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.

Neuropsychological development in children with early and continuously treated phenylketonuria : association with biochemical markers

PKU is a genetically inherited inborn error of metabolism caused by a deficiency of the enzyme phenylalanine hydroxylase. The failure of this enzyme causes incomplete metabolism of protein ingested in the diet, specifically the conversion of one amino acid, phenylalanine, to tyrosine, which is a precursor to the neurotransmitter dopamine. Rising levels of phenylalanine is toxic to the developing brain, disrupting the formation of white matter tracts. The impact of tyrosine deficiency is not as well understood, but is hypothesized to lead to a low dopamine environment for the developing brain. Detection in the newborn period and continuous treatment (a low protein phe-restricted diet supplemented with phenylalanine-free protein formulas) has resulted in children with early and continuously treated PKU now developing normal I.Q. However, deficits in executive function (EF) are common, leading to a rate of Attention Deficit Hyperactivity Disorder (ADHD) up to five times the norm. EF worsens with exposure to higher phenylalanine levels, however recent research has demonstrated that a high phenylalanine to tyrosine ratio (phenylalanine:tyrosine ratio), which is hypothesised to lead to poorer dopamine function, has a more negative impact on EF than phenylalanine levels alone. Research and treatment of PKU is currently phenylalanine-focused, with little investigation of the impact of tyrosine on neuropsychological development. There is no current consensus as to the veracity of tyrosine monitoring or treatment in this population. Further, the research agenda in this population has demonstrated a primary focus on EF impairment alone, even though there may be additional neuropsychological skills compromised (e.g., mood, visuospatial deficits). The aim of this PhD research was to identify residual neuropsychological deficits in a cohort of children with early and continuously treated phenylketonuria, at two time points in development (early childhood and early adolescence), separated by eight years. In addition, this research sought to determine which biochemical markers were associated with neuropsychological impairments. A clinical practice survey was also undertaken to ascertain the current level of monitoring/treatment of tyrosine in this population. Thirteen children with early and continuously treated PKU were tested at mean age 5.9 years and again at mean age 13.95 years on several neuropsychological measures. Four children with hyperphenylalaninemia (a milder version of PKU) were also tested at both time points and provide a comparison group in analyses. Associations between neuropsychological function and biochemical markers were analysed. A between groups analysis in adolescence was also conducted (children with PKU compared to their siblings) on parent report measures of EF and mood. Minor EF impairments were evident in the PKU group by age 6 years and these persisted into adolescence. Life-long exposure to high phenylalanine:tyrosine ratio and/or low tyrosine independent of phenylalanine were significantly associated with EF impairments at both time points. Over half the children with PKU showed severe impairment on a visuospatial task, and this was associated only with concurrent levels of tyrosine in adolescence. Children with PKU also showed a statistically significant decline in a language comprehension task from 6 years to adolescence (going from normal to subnormal), this deficit was associated with lifetime levels of phenylalanine. In comparison, the four children with hyperphenylalaninemia demonstrated normal function at both time points, across all measures. No statistically significant differences were detected between children with PKU and their siblings on the parent report of EF and mood. However, depressive symptoms were significantly correlated with: EF; long term high phe:tyr exposure; and low tyrosine levels independent of phenylalanine. The practice survey of metabolic clinics from 12 countries indicated a high level of variability in terms of monitoring/treatment of tyrosine in this population. Whilst over 80% of clinics surveyed routinely monitored tyrosine levels in their child patients, 25% reported treatment strategies to increase tyrosine (and thereby lower the phenylalanine:tyrosine ratio) under a variety of patient presentation conditions. Overall, these studies have shown that EF impairments associated with PKU provide support for the dopamine-deficiency model. A language comprehension task showed a different trajectory, serving a timely reminder that non-EF functions also remain vulnerable in this population; and that normal function in childhood does not guarantee normal function by adolescence. Mood impairments were associated with EF impairments as well as long term measures of phenylalanine:tyrosine and/or tyrosine. The implications of this research for enhanced clinical guidelines are discussed given varied current practice.

Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma

Background: The EphB4 receptor tyrosine kinase has been reported as increased in tumours originating from several different tissues and its expression in a prostate cancer xenograft model has been reported. Methods: RT-PCR, western blotting and immunohistochemical techniques were used to examine EphB4 expression and protein levels in human prostate cancer cell lines LNCaP, DU145 and PC3. Immunohistochemistry was also used to examine localisation of EphB4 in tissue samples from 15 patients with prostate carcinomas. Results: All three prostate cancer cell lines expressed the EphB4 gene and protein. EphB4 immunoreactivity in vivo was significantly greater in human prostate cancers as compared with matched normal prostate epithelium and there appeared to be a trend towards increased expression with higher grade disease. Conclusions: EphB4 is expressed in prostate cancer cell lines with increased expression in human prostate cancers when compared with matched normal tissue. EphB4 may therefore be a useful anti-prostate cancer target. © 2005 Lee et al., licensee BioMed Central Ltd.

Cellular settings mediating Src Substrate switching between Focal Adhesion Kinase Tyrosine 861 and CUB-domain-containing protein 1 (CDCP1) Tyrosine 734

Reciprocal interactions between Src family kinases (SFKs) and focal adhesion kinase (FAK) are critical during changes in cell attachment. Recently it has been recognized that another SFK substrate, CUB-domain-containing protein 1 (CDCP1), is differentially phosphorylated during these events. However, the molecular processes underlying SFK-mediated phosphorylation of CDCP1 are poorly understood. Here we identify a novel mechanism in which FAK tyrosine 861 and CDCP1-Tyr-734 compete as SFK substrates and demonstrate cellular settings in which SFKs switch between these sites. Our results show that stable CDCP1 expression induces robust SFK-mediated phosphorylation of CDCP1-Tyr-734 with concomitant loss of p-FAK-Tyr-861 in adherent HeLa cells. SFK substrate switching in these cells is dependent on the level of expression of CDCP1 and is also dependent on CDCP1-Tyr-734 but is independent of CDCP1-Tyr-743 and -Tyr-762. In HeLa CDCP1 cells, engagement of SFKs with CDCP1 is accompanied by an increase in phosphorylation of Src-Tyr-416 and a change in cell morphology to a fibroblastic appearance dependent on CDCP1-Tyr-734. SFK switching between FAK-Tyr-861 and CDCP1-Tyr-734 also occurs during changes in adhesion of colorectal cancer cell lines endogenously expressing these two proteins. Consistently, increased p-FAK-Tyr-861 levels and a more epithelial morphology are seen in colon cancer SW480 cells silenced for CDCP1. Unlike protein kinase Cδ, FAK does not appear to form a trimeric complex with Src and CDCP1. These data demonstrate novel aspects of the dynamics of SFK-mediated cell signaling that may be relevant during cancer progression.

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.

Association between migraine and a functional polymorphism at the dopamine β-hydroxylase locus

Migraine is a common neurological disorder with a significant genetic component. Although a number of linkage and association studies have been undertaken, the number and identity of all migraine susceptibility genes has yet to be defined. The existence of dopaminergic hypersensitivity in migraine has been recognised on a pharmacological basis and some studies have reported genetic association between migraine and dopamine-related gene variants. Our laboratory has previously reported association of migraine with a promoter STR marker in the dopamine beta hydroxylase (DBH) gene. In the present study, we analysed two additional DBH markers in two independent migraine case–control cohorts. These two markers are putative functional SNPs, one within the promoter (−1021C→T) and another SNP (+1603C→T) in exon 11 of the DBH gene. The results showed a significant association for allelic and genotypic frequency distribution between the DBH marker in the promoter and migraine in the first (P = 0.004 and P = 0.012, respectively) and the second (P = 0.013 and P = 0.031, respectively) tested cohorts. There was no association observed between either genotype and/or allelic frequencies for the DBH marker located in exon 11 and migraine (P ≥ 0.05). The promoter DBH marker, reported associated with migraine in this study, has been shown to affect up to 52% of plasma DBH activity. Varying DBH activity levels have been postulated to be involved in migraine process with an increase of dopamine, resulting from a lower DBH activity shown positively correlated with migraine severity. It is plausible that the functional promoter variant of DBH may play a role in the migraine disorder.

