Phosphoproteomic responses of TORC1 target kinases reveal discrete and convergent mechanisms that orchestrate the quiescence program in yeast

Dokládal, Ladislav; Stumpe, Michael; Hu, Zehan; Jaquenoud, Malika; Dengjel, Jörn; Virgilio, Claudio De

doi:10.1016/j.celrep.2021.110149

Cited by 25 publications

(29 citation statements)

References 132 publications

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“…Because serine–proline (SP)/threonine–proline (TP) motifs are among those reported to be frequently phosphorylated by kinases acting downstream of TORC1 ( 21 ), we initially focused on testing the five residues corresponding to SP and TP motifs found in Shp1 (S108, S315, S321, S322, and T331) as they have been previously identified by mass spectrometry as being phosphorylated ( 18 , 20 , 22 , 23 ). We generated mutant forms of Shp1-3xHA by changing candidate serine or threonine residues to alanine.…”

Section: Resultsmentioning

confidence: 99%

Multiple phosphorylation of the Cdc48/p97 cofactor protein Shp1/p47 occurs upon cell stress in budding yeast

et al. 2023

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The homohexameric p97 complex, composed of Cdc48 subunits in yeast, is a crucial component of protein quality control pathways including ER-associated degradation. The complex acts to segregate protein complexes in an ATP-dependent manner, requiring the engagement of cofactor proteins that determine substrate specificity. The function of different Cdc48 cofactors and how they are regulated remains relatively poorly understood. In this study, we assess the phosphorylation of Cdc48 adaptor proteins, revealing a unique and distinctive phosphorylation pattern of Shp1/p47 that changed in response to TORC1 inhibition. Site-directed mutagenesis confirmed that this pattern corresponded to phosphorylation at residues S108 and S315 of Shp1, with the double-phosphorylated form becoming predominant upon TORC1 inhibition, ER-stress, and oxidative stress. Finally, we assessed candidate kinases and phosphatases responsible for Shp1 phosphorylation and identified two regulators. We found that cells lacking the kinase Mpk1/Slt2 show reduced Shp1 phosphorylation, whereas impaired PP1 phosphatase catalytic subunit (Glc7) activity resulted in increased Shp1 phosphorylation. Overall, these findings identify a phosphoregulation of Shp1 at multiple sites by Mpk1 kinase and PP1 phosphatase upon various stresses.

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Section: Resultsmentioning

confidence: 99%

Multiple phosphorylation of the Cdc48/p97 cofactor protein Shp1/p47 occurs upon cell stress in budding yeast

et al. 2023

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“…To investigate the organization of major signaling hubs we chose to focus on the TOR signaling network. We selected 9 kinases and phosphatases associated with TOR signaling (Dokládal et al, 2021) and matched these with sets of phosphosite targets curated from the literature to produce a list of 934 total phosphosites with which we investigated dynamics within the TOR network (Figure S5A, Table S8). Given the number of phosphosites available to analyze, we employed Weighted Gene Correlation Network Analysis (WGCNA) (Zhang and Horvath, 2005) for determining groups of co-regulated phosphosites and their dynamics (Langfelder and Horvath, 2008).…”

Section: Activity and Architecture Of The Tor Signaling Networkmentioning

confidence: 99%

“…The categories used for the annotation of proteins were GO Biological Processes, GO Cellular Components, GO Molecular Functions, protein complexes and phenotypes of gene deletion mutants, all downloaded from SGD (https://www.yeastgenome.org/ on 04/05/2022. Kinases and phosphatase target annotation were downloaded from BioGrid (https://thebiogrid.org on 10/23/2020) and completed with data from (Dokládal et al, 2021;Goldman et al, 2014;Hu et al, 2019;Li et al, 2019;Rubenstein and Schmidt, 2007;Soste et al, 2014;Stark et al, 2010;Velázquez et al, 2020;Wagih et al, 2018). Cytoscape 3.9.1 was used to create and plot STRING networks of phosphoproteins at a minimum required STRING interaction score of 0.4.…”

Section: Other Bioinformatic Analysesmentioning

confidence: 99%

“…Numerous studies have characterized environmental stress responses in yeast, exploring gene expression programs (Causton et al, 2001;Costanzo et al, 2021;Gasch et al, 2000;Hohmann and Mager, 2007), signaling pathways (Bahn et al, 2007;Gutin et al, 2015) and metabolic (Oliveira et al, 2015;Schulz et al, 2014), proteomic (Gutin et al, 2019;Paulo et al, 2015), and phenotypic responses (Brauer et al, 2008;Hillenmeyer et al, 2008;Viéitez et al, 2022). Several studies have employed phosphoproteomics to elucidate phosphorylation responses to individual perturbations (Gruhler et al, 2005;Kanshin et al, 2015aKanshin et al, , 2015bLanz et al, 2021;Leutert et al, 2019;MacGilvray et al, 2018;Oliveira et al, 2012Oliveira et al, , 2015Saleem et al, 2010;Smolka et al, 2007;Vaga et al, 2014) or to the activation of specific pathways (Dokládal et al, 2021;Holt et al, 2009;Plank et al, 2020;Soste et al, 2014). Systems-level genetic perturbation studies have applied phosphoproteomics (Bodenmiller et al, 2010;Li et al, 2019) or orthogonal measurements (Schulz et al, 2014;da Silveira Dos Santos et al, 2014;Viéitez et al, 2022;van Wageningen et al, 2010) to uncover kinase-substrate relationships and functional phosphosites in yeast.…”

