The role of protein tyrosine phosphatases in colorectal cancer

Colorectal cancer is one of the most common oncogenic diseases in the Western world. Several cancer associated cellular pathways have been identified, in which protein phosphorylation and dephosphorylation, especially on tyrosine residues, are one of most abundant regulatory mechanisms. The balance between these processes is under tight control by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Aberrant activity of oncogenic PTKs is present in a large portion of human cancers. Because of the counteracting role of PTPs on phosphorylation-based activation of signal pathways, it has long been thought that PTPs must act as tumor suppressors. This dogma is now being challenged, with recent evidence showing that dephosphorylation events induced by some PTPs may actually stimulate tumor formation. As such, PTPs might form a novel attractive target for anticancer therapy. In this review, we summarize the action of different PTPs, the consequences of their altered expression in colorectal cancer, and their potential as target for the treatment of this deadly disease.     

Analysis of the human TrkB gene genomic organization reveals novel TrkB isoforms, unusual gene length, and splicing mechanism.

We determined the gene structure of the human TrkB gene. The gene is unusually large and spans at least 590 kbp. It contains 24 exons. Using alternative promoters, splicing, and polyadenylation sites, the gene can create at least 100 isoforms, that can encode 10 proteins. RT-PCR and Northern blot analysis reveals that only three major protein isoforms are generated by the gene: the full length receptor, an isoform lacking the tyrosine kinase domain, and a novel isoform lacking the tyrosine kinase domain but containing a Shc binding site. This novel isoform, TrkB-T-Shc is generated by the use of a new alternative exon 19. It is expressed only in brain. TrkB-T-Shc protein is located in the plasma membrane. Coimmunoprecipitation experiments show that TrkB-T-Shc is not phosphorylated by the full length receptor, indicating that it could be a negative regulator of TrkB signaling in the brain.     

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.     

Peptide probes containing a non-hydrolyzable phosphotyrosine-mimetic residue for enrichment of protein tyrosine phosphatases

We developed peptide probes containing a non-hydrolyzable phosphotyrosine mimetic, 4-[difluoro(phosphono)methyl]-L-phenylalanine (F₂Pmp) for the enrichment of protein tyrosine phosphatases (PTPs). We found that different F₂Pmp probes can enrich different PTPs, depending on the probe sequence. Furthermore, proteins containing a Src homology 2 (SH2) domain were enriched together. Importantly, probes containing phosphotyrosine instead of F₂Pmp failed to enrich PTPs due to dephosphorylation during the pulldown step. This enrichment approach using peptides containing F₂Pmp could be a generic tool for tyrosine phosphatome analysis without the use of antibodies.     

Tumour suppressor function of protein tyrosine phosphatase receptor-T

It has long been thought that PTPs (protein tyrosine phosphatases) normally function as tumour suppressors. Recent high-throughput mutational analysis identified loss-of-function mutations in six PTPs in human colon cancers, providing critical cancer genetics evidence that PTPs can act as tumour suppressor genes. PTPRT (protein tyrosine phosphatase receptor-T), a member of the family of type?IIB receptor-like PTPs, is the most frequently mutated PTP among them. Consistent with the notion that PTPRT is a tumour suppressor, PTPRT knockout mice are hypersensitive to AOM (azoxymethane)-induced colon cancer. The present review focuses on the physiological and pathological functions of PTPRT as well as the cellular pathways regulated by this phosphatase.     

5-Arylidene-2,4-thiazolidinediones as inhibitors of protein tyrosine phosphatases

4-(5-Arylidene-2,4-dioxothiazolidin-3-yl)methylbenzoic acids (2) were synthesized and evaluated in vitro as inhibitors of PTP1B and LMW-PTP, two protein tyrosine phosphatases (PTPs) which act as negative regulators of the metabolic and mitotic signalling of insulin. The synthesis of compounds 2 represents an example of utilizing phosphotyrosine-mimetics to identify effective low molecular weight nonphosphorus inhibitors of PTPs. Several thiazolidinediones 2 exhibited PTP1B inhibitory activity in the low micromolar range with moderate selectivity for human PTP1B and IF1 isoform of human LMW-PTP compared with other related PTPs.     

