Proteomic Analysis of Kinase Signaling
Eukaryotic cells respond to nutritional and environmental stress through complex regulatory programs controlling cell metabolism, growth, and morphology. In the budding yeast Saccharomyces cerevisiae, nutritional stress and environmental conditions can initiate a dramatic growth transition, wherein the yeast cells form extended multicellular filaments resembling the true hyphal tubes of filamentous fungi. The formation of these pseudohyphal filaments is governed by core regulatory pathways that have been studied for decades; however, the mechanism by which these signaling systems are integrated is less well understood. Our research indicates that the protein kinase Sks1p contributes to the integration of nitrogen stress and glucose stress signals, resulting in pseudohyphal growth. We recently implemented a mass spectrometry-based approach to profile phosphorylation events across the proteome dependent upon Sks1p kinase activity and identified phosphorylation sites important for mitochondrial function and pseudohyphal growth. Our studies place Sks1p in the regulatory context of the PKA pathway, a well-known signaling pathway responsive to the nutritional state of the cell. We further find that SKS1 is conserved and required for stress-responsive colony morphology in the principal opportunistic human fungal pathogen Candida albicans. Thus, Sks1p is part of the mechanism integrating glucose-responsive cell signaling and pseudohyphal growth, and its function is required for colony morphology linked with virulence in C. albicans.
Genetic Interaction Analysis of the RAM Network in Candida albicans
Candida albicans is the most common cause of fungal infections in humans. As a diploid yeast without a classical sexual cycle, many of the genetic approaches recently developed for large scale genetic interaction studies in the model yeast Saccharomyces cerevisiae cannot be applied to C. albicans. Genetic interaction studies have proven to be powerful genetic tools for the analysis of complex biological processes; so, we have adapted a synthetic genetic approach termed complex haploinsufficiency (CHI) for the analysis of regulatory pathways in Candida. CHI occurs when double heterozygous mutants show more severe phenotypes than strains with single heterozygous mutations; this CHI effect is indicative of a genetic interactions (e.g., genes within parallel pathways that regulate a common process).
We applied this approach to study the RAM (Regulation of Ace2 and Morphogenesis) signaling network in the morphogenetic transition of C. albicans from yeast to filamentous growth. Among the genes that interacted with CBK1, the key signaling kinase of the RAM pathway, were transcriptional targets of the RAM pathway and the protein kinase A pathway. Further analysis supports a model in which these two pathways co-regulate a common set of genes at different stages of filamentation.
Conditionally Controlling Protein Localization by Chemical-Induced Protein Dimerization
The functions of many proteins are strictly regulated by their subcellular localization. One of the most dramatic examples is the nucleo-cytoplasmic shuttling of certain transcription factors in response to physiological stimuli, which strictly governs their activity. Accordingly, the ability to selectively and reversibly control the subcellular localization of a protein is singularly useful as a tool in modulating its activity.
We have developed protocols for the utilization of chemically-directed protein dimerization as a platform for the rapid and reversible control of protein localization in the budding yeast Saccharomyces cerevisiae. This system is an expansion of methods for drug-induced protein dimerization first developed jointly by the Crabtree and Schrieber groups in the early 1990s. Building on these systems, we have developed the chemical-induced dimerization platform as a method for controlling nuclear trafficking (import/export) of target proteins in yeast. Specifically, this approach employs a pair of chimeras: one protein contains the small FRB domain fused to a target protein and the other consists of FKBP fused to a cellular address (nuclear localization sequence or nuclear export signal). Rapamycin and its analogs induces heterodimerization of these chimeras, conditionally placing a target protein under control of the cellular address. Thus, drug treatment can be used to direct nuclear import or export of the target protein. Target protein localization is rapid and reversible, with steady-state nuclear levels evident within 15 minutes of drug treatment. By this approach, target proteins can be conditionally sequestered from sites of activity to identify loss-of-function phenotypes, or conversely localized to sites of activity in the absence of endogenous cell signaling to identify if localization of the protein is sufficient for the relevant cell process.
Large-Scale Analysis of the Yeast Kinome During Filamentous Growth
Kinases and other signaling proteins are regulated such that their expression, modification, and localization are tightly controlled in response to cellular and environmental cues. While the former two regulatory mechanisms have been studied using high-throughput or global methods, differential protein localization has not been investigated systematically for any gene set in any organism. To consider this form of regulation more explicitly, we screened the full complement of known kinases in the budding yeast for differential localization under conditions inducing filamentous growth. Filamentous growth is an important stress response involving MAPK and PKA signaling modules, wherein certain strains of yeast form connected chains of elongated cells. As standard strains of yeast are non-filamentous, we constructed a unique set of 125 kinase-YFP chimeras in a filamentouss yeast strain for analysis of protein localization. In total, we identified five kinases (Fus3p, Ksp1p, Kss1p, Sks1p, Tpk2p) that shift their localization to the nucleus during filamentous growth. These kinases form part of an interdependent, localization-based regulatory network: deletion of each individual kinase disrupts the nuclear translocation of at least two other kinases. Utilizing kinase-dead alleles, we find that kinase activity is required for the observed localization shifts. We are currently using quantitative phosphoproteomics to identify the signaling networks of these and other filamentous growth kinases.