The human ubiquitin multigene family: some genes contain multiple directly repeated ubiquitin coding sequences.

Ubiquitin coding sequences were isolated from a human genomic library and two cDNA libraries. One human ubiquitin gene consists of 2055 nucleotides and codes for a polyprotein consisting of 685 amino acid residues. The polyprotein contains nine direct repeats of the ubiquitin amino acid sequence and the last ubiquitin sequence is extended with an additional valyl residue at the C-terminal end. No spacer sequences separate the ubiquitin repeats and the coding regions are not interrupted by intervening sequences. This particular gene is transcribed since cDNAs corresponding to the genomic sequence have been isolated. At least two more types of ubiquitin genes are encoded in the human genome, one coding for an ubiquitin monomer while another presumably codes for three or four direct repeats of the ubiquitin sequence. Human DNA contains many copies of the ubiquitin sequence. Ubiquitin is therefore encoded in the human genome as a multigene family.     

Drosophila protein tyrosine phosphatases

Seven protein tyrosine phosphatase (PTPase) genes have been identified in the fruit-fly Drosophila melanogaster. Four of these genes encode receptor-linked PTPases (R-PTPs) that are expressed on central nervous system axons in the embryo. Each axonal R-PTP has an extracellular domain that is homologous to vertebrate adhesion molecules and to identified mammalian R-PTPs. Two non-receptor PTPase genes have been isolated to date. One of these, corkscrew (csw), encodes an SH2 domain-containing PTPase that appears to be a homolog of mammalian PTP1D. Genetic evidence indicates that the csw PTPase is involved in the transduction of signals from receptor tyrosine kinases to their down-stream targets, which include Ras proteins.     

Protozoan protein tyrosine phosphatases

The aim of this review is to provide a synthesis of the published experimental data on protein tyrosine phosphatases from parasitic protozoa, in silico analysis based on the availability of completed genomes and to place available data for individual phosphatases from different unicellular parasites into the comparative and evolutionary context. We analysed the complement of protein tyrosine phosphatases (PTP) in several species of unicellular parasites that belong to Apicomplexa (Plasmodium; Cryptosporidium, Babesia, Theileria, and Toxoplasma), kinetoplastids (Leishmania and Trypanosoma spp.), as well as Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis and a microsporidium Encephalitozoon cuniculi. The analysis shows distinct distribution of the known families of tyrosine phosphatases in different species. Protozoan tyrosine phosphatases show considerable levels of divergence compared with their mammalian homologues, both in terms of sequence similarity between the catalytic domains and the structure of their flanking domains. This potentially makes them suitable targets for development of specific inhibitors with minimal effects on physiology of mammalian hosts.     

Ribosomal and protein coding gene based multigene phylogeny on the family Streptomycetaceae

The phylogenetic relationship among the three genera of the family Streptomycetaceae was examined using the small and large subunit ribosomal RNA genes, and the gyrB, rpoB, trpB, atpD and recA genes. The total stretches of the analyzed ribosomal genes were 4.2kb, and those of five protein coding genes were 4.5 kb. The resultant phylogenetic trees confirmed that each genus formed an independent clade in the majority of cases. The G+C contents of rRNA genes were 56.9-58.9 mol%, and those of protein coding genes were 65.4-72.4 mol%, the latter being closer to those of the genomic DNAs. The average nucleotide sequence identity between the organisms were 94.1-96.4% for rRNA genes and 85.7-90.6% for protein coding genes, thus indicating that protein coding genes can give higher resolution than rRNA genes. In addition, the protein coding gene trees were more stable than the rRNA gene trees, supported by higher bootstrap values and other treeing algorithms. Moreover, the genome data of six Streptomyces species indicated that many protein coding genes exhibited higher correlations with genome relatedness. The combined gene sequences were also shown to give a better resolution with higher stability than any single genes, though not necessarily more correlated with genome relatedness. It is evident from this study that the rRNA gene based phylogeny can be misleading, and also that protein coding genes have a number of advantages over the rRNA genes as the phylogenetic markers including a high correlation with the genome relatedness.     

Structural relationship among the members of a multigene family coding for the sweet potato tuberous root storage protein

Sporamin, the major soluble protein of the sweet potato tuberous root, is coded for by a multigene family. Fourty-nine essentially full-length sporamin cDNAs isolated from tuberous root cDNA library have been classified by cross hybridization, restriction endonuclease cleavage pattern and ribonuclease cleavage mapping. All the cDNAs fall into one of the two distinct homology groups, subfamilies A and B, which correspond to the polypeptide classes sporamin A and B, respectively. At least 5 different sequences are detected in both of the 22 sporamin A and 27 sporamin B cDNAs. Comparison of the nucleotide sequences of the coding region of three each of sporamin A and B subfamily members, four from cDNAs and two from genomic clones, indicates that intra-subfamily homologies (94 to 98%) are much higher than inter-subfamily homologies (82 to 84%), and there are deletions or insertions of one or two codons at three locations which characterize each subfamily. Large portions of base substitutions in the coding region accompany amino acid substitutions. In contrast to the coding region, most of the structural differences among the members in the 5 and 3 noncoding regions are deletions or insertions.     

