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At least 1,621 records · Page 90Linked to original sources
PubMed2006

FAK and Src kinases are required for netrin-induced tyrosine phosphorylation of UNC5.

During neuronal development, netrin and its receptors UNC5 and DCC (deleted in colorectal cancer) guide axonal growth cones in navigating to their targets. Netrin also plays important roles in the regulation of cell migration, tissue morphogenesis and tumor growth. Here, we show that netrin induces UNC5 tyrosine phosphorylation and that this effect of netrin is dependent on its co-receptor DCC. UNC5 tyrosine phosphorylation is known to be important for netrin to induce cell migration and axonal repulsion. Src tyrosine kinase activity is required for netrin to stimulate UNC5 tyrosine phosphorylation in neurons and transfected cells. The SH2 domain of Src kinase directly interacts with the cytosolic domain of UNC5 in a tyrosine-phosphorylation-dependent manner. Furthermore, the tyrosine kinase focal adhesion kinase (FAK) is also involved in netrin-induced UNC5 tyrosine phosphorylation. Both Src and FAK can phosphorylate UNC5. Our data suggest a model in which netrin stimulates UNC5 tyrosine phosphorylation and signaling in a manner dependent on the co-receptor DCC, through the recruitment of Src and FAK kinases.

Animals↗
PubMed1998

Growth hormone-induced tyrosine phosphorylation of EGF receptor as an essential element leading to MAP kinase activation and gene expression.

GH binding to its receptor, which belongs to the cytokine receptor superfamily, activates Janus kinase (JAK) 2 tyrosine kinase, thereby activating a number of intracellular key proteins such as STAT (signal transducers and activators of transcription) proteins and mitogen-activated protein (MAP) kinases, which finally lead to GH's biological actions including gene expression. In contrast to receptor tyrosine kinases, the signalling pathways leading to MAP kinase activation by GH are poorly understood but appear to involve Grb2 and Shc. We now show that GH stimulated tyrosine phosphorylation of epidermal growth factor receptor (EGFR) and its association with Grb2, and concomitantly stimulated MAP kinase activity in liver, a major target tissue. Expression of EGFR and its mutants into CHO-GH receptor (GHR) cells revealed that GH-induced full activation of MAP kinase and c-fos expression required tyrosine phosphorylation sites of EGFR but not its intrinsic tyrosine kinase activity. Moreover, by also using dominant negative JAK2 and in vitro kinase assay, we demonstrated that tyrosine 1068 of EGFR was evidently one of the major phosphorylation and Grb2 binding sites stimulated by GH via JAK2. These data suggest that the role of EGFR in GH signalling is to be phosphorylated by JAK2, thereby providing docking sites for Grb2 and activating MAP kinases and gene expression. This novel cross talk pathway may provide the first example of the hormone and cytokine receptor superfamily transducing signals via associated nonreceptor tyrosine kinase by phosphorylating growth factor receptor and utilizing it as a docking protein independent of its receptor tyrosine kinase activity.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Tyrosine kinases as targets in cancer therapy - successes and failures.

Protein kinases play a crucial role in signal transduction and also in cellular proliferation, differentiation and various regulatory mechanisms. The inhibition of growth-related kinases, especially tyrosine kinases, might therefore provide new therapies for diseases such as cancer. Due to the enormous progress that has been made in the past few years in the identification of the human genome, in molecular and cell biology technologies, in structural biology and in bioinformatics, the number of receptor and non-receptor tyrosine kinases that have been identified as valuable molecular targets has greatly increased. Currently, more than 20 different tyrosine kinase targets are under evaluation in drug discovery projects in oncology. The progress made in the crystallisation of protein kinases, in most cases complexed with ATP-site-directed inhibitors, has confirmed that the ATPbinding domain of tyrosine kinases is an attractive target for rational drug design; more than 20 ATP-competitive, low molecular weight inhibitors are in various phases of clinical evaluation. Meanwhile, clinical proof-of-concept (POC) has been achieved with several antibodies and small molecules targeted against tyrosine kinases. With Herceptin, Glivec and Iressa (registered in Japan), the first kinase drugs have entered the market. This review describes the preclinical and clinical status of low molecular weight drugs targeted against different tyrosine kinases (e.g., epidermal growth factor receptor [EGFR], vascular endothelial growth factor receptor [VEGFR], platelet-derived growth factor receptor [PDGFR], Kit, Fms-like tyrosine kinase [Flt]-3), briefly describes new targets, and provides a critical analysis of the current situation in the area of tyrosine kinase inhibitors.

