FBXW7-mediated ERK3 degradation regulates the proliferation of lung cancer cells.

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.

Kaempferol sensitizes cell proliferation inhibition in oxaliplatin-resistant colon cancer cells.

Resolution to chemoresistance is a major challenge in patients with advanced-stage malignancies. Thus, identification of action points and elucidation of molecular mechanisms for chemoresist human cancer are necessary to overcome this challenge. In this study, we provide important evidence that kaempferol targeting RSKs might be a strategy to reduce the oxaliplatin-resistant colon cancer cells. We found that MAPK and PI3K-AKT signaling were increased in oxaliplatin (Ox)-resistant HCT116 (HCT116-OxR) cells compared to Ox-sensitive HCT116 (HCT116-OxS) cells. Comparison of cell sensitivities using SP600125 (JNK inhibitor), SB206580 (p38 kinase inhibitor), or MK-2206 (AKT inhibitor) revealed that cell proliferation inhibition was strongly observed in HT29 cells compared to that in HCT116 cells in both OxS and OxR cells. Interestingly, SP600125, SB206580, and MK-2206 treatment showed higher cell proliferation inhibition in OxS cells than that in OxR cells in both HCT116 and HT29 cells, except following treatments with 10 µM of SP600125, and 30 µM of SB206580. In comparison to magnolin and aschantin, kaempferol showed the strongest inhibitory effect on cell proliferation in both HCT116 and HT29 cells. Importantly, HCT116- and HT29-OxR cells showed higher sensitivities to cell proliferation inhibition than those of HCT116- and HT29-OxS cells, resulting in the accumulation of cells at the G2/M-phases of the cell cycle. Finally, we showed that AP-1 transactivation activity was markedly decreased by kaempferol in HCT116- and HT29-OxR cells compared to the activity levels in HCT116- and HT29-OxS cells. Taken together, the results demonstrate that kaempferol-mediated AP-1 inhibition might be an important signaling mechanism to resolve the chemoresistance of Ox-resistant colon cancer cells.

F-box Protein ßTrCP1 Is a Substrate of Extracellular Signal-regulated Kinase 2.

F-box proteins, consisting of 69 members which are organized into the three subclasses FBXW, FBXL, and FBXO, are the substrate specific recognition subunits of the SKP1-Cullin 1-F-box protein E3 ligase complex. Although ßTrCP 1 and 2, members of the FBXW subfamily, are known to regulate some protein stability, molecular mechanisms by which these proteins can recognize proper substrates are unknown. In this study, it was found that ßTrCP1 showed strong interaction with members of mitogen-activated protein kinases. Although extracellular signal-regulated kinase (ERK) 3, p38ß, and p38δ showed weak interactions, ERK2 specifically interacted with ßTrCP1 as assessed by immunoprecipitation. In interaction domain determination experiments, we found that ERK2 interacted with two independent ERK docking sites located in the F-box domain and linker domain, but not the WD40 domain, of ßTrCP1. Notably, mutations of ßTrCP1 at the ERK docking sites abolished the interaction with ERK2. ßTrCP1 underwent phosphorylation by EGF stimulation, while the presence of the mitogen-activated protein kinase kinases inhibitor U0126, genetic silencing by sh-ERK2, and mutation of the ERK docking site of ßTrCP1 inhibited phosphorylation. This inhibition of ßTrCP1 phosphorylation resulted in a shortened half-life and low protein levels. These results suggest that ERK2-mediated ßTrCP1 phosphorylation may induce the destabilization of ßTrCP1.

Stat2 stability regulation: an intersection between immunity and carcinogenesis.

Signal transducer and activator of transcription (STAT2) is a member of the STAT family that plays an essential role in immune responses to extracellular and intracellular stimuli, including inflammatory reactions, invasion of foreign materials, and cancer initiation. Although the majority of STAT2 studies in the last few decades have focused on interferon (IFN)-α/ß (IFNα/ß) signaling pathway-mediated host defense against viral infections, recent studies have revealed that STAT2 also plays an important role in human cancer development. Notably, strategic research on STAT2 function has provided evidence that transient regulatory activity by homo- or heterodimerization induces its nuclear localization where it to forms a ternary IFN-stimulated gene factor 3 (ISGF3) complex, which is composed of STAT1 and/or STAT2 and IFN regulatory factor 9 (IEF9). The molecular mechanisms of ISGF3-mediated ISG gene expression provide the basic foundation for the regulation of STAT2 protein activity but not protein quality control. Recently, previously unknown molecular mechanisms of STAT2-mediated cell proliferation via STAT2 protein quality control were elucidated. In this review, we briefly summarize the role of STAT2 in immune responses and carcinogenesis with respect to the molecular mechanisms of STAT2 stability regulation via the proteasomal degradation pathway.