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PubMed1995

Analysis of an Escherichia coli mutant TyrR protein with impaired capacity for tyrosine-mediated repression, but still able to activate at sigma 70 promoters.

In Escherichia coli, TyrR represses and activates transcription of operons required for tyrosine, phenylalanine and tryptophan biosynthesis and uptake. The TyrR central domain is homologous with NtrC and some other bacterial regulatory proteins, although TyrR regulates sigma 70, not sigma 54, promoters. We isolated a central domain TyrR mutant (TyrR E274Q) by substitution of a normally conserved amino acid. The mutant was unable to bring about tyrosine-mediated repression of aroF, aroL, tyrB, and tyrP and had diminished capability for tyrosine- and phenylalanine-mediated repression of aroP. In contrast, it was able to effect wild-type levels of phenylalanine-mediated repression of aroG, tryptophan-mediated repression of aroP and transcriptional activation of mtr and tyrP. The binding of purified TyrR E274Q to ATP (a requirement for tyrosine binding) and to the strong TyrR box of tyrP operator DNA were normal, but tyrosine binding and tyrosine-dependent hexamerization were significantly impaired. These properties are consistent with the proposal that self association is essential for tyrosine-mediated repression by TyrR but not for tyrosine- or phenylalanine-mediated activation. E274 of TyrR must participate in either the binding of tyrosine, or the coupling of ATP binding with a conformational change that alters the affinity of the ATP-dependent aromatic amino acid-binding site.

Adenosine Triphosphatases↗
PubMed1985

Chemical modification and nuclear magnetic resonance studies on human plasminogen kringle 4. Assignment of tyrosine and histidine resonances to specific residues in the sequence.

Modification of kringle 4 with tetranitromethane leads to the selective nitration of tyrosine 40 but on prolonged incubation with reagent, reaction of tyrosine 49 is also observed. Nitration of tyrosines 40 and 49 had no influence on the lysine-Sepharose affinity of kringle 4, indicating that these residues are not important for the functional integrity of the ligand-binding site. Comparison of the NMR spectra of native kringle 4 with those of kringle 4 in which tyrosine 40 or tyrosines 40 and 49 are nitrated permitted the identification of the resonances of these residues. These NMR studies also showed that the chemical modifications caused little perturbation of the three-dimensional structure of the protein. Cross-linking of lysine 35 and tyrosine 40 with 1,3-difluoro-4,6-dinitrobenzene demonstrates that in the kringle-fold the reactive epsilon-amino and phenolic groups of these residues can approach each other to a distance of 0.5 nm. NMR spectra of this kringle 4 species also confirmed the assignment of the resonances to tyrosine 40. NMR spectra of a kringle 4 derivative in which the disulphide bridge between cysteines 1 and 79 has been broken by selective reduction and alkylation showed that the core structure of the kringle-fold and the lysine-binding site are unaltered by this modification. This observation is in agreement with earlier results which showed that the lysine-Sepharose affinity of kringle 4 is not affected by reduction and alkylation of this disulphide bridge. Comparison of the NMR spectra of native and disulphide-cleaved kringle 4 aided in the assignment of resonances to residues adjacent to the site of modification (tyrosine 2 and histidine 3) and permitted the tentative assignment of the resonances of tyrosines 9 and 73.

Amino Acids↗
PubMed1997

Participation of a stress-activated protein kinase cascade in the activation of tyrosine hydroxylase in chromaffin cells.

