In comparison with the well-studied conventional MAPKs, such as extracellular signal-regulated kinase (ERK)1/2, much less is known about the regulation and downstream targets of ERK3, an atypical MAPK. Whereas dual phosphorylation of the TXY activation motif in ERK1/2 is critical for their activation, it is unclear if phosphorylation of the single phospho-acceptor site (S189) in the SEG activation motif of ERK3 is critical for its kinase activity or function. In addition, little is known about the functions of the structurally distinct elongated C-terminus extension of ERK3. Recent studies have revealed important roles for ERK3 in promoting cancer progression. Of note, our previous study showed that ERK3 increases cancer cell invasiveness by phosphorylating the oncogenic protein steroid receptor co-activator 3 (SRC-3) and upregulating SRC-3-mediated matrix metalloproteinase gene expression. Here we aimed to elucidate the importance of the phospho-site S189 and the C-terminus in regulating ERK3 kinase activity and its functions in cancer cells. By performing in vitro kinase assays, we found that as compared to the wild type (WT) ERK3 protein, ERK3-S189A has remarkably reduced kinase activity towards auto-phosphorylation and phosphorylating SRC-3. In line with the critical role of S189 in ERK3 kinase activity, mutation of S189 to alanine greatly reduced ERK3’s activity in promoting the migration and invasion of lung cancer cells. To study the regulatory role of the C-terminus in ERK3 kinase activity, we expressed and purified the N-terminal ERK3 (aa 1-340) that retains the kinase domain but has deletion of the C-terminus. While it showed higher auto-phosphorylation ability than the full length ERK3 protein, ERK3 (aa 1-340) had greatly decreased ability to phosphorylate SRC-3. Interestingly, ERK3-S189A (aa 1-340) exhibited kinase activity equivalent to that of ERK3 (aa 1-340). These results suggest that on one hand, the C-terminus may play an auto-inhibitory role on ERK3 kinase activation through intramolecular interaction and that the phospho-S189 is required for relieving this auto-inhibition; on the other hand, C-terminus plays a critical role for ERK3 to recruit substrates, such as SRC-3. In agreement with its inability to phosphorylate SRC-3, ERK3 (aa 1-340) had decreased ability to stimulate migration and invasion of lung cancer cells as compared to full length ERK3. Taken together, our study unravels the importance of ERK3 S189 residue as well as the C-terminus for the intramolecular regulation of ERK3 kinase activity and invasiveness-promoting ability in cancer cells.

Citation Format: Lobna Elkhadragy, Hadel Alsaran, Weiwen Long. Novel insights into the regulation of ERK3’s kinase activity and its ability to promote cancer cell invasiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2376. doi:10.1158/1538-7445.AM2017-2376