FUS3 PHOSPHORYLATES MULTIPLE COMPONENTS OF THE MATING SIGNAL-TRANSDUCTION CASCADE - EVIDENCE FOR STE12 AND FAR1

The mitogen-activated protein (MAP) kinase homologue FUS3 mediates bot h transcription and G1 arrest in a pheromone-induced signal transducti on cascade in Saccharomyces cerevisiae. We report an in vitro kinase a ssay for FUS3 and its use in identifying candidate substrates. The ass ay requires catalytically active FUS3 and pheromone induction. STE7, a MAP kinase kinase homologue, is needed for maximal activity. At least seven proteins that specifically associate with FUS3 are phosphorylat ed in the assay. Many of these substrates are physiologically relevant and are affected by in vivo levels of numerous signal transduction co mponents. One substrate is likely to be the transcription factor STE12 . A second is likely to be FAR1, a protein required for G1 arrest. FAR 1 was isolated as a multicopy suppressor of a nonarresting fus3 mutant and interacts with FUS3 in a two hybrid system. Consistent with this FAR1 is a good substrate in vitro and generates a FUS3-associated subs trate of expected size. These data support a model in which FUS3 media tes transcription and G1 arrest by direct activation of STE12 and FAR1 and phosphorylates many other proteins involved in the response to ph eromone.