Nicholas K. Tonks

MKP-1 (3CH134), an immediate early gene product, is a dual specificity phosphatase that dephosphorylates MAP kinase in vivo

Mitogenic stimulation of cells induces rapid and transient activation of MAP kinases. Here we report that a growth factor-inducible gene, 3CH134, encodes a dual specificity phosphatase that dephosphorylates and inactivates p42MAPK both in vitro and in vivo. In vitro, 3CH134 protein dephosphorylates both T183 and Y185 in p42MAPK. In serum-stimulated normal fibroblasts, the kinetics of inactivation of p42MAPK coincides with the appearance of newly synthesized 3CH134 protein, and the protein synthesis inhibitor cycloheximide leads to persistent activation of MAP kinase. Expression of 3CH134 in COS cells leads to selective dephosphorylation of p42MAPK from the spectrum of phosphotyrosyl proteins. 3CH134 blocks phosphorylation and activation of p42MAPK mediated by serum, oncogenic Ras, or activated Raf, whereas the catalytically inactive mutant of the phosphatase, Cys-258-->Ser, augments MAP kinase phosphorylation under similar conditions. The mutant 3CH134 protein also forms a physical complex with the phosphorylated form of p42MAPK. These findings suggest that 3CH134 is a physiological MAP kinase phosphatase; we propose the name MKP-1 for this phosphatase.

Protein tyrosine phosphatases: from genes, to function, to disease

The protein tyrosine phosphatase (PTP) superfamily of enzymes functions in a coordinated manner with protein tyrosine kinases to control signalling pathways that underlie a broad spectrum of fundamental physiological processes. In this review, I describe recent breakthroughs in our understanding of the role of the PTPs in the regulation of signal transduction and the aetiology of human disease.

Reversible Oxidation and Inactivation of Protein Tyrosine Phosphatases In Vivo

We have investigated the regulation of protein tyrosine phosphatases (PTPs) by reactive oxygen species (ROS) in a cellular environment. We demonstrate that multiple PTPs were reversibly oxidized and inactivated following treatment of Rat-1 cells with H(2)O(2) and that inhibition of PTP function was important for ROS-induced mitogenesis. Furthermore, we show transient oxidation of the SH2 domain containing PTP, SHP-2, in response to PDGF that requires association with the PDGFR. Our results indicate that SHP-2 inhibits PDGFR signaling and suggest a mechanism by which autophosphorylation of the PDGFR occurs despite its association with SHP-2. The data suggest that several PTPs may be regulated by oxidation and that characterization of this process may define novel links between specific PTPs and particular signaling pathways in vivo.

Redox regulation of protein tyrosine phosphatase 1B involves a sulphenyl-amide intermediate.

The second messenger hydrogen peroxide is required for optimal activation of numerous signal transduction pathways, particularly those mediated by protein tyrosine kinases. One mechanism by which hydrogen peroxide regulates cellular processes is the transient inhibition of protein tyrosine phosphatases through the reversible oxidization of their catalytic cysteine, which suppresses protein dephosphorylation. Here we describe a structural analysis of the redox-dependent regulation of protein tyrosine phosphatase 1B (PTP1B), which is reversibly inhibited by oxidation after cells are stimulated with insulin and epidermal growth factor. The sulphenic acid intermediate produced in response to PTP1B oxidation is rapidly converted into a previously unknown sulphenyl-amide species, in which the sulphur atom of the catalytic cysteine is covalently linked to the main chain nitrogen of an adjacent residue. Oxidation of PTP1B to the sulphenyl-amide form is accompanied by large conformational changes in the catalytic site that inhibit substrate binding. We propose that this unusual protein modification both protects the active-site cysteine residue of PTP1B from irreversible oxidation to sulphonic acid and permits redox regulation of the enzyme by promoting its reversible reduction by thiols.

Redox Redux: Revisiting PTPs and the Control of Cell Signaling

The architecture of the active site of members of the protein tyrosine phosphatase (PTP) superfamily renders these enzymes sensitive to reversible oxidation and inactivation. The importance of reversible oxidation of PTP superfamily members in controlling the signal output following an extracellular stimulus is discussed.

Structural and Evolutionary Relationships among Protein Tyrosine Phosphatase Domains

With the current access to the whole genomes of various organisms and the completion of the first draft of the human genome, there is a strong need for a structure-function classification of protein families as an initial step in moving from DNA databases to a comprehensive understanding of human biology. As a result of the explosion in nucleic acid sequence information and the concurrent development of methods for high-throughput functional characterization of gene products, the genomic revolution also promises to provide a new paradigm for drug discovery, enabling the identification of molecular drug targets in a significant number of human diseases. This molecular view of diseases has contributed to the importance of combining primary sequence data with three-dimensional structure and has increased the awareness of computational homology modeling and its potential to elucidate protein function. In particular, when important proteins or novel therapeutic targets are identified—like the family of protein tyrosine phosphatases (PTPs) (reviewed in reference 53)—a structure-function classification of such protein families becomes an invaluable framework for further advances in biomedical science. Here, we present a comparative analysis of the structural relationships among vertebrate PTP domains and provide a comprehensive resource for sequence analysis of phosphotyrosine-specific PTPs.

