A new model of kinase regulation based on the assembly of hydrophobic spines has been proposed. Changes in their positions can explain the mechanism of kinase activation. Here, we examined mutations in human cancer for clues about the regulation of the hydrophobic spines by focusing initially on mutations to Phe. We identified a selected number of Phe mutations in a small group of kinases that included BRAF, ABL1, and the epidermal growth factor receptor. Testing some of these mutations in BRAF, we found that one of the mutations impaired ATP binding and catalytic activity but promoted noncatalytic allosteric functions. Other Phe mutations functioned to promote constitutive catalytic activity. One of these mutations revealed a previously underappreciated hydrophobic surface that functions to position the dynamic regulatory αC-helix. This supports the key role of the C-helix as a signal integration motif for coordinating multiple elements of the kinase to create an active conformation. The importance of the hydrophobic space around the αC-helix was further tested by studying a V600F mutant, which was constitutively active in the absence of the negative charge that is associated with the common V600E mutation. Many hydrophobic mutations strategically localized along the C-helix can thus drive kinase activation.

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.