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Effects of dietary protein, energy and tyrosine on central and peripheral norepinephrine turnover in mice.

Norepinephrine (NE) turnover rate and tyrosine concentrations were assessed in several tissues of 5-wk-old female lean mice that were fed diets of 20% protein (1.2% tyrosine), 40% protein (2.4% tyrosine) or tyrosine-supplemented diets of 40% protein with 4 or 8% tyrosine or 20% protein with 4% tyrosine for 4 d. Mice fed the 40% protein diet had significantly lower NE turnover rates in interscapular brown adipose tissue, heart and kidney (66, 64 and 49%, respectively) and nonsignificantly lower (84%) turnover rate in brain than did mice fed the 20% protein diet, but plasma and tissue tyrosine concentrations did not differ. When energy intake of the 20% protein-fed mice was restricted to 92% of the 40% protein-fed mice, tyrosine concentration in plasma and tissues was lower by half, but NE turnover rate did not differ between the two groups. Supplementation of the 40 or 20% diets with tyrosine, resulting in twofold higher plasma and tissue tyrosine concentrations, had no effect on NE turnover rate in any organ examined. We conclude that both energy intake and dietary protein concentration affect sympathetic nervous system activity in young mice. Tyrosine does not mediate or alter these changes.

Adipose Tissue↗

Studies on the transport of tyrosine, leucine, and methionine in cultured B-16 mouse melanoma cells.

1. Linear uptake of tyrosine lasted for 1 min in cultured B-16 mouse melanoma cells preloaded with an amino acid such as tyrosine. 2. The tyrosine uptake increased markedly on preincubating the cells with 0.1 mM methionine, tyrosine, histidine or tryptophan and moderately with 1 mM phenylalanine, valine, isoleucine, or leucine. The effects of the preincubation on leucine uptake were similar to those on tyrosine uptake. 3. The tyrosine uptake was Na-independent under the experimental conditions used (0.05-1.0 mM); Km 75 micrometers and Vmax 15 nmol/min/mg protein. The methionine uptake (0.1-0.5 mM) was also Na-independent (Km 150 micrometers and Vmax 25 nmol/min/mg protein), whereas Na-dependent uptake could contribute at 2 mM methionine. 4. Inhibitions of tyrosine uptake by tryptophan, phenylalanine, leucine, isoleucine, methionine, valine, and histidine were competitive, giving K1 values of 70, 80, 100, 130, 160, 350, and 900 micrometers, respectively. 5. Exchange between intracellular methionine and extracellular tyrosine and vice versa was equimolar. Potencies of amino acids in stimulating tyrosine efflux were in the following order: methionine greater than tyrosine greater than histidine greater than tryptophan greater than phenylalanine greater than leucine greater than isoleucine much greater than valine. 6. The amino acid affinity of the system in the intracellular surface was suggested to be different from that in the extracellular surface. 7. The theophylline-treated cells showed a marked increase in tyrosine-uptake rate with elevated Vmax and unchanged Km.

Animals↗

Modulation of the actions of tyrosine by alpha 2-adrenoceptor blockade.

1. Eight normal subjects were given, in double-blind, random order L-tyrosine 50, 250 and 500 mg kg-1 and placebo orally. Plasma tyrosine concentrations rose in a dose-dependent manner, without affecting the concentrations of the other large neutral amino acids. Tyrosine stimulated the secretion of prolactin and thyrotrophin (TSH) but had no effect on the plasma concentrations of adrenocorticotrophic hormone (ACTH), cortisol, growth hormone or the gonadotrophins. 2. The lack of a stimulant effect of tyrosine on ACTH secretion was presumed to be due to activation of one of the negative feedback mechanisms that control the rate of synthesis and release of the catecholamines, and this hypothesis was tested by examining the effects of the alpha 2-adrenoceptor antagonist idazoxan on the actions of tyrosine. 3. Seven normal males were given on 6 separate occasions tyrosine 250 and 500 mg kg-1 and placebo orally following pretreatment with saline and idazoxan (0.1 mg kg-1 i.v.). Following pretreatment with idazoxan, tyrosine stimulated the secretion of ACTH and noradrenaline in a dose-dependent manner, although neither tyrosine nor idazoxan on their own had any effect on the secretion of either substance. 4. The lack of effect of tyrosine when given on its own appears to be due, to partly, to activation of alpha 2-adrenoceptors, which inhibit the release of noradrenaline. Idazoxan caused a small increase in systolic blood pressure, both when given on its own and in combination with tyrosine. Neither tyrosine nor idazoxan had any significant effect on the state of behavioural arousal, as measured by visual analogue scales, or on the secretion of growth hormone or the gonadotrophins.

