Allosteric activation of functionally asymmetric RAF kinase dimers

Abstract

Although RAF kinases are critical for controlling cell growth, their mechanism of activation is incompletely understood. Recently, dimerization was shown to be important for activation. Here we show that the dimer is functionally asymmetric with one kinase functioning as an activator to stimulate activity of the partner, receiver kinase. The activator kinase did not require kinase activity but did require N-terminal phosphorylation that functioned allosterically to induce cis-autophosphorylation of the receiver kinase. Based on modeling of the hydrophobic spine assembly, we also engineered a constitutively active mutant that was independent of Ras, dimerization, and activation-loop phosphorylation. As N-terminal phosphorylation of BRAF is constitutive, BRAF initially functions to activate CRAF. N-terminal phosphorylation of CRAF was dependent on MEK, suggesting a feedback mechanism and explaining a key difference between BRAF and CRAF. Our work illuminates distinct steps in RAF activation that function to assemble the active conformation of the RAF kinase.

Figure 1. The ability of kinase-dead BRAF to activate ERK requires phosphorylation of the N-terminal acidic motif (NtA)

A. The NtA is required for BRAF transactivation. Kinase-dead full-length BRAF (A481F) (lane 2) or truncated forms of BRAF A481F (Δ434-lane 3, Δ454-lane 4) were transiently overexpressed in 293 cells and cell lysates immunoblotted for ERK activation (pERK) after 24 hours. B. Substitution of alanines (AAAA) for residues 446–449 of BRAF A481F impaired ERK activation. Expression and immunoblotting were as described in A. C. Substitution of acidic residues (DDEE) for residues 338–341 of kinase-dead CRAF (A373F) conferred the ability to transactivate. ERK activation was measured as described in A. D. Acidic residues substituted in dimerization-competent, kinase-dead KSR1 (A587F) confers the ability to transactivate. Overexpression of kinase-dead KSR1 (A587F) had no effect on ERK activation, while substitution of acidic residues (DDEE) for residues 552–555 of KSR1, for Y522 (Y522E), or for T549 and S550 (TESD) could confer the ability to transactivate. In all panels, the mutants were overexpressed as analyzed as in A. Transactivation by kinase-dead BRAF (E), CRAF (F) or KSR1 (G) involves dimerization. Replacement of an arginine residue critical for efficient dimerization impaired the ability of BRAF A481F (E) and mutated forms of CRAF (A383F/DDEE) (F) and KSR1 (A587F/DDEE) (G) to activate ERK. ERK activation was measured as in A.

Figure 2. Phosphorylation of the acidic motif is required on the activator but not the receiver RAF kinase

A. A mutated form of truncated, catalytically active CRAF (Δ1–322) with AAFF substituted for residues 338–341 was transiently co-expressed with kinase-dead BRAF (A481F, Δ1-434 – left panel) or with acidic, kinase-dead CRAF (A373F/DDEE, Δ1–322 – right panel) with and without dimerization mutations (R509H or R401H) in 293 cells. Cell lysates were immunoblotted with antibodies to pERK1/2, ERK2, MYC (receiver constructs) or FLAG (activator constructs). B. A mutated form of truncated, catalytically active BRAF (Δ1–434) with AAAA substituted for residues 446–449 was transiently co-expressed with kinase-dead BRAF (A481F, Δ1–434 – left panel) or with acidic, kinase-dead CRAF (A373F/DDEE, Δ1–322 – right panel). Impairing dimerization (BRAF/R509H, CRAF/R401H) impaired transactivation. See also Figure S1. Cell lysates were analyzed as described in A. C and D. Activation of receiver CRAF (C) or BRAF (D) kinase activity measured by in vitro kinase reaction. The CRAF (AAFF, Δ1–322) or BRAF (AAAA, Δ1–434) receiver constructs were co-expressed with empty vector, BRAF or CRAF activator constructs. Immunoprecipitates were prepared using an antibody to the MYC tag present on the receiver. In vitro kinase reactions were performed with purified MEK as the substrate and measured by immunoblotting with antibodies to pMEK. Immunoblotting with antibodies to MYC (receiver) or FLAG (activator) confirmed similar levels of expression.

