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PubMed2026

Dietary L-Tyrosine Sustains Egg Production and Quality by Modulating Promoter CpG Methylation, Gene Expression, and Hormone Profiles in Reproductive Signalling Pathways of Aged Laying Hens.

Managing prolactin (PRL)-mediated reproductive suppression is critical for maintaining egg production in commercial laying hens. L-tyrosine (TYR), a precursor of dopamine (DA), may counteract age-related reproductive decline by suppressing PRL, thereby sustaining productivity and egg quality in late-phase laying hens. The present study evaluated the effects of dietary L-tyrosine supplementation on laying performance, egg quality, reproductive hormone profiles, gene expression, and promoter CpG methylation in aged laying hens. A total of 180 Atak-S laying hens (71 weeks old) were randomly assigned to three dietary treatments, each with six replicates of 10 hens, and were fed a basal diet without L-tyrosine (control) or the basal diet supplemented with 0.5 g/kg (TYR0.5) or 1 g/kg (TYR1) L-tyrosine for 12 weeks. Hens in the TYR1 group exhibited higher hen-day egg production, improved feed conversion ratio, and significantly greater eggshell weight, shell thickness, Haugh unit, yolk index, and albumen index than the control group (p < 0.05). Dietary L-tyrosine increased plasma DA and serum estradiol while reducing serum PRL (p < 0.05). L-tyrosine also upregulated the expression of key dopaminergic and reproductive signalling genes, with corresponding promoter hypomethylation (p < 0.05), whereas PRL mRNA expression tended to decrease with concurrent promoter hypermethylation. A significant negative correlation was observed between target gene expression and promoter CpG methylation levels (p < 0.05). These findings indicate that dietary L-tyrosine modulates reproductive hormone profiles and molecular responses within reproductive signalling pathways, resulting in improved laying performance and egg quality. Therefore, L-tyrosine may represent a promising nutritional intervention to sustain reproductive function and productivity in late-phase laying hens.

Animals↗

PubMed1997

Internalization of Escherichia coli by human renal epithelial cells is associated with tyrosine phosphorylation of specific host cell proteins.

Human renal epithelial cells are capable of internalizing Escherichia coli regardless of whether the bacteria are isolated from individuals with pyelonephritis or from healthy volunteers. In this study, we investigated the role of host cell tyrosine kinase activity in internalization. We found that internalization of both fecal and pyelonephritis isolates is blocked by tyrosine kinase inhibitors. We found increased intensity of two tyrosine-phosphorylated proteins, with relative mobilities of approximately 123,000 and 110,000, in Western blots of extracts from human renal epithelial cells infected with E. coli. The increased intensity of these tyrosine-phosphorylated proteins was observed only in the Triton X-100-insoluble fraction, suggesting that these proteins could be associated with the cytoskeleton. Increased tyrosine phosphorylation of these proteins upon E. coli infection was observed in both transformed and primary human renal epithelial cells and in cells infected with several different strains of E. coli isolated from the feces of healthy individuals or from the blood or urine of patients with pyelonephritis. The increased tyrosine phosphorylation of these proteins required live bacteria and was blocked by tyrosine kinase inhibition but not by protein synthesis inhibitors or cytochalasin D. These experiments establish a strong link between E. coli internalization and host cell signaling through tyrosine kinases in human kidney cells and provide evidence that specific proteins are involved in these processes.

Cells, Cultured↗

PubMed2002

Impact of phosphorylation of specific residues in the tyrosine autokinase, Wzc, on its activity in assembly of group 1 capsules in Escherichia coli.