Adrenergic alpha-Antagonists↗

Metabolic influences on tyrosine excretion in Bacillus subtilis.

The biosynthetic pathway for tyrosine synthesis is regulated both by repression of enzyme synthesis and by feedback inhibition of enzyme activity in Bacillus subtilis. Nevertheless, wild-type cells produce significantly more tyrosine than is required for protein synthesis, and part of this is excreted into the medium. Alteration of nutritional and other environmental conditions of cultivation strongly influenced the amount of tyrosine excretion. The excretion of tyrosine by wild-type cells was compared with that of a regulatory mutant having a feedback-insensitive prephenate dehydrogenase. Tyrosine excretion varied directly with the in vitro activity of prephenate dehydrogenase and inversely with temperature in the two strains. The regulatory mutant excreted five times as much tyrosine as the wild type at all growth temperatures examined. The carbon source used for growth significantly influenced the level of tyrosine excretion. The specific activity of prephenate dehydrogenase was also affected by the carbon source. Incorporation studies with isotopic tyrosine and fluorometric determinations of tyrosine concentrations extractable in hot water were used to measure operationally the tyrosine pools in the mutant and wild-type strains. The effects of various environmental conditions on the synthesis and excretion of tyrosine led to the conclusion that metabolic controls governing end product contrations exist which are completely independent of regulation by feedback inhibition and repression.

Bacillus subtilis↗

Role of protein tyrosine phosphorylation in H2O2-induced activation of endothelial cell phospholipase D.

Oxidant-induced activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAEC) is independent of protein kinase C and calcium. In the present study, the effects of tyrosine kinase and protein tyrosine phosphatase (PTPase) inhibitors on hydrogen peroxide (H2O2)-induced PLD activation and protein tyrosine phosphorylation were examined in BPAEC. Pretreatment of BPAEC with putative tyrosine kinase inhibitors genistein, tyrphostin, and herbimycin attenuated H2O2 (1 mM)-induced PLD activation. The inhibitory effect of the tyrosine kinase inhibitors was highly specific for H2O2-induced modulation and showed no effect on PLD activation mediated by 12-O-tetradecanoylphorbol 13-acetate or bradykinin. Furthermore, addition of H2O2 increased in a time-dependent manner tyrosine phosphorylation of several proteins (17-200 kDa), as determined by immunoblot analysis with antiphosphotyrosine antibodies. H2O2-mediated protein tyrosine phosphorylation preceded PLD activation, and a good correlation was observed on the effect of genistein in H2O2-induced PLD activation and protein tyrosine phosphorylation. Addition of vanadate, a phosphotyrosine phosphatase inhibitor, synergistically increased both PLD activation and protein tyrosine phosphorylation mediated by H2O2. Moreover, vanadate by itself had minimal effect on basal PLD activity in BPAEC; however, at 10 microM vanadate, an increase in protein tyrosine phosphorylation was observed. In addition to vanadate, phenylarsine oxide and diamide potentiated H2O2-induced PLD activation. These results suggest that tyrosine kinase activation may be involved in H2O2-induced PLD activation in vascular endothelial cells.

Animals↗

Tyrosine transport in winter flounder intestine: interaction with Na+-K+-2Cl- cotransport.

