FUNCTIONAL BINDING BETWEEN G-BETA AND THE LIM DOMAIN OF STE5 IS REQUIRED TO ACTIVATE THE MEKK STE11

Background: In the budding yeast Saccharomyces cerevisiae, the pheromo nes that induce haploid cells of opposite cell types to mate activate the G beta and G gamma subunits of a heterotrimeric G protein. These s ubunits signal through the PAK kinase StePO to activate a mitogen-acti vated protein (MAP) kinase cascade comprising the MEKK Ste 11, the MEK Ste7 and two MAP kinases, Fus3 and Kss1. The pathway requires Ste5, a scaffold protein that tethers the MAP kinase cascade enzymes into a h igh molecular weight complex. Ste5 is thought to associate with G beta in a pheromone-independent manner, but it is not known if this intera ction affects signaling. Results: A ste5C180A mutant - which expresses Ste5 disrupted in the LIM domain, a putative metal-binding motif that has been proposed to be essential for Ste5 oligomerization - could no t transmit the pheromone signal from G beta through Ste20 to Ste11. Th e SteSC180A protein was impaired in binding G beta, although it could oligomerize, bind Ste11, Ste7 and Fus3, facilitate the basal activatio n of Ste11, and relay the Ste11 signal to MAP kinases, Ste5 bound to G beta in a pheromone-dependent manner and preferentially associated wi th a phosphorylated form of G beta in wild-type and ste20 Delta, but n ot in ste5C180A, strains. Conclusions: Pheromone induces binding of G beta to Ste5 through its LIM domain, This binding is essential for act ivation of Ste11 and is distinct from the ability of Ste5 to oligomeri ze or to serve as a scaffold and relay the signal from Ste11 to the MA P kinases. Pheromone also induces Ste5-dependent phosphorylation of G beta.