A novel phosphatase family, structurally related to dual-specificity phosphatases, that displays unique amino acid sequence and substrate specificity

Members of the superfamily of protein tyrosine phosphatases (PTPs) share the presence of an evolutionarily conserved PTP catalytic domain. Among them, the dual-specificity phosphatases (DSPs) constitute a diverse group of enzymes in terms of substrate specificity, including nonprotein substrates. In recent years, an increasing number of novel DSPs, whose functions and biological substrates are not well defined, have been discovered in a variety of organisms. In this study, we define the structural and functional properties of evolutionarily related atypical DSPs from different phyla. Sets of conserved motifs were defined that (i) uniquely segregated mammalian atypical DSPs from closely related enzymes and (ii) exclusively characterised a novel family of atypical DSPs present in plants, fungi, and kinetoplastids [plant and fungi atypical (PFA)-DSPs]; despite having different sequence “fingerprints,” the PTP tertiary structure of PFA-DSPs is conserved. Analysis of the catalytic properties of PFA-DSPs suggests the existence of a unique substrate specificity for these enzymes. Our findings predict characteristic functional motifs for the diverse members of the DSP families of PTPs and provide insights into the functional properties of DSPs of unknown function.     

CRYP-2: A receptor-type tyrosine phosphatase selectively expressed by developing vertebrate neurons

Axonal growth and guidance, like other aspects of neuronal differentiation, can be regulated by changes in tyrosine phosphorylation. Although much is known concerning the role of tyrosine kinases in these processes, relatively little is known about the nature and function of protein tyrosine phosphatases (PTPs) that may be involved. To identify the PTPs expressed in the embryonic chicken CNS at the time of axon growth, we performed a polymerase chain reaction based “screen” using degenerate primers directed against conserved regions of the PTP catalytic domain. We obtained five distinct PTP-related cDNAs, two of which code for novel PTPs. One, designated CRYP-2, is selectively expressed in the CNS. Full-length cloning of CRYP-2 revealed that it is a receptor-type PTP with an adhesion molecule-like extracellular region comprising fibronectin (FN) type III repeats and a single catalytic domain in the intracellular region. It is alternatively spliced in the juxtamembrane region, similar to other PTPs recently cloned. CRYP-2 mRNA is strongly expressed in the brain during the time of axon growth; it is downregulated toward the end of embryo-genesis. Western blot analysis identifies a 330-kDa glycoprotein as CRYP-2 and confirms that the protein is downregulated after hatching. Immunostaining of cerebellar neurons in vitro reveals that CRYP-2 is expressed on neuronal cell bodies and processes, but not on glia. The CAM-like structure, developmental pattern of expression, and neuron-specific localization of the CRYP-2 PTP suggest that it is involved in neuronal differentiation, particularly axon growth. © 1996 John Wiley & Sons, Inc.     

Protein tyrosine phosphatases as novel targets in breast cancer therapy

Breast cancer is linked to hyperactivation of protein tyrosine kinases (PTKs), and recent studies have unveiled that selective tyrosine dephosphorylation by protein tyrosine phosphatases (PTPs) of specific substrates, including PTKs, may activate or inactivate oncogenic pathways in human breast cancer cell growth-related processes. Here, we review the current knowledge on the involvement of PTPs in breast cancer, as major regulators of breast cancer therapy-targeted PTKs, such as HER1/EGFR, HER2/Neu, and Src. The functional interplay between PTKs and PTK-activating or -inactivating PTPs, and its implications in novel breast cancer therapies based on targeting of specific PTPs, are discussed.     

