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调，使细胞能够维持增殖。磷酸酪氨酸信号传导在正常和异常信号传导网络中起核心作用，其中大多数酪氨酸激酶与癌症有关。在包括肺癌、乳腺癌和结肠癌在内的许多癌症中已经观察到原癌基因Src的差异磷酸化，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，这是由于缺乏完全概括磷酸化残基化学的磷酸模拟物。最近的工作已经证明了磷酸丝氨酸直接共翻译掺入到促分裂原活化蛋白激酶激酶（Mek）中，使得能够在不共表达调节蛋白的情况下产生重组磷酸化MEK。在该方案中，非标准氨基酸如磷酸丝氨酸通过成对的氨酰基-tRNA合成酶和针对TAG终止密码子的tRNA掺入。磷酸酪氨酸没有类似的系统存在。我们通过基因组重新编码的E.大肠杆菌，这是优化的磷酸丝氨酸的掺入。我们推测，氨酰-tRNA合成酶的进化在重新编码的E。大肠杆菌将能够位点特异性地掺入磷酸酪氨酸。我们将应用这一技术进步来破译Src的调控，以前的检测方法受到不均匀磷酸化蛋白质的限制。我们假设Src仅在其C-末端结构域（Tyr 527）上磷酸化将显示完全失活，而在其SH 2结构域（Tyr 213）上获得第二次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。第一种是利用诱变来工程化优化的酪氨酰氨酰-tRNA合成酶，用于结构上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E.杆菌最后，我们将整合这些组分以产生含磷酸酪氨酸的蛋白质，通过质谱法确认掺入。在第二个目标中，我们将应用这种共翻译磷酸酪氨酸掺入系统的Src的生化特性。我们将产生Src磷酸化独特的C-末端抑制结构域和表征激酶活性对一系列已知的底物在体外。为了研究SH 2结构域磷酸化对抑制Src的影响，我们将表达在SH 2和抑制结构域上双重磷酸化的Src，假设SH 2磷酸化可以克服分子内Src抑制。相关性：本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Src上磷酸化事件的个体贡献，以揭示生理相关的激活机制。

 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.