Use of Dominant-Negative/Substrate Trapping PTP Mutations to Search for PTP Interactors/Substrates.

Abstract

Phosphorylation of proteins on tyrosine residues is the consequence of coordinated action of tyrosine kinases (TKs), and protein tyrosine phosphatases (PTPs). Together, they regulate intermolecular interactions, subcellular localization, and activity of a variety of proteins. The level of total protein-associated tyrosine phosphorylation in eukaryotic cells is only a small fraction of the total phosphorylation. PTPs, which have high specific activity compared to tyrosine kinases, play an important role in maintaining the tyrosine phosphorylation state of proteins and regulate signal transduction pathways and cellular responses. PTPs depend on specific invariant residues that enable binding to substrates phosphorylated at tyrosine and aid catalytic activity. Identification of PTP substrates has helped understand their role in distinct intracellular signaling pathways. Because of their high specific activity, the interaction between tyrosine phosphatases and their substrates is often very transient in the cellular context, and therefore identification of physiological substrates has been difficult. Single-site mutations in the enzymes stabilize interaction between the enzyme and its targets and have been used extensively to identify substrates. The mutations are either of the catalytic cysteine (Cys) residue or other invariant residues and have been classified as substrate-trapping mutants (STMs). These mutants often serve as dominant negatives that can inactivate effector functions of a specific PTP within cells. Considering their association with human disorders, inhibiting specific PTPs is important therapeutically. Since the catalytic domains are largely conserved, developing small-molecule inhibitors to a particular enzyme has proven difficult and therefore alternate strategies to block functions of individual enzymes are seriously being investigated. We provide a description of methods that will be useful to design strategies of using dominant-negative and substrate-trapping mutants for identifying novel interacting partners and substrates of PTPs.