Kyriakis J M, Avruch J
Medical Services and Diabetes Unit, Massachusetts General Hospital, Boston 02129.
Biochim Biophys Acta. 1990 Aug 13;1054(1):73-82. doi: 10.1016/0167-4889(90)90207-t.
The polypeptides which are phosphorylated at tyrosine residues in the murine muscle-like cell line, BC3H1, in response to insulin, epidermal growth factor (EGF) and fibroblast growth factor (FGF) were detected by immunoblotting with antiphosphotyrosine antibodies. Each ligand elicited the tyrosine phosphorylation of a characteristic, largely nonoverlapping set of polypeptide substrates, as classified by subunit Mr, pI, behavior on subcellular fractionation and adsorption to lectin (what germ agglutinin-Sepharose) columns. The dose-response curves for all stimulated tyrosine phosphorylations elicited by a single ligand were superimposable. By contrast, the temporal pattern of the responses elicited by each ligand differed in regard to speed of onset and persistence of the stimulation. Phosphorylation in response to insulin was maximal in a virtually instantaneous fashion and was fully maintained for at least 30 min. The response to EGF increased steadily over the initial 15-60 s to peak values, and fell progressively thereafter. FGF-stimulated phosphorylation was not detectable until 4 min after FGF addition, abruptly rose to maximal within the next 30 s, and declined subsequently. Exposure of BC3H1 cells to active phorbol esters prior to hormone addition altered the response to hormones in a differential fashion. FGF responses were abolished, EGF responses were partially inhibited, whereas the response to insulin was unaffected. Thus, acting on a single cell, insulin, EGF and FGF each mediate the tyrosine phosphorylation of a characteristic, largely nonoverlapping array of polypeptide substrates, indicating that each of these receptor tyrosine kinases exhibits a fundamentally distinct substrate specificity. Differences in the kinetic and regulatory properties of the response to each ligand are also apparent, and reflect the differing regulatory properties of each receptor tyrosine kinase acting in situ.
通过用抗磷酸酪氨酸抗体进行免疫印迹，检测了小鼠肌肉样细胞系BC3H1中响应胰岛素、表皮生长因子（EGF）和成纤维细胞生长因子（FGF）而在酪氨酸残基处发生磷酸化的多肽。根据亚基分子量、等电点、亚细胞分级行为以及对凝集素（麦胚凝集素-琼脂糖）柱的吸附情况分类，每种配体都会引发一组特征性的、基本不重叠的多肽底物的酪氨酸磷酸化。由单一配体引发的所有刺激酪氨酸磷酸化的剂量反应曲线是可叠加的。相比之下，每种配体引发的反应的时间模式在刺激的起始速度和持续时间方面有所不同。对胰岛素的磷酸化几乎以瞬时方式达到最大值，并至少持续维持30分钟。对EGF的反应在最初的15 - 60秒内稳步增加至峰值，此后逐渐下降。直到添加FGF后4分钟才检测到FGF刺激的磷酸化，在接下来的30秒内突然升至最大值，随后下降。在添加激素之前将BC3H1细胞暴露于活性佛波酯会以不同方式改变对激素的反应。FGF反应被消除，EGF反应被部分抑制，而对胰岛素的反应不受影响。因此，作用于单个细胞时，胰岛素、EGF和FGF各自介导一组特征性的、基本不重叠的多肽底物的酪氨酸磷酸化，表明这些受体酪氨酸激酶中的每一种都表现出根本不同的底物特异性。对每种配体反应的动力学和调节特性的差异也很明显，这反映了每种原位作用的受体酪氨酸激酶的不同调节特性。