Animals↗
PubMed1996

Role of tyrosine phosphorylation in radiation-induced cell cycle-arrest of leukemic B-cell precursors at the G2-M transition checkpoint.

Here we provide experimental evidence that ionizing radiation induces inhibitory tyrosine phosphorylation of the p34cdc2 kinase in human leukemic B-cell precursors. Herbimycin A markedly reduced tyrosine phosphorylation of p34cdc2 in irradiated leukemic B-cell precursors, thereby preventing radiation-induced cell cycle arrest at the G2-M transition checkpoint. Thus, tyrosine phosphorylation is directly responsible for the inactivation of p34cdc2 in irradiated human leukemic B-cell precursors and activation of protein tyrosine kinases is a proximal and mandatory step in radiation-induced G2-arrest arrest at the G2-M checkpoint. Human WEE1 kinase isolated from unirradiated or irradiated leukemic B-cell precursors had minimal tyrosine kinase activity towards p34cdc2. We detected no increase of human WEE1 kinase activity after radiation of leukemic B-cell precursors, as measured by (a) autophosphorylation, (b) tyrosine phosphorylation of a synthetic peptide derived from the p34cdc2 amino-terminal region or (c) recombinant human p34cdc2-cyclin B complex. Thus the signaling pathway leading to inhibitory tyrosine phosphorylation of p34cdc2 and G2-arrest in irradiated human leukemic B-cell precursors functions independent of p49 WEE1 HU and enzymes which augment the tyrosine kinase activity of p49 WEE 1HU.

B-Lymphocytes↗
PubMed1998

Cadherin mediated cell-cell adhesion is regulated by tyrosine phosphatases in human keratinocytes.

Normal Human Keratinocytes express on their cell surface E- and P-cadherins, two Ca2+ dependent homophilic cell adhesion molecules that mediate keratinocyte-keratinocyte adherens junctions. In other cell types, adherens-type junctions are reported to be major subcellular targets for tyrosine specific protein phosphorylation (Volberg et al. (1991) Cell Regul., 2, 105-120) involving tyrosine kinases and tyrosine phosphatases. We investigated the role of tyrosine phosphatases in the regulation of cadherin mediated keratinocyte-keratinocyte adhesion. We report the results of a wide tyrosine phosphatase inhibition using pervanadate, a modified vanadate derivative known to inhibit most tyrosine phosphatases. Keratinocytes treated with pervanadate, exhibit an important change in cellular morphology and cadherins/catenins localization as shown by phase contrast microscopy and immunocytochemistry. In this conditions, cadherins and catenins no longer colocalize with the actin cytoskeleton of cells and the amount of E-cadherin bound to the cytoskeleton decreases. A more intense phosphotyrosine labelling is seen at the edges of the treated cells, suggesting that an increase in the phosphorylation rate of some cadherin-catenin complex proteins induces a diminished intercellular adhesion. Finally immunoprecipitation experiments of the E-cadherin/catenin complex from pervanadate treated keratinocytes reveal an increase in the tyrosine phosphorylation rate of E-cadherin, beta catenin and probably gamma catenin. These data suggest an essential role for the protein tyrosine phosphatases in keratinocyte intercellular junctions.

Actins↗
PubMed1995

Inhibition by 5'-methylthioadenosine of cell growth and tyrosine kinase activity stimulated by fibroblast growth factor receptor in human gliomas.

