LOSS OF SUSTAINED FUS3P KINASE-ACTIVITY AND THE G(1) ARREST RESPONSE IN CELLS EXPRESSING AN INAPPROPRIATE PHEROMONE RECEPTOR

The yeast pheromone response pathway is mediated hy two G protein-link ed receptors, each of which is expressed only in its specific cell typ e, The STE3(DAF) mutation results in inappropriate expression of the a -factor receptor in MATa cells, Expression of this receptor in the ina ppropriate cell type confers resistance to pheromone-induced G(1) arre st, a phenomenon that we have termed receptor inhibition, The ability of STE3(DAF) cells to cycle in the presence of pheromone was found to correlate with reduced phosphorylation of the cyclin-dependent kinase inhibitor Far1p, Measurement of Fus3p mitogen-activated protein (MAP) kinase activity in wild-type and STE3(DAF) cells showed that induction of Fus3p activity was the same in both strains at times of up to 1 h after pheromone treatment, However, after 2 or more hours, Fus3p activ ity declined in STE3(DAF) cells but remained high in wild-type cells, The level of inducible FUS1 RNA paralleled the changes seen in Fus3p a ctivity, Short-term activation of the Fus3p MAP kinase is therefore su fficient for the early transcriptional induction response to pheromone , but sustained activation is required for cell cycle arrest, Escape f rom the cell cycle arrest response was not seen in wild-type cells tre ated with low doses of pheromone, indicating that receptor inhibition is not simply a result of weak signaling but rather acts selectively a t late times during the response, STE3(DAF) was found to inhibit the p heromone response pathway at a step between the G(beta) subunit and St e5p, the scaffolding protein that binds the components of the MAP kina se phosphorylation cascade, Overexpression of Ste20p, a kinase thought to act between the G protein and the MAP kinase cascade, suppressed t he STE3(DAF) phenotype. These findings are consistent with a model in which receptor inhibition acts by blocking the signaling pathway downs tream of G protein dissociation and upstream of MAP kinase cascade act ivation, at a step that could directly involve Ste20p.