Intramolecular Regulation of Protein Tyrosine Phosphatase SH-PTP1: A New Function for Src Homology 2 Domains

The steady-state kinetic properties of SH-PTP1 (PTP1C, SHP, HCP), a Src homology 2 (SH2) domain-containing protein tyrosine phosphatase (PTPase), were assessed and compared with those of three truncation mutants, using p-nitrophenyl phosphate, phosphotyrosyl (pY) peptides, and reduced, carboxyamido-methylated, maleylated, and tyrosyl-phosphorylated lysozyme as substrates. At physiological pH (7.4), truncation of the two N-terminal SH2 domains [SH-PTP1(ΔSH2) or the last 35 amino acids of the C-terminus [SH-PTP1(ΔC35) activated the phosphatase activity by 30-fold and 20–34-fold relative to the wild-type enzyme, respectively. Truncation of the last 60 amino acids resulted in a mutant [SHPTP1(ΔC60)] with wild-type activity. SH-PTP1 and SH-PTP1(ΔC60) displayed apparent saturation kinetics toward pNPP only at acidic pH (pH ≤ 5.4); as pH increased above 5.5, their apparent K_M values increased dramatically. In contrast, SH-PTP1(ΔSH2) obeyed normal Michaelis-Menten kinetics at all pH values tested (pH 5.1–7.4) with a constant K_M (10–14 mM). Furthermore, two synthetic pY peptides corresponding to known and potential phosphorylation sites on the erythropoietin (EPOR pY429) and interleukin-3 (IL-3R pY628) receptors bound specifically to the N-terminal SH2 domain of SH-PTP1 (K_D = 1.8-10 μM) and activated the catalytic activity of SH-PTP1 and SH-PTP1(ΔC60) but not SHPTP1(ΔSH2), in a concentration-dependent manner. Maximal activation (25–30-fold) of SH-PTP1 was achieved at 70 μM EPOR pY429, and the maximally activated enzyme approached the activity of SHPTP1(ΔSH2). Addition of EPOR pY429 peptide, which corresponds to the recently identified in vivo binding site for SH-PTP1, at 40 μM also completely restored the saturation kinetic behavior of SH-PTP1 (at pH 7.4) toward pNPP, with catalytic parameters (K_M = 12.8 mM, k_cat = 3.2 s⁻¹) similar to those of SH-PTP1(ΔSH2). These data suggest that the SH2 domains of SH-PTP1 serve to autoinhibit the phosphatase activity of the PTPase domain. A model is proposed in which the SH2 domains interact with the PTPase domain in a pY-independent fashion and drive the PTPase domain into an inactive conformation.