Wzc(CPS) is a tyrosine autokinase essential for the assembly of a high-molecular-weight (HMW) group 1 capsular polysaccharide (CPS) in Escherichia coli. Homologues of Wzc participate in the formation of CPS and exopolysaccharides in a variety of gram-positive and gram-negative bacteria. Phosphorylation of tyrosine residues in the Wzc(CPS) C terminus is essential for HMW CPS assembly. Overexpression of Wzb(CPS) (phosphatase) in a wild-type background caused a 3.7-fold decrease in the amount of cell-associated K30 CPS produced, confirming the importance of Wzc(CPS) phosphorylation for capsule assembly. In this study, the tyrosine-rich region was dissected in an attempt to identify residues critical for Wzc(CPS) phosphorylation and/or capsule expression. Site-directed mutagenesis demonstrated that no single tyrosine residue in this region is sufficient for detectable phosphorylation of Wzc(CPS) in vivo or for HMW CPS expression. Furthermore, no single tyrosine residue is essential for phosphorylation or capsule assembly, since removal of any one tyrosine residue has no detectable effect. Altering combinations of tyrosine residues (from two to five) led to Wzc(CPS) derivatives that were still competent for phosphorylation but that could not support assembly of HMW CPS, showing that phosphorylation of Wzc per se is not an accurate measure of its ability to function in capsule assembly. One interpretation of these data is that the overall level of phosphorylation in this region, rather than the precise combination of residues accessible to phosphorylation, is important for the activity of Wzc(CPS). Tyrosine 569, a residue shown to modulate the in vitro phosphorylation of Wzc(CA) from E. coli K-12, was also mutated. The derivative with this mutation still functioned in capsule assembly. Quantitation of K30(CPS) from this mutant revealed no difference in the amount of polymer produced. Finally, dithiobis(succinimidylpropionate) cross-linking was used to confirm that Wzc(CPS) forms complexes in vivo, independent of the phosphorylation state of the protein.

Bacterial Capsules↗

PubMed1997

Human immunodeficiency virus matrix tyrosine phosphorylation: characterization of the kinase and its substrate requirements.

During virus assembly, a subset of human immunodeficiency virus (HIV) matrix (MA) molecules is phosphorylated on C-terminal tyrosine. This modification facilitates infection of nondividing cells by allowing for the recruitment of the karyophilic MA into the viral core and preintegration complex. MA tyrosine phosphorylation is accomplished by a cellular protein kinase which is incorporated into virions. In this study, we have investigated the nature of this enzyme as well as the determinants of MA necessary for its phosphorylation. Employing an in vitro kinase assay, we found that the MA tyrosine kinase activity is present in various cultured cell lines including CEM and SupT1 T-lymphoid cells, Namalwa B cells, 293 and CV-1 kidney fibroblasts, and P4 HeLa cells. In addition, it could be detected in platelets, macrophages, and activated peripheral blood lymphocytes (PBLs) but not in erythrocytes and resting PBLs isolated from human blood. Subcellular localization of the kinase activity by cell fractionation demonstrated that it is enriched in cellular membranes. In HIV type 2 (HIV-2) particles, the MA tyrosine kinase is associated with the inner leaflet of the viral membrane, while the tyrosine-phosphorylated MA is localized to the core. Individual mutations of each of the last eight residues immediately upstream of the C-terminal tyrosine (Y132) of HIV-1 MA did not prevent Y132 phosphorylation, suggesting that the kinase does not require a highly specific sequence adjacent to the C-terminal tyrosine. Confirming this, we found that the MA of murine leukemia virus, the sequence of which is only moderately homologous to that of HIV-1 and HIV-2 MA, is also C-terminally tyrosine phosphorylated.

3T3 Cells↗

PubMed1991

Characterization of elk, a brain-specific receptor tyrosine kinase.

