Fig. 8: VF-motif mediated phosphorylation of RSK substrates.

a In vitro kinase assay results with VF-motif containing human proteins: BMF (full-length), SOS1(1101-1170), RHDF1 (1-289), and with a VF-motif lacking substrate (EIF4B). The panels below show the results of in vitro kinase assays: P³²-autoradiography based detection by phosphorimaging is shown on the left and the Western-blots using the anti-RxxpS/pT phosphorylation sensitive antibody on the right confirms AGC kinase-specific site (RxxS/T) phosphorylation for EIF4B and RHDF1 after 50 min. 0.5 µM activated RSK2 was mixed with 10 µM GST-fusion constructs (EIF4B: 411-ETQERERSRTGSESSQTGTS-430; full-length BMF, SOS1(1101-1171), and RHDF1(1-289)), and the substrate phosphorylation was monitored in the absence (-) or presence of 2 µM competitor: GST-ORF45(16-76) or the VF-motif mutated version of the latter (ORF45_F66A). The bar charts below show the initial phosphorylation rate normalized to the reaction containing no competitor. Error bars show SD calculated based on three independent experiments. P-values of two-sided paired t-tests were the following: BMF, p = 1.8E−4; RHDF1, p = 5.4E−4; SOS1; p = 6.4E−4; EIF4B: p = 0.5. b Simulation results showing the effect of ORF45 on the phosphorylation levels of VF-motif independent (SUBr) and VF-motif dependent (VF-SUBr) substrates after EGF stimulation. The plots were generated using the Network Model (Fig. 6). c Enhanced phosphorylation of the RxxS/T motif through VF-motif-based RSK docking. RxxS/T substrate phosphorylation sites bind in the negatively charged substrate-binding pocket of NTK (depicted with a red ellipsoid; RxxS_EIF4B indicates the 411-430 region from EIF4B, see Panel a). A VF-motif located N- or C-terminally—VF-RxxS_EIF4B or RxxS_EIF4B-VF, respectively—to this phosphorylation site may enhance the rate of phosphorylation through VF groove (shown with a green ellipsoid) based docking. The phosphorylation rate of 10 µM GST-fusion constructs containing the EIF4B RxxS/T phosphorylation site alone (RxxS_EIF4B) or in combination with the VF-motif from ORF45 (VF: ORF45_a, see Fig. 7b; VF-RxxS_EIF4B or RxxS_EIF4B-VF; where the F and S from these two motifs were separated by 34 or 20 amino acids, respectively) were examined in kinase assays using 100 nM activated RSK2. The upper panel shows the gel stained with a protein dye (Ponceau) and the lower panel shows the quantitative Western-blot signal obtained with the phosphorylation-specific Anti-RxxpS/pT antibody. Phosphorylation rates (mean) were calculated based on the linear phase of the kinetic plots and the SD was calculated based on three independent experiments (N = 3). The panels at the bottom show the diminished phosphorylation rate of the RxxS motifs in RHDF1 (5 µM RHDF1_1-289 with 0.25 µM activated RSK2) when the VF motif was mutated (F281A). Source data are provided as Source data file.