Project description:In Saccharomyces cerevisiae impairment of protein phosphatase PP2A-Rts1 leads to temperature and hyperosmotic stress sensitivity, yet the underlying mechanism and the scope of action of the phosphatase in the stress response remain elusive. Using quantitative mass spectrometry-based approaches we have identified a set of putative substrate proteins that show both, hyperosmotic stress- and PP2A-Rts1-dependent changes in their phosphorylation pattern. A comparative analysis with published MS-shotgun data revealed that the phosphorylation status of many of these sites is regulated by the MAPKAP kinase Rck2, suggesting a node of regulation. Detailed gel mobility shift assays and protein-protein interaction analysis strongly suggest that Rck2 activity is directly regulated by PP2A-Rts1 via a SLiM B56-family interaction motif, uncovering a previously unknown mechanism of how PP2A influences the response to hyperosmotic stress in Yeast.
Project description:The hyperosmotic stress response in budding yeast is a paradigm for cellular responses to physicochemical stimuli that is often used for modeling signal transduction pathways. Here, we describe the phosphatase PP2A-Cdc55 as a novel master regulator of hyperosmotic stress signaling. We show that its inhibition by the Greatwall kinase-Endosulfine signaling module at the onset of hyperosmotic stress is crucial for cellular survival with far-reaching consequences for the stress-regulated phospho-proteome. Indeed, this mechanism is required and sufficient to induce stress-specific phosphorylation patterns. This system operates in parallel and independently of the well-established Hog1 MAP kinase pathway, affecting up to 50% of the stress-induced S/T-P motifs. Many of these motifs appear to be direct substrates of PP2A-Cdc55. We exemplify the functional impact of stress-induced inhibition of PP2A-Cdc55 on the transcriptional regulation of stress-associated genes via the transcriptional regulators Rph1 and Gis1.
Project description:Hsp90 is one of the most abundant and conserved proteins in the cell. Reduced levels or activity of Hsp90 causes defects in many cellular processes and also reveals genetic or non-genetic variation in populations. Despite information about Hsp90 protein-protein interactions, a global view of the Hsp90 regulated proteome in yeast is unavailable. To investigate the degree of dependency of individual yeast proteins on Hsp90, we used the SILAC method coupled with mass spectrometry (MS) to quantify around 4000 proteins in low-Hsp90 cells and observed that 904 proteins were changed in their abundance by more than 1.5 fold. When compared with the transcriptome of the same population of cells, two-thirds of the mis-regulated proteins were observed to be affected post-transcriptionally, of which the majority were down-regulated. Further analyses indicated that the down-regulated proteins are highly conserved and assume central roles in cellular networks with a high number of interacting partners, suggesting that Hsp90 buffers genetic or non-genetic variation through regulating protein network hubs. The down-regulated proteins were enriched for essential proteins previously unknown to be Hsp90-dependent. Finally, we observed that down-regulation of transcription factors and mating pathway components by attenuating Hsp90 function led to decreased target gene expression and pheromone response respectively, providing a direct link between observed proteome regulation and cellular phenotypes.
Project description:To gain insights into the signalling pathways regulated by Leukocyte common antigen-related protein (LAR), including those that are PDGF-dependent, we have carried out the first systematic analysis of LAR-regulated signal transduction using SILAC-based quantitative proteomic and phosphoproteomic techniques. We have analysed differential phosphorylation between wild-type mouse embryo fibroblasts (MEFs) or MEFS in which the LAR cytoplasmic phosphatase domains had been deleted (LARΔP).
