Function Of Protein Tyrosine Kinases Research Articles

Role of Protein Tyrosine Phosphatase Receptor Type E (PTPRE) in Chemoresistant Retinoblastoma.

Protein tyrosine phosphatase receptor type E (PTPRE) is a member of the "classical" protein tyrosine phosphatase subfamily and regulates a variety of cellular processes in a tissue-specific manner by antagonizing the function of protein tyrosine kinases. PTPRE plays a tumorigenic role in different human cancer cells, but its role in retinoblastoma (RB), the most common malignant eye cancer in children, remains to be elucidated. Etoposide-resistant RB cell lines and RB patients display significant higher PTPRE expression levels compared to chemosensitive counterparts and the healthy human retina, respectively. PTPRE promotor methylation analyses revealed that PTPRE expression in RB is not regulated via this mechanism. Lentiviral PTPRE knockdown (KD) induced a significant decrease in growth kinetics, cell viability, and anchorage-independent growth of etoposide-resistant Y79 and WERI RB cells. Caspase-dependent apoptosis rates were significantly increased and a re-sensitization for etoposide could be observed after PTPRE depletion. In vivo chicken chorioallantoic membrane (CAM) assays revealed decreased tumor formation capacity as well as reduced tumor size and weight following PTPRE KD. Expression levels of miR631 were significantly downregulated in etoposide-resistant RB cells and patients. Transient miR631 overexpression resulted in significantly decreased PTPRE levels and concomitantly decreased proliferation and increased apoptosis levels in etoposide-resistant RB cells. These impacts mirror PTPRE KD effects, indicating a regulation of PTPRE via this miR. Additionally, PTPRE KD led to altered phosphorylation of protein kinase SGK3 and-dependent on the cell line-AKT and ERK1/2, suggesting potential PTPRE downstream signaling pathways. In summary, these results indicate an oncogenic role of PTPRE in chemoresistant retinoblastoma.

The anti‐inflammatory compound BAY‐11‐7082 is a potent inhibitor of protein tyrosine phosphatases

The families of protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) function in a coordinated manner to regulate signal transduction events that are critical for cellular homeostasis. Aberrant tyrosine phosphorylation, resulting from disruption of either PTP or PTK function, has been shown to be the cause of major human diseases, including cancer and diabetes. Consequently, the characterization of small-molecule inhibitors of these kinases and phosphatases may not only provide molecular probes with which to define the significance of particular signaling events, but also may have therapeutic implications. BAY-11-7082 is an anti-inflammatory compound that has been reported to inhibit IκB kinase activity. The compound has an α,β-unsaturated electrophilic center, which confers the property of being a Michael acceptor; this suggests that it may react with nucleophilic cysteine-containing proteins, such as PTPs. In this study, we demonstrated that BAY-11-7082 was a potent, irreversible inhibitor of PTPs. Using mass spectrometry, we have shown that BAY-11-7082 inactivated PTPs by forming a covalent adduct with the active-site cysteine. Administration of the compound caused an increase in protein tyrosine phosphorylation in RAW 264 macrophages, similar to the effects of the generic PTP inhibitor sodium orthovanadate. These data illustrate that BAY-11-7082 is an effective pan-PTP inhibitor with cell permeability, revealing its potential as a new probe for chemical biology approaches to the study of PTP function. Furthermore, the data suggest that inhibition of PTP function may contribute to the many biological effects of BAY-11-7082 that have been reported to date.

Ptk7 promotes non-canonical Wnt/PCP-mediated morphogenesis and inhibits Wnt/β-catenin-dependent cell fate decisions during vertebrate development

