Development of Co-translational Phosphotyrosine Incorporation to Elucidate Mechanisms of Src Activation

 描述(由申请人提供)：癌症的定义是信号失控，使细胞能够维持增殖。磷酸酪氨酸信号在正常和异常信号网络中起着中心作用，大多数酪氨酸激酶与癌症有关。原癌基因Src的差异磷酸化在包括肺癌、乳腺癌和结肠癌在内的许多癌症中都被观察到，但技术挑战限制了对特定磷酸化残基的研究。酪氨酸、丝氨酸和苏氨酸残基的磷酸化分析一直受到限制，因为我们无法在体内或体外控制这些化学修饰，这是因为缺乏完全概括磷酸化残基化学的磷酸仿制。最近的工作表明，磷酸丝氨酸直接共翻译结合到丝裂原激活的蛋白激酶(MEK)中，从而能够在不共表达调节蛋白的情况下生产重组的磷酸化的MEK。在这个方案中，非标准氨基酸如磷酸丝氨酸通过成对的氨酰-tRNA合成酶和指向标签终止密码子的tRNA结合。磷酸酪氨酸不存在类似的体系。我们通过基因重组的大肠杆菌的产生进一步推进了这项工作，它是为掺入磷酸丝氨酸而优化的。我们假设，在重新编码的大肠杆菌中的氨酰-tRNA合成酶的进化将使磷酸酪氨酸的位点特异性掺入成为可能。我们将把这一技术进步应用于破译Src的调控，此前的检测受到非均一磷酸化蛋白的限制。我们假设，仅在其C-末端结构域(Tyr527)上磷酸化的Src将显示完全失活，而在其SH2结构域(Tyr213)上获得第二个磷酸化事件将导致功能恢复。具体目标：该提案包括两个具体目标。第一种是利用诱变技术设计一种优化的酪氨酰氨基酰-tRNA合成酶，用于合成结构类似于磷酸酪氨酸的氨基酸。然后，我们将使用代谢工程在重新编码的大肠杆菌中产生稳定的磷酸酪氨酸库。最后，我们将整合这些成分来产生含有磷酸酪氨酸的蛋白质，通过质谱学确认掺入。在第二个目标中，我们将应用这个共翻译的磷酸酪氨酸掺入系统来研究Src的生化特性。我们将在其C端抑制结构域上产生唯一的磷酸化的Src，并在体外表征一系列已知底物的激酶活性。为了研究SH2结构域的磷酸化对抑制的Src的影响，我们将在SH2和抑制结构域上表达双磷酸化的Src，假设SH2的磷酸化可以克服分子内的Src抑制。相关性：这项提案的总体目标是开发和应用基础技术来解码磷酸酪氨酸介导的原癌基因酪氨酸激酶Src的调节。我们试图评估磷酸化事件对Src的个体贡献，以揭示生理上相关的激活机制。

 DESCRIPTION (provided by applicant): Cancer is defined by dysregulated signaling, enabling cells to sustain proliferation. Phosphotyrosine signaling plays a central role in normal and aberrant signaling networks with a majority of tyrosine kinases implicated in cancer. Differential phosphorylation of the proto-oncogene Src has been observed in numerous cancers including lung, breast, and colon, but technological challenges have limited study of specific phosphorylated residues. Analysis of phosphorylation on tyrosine, serine, and threonine residues has been limited by our inability to control these chemical modifications in vivo or in vitro due to a lack of phosphomimetics that fully recapitulate the chemistry of phoshorylated residues. Recent work has demonstrated direct co-translational incorporation of phosphoserine into mitogen-activated protein kinase kinase (Mek), enabling production of recombinant phosphorylated MEK without co-expression of regulatory proteins. In this scheme, non-standard amino acids like phosphoserine are incorporated via a paired aminoacyl-tRNA synthetase and tRNA directed to a TAG stop codon. No analogous system exists for phosphotyrosine. We have furthered this work through the generation of a genomically recoded E. coli, that is optimized for the incorporation of phosphoserine. We hypothesize that aminoacyl-tRNA synthetase evolution in the recoded E. coli will enable site-specific incorporation of phosphotyrosine. We will apply this technological advance to deciphering regulation of Src, where previous assays have been limited by inhomogeneously phosphorylated protein. We hypothesize that Src phosphorylated exclusively on its C-terminus domain (Tyr527) will show complete inactivation, while the acquisition of a second phosphorylation event on its SH2 domain (Tyr213) will lead to recovery of function. Specific Aims: This proposal includes two specific aims. The first is to utilize mutagenesis to engineer an optimized tyrosyl aminoacyl-tRNA synthetase for amino acids structurally similar to phosphotyrosine. We will then use metabolic engineering to produce a stable phosphotyrosine pool in the recoded E. coli. Finally, we will integrate these components to generate phosphotyrosine-containing proteins, confirming incorporation by mass spectrometry. In the second aim, we will apply this co-translational phosphotyrosine incorporation system to the biochemical characterization of Src. We will produce Src phosphorylated uniquely on its C-terminal inhibitory domain and characterize kinase activity against a series of known substrates in vitro. To study the effects of SH2 domain phosphorylation on inhibited Src, we will express Src dually phosphorylated on the SH2 and inhibitory domains, hypothesizing that SH2 phosphorylation can overcome intramolecular Src inhibition. Relevance: The overall aim of this proposal is to develop and apply foundational technologies to decoding phosphotyrosine-mediated regulation of the proto-oncogene tyrosine kinase Src. We seek to assess the individual contributions of phosphorylation events on Src to reveal physiologically relevant mechanisms of activation.