Functional binding between Gβ and the LIM domain of Ste5 is required to activate the MEKK Ste11

Background: In the budding yeast Saccharomyces cerevisiae, the pheromones that induce haploid cells of opposite cell types to mate activate the G beta and G gamma subunits of a heterotrimeric G protein. These subunits signal through the PAK kinase StePO to activate a mitogen-activated 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 high molecular weight complex. Ste5 is thought to associate with G beta in a pheromone-independent manner, but it is not known if this interaction 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 not transmit the pheromone signal from G beta through Ste20 to Ste11. The SteSC180A protein was impaired in binding G beta, although it could oligomerize, bind Ste11, Ste7 and Fus3, facilitate the basal activation of Ste11, and relay the Ste11 signal to MAP kinases, Ste5 bound to G beta in a pheromone-dependent manner and preferentially associated with a phosphorylated form of G beta in wild-type and ste20 Delta, but not in ste5C180A, strains. Conclusions: Pheromone induces binding of G beta to Ste5 through its LIM domain, This binding is essential for activation of Ste11 and is distinct from the ability of Ste5 to oligomerize or to serve as a scaffold and relay the signal from Ste11 to the MAP kinases. Pheromone also induces Ste5-dependent phosphorylation of G beta.