Using zebrafish, we have characterised the function of Protein tyrosine kinase 7 (Ptk7), a transmembrane pseudokinase implicated in Wnt signal transduction during embryonic development and in cancer. Ptk7 is a known regulator of mammalian neural tube closure and Xenopus convergent extension movement. However, conflicting reports have indicated both positive and negative roles for Ptk7 in canonical Wnt/β-catenin signalling. To clarify the function of Ptk7 in vertebrate embryonic patterning and morphogenesis, we generated maternal-zygotic (MZ) ptk7 mutant zebrafish using a zinc-finger nuclease (ZFN) gene targeting approach. Early loss of zebrafish Ptk7 leads to defects in axial convergence and extension, neural tube morphogenesis and loss of planar cell polarity (PCP). Furthermore, during late gastrula and segmentation stages, we observe significant upregulation of β-catenin target gene expression and demonstrate a clear role for Ptk7 in attenuating canonical Wnt/β-catenin activity in vivo. MZptk7 mutants display expanded differentiation of paraxial mesoderm within the tailbud, suggesting an important role for Ptk7 in regulating canonical Wnt-dependent fate specification within posterior stem cell pools post-gastrulation. Furthermore, we demonstrate that a plasma membrane-tethered Ptk7 extracellular fragment is sufficient to rescue both PCP morphogenesis and Wnt/β-catenin patterning defects in MZptk7 mutant embryos. Our results indicate that the extracellular domain of Ptk7 acts as an important regulator of both non-canonical Wnt/PCP and canonical Wnt/β-catenin signalling in multiple vertebrate developmental contexts, with important implications for the upregulated PTK7 expression observed in human cancers.

Concomitant downregulation of proliferation/survival pathways dependent on FGF-R3, JAK2 and BCMA in human multiple myeloma cells by multi-kinase targeting

The identification of proliferation/survival pathways constitutively activated by genetic alterations in multiple myeloma (MM), or sustained by the bone marrow (BM) microenvironment, provides novel opportunities for the development of targeted therapies. The deregulated function of protein tyrosine kinases plays a critical role in driving MM malignant phenotype. We investigated the effects of the multi-target tyrosine kinase inhibitor RPI-1 in a panel of human MM cell lines, including t(4;14) positive cell lines expressing the TK receptor FGF-R3. Cells harboring FGF-R3 activating mutations (KMS11 and OPM2) displayed the highest sensitivity to RPI-1 antiproliferative effect. The stimulating effect of the aFGF ligand was abrogated in cells harboring a non-constitutively active receptor. Drug treatment inhibited activation and expression of the FGF-R3 Y373C mutant as well as aFGF-dependent signaling involving AKT and ERKs. Inhibition of JAK2, an additional RPI-1 target, resulted in STAT3 inactivation. Blockade of these proliferation/survival pathways was associated with caspase-dependent apoptosis. Moreover, drug treatment abrogated proliferative and pro-invasive stimuli provided by conditioned medium from mesenchymal stromal cells. Gene expression profile of KMS11 cells showed 22 upregulated and 52 downregulated genes upon RPI-1 treatment, with an early modulation of genes implicated in MM pathobiology such as SAT-1, MYC, MIP-1α/β, FGF-R3, and the growth factor receptor B-cell maturation antigen ( BCMA). Thus, concomitant blockade of FGF-R3 and JAK2 results in inhibition of several MM-promoting pathways, including BCMA-regulated signaling, and downregulation of disease-associated proteins. These data may have therapeutic implications in the design of treatment strategies resulting in the concomitant inhibition of FGF-R3 and JAK2 signaling pathways in t(4;14) MM.

Tumor Suppressor FUS1 Signaling Pathway

FUS1 is a novel tumor suppressor gene identified in the human chromosome 3p21.3 region where allele losses and genetic alterations occur early and frequently for many human cancers. Expression of FUS1 protein is absent or reduced in the majority of lung cancers and premalignant lung lesions. Restoration of wt-FUS1 function in 3p21.3-deficient non-small cell lung carcinoma cells significantly inhibits tumor cell growth by induction of apoptosis and alteration of cell cycle kinetics. Here we present recent findings indicating that FUS1 induces apoptosis through the activation of the intrinsic mitochondrial-dependent and Apaf-1-associated pathways and inhibits the function of protein tyrosine kinases including EGFR, PDGFR, AKT, c-Abl, and c-Kit. Intravenous administration of a nanoparticle encapsulated FUS1 expression plasmid effectively delivers FUS1 to distant tumor sites and mediates an antitumor effect in orthotopic human lung cancer xenograft models. This approach is the rationale for an ongoing FUS1-nanoparticle-mediated gene delivery clinical trial for the treatment of lung cancer.

