Transglutaminases and receptor tyrosine kinases

Transglutaminases are confounding enzymes which are known to play key roles in various cellular processes. In this paper, we aim to bring together several pieces of evidence from published research and literature that suggest a potentially vital role for transglutaminases in receptor tyrosine kinases (RTK) signalling. We cite literature that confirms and suggests the formation of integrin:RTK:transglutaminase complexes and explores the occurrence and functionality of these complexes in a large fraction of the RTK family.

Signaling of receptor tyrosine kinases in the nucleus

Since the discovery of the first receptor tyrosine kinase (RTK) proteins in the late 1970s and early 1980s, many scientists have explored the functions of these important cell signaling molecules. The finding that these proteins are often deregulated or mutated in diseases such as cancers and diabetes, together with their potential as clinical therapeutic targets, has further highlighted the necessity for understanding the signaling functions of these important proteins. The mechanisms of RTK regulation and function have been recently reviewed by Lemmon & Schlessinger (2010) but in this review we instead focus on the results of several recent studies that show receptor tyrosine kinases can function from subcellular localisations, including in particular the nucleus, in addition to their classical plasma membrane location. Nuclear localisation of receptor tyrosine kinases has been demonstrated to be important for normal cell function but is also believed to contribute to the pathogenesis of several human diseases.

An investigation of the C77G and C772T variations within the human protein tyrosine phosphatase receptor type C gene for association with multiple sclerosis in an Australian population

Multiple sclerosis (MS) is a common cause of neurological disability in young adults. The disease generally manifests in early to middle adulthood and causes various neurological deficits. Autoreactive T lymphocytes and their associated antigens have long been presumed important features of MS pathogenesis. The Protein tyrosine phosphatase receptor type C gene (PTPRC) encodes the T-cell receptor CD45. Variations within PTPRC have been previously associated with diseases of autoimmune origin such as type 1 diabetes mellitus and Graves' disease. We set out to investigate two variants within the PTPRC gene, C77G and C772T in subjects with MS and matched healthy controls to determine whether significant differences exist in these markers in an Australian population. We employed high resolution melt analysis (HRM) and restriction length polymorphism (RFLP) techniques to determine genotypic and allelic frequencies. Our study found no significant difference between frequencies for PTPRC C77G by either genotype (Χ2 = 0.65, P = 0.72) or allele (Χ2 = 0.48, P = 0.49). Similarly, we did not find evidence to suggest an association between PTPRC C772T by genotype (Χ2 = 1.06, P = 0.59) or allele (Χ2 = 0.20, P = 0.66). Linkage disequilibrium (LD) analysis showed strong linkage disequilibrium between the two tested markers (D' = 0.9970, SD = 0.0385). This study reveals no evidence to suggest that these markers are associated with MS in the tested Australian Caucasian population. Although the PTPRC gene has a significant role in regulating CD4+ and CD8+ autoreactive T-cells, interferon-beta responsiveness, and potentially other important processes, our study does not support a role for the two tested variants of this gene in MS susceptibility in the Australian population.

Dynamic contrast-enhanced magnetic resonance imaging as a biomarker for the pharmacological response of PTK787/ZK 222584, an inhibitor of the vascular endothelial growth factor receptor tyrosine kinases, in patients with advanced colorectal cancer and liver metastases: Results from two phase I studies

Purpose: PTK787/ZK 222584 (PTK/ZK), an orally active inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, inhibits VEGF-mediated angiogenesis. The pharmacodynamic effects of PTK/ZK were evaluated by assessing changes in contrast-enhancement parameters of metastatic liver lesions using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in patients with advanced colorectal cancer treated in two ongoing, dose-escalating phase I studies. Patients and Methods: Twenty-six patients had DCE-MRI performed at baseline, day 2, and at the end of each 28-day cycle. Doses of oral PTK/ZK ranged from 50 to 2000 mg once daily. Tumor permeability and vascularity were assessed by calculating the bidirectional transfer constant (Ki). The percentage of baseline Ki (% of baseline Ki) at each time point was compared with pharmacokinetic and clinical end points. Results: A significant negative correlation exists between the % of baseline Ki and increase in PTK/ZK oral dose and plasma levels (P = .01 for oral dose; P = .0001 for area under the plasma concentration curve at day 2). Patients with a best response of stable disease had a significantly greater reduction in Ki at both day 2 and at the end of cycle 1 compared with progressors (mean difference in % of baseline Ki, 47%, P = .004%; and 51%, P = .006; respectively). The difference in % of baseline Ki remained statistically significant after adjusting for baseline WHO performance status. Conclusion: These findings should help to define a biologically active dose of PTK/ZK. These results suggest that DCE-MRI may be a useful biomarker for defining the pharmacological response and dose of angiogenesis inhibitiors, such as PTK/ZK, for further clinical development. © 2003 by American Society of Clinical Oncology.

