Distinct Binding Preferences between Ras and Raf Family Members and the Impact on Oncogenic Ras Signaling

Abstract

The Ras GTPases are frequently mutated in human cancer, and, although the Raf kinases are essential effectors of Ras signaling, the tumorigenic properties of specific Ras-Raf complexes are not well characterized. Here, we examine the ability of individual Ras and Raf proteins to interact in live cells using bioluminescence resonance energy transfer (BRET) technology. We find that C-Raf binds all mutant Ras proteins with high affinity, whereas B-Raf exhibits a striking preference for mutant K-Ras. This selectivity is mediated by the acidic, N-terminal segment of B-Raf and requires the K-Ras polybasic region for high-affinity binding. In addition, we find that C-Raf is critical for mutant H-Ras-driven signaling and that events stabilizing B-Raf/C-Raf dimerization, such as Raf inhibitor treatment or certain B-Raf mutations, can allow mutant H-Ras to engage B-Raf with increased affinity to promote tumorigenesis, thus revealing a previously unappreciated role for C-Raf in potentiating B-Raf function.

Figure 1.. Analysis of Raf-Binding Interactions with Activated K-Ras Mutants

(A) BRET saturation curves are shown examining the interaction of WT or RBD mutant (R > L) C-Raf^Reg-Rluc proteins with Venus-K-Ras^Q61R and the interaction of WT C-Raf^Reg-Rluc with the CAAX mutant (C185A) Venus-K-Ras^Q61R/C>A. BRET₅₀ values are listed. (B) K-Ras and C-Raf proteins analyzed in (A) were examined in co-immunoprecipitation assays. Venus-K-Ras proteins were also evaluated for GTP loading in Raf-RBD pull-down assays. (C) Live-cell imaging shows the intracellular localization of the indicated K-Ras and C-Raf proteins. (D) BRET saturation curves are shown examining the interaction of WT or R > L Raf^Reg-Rluc proteins with the indicated Venus-K-Ras mutants. (E) BRET₅₀ values from (D) are listed and the expression level of the K-Ras mutants is shown. (F) WT and R > L Raf^Reg-Rluc proteins were examined in co-immunoprecipitation assays for binding to Venus-K-Ras^Q61R. Lysates were also monitored for Raf^Reg-Rluc expression, and all experiments were conducted in 293FT cells.

Figure 2.. Binding Preferences between Ras and Raf Family Members

(A) BRET saturation curves examining the interaction of WT or R > L Raf^Re9-Rluc proteins with the Venus-Ras^G12V proteins are shown, and the BRET₅₀ values are listed. (B) WT Raf^Reg-Rluc proteins were examined in co-immunoprecipitation assays for binding to the Venus-Ras^G12V proteins. (C) Immunoprecipitated Venus-Ras^G12V complexes were probed for the presence of endogenous B-Raf, C-Raf, or A-Raf and Venus-Ras. Lysates were also examined for B-Raf, C-Raf, A-Raf, and pMEK levels (upper). Endogenous C-Raf complexes were isolated from cells expressing the indicated Venus-Ras^G12V proteins and examined for dimerization with B-Raf (lower). (D) HeLa cells expressing WT Venus-Ras proteins were treated or not with EGF prior to lysis. Immunoprecipitated Venus-Ras complexes were probed for the presence of endogenous B-Raf, C-Raf, or A-Raf and Venus-Ras. Lysates were also examined for Raf levels. (E) Ras complexes were immunoprecipitated from Ras-deficient MEFs re-expressing either K-Ras^Q61L or H-Ras^Q61L and probed for the presence of endogenous B-Raf, C-Raf, or A-Raf and Ras. Endogenous C-Raf was also isolated from the MEF lines and examined for dimerization with B-Raf. Lysates were examined for B-Raf, C-Raf, A-Raf, and pMEK levels. (F) MCF10A cells stably expressing Halo-tagged K-Ras^G12V or H-Ras^G12V and B-Raf-Cherry or C-Raf-Cherry were examined by live-cell imaging to visualize recruitment of the Rafs to the plasma membrane. Experiments shown in (A)–(C) were conducted in 293FT cells. See also Figure S1.

