蛋白（p36）先前已被鉴定为酪氨酸特异性蛋白激酶磷酸化的丰富底物。由于几种相关激酶与质膜相关，我们探究了p36的亚细胞分布。生化分级分离将p36定位在正常细胞和逆转录病毒转化细胞的质膜上。大约一半的p36以外周膜蛋白的亲和力与膜结合；其余部分结合得更紧密。p36在亚细胞组分中的分布及其对质膜的亲和力不受酪氨酸磷酸化的影响。我们确定p36在细胞的可溶性区室中合成，然后迅速移动到膜性区室。用针对p36的抗体进行免疫荧光显微镜检查发现抗原的两种不同分布：（i）在质膜内或紧挨着质膜下方的清晰界定的锯齿状模式，我们推测这与生化分级分离中p36的分布相关；（ii）在细胞质位置的弥漫性染色，这不能归因于细胞结构的特定特征，并且难以与生化分级分离的结果相协调。检测p36分泌的努力未成功。没有获得p36暴露在细胞表面的证据，并且未观察到由于肿瘤转化导致的定位变化。在本研究过程中，我们有机会追踪之前的一份报告，该报告称p36是苹果酸脱氢酶的一个组分（鲁布萨门等人，《美国国家科学院院刊》79:228 - 232, 1982）。我们无法证实这一说法。我们得出结论，至少相当一部分p36位于质膜的细胞质一侧，在那里它很适合作为几种已鉴定的酪氨酸特异性激酶的底物。但是p36的功能及其在具有酪氨酸特异性激酶的逆转录病毒对细胞的肿瘤转化中（如果有）所起的作用仍然是个谜。