The elk gene encodes a novel receptorlike protein-tyrosine kinase, which belongs to the eph subfamily. We have previously identified a partial cDNA encompassing the elk catalytic domain (K. Letwin, S.-P. Yee, and T. Pawson, Oncogene 3:621-678, 1988). Using this cDNA as a probe, we have isolated cDNAs spanning the entire rat elk coding sequence. The predicted Elk protein contains all the hallmarks of a receptor tyrosine kinase, including an N-terminal signal sequence, a cysteine-rich extracellular domain, a membrane-spanning segment, a cytoplasmic tyrosine kinase domain, and a C-terminal tail. In both amino acid sequence and overall structure, Elk is most similar to the Eph and Eck protein-tyrosine kinases, suggesting that the eph, elk, and eck genes encode members of a new subfamily of receptorlike tyrosine kinases. Among rat tissues, elk expression appears restricted to brain and testes, with the brain having higher levels of both elk RNA and protein. Elk protein immunoprecipitated from a rat brain lysate becomes phosphorylated on tyrosine in an in vitro kinase reaction, consistent with the prediction that the mammalian elk gene encodes a tyrosine kinase capable of autophosphorylation. The characteristics of the Elk tyrosine kinase suggest that it may be involved in cell-cell interactions in the nervous system.

Amino Acid Sequence↗

PubMed1994

Tyrosine 569 in the c-Fms juxtamembrane domain is essential for kinase activity and macrophage colony-stimulating factor-dependent internalization.

The receptor (Fms) for macrophage colony-stimulating factor (M-CSF) is a member of the tyrosine kinase class of growth factor receptors. It maintains survival, stimulates growth, and drives differentiation of the macrophage lineage of hematopoietic cells. Fms accumulates on the cell surface and becomes activated for signal transduction after M-CSF binding and is then internalized via endocytosis for eventual degradation in lysosomes. We have investigated the mechanism of endocytosis as part of the overall signaling process of this receptor and have identified an amino acid segment near the cytoplasmic juxtamembrane region surrounding tyrosine 569 that is important for internalization. Mutation of tyrosine 569 to alanine (Y569A) eliminates ligand-induced rapid endocytosis of receptor molecules. The mutant Fms Y569A also lacks tyrosine kinase activity; however, tyrosine kinase activity is not essential for endocytosis because the kinase inactive receptor Fms K614A does undergo ligand-induced endocytosis, albeit at a reduced rate. Mutation of tyrosine 569 to phenylalanine had no effect on the M-CSF-induced endocytosis of Fms, and a four-amino-acid sequence containing Y-569 could support endocytosis when transferred into the cytoplasmic juxtamembrane region of a glycophorin A construct. These results indicate that tyrosine 569 within the juxtamembrane region of Fms is part of a signal recognition sequence for endocytosis that does not require tyrosine phosphorylation at this site and that this domain also influences the kinase activity of the receptor. These results are consistent with a ligand-dependent step in recognition of the potential cryptic internalization signal.

Amino Acid Sequence↗

PubMed2000

Phosphorylation of tyrosine residues in the kinase domain and juxtamembrane region regulates the biological and catalytic activities of Eph receptors.

Members of the Eph family of receptor tyrosine kinases exhibit a striking degree of amino acid homology, particularly notable in the kinase and membrane-proximal regions. A mutagenesis approach was taken to address the functions of specific conserved tyrosine residues within these catalytic and juxtamembrane domains. Ligand stimulation of wild-type EphB2 in neuronal NG108-15 cells resulted in an upregulation of catalytic activity and an increase in cellular tyrosine phosphorylation, accompanied by a retraction of neuritic processes. Tyrosine-to-phenylalanine substitutions within the conserved juxtamembrane motif abolished these responses. The mechanistic basis for these observations was examined using the highly related EphA4 receptor in a continuous coupled kinase assay. Tandem mass spectrometry experiments confirmed autophosphorylation of the two juxtamembrane tyrosine residues and also identified a tyrosine within the kinase domain activation segment as a phosphorylation site. Kinetic analysis revealed a decreased affinity for peptide substrate upon substitution of activation segment or juxtamembrane tyrosines. Together, our data suggest that the catalytic and therefore biological activities of Eph receptors are controlled by a two-component inhibitory mechanism, which is released by phosphorylation of the juxtamembrane and activation segment tyrosine residues.

Amino Acid Motifs↗

PubMed2004

Tyrosine 813 is a site of JAK2 autophosphorylation critical for activation of JAK2 by SH2-B beta.

