Phosphoproteomic Analysis of Protein Kinase C Signaling in Saccharomyces cerevisiae Reveals Slt2 Mitogen-activated Protein Kinase (MAPK)-dependent Phosphorylation of Eisosome Core Components

Abstract

The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1-cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasmamembrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2

An Analog-sensitive Version of the Protein Kinase Slt2 Allows Identification of Novel Targets of the Yeast Cell Wall Integrity Pathway

“…In addition, our labeling experiments using Slt2-as allowed us to confirm Gga1, Caf20, and Rcn2 to be direct substrates of Slt2 among a number of putative candidates that were identified in our previous phosphoproteomic analysis (18). Although thiophosphorylation-based approaches have been used in other cell types, to our knowledge this is the first evidence to indicate that this methodology can be successfully applied to identify yeast MAPK substrates.…”

“…In the case of Rcn2, each individual mutation at Ser-152, Ser-160, or Ser-255 reduced the thiophosphorylation of this protein by Slt2-as (Fig. 7B), but the most intense effect was observed when Ser-255, the only of these Rcn2 phosphosites previously detected as up-phosphorylated (18), was mutated. However, labeling was totally lost only in the mutant protein with all three residues substituted by alanine (Fig.…”

“…Caf20 contains only one site, corresponding to Thr-102, which was found up-phosphorylated in our previous phosphoproteomic analysis (18). Rcn2 includes four potential sites at Thr-49, Ser-152, Ser-160 and Ser-255, whereas Gga1 comprises seven of these sites along the protein.…”

“…Identification of Rcn2, Caf20, and Gga1 as Slt2 SubstratesBy using a quantitative SILAC (stable isotope labeling by amino acids in cell culture)-based phosphoproteomic approach, we previously identified 43 proteins containing phosphopeptides that display enhanced phosphorylation upon CWI pathway stimulation triggered by Pkc1 hyperactivation (18). Among them, 25 proteins displayed differentially phosphorylated (S/ T)P residues, constituting putative MAPK targets.…”

“…By using a quantitative stable isotope labeling by amino acids in cell culture (SILAC)-based phosphoproteomic approach, we previously identified a number of putative Slt2 targets that displayed enhanced phosphorylation at (S/T)P sites upon CWI pathway stimulation triggered by Pkc1 hyperactivation (18). Here, by using an analog-sensitive version of this MAPK (Slt2-as) and an ATP␥S analog in in vitro kinase assays, we confirm that three of these candidates, Rcn2, Gga1, and Caf20, are bona fide substrates of Slt2.…”