Stimulation of three human glioma cell lines with basic fibroblast growth factor (bFGF) led to the enhancement of cell growth and the rapid tyrosine phosphorylation of cellular proteins, including major substrates of 90 kD. A methyltransferase inhibitor, 5'-methylthioadenosine (MTA), inhibited dose dependently the bFGF-stimulated cell growth and protein tyrosine phosphorylation in glioma cells by blocking both receptor autophosphorylation and substrate phosphorylation, as shown by immunoblotting with antiphosphotyrosine antibodies and cross-linking bFGF to receptors. The antiproliferative activity of MTA correlated quantitatively with its potency as an inhibitor of bFGF-stimulated protein tyrosine kinase activity. The methyltransferase inhibitor MTA had no effect on either epidermal growth factor- or platelet-derived growth factor-stimulated protein tyrosine phosphorylation in glioma cells, but inhibited specifically bFGF-stimulated protein tyrosine kinase activity. The concentration of MTA required for inhibition of protein methylation correlated well with the concentration required for inhibition of bFGF-stimulated cell growth and protein tyrosine phosphorylation. Because MTA had no effect on numbers and dissociation constants of high- and low-affinity bFGF receptors, the inhibition of bFGF-stimulated bFGF receptor tyrosine kinase activity is not likely to be the result of a reduction in bFGF receptor and bFGF binding capacity. In fact, MTA delayed and reduced the internalization and nuclear translocation of bFGF, and the internalized bFGF was submitted to a limited proteolysis that converted it to lower molecular peptides whose presence remained for at least 22 hours. The effect of MTA on bFGF-stimulated tyrosine phosphorylation was immediate and readily reversible.

Antibodies↗
PubMed1996

Relation of ornithine decarboxylase and tyrosine kinase activity in the jejunal mucosa in vivo.

Our aim was to study the relationship between jejunal mucosal activity of ornithine decarboxylase and tyrosine kinase during proliferation in adolescent rats in vivo. Their relationship in the proliferating intestinal mucosa under in vivo conditions has not been reported before. From the results of in vitro studies, it was speculated that tyrosine kinase activity modulated ornithine decarboxylase activity during colonic mucosal proliferation (Majumdar AP. Am J Physiol 259:G626-G630, 1990). Jejunal mucosal hyperplasia was induced by Type 1 diabetes and suppressed in both control and diabetic rats by administration of difluoromethylornithine. Jejunal mucosal weight and enzyme activity were determined after 3, 6, and 10 days, and tyrosine-specific phosphorylated proteins after 10 days of induction of diabetes. Difluoromethylornithine suppressed jejunal mucosal proliferation and tyrosine kinase activity after the 6- and 10- day study periods. After the 3-day study period although jejunal mucosal growth was suppressed, tyrosine kinase activity was not. Activity of tyrosine kinase and ornithine decarboxylase were highly significantly correlated at all time periods in both control and diabetic rats. Tyrosine-specific phosphorylated proteins of 34, 54, 80, and 200 kDa proteins were observed in jejunal mucosa of both control and diabetic rats. In the difluoromethylornithine-treated rats, phosphorylation of the above proteins was negligible while the phosphorylation of a 14-kDa protein was prominent. We speculate that in vivo ornithine decarboxylase activity may be modulating tyrosine kinase activity and that phosphorylation of a 14-kDa protein was associated with suppressed mucosal growth in difluoromethylornithine-treated rats.

Animals↗
PubMed2002

Mutational analysis of immunoreceptor tyrosine-based inhibition motifs of the Ig-like transcript 2 (CD85j) leukocyte receptor.

