FIG 2.

A mutation that fuses the C-spine and blocks ATP binding generates a pseudokinase, allosteric activator. (A) Model showing the relationship between the C-spine residues and ATP. Using PKA numbering, in the N-lobe, V57 and A70 interact with the upper surface of the adenine ring, while L173 interacts with the bottom surface. In BRAF, the C-spine residue below is a Phe. (B) Phe replacement of V471 of BRAF is an activating mutation that requires dimerization for its effect. BRAF V471F, A481F, or V471F plus R509H were overexpressed in 293 cells and immunoblotted with antibodies to pERK, ERK2, or an epitope tag, HA, that was fused to BRAF. (C) Phe replacement of V363 of CRAF is an activating mutation that requires dimerization for its effect. Because the allosteric function of CRAF requires N-terminal acidic residues, acidic residues (DDEE) were substituted for residues 338 to 341. CRAF V363F, A373F, or V363F plus R401H were overexpressed in 293 cells and immunoblotted with antibodies to pERK, ERK2, or an epitope tag, HA, that was fused to CRAF. (D) In vitro kinase reactions of BRAF C-spine mutants. The wild type (WT) or V471F or A481F mutants of BRAF were overexpressed in cells, and immunoprecipitates were tested for kinase activity against MEK by using purified MEK. Kinase reactions were analyzed by immunoblotting with antibodies against pMEK1/2, MEK1/2, and the HA epitope tag. (E) In vitro kinase reactions of CRAF C-spine mutants. To generate constitutively active CRAF kinases, we used a construct encoding acidic residues (TESD) for the two activation loop phosphorylation sites (T491E and S494D). The V363F and A373F mutants were compared to the wild-type CRAF construct with the two activation loop substitutions (TESD). (F) Activator/receiver assays demonstrated that the BRAF C-spine mutants function as allosteric activators. BRAF and BRAF C-spine mutants (A481F and V471F) were coexpressed with a CRAF construct that lacks N-terminal acidic residues (AAFF). This prevents the CRAF construct from being able to function as an activator, but its kinase activity was still stimulated when it dimerized with a RAF protein that has an acidic N-terminal acidic domain. Various constructs were expressed alone or together, as indicated, as cell lysates were immunoblotted with antibodies to pERK, ERK2, and epitope tags (HA and FLAG) that were present on the receiver or the activator constructs, respectively. The ability to activate ERK required dimerization, as mutation of R401H blocked this effect. (G) Activator/receiver assays demonstrated that the CRAF C-spine mutants function as allosteric activators. This experiment was performed similar to that shown in panel F, except that the C-spine CRAF mutants with DDEE substituted for the N-terminal acidic domain residues 338 to 341 was used. (H) Only Phe substitutions abrogate kinase activity. The V363F substitution in CRAF was compared with Met or Leu substitutions. CRAF constructs with the designated substitution for V363 that also contained acidic substitutions at positions 338 to 341 and an acidic substitution in the activation loop (DDEE/TESD) were overexpressed in cells, and kinase activity measured in vitro toward purified MEK was measured as described for panel E.