Pandey Akhilesh, Blagoev Blagoy, Kratchmarova Irina, Fernandez Minerva, Nielsen Mogens, Kristiansen Troels Zakarias, Ohara Osamu, Podtelejnikov Alexandre V, Roche Serge, Lodish Harvey F, Mann Matthias
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA.
Oncogene. 2002 Nov 14;21(52):8029-36. doi: 10.1038/sj.onc.1205988.
We have used a proteomic approach using mass spectrometry to identify signaling molecules involved in receptor tyrosine kinase signaling pathways. Using affinity purification by anti-phosphotyrosine antibodies to enrich for tyrosine phosphorylated proteins, we have identified a novel signaling molecule in the epidermal growth factor receptor signaling pathway. This molecule, designated Odin, contains several ankyrin repeats, two sterile alpha motifs and a phosphotyrosine binding domain and is ubiquitously expressed. Using antibodies against endogenous Odin, we show that it undergoes tyrosine phosphorylation upon addition of growth factors such as EGF or PDGF but not by cytokines such as IL-3 or erythropoietin. Immunofluorescence experiments as well as Western blot analysis on subcellular fractions demonstrated that Odin is localized to the cytoplasm both before and after growth factor treatment. Deletion analysis showed that the phosphotyrosine binding domain of Odin is not required for its tyrosine phosphorylation. Overexpression of Odin, but not an unrelated adapter protein, Grb2, inhibited EGF-induced activation of c-Fos promoter. Microinjection of wild-type or a mutant version lacking the PTB domain into NIH3T3 fibroblasts inhibited PDGF-induced mitogenesis. Taken together, our results indicate that Odin may play a negative role in growth factor receptor signaling pathways.
我们采用了一种蛋白质组学方法，利用质谱来鉴定参与受体酪氨酸激酶信号通路的信号分子。通过抗磷酸酪氨酸抗体进行亲和纯化以富集酪氨酸磷酸化蛋白，我们在表皮生长因子受体信号通路中鉴定出一种新型信号分子。这种分子命名为奥丁（Odin），含有多个锚蛋白重复序列、两个无活性α基序和一个磷酸酪氨酸结合结构域，且广泛表达。使用针对内源性奥丁的抗体，我们发现加入表皮生长因子（EGF）或血小板衍生生长因子（PDGF）等生长因子后它会发生酪氨酸磷酸化，但加入白细胞介素-3（IL-3）或促红细胞生成素等细胞因子后则不会。免疫荧光实验以及对亚细胞组分的蛋白质印迹分析表明，在生长因子处理前后，奥丁均定位于细胞质中。缺失分析表明，奥丁的磷酸酪氨酸结合结构域对其酪氨酸磷酸化并非必需。过表达奥丁而非无关的衔接蛋白Grb2会抑制EGF诱导的c-Fos启动子激活。将野生型或缺失PTB结构域的突变体微注射到NIH3T3成纤维细胞中会抑制PDGF诱导的有丝分裂。综上所述，我们的结果表明奥丁可能在生长因子受体信号通路中发挥负性作用。