From Form to Function: Signaling by Protein Tyrosine Phosphatases

Tyrosine phosphorylation, controlled by the coordinated actions of protein tyrosine phosphatases (PTPs) and kinases (PTKs), is a critical control mechanism for numerous physiological processes, including growth, differentiation, metabolism, cell cycle regulation and cytoskeletal function. Originally, PTKs were believed to be the key enzymes controlling the dynamic process of tyrosine phosphorylation in vivo, with a small number of PTPs playing largely housekeeping roles. Unexpected structural diversity within a large family of PTPs called this idea into question. Approximately 75 PTPs have been identified, including both receptor-like and nontransmembrane enzymes, with genome sequencing data predicting the existence of similar to 500 human PTPs. These enzymes are characterized by the presence of a conserved catalytic domain of similar to 240 residues, containing the unique signature motif, [I/V]HCxAGxxR[S/T]G that defines this enzyme family (see accompanying minireview by Denu et al., 1996 [this issue of Cell]), fused, at either the N- or C-terminal ends, to a variety of noncatalytic, regulatory sequences. Now, several studies have illustrated subtleties of regulation and diversity of function for the PTPs which at least match those of the PTKs. Furthermore, PTPs can have both positive and negative effects on cellular signaling. This minireview discusses selected recent examples in which insights have been gained into the physiological function of PTP family members.

Reactive oxygen species enhance insulin sensitivity

Chronic reactive oxygen species (ROS) production by mitochondria may contribute to the development of insulin resistance, a primary feature of type 2 diabetes. In recent years it has become apparent that ROS generation in response to physiological stimuli such as insulin may also facilitate signaling by reversibly oxidizing and inhibiting protein tyrosine phosphatases (PTPs). Here we report that mice lacking one of the key enzymes involved in the elimination of physiological ROS, glutathione peroxidase 1 (Gpx1), were protected from high-fat-diet-induced insulin resistance. The increased insulin sensitivity in Gpx1(-/-) mice was attributed to insulin-induced phosphatidylinositol-3-kinase/Akt signaling and glucose uptake in muscle and could be reversed by the antioxidant N-acetylcysteine. Increased insulin signaling correlated with enhanced oxidation of the PTP family member PTEN, which terminates signals generated by phosphatidylinositol-3-kinase. These studies provide causal evidence for the enhancement of insulin signaling by ROS in vivo.

Molecular basis for the dephosphorylation of the activation segment of the insulin receptor by protein tyrosine phosphatase 1B.

The protein tyrosine phosphatase PTP1B is responsible for negatively regulating insulin signaling by dephosphorylating the phosphotyrosine residues of the insulin receptor kinase (IRK) activation segment. Here, by integrating crystallographic, kinetic, and PTP1B peptide binding studies, we define the molecular specificity of this reaction. Extensive interactions are formed between PTP1B and the IRK sequence encompassing the tandem pTyr residues at 1162 and 1163 such that pTyr-1162 is selected at the catalytic site and pTyr-1163 is located within an adjacent pTyr recognition site. This selectivity is attributed to the 70-fold greater affinity for tandem pTyr-containing peptides relative to mono-pTyr peptides and predicts a hierarchical dephosphorylation process. Many elements of the PTP1B-IRK interaction are unique to PTP1B, indicating that it may be feasible to generate specific, small molecule inhibitors of this interaction to treat diabetes and obesity.

A MAP kinase-dependent spindle assembly checkpoint in Xenopus egg extracts.

Like early Xenopus embryos, extracts made from Xenopus eggs lack the cell cycle checkpoint that keeps anaphase from occurring before spindle assembly is complete. At very high densities of sperm nuclei, however, microtubule depolymerization arrests the extracts in mitosis. The arrested extracts have high levels of maturation-promoting factor activity, fail to degrade cyclin B, and contain activated ERK2/mitogen-activated protein (MAP) kinase. The addition of the purified MAP kinase-specific phosphatase MKP-1 demonstrates that MAP kinase activity is required for both the establishment and maintenance of the mitotic arrest induced by spindle depolymerization. Increased calcium concentrations, which release unfertilized frog eggs from their natural arrest in metaphase of meiosis II, have no effect on the mitotic arrest.