INTRAMOLECULAR REGULATION OF PROTEIN-TYROSINE-PHOSPHATASE SH-PTP1 - ANEW FUNCTION FOR SRC HOMOLOGY-2 DOMAINS

The steady-state kinetic properties of SH-PTP1 (PTP1C, SHP, HCP), a Sr c homology 2 (SH2) domain-containing protein tyrosine phosphatase (PTP ase), were assessed and compared with those of three truncation mutant s, using p-nitrophenyl phosphate, phosphotyrosyl (pY) peptides, and re duced, carboxyamido-methylated, maleylated, and tyrosyl-phosphorylated lysozyme as substrates. At physiological pH (7.4), truncation of the two N-terminal SH2 domains [SH-PTP1(Delta SH2)] or the last 35 amino a cids of the C-terminus [SH-PTP1(Delta C35)] activated the phosphatase activity by 30-fold and 20-34-fold relative to the wild-type enzyme, r espectively. Truncation of the last 60 amino acids resulted in a mutan t [SH-PTP1(Delta C60)] with wild-type activity. SH-PTP1 and SH-PTP1(De lta C60) displayed apparent saturation kinetics toward pNPP only at ac idic pH (pH less than or equal to 5.4); as pH increased above 5.5, the ir apparent K-M values increased dramatically. In contrast, SH-PTP1(De lta SH2) obeyed normal Michaelis-Menten kinetics at all pH values test ed (pH 5.1-7.4) with a constant K-M (10-14 mM). Furthermore, two synth etic pY peptides corresponding to known and potential phosphorylation sites on the erythropoietin (EPOR pY429) and interleukin-3 (IL-3R pY62 8) receptors bound specifically to the N-terminal SH2 domain of SH-PTP 1 (K-D = 1.8-10 mu M) and activated the catalytic activity of SH-PTP1 and SH-PTP1(Delta C60) but not SH-PTP1(Delta SH2), in a concentration- dependent manner. Maximal activation (25-30-fold) of SH-PTP1 was achie ved at 70 mu M EPOR pY429, and the maximally activated enzyme approach ed the activity of SH-PTP1(Delta SH2). Addition of EPOR pY429 peptide, which corresponds to the recently identified in vivo binding site for SH-PTP1, at 40 mu M also completely restored the saturation kinetic b ehavior 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(Delta SH2). These data suggest that the SH2 domains of SH-PTP1 serve to auto inhibit the phosphatase activity of the PTPase domain, A model is prop osed in which the SH2 domains interact with the PTPase domain in a pY- independent fashion and drive the PTPase domain into an inactive confo rmation.