Abstract

A multicellular organism must be able to coordinate the actions of its cells in order to function effectively. As such, cells have developed mechanisms to sense and respond to changes in their environment and signals from other cells. One common mechanism by which signals are transmitted within cells involves protein phosphorylation. For example, signal transmission from many extracellular signaling molecules is initiated by receptors that are themselves protein tyrosine kinases (PTKs) or have an associated PTK activity. SH2 domains and protein tyrosine phosphatases (PTPs) play essential roles in transmitting these signals. SH2 domains are small domains of V100 amino acids that bind to phospho-tyrosine (pY) in the context of adjacent amino acids. and PTPs counteract the activity of PTKs by removing phosphates from tyrosine residues. Many SH2 domains and PTPs exist, yet in spite of significant overlap in the intracellular machinery of tyrosine phosphorylation signaling pathways. specificity of the different extracellular signals is maintained. The goal of the work presented in this thesis was to explore the basis of SH2 domain and PTP specificity. In the first part, the SH2 domains of the PTP SHP-2 were characterized. The specificity of these SH2 domains was shown to involve two residues N-terminal to the target pY in addition to three C-terminal residues. A single residue of the SH2 domains was found to play a critical role in directing the specificity requirement for N-terminal residues. This unique binding specificity defines an additional class of SH2 domain binding with general implications for SH2 domain-protein interactions. In the second part, PTP specificity was studied. First, the mechanism of inhibition of two non-specific PTP inhibitors, vanadate and pervanadate, was elucidated. While little was revealed regarding PTP specificity, different mechanisms of inhibition were uncovered for these inhibitors having important implications for their use. Second, kinetic studies with pY-peptide substrates were used to attempt to uncover specificity determinants for three transmembrane PTPs. Finally. a general peptide affinity selection technique that was initially explored with the SH2 domains of SHP-2 was refined to define the in vitro sequence selectivities of PTPs. This technology should aid in predicting potential in vivo targets for PTPs and in the development of specific PTP inhibitors.