Association between a 19 bp deletion polymorphism at the dopamine beta-hydroxylase (DBH) locus and migraine with aura

Migraine is a debilitating neurological disorder, affecting 12% of Caucasian populations. It is well known that migraine has a strong genetic component, although the type and number of genes involved is unclear. Our previous work has investigated dopamine related migraine candidate genes and has reported a significant allelic association with migraine of a microsatellite localised to the promoter region of the dopamine beta-hydroxylase (DBH) gene. The present study performed an association analysis in a larger population of case-controls (275 unrelated Caucasian migraineurs versus 275 controls) examining two different genetic DBH polymorphisms (a functional insertion/deletion promoter and a coding SNP A444G polymorphism). Although no significant association was found for the SNP polymorphism, the results showed a significant association between the insertion/deletion variant and disease (chi(2)=8.92, P=0.011), in particular in migraine with aura (chi(2)=11.53, P=0.003) compared to the control group. Furthermore, the analysis of this polymorphism stratified by gender, revealed that male individuals with the homozygote deletion genotype had three times the risk of developing migraine, compared to females. The DBH insertion/deletion polymorphism is in linkage disequilibrium with the previously reported migraine associated DBH microsatellite and this insertion/deletion polymorphism is functional, which may explain a potential role in susceptibility to migraine.

Evidence for allelic association of the dopamine β-hydroxylase gene (DBH) with susceptibility to typical migraine

Migraine is a debilitating neurological disorder characterized by recurrent attacks of severe headache. The disorder is highly prevalent, affecting approximately 12% of Caucasian populations. It is well known that migraine has a strong genetic component, although the type and number of genes involved is not yet clear. However, the calcium channel gene, CACNA1A, on chromosome 19 contains mutations responsible for familial hemiplegic migraine, a rare and severe subtype of migraine. There is also evidence to suggest that serotonin- and dopamine-related genes may be involved in the pathogenesis of migraine. This study employed a linkage and association approach to investigate neurotransmitter-related migraine candidate genes. Polymorphisms within the dopamine beta-hydroxylase (DBH) gene, serotonin transporter gene (SERT), and dopamine receptor gene (DRD2) were tested in 177 unrelated Caucasian migraineurs and 182 control individuals. In addition, an independent sample of 82 families affected with migraine was examined. Unrelated case-control association analysis of a DBH intragenic dinucleotide polymorphism indicated altered allelic distribution between migraine and control groups (L2=16.53, P=0.019). Furthermore, the transmission/disequilibrium test, which was implemented on the family data, also indicated distortion of allele transmission for the same DBH marker (L2=4.44, P=0.035). Together, these results provide evidence for allelic association of the DBH gene with typical migraine susceptibility (Fisher's combined P value=0.006) and indicate that further research into the role of the DBH gene in the etiology of migraine is warranted.

Voltammetric monitoring of gold nanoparticle formation facilitated by glycyl-L-tyrosine : relation to electronic spectra and transmission electron microscopy images

Voltammetric techniques have been introduced to monitor the formation of gold nanoparticles produced via the reaction of the amino acid glycyl-L-tyrosine with Au(III) (bromoaurate) in 0.05 M KOH conditions. The alkaline conditions facilitate amino acid binding to Au(III), inhibit the rate of reduction to Au(0), and provide an excellent supporting electrolyte for voltammetric studies. Data obtained revealed that a range of time-dependent gold solution species are involved in gold nanoparticle formation and that the order in which reagents are mixed is critical to the outcome. Concomitantly with voltammetric measurements, the properties of gold nanoparticles formed are probed by examination of electronic spectra in order to understand how the solution environment present during nanoparticle growth affects the final distribution of the nanoparticles. Images obtained by the ex situ transmission electron microscopy (TEM) technique enable the physical properties of the nanoparticles isolated in the solid state to be assessed. Use of this combination of in situ and ex situ techniques provides a versatile framework for elucidating the details of nanoparticle formation.

