Fig. 4: Differential responses of RAF proteins to dephosphorylation by the SHOC2-MRAS-PP1C complex. | Nature Communications

Fig. 4: Differential responses of RAF proteins to dephosphorylation by the SHOC2-MRAS-PP1C complex.

From: Cryo-EM structures of CRAF/MEK1/14-3-3 complexes in autoinhibited and open-monomer states reveal features of RAF regulation

Fig. 4

In vitro dephosphorylation of autoinhibited BRAF/MEK1/14-3-3 (a), CRAFSSYY/MEK1/14-3-3 (b), and CRAFSSDD/MEK1/14-3-3 (c) complexes by the SHOC2-MRAS-PP1C holophosphatase complex. The purified full-length RAF complexes were incubated with increasing concentrations of the SHOC2-MRAS-PP1C complex. Aliquots of the dephosphorylation reactions were analyzed for phosphorylation at pSer365 (BRAF) or pSer259 (CRAF) by Western blotting with a phosphospecific antibody for this site and for total BRAF or CRAF as a loading control (upper panels). The kinase activity of aliquots of the dephosphorylation reactions was assessed using a TR-FRET-based assay (lower panels). Dephosphorylation reactions were stopped by addition of a phosphatase inhibitor and samples were then diluted in reaction buffer for the kinase assay. The FRET ratio at 665/620 nm is plotted over the time course upon addition of the reaction mixture containing wild-type MEK1 as a substrate and ATP. Each data point represents the mean of triplicates from one representative experiment. The experiments were independently repeated twice with similar results. Source data are provided as a Source Data file.

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