Oxidative activation of Src in smoke-induced epithelial mesenchymal transition

描述（由申请人提供）：我们将研究香烟烟雾中的氧化剂和其他亲电物质如何激活上皮-间充质转化（EMT）之前的Src激酶。我们的初步研究结果表明，香烟烟雾提取物（CSE）激活Src和EMT的原因，这两个抑制N-乙酰半胱氨酸（NAC），抗氧化剂和谷胱甘肽前体，或PP 2，Src抑制剂在人非小细胞肺癌细胞系，H358。受体介导的Src激活似乎需要Tyr 529去磷酸化，然而，我们观察到Src激活发生没有Tyr 529去磷酸化，这可能会影响活动的持续时间。 EMT与肺纤维化和癌症转移有关。它可以 通过激活多种细胞内信号通路，由不同的刺激触发。吸烟与特发性肺纤维化和肺癌有关。CSE含有数千种化合物，其中许多可能导致EMT。因此，找出EMT启动的机制似乎是一项艰巨的任务。幸运的是，至少有一个共同的因素，大多数生理激动剂和CSE的无数成分似乎都是通过这个因素起作用的。这是NAC抑制EMT所提示的氧化还原机制的共同参与。此外，Src，这是激活的大多数，如果不是所有的EMT诱导剂可能是一个共同的焦点，这种氧化还原调节。Src激活刺激受体结合的生长因子似乎需要脱磷酸化的Tyr 529，然后在Tyr 418产生的活性酶的自磷酸化。但是CSE如何激活Src在很大程度上仍然没有解决。我们的初步结果支持CSE通过氧化或烷基化而没有Try 529去磷酸化的Src活化机制。 目的1是测试CSE和两个CSE组分，过氧化氢（H2 O2）和丙烯醛，通过氧化或烷基化调节半胱氨酸残基启动Src激活的假设。为了测试这一点，将构建带his标签的Src质粒并在H358和HBE 1细胞中表达。在确认Src活化后，将分离Src蛋白并使用质谱法分析半胱氨酸氧化和/或烷基化。预期低剂量暴露于激活Src的CSE将 修饰Src上的九个半胱氨酸残基中的一些，而其他的将保持还原形式（-SH）。目标2然后将鉴定和确认单个半胱氨酸残基，其氧化还原修饰对于氧化Src活化至关重要。在目标1中鉴定的半胱氨酸残基将单独或成对突变为亮氨酸以模拟烷基化，并且突变的质粒将在细胞中表达。然后通过测量Tyr 418处的Src磷酸化和Src活性来确定CSE对Src的活化。目的3是确定是否Src激活CSE，H2 O2或丙烯醛是延长其激活相比，通过经典途径。我们将研究是否Src激活CSE，H2 O2或丙烯醛持续时间比Src激活的激动剂，通过Tyr 529去磷酸化。

DESCRIPTION (provided by applicant): We will investigate how oxidants and other electrophiles in cigarette smoke activate Src kinase, which precedes epithelial-mesenchymal transition (EMT). Our preliminary results show that cigarette smoke extract (CSE) activates Src and causes EMT and that both are inhibited by N-acetylcysteine (NAC), an antioxidant and glutathione precursor, or by PP2, a Src inhibitor in the human non-small cell lung carcinoma cell line, H358. Receptor-mediated Src activation appears to require Tyr529 dephosphorylation; however, we observed that Src activation occurs without Tyr529 dephosphorylation, which may affect duration of activity. EMT is implicated in both lung fibrosis and cancer metastasis. It can be triggered by diverse stimuli through activation of multiple intracellular signaling pathways. Cigarette smoking is implicated in idiopathic pulmonary fibrosis and lung cancer. CSE contains thousands of compounds, many of which could be responsible for inducing EMT. Thus, sorting out the mechanism for EMT initiation would seem a monumental task. Fortunately, there is at least one common factor through which most physiological agonists and the myriad components of CSE, appear to act. This is the common involvement of redox mechanisms suggested by NAC inhibition of EMT. Also, Src, which is activated by most if not all EMT inducers may be a common focal point for this redox regulation. Src activation stimulated by receptor binding of growth factors appears to require dephosphorylation of Tyr529 followed by autophosphorylation at Tyr418 that produces the active enzyme. But how CSE activates Src remains largely unresolved. Our preliminary results support a mechanism for Src activation by CSE through oxidation or alkylation without Try529 dephosphorylation. Aim 1 is to test the hypothesis that CSE and two CSE components, hydrogen peroxide (H2O2) and acrolein, initiate Src activation through oxidation or alkylation of regulatory cysteine residues. To test this, a his-tagged- Src plasmid will be constructed and expressed in H358 and HBE1 cells. After confirming Src activation, Src protein will be isolated and analyzed with mass spectrometry for cysteine oxidation and/or alkylation. It is expected that a low dose exposure to CSE that activates Src will modify some of the nine cysteine residues on Src while the others will remain in the reduced form (-SH). Aim 2 will then identify and confirm individual cysteine residue whose redox modification is critical for oxidative Src activation. Cysteine residues identified in Aim 1 will b mutated individually or in pairs to leucine to mimic alkylation and the mutated plasmid will be expressed in cells. Src activation by CSE will then be determined by measuring Src phosphorylation at Tyr418 and Src activity. Aim 3 is to determine if Src activation by CSE, H2O2 or acrolein is prolonged in comparison with its activation through the classical pathway. We will examine whether Src activation by CSE, H2O2 or acrolein lasts longer than Src activated by agonists that act through Tyr529 dephosphorylation.