Sébastien Wälchli

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

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 ζ subunit. To characterize these PTPases, we purified enzyme activities directed against the phosphorylated T cell receptor ζ 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 ζ substrate (specific activity ranging from 0.23 to 40 pmol/min/μg). 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-ζ. Subsequent transfection assays indicated that SHP-1 and PTPH1 are the two principal PTPases capable of regulating the phosphorylation state of the TCR ζ ITAMs, with PTPH1 directly dephosphorylating ζ. This is the first reported demonstration that PTPH1 is a candidate PTPase capable of interacting with and dephosphorylating TCR ζ.

Probing Protein-tyrosine Phosphatase Substrate Specificity Using a Phosphotyrosine-containing Phage Library

Protein tyrosine phosphatases (PTPs) play important, highly dynamic roles in signaling. Currently about 90 different PTP genes have been described. The enzymes are highly regulated at all levels of expression, and it is becoming increasingly clear that substrate specificity of the PTP catalytic domains proper contributes considerably to PTP functionality. To investigate PTP substrate selectivity, we have set up a procedure to generate phage libraries that presents randomized, phosphotyrosine-containing peptides. Phages that expressed suitable substrates were selected by immobilized, substrate-trapping GST-PTP fusion proteins. After multiple rounds of selection, positive clones were confirmed by SPOT analysis, dephosphorylation by wild-type enzyme, and Km determinations. We have identified distinct consensus substrate motifs for PTP1B, Sap-1, PTP-β, SHP1, and SHP2. Our results confirm substrate specificity for individual PTPs at the peptide level. Such consensus sequences may be useful both for identifying potential PTP substrates and for the development of peptidomimetic inhibitors.

MetaBlasts: tracing protein tyrosine phosphatase gene family roots from Man to Drosophila melanogaster and Caenorhabditis elegans genomes.

At increasing speed, sequencing data are being made public from both complex and simple life forms. Although biomedical interests tend to focus on mammalian genes, only simple organisms allow rapid genetic manipulation and functional analysis. A prerequisite for the meaningful extrapolation of gene functional studies from invertebrates to man is that the orthologs under study are unambiguously linked. However, identifying orthologs is not trivial, especially where large gene families are involved. We present here an automated sequence analysis procedure that allows a rapid visualization of most likely ortholog pairs. We illustrate the utility of this approach for the human gene family of protein tyrosine phosphatases (PTPs) as compared with the full set of Caenorhabditis elegans and Drosophila melanogaster conceptual ORFs. The approach is based on a reciprocal series of BLAST searches, which are automatically stored and represented in an HTML-formatted table. We have used this MetaBlast approach to compile lists of human PTPs and their worm and fly orthologs. Many of these PTP orthologs had not been previously identified as such.

Pulling strings below the surface: Hormone receptor signaling through inhibition of protein tyrosine phosphatases

Hormones, cytokines, and related proteins (such as soluble hormone receptors) play an important role as therapeutic agents. Most hormone receptors signal through a mechanism that involves phosphorylation of the receptors tyrosine residues. At any given moment, the receptors phosphorylation state depends on the balance of kinase and phosphatase activities. Recent findings point to the exciting possibility that receptor signaling can be regulated by inhibition of protein tyrosine phosphatases (PTPs) that specifically hydrolyze receptor tyrosine-phosphates, or their immediate downstream effectors. This strategy has now been firmly validated for the insulin receptor and PTP1B; inhibiting PTP1B activity results in stimulation of the insulin receptor and signaling, even in the absence of insulin. This and similar findings suggest that PTP inhibitors have potential as hormone mimetics. In the present review, we outline this new paradigm for therapeutic regulation of the insulin receptor and discuss evidence that hints at other specific receptor-PTP pairs.

Vector-based delivery of siRNAs: in vitro and in vivo challenges.

RNA interference (RNAi) induced by small interfering RNAs (siRNAs) has recently become a powerful tool to knock-down gene expression in a sequence-specific manner. In addition to chemically made siRNAs, stable expression of siRNA in the form of short hairpin RNAs (shRNAs) expressed from an RNA polymerase III (pol III) promoter is now widely used approach for the application of RNAi in mammalian cells. However, long-term suppression using constitutive promoters can be problematic and emerging evidence indicates that siRNAs can cause several side effects in human cells. Here we review the recent advances in developing controllable expression vectors in order to accelerate the therapeutic applications of RNAi.

Protein tyrosine phosphatases as drug targets: PTP1B and beyond

Protein tyrosine phosphatases (PTPs) control signal transduction pathways and have recently emerged as potential drug targets. Inhibition of individual PTPs can result in the activation of therapeutically relevant kinase cascades. This is particularly useful in cases where disease is associated with hormonal resistance, such as insensitivity to insulin or leptin. Currently, PTP1B is being investigated by a number of companies as a promising target for leptin/insulin mimetics and in the treatment of diabetes and obesity. Since all 90 – 100 PTPs have been identified in the human genome, the challenge now is to identify the function of these enzymes and the therapeutic indications that may exist for specific PTP inhibitors.

Pulling strings below the surface

Hormones, cytokines, and related proteins (such as soluble hormone receptors) play an important role as therapeutic agents. Most hormone receptors signal through a mechanism that involves phosphorylation of the receptors tyrosine residues. At any given moment, the receptors phosphorylation state depends on the balance of kinase and phosphatase activities. Recent findings point to the exciting possibility that receptor signaling can be regulated by inhibition of protein tyrosine phosphatases (PTPs) that specifically hydrolyze receptor tyrosine-phosphates, or their immediate downstream effectors. This strategy has now been firmly validated for the insulin receptor and PTP1B; inhibiting PTP1B activity results in stimulation of the insulin receptor and signaling, even in the absence of insulin. This and similar findings suggest that PTP inhibitors have potential as hormone mimetics. In the present review, we outline this new paradigm for therapeutic regulation of the insulin receptor and discuss evidence that hints at other specific receptor-PTP pairs.