Sodium arsenite and osmotic shock both stimulated stress-activated protein kinase-2 (SAPK2, also termed RK, p38, CSBP and Mxi2) and its downstream target mitogen-activated protein kinase (MAP kinase)-activated protein kinase-2 (MAPKAP-K2) in bovine adrenal chromaffin and rat PC12 cells. The same stimuli also increased tyrosine hydroxylase activity 2-3-fold and induced its phosphorylation at Ser19, a residue phosphorylated by MAPKAP-K2 in vitro. The arsenite-induced activation of tyrosine hydroxylase and its phosphorylation at Ser19 were prevented by SB 203580 at concentrations similar to those that inhibited SAPK2 in vitro. These results indicate that MAPKAP-K2 mediates the stress-induced activation of tyrosine hydroxylase. SB 203580 had no effect on the phosphorylation or activation of tyrosine hydroxylase induced by nerve growth factor or forskolin, which trigger the phosphorylation of Ser31 and Ser40, respectively. Stimulation of bovine adrenal chromaffin cells with acetylcholine activated SAPK2 and MAPKAP-K2, as well as p42/p44 MAP kinases and their downstream target MAPKAP-K1. The half-times for activation of MAPKAP-K1 and MAPKAP-K2 (1 min) were similar. In contrast, the activation of tyrosine hydroxylase by acetylcholine peaked within 1 min and gradually declined thereafter. Neither SB 203580 (which blocked the activation of MAPKAP-K2 by acetylcholine) nor PD 98059 (which prevented the activation of p42/p44 MAP kinases by acetylcholine) affected tyrosine hydroxylase activation after 1 min, but these compounds inhibited activation by 40-50% after 5 min. PD 98059 prevented the acetylcholine-induced phosphorylation of tyrosine hydroxylase at Ser31, the residue targetted by p42/p44 MAP kinases in vitro, but did not inhibit the phosphorylation of Ser40 (which is phosphorylated by MAPKAP-K1 in vitro). Our results establish that p42/p44 MAP kinases mediate the acetylcholine-induced phosphorylation of tyrosine hydroxylase at Ser31. SB 203580 did not suppress the phosphorylation of Ser19 or Ser40 by acetylcholine but, like PD 98059, this drug decreased the phosphorylation of Ser31. SAPK2 may therefore contribute to the acetylcholine-induced activation of tyrosine hydroxylase by facilitating (in an unknown way) its phosphorylation by MAP kinases.

Adrenal Glands↗
PubMed1980

An apparent paradox in the occurrence, and the in vivo turnover, of C-terminal tyrosine in membrane-bound tubulin of brain.

Tubulin tyrosine ligase catalyzes the reversible addition of tyrosine to the C-terminus of tubulin alpha chains. By using ligase and carboxypeptidase A in conjunction, we have previously shown that brain cytoplasmic tubulin exists in three forms: 15-40% already has C-terminal tyrosine, another 10-30% can accept additional tyrosine, and about one-half is an uncharacterized species which is not a ligase substrate. A membrane-bound fraction of brain tubulin, purified by vinblastine precipitation from a detergent extract, has been found to differ by the complete absence of preexisting tyrosine. The membrane fraction from which tubulin was extracted also contained masked forms of both ligase and a distinct detyrosylating enzyme, which can be released by detergent extraction. The turnover of alpha-chain C-terminal tyrosine in vivo was studied by incubating brain mince with labeled tyrosine, or injecting it intracerebrally, under conditions where protein synthesis was inhibited. Tyrosine appeared to turn over to about the same extent in membrane-bound, as in soluble, tubulin. This apparently paradoxical result was not due to ATPase in the membrane fraction, which might have allowed ligase-catalyzed exchange between free and fixed tyrosine. Authentic [14C]tyrosylated tubulin added to the brain membrane fraction was not detyrosylated or subject to endoprotease digestion during subsequent procedures to isolate tubulin. The unexpected finding that tubulin tyrosylated at the C-terminal in vivo appears to be in the "non-substrate" fraction points toward a possible resolution of the paradox.

Animals↗
PubMed1987

Activation of tyrosine hydroxylase in PC12 cells by the cyclic GMP and cyclic AMP second messenger systems.

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is subject to regulation by a variety of agents. Previous workers have found that cyclic AMP-dependent protein kinase and calcium-stimulated protein kinases activate tyrosine hydroxylase. We wanted to determine whether cyclic GMP might also be involved in the regulation of tyrosine hydroxylase activity. We found that treatment of rat PC12 cells with sodium nitroprusside (an activator of guanylate cyclase), 8-bromocyclic GMP, forskolin (an activator of adenylate cyclase), and 8-bromocyclic AMP all produced an increase in tyrosine hydroxylase activity measured in vitro or an increased conversion of [14C]tyrosine to labeled catecholamine in situ. Sodium nitroprusside also increased the relative synthesis of cyclic GMP in these cells. In the presence of MgATP, both cyclic GMP and cyclic AMP increased tyrosine hydroxylase activity in PC12 cell extracts. The heat-stable cyclic AMP-dependent protein kinase inhibitor failed to attenuate the activation produced in the presence of cyclic GMP. It eliminated the activation produced in the presence of cyclic AMP. Sodium nitroprusside also increased tyrosine hydroxylase activity in vitro in rat corpus striatal synaptosomes and bovine adrenal chromaffin cells. In all cases, the cyclic AMP-dependent activation of tyrosine hydroxylase was greater than that of the cyclic GMP-dependent second messenger system. These results indicate that both cyclic GMP and cyclic AMP and their cognate protein kinases activate tyrosine hydroxylase activity in PC12 cells.

