FIG 4.

L74 functions to couple the C-spine to the R-spine. (A) Model showing the position of L74 in PKA (left), how ATP interacts with both the C- and R-spines (center), and how L485 in BRAF potentially interacts with F498 in the C-helix. (B) L74 replacement by Phe in BRAF and CRAF results in a constitutively active kinase that is independent of dimerization and activation loop phosphorylation. The indicated mutants were overexpressed in 293 cells, and cell lysates were analyzed as described above. (C) Replacement of L74 with other hydrophobic residues also generates constitutively active forms of CRAF. (D) Mutation of F498 in the C-helix abrogates the ability of L485F to induce BRAF activation. (E) The F498A mutant of BRAF can still be activated. An activator/receiver assay was performed using BRAF V471F as the activator and BRAF F498A as the receiver. Similar amounts of active ERK were induced with either BRAF or F498A BRAF as the receiver. (F) Models showing the series of hydrophobic interactions along the surface of the C-helix facing the β3-strand in PKA (left) and BRAF (right). In PKA, R-spine residues (RS2 to -4) interact with the gatekeeper residue (M120), which interacts with C-spine residues V57 and A70. Note how F350 from the C-tail interacts with L74, filling the space between L74 and the C-spine. On the right is the same surface of BRAF. In this case, F485 interacts with F498, which is facing it from the C-helix. Also note the smaller gatekeeper residue (T529), which does not completely fill the space between the R-spine and the C-spine.