Development of Quantitative Proteomic Strategies to Identify Tyrosine Phosphatase Substrates

Peipei Zhu, Purdue University

Abstract

Protein tyrosine phosphorylation is an essential posttranslational modification that controls cell signaling involving various biological processes, including cell growth, proliferation, migration, survival, and death. Balancing tyrosine phosphorylation levels is necessary for normal and pathological states, and this reversible mechanism occurs through protein tyrosine kinases and phosphatases. Advancements in instrumentation and applying conventional biochemical and genetic methods have led to cell signaling studies and pharmaceutical development discoveries. However, there is still a lack of understanding of tyrosine phosphatases' mechanisms, substrates, and activities within complex networks. The challenges remain in the tyrosine phosphatase field due to the low abundance and dynamic nature, sample preparation steps, and sensitivity to detect tyrosine phosphorylation events. Although mass spectrometry (MS)-based phosphoproteomics has allowed the identification of thousands of phosphotyrosine sites in a single run, protein phosphorylation poses another analysis caveat of dissecting complex phosphorylation signaling pathways involved in healthy cellular processes similarly in disease pathogenesis. This dissertation discusses strategies for improving tyrosine phosphatase sample preparation and identifying the tyrosine phosphatases' direct substrates. Chapter one is an overview of current techniques to study tyrosine phosphatases. In contrast, chapters two and three highlight the work that has been done to identify the direct substrates of phosphatase SHP2 and PTP1B, respectively, whose dysregulation leads to the development of cancers. In chapter 2, we describe a novel method that incorporated three separate MS-based experiments to identify the direct substrates of phosphatase SHP2: immunoprecipitation of substrate trapping mutants complex, in vivoglobal phosphoproteomics, and in vitro dephosphorylation of SHP2 phosphatase substrates. With immunoprecipitation of substrate trapping mutant experiment, weak and transient phosphatase-substrate interactions were detected by mass spectrometry after being stabilized by substrate trapping mutant method. This experiment not only identified the interactions between phosphatase and substrates but also revealed phosphotyrosine sites that are potentially protected in the substrate trapping mutant. We identified 80 phosphotyrosine proteins that showed upregulated in SHP2 mutant samples, and GAB1, GAB2, IRS1, SIRPA, and MPZL1 were examined in our list, which are reported SHP2 substrates. In the second experiment in parallel, we explored the global phosphorylation in HEK293 cells stimulated by epidermal growth factor. Peptides containing phosphotyrosine residues were captured by immobilized anti-pY PT-66 antibody and analyzed by LC-MS/MS. The results provided information on how SHP2 regulates downstream protein tyrosine phosphorylation and global phosphotyrosine response initiated by EGF. We used SHP2 substrate trapping mutant to isolate phosphotyrosine-containing proteins to serve as a SHP2 substrate pool for an in vitro phosphatase assay, then analyzed by LC-MS/MS. Finally, the overlap of the three separate MS-based experiments gave us the final list of high-confidence SHP2 substrates. DOK1 was validated to be a direct SHP2 substrate. Chapter 3 describes a novel method that integrates in vivo global phosphoproteomics perturbed by PTP1B inhibitor and stimulated by insulin with in vitro kinetic profile of PTP1B phosphatase to identify its substrates. We were able to identify 114 phosphotyrosine proteins that showed upregulated in PTP1B inhibitor and insulin-treated sample in in vivoglobal phosphoproteomics experiment. CTTN, EGFR, FER, IRS1, PTPN11, SRC, TYK2, PKM, GAB1, GAB2, and INSR were examined, which are PTP1B reported substrates.

Degree

Ph.D.

Advisors

Tao, Purdue University.

Subject Area

Analytical chemistry|Bioinformatics|Cellular biology|Chemistry|Pharmaceutical sciences|Web Studies

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