Methods for analyzing MAPK cascades.
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Animals↗
Biomedical subjects
Elaine A Elion
Publications and source records attributed to Elaine A Elion.
Formin-induced actin cables are required for polarized recruitment of the Ste5 scaffold and high level activation of MAPK Fus3.
Little is known about how a mitogen-activated protein kinase (MAPK) cascade is targeted to specific sites at the plasma membrane during receptor stimulation. In budding yeast, the Ste5 scaffold is recruited to a receptor-coupled G protein during mating pheromone stimulation, allowing the tethered MAPK cascade to be activated by Ste20, a Cdc42-anchored kinase. Here we show that stable recruitment of Ste5 at cortical sites requires the formin Bni1, Bni1-induced actin cables, Rho1 and Myo2. Rho1 directs recruitment of Bni1 via the Rho-binding domain, and Bni1 mediates localization of Ste5 through actin cables and Myo2, which co-immunoprecipitates with Ste5 during receptor stimulation. Bni1 is also required for polarized recruitment and full activation of MAPK Fus3, which must bind Ste5 to be activated, and polarized recruitment of Cdc24, the guanine exchange factor that binds Ste5 and promotes its recruitment to the G protein. In contrast, Bni1 is not important for activation of MAPK Kss1, which can be activated while not bound to Ste5 and does not accumulate at cortical sites. These findings reveal that Bni1 mediates the formation of a Ste5 scaffold/Fus3 MAPK signaling complex at polarized sites, and suggests that a pool of Ste5 may translocate along formin-induced actin cables to the cell cortex.
Actins↗
Signal transduction. Signaling specificity in yeast.
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Cell Cycle Proteins↗
Cdc24 regulates nuclear shuttling and recruitment of the Ste5 scaffold to a heterotrimeric G protein in Saccharomyces cerevisiae.
The Saccharomyces cerevisiae guanine nucleotide exchange factor Cdc24 regulates polarized growth by binding to Cdc42, a Rho-type GTPase that has many effectors, including Ste20 kinase, which activates multiple MAPK cascades. Here, we show that Cdc24 promotes MAPK signaling during mating through interactions with Ste5, a scaffold that must shuttle through the nucleus and bind to the beta subunit (Ste4) of a G protein for Ste20 to activate the tethered MAPK cascade. Ste5 was basally recruited to growth sites of G1 phase cells independently of Ste4. Loss of Cdc24 inhibited nuclear import and blocked basal and pheromone-induced recruitment of Ste5. Ste5 was not basally recruited and the MAPK Fus3 was not basally activated in the presence of a Cdc24 mutant (G168D) that still activates Cdc42, suggesting that Cdc24 regulates Ste5 and the associated MAPK cascade through a function that is not dependent on its guanine nucleotide exchange factor activity. Consistent with this, Cdc24 bound Ste5 and coprecipitated with Ste4 independently of Far1 and Ste5. Loss of Cdc24 decreased Ste5-Ste4 complex formation, and loss of Ste4 stimulated Cdc24-Ste5 complex formation. Collectively, these findings suggest that Cdc24 mediates site-specific localization of Ste5 to a heterotrimeric G protein and may therefore ensure localized activation of the associated MAPK cascade.
Actins↗
Localized feedback phosphorylation of Ste5p scaffold by associated MAPK cascade.
Scaffold proteins play pivotal roles during signal transduction. In Saccharomyces cerevisiae, the Ste5p scaffold protein is required for activation of the mating MAPK cascade in response to mating pheromone and assembles a G protein-MAPK cascade complex at the plasma membrane. To serve this function, Ste5p undergoes a regulated localization event involving nuclear shuttling and recruitment to the cell cortex. Here, we show that Ste5p is also subject to two types of phosphorylation and increases in abundance as a result of MAPK activation. During vegetative growth, Ste5p is basally phosphorylated through a process regulated by the CDK Cdc28p. During mating pheromone signaling, Ste5p undergoes increased phosphorylation by the mating MAPK cascade. Multiple kinases of the mating MAPK cascade contribute to pheromone-induced phosphorylation of Ste5p, with the mating MAPKs contributing the most. Pheromone induction or overexpression of the Ste4p Gbeta subunit increases the abundance of Ste5p at a post-translational step, as long as the mating MAPKs are present. Increasing the level of MAPK activation increases the amount of Ste5p at the cell cortex. Analysis of Ste5p localization mutants reveals a strict requirement for Ste5p recruitment to the plasma membrane for the pheromone-induced phosphorylation. These results suggest that the pool of Ste5p that is recruited to the plasma membrane selectively undergoes feedback phosphorylation by the associated MAPKs, leading to an increased pool of Ste5p at the site of polarized growth. These findings provide evidence of a spatially regulated mechanism for post-activation control of a signaling scaffold that potentiates pathway activation.