Direct evidence that Gi-coupled receptor stimulation of mitogen-activated protein kinase is mediated by G beta gamma activation of p21ras.

Stimulation of Gi-coupled receptors leads to the activation of mitogen-activated protein kinases (MAP kinases). In several cell types, this appears to be dependent on the activation of p21ras (Ras). Which G-protein subunit(s) (G alpha or the G beta gamma complex) primarily is responsible for triggering this signaling pathway, however, is unclear. We have demonstrated previously that the carboxyl terminus of the beta-adrenergic receptor kinase, containing its G beta gamma-binding domain, is a cellular G beta gamma antagonist capable of specifically distinguishing G alpha- and G beta gamma-mediated processes. Using this G beta gamma inhibitor, we studied Ras and MAP kinase activation through endogenous Gi-coupled receptors in Rat-1 fibroblasts and through receptors expressed by transiently transfected COS-7 cells. We report here that both Ras and MAP kinase activation in response to lysophosphatidic acid is markedly attenuated in Rat-1 cells stably transfected with a plasmid encoding this G beta gamma antagonist. Likewise in COS-7 cells transfected with plasmids encoding Gi-coupled receptors (alpha 2-adrenergic and M2 muscarinic), the activation of Ras and MAP kinase was significantly reduced in the presence of the coexpressed G beta gamma antagonist. Ras-MAP kinase activation mediated through a Gq-coupled receptor (alpha 1-adrenergic) or the tyrosine kinase epidermal growth factor receptor was unaltered by this G beta gamma antagonist. These results identify G beta gamma as the primary mediator of Ras activation and subsequent signaling via MAP kinase in response to stimulation of Gi-coupled receptors.

Functional interaction of vascular endothelial-protein-tyrosine phosphatase with the angiopoietin receptor Tie-2

During development of the vertebrate vascular system essential signals are transduced via protein-tyrosine phosphorylation. Null-mutations of receptor-tyrosine kinase (RTK) genes expressed in endothelial cells (ECs) display early lethal vascular phenotypes. We aimed to identify endothelial protein-tyrosine phosphatases (PTPs), which should have similar importance in EC-biology. A murine receptor-type PTP was identified by a degenerated PCR cloning approach from endothelial cells (VE-PTP). By in situ hybridization this phosphatase was found to be specifically expressed in vascular ECs throughout mouse development. In experiments using GST-fusion proteins, as well as in transient transfections, trapping mutants of VE-PTP co-precipitated with the Angiopoietin receptor Tie-2, but not with the Vascular Endothelial Growth Factor receptor 2 (VEGFR-2/Flk-1). In addition, VE-PTP dephosphorylates Tie-2 but not VEGFR-2. We conclude that VE-PTP is a Tie-2 specific phosphatase expressed in ECs, and VE-PTP phosphatase activity serves to specifically modulate Angiopoietin/Tie-2 function. Based on its potential role as a regulator of blood vessel morphogenesis and maintainance, VE-PTP is a candidate gene for inherited vascular malformations similar to the Tie-2 gene.

Interplay between receptor tyrosine kinases and hypoxia signaling in cancer.

Deregulated signaling via receptor tyrosine kinase (RTK) pathways is prevalent in numerous types of human cancers and is commonly correlated with worst prognosis, resistance to various treatment modalities and increased mortality. Likewise, hypoxic tumors are often manifested by aggressive mode of growth and progression following an adaptive genetic reprogramming with consequent transcriptional activation of genes encoding proteins, which support tumor survival under low oxygen-related conditions. Consequently, both the hypoxia-inducible factor (HIF) system, which is the major mediator of hypoxia-related signaling, and numerous RTK systems are considered critical molecular targets in current cancer therapy. It is now evident that there is an intricate molecular crosstalk between RTKs and hypoxia-related signaling in the sense that hypoxia can activate expression of particular RTKs and/or their corresponding ligands, while some RTK systems have been shown to trigger activation of the HIF machinery. Moreover, signaling regulation of some RTK systems under hypoxic conditions has also been documented to take place in a HIF-independent manner. With this review we aim at overviewing the most current observations on that topic and highlight the importance of the potential co-drugging the HIF system along with particular relevant RTKs for better tumor growth control.

