Julian Cambronero (Committee Member), Weiwen Long (Advisor), Hongmei Ren (Committee Member)
Master of Science (MS)
Extracellular signal-regulated kinase 3 (ERK3) is an atypical protein kinase of the mitogen-activated protein kinase (MAPK) family. In comparison to well-investigated ERK1/2 (classical) MAPKs, much less has been discovered about ERK3 signaling and its cellular functions. Recent studies have shown that ERK3 is overexpressed in various types of cancers such as lung cancer and breast cancer and that ERK3 promotes cancer cell migration and invasion. How ERK3 regulates cancer cell motility and invasiveness, however, is still largely unknown. RhoGTPases, including Rho, Cdc42 and Rac1, play critical roles in regulating cell motility and invasiveness through activating downstream effectors such as p21-activated kinases (PAKs). Rho GTPase-GDP dissociation inhibitor 1 (RhoGDI1) inhibits RhoGTPases’ activity by binding to GDP-bound GTPases and preventing their activation. Cdc42 and Rac1 are well-known activators of group I PAKs, including PAK1, PAK2 and PAK3. They activate group I PAKs through binding to the latter’s auto-inhibitory domains and exposing the auto-phosphorylation residues. Interestingly, group I PAKs were shown to interact with and phosphorylate ERK3 at S189 of the SEG activation motif. In addition, proteomic analyses of ERK3-interacting proteins by Yeast-Two-Hybrid screening (Y2H) and Immunoprecipitation-Mass spectrometry (IP-MS) revealed that ERK3 interacts with RhoGDI1. These findings raise an intriguing hypothesis that ERK3 regulates RhoGDI1/RhoGTPases/PAKs signaling pathway, thereby promoting cancer cell migration. To test this hypothesis, first, we confirmed the interactions of ERK3 with RhoGDI1, PAKs and Rho GTPases by co-immunoprecipitation and co-immunofluorescent staining. Interestingly, co-immunofluorescence data showed that ERK3 co-localizes with RhoGDI1, PAK1 and Cdc42 at the leading edge of the plasma membrane, suggesting that ERK3 might regulate PAKs’ activation. Indeed, we found that in both A549 lung cancer cell and MDA-MB231 breast cancer cells, ERK3 greatly upregulates group I PAKs’ activating phosphorylations at Ser144/Ser141 and Thr423/Thr402, but has no effect on PAKs’ expression levels. We then sought to elucidate how ERK3 promotes PAKs’ phosphorylations (activation). By performing Cdc42/Rac1 activation assay we found that ERK3 upregulates the cellular levels of the active forms of Cdc42 and Rac1 (GTP-Cdc42 and GTP-Rac1), which accounts for the increase of PAKs’ activity. As RhoGDI1 inhibits the activation of Cdc42 and Rac1 via direct binding, we investigated the effect of ERK3 on their interactions. While exogenous overexpression of ERK3 did not show clear effect, expression of ERK3 kinase dead mutant (a potential dominant negative form of ERK3) greatly increased the interaction of RhoGDI1 with Rac, suggesting a potential role of ERK3 in facilitating the dissociation of RhoGDI1 off Rac. Mechanistically, our preliminary data of in vitro kinase assay suggests that ERK3 might phosphorylates RhoGDI1, which may decrease RhoGDI1’s binding with Rac1/Cdc42. Lastly, we found that ERK3-induced cancer cell migration was almost abolished by the inhibitors of group I PAKs. In conclusion, these results clearly suggest that ERK3 promotes cancer cell migration through upregulating group I PAKs signaling.
Department or Program
Department of Biochemistry and Molecular Biology
Year Degree Awarded
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