Tyrosine kinase and cooperative TGFbeta signaling in the reproductive organs of Schistosoma mansoni

Drug-induced suppression of female schistosome sexual maturation is an auspicious strategy to combat schistosomiasis since the eggs are the causative agent. The establishment of drug targets requires knowledge about the molecular mechanisms that regulate the development of the female reproductive organs, which include vitellarium and ovary. This review summarizes recent studies suggesting tyrosine kinases as important factors for the regulation of female gonad development. In this context, especially cytoplasmatic tyrosine kinases of the Src class seem to play dominant roles. Moreover, experimental data and theoretical concepts are provided supporting a crosstalk between tyrosine kinase and TGFbeta signaling in the production of vitellocytes. Finally, we take advantage from the schistosome genome project to propose a model for the regulation of vitelline-cell production and differentiation.     

Protein tyrosine nitration in hydrophilic and hydrophobic environments

Summary. In this review we address current concepts on the biological occurrence, levels and consequences of protein tyrosine nitration in biological systems. We focused on mechanistic aspects, emphasizing on the free radical mechanisms of protein 3-nitrotyrosine formation and critically analyzed the restrictions for obtaining large tyrosine nitration yields in vivo, mainly due to the presence of strong reducing systems (e.g. glutathione) that can potently inhibit at different levels the nitration process. Evidence is provided to show that the existence of metal-catalyzed processes, the assistance of nitric oxide-dependent nitration steps and the facilitation by hydrophobic environments, provide individually and/or in combination, feasible scenarios for nitration in complex biological milieux. Recent studies using hydrophobic tyrosine analogs and tyrosine-containing peptides have revealed that factors controlling nitration in hydrophobic environments such as biomembranes and lipoproteins can differ to those in aqueous compartments. In particular, exclusion of key soluble reductants from the lipid phase will more easily allow nitration and lipid-derived radicals are suggested as important mediators of the one-electron oxidation of tyrosine to tyrosyl radical in proteins associated to hydrophobic environments. Development and testing of hydrophilic and hydrophobic probes that can compete with endogenous constituents for the nitrating intermediates provide tools to unravel nitration mechanisms in vitro and in vivo; additionally, they could also serve to play cellular and tissue protective functions against the toxic effects of protein tyrosine nitration.     

Design, synthesis and evaluation of new 6-substituted-5-benzyloxy-4-oxo-4H-pyran-2-carboxamides as potential Src inhibitors

Src family kinases (SFKs) are nonreceptor tyrosine kinases that are reported to be critical for cancer progression. Inhibiting the catalytic activity of these proteins has become one of the major therapeutic concepts in contemporary drug discovery. We report here the design and the synthesis of novel 6-substituted-5-benzyloxy-4-oxo-4H-pyran-2-carboxamides as potential inhibitors of Src kinase. The synthesis of these derivatives and the preliminary results of biological activity will be discussed.     

Regulation of total and myofibrillar protein breakdown in rat extensor digitorum longus and soleus muscle incubated flaccid or at resting length.

The present study characterized total and myofibrillar protein breakdown rates in a muscle preparation frequently used in vitro, i.e. incubated extensor digitorum longus (EDL) and soleus (SOL) muscles of young rats. Total and myofibrillar protein breakdown rates were assessed by determining net production by the incubated muscles of tyrosine and 3-methylhistidine (3-MH) respectively. Both amino acids were determined by h.p.l.c. Both total and myofibrillar protein breakdown rates were higher in SOL than in EDL muscles and were decreased by incubating the muscles maintained at resting length, rather than flaccid. After fasting for 72 h, total protein breakdown (i.e. tyrosine release) was increased by 73% and 138% in EDL muscles incubated flaccid and at resting length respectively. Net production of tyrosine by SOL muscle was not significantly altered by fasting. In contrast, myofibrillar protein degradation (i.e. 3-MH release) was markedly increased by fasting in both muscles. When tissue was incubated in the presence of 1 munit of insulin/ml, total protein breakdown rate was inhibited by 17-20%, and the response to the hormone was similar in muscles incubated flaccid or at resting length. In contrast, myofibrillar protein breakdown rate was not altered by insulin in any of the muscle preparations. The results support the concepts of individual regulation of myofibrillar and non-myofibrillar proteins and of different effects of various conditions on protein breakdown in different types of skeletal muscle. Thus determination of both tyrosine and 3-MH production in red and white muscle is important for a more complete understanding of protein regulation in skeletal muscle.     