Adrenal Gland Neoplasms↗
PubMed1988

Long-term changes in rat brain tyrosine hydroxylase following reserpine treatment: a quantitative immunochemical analysis.

An immunoblot procedure was developed to quantify the amount of tyrosine hydroxylase protein in homogenate of small brain regions. With the use of this method we have studied the variations in tyrosine hydroxylase activity and protein levels in some catecholaminergic neurons at different times following a single reserpine injection (10 mg/kg s.c.) and reevaluated the anatomical specificity of tyrosine hydroxylase induction by this drug. Reserpine administration provoked a long-lasting increase in both tyrosine hydroxylase activity and protein levels within locus ceruleus neurons. This effect culminated at day 4 after injection. At this time, the enzyme activity and protein levels in treated animals were respectively 2.7 and 2.6 times that measured in vehicle-treated animals. Both parameters varied in parallel so that tyrosine hydroxylase specific activity did not change over time. In contrast, reserpine did not cause any changes in tyrosine hydroxylase activity in the dopaminergic neurons of the substantia nigra, but provoked a moderate increase in tyrosine hydroxylase protein level. This latter effect was maximal (1.5 times) 4 days after treatment. In the adjacent dopaminergic area, i.e., the ventral tegmental area, a small decrease in the enzyme activity was recorded at day 2 without any significant change in the level of the protein. In conclusion, first, our data show the capacity of our method to assay tyrosine hydroxylase protein amounts in small brain catecholaminergic nuclei. Second, our results confirm and extend previous studies on the effect of reserpine on the regulation of tyrosine hydroxylase level within brain noradrenergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Glands↗
PubMed1988

Protein tyrosine kinase activity and its endogenous substrates in rat brain: a subcellular and regional survey.

The rat CNS contains high levels of tyrosine-specific protein kinases that specifically phosphorylate the tyrosine-containing synthetic peptide poly(Glu80,Tyr20). The phosphorylation of this peptide is rapid and occurs with normal Michaelis-Menten kinetics. Using this peptide to assay for enzyme activity, we have measured the protein tyrosine kinase activity in homogenates from various regions of rat CNS. A marked regional distribution pattern was observed, with high activity present in cerebellum, hippocampus, olfactory bulb, and pyriform cortex, and low activity in the pons/medulla and spinal cord. The distribution of protein tyrosine kinase activity was examined in various subcellular fractions of rat forebrain. The majority of the activity was associated with the particulate fractions, with enrichment in the crude microsomal (P3) and crude synaptic vesicle (LP2) fractions. Moreover, the subcellular distribution of pp60csrc, a well-characterized protein tyrosine kinase, was examined by immunoblot analysis using an affinity-purified antibody specific for pp60csrc. The subcellular distribution of pp60csrc paralleled the overall protein tyrosine kinase activity. In addition, using an antibody specific for phosphotyrosine, endogenous substrates for protein tyrosine kinases were demonstrated on immunoblots of homogenates from the various regions and the subcellular fractions. The immunoblots revealed numerous phosphotyrosine-containing proteins that were present in many of the CNS regions examined and were associated with specific subcellular fractions. The differences in tyrosine-specific protein kinase activity, and in phosphotyrosine-containing proteins, observed in various regional areas and subcellular fractions may reflect specific functional roles for protein tyrosine kinase activity in mammalian brain.

Animals↗
PubMed1991

Phosphorylation of rat tyrosine hydroxylase and its model peptides in vitro by cyclic AMP-dependent protein kinase.

