Fig. 7: VF-motifs from RSK signaling client proteins.

a ORF45 VF-motif (in magenta) docks into a positively charged (blue) AGC kinase domain region via aspartates/glutamates (in yellow). The same surface area for a close RSK relative, MSK1 (PDB ID: 1VZO)⁸², and for the most distant AGC kinase relative, PDK1 (PDB ID: 4RRV)⁸³, is also shown with the ORF45(56-69) region overlaid on the smaller panels at the right. (Blue: +10 eV; Red: −10  eV). The panel below shows the result of an MBP-pull down experiment where binding of GST-ORF45(16-76) and -BMF was tested with MBP-NTK_RSK2, -NTK_MSK1, and MBP as the control (N = 1). The gel was stained with Coomassie protein dye. MBP: maltose binding protein. b Selected peptides with VF-motif-like sequences in disordered protein regions. The position of F in the full-length protein sequence (from Uniprot) is shown in brackets. Indicated protein fragments were produced as GST-fusions, purified and their binding affinity to the RSK2 NTK was measured by SPR. The panel on the left shows the schematic of the VF-motif containing human full-length proteins: disordered protein regions are shown as a dashed line and the relative position of VF-motifs and K/RxxS/T phosphorylation sites are indicated. (In BMF, the dynein light chain binding motif is indicated and boxed, BH3 – BCL2 binding region; SOS1 has a Ras GDP exchange factor domain, RAS-GEF; RHDF1, has an inactive rhomboid protease domain, iRhom.) Phosphorylation sensitive interacting partners are shown with black ellipsoids: DYNLL, dynein light chain binding affects cellular BMF localization; for SOS1, 14-3-3 protein binding affects cellular localization and EGF receptor adapter binding; for RHDF1/2, 14-3-3 binding affects TNFα converting enzyme (TACE) activity and ultimately cell surface receptor shredding. c MBP pull-downs with purified GST-fusion constructs containing VF-motif peptides (preys) from ORF45, YopM, POLG, SOS1, BMF, and RHDF1 (see Panel b). The gel was stained with Coomassie protein dye. Maltose resin was loaded with MBP-NTK or MBP only, where the latter was used to asses unspecific binding of the preys (N = 1). Source data are provided as Source data file.