Figure 3. The mechanism of transactivation involves a conserved tryptophan

A. Schematic diagram showing the position of the W342 in the CRAF dimer interface. Note the close proximity of W342 to R401, the residue critical for dimerization and the position of the unphosphorylated Y340 and Y341. Blue and green are used to distinguish the two components of the dimer. Residue numbers between each kinase are also distinguished with a “prime”. B. Alignment of the N-terminal motifs of human BRAF, CRAF, KSR1, KSR2, LCK, SRC, ABL, BTK, EGFR and PKA show the conservation of the tryptophan in all of the kinases except for PKA and EGFR. In EGFR, L680 is the functional equivalent to the tryptophan (Zhang et al., 2006). C and D. Mutation of the tryptophan on the activator kinase impairs ERK activation. Activator forms of BRAF (C) or CRAF (D) with and without the tryptophan mutation (BRAF W450A, or CRAF W342A) were transiently co-expressed in 293 cells with CRAF receiver (AAFF, Δ1–322) in cells and cell lysates immunoblotted with antibodies to pERK, ERK2, HA (activator) and Myc (receiver). E. Mutation of tryptophan on the receiver impairs ERK activation. CRAF activator construct (CRAF A373F/DDEE, Δ1–322) was transiently co-expressed in 293 cells with either empty vector, CRAF receiver (AAFF, Δ1–322) or with CRAF receiver with the tryptophan mutated (AAFF/W342A, Δ1–322). Cell lysates were prepared and immunoblotted with antibodies to pERK, ERK2, HA (activator) and Myc (receiver). F. Mutation of the tryptophan on the activator only modestly impairs dimerization. A CRAF receiver construct (AAFF, Δ1–322) was co-expressed with vector alone, a CRAF activator (A373F/DDEE) CRAF activator with the tryptophan mutation (A373F/DDEE/W342A), or the CRAF activator with the R401H dimerization mutation. Immunoprecipitates were made with antibodies to the activator (HA) and immunoblotted with antibodies to the receiver (Myc). See also Figure S1. G. Mutation of the tryptophan on the receiver only moderately impairs dimerization. The CRAF receiver construct (CRAF AAFF, Δ1–322), with the tryptophan mutation (W342A) or the dimerization mutation (R401H) was transiently expressed alone or co-expressed with a CRAF activator construct (A373F/DDEE, Δ1–322). Immunoprecipitates were made with antibodies to the activator (HA) and immunoblotted with antibodies to the receiver (Myc).

Figure 4. Constitutive assembly of the R-spine results in an active kinase that does not require dimerization or AL phosphorylation

A. Phosphorylation of the AL is induced on the receiver but not the activator. An HA-tagged CRAF activator (A373F/DDEE, Δ1–322) was co-expressed with a FLAG-tagged CRAF receiver (AAFF, Δ1–322). Immunoprecipitates were made to the receiver (anti-FLAG, lanes 1 and 2) or to the activator (anti-HA, lanes 3 and 4) and immunoblotted with a phospho-specific antibody to BRAF pT599 which also specifically reacts with pT491 of CRAF. Mutation of the AL phosphorylation sites (T491A, S494A) verified the specificity of the antibody (lanes 2 and 4). B. CRAF L397F and BRAF L505F mutants are constitutively active and do not require AL phosphorylation or dimerization. CRAF L397F and BRAF L505F mutants were transiently overexpressed in 293 cells and cell extracts immunoblotted with antibodies to pERK, ERK2 or to HA (CRAF or BRAF). CRAF mutant with mutations in the AL phosphorylation sites (T491A, S494A) had no effect on ERK activation. Similarly, dimerization mutants of the CRAF L397F mutant (R401H or RH) and BRAF L505F (R509H or RH) had only minor effects on ERK activation. C. Schematic diagram showing the positions of the R-spine residues in BRAF. In the left panel, the conformation of the R-spine in the active conformation is shown (PDB:4E26). In the middle panel, the position of the residues of the R-spine in the inactive, dimeric structure of BRAF is shown (PDB:1UWH). Note the rearward displacement of F595. In the right panel, a model predicting the structure of the L397F mutant is shown. This was modeled using TINKER (http://dasher.wustl.edu/tinker/). D. Kinase activity of bacterial expressed CRAF L397F/DDEE mutant. GST fusion protein of L397F/DDEE or wild-type CRAF/DDEE kinase domain was purified with GST and tested in vitro for kinase activity in vitro towards purified MEK. E. Co-expression of His-tagged CRAF L397F/DDEE construct with GST-CRAF/A373F mutant in bacteria. Bacteria were lysed and proteins purified with glutathione or Ni⁺⁺ beads and immunoblotted with BRAF pT599 antibody.

