Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods.

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental disorders. A characterization of disease-related RAF mutants will help us select appropriate therapeutic strategies for treating these diseases. Recent studies have shown that RAF family kinases have both catalytic and allosteric activities, which are tightly regulated by dimerization. Here, we constructed a set of practical and feasible methods to determine the catalytic and allosteric activities and the relative dimer affinity/stability of RAF family kinases and their mutants. Firstly, we amended the classical in vitro kinase assay by reducing the detergent concentration in buffers, utilizing a gentle quick wash procedure, and employing a glutathione S-transferase (GST) fusion to prevent RAF dimers from dissociating during purification. This enables us to measure the catalytic activity of constitutively active RAF mutants appropriately. Secondly, we developed a novel RAF co-activation assay to evaluate the allosteric activity of kinase-dead RAF mutants by using N-terminal truncated RAF proteins, eliminating the requirement of active Ras in current protocols and thereby achieving a higher sensitivity. Lastly, we generated a unique complementary split luciferase assay to quantitatively measure the relative dimer affinity/stability of various RAF mutants, which is more reliable and sensitive compared to the traditional co-immunoprecipitation assay. In summary, these methods have the following advantages: (1) user-friendly; (2) able to carry out effectively without advanced equipment; (3) cost-effective; (4) highly sensitive and reproducible.