Requirement for receptor-intrinsic tyrosine kinase activities during ligand-induced membrane ruffling of KB cells. Essential sites of src-related growth factor receptor kinases.
Requirement for receptor-intrinsic tyrosine kinase activities during ligand-induced membrane ruffling of KB cells. Essential sites of src-related growth factor receptor kinases.
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配体诱导 KB 细胞膜波动期间对受体内在酪氨酸激酶活性的需求。
DOI：
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发表时间：
1988
影响因子：
4.8
通讯作者：
Masato Kasuga
中科院分区：
文献类型：
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作者：
Tetsuro Izumi;Yoshiyuki SaekiS;Yasuo AkanumaQ;F. Takaku;Masato Kasuga
我们已经制备了针对人胰岛素、胰岛素样生长因子-I和表皮生长因子受体的位点特异性抗体，这些受体具有来自这些受体的cDNA预测氨基酸序列的化学合成肽。两类抗体产生对每个受体：一个对羧基末端和其他对激酶域含有序列同源的酪氨酸磷酸化位点的产品的src基因（pp 60 v-src）。这两类抗体特异性免疫沉淀适当的125 I-配体-受体复合物和[35 S]蛋氨酸标记的受体几乎相同的效力。抗体对激酶结构域抑制自磷酸化和酪氨酸激酶活性的相应受体在无细胞系统中，而抗体对羧基末端没有。将激酶抑制抗体显微注射到人表皮样癌KB细胞的细胞质中，阻断了相应配体诱导膜皱褶的能力。相反，这些抑制性抗体并不阻断非对应配体诱导相同反应的能力。此外，对照免疫球蛋白和针对羧基末端的抗体不阻断这种生物学应答。这些结果支持这些生长因子受体的酪氨酸特异性蛋白激酶活性在介导其生物学效应中的作用，并表明与pp 60 v-SRC的酪氨酰磷酸化位点同源的区域对于这些激酶活性在无细胞和完整细胞中都很重要系统。
We have prepared site-specific antibodies toward human insulin, insulin-like growth factor-I, and epidermal growth factor receptors with chemically synthesized peptides derived from the cDNA-predicted amino acid sequences of these receptors. Two classes of antibodies were produced toward each receptor: one toward the carboxyl termini and the other against the kinase domains containing sequences homologous to the tyrosyl phosphorylation site of the product of src gene (pp60v-src). Both classes of antibodies specifically immunoprecipitated the appropriate 125I-ligand-receptor complexes and [35S]methionine-labeled receptors with almost equal potencies. Antibodies toward the kinase domains inhibited both autophosphorylation and tyrosine kinase activity of the corresponding receptors in a cell-free system, whereas antibodies toward the carboxyl termini did not. Microinjection of the kinase-inhibitory antibodies into the cytoplasm of human epidermoid carcinoma KB cells blocked the ability of the corresponding ligand to induce membrane ruffling. In contrast, these inhibitory antibodies did not block the ability of noncorresponding ligands to induce the same response. Furthermore, control immunoglobulin and antibodies toward the carboxyl termini did not block this biological response. These results support a role for the tyrosine-specific protein kinase activities of these growth factor receptors in mediating their biological effects and suggest that the regions homologous to the tyrosyl phosphorylation site of pp60v-src are important for these kinase activities both in cell-free and intact cell systems.