Evolution of the multifunctional protein tyrosine phosphatase family

The protein tyrosine phosphatase (PTP) family plays a central role in signal transduction pathways by controlling the phosphorylation state of serine, threonine, and tyrosine residues. PTPs can be divided into dual specificity phosphatases and the classical PTPs, which can comprise of one or two phosphatase domains. We studied amino acid substitutions at functional sites in the phosphatase domain and identified putative noncatalytic phosphatase domains in all subclasses of the PTP family. The presence of inactive phosphatase domains in all subclasses indicates that they were invented multiple times in evolution. Depending on the domain composition, loss of catalytic activity can result in different consequences for the function of the protein. Inactive single-domain phosphatases can still specifically bind substrate and protect it from dephosphorylation by other phosphatases. The inactive domains of tandem phosphatases can be further subdivided. The first class is more conserved, still able to bind phosphorylated tyrosine residues and might recruit multiphosphorylated substrates for the adjacent active domain. The second has accumulated several variable amino acid substitutions in the catalytic center, indicating a complete loss of tyrosine-binding capabilities. To study the impact of substitutions in the catalytic center to the evolution of the whole domain, we examined the evolutionary rates for each individual site and compared them between the classes. This analysis revealed a release of evolutionary constraint for multiple sites surrounding the catalytic center only in the second class, emphasizing its difference in function compared with the first class. Furthermore, we found a region of higher conservation common to both domain classes, suggesting a new regulatory center. We discuss the influence of evolutionary forces on the development of the phosphatase domain, which has led to additional functions, such as the specific protection of phosphorylated tyrosine residues, substrate recruitment, and regulation of the catalytic activity of adjacent domains.     

UVA inactivates protein tyrosine phosphatases by calpain-mediated degradation

UV irradiation causes inflammatory and proliferative cellular responses. We have proposed previously that these effects are, to a large extent, caused by the ligand-independent activation of several receptor tyrosine kinases due to the inactivation of their negative control elements, the protein tyrosine phosphatases (PTPs). We examined the mechanism of this inactivation and found that, in addition to reversible oxidation of PTPs, UV triggers a novel mechanism: induced degradation of PTPs by calpain, which requires both calpain activation and substrate PTP oxidative modification. This as yet unrecognized effect of UV is irreversible, occurs predominantly with UVA and UVB, the range of wavelengths in sunlight that reach the skin surface, and at physiologically relevant doses.     

Crystal structure of a phosphoinositide phosphatase, MTMR2: insights into myotubular myopathy and Charcot-Marie-Tooth syndrome

Myotubularin-related proteins are a large subfamily of protein tyrosine phosphatases (PTPs) that dephosphorylate D3-phosphorylated inositol lipids. Mutations in members of the myotubularin family cause the human neuromuscular disorders myotubular myopathy and type 4B Charcot-Marie-Tooth syndrome. The crystal structure of a representative member of this family, MTMR2, reveals a phosphatase domain that is structurally unique among PTPs. A series of mutants are described that exhibit altered enzymatic activity and provide insight into the specificity of myotubularin phosphatases toward phosphoinositide substrates. The structure also reveals that the GRAM domain, found in myotubularin family phosphatases and predicted to occur in approximately 180 proteins, is part of a larger motif with a pleckstrin homology (PH) domain fold. Finally, the MTMR2 structure will serve as a model for other members of the myotubularin family and provide a framework for understanding the mechanism whereby mutations in these proteins lead to disease.     

Receptor protein-tyrosine phosphatases: origin of domains (catalytic domain, Ig-related domain, fibronectin type III module) based on the sequence of the sponge Geodia cydonium

