Relative dependence of different outputs of the Saccharomyces cerevisiae pheromone response pathway on the MAP kinase Fus3p.

Fus3p and Kss1p act at the end of a conserved signaling cascade that mediates numerous cellular responses for mating. To determine the role of Fus3p in different outputs, we isolated and characterized a series of partial-function fus3 point mutants for their ability to phosphorylate a substrate (Ste7p), activate Ste12p, undergo G1 arrest, form shmoos, select partners, mate, and recover. All the mutations lie in residues that are conserved among MAP kinases and are predicted to affect either enzyme activity or binding to Ste7p or substrates. The data argue that Fus3p regulates the various outputs assayed through the phosphorylation of multiple substrates. Different levels of Fus3p function are required for individual outputs, with the most function required for shmoo formation, the terminal output. The ability of Fus3p to promote shmoo formation strongly correlates with its ability to promote G1 arrest, suggesting that the two events are coupled. Fus3p promotes recovery through a mechanism that is distinct from its ability to promote G1 arrest and may involve a mechanism that does not require kinase activity. Moreover, catalytically inactive Fus3p inhibits the ability of active Fus3p to activate Ste12p and hastens recovery without blocking G1 arrest or shmoo formation. These results raise the possibility that in the absence of sustained activation of Fus3p, catalytically inactive Fus3p blocks further differentiation by restoring mitotic growth. Finally, suppression analysis argues that Kss1p contributes to the overall pheromone response in a wild-type strain, but that Fus3p is the critical kinase for all of the outputs tested.

Ste5 tethers multiple protein kinases in the MAP kinase cascade required for mating in S. cerevisiae.

Ste5 is a Zn2+ finger-like protein thought to function before three kinases, Ste11 (a MEKK), Ste7 (a MEK), and Fus3 (a MAPK), in a conserved MAP kinase cascade required for mating in S. cerevisiae. Here, we present evidence that Ste5 forms a multikinase complex that joins these kinases for efficient Fus3 activation. By two-hybrid analysis, Ste11, Ste7, and Fus3 associate with different domains of Ste5, while Kss1, another MAPK, associates with the same domain as Fus3, thus implying that Ste5 simultaneously binds a MEKK, MEK, and MAPK. Ste5 copurifies with Ste11, Fus3, and a hypophosphorylated form of Ste7, and all four proteins cosediment in a glycerol gradient as if in a large complex. Ste5 also increases the amount of Ste11 complexed to Ste7 and Fus3 and is required for Ste11 to function. These results substantiate a novel signal transduction component that physically links multiple kinases within a single cascade.

The MAP kinase Fus3 associates with and phosphorylates the upstream signaling component Ste5.

Activation of the Saccharomyces cerevisiae MAP kinase Fus3 is thought to occur via a linear pathway involving the sequential action of three proteins: Ste5, a protein of unknown function, Ste11, a MAPKK kinase homolog, and Ste7, a MAPK kinase homolog which phosphorylates and activates Fus3. In this report, we present evidence for a novel mechanism of Fus3 activation that involves a direct association with Ste5, a protein not predicted to interact with Fus3. First, overexpression of Ste5 suppresses fus3 point mutations in an allele-specific manner and increases Fus3 kinase activity in vitro. Second, Ste5 associates with Fus3 in vivo as demonstrated by the two-hybrid system and by two methods of copurification. Third, Ste5 and Fus3 associate prior to pheromone stimulation even when Fus3 is inactive, and in strains lacking Ste7 and Ste11. Fourth Ste5 is phosphorylated by Fus3 in purified complexes and copurifies with an additional protein kinase(s). These observations suggest the possibility that Ste5 promotes signal transduction by tethering Fus3 to its activating protein kinase(s).

FUS3 phosphorylates multiple components of the mating signal transduction cascade: evidence for STE12 and FAR1.

The mitogen-activated protein (MAP) kinase homologue FUS3 mediates both transcription and G1 arrest in a pheromone-induced signal transduction cascade in Saccharomyces cerevisiae. We report an in vitro kinase assay for FUS3 and its use in identifying candidate substrates. The assay 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 phosphorylated in the assay. Many of these substrates are physiologically relevant and are affected by in vivo levels of numerous signal transduction components. One substrate is likely to be the transcription factor STE12. A second is likely to be FAR1, a protein required for G1 arrest. FAR1 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 substrate of expected size. These data support a model in which FUS3 mediates transcription and G1 arrest by direct activation of STE12 and FAR1 and phosphorylates many other proteins involved in the response to pheromone.

A direct role for c-fos in AP-1-dependent gene transcription.

Transcription factor activator protein 1 (AP-1) is a protein fraction that contains c-fos, c-jun, and several other related proteins. Although this protein fraction can stimulate transcription in vitro, the relative contributions of c-fos and c-jun to the transcriptional effect of AP-1 are not clear. In order to approach this question, we have overexpressed both proteins using a baculovirus-mediated expression system and defined their DNA-binding and transcriptional enhancement activities in vitro. Gel mobility-shift and DNase 1 footprinting assays showed that c-jun protein specifically binds to DNA through an AP-1 binding site. Under the same conditions, no detectable binding of c-fos protein was observed. However, when the DNA binding assays were performed in the presence of both c-jun and c-fos, a marked increase in the affinity of c-jun for the AP-1 site was observed. An AP-1-dependent transcription assay was used to test the capability of both proteins to stimulate correctly initiated RNA synthesis in vitro. Under our conditions, c-jun protein was capable of stimulating specific RNA transcription in an AP-1 site-dependent manner. In contrast, c-fos protein showed no detectable transcriptional activation by itself. However, a transcription assay carried out in the presence of both c-fos and c-jun proteins showed that the c-fos/c-jun complex was more active as a transcriptional regulator than c-jun protein alone. These experimental results indicate that c-fos and c-jun proteins are required to reconstitute full AP-1-dependent transcriptional activation and directly demonstrate that c-fos is a regulator of gene expression.

Control of the adipsin gene in adipocyte differentiation. Identification of distinct nuclear factors binding to single- and double-stranded DNA.

The mouse adipsin gene encodes a serine protease with complement factor D activity that is expressed during adipocyte differentiation and is deficient in several animal models of obesity. We have investigated the regulation of adipsin expression by transfecting preadipocytes and adipocytes with plasmids containing the 5'-flanking region of the adipsin gene linked to a reporter gene. Constructions containing a -950 to +35 segment of the adipsin promoter were preferentially expressed in adipose cells. Deletion experiments identified a region from -114 to -38 which contains a large inverted repeat sequence and negatively regulated gene expression in preadipocytes and positively regulated expression in fat cells. Exonuclease III protection and gel retardation assays indicated that this region of duplex DNA had multiple binding sites for nuclear factors, several of which were preadipose specific. In addition, we also identified two distinct factors that bound symmetrically and sequence specifically to the inverted repeat sequences only when they were in single-stranded form; one of these factors was induced during adipocyte differentiation. These results suggest that the control of the adipsin promoter in differentiation may involve an interplay of multiple regulated DNA-binding proteins, including two that have preferential affinity for single-stranded DNA.