Elaine A. Elion

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.

MAP kinase pathways

Mitogen-activated protein kinase (MAPK) pathways regulate diverse processes ranging from proliferation and differentiation to apoptosis. Activated by an enormous array of stimuli, they phosphorylate numerous proteins, including transcription factors, cytoskeletal proteins, kinases and other enzymes

FUS3 encodes a cdc2+/CDC28-related kinase required for the transition from mitosis into conjugation

FUS3 is required for both the arrest of cells in G1 and mating. Upon exposure to mating pheromone, fus3-1 and fus3-2 mutants fail to arrest in G1 and continue to divide while undergoing the transcription induction and morphological changes typical of mating cells. The G1 arrest defect of these fus3 mutants is suppressed by a daf1/whi1 null mutation (also called cln3, a putative cyclin). FUS3 has a positive role in conjugation, because overexpression of FUS3 increases the pheromone sensitivity of wild-type cells, while the absence of FUS3 causes sterility. The suppression of a gpa1 null (G alpha subunit) by a fus3 null also suggests FUS3 is in the signal transduction pathway. The predicted FUS3 protein is 35% identical to the cdc2+/CDC28 kinases and 52% identical to the KSS1 predicted kinase.

Pheromone response, mating and cell biology.

Saccharomyces cerevisiae responds to mating pheromones by activating a receptor-G-protein-coupled mitogen-activated protein kinase (MAPK) cascade that is also used by other signaling pathways. The activation of the MAPK cascade may involve conformational changes through prebound receptor and heterotrimeric G-protein. G beta may then recruit Cdc42-bound MAPKKKK Ste20 to MAPKKK Ste11 through direct interactions with Ste20 and the Ste5 scaffold. Ste20 activates Ste11 by derepressing an autoinhibitory domain. An underlying nuclear shuttling machinery may be required for proper recruitment of Ste5 to G beta. Subsequent polarized growth is mediated by a similar mechanism involving Far1, which binds G beta in addition to Cdc24 and Bem1. Far1 and Cdc24 also undergo nuclear shuttling and the nuclear pool of Far1 may temporally regulate access of Cdc24 to the cell cortex.

Nuclear Shuttling of Yeast Scaffold Ste5 Is Required for Its Recruitment to the Plasma Membrane and Activation of the Mating MAPK Cascade

Localization of Ste5 to GP at the plasma membrane is essential for transmission of the pheromone signal to associated MAP kinase cascade enzymes. Here, we show that this crucial localization requires prior shuttling of Ste5 through the nucleus. Ste5 shuttles through the nucleus constitutively during vegetative growth. Pheromone enhances nuclear export of Ste5, and this pool translocates vectorially to the cell periphery. Remarkably, Ste5 that cannot transit the nucleus is unable to localize at the periphery and activate the pathway, while Ste5 with enhanced transit through the nucleus has enhanced ability to localize to the periphery and activate the pathway. This novel regulatory scheme may ensure that cytoplasmic Ste5 does not activate downstream kinases in the absence of pheromone and could be applicable to other membrane-recruited signaling proteins.

Functional binding between Gβ and the LIM domain of Ste5 is required to activate the MEKK Ste11

BACKGROUND In the budding yeast Saccharomyces cerevisiae, the pheromones that induce haploid cells of opposite cell types to mate activate the Gbeta and Ggamma subunits of a heterotrimeric G protein. These subunits signal through the PAK kinase Ste20 to activate a mitogen-activated protein (MAP) kinase cascade comprising the MEKK Ste11, the MEK Ste7 and two MAP kinases, Fus3 and Kss1. The pathway requires Ste5, a scaffold protein that tethers the MAP kinase cascade enzymes into a high molecular weight complex. Ste5 is thought to associate with Gbeta in a pheromone-independent manner, but it is not known if this interaction affects signaling. RESULTS A ste5C180A mutant - which expresses Ste5 disrupted in the LIM domain, a putative metal-binding motif that has been proposed to be essential for Ste5 oligomerization - could not transmit the pheromone signal from Gbeta through Ste20 to Ste11. The Ste5C180A protein was impaired in binding Gbeta, although it could oligomerize, bind Ste11, Ste7 and Fus3, facilitate the basal activation of Ste11, and relay the Ste11 signal to MAP kinases. Ste5 bound to Gbeta in a pheromone-dependent manner and preferentially associated with a phosphorylated form of Gbeta in wild-type and ste20Delta, but not in ste5C180A, strains. CONCLUSIONS Pheromone induces binding of Gbeta to Ste5 through its LIM domain. This binding is essential for activation of Ste11 and is distinct from the ability of Ste5 to oligomerize or to serve as a scaffold and relay the signal from Ste11 to the MAP kinases. Pheromone also induces Ste5-dependent phosphorylation of Gbeta.