Tyrosine absorption across the brush border of the intestinal epithelium of the winter flounder Pseudopleuronectes americanus was studied in Ussing chambers modified to determine early rates of uptake. At 0.1 mM tyrosine, the 4-min rate of uptake (influx) of tyrosine across the brush border averaged 37.5 nmol X cm-2 X h-1. Omission of Na decreased influx by 60%, indicating that tyrosine influx occurs, at least in part, by a Na-coupled process. Ouabain inhibited influx by 80%. Inhibition of brush border Na+-K+-2Cl- cotransport by bumetanide, 8-bromo-cyclic GMP, or Cl replacement stimulated tyrosine influx 2.5- to 4-fold. However, atriopeptin III, which also inhibits Na+-K+-2Cl- cotransport, did not stimulate tyrosine influx. Cyclic AMP, which does not appear to inhibit ion cotransport, did not stimulate tyrosine influx. Both cyclic GMP and bumetanide also stimulated the net mucosa-to-serosa tyrosine flux (43 and 29%, respectively) and increased the cellular concentration of tyrosine by 50%. Thus tyrosine's influx is increased to a greater extent than is its transmural flux or its cellular concentration, suggesting that the main change occurs at the brush border and represents large increases in both influx and efflux of tyrosine across this membrane.

Animals↗

Molecular mechanism and functional implications of thrombin-mediated tyrosine phosphorylation of PKCdelta in platelets.

Thrombin has been known to cause tyrosine phosphorylation of protein kinase C delta (PKCdelta) in platelets, but the molecular mechanisms and function of this tyrosine phosphorylation is not known. In this study, we investigated the signaling pathways used by protease-activated receptors (PARs) to cause tyrosine phosphorylation of PKCdelta and the role of this event in platelet function. PKCdelta was tyrosine phosphorylated by either PAR1 or PAR4 in a concentration- and time-dependent manner in human platelets. In particular, the tyrosine 311 residue was phosphorylated downstream of PAR receptors. Also the tyrosine phosphorylation of PKCdelta did not occur in Galpha(q)-deficient mouse platelets and was inhibited in the presence of a phospholipase C (PLC) inhibitor U73122 and calcium chelator BAPTA (5,5'-dimethyl-bis(o-aminophenoxy)ethane-N, N, N ', N '-tetraacetic acid), suggesting a role for Galpha(q) pathways and calcium in this event. Both PAR1 and PAR4 caused a time-dependent activation of Src (pp60c-src) tyrosine kinase and Src tyrosine kinase inhibitors completely blocked the tyrosine phosphorylation of PKCdelta. Inhibition of tyrosine phosphorylation or the kinase activity of PKCdelta dramatically blocked PAR-mediated thromboxane A2 generation. We conclude that thrombin causes tyrosine phosphorylation of PKCdelta in a calcium- and Src-family kinase-dependent manner in platelets, with functional implications in thromboxane A2 generation.

Adenosine Diphosphate↗

The insulin-like effect of vanadate on lipolysis in rat adipocytes is not accompanied by an insulin-like effect on tyrosine phosphorylation.

Tyrosine phosphorylation of the insulin receptor and other intracellular proteins in rat adipocytes was examined using an immunoblot technique with antiphosphotyrosine antibody. Insulin at 10(-7) M increased the tyrosine phosphorylation of the 95K subunit of the insulin receptor (15-fold) and proteins of 180K (7-fold) and 60K (23-fold). Increases in insulin-dependent phosphorylation of the three proteins were detectable at 10(-10) M insulin and attained steady state within 30 sec of insulin (10(-7) M) addition. Small effects of insulin (less than 30% increases) were observed on proteins of 120K and 53K. In contrast to insulin, the effects of vanadate on tyrosine phosphorylation were small and nonspecific. Vanadate increased tyrosine phosphorylation of the 95K insulin receptor beta-subunit and the 120K and 60K proteins similarly, with increases of 1.5- to 3-fold at 1 mM and 2-fold or less at 200 and 50 microM. Vanadate-dependent tyrosine phosphorylation of the 180K protein increased to a maximum of only 30% at 200 microM. Tyrosine phosphorylation of the 53K protein was somewhat larger, approaching 4-fold at 1 mM vanadate. The concentration of insulin and vanadate that inhibited isoproterenol-dependent lipolysis were not comparable to those that increased tyrosine phosphorylation. Vanadate at 1 mM was more potent as an antilipolytic agent than 10(-9) M insulin (93% vs. 81%), yet increased tyrosine phosphorylation of the 95K insulin receptor beta-subunit only as effectively as 10(-10) M insulin (which inhibited lipolysis only 42%). The dissimilar responses were even more pronounced when antilipolysis was compared to tyrosine phosphorylation of the 180K and 60K proteins. For example, insulin at 10(-9) M increased tyrosine phosphorylation of the 180K protein 2.9-fold, while 1 mM vanadate had a negligible effect (10% increase). Thus, vanadate exerts an insulin-like effect on lipolysis, yet its effects on tyrosine phosphorylation differ from those of insulin.

