The Noncatalytic C-terminal Segment of the T Cell Protein Tyrosine Phosphatase Regulates Activity via an Intramolecular Mechanism

Abstract

Human T cell protein tyrosine phosphatase (TCPTP) is a nontransmembrane enzyme, the first of the protein tyrosine phosphatase family to be cloned. Alternative mRNA splicing results in variation in the sequence at the extreme C terminus of TCPTP and generates a 45-kDa form (TC45) that is targeted to the nucleus and a 48-kDa variant (TC48) associated with membranes of the endoplasmic reticulum. In this report, we assessed the role of the C-terminal, noncatalytic segment of TCPTP in regulating activity, concentrating primarily on the TC45 variant. We have demonstrated that limited tryptic proteolysis of TC45 releases first a 42-kDa fragment, then a 33-kDa catalytic domain. Using reduced carboxyamidomethylated and maleylated lysozyme as substrate (RCML), the catalytic domain displays 20-100-fold more activity than the full-length enzyme. Analysis of the time course of limited trypsinolysis revealed that proteolytic activation occurred following cleavage of a protease-sensitive region (residues 353-387) located at the C terminus of TC45. The activity of truncation mutants illustrated that removal of 20 C-terminal residues was sufficient to activate the enzyme fully. The 33-kDa catalytic domain, but not the full-length enzyme, was inhibited in a concentration-dependent manner by addition of the noncatalytic C-terminal segment of TC45. A monoclonal antibody to TCPTP, CF4, which recognizes an epitope located between residues 350 and 363, was capable of fully activating TC45. These da…