Time-Resolved Phosphoproteomics Unravel the Dynamics of Intracellular Signaling

Protein phosphorylation is a highly abundant post translational modification primarily observed on the amino acids Serine, Threonine and Tyrosine. Phosphate residues are transferred through kinases and are removed by protein phosphatases. Phosphorylation regulates the structural conformation of proteins, therefore modifying their function. It functions as an intracellular signaling mediator that regulates key processes such as protein-protein interactions and the activity of enzymes. Cell proliferation, differentiation and morphology are some of the most important cellular machineries regulated through phosphorylation signaling. Knowledge about phosphorylation pathways therefore is of high importance for cancer research. The study of protein phosphorylation has greatly benefited from the implementation of mass spectrometry based proteomics techniques that allows for the profiling of thousands of protein phosphorylation sites in a matter of hours. When investigating the effects of extracellular stimulations on the phosphoproteome, mass spectrometry experiments are typically designed by taking a snapshot of the phosphoproteome at a given time post stimulation. This type of analysis, however, does not allow to follow the temporal characteristics of phosphorylation that could explain the functioning of signaling pathways. This thesis describes the development and application of phosphoproteomics techniques that monitor phosphorylation signaling at high temporal resolution. This approach allowed the discovery of stimulation dependent signaling pathways and aided the refinement of already characterized phosphorylation networks.   At first, temporal profiling of the phosphoproteome was applied to study the effects of heat and cold stress on Saccharomyces cerevisiae within the first 30 minutes post stimulation. Fifteen different kinetic trends were discovered. Kinase-substrate interactions could be unraveled based on kinetic patterns of phosphorylation. In more detail, temporal information allowed the characterization of the signaling network of the kinases Cdc28 and PKA following temperature stress. These responses were associated with extensive changes in phosphorylation on proteins involved in transcription, protein folding and cell cycle progression.   In a second study, human colon cancer cells harboring somatic BRAF or KRAS mutations were subjected to treatments with the BRAF kinase inhibitor Vemurafenib. Temporally resolved phosphoprotoemics aided the discovery of novel targets of the BRAF-RAS-ERK signaling pathway. The transcription factors MKL1/2 and TEAD3 were found to be direct target of the kinase ERK. ERK mediated phosphorylation of MKL1/2 could be associated to actin binding, regulating their activity.   Third, a study investigating the effects of protein phosphatase 2 A (PP2A) small molecule activators on the phosphoprotoeme is described. Hematopoietic mouse cells were treated with the anti-oncogenic sphingolipids C2-ceramide and SH-BC-893 as well as the specific PP2A inhibitor LB-100. The investigated sphingolipids are known activators of PP2A that starve cancer cells to death. Both sphingolipids cause nutrient transporter loss, leading to cell starvation. In addition, SH-BC-893 causes vacuolation, a phenotype that is linked to nutrient mobilization. Phosphoprotoeme dynamics were monitored within 60 minutes post stimulation and unraveled differential activation of PP2A with sphingolipids. Comparison of sphingolipid response to the LB-100 perturbed phosphoprotoeme revealed PP2A directed phosphorylation on numerous GTPase and actin cytoskeletal proteins including Arhgef2 and Paxilin. Cell starvation was found to be partially dependent on sphingolipid induced actin polymerization. Lysosomal phosphoproteins affiliated with the kinase AKT and endosome recycling were differentially regulated by SH-BC-893 and C2-ceramide, partially explaining why C2-ceramide does not induce vacuolation.   Overall, temporal profiling of the phosphoproteome improved the depth of knowledge gained from phosphoproteomics experiments compared to conventional studies, and lead to the discovery of yet unknown biological mechanisms that are of importance to cancer research.

Keyphrases: Cancer, Phosphoproteomics, Intracellular signaling, Mass spectrometry, Small Molecule Drugs