Chemical genetics of AGC-kinases reveals shared targets of Ypk1, Protein Kinase A and Sch9

Abstract

23Protein phosphorylation cascades play a central role in the regulation of cell growth and protein 24 kinases PKA, Sch9 and Ypk1 take centre stage in regulating this process in S. cerevisiae. To 25 understand how these kinases co-ordinately regulate cellular functions we compared the 26 phospho-proteome of exponentially growing cells without and with acute chemical inhibition of 27 PKA, Sch9 and Ypk1. Sites hypo-phosphorylated upon PKA and Sch9 inhibition were preferentially 28 located in RRxS/T-motifs suggesting that many are directly phosphorylated by these enzymes. 29Interestingly, when inhibiting Ypk1 we not only detected several hypo-phosphorylated sites in the 30 previously reported RxRxxS/T-, but also in an RRxS/T-motif. Validation experiments revealed that 31 neutral trehalase Nth1, a known PKA target, is additionally phosphorylated and activated 32 downstream of Ypk1. Signalling through Ypk1 is therefore more closely related to PKA-and Sch9-33 signalling than previously appreciated and may perform functions previously only attributed to 34 the latter kinases. 35 36 2

Systems level analysis of time and stimuli specific signaling through PKA

“…It is well established that cAMP levels spike when glucose is added to yeast growing in a non-fermentable carbon source (such as glycerol) and then drop to a level just above the pre-stimulus baseline over a period of 10-20 min (Botman et al, 2021; Ma et al, 1999; Purwin et al, 1982; Thevelein and Beullens, 1985). Despite this, previous studies of PKA signaling have focused, almost entirely, on measuring PKA-dependent phosphorylation during steady state growth in glucose (Budovskaya et al, 2005; Plank et al, 2020; Searle et al, 2004; Yu et al, 2002). Therefore, to determine if the pulse of cAMP seen in fresh glucose activates a different signaling program than that found during log phase growth, we measured the phosphorylation changes that occur at various time-points after 2% glucose is added to cells growing in glycerol using phosphoproteomics.…”

“…In other words, gene expression data suggest that PKA is primarily active during the initial response to glucose, when cAMP levels are high. In contrast, studies of protein phosphorylation—virtually all of which have examined PKA signaling during steady state growth in glucose—show that PKA is active even when cAMP levels are low (Budovskaya et al, 2005; Plank et al, 2020; Searle et al, 2004; Yu et al, 2002).…”

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

“…Whether Sch9 may antagonize the quiescence program by inhibiting protein kinases other than Rim15, Slt2, and Yak1 is currently not known. To address this question, we reanalyzed a recently published phosphoproteome dataset, which served to identify proximal Sch9 targets that are hypophosphorylated following acute inhibition of an ATP-analog-sensitive Sch9 as allele (Plank et al, 2020). Accordingly, we were able to extract 121 residues that were oppositely regulated (i.e., hyper-phosphorylated) upon inactivation of Sch9 and that we were able to match with phospho-residues in our current study (Table S3; Figure S4A).…”

Phosphorylation of Jhd2 by the Ras-cAMP-PKA(Tpk2) pathway regulates histone modifications and autophagy

“…3g ). To further verify that Tpk2 phosphorylates Jhd2, we constructed a Shokat allele of Tpk2 (Tpk2-as) by mutating M147A within its kinase active site, whose kinase activity can be inhibited by adding the bulky ATP analog, 1NM-PP1 42 , 43 . The intracellular Jhd2 phosphorylation was significantly reduced in Tpk2-as mutant but not WT cells upon treatment with 1NM-PP1 (Supplementary Fig.…”