B.S., University of California, Los Angeles, 1983.
Ph.D., University of California, San Francisco 1992.
Postdoctoral Fellow, University of Washington, Seattle,
Signal Transduction and Cell Polarity
Research in my laboratory is aimed at understanding how cellular behavior is dictated by external signals. Our studies stress three general topics: What is the role of subcellular compartmentalization in signal transduction? How do cells judge the location of extracellular cues and mount a directional response? How is signaling specificity ensured?
To investigate these issues, we use the simple eukaryotic cells of bakers yeast,Saccharomyces cerevisiae. The yeast mating reaction provides examples of signal transduction and cell shape control in a model system that is highly amenable to experimentation using genetics, biochemistry, and cell biology. Here, secreted pheromones stimulate fusion of partner cells in a process involving cell cycle arrest, transcriptional regulation, and cell polarization. These responses are mediated by two signaling modules found in all eukaryotes from yeast to humans: heterotrimeric G proteins and mitogen activated protein (MAP) kinase cascades. We also study two other ubiquitous proteins, the GTPase Cdc42 and a PAK-family kinase (Ste20), which control signaling and cytoskeletal rearrangements.
Subcellular localization can play a crucial role in signal transduction. Activation of the mating pathway involves plasma membrane recruitment of the MAP kinase cascade “scaffold” protein, Ste5 (Pryciak and Huntress, 1998). (see Figure 1 and Figure 2) This recruitment requires synergistic protein-protein and protein-membrane interactions (Winters et al, 2005). Furthermore, Ste5 localization is regulated during the cell cycle by cyclin-dependent kinases (CDKs), which phosphorylate Ste5 near its membrane-binding domain and thus inhibit membrane recruitment and signaling (Strickfaden et al, 2007). This allows cells that are beginning a new division cycle to ignore the antiproliferative and differentiation effects of pheromone, which otherwise would cause a catastrophic division arrest. The factor awaiting the MAP kinase cascade at the membrane is the PAK-family kinase Ste20, whose activity and localization are regulated by multiple factors including Cdc42 (Lamson et al, 2002), the SH3 domain protein Bem1 (Winters and Pryciak, 2005), and direct membrane interactions (Takahashi and Pryciak, 2007). (see Figure 3)
Another project is focused on how cells polarize toward localized signals. (see Figure 4) This behavior, exhibited by many cell types, implies communication between proteins that sense the signal and those that govern cell polarity. Indeed, in addition to its role in activating the MAP kinase cascade, the yeast heterotrimeric G protein has a separate function in orienting cell polarity along gradients of pheromone chemoattractants. This involves the formation of multiprotein complexes between Gbg and polarity-control proteins (Butty et al, 1998). Our recent work suggests that Gbg must be regulated in a spatially-asymmetric manner by the receptor and Ga subunit in order for it to properly guide cell polarization (Strickfaden and Pryciak, 2008).
We are also interested in the fidelity of signaling pathways. In yeast, as in human cells, independent pathways generate different responses to different stimuli. But some proteins can function in multiple pathways, raising the question of how the pathways avoid “crosstalk”. (see Figure 4) Earlier, we showed that pathway-specific binding interactions can route signaling toward specific pathways. We developed a method to force shared signaling proteins to associate with a subset of their possible partners, creating custom signaling molecules that are “steered” toward chosen pathways (Harris et al, 2001). More recently, we found that membrane recruitment has an enhancing, or "amplifying" effect on signal transmission, which helps ensure signaling fidelity because it acts only on factors that are included in the recruited signaling complex (Lamson et al, 2006). This effect also alters the input-output signal processing behavior of the MAP kinase cascade, and may help explain why this pathway shows "graded" rather than "switch-like" dose-response behavior (Takahashi and Pryciak, 2008).
Figure 1. (A) Yeast mating reaction. Mating pheromones (a factor and a factor) cause cells to stop dividing, polarize toward their mating partners, and fuse with each other to form a diploid zygote. (B) Pheromone response pathway, emphasizing the membrane recruitment of the "scaffold" protein, Ste5, by the heterotrimeric G proteinbg dimer. This allows activation of the MAP kinase cascade.
Figure 2. (A) Synergy between two weak interactions contols Ste5 membrane recruitment. (B) The Ste5 PM domain is a putative amphipathic alpha helix that binds acidic phospholipid membranes in vivo and in vitro. (C) Model for CDK inhibition of signaling through the mating MAP kinase pathway. Phosphorylation of multiple sites flanking the PM domain electrostatically interferes with membrane binding, and hence disrupts signaling. The inset at bottom shows cell cycle-dependent fluctuations in the electrophoretic mobility of Ste5, indicative of periodic phosphorylation.
Figure 3. Regulation and localization of Cdc42 targets. (A) The PAK-family kinase Ste20 is localized and activated by the GTPase Cdc42. Our recent results show that this also requires direct membrane interaction by Ste20. (B) Localization of isolated membrane-binding motifs called BR domains (for "basic-rich") from three different Cdc42 targets. (C) Domain structure of yeast Cdc42 targets, illustrating the presence of a membrane-binding motif (BR or PH domain) immediately adjacent to the Cdc42-binding motif in each protein.
Figure 4. (A) Yeast cell polarization in response to pheromone. A gradient of pheromone normally serves as a spatial cue for the direction of polarization; cells polarize up the chemoattractant gradient in order to find mating partners. However, these cells can polarize in random directions when exposed to a uniform field of chemoattractant, implying the existence of "symmetry breaking" mechanisms that can generate asymmetric responses to symmetric signals, and "directional persistence" mechanisms that allow for continual reinforcement of the initially chosen direction. (B) Common components are shared among three separate signaling pathways: mating, filamentous growth, and the high osmolarity glycerol (HOG) response. Though individual proteins are shared (e.g., Cdc42, Ste20, Ste50, Ste11), these pathways are insulated from each other so that each stimulus activates only a single pathway. Pathway-specific scaffold proteins such as Ste5 and Pbs2 are thought to help provide this insulation. (In contrast, no known scaffold exists for the filamentation pathway.) Membrane-localized assembly can also contribute to signaling fidelity, by limiting phosphorylation events to those substrates that are co-localized with the active signaling complex.