Fig. 4: Thr417/Thr421 of ERK3 acts as a degron motif to bind FBXW7.

a The ERK3 C34D-364–481 region interacted with FBXW7. The interaction of amino acids 364–481 of the ERK3 C34D with FBXW7 was determined by IP (HEK293T cell lysate, 300 μg/lane)/Western blotting. b Upper panels, Vector maps indicating the locations of putative degron motifs (PDMs) for FBXW7 in the ERK3 C34D. Bottom panels, Amino acid alignment of PDM1. c Ser386/Thr389 did not serve as an ERK3 degron for FBXW7. Ser386/Thr389 involvement in ERK3-FBXW7 binding was determined by IP (HEK293T cell lysate, 300 μg/lane)/Western blotting. d Amino acid alignments and the locations of PDM2-5 of ERK3 in the C34D. e Interaction between FBXW7 and ERK3-mtPDM2-5 mutants. The interaction of FBXW7 and each Myc-ERK3-FL-mtPDM was evaluated by IP (HEK293T cell lysate, 300 μg)/Western blotting. f Upper panels, ERK3-mtPDM2 attenuated protein degradation. The turnover of ERK3 in A549 cells stably expressing ERK3-wt and ERK3-mtPDM2 was determined by Western blotting (CHX; 10 μg/ml). Graphs, The ERK3 band intensities were normalized to the β-actin intensity. The data were obtained from three independent experiments. The values are shown with ±SEMs. Significance; **p < 0.05 vs. wt control by Student’s t test. g Myc-ERK3-FL-mtPDM2 reduced FBXW7-mediated ubiquitination. FBXW7-mediated ERK3-FL and ERK3-FL-mtPDM2 ubiquitination was compared by IP (HEK293T cell lysate, 300 μg/lane)/Western blotting. h ERK3-mtPDM2 decreased complex formation with FBXW7 ex vivo. Immunopurified Flag-FBXW7 proteins were subjected to complex formation assays with Myc-Ab-myc-ERK3-WT or Myc-Ab-myc-ERK3-mtPDM2 beads. The beads were washed, and ERK3 binding was determined by Western blotting. The input was used to compare the protein amounts for these reactions. i FBXW7 ubiquitinated ERK3 in vitro. The proteins prepared in (h) were subjected to confirmation of the ERK3 ubiquitination degree using an in vitro ubiquitination kit as described in the Materials and Methods. Ubiquitinated ERK3 protein levels were determined by Western blotting. a, c, e–g β-Actin was used as an internal control to ensure equal protein loading. WCL, whole-cell lysate.