Fig 1: DDVF-mediated modulation of ERK signaling in TMEV-infected cells.A. Immunoblot analysis of L929 cells infected with TMEV derivatives expressing either the wild-type L protein (L-DDVF) or its mutants (L-DDVA and L-M60V) compared to uninfected controls (n = 4). Phospho-ERK (pERK) and total ERK levels were assessed. Viral 3D polymerase detection served as an infection control. B. Quantification of the pERK/ERK ratio from the immunoblots shown in panel A (n = 4). C. Proposed model of RSK-mediated ERK-MAPK regulation by L during TMEV infection. The DDVF motif of L competes with host ERK-MAPK proteins containing DDVF motifs, thereby inhibiting DDVF-mediated negative feedback on the ERK-MAPK pathway. In the absence of a functional DDVF motif in L (mutant forms, shown in green), the virus does not dysregulate the MAPK pathway. Proteins in the MAPK pathway with DDVF motifs (e.g., SPRED2, GAB3, CNKSR2, FGFR1, SOS1, etc.) are shown in purple. Activated RSK and ERK kinases are represented by red shading.
Fig 2: The conserved DDVF-interacting region contributes to an RSK-mediated negative feedback of the ERK-MAPK pathway in HeLa cells.A. Several proteins from the RAS-MAPK pathway bear a DDVF-like motif (purple) [31]. In the case of SPRED2, FGFR1 and SOS1, indicated RSK-dependent negative feedbacks were described in [4–6,28]. Black and gray arrows distinguish previously reported RSK interactors from newly identified ones respectively. Black arrows: The SPRED2-RSK interaction was recently demonstrated by Lopez et al. [6]. Nadratowska-Wesolowska et al. [4] described the FGFR1-RSK2 interaction, though the role of the DDVF motif was not investigated. Póti et al. [30] recently reported the SOS1-RSK interaction. To our knowledge, the interactions of RSK with GAB3 and CNKSR2 have not been previously described. B-C. RSK tridimensional structure (B) and sequence alignment (C) showing the conservation of the RSK DDVF docking site across evolution (from purple = highly conserved to green = poorly conserved). B. RSK2 tridimensional structure colored according to conservation across evolution with the DDVF peptide colored in orange (PDB: 7OPO, [14]). C. Sequence alignment showing that the DDVF-interacting residues (indicated by upper black bars) in the KAKLGM region are well conserved across all four RSK isoforms but not in the closely related MSK1, justifying the KAKLGM-to-KSEPPY mutation. D-E. Immunoblotting of lysates from RSK-DKO cells transduced with an empty vector (orange), or with vectors expressing RSK1 KAKLGM WT (purple) or the KSEPPY mutant (green). Cells were starved for 14-16 hours and then stimulated with 100nM bFGF for indicated periods of time. Western blots (n = 3) were quantified to calculate phospho-ERK/ERK and phospho-RSK/RSK ratios (mean +/- SD).
Fig 3: Highly conserved DDVF motifs in SPRED2 and GAB3 mediate the interaction with RSK1.A-B. Conservation of SPRED2 (A) and GAB3 (B) DDVF motifs (purple) across evolution C-D. Immunoblots showing the detection of wild type (KAKLGM) or mutated (KSEPPY) HA-RSK1 and FLAG (SPRED or GAB3 variants) in lysates (INPUT) of transfected HEK293T cells or after co-immunoprecipitation (IP FLAG) with various FLAG-SPRED variants (SPRED2-DDVF, SPRED2-DDVA mutant, or SPRED1), (n = 3) (C) or FLAG-GAB3 variants (GAB3-DDVF or GAB3-DDVA mutant), (n = 2) (D).
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