Yeast Protein Kinase A Isoforms: A Means of Encoding Specificity in the Response to Diverse Stress Conditions? (original) (raw)
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Enzyme and Microbial Technology, 2000
Yeast cells growing in the presence of glucose or a related rapidly-fermented sugar differ strongly in a variety of physiological properties compared to cells growing in the absence of glucose. Part of these differences appear to be caused by the protein kinase A (PKA) and related signal transduction pathways. Addition of glucose to cells previously deprived of glucose triggers cAMP accumulation, which is apparently mediated by the Gpr1-Gpa2 G-protein coupled receptor system. However, the resulting effect on PKA-controlled properties is only transient when there is no complete growth medium present. When an essential nutrient is lacking, the cells arrest in the stationary phase G0. At the same time they acquire all characteristics of cells with low PKA activity, even if there is ample glucose present. When the essential nutrient is added again, a similar PKA-dependent protein phosphorylation cascade is triggered as observed after addition of glucose to glucosedeprived cells, but which is not cAMP-mediated. Because the pathway involved requires a fermentable carbon source and a complete growth medium, at least for its sustained activation, it has been called "fermentable growth medium (FGM)-induced pathway."
Cellular Signalling, 2006
Protein kinase A (PKA), in yeast, plays a major role in controlling metabolism and gene expression in connection with the available nutrient conditions. We here measure, for the first time, a transient change in the in vivo PKA activity, along a cAMP peak produced by 100 mM glucose addition to glycerol-growing cells as well as a change in the phosphorylation state of its catalytic subunit (Tpk1p) following PKA activation. PKA activity was measured in situ in permeabilized cells, preserving its intracellular localization. Comparison of total PKA activity, measured in situ in permeabilized cells with data obtained from in vitro assays in crude extracts, underscores the inhibitory potency of the regulatory subunit within the cell. Tpk1p phosphorylation was detected through non-denaturing gel electrophoresis. Phosphorylation of Tpk1p increases its specificity constant toward kemptide substrate. The use of mutants of the cAMP pathway showed that phosphorylation depends on the activation of PKA via the G-protein coupled receptor pathway triggered by glucose. The phosphorylation state of Tpk1p was followed during the diauxic shift. Tpk1p phosphorylation is dynamic and reversible: its up-regulation correlates with a fully fermentative metabolism, while its down-regulation with stationary phase or respiratory metabolism. Reversible phosphorylation can thus be considered a new control mechanism possibly pointing to a fine-tuning of PKA activity in response to environmental conditions.
Yeast, 2017
Yeast cells can adapt their growth in response to the nutritional environment. Glucose is the favorite carbon source of Saccharomyces cerevisiae that prefers a fermentative metabolism despite the presence of oxygen. When glucose is consumed, the cell switches to the aerobic metabolism of ethanol, during the so-called diauxic shift. The difference between fermentative and aerobic growth is in part mediated by a regulatory mechanism called glucose repression. During glucose derepression a profound gene transcriptional reprogramming occurs and genes involved in the utilization of alternative carbon sources are expressed. Protein kinase A (PKA) controls different physiological responses following the increment of cAMP as a consequence of a particular stimulus. cAMP-PKA is one of the major pathways involved in the transduction of glucose signaling. In this work the regulation of the promoters of the PKA subunits during respiratory and fermentative metabolism are studied. It is demonstrated that all these promoters are upregulated in the presence of glycerol as carbon source through the Snf1/Cat8 pathway. However, in the presence of glucose as carbon source, the regulation of each PKA promoter subunits is different and only TPK1 is repressed by the complex Hxk2/Mig1 in the presence of active Snf1.