Project description:The isotopic dimethyl labeling-based quantitative post-translational modification proteomics was applied to study the phosphoproteomic changes associated with the drought responses of two contrasting soybean cultivars. A total of 9,457 non-redundant, unambiguous, label-independent and repeatable phosphopeptides were subsequently identified from six experimental replicates, corresponding to 9,234 of deduced unique phosphoproteins. These soybean proteins contain a total of 20,880 phosphosites, 84.9% of which were found to be novel phosphosites of the soybean phosphoproteome. The overly post-translationally modified proteins is 2.05% of the phosphoproteins identified. Most of these extensively phosphorylated proteins are photoreceptors, mRNA-, histone- and phospholipid-binding as well as serine/threonine/tyrosine protein kinase/phosphatases. The subgroup population distribution of phosphoproteins over the number of the phosphosite of phosphoprotein follows the exponential decay law, Y=4.13e^(-0.098X)-0.04. From 218 significantly regulated unique phosphopeptide groups, 188 significantly regulated phosphoproteins were found, and they are enriched in biological functions of water transport and deprivation, methionine metabolic process, photosynthesis/light reaction, and response to cadmium ion, osmotic stress and ABA under drought treatment. A total of 15 and 20 drought-tolerant cultivar significantly regulated phosphoproteins are transcription factors and protein kinases/phosphatases, respectively. More than 50% of these phosphoproteins are considered to be novel regulatory components, presumably mediating the development of the soybean drought tolerance under water deprivation process. The association of five randomly selected phosphoproteins with the drought response was corroborated with the qRT-PCR method.
Project description:The isotopic dimethyl labeling-based quantitative post-translational modification proteomics was applied to study the phosphoproteomic changes associated with the drought responses of two contrasting soybean cultivars. A total of 9,457 non-redundant, unambiguous, label-independent and repeatable phosphopeptides were subsequently identified from six experimental replicates, corresponding to 9,234 of deduced unique phosphoproteins. These soybean proteins contain a total of 20,880 phosphosites, 84.9% of which were found to be novel phosphosites of the soybean phosphoproteome. The overly post-translationally modified proteins is 2.05% of the phosphoproteins identified. Most of these extensively phosphorylated proteins are photoreceptors, mRNA-, histone- and phospholipid-binding as well as serine/threonine/tyrosine protein kinase/phosphatases. The subgroup population distribution of phosphoproteins over the number of the phosphosite of phosphoprotein follows the exponential decay law, Y=4.13e^(-0.098X)-0.04. From 218 significantly regulated unique phosphopeptide groups, 188 significantly regulated phosphoproteins were found, and they are enriched in biological functions of water transport and deprivation, methionine metabolic process, photosynthesis/light reaction, and response to cadmium ion, osmotic stress and ABA under drought treatment. A total of 15 and 20 drought-tolerant cultivar significantly regulated phosphoproteins are transcription factors and protein kinases/phosphatases, respectively. More than 50% of these phosphoproteins are considered to be novel regulatory components, presumably mediating the development of the soybean drought tolerance under water deprivation process. The association of five randomly selected phosphoproteins with the drought response was corroborated with the qRT-PCR method.
Project description:Hyperhomocysteinemia (HHcy) inhibits growth and is cytotoxic to bacterial, yeast, and mammalian cells. The aim of this study was to determine the changes in proteome of the yeast induced by HHcy and map N-homocysteinylated sites. We identified 38 up- and 32-down-regulated proteins as well as 244 N-homocysteinylation sites in 98 proteins in Saccharomyces cerevisiae; with 57 sites in 34 proteins occurring in vivo. The bioinformatics analysis indicated that the N-homocysteinylated proteins were involved in a wide range of cellular functions with mostly cytosolic and ribosomal localizations. Furthermore, we discovered that lysine N-homocysteinylation sites are surrounded by neutral, hydrophobic and buried amino acid residues, and 60% of them occur within helix. The KEGG enrichment pathway analysis of these N-Hcy-proteins suggested that N-homocysteinylation disrupts metabolism of amino acids, ribosome biogenesis and glycolysis/gluconeogenesis. These findings suggest that protein N-homocysteinylation and dysregulation of cellular proteostasis affecting ribosomal proteins, biosynthesis of amino acids and changes in basic cellular pathways signaling are involved in the toxicity of HHcy in yeast. Homologous proteins are likely to be involved in HHcy toxicity in human and animal cells. We believe that the collection of N-homocysteinylation sites presented here is an important resource for future functional studies of N-homocysteinylation in yeast.