Functional Characterization of the Kinase Activation Loop in Nucleophosmin (NPM)-Anaplastic Lymphoma Kinase (ALK) Using Tandem Affinity Purification and Liquid Chromatography-Mass Spectrometry

Previous studies have shown that the kinase activation loop (KAL) of the oncogenic fusion protein NPM-ALK regulates its overall tyrosine phosphorylation status and tumorigenicity. Using tandem affinity purification-mass spectrometry, we assessed how the KAL of NPM-ALK regulates the phosphorylation status of its individual tyrosines. Using the lysates of GP293 cells transfected with NPM-ALK, our highly reproducible results showed evidence of phosphorylation in all 3 tyrosines in KAL and 8 tyrosines outside KAL. We created 7 KAL mutants, each of which carried a Tyr-to-Phe mutation of ≥1 of the 3 tyrosines in KAL. A complete loss of the 8 phosphotyrosines outside KAL was found in 3 KAL mutants, and their oncogenicity (assessed by cell viability, colony formation, and the ability to phosphorylate effector proteins) was abrogated. A partial loss of the 8 phosphotyrosines was found in 4 KAL mutants, but their oncogenicity did not show simple correlation with the number of residual phosphotyrosines. Tyr-to-Phe mutations of each of the 8 phosphotyrosines outside KAL did not result in a significant decrease in the oncogenicity. In conclusion, we have provided details of how the KAL in NPM-ALK regulates its tyrosine phosphorylation pattern. Our results challenge some of the current concepts regarding the relationship between the tyrosine phosphorylation and oncogenicity of NPM-ALK.     

Design,synthesis and evaluation of new 6-substituted-5-benzyloxy-4-oxo-4H-pyran-2-carboxamides as potential Src inhibitors

Src family kinases (SFKs) are nonreceptor tyrosine kinases that are reported to be critical for cancer progression. Inhibiting the catalytic activity of these proteins has become one of the major therapeutic concepts in contemporary drug discovery. We report here the design and the synthesis of novel 6-substituted-5-benzyloxy-4-oxo-4H-pyran-2-carboxamides as potential inhibitors of Src kinase. The synthesis of these derivatives and the preliminary results of biological activity will be discussed.     

Focal adhesion kinase (FAK) perspectives in mechanobiology: implications for cell behaviour

Mechanobiology is a scientific interface discipline emerging from engineering and biology. With regard to tissue-regenerative cell-based strategies, mechanobiological concepts, including biomechanics as a target for cell and human mesenchymal stem cell behaviour, are on the march. Based on the periodontium as a paradigm, this mini-review discusses the key role of focal-adhesion kinase (FAK) in mechanobiology, since it is involved in mediating the transformation of environmental biomechanical signals into cell behavioural responses via mechanotransducing signalling cascades. These processes enable cells to adjust quickly to environmental cues, whereas adjustment itself relies on the specific intramolecular phosphorylation of FAK tyrosine residues and the multiple interactions of FAK with distinct partners. Furthermore, interaction-triggered mechanotransducing pathways govern the dynamics of focal adhesion sites and cell behaviour. Facets of behaviour not only include cell spreading and motility, but also proliferation, differentiation and apoptosis. In translational terms, identified and characterized biomechanical parameters can be incorporated into innovative concepts of cell- and tissue-tailored clinically applied biomaterials controlling cell behaviour as desired.     