The tyrosine kinase Janus kinase 2 (JAK2) binds to the majority of the known members of the cytokine family of receptors. Ligand-receptor binding leads to activation of the associated JAK2 molecules, resulting in rapid autophosphorylation of multiple tyrosines within JAK2. Phosphotyrosines can then serve as docking sites for downstream JAK2 signaling molecules. Despite the importance of these phosphotyrosines in JAK2 function, only a few sites and binding partners have been identified. Using two-dimensional phosphopeptide mapping and a phosphospecific antibody, we identified tyrosine 813 as a site of JAK2 autophosphorylation of overexpressed JAK2 and endogenous JAK2 activated by growth hormone. Tyrosine 813 is contained within a YXXL sequence motif associated with several other identified JAK2 phosphorylation sites. We show that phosphorylation of tyrosine 813 is required for the SH2 domain-containing adapter protein SH2-B beta to bind JAK2 and to enhance the activity of JAK2 and STAT5B. The homologous tyrosine in JAK3, tyrosine 785, is autophosphorylated in response to interleukin-2 stimulation and is required for SH2-B beta to bind JAK3. Taken together these data strongly suggest that tyrosine 813 is a site of autophosphorylation in JAK2 and is the SH2-B beta-binding site within JAK2 that is required for SH2-B beta to enhance activation of JAK2.

3T3 Cells↗

PubMedDate not supplied

Pharmacological doses of insulin equalize insulin receptor phosphotyrosine content but not tyrosine kinase activity in plasmalemmal and endosomal membranes.

Following insulin administration to intact rats, the insulin receptor kinase activity of subsequently isolated cell fractions was significantly augmented. Of interest was the observation that the endosomal insulin receptor tyrosine kinase displayed four- to six-fold greater autophosphorylation activity than that of plasma membrane. Surprisingly, the endosomal insulin receptor tyrosine kinase displayed a decrease in beta-subunit phosphotyrosine content compared with that seen in the plasma membrane. These observations prompted the suggestion that insulin receptor tyrosine kinase phosphotyrosine dephosphorylation mediated by an endosome-specific phosphotyrosine phosphatase(s) yields activation of the endosomal insulin receptor tyrosine kinase. In a previous study we examined the effect of subsaturating doses of injected insulin. In this work we evaluated insulin receptor tyrosine kinase activity and phosphotyrosine content in plasma membrane and endosomes after a receptor-saturating pharmacological dose of insulin (150 micrograms/100 g body weight). At this dose the phosphotyrosine content per receptor was reduced compared with that seen earlier at insulin doses of 1.5 and 15 micrograms/100 g body weight. Endosomal insulin receptor tyrosine kinase was greater than that seen at the lower nonsaturating insulin doses. Furthermore, endosomal insulin receptor tyrosine kinase activity exceeded that of the plasma membrane, despite retaining about the same phosphotyrosine content per receptor. These data are consistent with the view that insulin receptor tyrosine kinase activity may be regulated by a particular pattern of phosphotyrosine content on the beta-subunit wherein both activating and inhibitory phosphotyrosine residues play a role.

Amino Acid Sequence↗

PubMed2005

Constitutive secretion of serum albumin requires reversible protein tyrosine phosphorylation events in trans-Golgi.