Adaptor Proteins, Signal Transducing↗
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.
1-Butanol↗
Making protein immunoprecipitates.
A wide variety of methods used in the study of signal transduction in eukaryotes rely on the ability to precipitate proteins from whole cell extracts. Immunoprecipitation and related methods of affinity purification are routinely used to assess binding partner interactions and enzyme activity in addition to the size of a protein, rates of protein synthesis and turnover, and protein abundance, thus making it a mainstay of a wide variety of protocols. This chapter will provide starting-point methods for immunoprecipitation of proteins under denaturing and nondenaturing conditions and the detection of protein-protein interactions by co-precipitation. The Notes section gives recommendations on how to troubleshoot potential problems that can arise while doing these methodologies.
Cell Extracts↗
A novel functional link between MAP kinase cascades and the Ras/cAMP pathway that regulates survival.
In mammalian cells, Ras regulates multiple effectors, including activators of mitogen-activated protein kinase (MAPK) cascades, phosphatidylinositol-3-kinase, and guanine nucleotide exchange factors (GEFs) for RalGTPases. In S. cerevisiae, Ras regulates the Kss1 MAPK cascade that promotes filamentous growth and cell integrity, but its major function is to activate adenylyl cyclase and control proliferation and survival ([; see Figure S1 in the Supplemental Data available with this article online). Previous work hints that the mating Fus3/Kss1 MAPK cascade cross-regulates the Ras/cAMP pathway during growth and mating, but direct evidence is lacking. Here, we report that Kss1 and Fus3 act upstream of the Ras/cAMP pathway to regulate survival. Loss of Fus3 increases cAMP and causes poor long-term survival and resistance to stress. These effects are dependent on Kss1 and Ras2. Activation of Kss1 by a hyperactive Ste11 MAPKKK also increases cAMP, but mating receptor/scaffold activation has little effect and may therefore insulate the MAPKs from cross-regulation. Catalytically inactive Fus3 represses cAMP by blocking accumulation of active Kss1 and by another function also shared by Kss1. The conserved RasGEF Cdc25 is a likely control point, because Kss1 and Fus3 complexes associate with and phosphorylate Cdc25. Cross-regulation of Cdc25 may be a general way that MAPKs control Ras signaling networks.
Cell Cycle Proteins↗
Nuclear export and plasma membrane recruitment of the Ste5 scaffold are coordinated with oligomerization and association with signal transduction components.
The Ste5 scaffold activates an associated mitogen-activated protein kinase cascade by binding through its RING-H2 domain to a Gbetagamma dimer (Ste4/Ste18) at the plasma membrane in a recruitment event that requires prior nuclear shuttling of Ste5. Genetic evidence suggests that Ste5 must oligomerize to function, but its impact on Ste5 function and localization is unknown. Herein, we show that oligomerization affects Ste5 activity and localization. The majority of Ste5 is monomeric, suggesting that oligomerization is tightly regulated. Increasing the pool of Ste5 oligomers increases association with Ste11. Remarkably, Ste5 oligomers are also more efficiently exported from the nucleus, retained in the cytoplasm by Ste11 and better recruited to the plasma membrane, resulting in constitutive activation of the mating mitogen-activated protein kinase cascade. Coprecipitation tests show that the RING-H2 domain is the key determinant of oligomerization. Mutational analysis suggests that the leucine-rich domain limits the accessibility of the RING-H2 domain and inhibits export and recruitment in addition to promoting Ste11 association and activation. Our results suggest that the major form of Ste5 is an inactive monomer with an inaccessible RING-H2 domain and Ste11 binding site, whereas the active form is an oligomer that is more efficiently exported and recruited and has a more accessible RING-H2 domain and Ste11 binding site.
Adaptor Proteins, Signal Transducing↗
How to monitor nuclear shuttling.
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Active Transport, Cell Nucleus↗