Abstract
Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase (MAPK) family, members of which play essential roles in diverse cellular processes during carcinogenesis, including cell proliferation, differentiation, migration, and invasion. Unlike other MAPKs, ERK3 is an unstable protein with a short half-life. Although deubiquitination of ERK3 has been suggested to regulate the activity, its ubiquitination has not been described in the literature. Here, we report that FBXW7 (F-box and WD repeat domain-containing 7) acts as a ubiquitination E3 ligase for ERK3. Mammalian two-hybrid assay and immunoprecipitation results demonstrated that ERK3 is a novel binding partner of FBXW7. Furthermore, complex formation between ERK3 and the S-phase kinase-associated protein 1 (SKP1)-cullin 1-F-box protein (SCF) E3 ligase resulted in the destabilization of ERK3 via a ubiquitination-mediated proteasomal degradation pathway, and FBXW7 depletion restored ERK3 protein levels by inhibiting this ubiquitination. The interaction between ERK3 and FBXW7 was driven by binding between the C34D of ERK3, especially at Thr417 and Thr421, and the WD40 domain of FBXW7. A double mutant of ERK3 (Thr417 and Thr421 to alanine) abrogated FBXW7-mediated ubiquitination. Importantly, ERK3 knockdown inhibited the proliferation of lung cancer cells by regulating the G1/S-phase transition of the cell cycle. These results show that FBXW7-mediated ERK3 destabilization suppresses lung cancer cell proliferation in vitro.
Highlights
Extracellular signal-regulated kinase 3 (ERK3) family members are atypical mitogen-activated protein kinase (MAPK) and include ERK3, ERK4, ERK7, and Nemo-like kinase (NLK)[1]
ERK3 is a novel binding partner of FBXW7 To identify the binding partner of FBXW7, Gal4-FBXW7 in the pBIND vector was used as a bait to screen for novel binding sites using MAPK kinases, which were recombined into the pACT expression vector
Since our laboratory possessed many constructs that allowed a detailed investigation of the molecular mechanisms of FBXW725, we focused on the interaction between FBXW7 and ERK3
Summary
Extracellular signal-regulated kinase 3 (ERK3) family members are atypical MAPKs and include ERK3, ERK4, ERK7, and Nemo-like kinase (NLK)[1]. The biological activities of these members are mainly regulated by phosphorylation at the activation loop motif Thr-Xaa-Tyr that is catalyzed by a family of dual-specificity protein kinases[2] and by subcellular localization[3,4]. Glu-Gly (rather than Thr-Xaa-Tyr) is responsible for the functional characteristics of ERK3 that distinguish it from other MAPKs5. Turnover experiments using deletion and truncation mutants of ERK3 have indicated that neither kinase activity nor the C-terminal extension of ERK3 is required for its high turnover[5]. ERK3 harbors two degradation domains in its N-terminal lobe that are both necessary and sufficient to target ERK3 for ubiquitination and degradation via the proteasomal degradation pathway[5,6]. The domain(s) of ERK3 that interact with E3 ubiquitin ligase(s) and the identities of the responsible E3 ligase(s) have not been elucidated
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