Steven Berberich (Committee Member), Michael Markey (Committee Member), Weiwen Long (Advisor)
Master of Science (MS)
Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase (MAPK) family. Recent studies have shown that ERK3 is highly upregulated in multiple cancers, such as lung cancer and colon cancer. Importantly, ERK3 promotes cancer cell migration and invasion by phosphorylating steroid receptor activator 3 (SRC-3), hence upregulating pro-invasive matrix metalloproteinase genes. While the link between ERK3 and cancers has been recognized, little is known about ERK3 mutations in cancer progression. In this study, we have investigated ERK3 mutations on arginine 64 (arginine 64 mutated to cysteine or histidine, R64C or R64H) and leucine 290 (leucine 290 mutated to proline or valine, L290P or L290V) that are found in cancers of lung, large intestine and skin (COSMIC database). Interestingly, both R64 and L290 residues are located in the kinase domain of ERK3 and are conserved in all ERK isoforms (ERKs1-4). In order to characterize these mutations, we generated expression vectors coating plasmids of ERK3 and each ERK3 constructs containing point mutation, and then overexpressed them in HeLa cells and lung cancer cell lines with or without stable knockdown of endogenous ERK3. Notably, we found that all of these cancer-related mutations lead to reduction of ERK3 phosphorylation at S189 within the activation loop. To study the functional impact of these ERK3 mutations on cancer cell invasiveness, we expressed each of these mutants or the wild type (WT) ERK3 in HeLa cells and lung cancer cell lines with or without stable knockdown of endogenous ERK3 and performed trans-well migration and invasion assays. Interestingly, we found that in comparison with WT ERK3, both L290P and L290V mutations significantly increased ERK3’s ability in promoting cell migration and invasion, whereas R64C and R64H mutations resulted in a decrease in cell migration. Given that all these cancer-related mutations led to reduction of ERK3 phosphorylation at S189, these results suggest that S189 phosphorylation within the activation loop is not associated with ERK3’s function in promoting cancer cell motility. To elucidate the underlying mechanism by which ERK3 L290 mutants increase cancer cell invasiveness, we examined the kinase activity of WT and mutant ERK3 in vitro by assessing the phosphorylation of SRC-3, a substrate for ERK3. We found that ERK3 L290P and L290V mutants have similar kinase activity to that of WT ERK3. ERK3 protein is known to shuttle between the nucleus and the cytoplasm, which may alter its function. Interestingly, by immunofluorescent staining, we found that both L290P and L290V mutations greatly increased the cytoplasmic localization of ERK3 proteins, whereas WT ERK3 is mostly nuclear. In conclusion, this study demonstrates that cancer-related L290P and L290V mutations lead to increased ability of ERK3 to promote cancer cell migration and invasion, possibly through their increased cytoplasmic localization.
Department or Program
Department of Biochemistry and Molecular Biology
Year Degree Awarded
Copyright 2016, all rights reserved. My ETD will be available under the "Fair Use" terms of copyright law.