Serum albumin secretion from rat hepatocytes proceeds via the constitutive pathway. Although much is known about the role of protein tyrosine phosphorylation in regulated secretion, nothing is known about its function in the constitutive process. Here we show that albumin secretion is inhibited by the tyrosine kinase inhibitor genistein but relatively insensitive to subtype-selective inhibitors or treatments. Secretion is also blocked in a physiologically identical manner by the tyrosine phosphatase inhibitors pervanadate and bisperoxo(1,10-phenanthroline)-oxovanadate. Inhibition of either the kinase(s) or phosphatase(s) leads to the accumulation of albumin between the trans-Golgi and the plasma membrane, whereas the immediate precursor proalbumin builds up in a proximal compartment. The trans-Golgi marker TGN38 is rapidly dispersed under conditions that inhibit tyrosine phosphatase action, whereas the distribution of the cis-Golgi marker GM130 is insensitive to genistein or pervanadate. By using a specifically reactive biotinylation probe, we detected protein tyrosine phosphatases in highly purified rat liver Golgi membranes. These membranes also contain both endogenous tyrosine kinases and their substrates, indicating that enzymes and substrates for reversible tyrosine phosphorylation are normal membrane-resident components of this trafficking compartment. In the absence of perturbation of actin filaments and microtubules, we conclude that reversible protein tyrosine phosphorylation in the trans-Golgi network is essential for albumin secretion and propose that the constitutive secretion of albumin is in fact a regulated process.

Animals↗

PubMed2000

Tyrosine phosphorylation modulates arteriolar tone but is not fundamental to myogenic response.

The present study investigated the role of protein tyrosine phosphorylation in myogenic responsiveness of rat skeletal muscle arterioles. Arteriolar segments were cannulated and pressurized without intraluminal flow. All vessels studied developed spontaneous tone and demonstrated significant myogenic constriction to step changes in pressure with a resultant increase in myogenic tone over an intraluminal pressure range of 50-150 mmHg. Step increases in intraluminal pressure from 50 to 120 mmHg caused a rapid and sustained elevation in intracellular [Ca(2+)], as measured using fura 2. Vessels with myogenic tone dilated in response to tyrosine kinase inhibitors genistein (10 or 30 microM) and tyrphostin A47 (10 or 30 microM) and constricted to the tyrosine phosphatase inhibitor pervanadate (1 or 10 microM). Despite the dilator effect, myogenic reactivity was not blocked by the inhibitors. Daidzein (10 microM), a compound structurally similar to genistein but without tyrosine kinase-inhibiting activity, did not alter vessel tone or myogenic responses. Preincubation of arterioles with genistein or tyrphostin A47 did not significantly alter baseline arteriolar [Ca(2+)], and neither drug reduced the increase in [Ca(2+)] following an acute increase in intraluminal pressure. Constriction induced by pervanadate (10 microM) was not accompanied by a significant increase in intracellular [Ca(2+)], even though removal of extracellular Ca(2+) reversed the constriction. Examination of smooth muscle tyrosine phosphorylation, using a fluorescent phosphotyrosine antibody and confocal microscopy, showed that increased intraluminal pressure resulted in an increase in anti-phosphotyrosine fluorescence. Because manipulation of tyrosine kinase activity was found to alter vessel diameter, these data support a role for tyrosine phosphorylation in modulation of arteriolar tone. However, the results indicate that acute arteriolar myogenic constriction does not require tyrosine phosphorylation.

Animals↗

PubMed1998

Effect of tyrosine kinase inhibition on surfactant protein A gene expression during human lung development.

Epidermal growth factor (EGF) stimulates surfactant protein (SP) A synthesis in human fetal lung explants. Ligand binding to the EGF receptor stimulates an intrinsic receptor tyrosine kinase with subsequent activation of second messengers. We hypothesized that inhibition of EGF-receptor tyrosine kinase activity would block SP-A expression in spontaneously differentiating cultured human fetal lung tissue. Midtrimester fetal lung explants were exposed for 4 days to genistein (a broad-range inhibitor of tyrosine kinases) and tyrphostin AG-1478 (a specific inhibitor of EGF-receptor tyrosine kinase). Genistein significantly decreased SP-A and SP-A mRNA levels without affecting either tissue viability or the morphological differentiation of alveolar type II cells. Tyrphostin AG-1478 also decreased SP-A content and SP-A mRNA levels in cultured fetal lung explants. Treatment with EGF could not overcome the inhibitory effects of either genistein or tyrphostin on SP-A; however, only tyrphostin inhibited EGF-receptor tyrosine phosphorylation. We conclude that specific inhibition of EGF-receptor tyrosine kinase with tyrphostin AG-1478 blocks the expression of SP-A during spontaneous differentiation of cultured human fetal lung tissue. Furthermore, exposure to genistein also decreases SP-A expression and blocks the effects of EGF in human fetal lung tissue without inhibiting EGF-receptor tyrosine phosphorylation. These findings support the importance of tyrosine kinase-dependent signal transduction pathways in the regulation of SP-A during human fetal lung development.

