PTPH1 Is a Predominant Protein-tyrosine Phosphatase Capable of Interacting with and Dephosphorylating the T Cell Receptor ζ Subunit

Abstract

Protein-tyrosine phosphatases (PTPases) play key roles in regulating tyrosine phosphorylation levels in cells, yet the identity of their substrates remains limited. We report here on the identification of PTPases capable of dephosphorylating the phosphorylated immune tyrosine-based activation motifs present in the T cell receptor subunit. To characterize these PTPases, we purified enzyme activities directed against the phosphorylated T cell receptor subunit by a combination of anion and cation chromatography procedures. A novel ELISA-based PTPase assay was developed to rapidly screen protein fractions for enzyme activity following the various chromatography steps. We present data that SHP-1 and PTPH1 are present in highly enriched protein fractions that exhibit PTPase activities toward a tyrosine-phosphorylated TCR substrate (specific activity ranging from 0.23 to 40 pmol/min/g). We also used a protein-tyrosine phosphatase substrate-trapping library comprising the catalytic domains of 47 distinct protein-tyrosine phosphatases, representing almost all the tyrosine phosphatases identified in the human genome. PTPH1 was the predominant phosphatase capable of complexing phospho-. Subsequent transfection assays indicated that SHP-1 and PTPH1 are the two principal PTPases capable of regulating the phosphorylation state of the TCR ITAMs, with PTPH1 directly dephosphorylating . This is the first reported demonstration that PTPH1 is a candidate PTPase capable of interacting with and dephosphorylating TCR .The T and B cell antigen receptors contain multiple copies of an immune tyrosine-based activation motif (ITAMs), 1 which initiate intracellular signals by coupling to several families of protein-tyrosine kinases (PTKs) (1). The activation of this pathway following receptor-ligand interactions results in a transient accumulation of tyrosine-phosphorylated proteins, leading to the induction of cellular effector functions (2, 3). Following TCR engagement, two tyrosine residues within each ITAM are specifically phosphorylated by the Src family of PTKs (2, 4). Once bi-phosphorylated, the ITAMs serve as high affinity binding sites for the ZAP-70/Syk family of PTKs, with each of two Src homology 2 (SH2) domains of Syk/ZAP-70 binding to one of the two phospho-tyrosine sequences (5, 6). The ␣␤ T cell receptor (TCR) complex actually comprises up to ten ITAMs that are distributed among the TCR and CD3 ␥, ␦, and ⑀ subunits (3). The presence of ten ITAMs is proposed to support signal amplification and/or signal bifurcation (reviewed in Refs. 3 and 4). 6 of 10 ITAMs in the TCR complex are localized in the TCR homodimer (three per chain), facilitating the recruitment of multiple ZAP-70 molecules following ITAM phosphorylations (3,7,8). There are two predominant tyrosinephosphorylated forms of that migrate with distinct molecular masses of 21 and 23 kDa, and their differential induction are linked to multiple critical biological functions for T cells (3, 8) (reviewed in Refs. 3,4,and 9).It is well established that intracellular protei...

Normal TCR Signal Transduction in Mice That Lack Catalytically Active PTPN3 Protein Tyrosine Phosphatase

“…PTPN3 has been suggested to function as an important negative regulator of TCR signal transduction acting to dephosphorylate the TCR chain (13,18,19). However, this conclusion has been based upon studies which have examined the effect of overexpression of PTPN3 in the Jurkat T leukemia cell line and upon biochemical characterization of PTPN3 activity in vitro.…”

“…A recent study indicates that one important target of PTPN3 is the TCR chain. In this regard, PTPN3 was identified as the only PTP from a large tested panel of rPTPs that is capable of interacting physically with TCR and dephosphorylating TCR ITAMs in vitro (19). In addition, using an unbiased biochemical fractionation approach, PTPN3 and SHP-1 were identified as essentially the only PTPs expressed in Jurkat that are able to dephosphorylate TCR (19).…”

“…In this regard, PTPN3 was identified as the only PTP from a large tested panel of rPTPs that is capable of interacting physically with TCR and dephosphorylating TCR ITAMs in vitro (19). In addition, using an unbiased biochemical fractionation approach, PTPN3 and SHP-1 were identified as essentially the only PTPs expressed in Jurkat that are able to dephosphorylate TCR (19). Together with the finding that PTPN3 dephosphorylates TCR when both are transfected into COS-7 cells, these studies have contributed to the notion that PTPN3 acts a negative regulator of TCR signaling by dephosphorylating the TCR chain.…”

“…PTPN3 has been proposed to function at an early step in the TCR signal transduction cascade acting to dephosphorylate the TCR chain (19). As such, it would be predicted that in T cells from PTPN3 ⌬PTP mice a number of signaling proteins should become hyperphosphorylated on tyrosine residues in response to CD3/ CD28 stimulation.…”

TCR-induced downregulation of protein tyrosine phosphatase PEST augments secondary T cell responses

“…For example, HePTP dephosphorylates Y185 in the activation loop of Erk and the equivalent site on p38 kinase, but does not dephosphorylate any other signaling molecules (Saxena et al, 1999a, b;Saxena and Mustelin, 2000;Nika et al, 2006). PTPH1 has been proposed to dephosphorylate the TCR-ζ (Sozio et al, 2004) and VCP (Zhang et al, 1999) (although both remain unverified in T cells). In our hands, SHP1 efficiently dephosphorylates Y493 of ZAP-70, but has minimal effects on Src family kinases , while LMPTP-B has a positive effect on TCR signaling by dephosphorylating ZAP-70 at the inhibitory site Y292 (Bottini et al, 2002).…”