Identification of a large multigene family encoding the major sperm protein of Caenorhabditis elegans

DNA fragments corresponding to genes encoding the MSP of Caenorhabditis elegans sperm have been isolated by recombinant DNA techniques. Analyses of individual genomic clones suggest that there are multiple MSP genes that are dispersed in the genome. From restriction enzyme digests of genomic DNA fractionated and hybridized with an MSP complementary DNA probe, there appear to be more than 30 MSP genes in the genome. Despite the occurrence of this large dispersed multigene family, the MSP messenger RNA from both males and hermaphrodites is homogene in size. There are at least three different proteins of identical molecular weight but different isoelectric point that cross-react with anti-MSP antisera. Each protein is a primary translation product with no detectable post-translational modifications, suggesting that at least three of the MSP genes are expressed.     

Receptor protein tyrosine phosphatases and cancer

There is general agreement that many cancers are associated with aberrant phosphotyrosine signaling, which can be caused by the inappropriate activities of tyrosine kinases or tyrosine phosphatases. Furthermore, incorrect activation of signaling pathways has been often linked to changes in adhesion events mediated by cell surface receptors. Among these receptors, receptor protein tyrosine phosphatases (RPTPs) both antagonize tyrosine kinases as well as engage extracellular ligands. A recent wealth of data on this intriguing family indicates that its members can fulfill either tumor suppressing or oncogenic roles. The interpretation of these results at a molecular level has been greatly facilitated by the recent availability of structural information on the extra- and intracellular regions of RPTPs. These structures provide a molecular framework to understand how alterations in extracellular interactions can inactivate RPTPs in cancers or why the overexpression of certain RPTPs may also participate in tumor progression.     

Bacterial and viral protein tyrosine phosphatases

Unrestricted protein tyrosine phosphatase (PTPase) activity may play a role in pathogenesis. For instance, the virulence determinant gene, yopH, of Yersinia pseudotuberculosis encodes a PTPase. The phosphatase activity of the YopH protein is essential for the pathogenesis of Y. pseudotuberculosis. Yersinia pestis, the bacterium which causes the bubonic plague, also contains a gene closely related to yopH. The action of YopH on host proteins appears to break down signal transduction mechanisms in many cell types including those of the immune system. This may contribute to the ability of the bacterium to escape effective surveillance by the immune system. The vaccinia virus VH1 gene, like yopH in the Yersinia bacteria, encodes a protein phosphatase. The VH1 PTPase defines a new class of phosphatases capable of dephosphorylating both phosphoserine/threonine and tyrosine containing substrates. Proteins sharing sequence identity to this dual-specificity phosphatase have been identified from other viruses, yeast and man. Although a complete understanding of the function of these dual-specificity phosphatases is not presently available, they clearly play important roles in cell cycle regulation, growth control and mitogenic signaling mechanisms. The unique catalytic properties of the dual specificity phosphatases suggest that these catalysts constitute a distinct subfamily of phosphatases.     

An affinity-based fluorescence polarization assay for protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) are important signaling enzymes that control such fundamental processes as proliferation, differentiation, survival/apoptosis, as well as adhesion and motility. Potent and selective PTP inhibitors serve not only as powerful research tools, but also as potential therapeutics against a variety illness including cancer and diabetes. PTP activity-based assays are widely used in high throughput screening (HTS) campaigns for PTP inhibitor discovery. These assays suffer from a major weakness, in that the reactivity of the active site Cys can cause serious problems as highly reactive oxidizing and alkylating agents may surface as hits. We describe the development of a fluorescence polarization (FP)-based displacement assay that makes the use of an active site Cys to Ser mutant PTP (e.g., PTP1B/C215S) that retains the wild-type binding affinity. The potency of library compounds is assessed by their ability to compete with the fluorescently labeled active site ligand for binding to the Cys to Ser PTP mutant. Finally, the substitution of the active site Cys by a Ser renders the mutant PTP insensitive to oxidation and alkylation and thus will likely eliminate "false" positives due to modification of the active site Cys that destroy the phosphatase activity.     