Adipose Tissue↗

pp125FAK tyrosine kinase activity is not required for the assembly of F-actin stress fibres and focal adhesions in cultured mouse aortic smooth muscle cells.

The observed increase in phosphotyrosine content of focal adhesion-associated proteins, in response to integrin engagement, indicates a role for integrin-regulatable tyrosine kinase(s) in cytoskeletal re-organisation. The tyrosine kinase pp125FAK, by virtue of its focal adhesion localisation in fibroblasts, represents a prime candidate to perform this function. We have investigated whether pp125FAK performs a similar function in mouse aortic smooth muscle cells (MASMC). MASMC cultured for 16 hours exhibit F-actin stress fibres and focal adhesions. We have shown that vinculin, pp125FAK and tyrosine-phosphorylated proteins are localised in focal adhesions during this time period. MASMC, under these culture conditions exhibit elevated pp125FAK tyrosine kinase activity, as measured by an increased autophosphorylation potential. We investigated the development of F-actin stress fibres and focal adhesions in MASMC in response to adherence to fibronectin, conditions shown to promote cytoskeletal reorganisation in fibroblasts. Within 30 minutes, MASMC exhibited well-developed F-actin stress fibres and prominent focal adhesions which immunostained intensely for vinculin, pp125FAK and phosphotyrosine. Adherence to fibronectin has been reported to activate pp125FAK tyrosine kinase in fibroblasts, leading to the proposal that pp125FAK plays a critical role in focal adhesion formation. Therefore pp125FAK activation, in response to adherence to fibronectin, was investigated in MASMC. Anti-phosphotyrosine immunoblotting and in vitro kinase assays of MASMC lysates have revealed that, under conditions which promote focal adhesion formation, pp125FAK remains inactive. Since overnight cultures of MASMC exhibited elevated pp125FAK tyrosine kinase activity, we investigated whether these cells deposit their own combination of extracellular matrix (ECM) molecules and/or secrete factors into their conditioned medium which are capable of activating pp125FAK tyrosine kinase. Our results indicate that MASMC-elaborated ECM, but not their conditioned medium, supported pp125FAK tyrosine kinase activation. Furthermore, MASMC exposed to MASMC-ECM displayed a poorly defined F-actin stress fibre network and rudimentary focal adhesions. Thus we have demonstrated the existence of two adhesion-mediated situations in MASMC; one in which fibronectin promotes cytoskeletal reorganisation in the absence of pp125FAK tyrosine kinase activity and the other in which cells adhering to MASMC-ECM display elevated pp125FAK tyrosine kinase activity in association with an impaired ability to promote F-actin stress fibre and focal adhesion formation. These results indicate that in MASMC, pp125FAK tyrosine kinase activity is not involved in F-actin stress fibre assembly and focal adhesion formation.

Actins↗

Protein kinase C-alpha produces reciprocal effects on the phorbol ester stimulated tyrosine phosphorylation of a 50 kDa kinase in Jurkat cells.