Figure 3.. Raf N-Terminal Segment Determines the Ras Binding Selectivity

(A) Schematic depiction of the B-Raf and C-Raf regulatory domains with the RBD, CRD, and N′-segment indicated. (B) Raf^Reg-Rluc proteins were generated in which the RBD/CRD or N′-segment of B-Raf and C-Raf were exchanged. BRET (upper) and co-immunoprecipitation assays (lower) were performed examining the interaction of WT or domain-exchanged Raf^Reg-Rluc proteins with Venus-tagged H-Ras^Q61R or K-Ras^Q61R. BRET₅₀ values are listed. (C) WT full-length B-Raf^FL-Rluc or B-Raf^FL-Rluc proteins lacking the N′-segment (Δ-N′) or containing the N′-segment of C-Raf (C-N′) were examined for their ability to interact with the indicated Venus-Ras^Q61R proteins in co-immunoprecipitation assays. Lysates were also monitored for Raf-Rluc expression in (B and C), and all experiments were conducted in 293FT cells. See also Figure S2.

Figure 4.. The Ras HVRs Contribute to the Ras/Raf Binding Preferences

(A) Shown are the HVR sequences of the various Ras proteins. (B) Venus-Ras^Q61R proteins were generated in which the HVRs of H-Ras and K-Ras4B were exchanged. BRET (upper) and co-immunoprecipitation assays (lower) were performed examining the interaction of B-Raf^Reg or C-Raf^Reg-Rluc with WT or HVR-exchanged Venus-Ras^Q61R proteins. BRET₅₀ values are listed. (C) Co-immunoprecipitation assays were performed examining the interaction of B-Raf^Reg or C-Raf^Reg-Rluc with Venus-tagged K-Ras4A^Q61R or K-Ras4B^Q61R. (D) Cells co-expressing the indicated GFP-Ras^G12V proteins and B-Raf^Reg or C-Raf^Reg-Rluc were lysed, and GFP-Ras complexes were immunoprecipitated from the cell lysates and examined for Raf^Reg-Rluc binding. (E) WT or N′-segment mutant B-Raf^Reg-Rluc proteins were examined in co-immunoprecipitation assays for binding to Venus-tagged K-Ras^Q61R or H-Ras^Q61R. Lysates were monitored for the indicated proteins in (B–E), and all experiments were conducted in 293FT cells. See also Figure S3.

Figure 5.. B-Raf/C-Raf Dimerization Can Modulate the B-Raf/H-Ras^Q61R Interaction

