Synthesis of the ancestral PTP catalytic domain and its characterization both in vitro and in vivo.
Receptor protein tyrosine phosphatases (RPTPs) have a pivotal role in regulating both normal and pathological cellular processes. Most RPTPs have two homologous intracellular domains but only the membrane proximal one is catalytically active. It has been broadly accepted that during evolution a hypothetical ancestral PTP domain duplicated leading to extant RPTPs. We propose in this project to reconstruct and characterize the catalytic domain of an ancestor of RPTPs. Initially, the ancestor gene will be synthesized then coded protein will be expressed in a prokaryotic system and purified to high purity. Similarly, several truncated and mutant forms will be obtained. The catalytic efficiency and substrate specificity under in vitro and in vivo conditions for all constructs will be evaluated and compared. It is expected that sequence of the ancestor PTP catalytic domain and its characteristics will consistently contribute to our understanding of the evolution of PTPs and the physiological function of the ancestor PTP. In addition, new insights into the role played by the molecular determinants of PTPs’ catalytic efficiency and substrate specificity will be generated. We expect that our results will help designing PTPs with defined substrate specificity.