All MAPK cascades are characterized by a core of three levels protein module (MAP3K, MAP2K and MAPK) that activates each other by phosphorylation in a specific hierarchy. MAPK activation requires phosphorylation of a unique motif consists of two adjacent Thr and Tyr residues. This unusual dual phosphorylation is catalyzed by MAP2Ks. Several recent studies described alternative, MAP2K-independent pathways for p38 activation. All these alternative pathways induce autophosphorylation of p38.
The yeast MAPK Hog1 is an ortholog of the mammalian MAPKs p38 and JNK. It is activated in response to osmotic stress and upon activation affects osmolite synthesis, gene expression, and cell cycle regulation which allow the cells to adapt to the high osmotic pressure. Recently, we found that Hog1 is activated to some degree in pbs2Δ cells via a pathway that is osmostress-dependent and evokes autophosphorylation and autoactivation of Hog1. The components of this pathway are not known. The goal of the current work is to identify these components.
Using robotic-assisted system we constructed a library of about 5,500 yeast strains, each knocked out for PBS2 and for one more gene. In addition, all strains expressed the intrinsically active Hog1D170A mutant that allows growth of pbs2Δ cells under osmotic pressure. The rationale was to isolate pbs2ΔxxxΔ strains that cannot survive osmostress in spite of the presence of Hog1D170A. Such strains should lack genes that participate in the Pbs2-independent cascade of Hog1 activation. Several such strains have been isolated and are now under investigation.