The inhibitory receptor Ig-like transcript (ILT)2 (leukocyte Ig-like receptor or CD85j) is a type I transmembrane protein expressed by different leukocyte lineages. The extracellular region of ILT2 binds HLA class I molecules, and its cytoplasmic domain displays four immunoreceptor tyrosine-based inhibition motifs. Upon tyrosine phosphorylation ILT2 recruits the Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) that is involved in negative signaling. To address the structural basis of ILT2-mediated inhibitory signaling, deletion and single tyrosine mutants were generated and transfected in the COS-7 and rat basophilic leukemia cell lines; their abilities to bind SHP-1 and to inhibit FcepsilonR-induced serotonin release in rat basophilic leukemia cells were studied. Both biochemical and functional analyses revealed tyrosines 644 (SIYATL) and 614 (VTYAQL) as the SHP-1 docking sites required for ILT2 inhibitory function. Substitution of tyrosine 562 (VTYAEV) did not alter receptor function. By contrast, mutation of tyrosine 533 (NLYAAV) interfered with ILT2 tyrosine phosphorylation and the subsequent SHP-1 recruitment, thus supporting a regulatory role for this motif.

Amino Acid Motifs↗
PubMed2001

Molecular regulation of tumor cell vasculogenic mimicry by tyrosine phosphorylation: role of epithelial cell kinase (Eck/EphA2).

During embryogenesis, blood vessels are formed initially by the process of vasculogenesis, the in situ differentiation of mesenchymal cells into endothelial cells, which form a primitive, patterned vasculogenic network. This is followed by angiogenesis, the sprouting of new vessels from preexisting vasculature, to yield a more refined microcirculation. However, we and our collaborators have recently described a process termed "vasculogenic mimicry," which consists of the formation of patterned, tubular networks by aggressive melanoma tumor cells (in three-dimensional cultures in vitro), that mimics endothelial-formed vasculogenic networks and correlates with poor clinical prognosis in patients. Previous microarray analysis from our laboratory comparing the highly aggressive versus the poorly aggressive melanoma cells revealed a significant increased expression of tyrosine kinases associated with the aggressive melanoma phenotype. Because of the important role of protein tyrosine kinases in phosphorylating various signal transduction proteins that are critical for many cellular processes (e.g., cell adhesion, migration, and invasion), we examined whether protein tyrosine kinases are involved in melanoma vasculogenic mimicry. Immunofluorescence analysis of aggressive melanoma cells forming tubular networks in vitro showed that tyrosine phosphorylation activity colocalized specifically within areas of tubular network formation. A phosphotyrosine profile of the aggressive melanoma cells capable of forming tubular networks indicated differences in tyrosine phosphorylated proteins compared with the poorly aggressive melanoma cells (incapable of forming tubular networks). Most notably, we identified epithelial cell kinase (EphA2) as being one receptor tyrosine kinase expressed and phosphorylated exclusively in the aggressive metastatic melanoma cells. Furthermore, general inhibitors of protein tyrosine kinases hindered tube formation, and transient knockout of EphA2 abrogated the ability of tumor cells to form tubular structures. These results suggest that protein tyrosine kinases, particularly EphA2, are involved in the formation of tubular networks by aggressive melanoma tumor cells in vitro, which may represent a novel therapeutic target for further clinical investigation.

Humans↗
PubMedDate not supplied

Puromycin is a potent and specific inhibitor of tyrosine kinase activity in HL-60 cells.

The search for specific tyrosine kinase inhibitors constitutes a novel approach to the development of anticancer agents. Puromycin (PM) inhibits protein synthesis by causing the premature release of truncated PM-peptide complexes (PMPs), the structure of which predicts an inhibitory effect on tyrosine kinase activity (21). The present study was undertaken to investigate the effects of a low dose of PM (0.9 microM) on tyrosine kinase activity in HL-60 leukaemic cells. Experiments were specifically controlled to exclude inhibitory effects consequent on protein synthesis blockade. Soluble enzyme extracts derived from PM-treated cells showed attenuated phosphorylation of two independent synthetic tyrosine kinase-specific substrates, the polymer Poly(Glu:Tyr; 4:1) and the peptide substrate Raytide. In contrast, Protein Kinase C-specific activity was unaffected by PM-exposure. It is possible that inhibition was due to metabolites of PM, perhaps PMPs, rather than to PM itself. Whether the inhibition of tyrosine kinase activity observed with the substrates used in this study is equally applicable to all tyrosine kinases, is not yet clear. Nevertheless, low-dose PM could provide a useful tool for dissecting out the role of tyrosine phosphorylation in the regulation and dysregulation of cell growth, and could conceivably prove beneficial in the treatment of tumours in which tyrosine kinase overactivity is linked to oncogenesis.

