Regulation of Cell Signaling Dynamics by the Protein Kinase-Scaffold Ste5

Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Molecular cell (Impact Factor: 14.02). 07/2008; 30(5):649-56. DOI: 10.1016/j.molcel.2008.04.016
Source: PubMed


Cell differentiation requires the ability to detect and respond appropriately to a variety of extracellular signals. Here we investigate a differentiation switch induced by changes in the concentration of a single stimulus. Yeast cells exposed to high doses of mating pheromone undergo cell division arrest. Cells at intermediate doses become elongated and divide in the direction of a pheromone gradient (chemotropic growth). Either of the pheromone-responsive MAP kinases, Fus3 and Kss1, promotes cell elongation, but only Fus3 promotes chemotropic growth. Whereas Kss1 is activated rapidly and with a graded dose-response profile, Fus3 is activated slowly and exhibits a steeper dose-response relationship (ultrasensitivity). Fus3 activity requires the scaffold protein Ste5; when binding to Ste5 is abrogated, Fus3 behaves like Kss1, and the cells no longer respond to a gradient or mate efficiently with distant partners. We propose that scaffold proteins serve to modulate the temporal and dose-response behavior of the MAP kinase.

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    • "MATa ptp2::URA3 ptp3::LEU2 msg5::kanMX This study ste5 ND MATa ste5 ND Hao et al. (2008) "
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    ABSTRACT: Protein kinases regulate a broad array of cellular processes and do so through the phosphorylation of one or more sites within a given substrate. Many protein kinases are themselves regulated through multisite phosphorylation, and the addition or removal of phosphates can occur in a sequential (processive) or a stepwise (distributive) manner. Here we measured the relative abundance of the monophosphorylated and dual-phosphorylated forms of Fus3, a member of the mitogen-activated protein kinase (MAPK) family in yeast. We found that upon activation with pheromone, a substantial proportion of Fus3 accumulates in the monophosphorylated state. Introduction of an additional copy of Fus3 lacking either phosphorylation site leads to dampened signaling. Conversely, cells lacking the dual-specificity phosphatase (msg5Δ) or that are deficient in docking to the MAPK-scaffold (Ste5(ND)) accumulate a greater proportion of dual-phosphorylated Fus3. The double mutant exhibits a synergistic, or "synthetic," supersensitivity to pheromone. Finally, we present a predictive computational model that combines MAPK scaffold and phosphatase activities and is sufficient to account for the observed MAPK profiles. These results indicate that the monophosphorylated and dual-phosphorylated forms of the MAPK act in opposition to one another. Moreover, they reveal a new mechanism by which the MAPK scaffold acts dynamically to regulate signaling.
    Molecular biology of the cell 09/2015; 26(18):3359-71. DOI:10.1091/mbc.E15-01-0037 · 4.47 Impact Factor
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    • "The second dilution was made in 5 milliliter of synthetic complete medium with dextrose (SCD) and the cells were grown in an incubator shaker at 30ºC till the OD 600 reached 0.2. The live-cell imaging experiment was carried out in a microfluidic device described previously (Hao et al., 2008). The microfluidic device is mounted on an Olympus IX81 microscope with a Yokogawa "
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    ABSTRACT: G-protein coupled receptor (GPCR) signaling is fundamental to physiological processes, such as vision, the immune response and wound healing. In the budding yeast Saccharomyces cerevisiae, GPCRs detect and respond to gradients of pheromone during mating. Following pheromone stimulation, the GPCR Ste2 is removed from the cell membrane and new receptors are delivered to the growing edge. The Regulator of G-protein Signaling (RGS) protein, Sst2, acts by accelerating GTP hydrolysis and facilitating pathway desensitization. Sst2 is also known to interact with the receptor Ste2. Here we show that Sst2 is required for proper receptor recovery at the growing edge of pheromone-stimulated cells. Mathematical modeling suggested pheromone-induced synthesis of Sst2 and its interaction with the receptor function together to reestablish a receptor pool at the site of polarized growth. To validate the model we used targeted genetic perturbations to selectively disrupt key properties of Sst2 and disrupt its induction by pheromone. Together, our results reveal that a regulator of G protein signaling can also regulate the G protein-coupled receptor. Whereas Sst2 negatively regulates G protein signaling, it acts in a positive manner to promote receptor retention at the growing edge.
    Molecular biology of the cell 08/2015; DOI:10.1091/mbc.E14-12-1635 · 4.47 Impact Factor
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    • "As cells reenter the cell cycle, they exhibit a polar rather than an axial budding pattern (Erdman and Snyder, 2001). We call this cell type " chemotropic " because growth is hyperpolarized in the direction of increasing pheromone concentration (Segall, 1993; Paliwal et al., 2007; Hao et al., 2008). "
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    ABSTRACT: Mitogen activated protein kinase (MAPK) pathways control many cellular processes, including differentiation and proliferation. These pathways commonly activate MAPK isoforms that have redundant or overlapping function. However, recent studies have revealed circumstances where MAPK isoforms have specialized, non-overlapping roles in differentiation. The mechanisms that underlie this specialization are not well understood. To address this question, we sought to establish regulatory mechanisms that are unique to the MAPK Fus3 in pheromone induced mating and chemotropic fate transitions of the budding yeast, Saccharomyces cerevisiae. Our investigations reveal a previously unappreciated role for inactive Fus3 as a potent negative regulator of pheromone-induced chemotropism. We show that this inhibitory role is dependent on inactive Fus3 binding to the α subunit of the heterotrimeric G-protein. Further analysis revealed that the binding of catalytically active Fus3 to the G-protein is required for gradient tracking and serves to suppress cell-to-cell variability between mating and chemotropic fates in a population of pheromone responding cells. © 2015 by The American Society for Cell Biology.
    Molecular biology of the cell 07/2015; 26(18). DOI:10.1091/mbc.E15-03-0176 · 4.47 Impact Factor
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