The enzyme tyrosine hydroxylase catalyzes the first step in the biosynthesis of dopamine, norepinephrine, and epinephrine. Tyrosine hydroxylase is a substrate for cyclic AMP-dependent protein kinase as well as other protein kinases. We determined the Km and Vmax of rat pheochromocytoma tyrosine hydroxylase for cyclic AMP-dependent protein kinase and obtained values of 136 microM and 7.1 mumol/min/mg of catalytic subunit, respectively. These values were not appreciably affected by the substrates for tyrosine hydroxylase (tyrosine and tetrahydrobiopterin) or by feedback inhibitors (dopamine and norepinephrine). The high Km of tyrosine hydroxylase correlates with the high content of tyrosine hydroxylase in catecholaminergic cells. We also determined the kinetic constants for peptides modeled after actual or potential tyrosine hydroxylase phosphorylation sites. We found that the best substrates for cyclic AMP-dependent protein kinase were those peptides corresponding to serine 40. Tyrosine hydroxylase (36-46), for example, exhibited a Km of 108 microM and a Vmax of 6.93 mumol/min/mg of catalytic subunit. The next best substrate was the peptide corresponding to serine 153. The peptide containing the sequence conforming to serine 19 was a very poor substrate, and that conforming to serine 172 was not phosphorylated to any significant extent. The primary structure of the actual or potential phosphorylation sites is sufficient to explain the substrate behavior of the native enzyme.

Animals↗
PubMedDate not supplied

L-tyrosine, L-dopa, and tyrosinase as positive regulators of the subcellular apparatus of melanogenesis in Bomirski Ab amelanotic melanoma cells.

In cultured cells of the Bomirski Ab amelanotic hamster melanoma line, the substrates of tyrosinase, L-tyrosine, and L-DOPA induce the melanogenic pathway. In this report, we demonstrate that these substrates regulate the subcellular apparatus involved in their own metabolism and that this regulation is under the dynamic control of one of the components of this apparatus, tyrosinase, via tyrosine hydroxylase activity. Culturing cells with nontoxic but melanogenically inhibitory levels of phenylthiourea (PTU; 100 microM) strongly inhibits induction of both the tyrosine hydroxylase and DOPA oxidase activities of tyrosinase by L-tyrosine (200 microM) but has no effect on the induction of either activity by L-DOPA (50 microM). De novo synthesis of premelanosomes precedes the onset of tyrosine-induced melanogenesis. Thereafter, increases in the population of melanosomes (likewise inhibited by PTU) correlate positively with increases in tyrosinase activity induced by L-tyrosine. Melanogenesis induced by L-DOPA in the absence of L-tyrosine is rate-limited not by tyrosinase but by inadequate melanosome synthesis. Our findings indicate that in Bomirski Ab amelanotic hamster melanoma cells the synthesis of the subcellular apparatus of melanogenesis is initiated by L-tyrosine and is regulated further by tyrosinase and L-DOPA, which serves as a second messenger subsequent to tyrosine hydroxylase activity.

Animals↗
PubMed2003

Tetrahydrobiopterin prevents nitration of tyrosine hydroxylase by peroxynitrite and nitrogen dioxide.

Tyrosine hydroxylase (TH) is the initial and rate-limiting enzyme in the synthesis of the neurotransmitter dopamine. TH is inhibited and nitrated at tyrosine residues in vitro by the reactive nitrogen species peroxynitrite and nitrogen dioxide (NO2) and in vivo by drugs that damage dopamine neurons. Tetrahydrobiopterin, which is the essential cofactor for TH and is concentrated in dopamine neurons, completely blocks nitration of tyrosine residues in TH caused by peroxynitrite or NO2. Various tetrahydro- and dihydro-analogs of tetrahydrobiopterin, including 6,7-dimethyl-tetrahydropterin, 6-methyl-tetrahydropterin, 6-hydroxymethyl-tetrahydropterin, tetrahydropterin, 7,8-dihydrobiopterin, 7,8-dihydroxanthopterin, and sepiapterin, also prevent nitration of tyrosines caused by the reactive nitrogen species. Biopterin and pterin, the fully oxidized forms of the pterin molecule, fail to block peroxynitrite- or NO2-induced nitration of TH. Reduced pterins prevent neither the inhibition of TH activity nor cysteine modification caused by peroxynitrite or NO2, despite blocking tyrosine nitration. However, dithiothreitol prevents and reverses these effects on TH of tetrahydrobiopterin and reactive nitrogen species. Using an enhanced green fluorescent protein-TH fusion construct as a real-time reporter of intracellular tyrosine nitration, tetrahydrobiopterin was found to prevent NO2-induced tyrosine nitration in intact cells but to leave TH activity inhibited. These results indicate that tetrahydrobiopterin prevents the tyrosine-nitrating properties of peroxynitrite and NO2. Tetrahydrobiopterin-derived radical species formed by reaction with reactive nitrogen species may account for inhibition of TH via mechanisms that do not involve tyrosine nitration.