Nonreceptor Protein-Tyrosine Phosphatases in Immune Cell Signaling

Tyrosyl phosphorylation plays a critical role in multiple signaling pathways regulating innate and acquired immunity. Although tyrosyl phosphorylation is a reversible process, we know much more about the functions of protein-tyrosine kinases (PTKs) than about protein-tyrosine phosphatases (PTPs). Genome sequencing efforts have revealed a large and diverse superfamily of PTPs, which can be subdivided into receptor-like (RPTPs) and nonreceptor (NRPTPs). The role of the RPTP CD45 in immune cell signaling is well known, but those of most other PTPs remain poorly understood. Here, we review the mechanism of action, regulation, and physiological functions of NRPTPs in immune cell signaling. Such an analysis indicates that PTPs are as important as PTKs in regulating the immune system.

Protein-tyrosine Phosphatase (PTP) Wedge Domain Peptides: A NOVEL APPROACH FOR INHIBITION OF PTP FUNCTION AND AUGMENTATION OF PROTEIN-TYROSINE KINASE FUNCTION

Inhibition of protein-tyrosine phosphatases (PTPs) counterbalancing protein-tyrosine kinases (PTKs) offers a strategy for augmenting PTK actions. Conservation of PTP catalytic sites limits development of specific PTP inhibitors. A number of receptor PTPs, including the leukocyte common antigen-related (LAR) receptor and PTPmu, contain a wedge-shaped helix-loop-helix located near the first catalytic domain. Helix-loop-helix domains in other proteins demonstrate homophilic binding and inhibit function; therefore, we tested the hypothesis that LAR wedge domain peptides would exhibit homophilic binding, bind to LAR, and inhibit LAR function. Fluorescent beads coated with LAR or PTPmu wedge peptides demonstrated PTP-specific homophilic binding, and LAR wedge peptide-coated beads precipitated LAR protein. Administration of LAR wedge Tat peptide to PC12 cells resulted in increased proliferation, decreased cell death, increased neurite outgrowth, and augmented Trk PTK-mediated responses to nerve growth factor (NGF), a phenotype matching that found in PC12 cells with reduced LAR levels. PTPmu wedge Tat peptide had no effect on PC12 cells but blocked the PTPmu-dependent phenotype of neurite outgrowth of retinal ganglion neurons on a PTPmu substrate, whereas LAR wedge peptide had no effect. The survival- and neurite-promoting effect of the LAR wedge peptide was blocked by the Trk inhibitor K252a, and reciprocal co-immunoprecipitation demonstrated LAR/TrkA association. The addition of LAR wedge peptide inhibited LAR co-immunoprecipitation with TrkA, augmented NGF-induced activation of TrkA, ERK, and AKT, and in the absence of exogenous NGF, induced activation of TrkA, ERK, and AKT. PTP wedge domain peptides provide a unique PTP inhibition strategy and offer a novel approach for augmenting PTK function.

Imprint of evolutionary conservation and protein structure variation on the binding function of protein tyrosine kinases

According to the models of divergent molecular evolution, the evolvability of new protein function may depend on the induction of new phenotypic traits by a small number of mutations of the binding site residues. Evolutionary relationships between protein kinases are often employed to infer inhibitor binding profiles from sequence analysis. However, protein kinases binding profiles may display inhibitor selectivity within a given kinase subfamily, while exhibiting cross-activity between kinases that are phylogenetically remote from the prime target. The emerging insights into kinase function and evolution combined with a rapidly growing number of publically available crystal structures of protein kinases complexes have motivated structural bioinformatics analysis of sequence-structure relationships in determining the binding function of protein tyrosine kinases. In silico profiling of Imatinib mesylate and PD-173955 kinase inhibitors with protein tyrosine kinases is conducted on kinome scale by using evolutionary analysis and fingerprinting inhibitor-protein interactions with the panel of all publically available protein tyrosine kinases crystal structures. We have found that sequence plasticity of the binding site residues alone may not be sufficient to enable protein tyrosine kinases to readily evolve novel binding activities with inhibitors. While evolutionary signal derived solely from the tyrosine kinase sequence conservation can not be readily translated into the ligand binding phenotype, the proposed structural bioinformatics analysis can discriminate a functionally relevant kinase binding signal from a simple phylogenetic relationship. The results of this work reveal that protein conformational diversity is intimately linked with sequence plasticity of the binding site residues in achieving functional adaptability of protein kinases towards specific drug binding. This study offers a plausible molecular rationale to the experimental binding profiles of the studied kinase inhibitors and provides a theoretical basis for constructing functionally relevant kinase binding trees.

