Abstract 2391: Intramolecular regulation of ERK3's kinase activity and migration/invasion-promoting ability by the C-terminus

Extracellular signal-regulated kinase 3 (ERK3) is an atypical MAPK that has recently gained interest for its role in promoting cancer cell migration and invasion. However, little is known about the functions of ERK39s domains in its oncogenic signaling. In contrast to most of the conventional MAPKs, ERK3 has an elongated C-terminus extension beyond its N-terminal kinase domain. The first region of this extension (aa 341 - 481) is highly conserved between ERK3 and ERK4 and is called the C34 domain, whereas the remaining C-terminal tail (aa 482-721) is unique for ERK3. Here, we have investigated the roles of the C-terminal extension in ERK39s kinase activity and functions in cancer cells. We generated two C-terminal deletion mutants of ERK3: ΔCT1, which contains the kinase domain and the C34 domain, and ΔCT2 which comprises the kinase domain alone. By expressing and purifying ERK3, ΔCT1 and ΔCT2 from 293T cells and performing in vitro kinase assays, we found that ERK39s kinase activity towards the substrates myelin basic protein (MBP) and steroid receptor co-activator 3 (SRC-3) decreases gradually with the deletion of the C-terminal tail and the whole C-terminal extension. Next, we wanted to test whether the C-terminus extension was important for ERK39s interaction with SRC-3, an oncogenic protein recently demonstrated to be downstream of ERK3 signaling in cancer. By co-immunoprecipitation experiments, we found that neither the C34 domain nor the C-terminal tail is required for ERK39s interaction with SRC-3. Hence, we hypothesized that the C-terminus extension rather regulates ERK39s kinase activity via an intramolecular interaction with the kinase domain. Indeed, by co-expressing ERK39s kinase domain and the C-terminus extension, we revealed an interaction between the N- and C-termini of ERK3. To test the role of the C-terminal extension on ERK39s ability to promote cancer cell migration and invasion, we overexpressed ERK3, ΔCT1 and ΔCT2 in lung cancer cells and performed transwell cell migration and invasion assays. As compared to ERK3, both the deletion mutants similarly showed a significant reduction in migration and invasion. To test if the effect of the deletion mutants on migration/invasion was due to their decreased kinase activity, we also tested the ability of an ERK3 kinase-dead (KD) mutant (K49/50A) to promote cancer cell invasiveness. Interestingly, while the migration/invasion-promoting ability of ERK3-KD mutant was significantly lower than that of ERK3, it was significantly higher than that of the vector control and those of ΔCT1 and ΔCT2 mutants, suggesting a kinase-independent role for the C-terminal tail in ERK39s invasiveness-promoting ability. Taken together, our study unravels the importance of the C-terminus extension for ERK39s kinase activity and ability to promote migration and invasion. Citation Format: Lobna Elkhadragy, Hadel Alsaran, Weiwen Long. Intramolecular regulation of ERK39s kinase activity and migration/invasion-promoting ability by the C-terminus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2391.