The opposing activities of phosphatases and kinases determine the phosphorylation status of proteins, yet kinases have received disproportionate attention in studies of cellular processes, with the roles of phosphatases remaining less understood. This dissertation describes the use of self-assembled monolayer laser desorption/ionization mass spectrometry (SAMDI-MS) together with peptide arrays to directly assay phosphatase activity, profile their substrate specificity and reveal novel cellar regulatory mechanisms protein tyrosine phosphatases (PTPs) are critically involved in. SHP2 is the first identified oncogenic phosphatase. In the first part of this work, we applied high-throughput SAMDI-MS assays for SHP2 drug discovery and reported an FDA-approved compound, adapalene, as a potent SHP2 inhibitor. We identified that the adamantyl functional group is crucial for its inhibition. SHP2 disease mutants were profiled with phosphotyrosine-containing peptide arrays to study the alteration of substrate specificity. We found that some mutants particularly favored aromatic residues at the -1 position adjacent to a phosphotyrosine. Extending the study to the whole PTP family in human proteome, twenty-two tyrosine phosphatases were characterized with the arrays to give a profile of their specificities. An analysis of the data revealed that certain residues in the substrates had a conserved effect on activity for all enzymes tested, including the general rule that inclusion of a basic lysine or arginine residue on either side of the phosphotyrosine decreased activity. This insight also provides a new perspective on the role of an R1152Q mutant in the insulin receptor, which is known to exhibit a lower phosphorylation level, and which our work suggests may be due to an increased activity towards phosphatase enzymes. We then identified more than 6,000 cancer mutations involving basic residues adjacent to known phosphotyrosine sites through a database search. Using two β-catenin mutants associated with cancer (T653R/K) and a mouse model for intellectual disability (T653K), we showed that T653-basic mutant β-catenins are less efficiently dephosphorylated by SHP1 phosphatase, leading to sustained Y654 phosphorylation and elevated downstream Wnt signal. This example rationalized how basic mutations proximal to phosphotyrosines can restrict counter-regulation by phosphatases, providing new mechanismistic and treatment insights for 6,000+ potentially relevant cancer mutations. Lastly, we showed that the same principle can be applied for a phosphorylation/chare-altering modification crosstalk via PTP. Using citrullination for example, we found the modification neutralized the basic arginine residue and increased the peptide dephosphorylation kcat/KM by 2.3-fold. This novel regulatory mechanism is generalizable because all PTPs lack activity towards substrates that have a basic residue proximal to the phosphotyrosine. This dissertation demonstrates the use of SAMDI-MS to provide a rapid and quantitative assay of phosphatase enzymes will be important to gaining a more complete understanding of the biochemistry and biology of this important enzyme class.

Creator

• Huang, Che-Fan

DOI

• https://doi.org/10.21985/n2-jwyq-3s46

Subject

• Chemistry
• Biochemistry
• Cellular biology

Language

• en

Alternate Identifier

• etdadmin_upload_819016
• http://dissertations.umi.com/northwestern:15570

Keyword

• SAMDI-MS
• Phosphatases
• High-Throughput Experiments
• Enzyme Specificity
• β-Catenin
• Peptide Arrays

Date created

• 2021-01-01

Resource type

• Dissertation

Rights statement

• In Copyright