Mutational and Functional Analysis in Human Ras/MAP Kinase Genetic Syndromes

The Ras/mitogen-activated protein kinase (MAPK) pathway is essential in regulation of the cell cycle, cell differentiation, growth, and cell senescence, each of which are critical to normal development. A class of developmental disorders, the “RASopathies,” is caused by germline mutations in genes that encode protein components of the Ras/MAPK pathway which result in dysregulation of the pathway and profound deleterious effects on development. One of these syndromes, cardiofaciocutaneous (CFC) syndrome, is caused by germline mutations in BRAF , MAP2K1 (MEK1 ) and MAP2K2 (MEK2 ), and possibly KRAS genes. Here, we describe the laboratory protocols and methods that we used to identify mutations in BRAF and MEK1/2 genes as causative for CFC syndrome. In addition, we present the techniques used to determine the effect these mutations have on activity of the Ras/MAPK pathway through Western blot analysis of the phosphorylation of endogenous ERK1/2, as well as through the use of an in vitro kinase assay that measures the phosphorylation of Elk-1.