Grossmann Arndt, Benlasfer Nouhad, Birth Petra, Hegele Anna, Wachsmuth Franziska, Apelt Luise, Stelzl Ulrich
Otto-Warburg Laboratory, Max-Planck Institute for Molecular Genetics (MPIMG), Berlin, Germany.
Otto-Warburg Laboratory, Max-Planck Institute for Molecular Genetics (MPIMG), Berlin, Germany
Mol Syst Biol. 2015 Mar 26;11(3):794. doi: 10.15252/msb.20145968.
Post-translational protein modifications, such as tyrosine phosphorylation, regulate protein-protein interactions (PPIs) critical for signal processing and cellular phenotypes. We extended an established yeast two-hybrid system employing human protein kinases for the analyses of phospho-tyrosine (pY)-dependent PPIs in a direct experimental, large-scale approach. We identified 292 mostly novel pY-dependent PPIs which showed high specificity with respect to kinases and interacting proteins and validated a large fraction in co-immunoprecipitation experiments from mammalian cells. About one-sixth of the interactions are mediated by known linear sequence binding motifs while the majority of pY-PPIs are mediated by other linear epitopes or governed by alternative recognition modes. Network analysis revealed that pY-mediated recognition events are tied to a highly connected protein module dedicated to signaling and cell growth pathways related to cancer. Using binding assays, protein complementation and phenotypic readouts to characterize the pY-dependent interactions of TSPAN2 (tetraspanin 2) and GRB2 or PIK3R3 (p55γ), we exemplarily provide evidence that the two pY-dependent PPIs dictate cellular cancer phenotypes.
翻译后蛋白质修饰，如酪氨酸磷酸化，可调节对信号处理和细胞表型至关重要的蛋白质-蛋白质相互作用（PPI）。我们扩展了一个已建立的酵母双杂交系统，该系统使用人类蛋白激酶，以直接实验的大规模方法分析磷酸酪氨酸（pY）依赖性PPI。我们鉴定出292种大多为新型的pY依赖性PPI，它们在激酶和相互作用蛋白方面表现出高度特异性，并在来自哺乳动物细胞的共免疫沉淀实验中验证了大部分。约六分之一的相互作用由已知的线性序列结合基序介导，而大多数pY-PPI由其他线性表位介导或受替代识别模式支配。网络分析表明，pY介导的识别事件与一个高度连接的蛋白质模块相关，该模块致力于与癌症相关的信号传导和细胞生长途径。通过结合测定、蛋白质互补和表型读数来表征TSPAN2（四跨膜蛋白2）与GRB2或PIK3R3（p55γ）的pY依赖性相互作用，我们示例性地提供了证据，证明这两种pY依赖性PPI决定细胞癌表型。
