Liu L, Damen J E, Cutler R L, Krystal G
Terry Fox Laboratory, University of British Columbia, Vancouver, Canada.
Mol Cell Biol. 1994 Oct;14(10):6926-35. doi: 10.1128/mcb.14.10.6926-6935.1994.
We recently reported that interleukin-3, Steel factor, and erythropoietin all induce the tyrosine phosphorylation of Shc and its association with Grb2 in hemopoietic cell lines. We have now further characterized the proteins that become associated with Shc following stimulation with these cytokines and found that, in response to all three, the tyrosine-phosphorylated form of Shc binds to common 145- and 52-kDa proteins which also become tyrosine phosphorylated in response to these growth factors. The 145-kDa protein, which appears, from antiphosphotyrosine blots of two-dimensional O'Farrell gels, to exist in four different phosphorylation states following cytokine stimulation (with isoelectric points ranging from 7.2 to 7.8), does not appear to be immunologically related to the beta subunit of the interleukin-3 receptor, c-Kit, BCR, ABL, JAK1, JAK2, Sos1, eps15, or insulin receptor substrate 1 protein. Silver-stained sodium dodecyl sulfate gels indicate that the association of the 145-kDa protein with Shc occurs only after cytokine stimulation and that it can bind to the tyrosine-phosphorylated form of Shc in its non-tyrosine-phosphorylated state. The latter finding, in conjunction with the observations that p145 does not bind, in vitro, to the Src homology 2 (SH2) domain of Shc, that it is not present in anti-Grb2 immunoprecipitates, and that a phosphopeptide which blocks the binding of Shc to the SH2 domain of Grb2 also blocks the binding of Shc to p145, suggests that p145 contains an SH2 domain and competes with Grb2 for the same tyrosine-phosphorylated site on Shc. This implicates p145 as a potential regulator of Ras activity and, perhaps, of other as yet unidentified functions of Shc.
我们最近报道，白细胞介素-3、Steel因子和促红细胞生成素均可在造血细胞系中诱导Shc的酪氨酸磷酸化及其与Grb2的结合。我们现在进一步鉴定了这些细胞因子刺激后与Shc结合的蛋白质，发现响应这三种细胞因子时，酪氨酸磷酸化形式的Shc会与常见的145 kDa和52 kDa蛋白质结合，这些蛋白质在响应这些生长因子时也会发生酪氨酸磷酸化。从二维O'Farrell凝胶的抗磷酸酪氨酸印迹来看，145 kDa蛋白质在细胞因子刺激后似乎存在四种不同的磷酸化状态（等电点范围为7.2至7.8），它似乎与白细胞介素-3受体的β亚基、c-Kit、BCR、ABL、JAK1、JAK2、Sos1、eps15或胰岛素受体底物1蛋白无免疫相关性。十二烷基硫酸钠银染凝胶表明，145 kDa蛋白质与Shc的结合仅在细胞因子刺激后发生，并且它可以在非酪氨酸磷酸化状态下与酪氨酸磷酸化形式的Shc结合。后一发现，结合p145在体外不与Shc的Src同源2（SH2）结构域结合、它不存在于抗Grb2免疫沉淀物中以及一个阻断Shc与Grb2的SH2结构域结合的磷酸肽也阻断Shc与p145结合的观察结果，表明p145含有一个SH2结构域，并与Grb2竞争Shc上相同的酪氨酸磷酸化位点。这意味着p145可能是Ras活性的潜在调节因子，也许也是Shc其他尚未确定功能的调节因子。