Receptor tyrosine kinases and their activation in melanoma

Receptor tyrosine kinases (RTKs) and their downstream signalling pathways have long been hypothesized to play key roles in melanoma development. A decade ago, evidence was derived largely from animal models, RTK expression studies and detection of activated RAS isoforms in a small fraction of melanomas. Predictions that overexpression of specific RTKs implied increased kinase activity and that some RTKs would show activating mutations in melanoma were largely untested. However, technological advances including rapid gene sequencing, siRNA methods and phospho-RTK arrays now give a more complete picture. Mutated forms of RTK genes including KIT, ERBB4, the EPH and FGFR families and others are known in melanoma. Additional over- or underexpressed RTKs and also protein tyrosine phosphatases (PTPs) have been reported, and activities measured. Complex interactions between RTKs and PTPs are implicated in the abnormal signalling driving aberrant growth and survival in malignant melanocytes, and indeed in normal melanocytic signalling including the response to ultraviolet radiation. Kinases are considered druggable targets, so characterization of global RTK activity in melanoma should assist the rational development of tyrosine kinase inhibitors for clinical use. © 2011 John Wiley & Sons A/S.

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.

Tyrosine phosphorylation mediates ConA-induced membrane type 1-matrix metalloproteinase expression and matrix metalloproteinase-2 activation in MDA-MB-231 human breast carcinoma cells

ConA-induced cell surface activation of pro-matrix metalloproteinase-2 (pro-MMP-2) by MDA-MB-231 human breast cancer cells is apparently mediated by up-regulation of membrane type 1 MMP (MT1-MMP) through transcriptional and posttranscriptional mechanisms. Here, we have explored the respective roles of cell surface clustering and protein tyrosine phosphorylation in the ConA- induction effects. Treatment with succinyl-ConA, a variant lacking significant clusterability, partially stimulated MT1-MMP mRNA and protein levels but did not induce MMP-2 activation, suggesting that clustering contributes to the transcriptional regulation by ConA but appears to be critical for the nontranscriptional component. We further found that genistein, an inhibitor of tyrosine phosphorylation, blocked ConA-induced pro-MMP-2 activation and ConA-induced MT1-MMP mRNA level in a dose-dependent manner, implicating tyrosine phosphorylation in the transcriptional aspect. This was confirmed by the dose-dependent promotion of pro-MMP-2 activation by sodium orthovanadate in the presence of suboptimal concentrations of ConA (7.5 μg/ml), with optimal effects seen at 25 μg/g orthovanadate. Genistein did not inhibit the ConA potentiation of MMP-2 activation in MCF-7 cells, in which transfected MT1-MMP is driven by a heterologous promoter, supporting the major implication of phosphotyrosine in the transcriptional component of ConA regulation. These data describe a major signaling event upstream of MT1- MMP induction by ConA and set the stage for further analysis of the nontranscriptional component.

A collagen prolyl 4-hydroxylase inhibitor reduces adhesions after tendon injury

Collagen synthesis inhibition potentially can reduce adhesion formation after tendon injury but also may affect cutaneous wound healing. We hypothesized that a novel orally administered collagen synthesis inhibitor (CPHI-I) would substantially reduce flexor tendon adhesions after injury, without any clinically important effect on cutaneous wound healing. The experiments were performed in a rat model with an in-continuity crush injury model in the rat hindfoot flexor tendon to provoke adhesion formation. Assays of dermal collagen production and the rate of healing of an excised wound were performed to assess cutaneous wound healing. Animals in the treatment groups received CPHI-I for 1, 2, or 6 weeks and were assessed at either 2 or 6 weeks. The work of flexion in the injured digit was reduced in the CPHI-I-treated animals compared with control animals, (0.188 J versus 0.0307 J at 2 weeks, and 0.0231 J versus 0.0331 J at 6 weeks) The cutaneous wound healing rate was similar in all animals, but dermal collagen synthesis was reduced in the treated animals. The CPHI-I seems to reduce tendon adhesion, and although collagen synthesis was reduced in cutaneous wounds, CPHI-I did not retard wound healing.