Block of the cell cycle of the yeast Saccharomyces cerevisiae by tyrphostin, an inhibitor of protein tyrosine kinase.

Tyrphostins are inhibitors of the epidermal growth factor receptor tyrosine kinase. To elucidate the biological function of protein tyrosine kinases in yeast cells, a mutant hypersensitive to tyrphostin was isolated and investigated for its response to the drug. The mutation was recessive and was designated tpt1 for tyrphostin hypersensitive. A tpt1 strain cannot grow in the presence of tyrphostin, implying that a biological process sensitive to tyrphostin is essential for cell growth. Microscopic observation indicated that large-budded cells were accumulated in the presence of the inhibitor. The results suggest the involvement of protein tyrosine phosphorylation in the cell cycle progression of Saccharomyces cerevisiae.

Integrin-mediated Invasion of Staphylococcus aureus into Human Cells Requires Src Family Protein-tyrosine Kinases

Staphylococcus aureus, a common cause of nosocomial infections, is able to invade eukaryotic cells by indirectly engaging beta1 integrin-containing host receptors, whereas non-pathogenic Staphylococcus carnosus is not invasive. Here, we identify intracellular signals involved in integrin-initiated internalization of S. aureus. In particular, the host cell actin cytoskeleton and Src family protein-tyrosine kinases (PTKs) are essential to mediate S. aureus invasion. Src PTKs are activated in response to pathogenic S. aureus, but not S. carnosus. In addition, pharmacological and genetic interference with Src PTK function reduces bacterial internalization. Importantly, Src PTK-deficient cells are resistant to S. aureus invasion, demonstrating the essentiality of host Src PTKs in integrin-mediated uptake of this pathogen.

The protein tyrosine kinases of the sea urchin Anthocidaris crassispina.

In order to know the function of protein tyrosine kinases (PTKs) in the development of sea urchin embryos, we performed reverse transcription-polymerase chain reaction (RT-PCR) to obtain partial cDNA fragments for PTK genes using primers to highly conserved regions of the PTK family. A total of seven PTK sequences were identified, two of which represented receptor PTK (RTK1 and RTK2), and five of which were non-receptor PTKs (NRTK1-5). RTK1 was highly similar to FGF receptor and Ret kinase, while RTK2 showed features of the insulin receptor family. NRTK1 and 2 belonged to the Src family and could be involved in egg activation at fertilization. NRTK3 showed the features of the Btk family kinases, while NRTK4 seemed to be a member of the Syk/ZAP70 family. NRTK5 is the Csk-type kinase of the sea urchin, which is known to negatively regulate the Src family kinases. RTK1 was not detected in unfertilized eggs and was activated after blastula stage. All the other PTK genes were expressed both maternally in unfertilized eggs and zygotically after fertilization, though each gene showed distinct temporal patterns.

Block of the cell cycle of the yeast Saccharomyces cerevisiae by tyrphostin, an inhibitor of protein tyrosine kinase

Tyrphostins are inhibitors of the epidermal growth factor receptor tyrosine kinase. To elucidate the biological function of protein tyrosine kinases in yeast cells, a mutant hypersensitive to tyrphostin was isolated and investigated for its response to the drug. The mutation was recessive and was designated tpt1 for t yr p hos t in hypersensitive. A tpt1 strain cannot grow in the presence of tyrphostin, implying that a biological process sensitive to tyrphostin is essential for cell growth. Microscopic observation indicated that large-budded cells were accumulated in the presence of the inhibitor. The results suggest the involvement of protein tyrosine phosphorylation in the cell cycle progression of Saccharomyces cerevisiae.