Embryonic and Fetal Development↗

PubMed2000

Diperoxovanadate alters endothelial cell focal contacts and barrier function: role of tyrosine phosphorylation.

Diperoxovanadate (DPV), a potent tyrosine kinase activator and protein tyrosine phosphatase inhibitor, was utilized to explore bovine pulmonary artery endothelial cell barrier regulation. DPV produced dose-dependent decreases in transendothelial electrical resistance (TER) and increases in permeability to albumin, which were preceded by brief increases in TER (peak TER effect at 10-15 min). The significant and sustained DPV-mediated TER reductions were primarily the result of decreased intercellular resistance, rather than decreased resistance between the cell and the extracellular matrix, and were reduced by pretreatment with the tyrosine kinase inhibitor genistein but not by inhibition of p42/p44 mitogen-activating protein kinases. Immunofluorescent analysis after DPV challenge revealed dramatic F-actin polymerization and stress-fiber assembly and increased colocalization of tyrosine phosphoproteins with F-actin in a circumferential pattern at the cell periphery, changes that were abolished by genistein. The phosphorylation of focal adhesion and adherens junction proteins on tyrosine residues was confirmed in immunoprecipitates of focal adhesion kinase and cadherin-associated proteins in which dramatic dose-dependent tyrosine phosphorylation was observed after DPV stimulation. We speculate that DPV enhances endothelial cell monolayer integrity via focal adhesion plaque phosphorylation and produces subsequent monolayer destabilization of adherens junctions initiated by adherens junction protein tyrosine phosphorylation catalyzed by p60(src) or Src-related tyrosine kinases.

Actins↗

PubMed1995

Angiotensin II and other hypertrophic stimuli mediated by G protein-coupled receptors activate tyrosine kinase, mitogen-activated protein kinase, and 90-kD S6 kinase in cardiac myocytes. The critical role of Ca(2+)-dependent signaling.

Many hypertrophic stimuli such as angiotensin II (Ang II) activate phospholipases through G protein-coupled receptors in cardiac myocytes. However, it is not known whether these stimuli also activate the tyrosine phosphorylation-dependent signaling pathway, which plays an essential role in growth factor-induced mitogenic responses in other cell types. Serine/threonine kinases such as mitogen-activated protein (MAP) kinases and 90-kD S6 kinase (RSK) are activated in response to many growth stimuli and are important downstream signaling pathways of tyrosine kinases. Therefore, we examined whether Ang II activates these protein kinases in primary cultures of cardiac myocytes and fibroblasts from neonatal rats. Ang II rapidly induced tyrosine phosphorylation of multiple proteins, including 42-, 44-, 75- to 80-, and 120- to 130-kD proteins, in both cardiac myocytes and fibroblasts. This was accompanied by an increase in tyrosine kinase activity. The 42- and 44-kD proteins were immunologically related to an extracellular signal-regulated kinase family (MAP kinases). Ang II rapidly increased kinase activity of MAP kinases and their downstream kinase, RSK. The Ang II-induced tyrosine phosphorylation and activation of MAP kinases and RSK were AT1 receptor-mediated. Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate or an increase in intracellular Ca2+ by the Ca2+ ionophore A23187 was sufficient to cause tyrosine phosphorylation of multiple proteins and activation of MAP kinase and RSK. Although downregulation of PKC did not suppress Ang II-induced activation of MAP kinase and RSK, chelating intracellular Ca2+ by BAPTA-AM completely abolished Ang II-induced activation of these kinases. Activation of MAP kinases and RSK was also observed in myocytes stimulated with other agonists for Gq protein-coupled receptors, such as phenylephrine, norepinephrine, and endothelin 1, but not with agonists to Gs protein-coupled receptors, such as isoproterenol. These results suggest that Ang II and other hypertrophic stimuli, known to act through Gq protein-coupled receptors, rapidly cause tyrosine phosphorylation of several intracellular substrates through activation of tyrosine kinase and activate MAP kinases and RSK in cardiac myocytes as well as in cardiac fibroblasts. Furthermore, intracellular Ca2+, rather than PKC, seems to be critical for Ang II-induced activation of these protein kinases in cardiac myocytes.