Receptor-associated constitutive protein tyrosine phosphatase activity controls the kinase function of JAK1

Exposure of cells to protein tyrosine phosphatase (PTP) inhibitors causes an increase in the phosphotyrosine content of many cellular proteins. However, the level at which the primary signaling event is affected is still unclear. We show that Jaks are activated by tyrosine phosphorylation in cells that are briefly exposed to the PTP inhibitor pervanadate (PV), resulting in tyrosine phosphorylation and functional activation of Stat6 (in addition to other Stats). Mutant cell lines that lack Jak1 activity fail to support PV-mediated [or interleukin 4 (IL-4)-dependent] activation of Stat6 but can be rescued by complementation with functional Jak1. The docking sites for both Jak1 and Stat6 reside in the cytoplasmic domain of the IL-4 receptor α-chain (IL-4Rα). The glioblastoma-derived cell lines T98G, GRE, and M007, which do not express the IL-4Rα chain, fail to support Stat6 activation in response to either IL-4 or PV. Complementation of T98G cells with the IL-4Rα restores both PV-mediated and IL-4-dependent Stat6 activation. Murine L929 cells, which do not express the γ common chain of the IL-4 receptor, support PV-mediated but not IL-4-dependent Stat6 activation. Thus, Stat6 activation by PV is an IL-4Rα-mediated, Jak1-dependent event that is independent of receptor dimerization. We propose that receptor-associated constitutive PTP activity functions to down-regulate persistent, receptor-linked kinase activity. Inhibition or deletion of PTP activity results in constitutive activation of cytokine signaling pathways.

The Syk and ZAP-70 SH2-containing tyrosine kinases are implicated in pre-T cell receptor signaling

An early stage in thymocyte development, after rearrangement of the β chain genes of the T cell receptor (TCR), involves expression of the pre-TCR complex and accompanying differentiation of CD4−CD8− double negative (DN) cells to CD4+CD8+ double positive (DP) cells. The ZAP-70 and Syk tyrosine kinases each contain two N-terminal SH2 domains that bind phosphorylated motifs in antigen receptor subunits and are implicated in pre-T receptor signaling. However, mice deficient in either ZAP-70 or Syk have no defect in the formation of DP thymocytes. Here we show that, in mice lacking both Syk and ZAP-70, DN thymocytes undergo β chain gene rearrangement but fail to initiate clonal expansion and are incapable of differentiating into DP cells after expression of the pre-TCR. These data suggest that the ZAP-70 and Syk tyrosine kinases have crucial but overlapping functions in signaling from the pre-TCR and hence in early thymocyte development.

Ceramide-induced inhibition of T lymphocyte voltage-gated potassium channel is mediated by tyrosine kinases

The n-type K+ channel (n-K+, Kv1.3) in lymphocytes has been recently implicated in the regulation of Fas-induced programmed cell death. Here, we demonstrate that ceramide, a lipid metabolite synthesized upon Fas receptor ligation, inhibits n-K+ channel activity and induces a tyrosine phosphorylation of the Kv1.3 protein in Jurkat T lymphocytes. Tyrosine phosphorylation of the n-K+ channel correlated with an activation of the Src-like tyrosine kinase p56lck upon cellular treatment with the ceramide analog C6-ceramide. Because genetic deficiency of p56lck or inhibition of Src-like tyrosine kinases by herbimycin A prevented ceramide-mediated n-K+ channel inhibition and tyrosine phosphorylation, we propose a ceramide-initiated activation of p56lck resulting in tyrosine phosphorylation and inhibition of the n-K+ channel protein.