Reversible tyrosine phosphorylation of proteins is one of the major regulatory physiological events in response to cell-cell- and cell-matrix contact in Metazoa. Previously it was documented that the tyrosine phosphorylating enzymes, the tyrosine kinases (TKs), are autapomorphic characters of Metazoa, including sponges. In this paper the tyrosine dephosphorylating enzymes, the protein-tyrosine phosphatases (PTPs), are studied which can be grouped into two subfamilies, the soluble PTPs and the receptor PTPs (RPTPs). PTPs are characterized by one PTPase domain which interestingly comprises sequence similarity to yeast PTPs. In contrast to the PTPs, the RPTPs - which have been found only in Metazoa - are provided with two PTPase domains. To study the evolution of the RPTPs the full-length size RPTP was cloned from the marine demosponge Geodia cydonium, the phylogenetic oldest metazoan taxon. The 3253 bp long sequence has a putative open reading frame coding for a 999 aa long RPTP which is characterized by two fibronectin (type III; FN-III) domains in the extracellular portion, one intracellular immunoglobulin (Ig)-related domain, and two PTPase domains. Phylogenetic analysis revealed that the sponge FN-III domains form the basis of the metazoan FN-III domain with the common metazoan ancestor. The Ig-related, typical metazoan, module is classified to the disulphide lacking Ig members and represents the phylogenetic earliest member of this group. The beta-sheet propensity was calculated and the characteristic amino acids are present in the seven beta-sheets. The analysis of the two PTPase domains of the sponge RPTP demonstrates that the first domain is closely related to the PTPase domains present in the soluble PTPs, while the second PTPase domain is only distantly related to them. By constructing a rooted phylogenetic cladogram it became overt that the duplication of the PTPase domains must have occurred already in yeast. This interesting finding indicates that two conserved PTPase domains originated from a common ancestor in yeast while the evolutionary novelties, the FN-III domains and the Ig-related module, were added during the transition to the Metazoa. Hence, the tyrosine dephosphorylating enzyme, RPTP, is an example for a modular protein which is composed of ancient modules (PTPase domain[s]) and two metazoan novelties, while the tyrosine phosphorylating enzymes, the TKs, evolved only in Metazoa.     

Targeting protein tyrosine phosphatases for the development of antivirulence agents: Yersinia spp. and Mycobacterium tuberculosis as prototypes

Protein phosphorylation mediated by protein kinases and phosphatases has a central regulatory function in many cellular processes in eukaryotes and prokaryotes. As a result, several diseases caused by imbalance in phosphorylation levels are known, especially due to protein tyrosine phosphatases (PTPs) activity, an important family of signaling enzymes. Furthermore, over the last decades several studies have shown the main role of PTPs in pathogenic bacteria: they are associated with growth, cell division, cell wall biosynthesis, biofilm formation, metabolic processes, as well as virulence factor. In this way, PTPs have ascended as targets for antibacterial drug design, particularly in view of the antibiotic resistance in pathogenic bacteria, which demands novel therapeutics strategies. Targeting secreted PTPs is an antivirulence strategy to combat the emergence of antimicrobial resistance (AMR). This review focuses on the recent advances in understanding the role of PTPs and the approaches to target them, with an emphasis in Yersinia spp. and Mycobacterium tuberculosis pathogenesis.     

Regulation of receptor tyrosine kinase signaling by protein tyrosine phosphatases

Signaling through receptor tyrosine kinases (RTKs) is a major mechanism for intercellular communication during development and in the adult organism, as well as in disease-associated processes. The phosphorylation status and signaling activity of RTKs is determined not only by the kinase activity of the RTK but also by the activities of protein tyrosine phosphatases (PTPs). This review discusses recently identified PTPs that negatively regulate various RTKs and the role of PTP inhibition in ligand-induced RTK activation. The contributions of PTPs to ligand-independent RTK activation and to RTK inactivation by other classes of receptors are also surveyed. Continued investigation into the involvement of PTPs in RTK regulation is likely to unravel previously unrecognized layers of RTK control and to suggest novel strategies for interference with disease-associated RTK signaling.     

PTP inhibitor IV protects JNK kinase activity by inhibiting dual-specificity phosphatase 14 (DUSP14)

Protein phosphorylation plays critical roles in the regulation of protein activity and cell signaling. The level of protein phosphorylation is controlled by protein kinases and protein tyrosine phosphatases (PTPs). Disturbance of the equilibrium between protein kinase and PTP activities results in abnormal protein phosphorylation, which has been linked to the etiology of several diseases, including cancer. In this study, we screened protein tyrosine phosphatases (PTPs) by in vitro phosphatase assays to identify PTPs that are inhibited by bis (4-trifluoromethyl-sulfonamidophenyl, TFMS)-1,4-diisopropylbenzene (PTP inhibitor IV). PTP inhibitor IV inhibited DUSP14 phosphatase activity. Kinetic studies with PTP inhibitor IV and DUSP14 revealed a competitive inhibition, suggesting that PTP inhibitor IV binds to the catalytic site of DUSP14. PTP inhibitor IV effectively and specifically inhibited DUSP14-mediated dephosphorylation of JNK, a member of the mitogen-activated protein kinase (MAPK) family.