Tyrosine kinases in megakaryocytopoiesis.

Protein-tyrosine kinases (PTKs) are of vital importance in a variety of cell functions. Recent studies have provided considerable insight into the binding of growth factors to tyrosine kinase receptors and the consequent induction of signal pathways that lead to a biologic response. Future studies will further delineate the signals that result in a proliferative response and those that induce a differentiation response. Current studies, reviewed here, indicate an important biologic role for PTKs in the regulation of megakaryocyte development and maturation. Whether PTKs function in megakaryocytes in signaling pathways that are similar to pathways in other cells will need to be examined in future studies.

Nondissociation of GAL4 and GAL80 in vivo after galactose induction : K.K. LEUTHER AND S.A. JOHNSTON Science 256, 1333–1335

This chapter discusses the structure and function of protein-tyrosine kinases (PTK) receptor (PTKR). It focuses on the role of the PTKs as integral components of membrane signaling complexes. All receptor PTKs identified to date possess an N-terminal extracellular domain that binds to its cognate ligand. Potential ligands might be secreted peptide growth factors, hormones, membrane-associated tethered ligands, extracellular matrix proteins, or cell-surface adhesion molecules. The extracellular domain is often extensively glycosylated. This extracellular domain that provides the specificity of response to external signals. The distinct structural motifs found within this domain are used as the major basis for grouping of receptors. Common re-occurring structural motifs found within extracellular domains include clusters of cysteine residues with distinct spacing patterns, immunoglobulin (Ig)-like domains, epidermal growth factor (EGF)-like repeats, and fibronectin III-like repeats. Studies have shown that PTKRs plays an important role in the regulation of intracellular signal transduction pathways. Studies suggest that the concept of PTKR-mediated signaling complexes involving homology 2 (SH2) and homology 3 (SH3) domain-containing proteins are major mechanism underlying receptor function.

Methyl-nitrosourea (MNU)-induced rat mammary carcinoma expresses both particulate and cytosolic forms off protein tyrosine kinases

Protein tyrosine kinases (PTKs) coordinate a broad spectrum of cellular responses to extracellular stimuli and cell–cell interactions during development, tissue homeostasis, and responses to environmental challenges. Thus, an understanding of the regulatory mechanisms that ensure physiological PTK function and potential aberrations of these regulatory processes during diseases such as cancer are of broad interest in biology and medicine. Aside from the expected role of phospho-tyrosine phosphatases, recent studies have revealed a critical role of covalent modification of activated PTKs with ubiquitin as a critical mechanism of their negative regulation. Members of the Cbl protein family (Cbl, Cbl-b and Cbl-c in mammals) have emerged as dominant “activated PTK-selective” ubiquitin ligases. Structural, biochemical and cell biological studies have established that Cbl protein-dependent ubiquitination targets activated PTKs for degradation either by facilitating their endocytic sorting into lysosomes or by promoting their proteasomal degradation. This mechanism also targets PTK signaling intermediates that become associated with Cbl proteins in a PTK activation-dependent manner. Cellular and animal studies have established that the relatively broadly expressed mammalian Cbl family members Cbl and Cbl-b play key physiological roles, including their critical functions to prevent the transition of normal immune responses into autoimmune disease and as tumor suppressors; the latter function has received validation from human studies linking mutations in Cbl to human leukemia. These newer insights together with embryonic lethality seen in mice with a combined deletion of Cbl and Cbl-b genes suggest an unappreciated role of the Cbl family proteins, and by implication the ubiquitin-dependent control of activated PTKs, in stem/progenitor cell maintenance. Future studies of existing and emerging animal models and their various cell lineages should help test the broader implications of the evolutionarily-conserved Cbl family protein-mediated, ubiquitin-dependent, negative regulation of activated PTKs in physiology and disease.