Cycloheximide↗
PubMed1992

A synthetic peptide derived from p34cdc2 is a specific and efficient substrate of src-family tyrosine kinases.

A peptide derived from p34cdc2, cdc2(6-20)NH2 with the amino acid sequence of KVEKIGEGTYGVVYK-amide, was found to be a specific and efficient substrate for a pp60c-src-related protein tyrosine kinase from bovine spleen (STK). Glu-12 and Thr-14 were identified to be substrate specificity determinants in this peptide (Cheng, H.-C., Litwin, C. M. E., Hwang, D. M., and Wang, J. H. (1991) J. Biol. Chem. 266, 17919-17925). In this study, we demonstrated the presence of cdc2(6-20)NH2 peptide tyrosine kinase activity in the membrane fractions of bovine brain, spleen, thymus, lung, liver, and kidney. Hydroxylapatite column chromatography of thymus membrane extract revealed four protein tyrosine kinases, TK-I, TK-II, TK-III, and TK-IV, with different relative activities toward cdc2(6-20)NH2 and a general tyrosine kinase substrate, poly(Glu/Tyr). Only TK-I and TK-II showed significant activity toward cdc2(6-20)NH2, they were suggested as belonging to the src-family by virtue of their cross-reactivity with an antibody against a synthetic peptide corresponding to a conserved sequence of src-family kinases. Further immunological characterization using antibodies specific to individual src-related protein tyrosine kinases suggested that TK-I, TK-II, and STK are bovine homologs of p56lck, p55fyn, and p56lyn, respectively. Substrate specificity and kinetic characterization of src-family tyrosine kinases including human platelet pp60c-src, bovine p56lyn, p56lck, and p55fyn, as well as several non-src-related tyrosine kinases including epidermal growth factor receptor, p43v-abl, TK-III, and TK-IV showed that all the src-family tyrosine kinases but none of the other kinases displayed efficient cdc2(6-20)NH2 phosphorylation. In all cases, the high efficiency of cdc2(6-20)NH2 peptide phosphorylation could be markedly attenuated when Glu-12 and Thr-14 of the peptide were substituted, respectively, by valine and serine.

Amino Acid Sequence↗
PubMed1990

The amino-terminal region of pp60c-src has a modulatory role and contains multiple sites of tyrosine phosphorylation.

The c-src gene product from either platelet-derived growth factor treated cells or from polyomavirus-infected cells migrates anomalously on gels, displays enhanced kinase activity, and contains additional sites of tyrosine phosphorylation within its amino-terminus. To probe the importance of these post-translational modifications, each of the five amino-terminal tyrosine residues (residues 90, 92, 131, 136, and 149) was altered to phenylalanine using site-directed mutagenesis. Except for the 136F variant, the mutants displayed enhanced kinase activity, albeit at a low level shown insufficient to induce focus formation in NIH3T3 cells. Mutagenesis of residues adjacent to tyrosines 90 and 92 also resulted in variants with enhanced kinase activity indicating that the region and not the tyrosines per se, may be involved in regulating this activity. Fingerprinting analysis demonstrated that the enhanced kinase activity brought about by these mutations occurred through a mechanism which appears to be independent of the phosphorylation state of Tyr 416 and possibly Tyr 527. Upon treatment of cells with vanadate both amino-terminal variants and transforming mutants of pp60c-src displayed a slower migrating form, designated p60+. Hence, the appearance of these p60+ proteins may be elicited either by mutations that enhance the transforming activity of pp60c-src or by perturbations within the amino-terminal region of the enzyme. The retarded mobility of p60+ was shown to be due in part to additional tyrosine phosphorylations residing at its amino-terminus. The demonstration that p60+ could be resolved into multiple bands and that amino-terminal fragments containing phosphorylated tyrosine residues were obtained regardless of which of the five tyrosines in the region was altered to phenylalanine indicates that there are multiple sites of tyrosine phosphorylation within the amino-terminal region of pp60c-src.

