To study the biochemistry and the biology of MAP kinases we have been developing intrinsically active mutants of these enzymes. Active MAPKs are being isolated via genetic screens in yeast that look for MAPK molecules that function biologically in the complete absence of their MAPKK.
The location of the “activating” mutations within the enzyme and their mechanism of action disclose valuable aspects of the structure-function relationships of MAP kianses. The active mutants also serve as powerful tools for revealing the exact and specific biological functions of each MAPK.
So far we were able to produce a battery of mutants of the Hog1/p38 and Mpk1/ERK families. Mechanistic, structure-function and biological studies with these mutants will be reported.
Briefly, some of the mutants manifest an intrinsic catalytic activity in vitro and a spontaneous catalytic activity in vivo (in yeast and mammalian cells). These mutants have acquired an efficient autophosphorylation capability. Other mutants show no increase in catalysis, raising curiosity about their mechanism. Furthermore, one of the mutations in the ERK family occurred in the DEF pocket and seems to reduce catalysis.
For studying the function of each MAPK in cells, the active mutants are expressed in an inducible manner, followed by genomic and proteomic analysis.