Functional Interactions and Evolution of cAMP-PKA Signaling in Saccharomyces
2013
In an attempt to gain more insight on functional evolution of cAMP-PKA pathway I have taken a comparative approach and examined functional interactions of cAMP-PKA signaling in well-studied yeast developmental programs and closely related Saccharomyces sensu stricto species. I have shown that variation in cAMP-PKA signaling contributes significantly to variation in developmental responses in Saccharomyces cerevisiae. Variation in pseudohyphal growth and sporulation, two inversely correlated developmental strategies to nutrient limitation in yeast, proportional to variation in intracellular cAMP levels. S. cerevisiae strains proficient in pseudohyphal growth have higher intracellular cAMP concentrations relative to strains that sporulate efficiently. Phenotypic, genetic and signaling data presented here suggest that the cAMP-PKA signaling underlies a phenotypic trade-off between sporulation and pseudohyphal growth in S. cerevisiae. Further investigation into the role of cAMP-PKA signaling in closely related Saccharomyces paradoxus and Saccharomyces bayanus revealed an antagonistic function of cAMP-PKA signaling for developmental responses in S. bayanus. Unlike in S. cerevisiae, increased cAMP concentrations surprisingly inhibit pseudohyphal response in S. bayanus. Another unanticipated finding in this work is that in S. bayanus, Flo11, required for pseudohyphal differentiation in S. cerevisiae, is dispensable. Additionally, interactions of cAMP-PKA signaling and the general-stress response mechanism appear v reversed in S. bayanus. As shown by deletion mutation, gene expression and pharmacological treatment data, altered interactions and alternative targets downstream of cAMP-PKA could critically contribute to alternative regulation of nutrient-induced development in S. bayanus. Intracellular cAMP concentrations show decaying oscillations upon glucose replenishment in derepressed yeast cells. The quantitative characteristics of oscillations are distinct within and between Saccharomyces species. Given the tight regulation of cAMP levels and its critical role, the variation in cAMP oscillatory dynamics could be reflective of differential interactions of cAMP-PKA signaling that also underlie induction of developmental programs to changing environments. As such, intracellular cAMP levels and dynamics could potentially be used as molecular phenotypes.. vi Dedication I dedicate this thesis to my cherished grandparents, Cemile and Zeki Ural.
Molecular and Cellular Biology, 1990
Addition of glucose or related fermentable sugars to derepressed cells of the yeast Saccharomyces cerevisiae triggers a RAS-mediated cyclic AMP (cAMP) signal that induces a protein phosphorylation cascade. In yeast mutants (tpk1w1, tpk2w1, and tpk3w1) containing reduced activity of cAMP-dependent protein kinase, fermentable sugars, as opposed to nonfermentable carbon sources, induced a permanent hyperaccumulation of cAMP. This finding confirms previous conclusions that fermentable sugars are specific stimulators of cAMP synthesis in yeast cells. Despite the huge cAMP levels present in these mutants, deletion of the gene (BCY1) coding for the regulatory subunit of cAMP-dependent protein kinase severely reduced hyperaccumulation of cAMP. Glucose-induced hyperaccumulation of cAMP was also observed in exponential-phase glucose-grown cells of the tpklw1 and tpk2w1 strains but not the tpk3w1 strain even though addition of glucose to glucose-repressed wild-type cells did not induce a cAMP ...
G3: Genes|Genomes|Genetics
The cyclic AMP – Protein Kinase A (cAMP–PKA) pathway is an evolutionarily conserved eukaryotic signaling network that is essential for growth and development. In the fungi, cAMP–PKA signaling plays a critical role in regulating cellular physiology and morphological switches in response to nutrient availability. We undertook a comparative investigation of the role that cAMP-PKA signaling plays in the regulation of filamentous growth in two closely related budding yeast species, Saccharomyces cerevisiae and Saccharomyces bayanus. Using chemical and genetic perturbations of this pathway and its downstream targets we discovered divergent roles for cAMP-PKA signaling in the regulation of filamentous growth. While cAMP-PKA signaling is required for the filamentous growth response in both species, increasing or decreasing the activity of this pathway leads to drastically different phenotypic outcomes. In S. cerevisiae, cAMP-PKA inhibition ameliorates the filamentous growth response while h...
Fems Yeast Research, 2016
The cAMP-dependent protein kinase (PKA) signaling is a broad pathway that plays important roles in the transduction of environmental signals triggering precise physiological responses. However, how PKA achieves the cAMP-signal transduction specificity is still in study. The regulation of expression of subunits of PKA should contribute to the signal specificity. Saccharomyces cerevisiae PKA holoenzyme contains two catalytic subunits encoded by TPK1, TPK2 and TPK3 genes, and two regulatory subunits encoded by BCY1 gene. We studied the activity of these gene promoters using a fluorescent reporter synthetic genetic array screen, with the goal of systematically identifying novel regulators of expression of PKA subunits. Gene ontology analysis of the identified modulators showed enrichment not only in the category of transcriptional regulators, but also in less expected categories such as lipid and phosphate metabolism. Inositol, choline and phosphate were identified as novel upstream signals that regulate transcription of PKA subunit genes. The results support the role of transcription regulation of PKA subunits in cAMP specificity signaling. Interestingly, known targets of PKA phosphorylation are associated with the identified pathways opening the possibility of a reciprocal regulation. PKA would be coordinating different metabolic pathways and these processes would in turn regulate expression of the kinase subunits.