Angiotensin II↗

PubMed1997

Angiotensin II signal transduction in vascular smooth muscle: role of tyrosine kinases.

In this review, the role of tyrosine kinases in angiotensin II-mediated signal transduction pathways in vascular smooth muscle is discussed. Angiotensin II was isolated by virtue of its vasoconstrictor abilities and has long been thought to play a critical role in hypertension. However, recent studies indicate important roles for angiotensin II in inflammation, atherosclerosis, and congestive heart failure. The expanding role of angiotensin II indicates that multiple signal transduction pathways are likely to be activated in a tissue-specific manner. Exciting recent data show that angiotensin II directly stimulates tyrosine kinases, including pp60(c-src) kinase (c-Src), focal adhesion kinase (FAK), and Janus kinases (JAK2 and TYK2). Angiotensin II may activate receptor tyrosine kinases, such as Axl and platelet-derived growth factor, by as-yet-undefined autocrine mechanisms. Finally, unknown tyrosine kinases may mediate tyrosine phosphorylation of Shc, Raf, and phospholipase C-gamma after angiotensin II stimulation. These angiotensin II-regulated tyrosine kinases appear to be required for angiotensin II effects, such as vasoconstriction, proto-oncogene expression, and protein synthesis, on the basis of studies with tyrosine kinase inhibitors. Thus, understanding angiotensin II-stimulated signaling events, especially those related to tyrosine kinase activity, may form the basis for the development of new therapies for cardiovascular diseases.

Angiotensin II↗

PubMed1998

Ca2+-independent activation of the endothelial nitric oxide synthase in response to tyrosine phosphatase inhibitors and fluid shear stress.

Fluid shear stress enhances NO formation via a Ca2+-independent tyrosine kinase inhibitor-sensitive pathway. In the present study, we investigated the effects of the protein tyrosine phosphatase inhibitor phenylarsine oxide and of fluid shear stress on endothelial NO production as well as on the membrane association and phosphorylation of the NO synthase (NOS) III. Phenylarsine oxide (10 micromol/L) induced an immediate and maintained NO-mediated relaxation of isolated rabbit carotid arteries, which was insensitive to the removal of extracellular Ca2+ and the calmodulin antagonist calmidazolium. This phenylarsine oxide-induced vasodilatation was unaffected by genistein but abrogated by the tyrosine kinase inhibitor erbstatin A. Incubation of native or cultured endothelial cells with phenylarsine oxide resulted in a time-dependent tyrosine phosphorylation of mainly Triton X-100-insoluble (cytoskeletal) proteins, along with a parallel change in the detergent solubility of NOS III, such that the enzyme was recovered in the cytoskeletal fraction. A similar, though slightly delayed, phenomenon was also observed after the application of fluid shear stress but not in response to any receptor-dependent agonist. Although Ca2+-independent NO formation was sensitive to erbstatin A, phenylarsine oxide treatment was associated with the tyrosine dephosphorylation of NOS III rather than its hyperphosphorylation. Proteins that also underwent redistribution in response to the tyrosine phosphatase inhibitor included paxillin, phospholipase C-gamma1, mitogen-activated protein kinase, and the tyrosine kinases Src and Fyn. We envisage that fluid shear stress and tyrosine phosphatase inhibitors may alter the conformation and/or protein coupling of NOS III, facilitating its interaction with specific phospholipids, proteins, and/or protein kinases that enhance/maintain its Ca2+-independent activation.

