Combination of Chemical Genetics and Phosphoproteomics for Kinase Signaling Analysis Enables Confident Identification of Cellular Downstream Targets

Abstract

Delineation of phosphorylation-based signaling networks requires reliable data about the underlying cellular kinasesubstrate interactions. We report a chemical genetics and quantitative phosphoproteomics approach that encompasses cellular kinase activation in combination with comparative replicate mass spectrometry analyses of cells expressing either inhibitor-sensitive or resistant kinase variant. We applied this workflow to Plk1 (Polo-like kinase 1) in mitotic cells and induced cellular Plk1 activity by wash-out of the bulky kinase inhibitor 3-MB-PP1, which targets a mutant kinase version with an enlarged catalytic pocket while not interfering with wild-type Plk1. We quantified more than 20,000 distinct phosphorylation sites by SILAC, approximately half of which were measured in at least two independent experiments in cells expressing mutant and wild-type Plk1. Based on replicate phosphorylation site quantifications in both mutant and wild-type Plk1 cells, our chemical genetic proteomics concept enabled stringent comparative statistics by significance analysis of microarrays, which unveiled more than 350 cellular downstream targets of Plk1 validated by full concordance of both statistical and experimental data. Our data point to hitherto poorly characterized aspects in Plk1-controlled mitotic progression and provide a largely extended resource for functional studies. We anticipate the described strategies to be of general utility for systematic and confident identification of cellular protein kinase substrates. Molecular & Cellular Proteomics 11: 10.1074/mcp.O111.012351, 1-12, 2012.Reversible protein phosphorylation by protein kinases represents a key control mechanism in signal transmission and controls nearly all aspects of cellular physiology. Quantitative proteomics approaches that incorporate techniques such as stable isotope labeling by amino acids in cell culture (SILAC), 1 phosphopeptide fractionation and enrichment by strong cation exchange (SCX), and ion metal affinity chromatography (IMAC) together with sensitive high resolution MS analysis and automated peptide identification and quantification have made it possible to monitor phosphorylation-based signaling on a global scale (1-4). Because signaling networks are defined by the underlying kinase-substrate relationships, systematic approaches are required for the comprehensive and confident assignment of cellular kinase substrates (5). To identify cellular substrates, the catalytic activity of a kinase of interest needs to be rapidly regulated to capture a high fraction of direct phosphorylation events. This implies that protein kinase ablation by genetic knockout or RNA interference can be of limited utility, because of secondary changes that can accumulate during the time required for cellular kinase depletion (3, 5). In contrast, pharmacological interference by small molecules allows for rapid modulation of kinase activity and should therefore enable unbiased monitoring of signaling perturbations when combined with advanced MS-based proteomics. S...

Mps1 Regulates Kinetochore-Microtubule Attachment Stability via the Ska Complex to Ensure Error-Free Chromosome Segregation

“…Next we used chemical genetics (Maciejowski et al, 2010) and global phosphoproteomics (Oppermann et al, 2012) to identify Mps1’s downstream targets with high confidence. This approach takes advantage of MPS1 -null retinal pigment epithelial (RPE) cells reconstituted with the wildtype kinase (Mps1 wt ) or an analog-sensitive allele (Mps1 as ) (Maciejowski et al, 2010).…”

“…2A). After pooling the SILAC-encoded cell lysates, chromatographically separated and enriched phosphopeptide fractions were prepared and analyzed by mass spectrometry (Oppermann et al, 2012). Over 27,000 phosphopeptides and 23,000 phosphosites were detected in four biological replicates of each cell line (Fig.…”

“…We leveraged the high level of phosphoproteome coverage to distinguish on-target (i.e., Mps1 as -specific) effects of 3MB-PP1 from potential off-target effects of the compound, which should manifest in both cell lines. For this purpose phosphorylation events quantified in at least two replicates of each cell line were statistically evaluated using SAM (Oppermann et al, 2012), revealing 19 phosphosites and 24 phosphopeptides that were significantly and greater than two-fold regulated in Mps1 as cells but not in Mps1 wt cells (estimated false-discovery rate (FDR) = 0%; Fig. S2A–B and Tables S1–S3).…”

High Mitotic Activity of Polo-like Kinase 1 Is Required for Chromosome Segregation and Genomic Integrity in Human Epithelial Cells

“…Although hundreds of Plk1 interactors and substrates have been identified (5)(6)(7), strong links between function and substrates are established for only a handful. Compounding this difficulty, its activities occur almost exclusively within the ϳ60 min of mitosis, making it difficult to isolate individual functional events.…”

Identification of Significant Features by the Global Mean Rank Test

“…Data originally published by Oppermann et al [21] with additional significance testing performed using the parametric MeanRank test, SAM and LIMMA.…”