Combination
of Two Activating Mutations in One HOG1 Gene Forms
Hyperactive Enzymes that Induce Growth Arrest.
Gilad Yaakov,
Michal Bell, David Engelberg
Dept. of Biological Chemistry, Hebrew University of Jerusalem, Givat Ram,
Jerusalem, 91904, Israel (gyaakov@pob.huji.ac.il)
Mitogen-Activated Protein Kinases (MAPKs) play key roles in differentiation, growth, proliferation and apoptosis. Although extensively studied, the precise function, specific substrates and target genes of each MAPK are not known. These issues could be addressed by sole activation of a given MAPK through the use of constitutively active MAPK enzymes. We have recently reported the isolation of eight hyperactive mutants of the yeast MAPK Hog1, each of which bears a distinct single point mutation. These mutants acquired high intrinsic catalytic activity, but did not impose the full biological potential of the HOG1 pathway, namely, lethality upon the yeast. Here we describe our attempt to obtain a MAPK catalytically and biologically more active. To this end, we combined two different activating point mutations in the same gene. A variety of combinations was tested, two of which acquired unusual properties. These alleles, HOG1D170A,F318L and HOG1D170A,F318S , induced a severe growth inhibition when expressed in yeast and had to be studied through an inducible expression system. This growth inhibition correlated with very high spontaneous (in the absence of stimulation) catalytic activity and induction of Hog1 target genes. We show that the effect on cell growth is not a result of cell death, but rather elongation of the G1 and S phases of the cell cycle. This study provides the first example of a MAPK intrinsically activated by mutations that induces a strong biological effect.