The role of phosphorylation in the cellular organization of the proteome

PROJECT SUMMARY Protein phosphorylation is the most important and most extensively studied mechanism by which cells rapidly sense signals, transduce signals, and execute decisions. Protein phosphorylation provides a means to dynamically regulate protein function, is integral to all cellular processes, and abnormal phosphorylation is

associated with many diseases. In our previous research, we developed and applied experimental and computational phosphoproteomics methods to systematically interrogate the signaling network and reveal its architecture and design principles. Our rigorous research delivered reproducible methods, which are essential

to analyze phosphoproteomes in multi-perturbation studies. Our applications delivered knowledge that is crucially important to understand why different cell types respond differently to the same stimuli to accomplish different functions. Building on the methods we developed and knowledge acquired, we propose to focus our research for the next

5-years on the functional aspects of protein phosphorylation, addressing the outstanding question of how phosphorylation regulates the organization of the cellular proteome. We will expand our studies in multiple directions that represent the next frontier in signaling biology. First, we will identify phosphosites that are

relevant to protein functions at the proteomic scale. Second, we will study how phosphorylation regulates the multi-level organization of the cellular proteome from transient protein interactions to subcellular organelles. Third, we will study the differences in signaling of morphologically distinct cellular phenotypes. Fourth, we will

develop methods to include phosphorylation of tyrosine and histidine in phosphoproteomic studies. Fifth, we will assess the impact of mutations on protein functions at the proteomic scale. To achieve these goals, we will combine mass spectrometry-based proteomics, high throughput biochemistry, molecular biology, advanced microscopy, cell sorting, and computational and statistical methods, placing a

strong emphasis on expanding the capabilities of current proteomic methods. This research will deliver new methods and a wealth of knowledge of protein phosphorylation in a spatial, temporal, and functional context; which can be expanded in many new directions. Our discoveries on the basic functioning and regulation of

proteins can have an immediate translational impact by informing us about the functional consequences of mutations and changes in phosphorylation in disease states.