Receptor-mediated endocytosis and cytoskeleton

Receptor-mediated endocytosis is the most specific pathway for macromolecules and macromolecular complexes generally designated as ligands to enter cells. Upon binding to their transmembrane receptors, the ligands enter endocytic vesicles that fuse with each other giving rise to the so-called early endosomes. The sorting of ligand-receptor complexes internalized in these endosomes depends on their nature: metabolic receptors are recycled back to the plasma membrane, while signaling receptors and their ligands (e.g. receptor tyrosine kinases or receptors associated with tyrosine kinase) are delivered to internal vesicles of the multivesicular late endosomes and finally are degraded after interaction with lysosomes. During these processes, endosomes undergo translocation from the cell periphery to the juxtanuclear region, which is accompanied by multiple fusion, invagination, tabulation, and membrane fission events. This review considers modern concepts of the sorting mechanisms of ligand-receptor complexes, the crosstalk between endosomes, microtubules, and actin, and the role of this crosstalk in endosome maturation.     

Protein tyrosine phosphatases and neural development

During neural development, cells interact dynamically with each other and with the extracellular matrix, using cell signaling to control differentiation, axonogenesis, and survival. Enzymes that regulate protein tyrosine phosphorylation often lie at the core of such cell signaling. Protein tyrosine phosphatases (PTPases) are recognized as being of central importance here, and a growing family of PTPases are now known to be expressed in embryonic neurons and glia. Both receptor-like and cytoplasmic enzymes have been identified. The receptor family includes immunoglobulin superfamily members that influence cell–cell adhesion, proteoglycans that control neurite growth, and enzymes in Drosophila that regulate axon guidance and target cell recognition. Cytoplasmic PTPases are implicated in nerve cell commitment and potentially in the regulation of cell survival. This review outlines what we currently know about PTPases in the nervous system and presents concepts concerning their possible modes of action. BioEssays 20 :463–472, 1998. © 1998 John Wiley & Sons, Inc.     

酪氨酸磷酸化蛋白质组学技术研究进展及其在生物医学研究中的应用

在酪氨酸磷酸化蛋白质组学的研究过程中,酪氨酸磷酸化位点的富集是最重要的一步。目前常用的富集方法是抗体亲和富集或SH2 superbinder富集。此外,通过质谱与生物信息学等技术,可实现大规模酪氨酸磷酸化位点的鉴定。对酪氨酸磷酸化蛋白质组学进行深度覆盖研究,揭示癌症发生发展过程中失调的激酶,将有助于深入理解癌症的发生发展过程;且由于75%的致癌基因是酪氨酸激酶基因,酪氨酸激酶抑制剂作为抗癌药物受到了越来越多的关注。应用酪氨酸磷酸化蛋白质组学技术,可以鉴定与癌症等重大疾病相关的酪氨酸激酶,从而帮助找到酪氨酸激酶抑制剂。总之,酪氨酸磷酸化蛋白质组学技术可以在酪氨酸激酶鉴定、酪氨酸激酶抑制剂研究及酪氨酸磷酸化信号通路研究等生物医学领域中得到很好的应用。     

Regulation of mitochondrial oxidative phosphorylation through cell signaling

The mitochondrial oxidative phosphorylation (OxPhos) system plays a key role in energy production, the generation of free radicals, and apoptosis. A lack of cellular energy, excessive radical production, and dysregulated apoptosis are found alone or in combination in most human diseases, including neurodegenerative diseases, stroke, cardiovascular disorders, ischemia/reperfusion, and cancer. In the context of its relevance to human disease, this article reviews current knowledge about the regulation of OxPhos with a focus on cell signaling and discusses identified phosphorylation sites with the aid of crystal structures of OxPhos complexes. Several recent studies have shown that all OxPhos components can be phosphorylated; even the small electron carrier cytochrome c is tyrosine phosphorylated in vivo. We propose that in higher organisms, in contrast to bacteria, cell signaling pathways are the main regulator of energy production, triggered for example by hormones. Pathways that have been identified to act on OxPhos include protein kinases A and C and growth factor activated receptor tyrosine kinase signaling. Present knowledge about kinases and phosphatases that execute signals at the level of the mitochondrial OxPhos system, and newly emerging concepts, such as the translocation of kinases to the mitochondria upon stimulation of a signaling pathway, are discussed.     

