Reversible phosphorylation of proteins on tyrosine controls the execution and regulation of many cellular processes. A proper level of tyrosyl phosphorylation is critical for these processes and is controlled by the opposing actions of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). A large number of PTPs (>100) have been identified and their importance in physiological and pathological processes has been demonstrated unambiguously. However, their mechanisms of action in these processes remain unclear. To understand their mechanisms of action, a critical first step is to identify their protein substrates involved in these processes, a currently very challenge task. This project describes the development and application of a chemical/bioinformatics approach to the identification of PTP substrates. In this approach, the substrate specificity of PTP is systematically determined by screening a combinatorial peptide library and the consensus motif(s) is used to search protein and genomic databases to identify potential protein substrates. The candidate proteins are subsequently validated as genuine PTP substrates (or rejected as false positives) by conventional cellular assays. In this project, we will focus our studies on three classical, non-receptor PTPs: the prototypical PTP1B and two Src homology 2 (SH2) domain-containing PTPs, SHP-1 and SHP-2. It consists of three specific aims.
Specific Aim 1 is to further develop the combinatorial peptide library method and determine the substrate specificity of PTP1B, SHP-1, and SHP-2.
Specific Aim 2 is to identify the in vivo protein substrates of PTP1B, SHP-1, and SHP-2.
Specific Aim 3 is to characterize SHP-2 mutants that are involved in human diseases. The binding and substrate specificity of the SH2 and PTP domains in SHP-2 mutants will be determined and the resulting information will be utilized to identify any protein substrates abnormally acted upon by SHP-2 mutants.

Public Health Relevance

PTPs play critical roles in both physiological and pathological processes and are therefore an important class of targets for chemotherapeutic intervention. PTP1B is currently being pursued as a target for treatment of type 2 diabetes. Mutations in SHP-2 cause Noonan syndrome, LEOPARD syndrome, and several leukemias. Catalytically defective SHP-1 mutants cause motheaten mice, which have a dysregulated immune system and die prematurely of inflammatory syndrome 2-3 weeks after birth. This project will investigate the molecular mechanisms by which these three PTPs function in physiological and pathological processes.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA132855-03
Application #
7995167
Study Section
Synthetic and Biological Chemistry B Study Section (SBCB)
Program Officer
Knowlton, John R
Project Start
2008-12-01
Project End
2013-11-30
Budget Start
2010-12-01
Budget End
2011-11-30
Support Year
3
Fiscal Year
2011
Total Cost
$259,419
Indirect Cost
Name
Ohio State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
832127323
City
Columbus
State
OH
Country
United States
Zip Code
43210
Upadhyaya, Punit; Bedewy, Walaa; Pei, Dehua (2016) Direct Inhibitors of Ras-Effector Protein Interactions. Mini Rev Med Chem 16:376-82
Trinh, Thi B; Pei, Dehua (2016) Screening One-Bead-One-Compound Peptide Libraries for Optimal Kinase Substrates. Methods Mol Biol 1360:169-81
Qian, Ziqing; Upadhyaya, Punit; Pei, Dehua (2015) Synthesis and screening of one-bead-one-compound cyclic peptide libraries. Methods Mol Biol 1248:39-53
Qian, Ziqing; Dougherty, Patrick G; Pei, Dehua (2015) Monitoring the cytosolic entry of cell-penetrating peptides using a pH-sensitive fluorophore. Chem Commun (Camb) 51:2162-5
Upadhyaya, Punit; Qian, Ziqing; Selner, Nicholas G et al. (2015) Inhibition of Ras signaling by blocking Ras-effector interactions with cyclic peptides. Angew Chem Int Ed Engl 54:7602-6
Qian, Ziqing; Xu, Xiaohua; Amacher, Jeanine F et al. (2015) Intracellular Delivery of Peptidyl Ligands by Reversible Cyclization: Discovery of a PDZ Domain Inhibitor that Rescues CFTR Activity. Angew Chem Int Ed Engl 54:5874-8
Qian, Ziqing; Dougherty, Patrick G; Liu, Tao et al. (2014) Structure-based optimization of a peptidyl inhibitor against calcineurin-nuclear factor of activated T cell (NFAT) interaction. J Med Chem 57:7792-7
Upadhyaya, Punit; Qian, Ziqing; Habir, Nurlaila A A et al. (2014) Direct Ras Inhibitors Identified from a Structurally Rigidified Bicyclic Peptide Library. Tetrahedron 70:7714-7720
Selner, Nicholas G; Luechapanichkul, Rinrada; Chen, Xianwen et al. (2014) Diverse levels of sequence selectivity and catalytic efficiency of protein-tyrosine phosphatases. Biochemistry 53:397-412
Lian, Wenlong; Jiang, Bisheng; Qian, Ziqing et al. (2014) Cell-permeable bicyclic peptide inhibitors against intracellular proteins. J Am Chem Soc 136:9830-3

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