A detergent extract isolated from the enriched fraction of integral membrane proteins of Jurkat cells showed an enhanced tyrosine phosphate level when phosphorylated in the presence of phorbol 12-myristate 13-acetate (TPA) and phorbol 12,13-dibutyrate (PDBu). The enhanced tyrosine phosphorylation was observed when the reaction time exceeded 6 min; at shorter incubation times, however, TPA inhibited tyrosine phosphorylation. When the reaction proceeded for a constant time period longer than 6 min and phorbol esters were added at different times after the start of the reaction, two phases of an enhanced tyrosine phosphorylation of a 50 kDa protein were observed. An increased phosphorylation of the 50 kDa protein was correlated with an enhanced phosphorylation of poly(Glu4,Tyr1). The two phases of enhanced phosphorylation differed in their TPA and PDBu requirement and in the proteins that were tyrosine phosphorylated. Studies with protein kinase C (PKC) inhibitors showed a negatively correlated effect on the enhanced tyrosine phosphorylation in phase I; tyrosine phosphorylation was further augmented. In phase II the regulation of tyrosine phosphorylation correlated with the efficiency of the PKC inhibitors on the alpha-isoform of PKC which was found in the cell extract. Separation of the proteins present in the investigated cell extract by gel filtration revealed a co-migration of the alpha-PKC and the 50 kDa protein. The metabolic labeling of intact Jurkat cells with 32Pi indicated that phorbol esters are also able to induce tyrosine phosphorylation of the 50 kDa protein underin vivo conditions. These data suggest an activation of two different tyrosine phosphorylation pathways by phorbol esters involving tyrosine phosphorylation/autophosphorylation of a 50 kDa kinase, as confirmed by 5'-p-fluorosulfonylbenzoyladenosine (FSBA) labeling, that are accurately regulated by alpha-PKC.

Adenosine↗

Protein tyrosine phosphorylation in smooth muscle: a potential coupling mechanism between receptor activation and intracellular calcium.

This review addresses a rapidly growing body of evidence suggesting that enhanced protein tyrosine phosphorylation may be a previously unrecognized mechanism for coupling receptor activation of vascular smooth muscle cells to increases In the intracellular concentration of Ca2+ and contraction. The hypothesis proposes that activation of diverse types of receptors that are not tyrosine kinase promotes stimulation of a cytosolic tyrosine kinase. In turn, the activated kinase induces tyrosine phosphorylation of substrates that are linked to regulatory mechanisms for release of intracellular Ca2+ stored in the sarcoplasmic reticulum and to regulatory mechanisms for influx of extracellular Ca2+. Within this framework, we examine some relevant functional aspects of receptor and nonreceptor tyrosine kinases in different types of cells, the emerging relationships between tyrosine kinase activity and regulation of intracellular Ca2+. We review studies of nonreceptor tyrosine kinase activity in vascular smooth muscle cells suggesting that a physiologically relevant kinase may be the enzyme called pp60. Data that appear to link tyrosine phosphorylation to contraction of smooth muscle are examined, particularly with respect to results obtained with tyrosine kinase inhibitors and measures of changes in tyrosine phosphorylation. Next, we review studies with cultured vascular smooth muscle cells that point to potential coupling between receptor activation, enhanced tyrosine phosphorylation of substrates such as the GTPase activating protein for ras, and the gamma-1 isoform of phospholipase C, and mechanisms controlling Ca2+ influx and release. Emphasis is placed on examining the strengths and weaknesses of different experimental approaches. Lastly, a summary of the data is provided which calls attention to some major issues requiring resolution to permit acceptance or rejection of the underlying hypothesis, and we briefly address some of its possible pathophysiological implications.

Animals↗

CD2/LFA-3 ligation induces phospholipase-C gamma 1 tyrosine phosphorylation and regulates CD3 signaling.