Current Genetics, 2006
The target of rapamycin (TOR) signaling pathway is an essential regulator of cell growth in eukaryotic cells. In Saccharomyces cerevisiae, TOR controls the expression of many genes involved in a wide array of distinct nutrient-responsive metabolic pathways. By exploring the TOR pathway under different growth conditions, we have identified novel TOR-regulated genes, including genes required for branched-chain amino acid biosynthesis as well as lysine biosynthesis (LYS genes). We show that TOR-dependent control of LYS gene expression occurs independently from previously identified LYS gene regulators and is instead coupled to cAMP-regulated protein kinase A (PKA). Additional genome-wide expression analyses reveal that TOR and PKA coregulate LYS gene expression in a pattern that is remarkably similar to genes within the ribosomal protein and ''Ribi'' regulon genes required for ribosome biogenesis. Moreover, this pattern of coregulation is distinct from other clusters of TOR/PKA coregulated genes, which includes genes involved in fermentation as well as aerobic respiration, suggesting that control of gene expression by TOR and PKA involves multiple modes of crosstalk. Our results underscore how multiple signaling pathways, general growth conditions, as well as the availability of specific nutrients contribute to the maintenance of appropriate patterns of gene activity in yeast.
FEMS Yeast Research, 2000
Protein kinase A (PKA) activity was measured in situ in permeabilised Saccharomyces cerevisiae cells in the absence and the presence of cAMP. Four strains genetically predicted to have differential PKA-dependent phenotypes were used: a wild-type strain and a strain containing a bcy1-14 mutation (with almost constitutively active PKA), and the same strains with overexpression of the wild-type or mutant BCY1 gene, respectively. Cells were grown on galactose or glucose. The measured phenotypic characteristics were: trehalose and glycogen levels and the activity of a reporter gene under control of the NTH1 promoter. The 'endogenous' PKA activity (measured in situ in the absence of cAMP) showed the best correlation with the PKA-dependent phenotypes determined in vivo. We propose that this parameter offers a good estimate for the degree of activation of PKA in vivo.
1991
The regulatory subunit of CAMP-dependent protein kinase in yeast, encoded by the BCYl gene, is known to be required under certain conditions such as growth on nonfermentable carbon sources and entry into stationary phase. We have identified novel isoforms of Bcyl in cells under these conditions. The isoforms are distinguishable by their migration on one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2-dimensional nonequilibrium pH gradient gel electrophoresis. The isoforms observed by one-dimensional SDS-PAGE bind CAMP, as determined by [S2P]8-azido-cAMP labeling (diagnostic of Bcyl protein). Proteins isolated from cells grown to stationary phase in rich medium exhibit five antibodyreactive bands, by one-dimensional SDS-PAGE immunoblot analysis, with apparent molecular masses of SO, 52, 66, 69 and 6 1 kDa. Total Bcyl protein increases at least 8-fold between exponential and stationary phase. Analysis of proteins from a variety of yeast mutants indicated that 1) many of the observed modifications of Bcyl are dependent upon the presence of the Ser-146 phosphorylation site; 2) the appearance of the 59-and 61-kDa bands is dependent upon the presence of Yak1 kinase; and 3) Bcyl protein is modified even in the absence of CAMP-dependent protein kinase catalytic subunits. Cells carrying the b~y l (a l a "~) allele exhibit non-wild type growth, indicating that these modifications may be functionally significant. During the yeast life cycle, the cell's ability to modulate CAMP-dependent protein kinase activity is essential for viability (Matsumoto et al., 1985). Analysis of mutants defective in various steps in the CAMP-dependent protein kinase pathway demonstrates that correct regulation of kinase activity is required for normal progression through the cell cycle, induction of thermotolerance, sporulation, and survival during starvation-induced arrest (Matsumoto et al., 1985; Shin et al., 1987; Uno et al., 1982). The specific role of the CAMPdependent protein kinase under these diverse physiological conditions is not known. Our laboratory is interested in characterizing the regulatory mechanisms involved in starvation-induced arrest or entry into stationary phase. Toward this end, we have begun to examine the dynamics of the CAMP-dependent protein kinase pathway under a variety of DCB-9000556 and PYI-9058136 (to M. W.-W.