The osmoregulatory pathway represses mating pathway activity in Saccharomyces cerevisiae: isolation of a FUS3 mutant that is insensitive to the repression mechanism.

Mitogen-activated protein (MAP) kinase cascades are conserved signal transduction pathways that are required for eukaryotic cells to respond to a variety of stimuli. Multiple MAP kinase pathways can function within a single cell type; therefore, mechanisms that insulate one MAP kinase pathway from adventitious activations by parallel pathways may exist. We have studied interactions between the mating pheromone response and the osmoregulatory (high-osmolarity glycerol response [HOG]) pathways in Saccharomyces cerevisiae which utilize the MAP kinases Fus3p and Hog1p, respectively. Inactivating mutations in HOG pathway kinases cause an increase in the phosphotyrosine content of Fus3p, greater expression of pheromone-responsive genes, and increased sensitivity to growth arrest by pheromone. Therefore, the HOG pathway represses mating pathway activity. In a HOG1+ strain, Fus3p phosphotyrosine increases modestly and transiently following an increase in the extracellular osmolarity; however, it increases to a greater extent and for a sustained duration in a hog1-delta strain. Thus, the HOG-mediated repression of mating pathway activity may insulate the mating pathway from activation by osmotic stress. A FUS3 allele whose gene product is resistant to the HOG-mediated repression of its phosphotyrosine content has been isolated. This mutant encodes an amino acid substitution in the highly conserved DPXDEP motif in subdomain XI. Other investigators have shown that the corresponding amino acid is also mutated in a gain-of-function allele of the MAP kinase encoded by the rolled locus in Drosophila melanogaster. These data suggest that the DPXDEP motif plays a role in the negative regulation of MAP kinases.

Ste5: a meeting place for MAP kinases and their associates.

Growth and differentiation of the budding yeast Saccharomyces cerevisiae is regulated by six functionally distinct but structurally similar MAP kinase cascades. Three of the protein kinases in the cascade that regulates G1-phase arrest and mating have recently been shown to form a multikinase complex with a LIM-domain-containing protein called Ste5. These studies implicate Ste5 as a tethering protein that physically links protein kinases operating sequentially in a cascade. The significance of this complex for the regulation and specificity of signal transduction is explored in this review.

The SH3-domain protein Bem1 coordinates mitogen-activated protein kinase cascade activation with cell cycle control in Saccharomyces cerevisiae.

The mating mitogen-activated protein kinase (MAPK) cascade has three major outputs prior to fusion: transcriptional activation of many genes, cell cycle arrest in the G1 phase, and polarized growth. Bem1 localizes near the cortical actin cytoskeleton and is essential for polarized growth during mating. Here we show that Bem1 is required for efficient signal transduction and coordinates MAPK cascade activation with G1 arrest and mating. bem1delta null mutants are defective in G1 arrest and transcriptional activation in response to mating pheromone. Bem1 protein stimulates Fus3 (MAPK) activity and associates with Ste5, the tethering protein essential for activation of the MAPK kinase kinase Ste11. Bem1-Ste5 complexes also contain Ste11, Ste7 (MAPK kinase), and Fus3, suggesting that Ste5 localizes the MAPK cascade to Bem1. Strikingly, Bem1 also copurifies with Far1, a Fus3 substrate required for G1 arrest and proper polarized growth during mating. These and other results suggest that Bem1 may cross-link the Ste5-MAPK cascade complex to upstream activators and specific downstream substrates at the shmoo tip, thus enabling efficient circuitry for G1 arrest and mating.

Differential input by Ste5 scaffold and Msg5 phosphatase route a MAPK cascade to multiple outcomes

Pathway specificity is poorly understood for mitogen‐activated protein kinase (MAPK) cascades that control different outputs in response to different stimuli. In yeast, it is not known how the same MAPK cascade activates Kss1 MAPK to promote invasive growth (IG) and proliferation, and both Fus3 and Kss1 MAPKs to promote mating. Previous work has suggested that the Kss1 MAPK cascade is activated independently of the mating G protein (Ste4)–scaffold (Ste5) system during IG. Here we demonstrate that Ste4 and Ste5 activate Kss1 during IG and in response to multiple stimuli including butanol. Ste5 activates Kss1 by generating a pool of active MAPKKK (Ste11), whereas additional scaffolding is needed to activate Fus3. Scaffold‐independent activation of Kss1 can occur at multiple steps in the pathway, whereas Fus3 is strictly dependent on the scaffold. Pathway specificity is linked to Kss1 immunity to a MAPK phosphatase that constitutively inhibits basal activation of Fus3 and blocks activation of the mating pathway. These findings reveal the versatility of scaffolds and how a single MAPK cascade mediates different outputs.