Activation of T cells through the TCR/CD3 receptor complex with either specific Ag or antibody results in tyrosine phosphorylation of intracellular protein substrates and phosphatidylinositol-phospholipase C (PLC) signaling, leading to the generation of PI breakdown products and the mobilization of intracellular calcium. Stimulation of the T cell surface receptor CD2 similarly propagates early signals through phosphatidylinositol-PLC activation. Previous reports have shown that CD3 activation leads to tyrosine phosphorylation of the PLC isozyme PLC gamma 1. In this report, we investigated the potential similarity between CD3-induced signaling through PLC gamma 1 and that induced by CD2. We show that stimulation of CD2 receptors on T cells caused tyrosine phosphorylation of PLC gamma 1. Cross-linking of CD2 with CD3 receptors augmented the phosphorylation of PLC gamma 1 on tyrosine, whereas ligation of the CD45 tyrosine phosphatase with CD2 receptors prevented PLC gamma 1 tyrosine phosphorylation. T cells stimulated by ligation of CD2 with its counter-receptor in the form of a soluble LFA-3/Ig fusion protein cross-linked on the cell surface, resulted in a low, but detectable level of PLC gamma 1 phosphorylation with prolonged kinetics, whereas that induced by cross-linking with anti-CD2 was stronger but transient. Co-ligation of LFA-3/Ig with suboptimal concentrations of anti-CD3 resulted in profound augmentation of PLC gamma 1 tyrosine phosphorylation, mobilization of intracellular calcium and T cell proliferation. To explore the relationship between CD3- and CD2-stimulated signaling, T cells were desensitized through 1 h incubation with anti-CD3. CD3 receptor modulation potently down-regulated CD2-induced PLC gamma 1 tyrosine phosphorylation and calcium mobilization. In contrast, PMA or ionomycin treatment did not alter CD2-stimulated tyrosine phosphorylation of PLC gamma 1, suggesting that tyrosine kinase inhibition by CD3 receptor modulation was not caused by signaling events downstream of PLC gamma 1. Taken together, these results support the hypothesis that CD2 provides a potent co-stimulatory signal for CD3-induced T cell activation that is associated with tyrosine kinase(s) and PLC gamma 1.

Antigens, Differentiation, T-Lymphocyte↗

Regulation of CD3-induced phospholipase C-gamma 1 (PLC gamma 1) tyrosine phosphorylation by CD4 and CD45 receptors.

Stimulation of the signal transduction cascade in T cells through the T-cell receptor (CD3) coincides with activation of the phosphatidylinositol-phospholipase C (PI-PLC) pathway. activation of phospholipase C-gamma 1 (PLC gamma 1) occurs through tyrosine phosphorylation in T cells following surface ligation of CD3 receptors with CD3-specific monoclonal antibodies (mAb). Here we show that cross-linking of CD4 molecules with CD3 augments the tyrosine phosphorylation of PLC gamma 1, while co-ligation of CD3 with CD45 (a receptor tyrosine phosphatase) results in reduced PLC gamma 1 tyrosine phosphorylation. Mobilization of intracellular calcium correlated with the extent of PLC gamma 1 tyrosine phosphorylation, indicating that PLC gamma 1 enzymatic activity in T cells may be regulated by its phosphorylation state. The time-course of PLC gamma 1 tyrosine phosphorylation in cells stimulated by soluble anti-CD3 was transient and closely paralleled that of calcium mobilization, while the kinetics in cells stimulated by immobilized anti-CD3 were prolonged. The PI-PLC pathway in T cells was not stimulated by tyrosine phosphorylation of PLC gamma 2, a homologue of PLC gamma 1, demonstrating the strict regulation of PLC gamma isoform usage in CD3-stimulated T cells. A 35,000/36,000 MW tyrosine phosphorylated protein in T cells formed stable complexes with PLC gamma 1, and its tyrosine phosphorylation was co-regulated with that of PLC gamma 1 by CD4 and CD45 receptors. Enzymatic activation and tyrosine phosphorylation of PLC gamma 1 occurs during growth factor stimulation of fibroblasts, where PLC gamma 1 exists in multi-component complexes. The observation that PLC gamma 1 exists in complexes with unique tyrosine phosphorylated proteins in T cells suggests that haematopoietic lineage-specific proteins associated with PLC gamma 1 may play roles in cellular signalling.