Project description:The formation of condensates in membraneless organelles is thought to be driven by protein phase separation. Arginine methylation and serine/threonine phosphorylation are important in the phase separation process, however these post-translational modifications are often present in intrinsically disordered regions that are difficult to analyse with standard proteomic techniques. Here we use a multi-protease and multi-MS/MS fragmentation approach, coupled with heavy methyl SILAC and phospho- or methyl-peptide enrichment, for the analysis of arginine methylation and serine/threonine phosphorylation, and to understand their co-occurrence in condensate-associated proteins. For Saccharomyces cerevisiae, we report a 50% increase in the known arginine methylproteome, involving 15 proteins that are almost all condensate-associated. Importantly, some of these proteins have arginine methylation on all predicted sites – providing evidence that this modification can be pervasive. We explored whether arginine methylated condensate-associated proteins are also phosphorylated, and found 12 such proteins to carry phosphoserine or phosphothreonine. In Npl3, Ded1 and Ssbp1, single peptides were found to carry both modifications, indicating a co-occurrence in close proximity and on the same protein molecule. We show that these co-modifications occur in regions of disorder and that arginine methylation is typically on basic regions of disorder. For phosphorylation, its association with charged regions of condensate-associated proteins was less consistent, although some regions with multisite phosphorylation sites were strongly acidic. We conclude that arginine-methylated proteins associated with condensates are typically co-modified with protein phosphorylation.
Project description:Cells arrest growth and enter a quiescent state upon nutrient deprivation. However, the molecular processes by which cells respond to different starvation signals to regulate exit from the cell division cycle and initiation of quiescence remains poorly understood. To study the role of protein expression and signaling in quiescence we combined temporal profiling of the proteome and phosphoproteome using stable isotope labeling with amino acids in cell culture (SILAC) in Saccharomyces cerevisiae (budding yeast). We find that carbon and phosphorus starvation signals activate quiescence through largely distinct proteome and phosphoproteome remodeling. However, increased expression of mitochondrial proteins is essential for quiescence establishment in response to both starvation signals. Whereas the quiescent proteome is established within 6 hours of starvation the quiescent phosphoproteome undergoes continuous changes for at least 30 hours following initial starvation. Deletion of the putative quiescence regulator RIM15, which encodes a serine-threonine kinase, results in reduced survival of cells starved for phosphorus and nitrogen, but not carbon. However, we identified common protein phosphorylation roles for RIM15 in quiescence that are enriched for RNA metabolism and translation. We also find evidence for RIM15-mediated phosphorylation of some targets, including IGO1, prior to starvation consistent with a functional role for RIM15 beyond quiescence regulation. Finally, we find evidence for widespread catabolism of amino acids in response to nitrogen starvation, indicating widespread amino acid recycling via salvage pathways in conditions lacking environmental nitrogen. Our study defines an expanded quiescent proteome and phosphoproteome in yeast, and highlights the multiple coordinated molecular processes at the level of protein expression that are required for quiescence.
Project description:Multiple discrete regions at 8q24 were recently shown to contain alleles that predispose to many cancers including prostate, breast, and colon. These regions are far from any annotated gene and their biological activities have been unknown. Here we profiled a 5-megabase chromatin segment encompassing all the risk regions for RNA expression, histone modifications, and locations occupied by RNA polymerase II and androgen receptor (AR). This led to the identification of several transcriptional enhancers, which were verified using reporter assays. Two enhancers in one risk region were occupied by AR and responded to androgen treatment; one contained a single nucleotide polymorphism (rs11986220) that resides within a FoxA1 binding site, with the prostate cancer risk allele facilitating both stronger FoxA1 binding and stronger androgen responsiveness. The study reported here exemplifies an approach that may be applied to any risk-associated allele in non-protein coding regions as it emerges from genome-wide association studies to better understand the genetic predisposition of complex diseases. 20 Normal Prostate cDNA samples (no replicates), 4 cell line cDNA samples (each cell line in duplicate), 4 cell line Acetylated H3 ChIP (each line in duplicate), 2 cell line various histone modification and protein ChIPs (6 Abs LNCaP, 5 Abs PC3, each in duplicate). All samples were hybridized with matching genomic DNA as reference.