Animals↗
PubMed1970

Molecular events in the growth inhibition of Bacillus subtilis by D-tyrosine.

The transformable strain of Bacillus subtilis strain 168 is extremely susceptible to growth inhibition by d-tyrosine. The molecular events associated with the inhibition of growth by d-tyrosine in this strain include the false feedback inhibition and probably the false repression of prephenate dehydrogenase. These effects were found to contribute to the formation of d-tyrosine-containing proteins by decreasing the intracellular concentration of l-tyrosine. Accordingly, growth inhibition of strain 168 by the d isomer of tyrosine was shown to be progressive, enduring, and delayed by prior growth on l-tyrosine. The synthesis of cellular macromolecules and viable cell count were progressively diminished in d-tyrosine-inhibited cultures. Several different enzyme activities were reduced after growth in the presence of d-tyrosine. Isotopic d-tyrosine was incorporated into cellular proteins without change of optical configuration. Long chains of cells with completed septa were observed microscopically, and therefore some cell wall effect may also be implicated.

Bacillus subtilis↗
PubMed1991

Cell cycle tyrosine phosphorylation of p34cdc2 and a microtubule-associated protein kinase homolog in Xenopus oocytes and eggs.

We have examined the time course of protein tyrosine phosphorylation in the meiotic cell cycles of Xenopus laevis oocytes and the mitotic cell cycles of Xenopus eggs. We have identified two proteins that undergo marked changes in tyrosine phosphorylation during these processes: a 42-kDa protein related to mitogen-activated protein kinase or microtubule-associated protein-2 kinase (MAP kinase) and a 34-kDa protein identical or related to p34cdc2. p42 undergoes an abrupt increase in its tyrosine phosphorylation at the onset of meiosis 1 and remains tyrosine phosphorylated until 30 min after fertilization, at which point it is dephosphorylated. p42 also becomes tyrosine phosphorylated after microinjection of oocytes with partially purified M-phase-promoting factor, even in the presence of cycloheximide. These findings suggest that MAP kinase, previously implicated in the early responses of somatic cells to mitogens, is also activated at the onset of meiotic M phase and that MAP kinase can become tyrosine phosphorylated downstream from M-phase-promoting factor activation. We have also found that p34 goes through a cycle of tyrosine phosphorylation and dephosphorylation prior to meiosis 1 and mitosis 1 but is not detectable as a phosphotyrosyl protein during the 2nd through 12th mitotic cell cycles. It may be that the delay between assembly and activation of the cyclin-p34cdc2 complex that p34cdc2 tyrosine phosphorylation provides is not needed in cell cycles that lack G2 phases. Finally, an unidentified protein or group of proteins migrating at 100 to 116 kDa increase in tyrosine phosphorylation throughout maturation, are dephosphorylated or degraded within 10 min of fertilization, and appear to cycle between low-molecular-weight forms and high-molecular-weight forms during early embryogenesis.

Animals↗
PubMed1996

Enhancement of antiproliferative activity of gamma interferon by the specific inhibition of tyrosine dephosphorylation of Stat1.

Gamma interferon (IFN-gamma) signals to the nucleus through the activation, by tyrosine phosphorylation, of the latent cytoplasmic transcription factor Stat1 (signal transducer and activator of transcription). It has been demonstrated that the activity of Stat1 is dependent on tyrosine phosphorylation which is regulated by Jak tyrosine kinases as well as by the as-yet-unidentified protein tyrosine phosphatase. We report that the N-terminal domain of Stat1, which is highly conserved among all STAT family members, is required for its tyrosine dephosphorylation. A single amino acid substitution (Arg-31 to Ala) in the Stat1 N-terminal domain inhibited Stat1 tyrosine dephosphorylation. The deletion of the Stat1 N-terminal domain resulted in a mutant Stat1 protein which was constitutively phosphorylated on Tyr-701. Upon IFN-gamma stimulation, the tyrosine phosphorylation of this mutant protein was further enhanced but was not down-regulated by protein tyrosine phosphatase in vivo. When expressed in NIH 3T3 cells, this mutant protein greatly enhanced the antiproliferative activity of IFN-gamma. We suggest that the N-terminal domains of STATs are crucial for modulating STAT activities through regulating the tyrosine dephosphorylation of STATs.