Four states of tyrosine residues in the fibrinogen molecule

The ionization of tyrosine residues in fibrinogen was studied by a spectrophotometric method. The total of 100 tyrosine residues in the fibrinogen molecule was classified into four states: (1) 28 tyrosine residues with pK 10.1 (m = 1.0). (2) tyrosine residues with pK 11.5 (m = 1.0), (3) 20 tyrosine residues with pK 12.2 (m = 3.0) and (4) 10 tyrosine residues non-ionizable. When fibrinogen was treated with 4 M guanidine . HCl, all of the tyrosine residues became ionizable with the ionization characteristics of pK 10.1 (m = 1.0). The ionization characteristics of tyrosine residues in plasmin-digested fibrinogen were similar to those of fibrinogen, while in CNBr-treated fibrinogen they were fairly different. The value, m, stands for the number of hydroxyl ions involved in the ionization of a tyrosine residue.     

Protein tyrosine phosphorylation in sperm during gamete interaction in the mouse: the influence of glucose

A key intracellular event during capacitation is protein tyrosine phosphorylation, but its involvement during sperm interaction with the oocyte has not been investigated. Glucose is necessary to achieve fertilization and thus may have an influence on sperm protein tyrosine phosphorylation. The objectives of this study were to 1) visualize protein tyrosine phosphorylation patterns in sperm during capacitation and interaction with the oocyte and 2) determine the influence of glucose. Protein tyrosine phosphorylation was investigated by Western analysis and immunofluorescence. Protein tyrosine phosphorylation was increased during capacitation, and immunofluorescence revealed that zona binding and gamete fusion were correlated with an increase in tyrosine phosphorylation of proteins in the midpiece. During capacitation, the absence of glucose led to a delay in the appearance of protein tyrosine phosphorylation. Following binding to the zona pellucida and the oolemma, tyrosine phosphorylation in the flagellum was also delayed in the absence of glucose and resulted in a significant inhibition of the midpiece phosphorylation. The correlation between successful gamete fusion and the tyrosine phosphorylation of midpiece proteins suggests that the effect of glucose on sperm-oocyte interaction is mediated through regulation of protein tyrosine phosphorylation in a specific area of the fertilizing sperm.     

Pharmacokinetics of systemically administered tyrosine: a comparison of serum, brain tissue and in vivo microdialysate levels in the rat

Tyrosine uptake has been reported to differ across brain regions. However, such studies have typically been conducted over brief intervals and in anesthetized rats; anesthesia itself affects amino acid transport across the blood-brain barrier. To address these concerns, serum, brain tissue and in vivo microdialysate tyrosine levels were compared for 0-3 h after administration of tyrosine [0.138-1.10 mmol/kg intraperitoneally (i.p.)] to groups of awake rats. Serum and brain tissue tyrosine levels increased linearly with respect to dose. Basal tissue tyrosine levels varied significantly across brain regions [medial prefrontal cortex (MPFC), striatum, hypothalamus, and cerebellum], but the rate of tyrosine uptake was similar for hypothalamus, striatum and MPFC. For brain regions in which tyrosine levels in both microdialysate and tissue were assayed, namely MPFC and striatum, there was a high degree of correlation between tyrosine levels in tissue and in microdialysate. Increasing brain tyrosine levels had no effect on DA levels in MPFC microdialysate. We conclude that (i) regional differences in the response of dopamine neurons to systemic tyrosine administration cannot be attributed to pharmacokinetic factors; (ii) in vivo microdialysate provides an excellent index over time and across a wide range of tyrosine doses, of brain tissue tyrosine levels; and (iii) increases in brain tyrosine levels do not affect basal DA release in the MPFC.     