Amino Acid Sequence↗
PubMed1990

Platelet-activating factor stimulation of tyrosine kinase and its relationship to phospholipase C in rabbit platelets: studies with genistein and monoclonal antibody to phosphotyrosine.

Platelet-activating factor (PAF) is a proinflammatory lipid that has platelet-stimulating property. PAF receptor-coupled activation of phosphoinositide-specific phospholipase C (PLC) and phosphorylation of several proteins has already been established in our laboratory. To investigate further the molecular mechanism and relationship between activation of PLC and protein phosphorylation, we have used Genistein (a putative inhibitor of tyrosine-specific protein kinases), phosphotyrosine antibody, and phosphoamino acid analysis to probe the involvement of tyrosine kinase in this process. Washed rabbit platelets were loaded with myo-[2-3H]inositol and challenged with PAF (100 nM) after pretreatment with Genistein. PLC-mediated production of radioactive inositol monophosphate, inositol diphosphate, and inositol triphosphate was monitored. PAF alone caused stimulation of PLC activity [( 3H]inositol triphosphate production), whereas pretreatment with Genistein (0.5 mM) diminished PAF-stimulated PLC activity to basal level. Genistein also blocked PAF-stimulated platelet aggregation at this dose. In contrast to Genistein, staurosporine which inhibits protein kinase C, potentiated PAF-stimulated [3H]inositol triphosphate production. Genistein substantially inhibited the combined effects of staurosporine and PAF on inositol triphosphate production. Genistein also reduced PAF-induced phosphorylation of Mr 20,000 and 50,000 proteins. Phorbol 12-myristate 13-acetate-induced Mr 40,000 protein phosphorylation was also affected by Genistein. The above results suggested that Genistein inhibited tyrosine kinase at an early stage of signal transduction by inhibiting PLC. This, in turn, decreased the activation of protein kinase C and, therefore, caused a reduction in Mr 40,000 protein phosphorylation. The inhibition of PLC by Genistein raised the possibility of involvement of tyrosine kinase in PAF receptor-coupled PLC activation. Western blot analysis using monoclonal antibody to phosphotyrosine demonstrated that PAF stimulated the tyrosine phosphorylation of two major proteins of 50,000 and 60,000 molecular weight. When platelets were challenged with PAF after treatment with either Genistein or CV-6209 (a PAF receptor antagonist), the reactivity of these proteins to monoclonal antibody was inhibited. Phosphoamino acid analysis of Mr 50,000 and 60,000 proteins confirmed that PAF increased the phosphorylation of tyrosine residues in both Mr 50,000 and 60,000 proteins and that this was inhibited by Genistein. Thus, PAF caused a receptor-dependent phosphorylation of tyrosine residues on Mr 50,000 and 60,000 proteins. Based on these observations, it is concluded that tyrosine kinase is involved in the PAF receptor-coupled PLC activation and signal transduction mechanism.

Animals↗
PubMed1991

Tyrosine phosphorylation is an essential event in the stimulation of B lymphocytes by Staphylococcus aureus Cowan I.

