Fig 2: Crabp1-selective compound C3 enhances Crabp1 interaction with the Raf-RBD and competition with active Ras for RBD binding. (A) In vitro interaction assay. Compound C3 increases Crabp1 interaction with the Raf-RBD. (B) Compound C3/Crabp1 reduces active Ras interaction with the Raf-RBD. (C) HEK293T cells reconstituted with the indicated expression vectors and treated with, or without, C3 for 30 min were monitored for Raf activation in cells, indicated with p-cRaf (S338).

Fig 3: Crabp1 sequentially forms complexes with the components of Raf-Mek signaling pathway to modulate Erk1/2 phosphorylation. (A) PLA (Duolink) assays detect atRA-induced sequential formation of Crabp1, first with cRaf (top panel), then Mek1/2 (middle panel) and finally p-Erk1/2 (bottom panel). Nuclei are stained with DAPI. The red punctate represents complex formed with Crabp1. Representative microphotographs of various fields are shown, and scale bar refers to 20 µm. (B) Quantitation of PLA signals. The kinetics of Crabp1 forming complexes with the three key components in the MAPK pathway are plotted for relative numbers of PLA signal/cell each with a standard deviation. Experiments were performed three times. (C) Crabp1 is a scaffold protein for cRaf-Mek2-Erk1/2, and atRA induces scaffolding. HEK293T cells expressing Flag-Crabp1-HA were treated with 100 nM atRA for 15 min, and lysates were immunoprecipitated with M2 flag beads. WT cells were used as negative control. Inp refers to input control. (D) Semi in vitro kinase assay. Recombinant Mek1 is phosphorylated by TnT generated HA-cRaf and ATP in the absence or presence of His-Crabp1. Phosphorylation is increased by the addition of 100 nM atRA. Densitometric analyses western blot for lanes 3 and 4 are depicted at right (*P < 0.05, student t-test). (E) WT (CJ7) and CKO ESCs were treated with 100 nM atRA for 30 min, and relevant endogenous proteins in cell lysates were monitored. (F) atRA activates cRaf-Mek1/2-Erk1/2. WT and Flag-Crabp1-HA expressing HEK293T cells were treated with atRA for the indicated time, and relevant endogenous proteins in cell lysates were monitored via western blot. (G) Crabp1 competes with Ksr1. Lysates of HEK293T expressing Flag-Crabp1-HA and cMyc-cRaf were treated with atRA for 15 min followed by immunoprecipitation with anti-cMyc antibody and western blot analysis. Relative intensity is numerically marked.

Fig 4: NMR analyses showing Crabp1 direct interaction with the RBD of cRaf. (A) An expanded region of 1H,15N HSQC spectra of the 19 µM Crabp1 in the absence (black peaks) and presence (red peaks) of 126 µM cRaf-(R) (aa 51–187). (B) Titration curves of selected Crabp1 residues (C96, T97, Q98) in which the chemical shift change is plotted vs. the cRaf-(R) concentration. (C) Maximal chemical shift changes (?d) induced by cRaf-(R) on Crabp1. (D) Maximal resonance intensity changes (?Intensity) induced by cRaf-(R) on Crabp1. (E) Cartoon representation of cRaf-(R) interaction sites with marked spectral changes on Crabp1 in the presence of cRaf-(R): 1 SD – orange, 2 SD – red. PDB: 1CBR. Chemical shifts were internally referenced to DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid), and chemical shift differences in panels (C,D) were calculated as [(?d 1H)2 + (0.25 ?d 15N)2]1/2. NMR acquisition and sample conditions are described in the “Material and Methods”.

Fig 5: atRA/Crabp1 dampens canonical EGF and Ras mutation elicited Erk1/2 activation. (A) Dampening effects in ESCs. ESCs were pre-treated with atRA (100 nM, 30 min) and then with EGF (25 ng/ml, 5 min). Erk1/2 phosphorylation, the final signal of MAPK cascade, is shown (top panel). Blots are representatives of three independent experiments. Band intensity is statistically analyzed at bottom: *P < 0.01, #P < 0.01 and **P < 0.05 (n = 4). (B,C) Dampening effects in Crabp1-positive HEK293T clones over-expressing other components as indicated, treated with, or without, atRA for 30 min. Anti-Flag antibody recognizes Flag-tagged Ras and Crabp1. (B) Phosphorylation of cRaf (at S338) and Erk1/2 was dampened by atRA in cells harboring the constitutively active mutant HRas (G12V), *P < 0.05 (n = 4). (C) Ras interaction with Raf (indicating activated Ras) in cells, monitored using GST-Raf-RBD beads (Millipore Sigma) to pulldown cell lysates in (B). The active Ras bound to the RBD of Raf is monitored. RBD: Ras binding domain. *P < 0.05 (n = 3).