The contribution of phenylalanine to tyrosine metabolism in vivo. Studies in the post-absorptive and phenylalanine-loaded rat.

1. Rates of appearance and oxidation of plasma L-leucine, L-phenylalanine and L-tyrosine, as well as conversion of plasma phenylalanine into plasma tyrosine, were determined in 90-120 g rats after overnight starvation and while receiving 115-120 mumol of L-phenylalanine/h. 2. In the post-absorptive state, plasma tyrosine and phenylalanine appearances were similar, despite the fact that 22% of plasma tyrosine appearance could be attributed to the hydroxylation of phenylalanine. 3. A constant infusion of 115-120 mumol of L-phenylalanine/h did not significantly alter plasma leucine kinetics, but increased appearance of plasma phenylalanine and tyrosine. The percentage of phenylalanine and tyrosine appearance that was oxidized increased from 12.1% and 24.4% to 37.3% and 48.0% respectively. In phenylalanine-loaded rats, 72% of plasma tyrosine appearance could be attributed to the conversion of phenylalanine. 4. Whole-body tyrosine oxidation measured from a continuous infusion of either L-[14C]tyrosine or L-[14C]phenylalanine differed by 165%. 5. It can be concluded that, in the post-absorptive state, phenylalanine hydroxylation makes a substantial contribution to the plasma appearance of tyrosine and is significantly increased when phenylalanine is administered. The disposal of excess infused phenylalanine is a result of a greater percentage of plasma phenylalanine being converted into tyrosine and a greater proportion of tyrosine being further oxidized. However, apparent tyrosine oxidation rates estimated from plasma tyrosine specific radioactivities and appearance of expired 14CO2 during administration of [14C]tyrosine are underestimates of true rates, in part because tyrosine generated from phenylalanine hydroxylation is catabolized without freely equilibrating with the plasma compartment.     

Dectin-1: a signalling non-TLR pattern-recognition receptor

Dectin-1 is a natural killer (NK)-cell-receptor-like C-type lectin that is thought to be involved in innate immune responses to fungal pathogens. This transmembrane signalling receptor mediates various cellular functions, from fungal binding, uptake and killing, to inducing the production of cytokines and chemokines. These activities could influence the resultant immune response and can, in certain circumstances, lead to autoimmunity and disease. As I discuss here, understanding the molecular mechanisms behind these functions has revealed new concepts, including collaborative signalling with the Toll-like receptors (TLRs) and the use of spleen tyrosine kinase (SYK), that have implications for the role of other non-TLR pattern-recognition receptors in immunity.     

Tyrosine content, influx and accumulation rate, and catecholamine biosynthesis measured in vivo, in the central nervous system and in peripheral organs of the young rat. Influence of neonatal hypo- and hyperthyroidism

The influence of neonatal hypo- and hyperthyroidism on different aspects of tyrosine metabolism in the hypothalamus, striatum, brainstem, adrenal glands, heart and brown adipose tissue (BAT) were studied in 14-day old rats. The synthesis rate of catecholamines (CA) was also determined in vivo after the injection of labelled tyrosine. Hypothyroidism increases tyrosinaemia and endogenous tyrosine concentration in the hypothalamus and BAT. Hyperthyroidism decreases tyrosinaemia and endogenous tyrosine levels in the striatum, adrenals and heart. The accumulation rate of tyrosine determined 30 min after an intravenous injection of the labelled amino acid has been determined in the organs, together with the influx of the amino acid, determined within 20s. Hypothyroidism increases tyrosine accumulation rate in all the organs studied, and tyrosine clearance is decreased in the striatum and brainstem; together with an increased tyrosinaemia, this leads to a normal influx. The influx of tyrosine is increased in the hypothalamus. Hyperthyroidism decreases tyrosine accumulation rate in all the organs except the adrenals. These results indicate that the thyroid status of the young rat can influence tyrosine uptake mechanisms, without modifying an organ's tyrosine content. The fact that hypothyroidism increases tyrosine influx in the hypothalamus without modifying it in the brainstem and striatum reflects an heterogeneous reactivity to the lack of thyroid hormones in different brain structures. Neonatal hypothyroidism decreases the CA synthesis rate in the striatum, the heart and the interscapular brown adipose tissue, while synthesis was enhanced in the brainstem and the adrenals. It is likely that these variations in CA synthesis are due to thyroid hormone modulation of tyrosine hydroxylase activity, the enzyme which catalyses the rate limiting step in CA biosynthesis.     