(A) BRET (left) and co-immunoprecipitation assays (right) are shown examining the interaction of WT, R509H (dimer-defective), or E586K (dimer-enhanced) B-Raf^FL-Rluc proteins with Venus-H-Ras^Q61R. The B-Raf^FL-Rluc proteins were also monitored for dimerization with C-Raf. (B) BRET saturation curves were performed examining the effect of 1 h DMSO or Raf inhibitor SB590885 (SB) treatment on the interaction of B-Raf^FL-Rluc with Venus-tagged H-Ras^Q61R or K-Ras^Q61R. BRET₅₀ values are listed. (C) MCF10A cells stably expressing Halo-H-Ras^G12V and B-Raf^FL-Cherry were treated for 1 h with DMSO or SB590885 prior to live-cell imaging. Recruitment of B-Raf to the plasma membrane in SB590885-treated cells is indicated by white arrows. (D) 293FT cells expressing B-Raf^FL-Rluc with Venus-H-Ras^Q61R or Venus-K-Ras^Q61R or expressing B-Raf^Reg-Rluc with Venus-H-Ras^Q61R were treated for 1 h with DMSO or SB590885 prior to lysis. Immunoprecipitated Venus-Ras complexes were probed for B-Raf-Rluc and Venus-Ras. (E) BRET (left) and co-immunoprecipitation (right) assays were performed examining the effect of various Raf inhibitors on the interaction of B-Raf^FL-Rluc with Venus-H-Ras^Q61R. BRET₅₀ values are listed. B-Raf^FL-Rluc proteins were also examined for dimerization with C-Raf. (F) BRET (left) and co-immunoprecipitation (right) assays were performed examining the effect of SB590885 treatment on the interaction of B-Raf^FL-Rluc and Venus-H-Ras^Q61R in control (NT) or C-Raf-depleted (C-Raf-T) 293FT cells. BRET₅₀ values are listed. Co-immunoprecipitation of endogenous C-Raf with Venus-H-Ras^Q61R is also shown. (G) BRET (left) and co-immunoprecipitation (right) assays were performed examining the effect of SB590885 treatment on the interaction of WT or R > L B-Raf^FL-Rluc with Venus-H-Ras^Q61R. BRET₅₀ values are listed. Co-immunoprecipitation of endogenous C-Raf with Venus-H-Ras^Q61R is also shown. Lysates were monitored for the indicated protein levels in (A, D, and E–G). See also Figures S4 and S5.

Figure 6.. Co-occurring B-Raf and H-Ras Mutations in Cancer

(A) BRET (left) and co-immunoprecipitation assays (right) were performed comparing the interaction of WT and mutant B-Raf^FL-Rluc proteins with Venus- H-Ras^Q61R. BRET₅₀ values are listed. Endogenous C-Raf was also examined for dimerization with the B-Raf^FL-Rluc mutants. Lysates were monitored for pMEK and B-Raf-Rluc levels. (B) Control (NT) or C-Raf-depleted (C-Raf-T) 293FT cells expressing WT, G466V, or D594G B-Raf^FL-Rluc with Venus-H-Ras^Q61R were examined in co-immunoprecipitation assays for binding of B-Raf^FL-Rluc to Venus-H-Ras^Q61R. (C) Control (sh-Neg) or C-Raf-depleted (sh-C-Raf) NIH 3T3 cells were infected with retroviruses expressing the indicated Ras proteins. After two weeks of culture, focus formation was visualized by methylene blue staining. Shown are focus plates from a representative experiment. (D) T24 and RL95–2 cells were infected with lentiviruses expressing Cas9 and either a non-targeting sgRNA (NT) or sgRNAs targeting A-Raf, B-Raf, C-Raf, or H-Ras. Cells were assessed for 2D proliferation (left), 3D growth (middle), and expression of A-Raf, B-Raf, C-Raf, or H-Ras proteins (right). Data are represented as mean ± SD. ***p < 0.001. (E) Control (NT) or C-Raf-depleted (C-Raf-T) lines were serum-starved for 18 h and then treated for 1 h with DMSO or SB590885 prior to lysis. Endogenous mutant H-Ras proteins from T24 and RL95–2 cells and endogenous mutant K-Ras proteins from H358 and SW480 were immunoprecipitated and examined for the presence of endogenous B-Raf. Lysates were monitored for the indicated protein levels in (A–E). See also Figure S6.

Figure 7.. Model for Ras/Raf Binding Preferences

The C-Raf kinase exhibits high-affinity binding to all Ras family members. In contrast, B-Raf, whose N-terminal segment is larger and possesses an overall acidic charge, only binds with high affinity to mutant K-Ras, whose HVR contains a series of polybasic lysine residues (upper). In the context of H-Ras or N-Ras, the B-Raf N′-segment might occlude the RBD or act to repel B-Raf from the negatively charged plasma membrane. However, events that promote stable B-Raf/C-Raf dimer formation, such as B-Raf mutations (depicted as yellow star) or treatment with B-Raf inhibitors (black box containing the letter I), allow mutant H-Ras to engage B-Raf with increased affinity to upregulate ERK cascade signaling (lower).