BCR-ABL and v-SRC tyrosine kinase oncoproteins support normal erythroid development in erythropoietin receptor-deficient progenitor cells

Erythropoietin (Epo)-independent differentiation of erythroid progenitors is a major characteristic of myeloproliferative disorders, including chronic myeloid leukemia. Epo receptor (EpoR) signaling is crucial for normal erythroid development, as evidenced by the properties of Epo−/− and EpoR−/− mice, which contain a normal number of fetal liver erythroid progenitors but die in utero from a severe anemia attributable to the absence of red cell maturation. Here we show that two constitutively active cytoplasmic protein tyrosine kinases, P210BCR-ABL and v-SRC, can functionally replace the EpoR and support full proliferation, differentiation, and maturation of fetal liver erythroid progenitors from EpoR−/− mice. These protein tyrosine kinases can also partially complement the myeloid growth factors IL-3, IL-6, and Steel factor, which are normally required in addition to Epo for erythroid development. Additionally, BCR-ABL mutants that lack residues necessary for transformation of fibroblasts or bone marrow cells can fully support normal erythroid development. These results demonstrate that activated tyrosine kinase oncoproteins implicated in tumorigenesis and human leukemia can functionally complement for cytokine receptor signaling pathways to support normal erythropoiesis in EpoR-deficient cells. Moreover, terminal differentiation of erythroid cells requires generic signals provided by activated protein tyrosine kinases and does not require a specific signal unique to a cytokine receptor.

Oncogenic mutation in the Kit receptor tyrosine kinase alters substrate specificity and induces degradation of the protein tyrosine phosphatase SHP-1

Activating mutations in the Kit receptor tyrosine kinase have been identified in both rodent and human mast cell leukemia. One activating Kit mutation substitutes a valine for aspartic acid at codon 816 (D816V) and is frequently observed in human mastocytosis. Mutation at the equivalent position in the murine c-kit gene, involving a substitution of tyrosine for aspartic acid (D814Y), has been described in the mouse mastocytoma cell line P815. We have investigated the mechanism of oncogenic activation by this mutation. Expression of this mutant Kit receptor tyrosine kinase in a mast cell line led to the selective tyrosine phosphorylation of a 130-kDa protein and the degradation, through the ubiquitin-dependent proteolytic pathway, of a 65-kDa phosphoprotein. The 65-kDa protein was identified as the src homology domain 2 (SH2)-containing protein tyrosine phosphatase SHP-1, a negative regulator of signaling by Kit and other hematopoietic receptors, and the protein product of the murine motheaten locus. This mutation also altered the sites of receptor autophosphorylation and peptide substrate selectivity. Thus, this mutation activates the oncogenic potential of Kit by a novel mechanism involving an alteration in Kit substrate recognition and the degradation of SHP-1, an attenuator of the Kit signaling pathway.

Long-term potentiation increases tyrosine phosphorylation of the N-methyl-D-aspartate receptor subunit 2B in rat dentate gyrus in vivo.

Long-term potentiation (LTP) is a form of synaptic memory that may subserve developmental and behavioral plasticity. An intensively investigated form of LTP is dependent upon N-methyl-D-aspartate (NMDA) receptors and can be elicited in the dentate gyrus and hippocampal CA1. Induction of this type of LTP is triggered by influx of Ca2+ through activated NMDA receptors, but the downstream mechanisms of induction, and even more so of LTP maintenance, remain controversial. It has been reported that the function of NMDA receptor channel can be regulated by protein tyrosine kinases and protein phosphatases and that inhibition of protein tyrosine kinases impairs induction of LTP. Herein we report that LTP in the dentate gyrus is specifically correlated with tyrosine phosphorylation of the NMDA receptor subunit 2B in an NMDA receptor-dependent manner. The effect is observed with a delay of several minutes after LTP induction and persists in vivo for several hours. The potential relevance of this post-translational modification to mechanisms of LTP and circuit plasticity is discussed.

Structural analysis of p185c-neu and epidermal growth factor receptor tyrosine kinases: oligomerization of kinase domains.

The epidermal growth factor receptor (EGFR) and p185c-neu proteins associate as dimers to create an efficient signaling assembly. Overexpression of these receptors together enhances their intrinsic kinase activity and concomitantly results in oncogenic cellular transformation. The ectodomain is able to stabilize the dimer, whereas the kinase domain mediates biological activity. Here we analyze potential interactions of the cytoplasmic kinase domains of the EGFR and p185c-neu tyrosine kinases by homology molecular modeling. This analysis indicates that kinase domains can associate as dimers and, based on intermolecular interaction calculations, that heterodimer formation is favored over homodimers. The study also predicts that the self-autophosphorylation sites located within the kinase domains are not likely to interfere with tyrosine kinase activity, but may regulate the selection of substrates, thereby modulating signal transduction. In addition, the models suggest that the kinase domains of EGFR and p185c-neu can undergo higher order aggregation such as the formation of tetramers. Formation of tetrameric complexes may explain some of the experimentally observed features of their ligand affinity and hetero-receptor internalization.