Fluorescein monophosphates as fluorogenic substrates for protein tyrosine phosphatases

A series of novel fluorescein monophosphates aimed as substrates for protein tyrosine phosphatases (PTPs) were synthesized and evaluated against fluorescein diphosphate (FDP), the currently used fluorescent substrate for PTPs. In contrast to FDP, which is dephosphorylated to monophosphate and then to fluorescein in a sequential reaction, these monophosphates are dephosphorylated in a single step. This eliminates the complication in assaying PTPs due to the cleavage of the second phosphate group. The kinetic studies of these substrates with PTPs were performed and Michaelis-Menten parameters were obtained. These designed substrates have Km 0.03-0. 35 mM, kcat/Km of 3-100 mM-1 s-1 with CD45 and PTP1B. The results showed that the substrates with negative charge groups on the fluorescein have higher affinities for PTP1B, which are consistent with other observations. In this series, fluorescein monosulfate monophosphate (FMSP) was the best substrate observed. Since FMSP showed large increases in both absorption and fluorescence upon dephosphorylation by PTPs at pH>6.0, it is one of the most sensitive, stable and high affinity substrates reported for PTPs.     

Proteomic approaches to studying protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) constitute a large family of enzymes that play key roles in cell signaling. Deregulation of PTP activity results in aberrant tyrosine phosphorylation, which has been linked to the etiology of several human diseases, including cancer. Since phosphate removal by the PTPs can both enhance and antagonize cellular signaling, it is essential to elucidate the physiological context in which PTPs operate. Two powerful proteomic approaches have been developed to rapidly establish the exact functional roles for every PTP, both in normal cellular physiology and in pathogenic conditions. In the first, an affinity-based substrate-trapping approach has been employed for PTP substrate identification. Identification and characterization of specific PTP-substrate interactions will associate functions with PTP as well as implicate PTP to specific signaling pathways. In the second, a number of activity-based PTP probes have been developed that can provide a direct readout of the functional state of the PTPs in complex proteomes. The ability to profile the entire PTP family on the basis of changes in their activity is expected to yield new functional insights into pathways regulated by the PTPs and contribute to the discovery of PTPs as novel therapeutic targets. Effective application of these proteomic techniques will accelerate the functional characterization of PTPs, thereby facilitating our understanding of PTPs in cell signaling and in diseases.     

Enzyme kinetic characterization of protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) play a central role in cellular signaling processes, resulting in an increased interest in modulating the activities of PTPs. We therefore decided to undertake a detailed enzyme kinetic evaluation of various transmembrane and cytosolic PTPs (PTPalpha, PTPbeta, PTPepsilon, CD45, LAR, PTP1B and SHP-1), using pNPP as substrate. Most noticeable is the increase in the turnover number for PTPbeta with increasing pH and the weak pH-dependence of the turnover number of CD45. The kinetic data for PTPalpha-D1 and PTPalpha-D1D2 suggest that D2 affects the catalysis of pNPP. PTPepsilon and the closely homologous PTPalpha behave differently. The K(m) data were lower for PTPepsilon than those for PTPalpha, while the inverse was observed for the catalytic efficiencies.     

Transmembrane homodimerization of receptor-like protein tyrosine phosphatases

Receptor-like protein tyrosine phosphatases (RPTPs) are type I integral membrane proteins. Together with protein tyrosine kinases, RPTPs regulate the phosphotyrosine levels in the cell. Studies of two RPTPs, CD45 and PTPalpha, have provided strong evidence that dimerization leads to inactivation of the receptors, and that the dimerization of PTPalpha involves interactions in the transmembrane domain (TMD). Using the TOXCAT assay, a genetic approach for analyzing TM interactions in Escherichia coli membranes, we show that the TMD of RPTPs interact in the membrane, albeit to different extents. Using fusion proteins of TMDs, we also observe an equilibrium between monomer and dimer in sodium dodecyl sulfate (SDS) micelles. Through a mutational study of the DEP1 TMD, we demonstrate that these interactions are specific. Taken together, our results define a subset of the RPTP family in which TM homodimerization may act as a mediator of protein function.     

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.     

Targeting protein tyrosine phosphatases for CDK6-induced immunotherapy resistance

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A gatekeeper residue for inhibitor sensitization of protein tyrosine phosphatases

Allele-specific enzyme inhibitors are powerful tools in chemical biology. However, few general approaches for the discovery of such inhibitors have been described. Herein is reported a method for the sensitization of protein tyrosine phosphatases (PTPs) to small-molecule inhibition. It is shown that mutation of an active-site isoleucine to alanine (I219A) sensitizes PTP1B to inhibition by a class of thiophene-based inhibitors. This sensitization strategy succeeds for both 'orthogonal' inhibitors, designed to be incompatible with wild-type PTP active sites, and previously optimized wild-type PTP inhibitors. The finding that the I219A mutation sensitizes phosphatase domains to a variety of compounds suggests that isoleucine 219 may act as a 'gatekeeper' residue that can be widely exploited for the chemical-genetic analysis of PTP function.     

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.