SUMMARY The spindle assembly checkpoint (SAC) kinase Mps1 not only inhibits anaphase but also corrects erroneous attachments that could lead to missegregation and aneuploidy. However, Mps1’s error correction-relevant substrates are unknown. Using a chemically tuned kinetochore-targeting assay, we show that Mps1 destabilizes microtubule attachments (K-fibers) epistatically to Aurora B, the other major error-correcting kinase. Through quantitative proteomics, we identify multiple sites of Mps1-regulated phosphorylation at the outer kinetochore. Substrate modification was microtubule-sensitive and opposed by PP2A-B56 phosphatases that stabilize chromosome-spindle attachment. Consistently, Mps1 inhibition rescued K-fiber stability after depleting PP2A-B56. We also identify the Ska complex as a key effector of Mps1 at the kinetochore-microtubule interface, as mutations that mimic constitutive phosphorylation destabilized K-fibers in vivo and reduced the efficiency of the Ska complex’s conversion from lattice diffusion to end-coupled microtubule binding in vitro. Our results reveal how Mps1 dynamically modifies kinetochores to correct improper attachments and ensure faithful chromosome segregation.

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...

Protein phosphorylation is the most important type of reversible post-translational modification involved in the regulation of cellular signal-transduction processes. In addition to controlling normal cellular physiology on the molecular level, perturbations of phosphorylation-based signaling networks and cascades have been implicated in the onset and progression of various human diseases. Recent advances in mass spectrometry-based proteomics helped to overcome many of the previous limitations in protein phosphorylation analysis. Improved isotope labeling and phosphopeptide enrichment strategies in conjunction with more powerful mass spectrometers and advances in data analysis have been integrated in highly efficient phosphoproteomics workflows, which are capable of monitoring up to several thousands of site-specific phosphorylation events within one large-scale analysis. Combined with ongoing efforts to define kinase-substrate relationships in intact cells, these major achievements have considerable potential to assess phosphorylation-based signaling networks on a system-wide scale. Here, we provide an overview of these exciting developments and their potential to transform signal-transduction research into a technology-driven, high-throughput science.

Protein phosphorylation controls the activity of signal transduction pathways regulated by kinases and phosphatases. Little is known, however, about the impact of preanalytical factors, for example, delayed times to tissue fixation, on global phosphoprotein levels in tissues. The aim of this study was to characterize the potential effects of delayed tissue preservation (cold ischemia) on the levels of phosphoproteins using targeted and nontargeted proteomic approaches. Rat and murine liver samples were exposed to different cold ischemic conditions ranging from 10 to 360 min prior to cryopreservation. The phosphoproteome was analyzed using reverse phase protein array (RPPA) technology and phosphoprotein-enriched quantitative tandem mass spectrometry (LC-MS/MS). RPPA analysis of rat liver tissues with long (up to 360 min) cold ischemia times did not reveal statistically significant alterations of specific phosphoproteins even though nonphosphorylated cytokeratin 18 (CK18) showed increased levels after 360 min of delay to freezing. Keeping the samples on ice prior to cryopreservation prevented this effect. LC-MS/MS-based quantification of 1684 phosphorylation sites in rat liver tissues showed broadening of their distribution compared to time point zero, but without reaching statistical significance for individual phosphosites. Similarly, RPPA analysis of mouse liver tissues with short (<60 min) cold ischemia times did not reveal directed or predictable changes of protein and phosphoprotein levels. Using LC-MS/MS and quantification of 791 phosphorylation sites, we found that the distribution of ratios compared to time point zero broadens with prolonged ischemia times, but these were rather undirected and diffuse changes, as we could not detect significant alterations of individual phosphosites. On the basis of our results from RPPA and LC-MS/MS analysis of rat and mouse liver tissues, we conclude that prolonged cold ischemia results in unspecific phosphoproteome changes that can be neither predicted nor assigned to individual proteins. On the other hand, we identified a number of phosphosites which were extraordinarily stable even after 360 min of cold ischemia and, therefore, may be used as general reference markers for future companion diagnostics for kinase inhibitors.