Revised Manuscript Received September 13, 1993® abstract: Full-length protein tyrosine phosphatase 1C (PTPlC), thecatalyticdomainofPTPlC (APTP1C), and the N-terminal SH2domain truncated PTP1C (ANPTP1C) were overexpressed in Escherichia coli and purified to near homogeneity. Various phosphorylated states of the synthetic phosphotyrosyl peptide TRDIYETDYYRK (IRP), corresponding to the major insulin receptor autophosphorylation sites, were used as substrates for the PTPs. There was no indication for selective dephosphorylation of any of the three phosphotyrosyl residues from the triphosphotyrosyl IRP. Kinetic studies were carried out using all seven different phosphotyrosyl IRPs. Saturation kinetics were observed for PTP1C using the triphosphotryosyl IRP only. In contrast, for APTP1C, saturation was achieved for all seven phosphotyrosyl IRPs. The best substrate for APTP1C was the triphosphotyrosyl IRP possessing a Km of approximately 1.6 pM, about 3-4-fold lower than either the mono-or diphosphotyrosyl IRPs. However, in contrast to APTP1C, PTP1C had a 22-fold lower affinity for triphosphotyrosyl IRP. Furthermore, deletion of a single N-terminal SH2 domain increased the affinity of the enzyme for the triphosphotyrosyl IRP to a value similar to that obtained with APTP1C. The pH optima for all three enzyme constructs were very similar and could not account for the observed change in substrate affinity between the three enzymes. These results suggest that the SH2 domain of PTP1C exerts an inhibitory effect on its PTP activity.

The phosphorylation of proteins on tyrosyl residues plays a central role in the regulation of a variety of cellular processes (Hunter, 1986, 1989; Ulrich & Schlessinger, 1990; Glenney, 1992). The phosphorylation status of a protein in the cell reflects a balance between two competing processes, namely, phosphorylation catalyzed by protein tyrosine kinases (PTKs) 1 and dephosphorylation mediated by protein tyrosine phosphatases (PTPs). It is currently known that the PTPs are a large family of enzymes specific for the dephosphorylation of phosphotyrosyl residues (Fischer et al., 1991; Saito & Streuli, 1991; Brautigan, 1992; Pot & Dixon, 1992). PTPs are divided into two groups. The first group of PTPs are transmembrane molecules having a variable extracellular domain (in some cases they may possess the hallmarks of a ligand binding