Section: Introductionmentioning

confidence: 99%

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The regulatory landscape of the yeast phosphoproteome

Leutert

Barente

Fukuda

et al. 2022

Preprint

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The cellular ability to react to environmental fluctuations depends on signaling networks that are controlled by the dynamic activities of kinases and phosphatases. To gain insight into these stress-responsive phosphorylation networks, we generated a quantitative mass spectrometry-based atlas of early phosphoproteomic responses in Saccharomyces cerevisiae exposed to 101 environmental and chemical perturbations. We report phosphosites on 59% of the yeast proteome, with 18% of the proteome harboring a phosphosite that is regulated within 5 minutes of stress exposure. We identify shared and perturbation-specific stress response programs, uncover dephosphorylation as an integral early event, and dissect the interconnected regulatory landscape of kinase-substrate networks, as we exemplify with TOR signaling. We further reveal functional organization principles of the stress-responsive phosphoproteome based on phosphorylation site motifs, kinase activities, subcellular localizations, shared functions, and pathway intersections. This information-rich map of 25,000 regulated phosphosites advances our understanding of signaling networks.

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“…This results in Tps2 dependent dephosphorylation of Rim15 permitting its release from Bmh2 enabling it to accumulate in the nucleus ( Kim et al, 2021 ). Here it directs the quiescence program predominantly by upregulating the partially redundant transcription activators Msn2/Msn4 and Hsf1 ( Cameroni et al, 2004 ; Roosen et al, 2005 ; Bontron et al, 2013 ; Lee et al, 2013 ; Dokladal et al, 2021 )} whose targets include genes required for glycogen and trehalose accumulation ( Boy-Marcotte et al, 1998 ; Pfanzagl et al, 2018 ). In response to nitrogen starvation, which both induces quiescence and upregulates macro-autophagy (hereon in referred to as autophagy) ( An et al, 2014 ), Rim15 also stimulates transcription of a subset of AuTophaGy ( ATG ) genes ( Delorme-Axford and Klionsky, 2018 ) by inactivating the Ume6 and Rph1 repressors ( Bartholomew et al, 2012 ; Bernard et al, 2015 ) although it remains unknown if the Rim15 induced phosphorylation of these repressors is direct.…”

Section: Introductionmentioning

confidence: 99%

Cyclin C-Cdk8 Kinase Phosphorylation of Rim15 Prevents the Aberrant Activation of Stress Response Genes

Willis

Hanley

Doyle

et al. 2022

Front. Cell Dev. Biol.

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Cells facing adverse environmental cues respond by inducing signal transduction pathways resulting in transcriptional reprograming. In the budding yeast Saccharomyces cerevisiae, nutrient deprivation stimulates stress response gene (SRG) transcription critical for entry into either quiescence or gametogenesis depending on the cell type. The induction of a subset of SRGs require nuclear translocation of the conserved serine-threonine kinase Rim15. However, Rim15 is also present in unstressed nuclei suggesting that additional activities are required to constrain its activity in the absence of stress. Here we show that Rim15 is directly phosphorylated by cyclin C-Cdk8, the conserved kinase module of the Mediator complex. Several results indicate that Cdk8-dependent phosphorylation prevents Rim15 activation in unstressed cells. First, Cdk8 does not control Rim15 subcellular localization and rim15∆ is epistatic to cdk8∆ with respect to SRG transcription and the execution of starvation programs required for viability. Next, Cdk8 phosphorylates a residue in the conserved PAS domain in vitro. This modification appears important as introducing a phosphomimetic at Cdk8 target residues reduces Rim15 activity. Moreover, the Rim15 phosphomimetic only compromises cell viability in stresses that induce cyclin C destruction as well as entrance into meiosis. Taken together, these findings suggest a model in which Cdk8 phosphorylation contributes to Rim15 repression whilst it cycles through the nucleus. Cyclin C destruction in response to stress inactivates Cdk8 which in turn stimulates Rim15 to maximize SRG transcription and cell survival.

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Phosphoproteomic responses of TORC1 target kinases reveal discrete and convergent mechanisms that orchestrate the quiescence program in yeast

Cited by 25 publications

References 132 publications

Multiple phosphorylation of the Cdc48/p97 cofactor protein Shp1/p47 occurs upon cell stress in budding yeast

Multiple phosphorylation of the Cdc48/p97 cofactor protein Shp1/p47 occurs upon cell stress in budding yeast

The regulatory landscape of the yeast phosphoproteome

Cyclin C-Cdk8 Kinase Phosphorylation of Rim15 Prevents the Aberrant Activation of Stress Response Genes

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