3T3 Cells↗
PubMed1996

Mutational analysis of Lck in CD45-negative T cells: dominant role of tyrosine 394 phosphorylation in kinase activity.

The CD45 tyrosine phosphatase has been reported to activate the src family tyrosine kinases Lck and Fyn by dephosphorylating regulatory COOH-terminal tyrosine residues 505 and 528, respectively. However, recent studies with CD45- T-cell lines have found that despite the fact that Lck and Fyn were constitutively hyperphosphorylated, the tyrosine kinase activity of both enzymes was actually increased. In the present study, phosphoamino acid analysis revealed that the increased phosphorylation of Lck in CD45- YAC-1 T cells was restricted to tyrosine residues. To understand the relationship between tyrosine phosphorylation and Lck kinase activity, CD45- YAC-1 cells were transfected with forms of Lck in which tyrosines whose phosphorylation is thought to regulate enzyme activity (Tyr-192, Tyr-394, Tyr-505, or both Tyr-394 and Tyr-505) were replaced with phenylalanine. While the Y-to-F mutation at position 192 (192-Y-->F) had little effect, the 505-Y-->F mutation increased enzymatic activity. In contrast, the 394-Y-->F mutation decreased the kinase activity to very low levels, an effect that the double mutation, 394-Y-->F and 505Y-->F, could not reverse. Phosphopeptide analysis of tryptic digests of Lck from CD45- YAC-1 cells revealed that it is hyperphosphorylated on two tyrosine residues, Tyr-505 and, to a lesser extent, Tyr-394. The purified and enzymatically active intracellular portion of CD45 dephosphorylated Lck Tyr-394 in vitro. These results demonstrate that in addition to Tyr-505, CD45 can dephosphorylate Tyr-394, and that in the absence of CD45 the hyperphosphorylation of Tyr-394 can cause an increase in the kinase activity of Lck despite the inhibitory hyperphosphorylation of Tyr-505. Therefore, Lck kinase activity is determined by the balance of activating and inhibitory tyrosine phosphorylations that are, in turn, regulated by CD45.

Amino Acid Sequence↗
PubMed1998

Tyrosine phosphorylation of platelet derived growth factor beta receptors in coronary artery lesions: implications for vascular remodelling after directional coronary atherectomy and unstable angina pectoris.

BACKGROUND: Growth factors such as platelet derived growth factor (PDGF) have been postulated to be important mediators of neointimal proliferation observed in atherosclerotic plaques and restenotic lesions following coronary interventions. Binding of PDGF to its receptor results in intrinsic receptor tyrosine kinase activation and subsequent cellular migration, proliferation, and vascular contraction. AIMS: To investigate whether the concentration of PDGF beta receptor tyrosine phosphorylation obtained from directional coronary atherectomy (DCA) samples correlate with atherosclerotic plaque burden, the ability of diseased vessels to remodel, coronary risk factors, and clinical events. METHODS: DCA samples from 59 patients and 15 non-atherosclerotic left internal thoracic arteries (LITA) were analysed for PDGF beta receptor tyrosine phosphorylation content by receptor immunoprecipitation and antiphosphotyrosine western blot. The amount of PDGF beta receptor phosphorylation was analysed in relation to angiographic follow up data and clinical variables. RESULTS: PDGF beta receptor tyrosine phosphorylation in the 59 DCA samples was greater than in the 15 non-atherosclerotic LITA (mean (SD) 0.84 (0.67) v 0.17 (0.08) over a control standard, p < 0.0001). As evaluated by stepwise regression analysis, incorporation of both PDGF beta receptor tyrosine phosphorylation and immediate gain correlated strongly (adjusted r2 = 0.579) with late loss, although PDGF beta receptor tyramine phosphorylation alone correlated poorly with late loss. Multivariate regression analysis of coronary risk factors and clinical events revealed unstable angina as the most significant correlate of PDGF beta receptor tyrosine phosphorylation (F value 20.009, p < 0.0001). CONCLUSIONS: PDGF beta receptor tyrosine phosphorylation in atherosclerotic lesions is increased compared with non-atherosclerotic arterial tissues. The association of PDGF beta receptor tyrosine phosphorylation with immediate gain strongly correlates with vascular remodelling. PDGF beta receptor tyrosine phosphorylation correlates with unstable angina pectoris.

