Regulation of the Low Molecular Weight Phosphotyrosine Phosphatase by Phosphorylation at Tyrosines 131 and 132

Abstract

Activation of resting T lymphocytes is initiated by rapid but transient tyrosine phosphorylation of a number of cellular proteins. Several protein tyrosine kinases and protein tyrosine phosphatases are known to be important for this response. Here we report that normal T lymphocytes express the B isoform of low molecular weight protein tyrosine phosphatase B (LMPTP-B). The cDNA was cloned from Jurkat T cells, and an antiserum was raised against it. LMPTP immunoprecipitated from resting Jurkat T cells was found to be tyrosine phosphorylated. On stimulation of the cells through their T cell antigen receptor, the phosphotyrosine content of LMPTP-B declined rapidly. In co-transfected COS cells, Lck and Fyn caused phosphorylation of LMPTP, whereas Csk, Zap, and Jak2 did not. Most of the phosphate was located at Tyr-131, and some was also located at Tyr-132. Incubation of wild-type LMPTP with Lck and adenosine 5-O-(thiotriphosphate) caused a 2-fold increase in the activity of LMPTP. Site-directed mutagenesis showed that Tyr-131 is important for the catalytic activity of LMPTP, and that thiophosphorylation of Tyr-131, and to a lesser degree Tyr-132, is responsible for the activation.One of the earliest biochemical events seen in T lymphocytes triggered through the T cell antigen receptor complex is the enhanced phosphorylation of a number of cellular proteins on tyrosine residues (1, 2). Inhibition of this event by pharmacological agents prevents T cell activation as measured by both functional readouts and biochemical assays (3, 4). It has become evident that several protein tyrosine kinases (PTKs) 1 and the CD45 protein tyrosine phosphatase (PTPase) play crucial roles (reviewed in Refs. 5-7), and that the T cell antigen receptor-induced cascade of transient tyrosine phosphorylation events depends on a dynamic interplay between these and, presumably, many additional PTKs and PTPases. In addition to CD45 (8 -10), only two other PTPases have been implicated in T cell activation, namely SHP1 (11) and SHP2 (12).The low molecular weight PTPases LMPTP-A and LMPTP-B constitute a class of PTPases with limited sequence homology to the other PTPases (13-16). Nevertheless, these enzymes are highly specific for PTyr (14). Chemical modifications and mutagenesis experiments have shown that their catalytic mechanism involves a cysteine residue, Cys-12, which participates in phosphoenzyme intermediate formation (15), as in other PTPases. The recent crystallization of LMPTP (16) showed that the catalytic center is quite similar to that of "classical" PTPases, with Cys-12 residing in the bottom of the catalytic pocket.The physiological functions of LMPTP are unknown. Overexpression of LMPTP in cells transformed by PTK oncogenes leads to decreased proliferation and the ability to form colonies in soft agar (17). Thus, a potential physiological function of the LMPTPs is to control normal cell growth by interacting directly or indirectly with the PTK signaling network. Reportedly, LMPTP can interact directly with the platelet-derived grow...

Autoimmune-associated PTPN22 R620W Variation Reduces Phosphorylation of Lymphoid Phosphatase on an Inhibitory Tyrosine Residue

“…2F and data not shown). Inactive PTP mutants are often used to study their regulation by phosphorylation on tyrosine residues, because PTPs often show a strong tendency toward auto-dephosphorylation (34,36). Indeed, we observed that TCR-induced phosphorylation of transfected inactive C227S mutants of LYP and PEP was severalfold more prominent than that of the wild type phosphatases (data not shown).…”

“…Given that LYP forms a complex with Csk, a PTK, and the polymorphism affects the PTP/PTK interaction, we considered the possibility that the phosphatase could be phosphorylated on tyrosine. Several PTPs are regulated by tyrosine phosphorylation (34,35), including PTP20 (36), which is homolog to LYP. We found that treatment of human JTAg cells (24) with pervanadate, a powerful PTP inhibitor, induced phosphorylation of LYP on tyrosine ( Fig.…”

The Role of C-terminal Tyrosine Phosphorylation in the Regulation of SHP-1 Explored via Expressed Protein Ligation

“…Thiophosphates, resistant to enzymatic hydrolysis, have been introduced into signaling proteins by enzymatic modification of recombinant proteins with ATP␥S. These are often sluggish reactions that generally afford low stoichiometries and often show poor regioselectivity (33). Moreover, thiophosphates can indeed be hydrolyzed chemically and enzymatically, by some phosphatases almost as rapidly as phosphate substrates (34), in contrast to the nonhydrolyzable analogues used here.…”

Two Vicinal Cysteines Confer a Peculiar Redox Regulation to Low Molecular Weight Protein Tyrosine Phosphatase in Response to Platelet-derived Growth Factor Receptor Stimulation

“…These two LMW-PTP pools are differentially regulated because only the cytoskeleton-associated LMW-PTP fraction is specifically phosphorylated by c-Src after PDGF stimulation (6). As a consequence of its phosphorylation, LMW-PTP shows an average 20-fold increase in its in vitro catalytic activity (7,8,9). Cytoskeleton-associated LMW-PTP influences cell adhesion, spreading, and migration, controlling the phosphorylation level of p190Rho-GAP, a protein that is able to regulate Rho activity and, consequently, cytoskeleton rearrangement in response to PDGF stimulation.…”