<p>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.</p>
<p>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, <i>in vivo </i>global phosphoproteomics, and <i>in
vitro</i> 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 <i>in
vitro </i>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. </p>
Chapter 3 describes a novel method that integrates <i>in
vivo </i>global phosphoproteomics perturbed by PTP1B inhibitor and stimulated
by insulin with <i>in vitro </i>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 <i>in
vivo </i>global phosphoproteomics experiment. CTTN, EGFR, FER, IRS1, PTPN11,
SRC, TYK2, PKM, GAB1, GAB2, and INSR were examined, which are PTP1B reported
substrates. <a>In <i>in vitro </i>kinetic profile of the
PTP1B phosphatase experiment, we utilized dimethyl labeling to quantify the
PTP1B dephosphorylation rate. No PTP1B substrate motif consensus was observed
in the labeling experiments. We finally overlapped <i>in vivo </i>and <i>in
vitro </i>experiments to identify PTP1B <i>bona fide </i>substrates with high
confidence.</a>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/17147843 |
| Date | 19 December 2021 |
| Creators | Peipei Zhu (11813591) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/thesis/DEVELOPMENT_OF_QUANTITATIVE_PROTEOMIC_STRATEGIES_TO_IDENTIFY_TYROSINE_PHOSPHATASE_SUBSTRATES/17147843 |
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