Identification of novel pheromone-response regulators through systematic overexpression of 120 protein kinases in yeast

Protein kinases are well known to transmit and regulate signaling pathways. To identify additional regulators of the pheromone signaling apparatus in yeast, we evaluated an array of 120 likely protein kinases encoded by the y east genome. Each kinase was fused to glutathione S-transferase, overexpres sed, and tested for changes in pheromone responsiveness in vivo, As expecte d, several known components of the pathway (YCK1, STE7 STE11, FUS3, and KSS 1) impaired the growth arrest response. Seven other kinases also interfered with pheromone-induced growth arrest; in rank order they are as follows: Y KL116c (renamed PRR1) = YDL214c (renamed PRR2) > YJL141c (YAK1, SRA1) > YNR 047w = YCR091w (KIN82) = YIL095w (PRK1) > YCL024w (KCC4). Inhibition of phe romone signaling by PRR1, but not PRR2, required the glutathione S-transfer ase moiety, Both kinases inhibited gene transcription after stimulation wit h pheromone, a constitutively active kinase mutant STE11-4, or overexpressi on of the transcription factor STE12. Neither protein altered the ability o f the mitogen-activated protein kinase (MAPK) Fus3 to feedback phosphorylat e a known substrate, the MAPK kinase Ste7, These results reveal two new com ponents of the pheromone-signaling cascade in yeast, each acting at a point downstream of the MAPK.