ISOLATION AND CHARACTERIZATION OF MUTANTS OF HUMAN MITOGEN-ACTIVATED PROTEIN-KINASE (ERK2)

Site directed mutagenesis/charged-to-alanine scanning mutagenesis of t he amino terminal portion of human ERK2 (from amino acids 1 to 150) pu rified as a glutathione-S-transferase fusion protein (GST-ERK2) from E . coli has been done to determine regions/amino acids important for ac tivation by rabbit skeletal muscle MAP kinase kinase (rMEK) and kinase activity towards myelin basic protein (MBP). Five classes of mutants have been isolated. The first class of mutants comprises of G30A/G32A, A50D and R65A/R68A/E69A, that can be phosphorylated by rMEK and have no kinase activity towards MBP, the second class includes mutants D122 A/H123A and N142A which have lower kinase activities but no change in their activation by rMEK; third class being Y34A, E58A/H59A, which hav e neutral effect towards either activity, the fourth class that includ es completely inactive mutants D42A/K46A/R48A, the deletion mutant in the same region (-9aa[40-48]) and D104A/E107A/D109A and finally the fi fth class that include K53A, E94A/K97A/D99A, K112A/K115A and R133A/K13 6A that are phosphorylated 140-240% but with kinase activity toward MB P ranging from 50-100% of the wild type.