Figure 5. CRAF S338 phosphorylation is stimulated by MEK activation

A. MEK inhibitors inhibit CRAF S338 phosphorylation induced by EGF. Untransfected 293 cells were pre-treated with MEK inhibitor (UO126-20uM or PD98059-1uM), PAK inhibitor (IPA3-20uM), CAMKII inhibitor (KN-93-10uM) or vehicle for 2 hours before addition of EGF (100ng/ml). Cells were lysed after 5 minutes and CRAF S338 and MEK phosphorylation assessed using phosphor-specific antibodies. See also Figure S2. B. Quantification of the effect of kinase inhibitors on CRAF S338 phosphorylation. Results from 4 separate experiments are presented as mean −/+ SEM. The amount of S338 phosphorylation from the EGF treatment alone was set as 1. C. Co-expression of constitutively active MEK with CRAF induces S338 phosphorylation. Wild-type HA-CRAF was co-expressed with constitutively active MEK1 (DD) with and without dominant negative RAS (RasN17). Cells were lysed after 24 hours and immunoprecipitates prepared with antibodies to HA. CRAF S338 phosphorylation was assessed using a phosphor-specific antibody. D. MEK inhibition blocks CRAF S338 phosphorylation induced by co-expression with constitutively active MEK. Cells were prepared as described in B, except that MEK (UO126) or ERK inhibitor (ERK inhibitor II) was added 24 hours after transfection. Cells were lysed 2 hours after addition of the inhibitor. Immunoprecipitates were analyzed as in B.

Figure 6. Model of RAF transactivation

A–F: BRAF (activator) activates CRAF (receiver). A. Domains of the RAF monomer. The two lobes of the kinase, the Ras binding domain (RBD) and the N-terminal acidic domain (NtA) are shown. BRAF is shown here with its constitutively acidic NtA (pS445/D448) depicted as a red circle. B. Recruitment of BRAF to the plasma membrane (PM) by GTP-RAS induces a new conformation in the N-terminal half of BRAF. C. RAS binding allows two RAF molecules to dimerize. A BRAF/CRAF dimer is shown here, with BRAF serving as the “activator” and CRAF as the “receiver”. D. For BRAF, the NtA is constitutively phosphorylated (red dot) and lies within the dimer interface. This allows it to transactivate the RAF receiver, in this case CRAF. E. Transactivation results in cis-autophosphorylation producing an active CRAF kinase that can then phosphorylate MEK. F. The S338 of the CRAF receiver can be phosphorylated in a MEK-dependent manner. CRAF-pS338 is designated as “CRAF*”. G-J: CRAF* activates CRAF. G. Monomeric CRAF* dissociates from BRAF. H. CRAF* (the “activator”) dimerizes withCRAF (the “receiver”). CRAF * may also dimerize with, and activate, a molecule ofBRAF. I. The phosphorylated NtA of the CRAF* activator lies within the dimer interface and transactivates the CRAF receiver. J. Transactivation results in cis-autophosphorylation producing an active CRAF kinase that can then phosphorylate MEK.