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Phosphoproteomic analysis reveals interconnected system-wide responses to perturbations of kinases and phosphatases in yeast - PubMed

  • ️Fri Jan 01 2010

Comparative Study

Phosphoproteomic analysis reveals interconnected system-wide responses to perturbations of kinases and phosphatases in yeast

Bernd Bodenmiller et al. Sci Signal. 2010.

Abstract

The phosphorylation and dephosphorylation of proteins by kinases and phosphatases constitute an essential regulatory network in eukaryotic cells. This network supports the flow of information from sensors through signaling systems to effector molecules and ultimately drives the phenotype and function of cells, tissues, and organisms. Dysregulation of this process has severe consequences and is one of the main factors in the emergence and progression of diseases, including cancer. Thus, major efforts have been invested in developing specific inhibitors that modulate the activity of individual kinases or phosphatases; however, it has been difficult to assess how such pharmacological interventions would affect the cellular signaling network as a whole. Here, we used label-free, quantitative phosphoproteomics in a systematically perturbed model organism (Saccharomyces cerevisiae) to determine the relationships between 97 kinases, 27 phosphatases, and more than 1000 phosphoproteins. We identified 8814 regulated phosphorylation events, describing the first system-wide protein phosphorylation network in vivo. Our results show that, at steady state, inactivation of most kinases and phosphatases affected large parts of the phosphorylation-modulated signal transduction machinery-and not only the immediate downstream targets. The observed cellular growth phenotype was often well maintained despite the perturbations, arguing for considerable robustness in the system. Our results serve to constrain future models of cellular signaling and reinforce the idea that simple linear representations of signaling pathways might be insufficient for drug development and for describing organismal homeostasis.

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Figures

Fig. 1
Fig. 1

Integrated experimental and computational pipeline to determine in vivo kinase-substrate and phosphatase-substrate relationships. Yeast kinase and phosphatase genes were systematically deleted one by one and the phosphoproteomes were systematically compared between mutant and wild-type strains. To achieve this, for each mutant strain and wild-type, we grew and processed three independent biological replicates by proteome isolation, protein digestion with trypsin, phosphopeptide enrichment by applying a TiO2 resin, and quantification and identification of the phosphopeptides with LC-MS/MS. Observed phosphopeptide ion features were aligned, quantified, and tested for statistical significance. For the example phosphopeptide shown, IAS*PIQHEHDSGSR, the resulting matrix gives the intensity values measured in the wild-type and mutant samples, as well as the corresponding log2 fold change (here −3.76) with its associated significance. Abbreviations for the amino acids are as follows: A, Ala; D, Asp; E, Glu; G, Gly; H, His; I, Ile; P, Pro; Q, Gln; R, Arg; and S, Ser.

Fig. 2
Fig. 2

Matrix of kinases and phosphatases analyzed in this study and their effects on the phosphoproteome. Overall, 124 kinases and phosphatases were interrogated through our experimental and computational pipeline. Each row (y axis) corresponds to a regulated phosphopeptide and each column (x axis) summarizes the responders of a given kinase or phosphatase. Phosphopeptides with a directionality as expected (that is, kinase deletion resulted in a decrease in peptide abundance, whereas phosphatase deletion resulted in an increase in peptide abundance) are shown in graded blue, and phosphopeptides with an inverted directionality (evidence for indirect effect, not compatible with direct molecular target) are displayed in graded gold, according to the observed fold change for each peptide. Phosphopeptides observed but not regulated or not detected are displayed in gray. At the bottom, the total numbers of events observed in this study are listed. “Full response” corresponds to phosphopeptides that appeared or vanished when wild-type and mutant strains were compared, and “partial response” corresponds to phosphopeptides that showed a statistically significant change in abundance, but were detected in both wild-type and mutant samples. Abbreviations for the amino acids are as follows: A, Ala; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; and V, Val.

Fig. 3
Fig. 3

(A) Phosphoproteome-wide impact of each kinase and phosphatase. For all kinases and phosphatases, we computed the fraction of phosphopeptides affected relative to the total number of phosphopeptides affected by all kinases and phosphatases. The kinases and phosphatases were then ranked accordingly. Blue circles represent kinases, light blue circles represent essential kinases, and golden circles represent phosphatases. A large golden triangle indicates a strong growth or morphological phenotype of a given mutant, whereas a small blue triangle represents a weak growth or morphological phenotype of a given mutant. Right side: examples of kinases that showed either a low or a high effect on the phosphoproteome regions, together with their known cellular functions. (B) For each kinase and phosphatase, the biological processes enriched among their regulated phosphoproteins were computed. Each column corresponds to a biological process, whereas each row corresponds to a given kinase or phosphatase (kinases are depicted in blue, essential kinases in light blue, and phosphatases in gold). The color scale denotes the statistical significance of the observed enrichment. Magnified inset: an example for three clustered kinases, for which a related set of processes is observed enriched among their substrates.

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