Chemical Genetics of AGC-kinases Reveals Shared Targets of Ypk1, Protein Kinase A and Sch9

Plank, Michaël; Perepelkina, M. P.; Müller, Markus; Vaga, Stefania; Zou, Xiaoming; Bourgoint, Clélia; Berti, Marina; Saarbach, Jacques; Haesendonckx, Steven; Winssinger, Nicolas; Aebersold, Ruedi; Loewith, Robbie

doi:10.1074/mcp.ra120.001955

Cited by 19 publications

(12 citation statements)

References 77 publications

(117 reference statements)

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“…Importantly, however, not all known PKA targets were affected by rapamycin treatment, which was validated for a subset [ 25 ]. Similarly, we found that some, but not all sites hypo-phosphorylated upon PKA inhibition were also hypo-phosphorylated upon Sch9 inhibition [ 163 ]. No bona fide unique Sch9 site is known to date and the sites shared with PKA reside in the R[R/K]x[S/T] motif [ 94 , 120 ].…”

Section: Substrate Specificity Of Pka and Sch9mentioning

confidence: 85%

Interaction of TOR and PKA Signaling in S. cerevisiae

Plank

2022

Biomolecules

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TOR and PKA signaling are the major growth-regulatory nutrient-sensing pathways in S. cerevisiae. A number of experimental findings demonstrated a close relationship between these pathways: Both are responsive to glucose availability. Both regulate ribosome production on the transcriptional level and repress autophagy and the cellular stress response. Sch9, a major downstream effector of TORC1 presumably shares its kinase consensus motif with PKA, and genetic rescue and synthetic defects between PKA and Sch9 have been known for a long time. Further, studies in the first decade of this century have suggested direct regulation of PKA by TORC1. Nonetheless, the contribution of a potential direct cross-talk vs. potential sharing of targets between the pathways has still not been completely resolved. What is more, other findings have in contrast highlighted an antagonistic relationship between the two pathways. In this review, I explore the association between TOR and PKA signaling, mainly by focusing on proteins that are commonly referred to as shared TOR and PKA targets. Most of these proteins are transcription factors which to a large part explain the major transcriptional responses elicited by TOR and PKA upon nutrient shifts. I examine the evidence that these proteins are indeed direct targets of both pathways and which aspects of their regulation are targeted by TOR and PKA. I further explore if they are phosphorylated on shared sites by PKA and Sch9 or when experimental findings point towards regulation via the PP2ASit4/PP2A branch downstream of TORC1. Finally, I critically review data suggesting direct cross-talk between the pathways and its potential mechanism.

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Section: Substrate Specificity Of Pka and Sch9mentioning

confidence: 85%

Interaction of TOR and PKA Signaling in S. cerevisiae

Plank

2022

Biomolecules

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“…Therefore, in addition to Ypk1, some other as yet unidentified protein kinase in the Ypk1-as-(His) 6 -HA preparation is capable of phosphorylating these proteins at these specific sites. In this regard, it has been reported recently that some targets that contain sequences that harbor within them the Ypk1 consensus can also be phosphorylated by other AGC family protein kinases, including•PKA and Sch9 [59]. We have not yet tested the Ypk1-as-(His) 6 -HA preparation for the presence of either of these potential contaminants.…”

Section: Localization Of β-Propeller Proteins Laf1 and Dgr2 At Er-pm mentioning

confidence: 98%

TORC2-Dependent Ypk1-Mediated Phosphorylation of Lam2/Ltc4 Disrupts Its Association with the β-Propeller Protein Laf1 at Endoplasmic Reticulum-Plasma Membrane Contact Sites in the Yeast Saccharomyces cerevisiae

et al. 2020

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Membrane-tethered sterol-binding Lam/Ltc proteins localize at junctions between the endoplasmic reticulum (ER) membrane and other organelles. Two of the six family members—Lam2/Ltc4 (initially Ysp2) and paralog Lam4/Ltc3—localize to ER-plasma membrane (PM) contact sites (CSs) and mediate retrograde ergosterol transport from the PM to the ER. Our prior work demonstrated that Lam2 and Lam4 are substrates of TORC2-regulated protein kinase Ypk1, that Ypk1-mediated phosphorylation inhibits their function in retrograde sterol transport, and that PM sterol retention bolsters cell survival under stressful conditions. At ER-PM CSs, Lam2 and Lam4 associate with Laf1/Ymr102c and Dgr2/Ykl121w (paralogous WD40 repeat-containing proteins) that reportedly bind sterol. Using fluorescent tags, we found that Lam2 and Lam4 remain at ER-PM CSs when Laf1 and Dgr2 are absent, whereas neither Laf1 nor Dgr2 remain at ER-PM CSs when Lam2 and Lam4 are absent. Loss of Laf1 (but not Dgr2) impedes retrograde ergosterol transport, and a laf1∆ mutation does not exacerbate the transport defect of lam2∆ lam4∆ cells, indicating a shared function. Lam2 and Lam4 bind Laf1 and Dgr2 in vitro in a pull-down assay, and the PH domain in Lam2 hinders its interaction with Laf1. Lam2 phosphorylated by Ypk1, and Lam2 with phosphomimetic (Glu) replacements at its Ypk1 sites, exhibited a marked reduction in Laf1 binding. Thus, phosphorylation prevents Lam2 interaction with Laf1 at ER-PM CSs, providing a mechanism by which Ypk1 action inhibits retrograde sterol transport.

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“…The roles of PKA and TORC1 seem to be rather temporally than functionally distinct (Kunkel et al ., 2019). We did not specifically investigate the role of TORC2 signaling for the program reflected by the PT score but do not exclude a potential influence, since TORC2 was recently shown to share common targets with PKA and TORC1 via its downstream kinase Ypk1 (Plank et al, 2020). Other pathways, including AMPK/Snf1 signaling, regulate overlapping processes and cross-talk with PKA and TOR but affect limited sets of proteins if modulated independently (Kingsbury et al ., 2015; Malecki et al ., 2020; Zaman et al, 2009).…”

Section: Discussionmentioning

confidence: 99%

Large-scale changes of molecular network states explain complex traits

Weith

Großbach

Clément-Ziza

et al. 2022

Preprint

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The complexity of many cellular and organismal traits results from poorly understood mechanisms integrating genetic and environmental factors via molecular networks. Here, we show when and how genetic perturbations lead to molecular changes that are confined to small parts of a network versus when they lead to large-scale adaptations of global network states. Integrating multi-omics profiling of genetically heterogeneous budding and fission yeast strains with an array of cellular traits identified a central state transition of the yeast molecular network that is related to PKA and TOR (PT) signaling. Genetic variants affecting this PT state globally shifted the molecular network along a single-dimensional axis, thereby modulating processes including energy- and amino acid metabolism, transcription, translation, cell cycle control and cellular stress response. We propose that genetic effects can propagate through large parts of molecular networks because of the functional requirement to centrally coordinate the activity of fundamental cellular processes.

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Chemical Genetics of AGC-kinases Reveals Shared Targets of Ypk1, Protein Kinase A and Sch9

Cited by 19 publications

References 77 publications

Interaction of TOR and PKA Signaling in S. cerevisiae

Interaction of TOR and PKA Signaling in S. cerevisiae

TORC2-Dependent Ypk1-Mediated Phosphorylation of Lam2/Ltc4 Disrupts Its Association with the β-Propeller Protein Laf1 at Endoplasmic Reticulum-Plasma Membrane Contact Sites in the Yeast Saccharomyces cerevisiae

Large-scale changes of molecular network states explain complex traits

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