PROJECT SUMMARY/ABSTRACT From a human physiological perspective, heavy metals (HM) are divided into two groups: essential (eHM) and toxic (tHM). eHM and tHM overexposure are both linked to numerous pathophysiological conditions; however, the signal transduction pathways and genomic activities stimulated by eHM dyshomeostasis and tHM exposure have yet to be adequately established. For example, the mammalian Cu-sensor protein responsible for relaying changes in extracellular Cu levels into signaling responses has not been identified. The goals of this application are to define (i) eHM- and tHM-stimulated and -regulatory signaling networks (including resultant genomic activities), and (ii) the underlying molecular biological, biochemical, and biophysical mechanisms. Through the work in this application, I have characterized the signaling and genomic responses induced by Cu exposure and identified a mechanism of Cu-stimulated epidermal growth factor receptor (EGFR, a cell surface receptor tyrosine kinase (RTK)) signal transduction along the MAPK/ERK pathway, thereby identifying EGFR as a candidate Cu- sensor protein. Biochemical experiments are underway investigating the EGFR-Cu binding site and geometry. Relatedly, through application of newly-developed next generation sequencing (NGS), molecular biological, and transcriptomic (in vitro and in vivo) techniques, I discovered a novel mechanism by which intracellular Cu levels are regulated. Elevated extracellular Cu levels stimulate EGFR/MAPK/ERK activation, which induces expression of the transcription factor (TF) EGR1 and EGR1-corepressor NAB1/2; the EGR1-NAB1/2 complex subsequently represses transcription of the mammalian Cu-importer, CTR1. EGR1 ChIP-seq experiments are underway for further corroboration. This work will pair with experiments assessing synergism of an inhibitor of Cu-chaperones and MAPK/ERK signaling (to increase CTR1 levels) with a platinum anticancer drug (cisplatin, imported via CTR1), as well as genomic insights into the TF activities of EGR1 in EGFR-driven lung cancer (a role that is currently unknown). My research establishing the Cu-regulatory network lays the foundation for my planned independent career (i) evaluating eHM- and tHM-regulatory and -responsive mechanisms and (ii) characterizing disruption of native signaling by tHM. In my independent career, I will apply phosphorylation arrays and NGS techniques to cells stimulated with non-Cu eHM and high-priority environmental pollutant tHM, with emphasis on the effects of tHM exposure in disrupting the eHM-regulatory and -responsive signal transduction networks. In summary, my identification of EGFR as a conduit for relaying elevated extracellular Cu levels into genomic activities via the MAPK/ERK pathway has linked a major signal transduction network (implicated in cancer pathophysiology, for example) to eHM-regulation, and thereby opens new avenues of understanding HM- regulatory networks in general. More broadly, the comp...