Staphylococcus aureus Cowan I (SAC) is a potent mitogen for purified human B cells. By using Western blotting with antiphosphotyrosine antibodies, we demonstrated that the mitogenic effect of SAC is associated with rapid tyrosine phosphorylation of proteins of 45, 68, 75, 97, and 145 kDa. This tyrosine phosphorylation was detected within 30 s of the addition of SAC; it reached a maximum within 10 min, after which it declined gradually. In contrast to SAC, most soluble anti-IgM antibodies do not induce proliferation of isolated human B cells. As indicated by Western blotting, soluble anti-IgM antibodies induced a similar pattern of tyrosine phosphorylation, with the exception of the 68-kDa protein, which was the most heavily phosphorylated protein in SAC-treated cells. A similar but less intense 68-kDa band was also induced by mitogenic anti-IgM bound to beads. This suggested that tyrosine phosphorylation, especially of p68, may play an important role in B cell mitogenesis. To test this hypothesis, we determined the effect of specific tyrosine kinase inhibitors (tyrphostins) on SAC-induced tyrosine phosphorylation, oncogene expression, and B cell proliferation. The concentration dependencies of inhibition of these processes suggested that they were linked. Nonspecific toxic effects of the tyrphostins were ruled out by the demonstration that the tyrphostins did not alter cell viability and did not inhibit B cell proliferation induced by phorbol esters, which do not induce tyrosine phosphorylation. For maximal inhibition of SAC-induced cell proliferation, the tyrophostins needed to be added before or shortly after addition of SAC. Taken together, these data indicate that tyrosine phosphorylation is an obligatory early signal in B cell proliferation.

Antibodies, Anti-Idiotypic↗
PubMed1975

The activity of tyrosine hydroxylase in intact adrenergic neurons of the mouse vas deferens.

A procedure is described for measuring the activity of tyrosine hydroxylase in the intact adrenergic neurons of the mouse vas deferens. In this procedure, the L-dopa-1-14C which is formed from L-tyrosine-1-14C by the action of tyrosine hydroxylase is selectively and quantitatively decarboxylated by endogenous aromatic-L-amino acid decarboxylase. Although considerable tyrosine hydroxylase activity can be demonstrated with the intact mouse vas deferens in the absence of exogenous tetrahydropterin cofactor, the addition of 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine results in increased activity. Exogenous norepinephrine inhibits the activity of tyrosine hydroxylase in the intact mouse vas deferens and this inhibitory effect is competitively antagonized by 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine. When the vesicular catecholamine uptake mechanism is blocked by treatment of mice with 5 mg/kg of reserpine 24 hours prior to sacrifice, the activity of tyrosine hydroxylase in intact vas deferens is reduced and the inhibitory effect of exogenous norepinephrine is enhanced. Inhibition of monoamine oxidase with 0.15 mM pargyline does not affect the activity of tyrosine hydroxylase in the intact mouse vas deferens when the vesicular catecholamine uptake mechanism is intact but has a pronounced inhibitory effect following reserpine treatment. These observations lend further support to the conclusion that the activity of tyrosine hydroxylase in the intact adrenergic neuron is inversely related to the catecholamine concentration within an extravesicular pool. They also suggest that the catecholamines tend to accumulate within this extravesicular pool and thus become accessible to the action of monoamine oxidase when the vesicular uptake mechanism is inactivated or when the vesicular stores are filled to capacity.

Animals↗
PubMed1986

[Tyrosine inhibition of anaphylactic shock in guinea pigs].

Experimental investigations on guinea-pigs were carried out to study the role of tyrosine in the prevention of anaphylaxis. It should be noted that the intensity of clinical manifestations of anaphylactic shock was reduced in 5 out of 24 L-tyrosine fed guinea-pigs (20.83%), while in 9 animals (37.5%) anaphylactic shock did not develop. 100% death of control animals was registered. The effect of tyrosine was absent only in 10 guinea-pigs. Positive effect of tyrosine was followed by tyrosine-aminotransferase activation with a tendency towards the decrease in tyrosine plasma level and the rise in cortisol blood level. It is suggested that tyrosine increases glucocorticoid secretion of the adrenal cortex: Glucocorticoids in their turn, enhance tyrosine-aminotransferase activity in the animal liver.