Analysis of Variance↗

PubMed1998

Role of tyrosine kinase in serotonin-induced constriction of the basilar artery in vivo.

BACKGROUND AND PURPOSE: Serotonin is one of the most potent constrictors of cerebral blood vessels and is implicated in several pathological conditions, including migraine and cerebral ischemia. Recent evidence has suggested that tyrosine kinase is involved in vasocontractile responses. The objective of this study was to test the hypothesis that activation of tyrosine kinase contributes to serotonin-induced constriction of the basilar artery in vivo. METHODS: Using a cranial window in anesthetized Sprague-Dawley rats, we examined effects of inhibitors of tyrosine kinase and tyrosine phosphatase on constrictor responses of the basilar artery to serotonin in vivo. RESULTS: Serotonin (10(-8), 10(-7), and 10(-6) mol/L) produced constriction of the basilar artery by 12+/-2%, 27+/-2%, and 37+/-3%, respectively. Genistein (3 x 10(-6) mol/L), an inhibitor of tyrosine kinase, did not affect baseline diameter of the basilar artery but attenuated serotonin-induced vasoconstriction (P<.05 versus control responses). Daidzein, an inactive analogue of genistein, did not affect serotonin-induced constriction of the basilar artery. Tyrphostin 47 (10(-5) mol/L), another inhibitor of tyrosine kinase, also attenuated serotonin-induced vasoconstriction, and tyrphostin 63, an inactive analogue of tyrphostin 47, did not affect the vasoconstriction. Sodium orthovanadate (10(-5) mol/L), an inhibitor of tyrosine phosphatase, enhanced serotonin-induced vasoconstriction. Phorbol 12,13-dibutyrate, a direct activator of protein kinase C, also caused constriction of the basilar artery, which was not affected by genistein or sodium orthovanadate. CONCLUSIONS: These results suggest that serotonin-induced constriction of the basilar artery is mediated, at least in part, by activation of tyrosine kinase in vivo.

Analysis of Variance↗

PubMed2002

Low density detergent-insoluble membrane of Xenopus eggs: subcellular microdomain for tyrosine kinase signaling in fertilization.

Protein-tyrosine phosphorylation plays an important role in egg activation signaling at fertilization. We show that in Xenopus, fertilization stimulates a rapid and transient tyrosine phosphorylation of egg proteins, as revealed by immunoblotting with anti-phosphotyrosine antibody. Immunofluorescent microscopic analysis demonstrated that the phosphorylation occurs in cortical area of the egg animal hemisphere. To further characterize subcellular compartment for fertilization-dependent tyrosine kinase signaling, we isolated low density detergent-insoluble membrane (LD-DIM) fraction from Xenopus eggs. The egg LD-DIM was enriched in cholesterol and GM1 ganglioside. It also contained signaling molecules such as Xyk (Xenopus egg Src), Gq alpha, Ras, integrin beta 1 and CD9. Fertilization stimulated tyrosine phosphorylation of Xyk and some other LD-DIM proteins. Remarkably, sperm stimulated tyrosine phosphorylation of the LD-DIM proteins in vitro. The sperm-dependent phosphorylation was sensitive to the tyrosine kinase inhibitors PP2 and genistein. We found that pretreatment of eggs with methyl-beta-cyclodextrin, a cholesterol-binding substance, led to a decrease in cholesterol, Xyk and sperm-induced tyrosine phosphorylation in LD-DIM. In methyl-beta-cyclodextrin-treated eggs, sperm-induced Ca(2+) transient and first cell division were also inhibited. These findings suggest that the egg LD-DIM might serve as subcellular microdomain for tyrosine kinase signaling in Xenopus egg fertilization.

Animals↗