Ste7 variants that promote pathway specificity in S. cerevisiae

The yeast S. cerevisiae must respond to a wide variety of external stimuli with the appropriate differential program to ensure survival. Yeast cells transmit signals from external environmental cues via signaling cascades to bring about proper transcriptional programs. One such signaling cascade, the Mitogen Activated Protein Kinase (MAPK) cascade, is conserved in all organisms. The MAPK cascade that contains the sequentially acting kinases Ste20, Ste11, Ste7 and Kss1 is used in both filamentous growth differentiation in response to limiting nutrients as well as differentiation in response to mating pheromone. Despite the shared components signaling fidelity remains. The central kinase in both the mating and filamentous growth differentiation pathways is Ste7. We used a constitutively active version of this kinase to examine if biological specificity is lost by removing the need for external stimulus. We found that the constitutively active Ste7 promoted filamentous growth while simultaneously failing to support mating differentiation. One version of constitutively active Ste7 that could not be feedback phosphorylated in a MAPK dependent manner was unable to bind to the MAPK Fus3 but retained the ability to bind to Ste5 and Kss1. Despite being able to bind to Ste5 this version of Ste7 was unable to activate Fus3 but retained the ability to activate Kss1. The activation of Kss1 by constitutively active Ste7 is independent of the scaffold protein Ste5. Unlike Kss1, Fus3 activation requires Ste5. We isolated a substitution mutation variant of Ste7 that was impaired for binding to Ste5 but not impaired for activation of the transcriptional reporter gene for filamentous growth. This variant of Ste7 neither activated Fus3 nor supported mating differentiation in response to pheromone. Yeast cells expressing this variant of Ste7 displayed higher levels of phosphorylated Mpk1, the MAPK that functions in response to plasma membrane distortions. Our findings uncovered a role for Ste5 in maintaining signal specificity by allowing for specific activation of Fus3 by Ste7 in response to mating pheromone. We also uncovered a novel role that Mpk1 may play in the transcriptional response during filamentous growth. These data suggest MAPK signaling occurs through a network rather than linear pathways