Fig 1.
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).
Fig 2.
FGFR1 interacts with RSK1 via a DSVF motif whose phosphorylation increases interaction.
A. The DSVF motif from FGFR1 is highly conserved in all 4 FGFR isoforms and across evolution. B. Co-immunoprecipitation of HA-FGFR1 DSVF or the DSVA mutant with FLAG-RSK1 KAKLGM or the KSEPPY mutant, from HeLa cells co-transfected with plasmids expressing indicated proteins. (INPUT = cell lysates; IP = immunoprecipitation), (n = 3). C. Assessment of the interaction between biotinylated peptides derived from FGFR1, bearing DSVF, DDVF, DSVA, or DpSVF (phosphoserine) motifs, and purified RSK2 (residues 44-367) using streptavidin pull-down assays (n = 3). D. Same experiment as in C comparing the pull-down efficiency of DSVF and DpSVF peptides on RSK2 with or without treatment of the peptides with alkaline phosphatase (n = 3). C-D. Quantitative analyses show the mean +/- SD of relative RSK pull-down efficiencies calculated from three independent experiments; P-values: *, p≤ 0.05; **, p≤0.01; ***, p≤0.001.
Fig 3.
CNKSR2 interacts with RSK1 through a DSVF motif.
A. The DSVF motif of CNKSR2 is conserved across evolution. Note that a potential RSK binding motif (EDVF) is present at another position in CNKSR1. B. Co-immunoprecipitation of FLAG-CNKSR2 DSVF or the DSVA mutant with FLAG-RSK1 KAKLGM or the KSEPPY mutant, from HEK293T cells co-transfected with plasmids expressing indicated proteins (n = 2).
Fig 4.
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 5.
YopM competes with SPRED2 for RSK binding via the DDVF motif.
A. Immunoblots showing the co-immunoprecipitation of endogenous RSK with FLAG-SPRED2 in the presence or absence of HA-YopM (wild-type or DDVA mutant), which acts as a competitor (n = 3). FLAG-SPRED2 was immunoprecipitated, and co-immunoprecipitation of RSK and phosphorylation of SPRED2 at pRxxS*/T* sites were assessed (*: Phosphorylated residues; x: any amino acid). B. Quantification of SPRED2 phosphorylation (pRxxS*/T*) in the presence or absence of HA-YopM, based on data from panel A (n = 3). C. Proposed model of YopM-mediated regulation of SPRED2 phosphorylation by RSK. Under physiological conditions, RSK binds to SPRED2 via its DDVF motif, leading to SPRED2 phosphorylation. Upon YopM expression, YopM enhances RSK activation (depicted as increased red shading) while it competes with SPRED2 for RSK binding using its DDVF motif, reducing SPRED2 phosphorylation.
Fig 6.
The L protein competes with SPRED2 for RSK binding via the DDVF motif in TMEV-infected cells. Immunoblots showing the co-immunoprecipitation of FLAG-SPRED2 with endogenous RSK in presence or not of the L competitor (n=2). HeLa cells were transduced with either FLAG-SPRED2(WT) or the FLAG-SPRED2(DDVA) mutant and infected with TMEV expressing wild-type L (DDVF) or L mutants: DDVA which lost RSK binding or M60V which conserved RSK binding but lost nucleo-cytoplasmic trafficking perturbation ability. Viral 3D polymerase served as an infection marker.
Fig 7.
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.