Rubin R A, O'Keefe E J, Earp H S
Proc Natl Acad Sci U S A. 1982 Feb;79(3):776-80. doi: 10.1073/pnas.79.3.776.
Epidermal growth factor (EGF) stimulates membrane protein phosphorylation in a human cell line, A-431. The known hepatic mitogenic action of EGF and the reduction in EGF receptor number that occurs during liver regeneration led us to study whether EGF-dependent protein kinase activity was present in rat liver and whether its activity was altered after partial hepatectomy. Liver membranes, preincubated with or without EGF, were phosphorylated (0 degrees C, 15 sec) and subjected to NaDodSO4/polyacrylamide gel electrophoresis and autoradiography. In microsomal fractions, EGF at 5-2000 ng/ml produced a dose-related stimulation of 32P incorporation into a single 170,000-dalton protein (p170). In plasma membranes, a similar EGF-dependent phosphorylation was present and was substantially enriched relative to the microsomal fraction. Acid hydrolysis of labeled microsomal fraction followed by phosphoamino acid determination revealed that EGF stimulated 32P incorporation into phosphotyrosine residues. The EGF-dependent phosphorylation of p170 was compared in microsomal fractions isolated from rats 36 hr after partial hepatectomy or sham operation. In the absence of EGF, in vitro labeling of p170 was similar. EGF stimulated the labeling of p170 in both groups, but the response was clearly diminished after partial hepatectomy. In the presence of EGF, the labeling of p170 in microsomal fraction from regenerating livers was only 47 +/- 6% of that observed in membranes from sham-operated rats (p less than 0.005). Reduction of EGF-dependent phosphorylation during liver regeneration paralleled the loss of binding of 125I-labeled EGF. An increase in the EGF-independent phosphorylation of a 130,000-dalton protein was also observed after partial hepatectomy. The increase in the amount of this phosphoprotein was roughly equal to the loss of EGF-stimulated p170 phosphorylation. Several additional proteins showed increased phosphorylation in membranes from partially hepatectomized rats. These findings indicate that alterations in membrane tyrosine residue phosphorylation occur during regulated growth in vivo.
表皮生长因子（EGF）可刺激人细胞系A - 431中的膜蛋白磷酸化。已知EGF具有肝脏促有丝分裂作用，且肝脏再生过程中EGF受体数量会减少，这促使我们研究大鼠肝脏中是否存在依赖EGF的蛋白激酶活性，以及部分肝切除术后其活性是否会发生改变。将肝细胞膜在有无EGF的情况下预孵育，然后进行磷酸化反应（0℃，15秒），接着进行十二烷基硫酸钠/聚丙烯酰胺凝胶电泳和放射自显影。在微粒体部分，5 - 2000 ng/ml的EGF可产生与剂量相关的32P掺入到单一170,000道尔顿蛋白（p170）中的刺激作用。在质膜中，存在类似的依赖EGF的磷酸化作用，且相对于微粒体部分其含量显著富集。对标记的微粒体部分进行酸水解，然后测定磷酸氨基酸，结果显示EGF可刺激32P掺入到磷酸酪氨酸残基中。比较了部分肝切除或假手术后36小时大鼠分离出的微粒体部分中p170的依赖EGF磷酸化情况。在无EGF时，p170的体外标记相似。EGF刺激两组中p170的标记，但部分肝切除术后反应明显减弱。在有EGF存在的情况下，再生肝脏微粒体部分中p170的标记仅为假手术大鼠膜中观察到标记的47±6%（p＜0.005）。肝脏再生过程中依赖EGF的磷酸化减少与125I标记的EGF结合丧失平行。部分肝切除术后还观察到一种130,000道尔顿蛋白的非依赖EGF磷酸化增加。这种磷蛋白量的增加大致等于EGF刺激的p170磷酸化的丧失。部分肝切除大鼠的膜中还有几种其他蛋白显示磷酸化增加。这些发现表明，体内调节生长过程中膜酪氨酸残基磷酸化会发生改变。