Regulation of hepatic tyrosine aminotransferase in genetically obese rats

The activities of hepatic tyrosine aminotransferase, tryptophan oxygenase and serine dehydratase were increased in obese rats shortly after weaning. Immunotitration experiments showed that the increase in tyrosine aminotransferase activity resulted from an increase in enzyme protein in obese rats. No increase in hepatic tyrosine aminotransferase was observed in suckling pre-obese rats. The post-weaning increase in hepatic tyrosine aminotransferase of obese rats was only observed during the light phase of the diurnal cycle, but was prevented by pair-feeding and by starvation. Tryptophan increased hepatic tyrosine aminotransferase of lean rats to obese levels but had no effect in obese rats until tyrosine aminotransferase levels were reduced by starvation or adrenalectomy. Adrenalectomy abolished the increase in hepatic tyrosine aminotransferase activity in obese rats although serum corticosterone was normal in these animals. Hepatic and brain tyrosine concentrations were decreased in obese rats but normalized after adrenalectomy. The results suggest that the corticosteroid-dependent increase in food and tryptophan intake may be the primary cause of the increased hepatic amino acid catabolism of obese rats.     

Effect of Additional Protein or Methionine and Threonine on Tyrosine Catabolism in Rats Fed Diets High in Tyrosine

The supplementation of additional protein or methionine and threonine to a high tyrosinc-low protein diet has previously been shown to prevent the tyrosine toxicity. To elucidate the mechanism, studies were performed on the effect of these supplements on the capacity to oxidize excessive tyrosine. Male weanling rats were ad libitum fed a 10% casein diet containing 5% tyrosine with and without extra casein or methionine plus threonine for 7 days, then animals were injected intraperitoneally with l-tyrosine-U-¹⁴C and the oxidation rate to ¹⁴CO₂ determined in vivo at an interval of several hours throughout a 24-hour period. The addition of extra casein or methionine and threonine to the high tyrosine diet enhanced the ability of tyrosine oxidation, and decreased the radioactivities of the TCA-soluble fractions in plasma, liver and muscle. A high level of free tyrosine in blood and tissues was also lowered by the addition of these supplements. The diurnal chenges in free tyrosine concentration of various tissues were observed. The data suggest that the beneficial effect of extra casein or methionine and threonine supplementation on tyrosine toxicity is due to an increased rate of tyrosine catabolism which results in lower tyrosine concentrations in body fluids which overcomes tyrosine toxicity.     

Inactivation of tyrosine aminotransferase in neutral homogenates and rat liver slices

Inactivation of tyrosine aminotransferase induced in vivo by triamcinolone was studied in a homogenate incubated at neutral pH values. The integrity and the presence of subcellular particles together with a compartment of acidic pH are necessary for inactivation of tyrosine aminotransferase. It is suggested that tyrosine aminotransferase is inactivated inside lysosomes. The system responsible for inactivation of tyrosine aminotransferase was partially purified and identified with lysosomal cathepsins B and B(1). Inactivation of tyrosine aminotransferase in liver slices is controlled by the amino acid concentration and strongly stimulated by cysteine. 3,3',5-Tri-iodo-l-thyronine reversibly and strongly decreases the rate of inactivation of tyrosine aminotransferase. The effect is not due to an increased rate of tyrosine aminotransferase synthesis.