Angina, Unstable↗
PubMed2001

Modulation of PKCdelta tyrosine phosphorylation and activity in salivary and PC-12 cells by Src kinases.

Protein kinase C (PKC) delta becomes tyrosine phosphorylated in rat parotid acinar cells exposed to muscarinic and substance P receptor agonists, which initiate fluid secretion in this salivary cell. Here we examine the signaling components of PKCdelta tyrosine phosphorylation and effects of phosphorylation on PKCdelta activity. Carbachol- and substance P-promoted increases in PKCdelta tyrosine phosphorylation were blocked by inhibiting phospholipase C (PLC) but not by blocking intracellular Ca2+ concentration elevation, suggesting that diacylglycerol, rather than D-myo-inositol 1,4,5-trisphosphate production, positively modulated this phosphorylation. Stimuli-dependent increases in PKCdelta activity in parotid and PC-12 cells were blocked in vivo by inhibitors of Src tyrosine kinases. Dephosphorylation of tyrosine residues by PTP1B, a protein tyrosine phosphatase, reduced the enhanced PKCdelta activity. Lipid cofactors modified the tyrosine phosphorylation-dependent PKCdelta activation. Two PKCdelta regulatory sites (Thr-505 and Ser-662) were constitutively phosphorylated in unstimulated parotid cells, and these phosphorylations were not altered by stimuli that increased PKCdelta tyrosine phosphorylation. These results demonstrate that PKCdelta activity is positively modulated by tyrosine phosphorylation in parotid and PC-12 cells and suggest that PLC-dependent effects of secretagogues on salivary cells involve Src-related kinases.

Animals↗
PubMed1986

In vivo inhibition of tyrosine uptake into rat retina by large neutral but not acidic amino acids.

The uptake of tyrosine into rat retina and brain was studied in vivo after its peripheral injection alone or in combination with other amino acids. Both retinal and brain tyrosine levels increased monotonically for at least 60 min after tyrosine administration. When tyrosine was injected along with branched-chain amino acids, but not with acidic amino acids, such increments in retinal and brain tyrosine levels were significantly attenuated. The postinjection tyrosine levels in retina and brain paralleled better the serum ratio of tyrosine to the sum of the other large neutral amino acids (which include the branched-chain amino acids) than the serum tyrosine level alone. These results suggest that tyrosine uptake into rat retina, like that into brain, is mediated by a competitive transport system shared among the large neutral amino acids.

Amino Acids↗
PubMed2002

ACh and adenosine activate PI3-kinase in rabbit hearts through transactivation of receptor tyrosine kinases.

Adenosine and acetylcholine (ACh) trigger preconditioning through different signaling pathways. We tested whether either could activate myocardial phosphatidylinositol 3-kinase (PI3-kinase), a putative signaling protein in ischemic preconditioning. We used phosphorylation of Akt, a downstream target of PI3-kinase, as a reporter. Exposure of isolated rabbit hearts to ACh increased Akt phosphorylation 2.62 +/- 0.33 fold (P = 0.001), whereas adenosine caused a significantly smaller increase (1.52 +/- 0.08 fold). ACh-induced activation of Akt was abolished by the tyrosine kinase blocker genistein indicating at least one tyrosine kinase between the muscarinic receptor and Akt. ACh-induced Akt activation was blocked by the Src tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and by 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG-1478), an epidermal growth factor receptor (EGFR) inhibitor, suggesting phosphorylation of a receptor tyrosine kinase in an Src tyrosine kinase-dependent manner. ACh caused tyrosine phosphorylation of the EGFR, which could be blocked by PP2, thus supporting this receptor hypothesis. AG-1478 failed to block the cardioprotection of ACh, however, suggesting that other receptor tyrosine kinases might be involved. Therefore, G(i) protein-coupled receptors can activate PI3-kinase/Akt through transactivation of receptor tyrosine kinases in an Src tyrosine kinase-dependent manner.

Acetylcholine↗