Oxidative activation of Src in smoke-induced epithelial mesenchymal transition

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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. PUBLIC HEALTH RELEVANCE: Src is an enzyme that is critical to the transformation of normal epithelial cells into other cell types that are associated with fibrosis and metastatic cancer. Cigarette smoking can cause this transformation and our results suggest that oxidizing and alkylating compounds in smoke are the cause of Src activation and cell transformation. We will investigate how Src is activated at a molecular level and whether this kind of activation also causes prolonged Src activity.