Anaphylaxis↗
PubMed1995

Involvement of a tyrosine kinase pathway in the growth-promoting effects of angiotensin II on aortic smooth muscle cells.

Angiotensin II (AII) is a growth factor that stimulates protein synthesis and induces cellular hypertrophy in aortic smooth muscle cells (SMC). This trophic effect is mediated by the AT1 subtype of AII receptors. However, very little is known about the cellular signaling pathways involved in this response. In the present study, we examined the role of protein tyrosine phosphorylation in the growth-promoting effects of AII on rat aortic SMC. The addition of AII to quiescent aortic SMC induced tyrosine phosphorylation of multiple substrates, as revealed by antiphosphotyrosine immunoblotting. This response was blocked by preincubation with the AT1-selective antagonist losartan. To explore the functional role of this signaling pathway, we performed experiments with two mechanistically distinct tyrosine kinase inhibitors. Treatment of quiescent aortic SMC with genistein and herbimycin A abolished the stimulatory effect of AII on overall protein tyrosine phosphorylation. Similarly, the two inhibitors prevented AII-induced tyrosine phosphorylation of the cytoskeletal protein paxillin. Under the same conditions, incubation with genistein or herbimycin A did not interfere with AII binding to the AT1 receptor and did not significantly affect AII-stimulated inositol-1,4,5-trisphosphate production and Ca2+ mobilization. In parallel to their selective action on tyrosine phosphorylation, both genistein and herbimycin A completely inhibited AII-stimulated protein synthesis in a dose-dependent manner. In contrast, the two inhibitors were much less potent in preventing the trophic effect of phorbol-12-myristate 13-acetate in these cells. We further demonstrate that genistein and herbimycin A did not prevent mitogen-activated protein kinase activation and c-fos gene induction, which is consistent with the notion that these downstream effectors do not link AII-induced tyrosine phosphorylation to protein synthesis. These results provide evidence that tyrosine phosphorylation has a critical role in cellular hypertrophy and is involved in AII action in vascular SMC.

Angiotensin II↗
PubMed1994

Identification of a protein, SPY75, with repetitive helix-turn-helix motifs and an SH3 domain as a major substrate for protein tyrosine kinase(s) activated by Fc epsilon RI cross-linking.

Cross-linking of the high affinity receptor for IgE (Fc epsilon RI) initiates various biochemical and morphologic changes leading to degranulation and synthesis and release of cytokines and lipid mediators. Tyrosine phosphorylation of several cellular proteins was previously reported as the earliest signaling event for the Fc epsilon RI signal transduction pathway. By amino acid sequence determination and cDNA cloning analysis, a 75-kDa protein, termed SPY75, was identified as a major tyrosine-phosphorylated protein in activated mouse mast cells. SPY75, barely tyrosine phosphorylated in resting cells, was rapidly and transiently tyrosine phosphorylated on Fc epsilon RI cross-linking in an Ag concentration-dependent manner. Similar SPY75 tyrosine phosphorylation was observed when Ag receptors on B and T lymphocytes were cross-linked by appropriate antibodies. However, IL-3, granulocyte macrophage-CSF, or stem cell factor did not induce tyrosine phosphorylation of SPY75 in PT-18 mast cells, despite their responsiveness to these cytokines. SPY75 was not physically associated with the receptor or other known signaling molecules. This protein, the mouse homologue of the human HS1 gene product, has putative repetitive helix-turn-helix motifs found in many DNA-binding proteins and a putative nuclear transport signal. It also has a Src homology 3 domain, which is found in many signaling molecules and cytoskeletal proteins. These structural features and the rapid tyrosine phosphorylation on Fc epsilon RI cross-linking suggest that the signal generated by Fc epsilon RI cross-linking is transmitted through tyrosine phosphorylation of SPY75.

Amino Acid Sequence↗