Antigens, CD↗

Purification and characterization of a pp60c-src-related tyrosine kinase that effectively phosphorylates a synthetic peptide derived from p34cdc2.

A protein tyrosine kinase has been purified from the particulate fraction of bovine spleen to a specific activity of 0.217 mumol/min/mg at 100 microM ATP and 3 mM [Val5] angiotensin II. Both the angiotensin phosphorylation activity and immunoreactivity towards an antibody preparation raised against a synthetic peptide containing the autophosphorylation site of pp60c-src, Cys-src(403-421), were monitored during the purification. The purified sample displayed three closely spaced protein bands with molecular weights of 50-55 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All bands could be phosphorylated exclusively on tyrosine residues under autophosphorylation conditions. All reacted on immunoblots with an antibody raised against a synthetic peptide corresponding to the consensus autophosphorylation site of members of the pp60c-src family of tyrosine kinases. Tryptic phosphopeptide maps of the three proteins were essentially indistinguishable. The results suggest that the purified enzyme preparation contained mainly three closely related pp60c-src-family protein tyrosine kinases or a pp60src-family protein tyrosine kinase modified posttranslationally to give three closely spaced protein bands on sodium dodecyl sulfate gel. Neither of these proteins appears to be pp60c-src or p56lck. The spleen protein tyrosine kinase was found to phosphorylate a p34cdc2 kinase peptide, Cys-cdc2(8-20), which contained the regulatory tyrosine residue Tyr-15 about 20 times better than [Val5]angiotensin II or Cys-src(403-421) peptide at a peptide substrate concentration of 1 mM. In contrast, epidermal growth factor receptor kinase partially purified from A431 cells did not show preference for Cys-cdc2(8-20) as its substrate. Although Cys-cdc2(8-20) contained two tyrosine residues, only the tyrosine corresponding to Tyr-15 in p34cdc2 was phosphorylated by the spleen tyrosine kinase. The observation suggests that the primary structure surrounding Tyr-15 of p34cdc2 contains substrate structural determinants specific for the spleen tyrosine kinase.

Amino Acid Sequence↗

Endothelin, vasopressin, and angiotensin II enhance tyrosine phosphorylation by protein kinase C-dependent and -independent pathways in glomerular mesangial cells.

Protein tyrosine phosphorylation has not been considered to be important for cellular activation by phospholipase C-linked vasoactive peptides. We found that endothelin, angiotensin II, and vasopressin (AVP), peptides that signal via phospholipase C activation, rapidly enhanced tyrosine phosphorylation of proteins of approximate molecular mass 225, 190, 135, 120, and 70 kDa in rat renal mesangial cells. The phosphorylated proteins were cytosolic or membrane-associated, and none were integral to the membrane, suggesting that the peptide receptors are not phosphorylated on tyrosine. Epidermal growth factor (EGF), which does not activate phospholipase C in these cells, induced the tyrosine phosphorylation of its own 175-kDa receptor, in addition to five proteins of identical molecular mass to those phosphorylated in response to endothelin, AVP, and angiotensin II. This suggests that in mesangial cells there is a common signaling pathway for phospholipase C-coupled agonists and agonists classically assumed to signal via receptor tyrosine kinase pathways, such as EGF. The phorbol ester, phorbol 12-myristate 13-acetate, and the synthetic diacylglycerol, oleoyl acetylglycerol, stimulated the tyrosine phosphorylation of proteins identical to those phosphorylated by the phospholipase C-linked peptides, suggesting that protein kinase C (PKC) activation is sufficient to active tyrosine phosphorylation. However, the PKC inhibitor, staurosporine, and down-regulation of PKC activity by prolonged exposure to phorbol esters completely inhibited tyrosine phosphorylation in response to PMA but not to endothelin, AVP, or EGF. In conclusion, endothelin, angiotensin II, and AVP enhances protein tyrosine phosphorylation via at least two pathways, PKC-dependent and PKC-independent. Although activation of PKC may be sufficient to enhance protein tyrosine phosphorylation, PKC is not necessary and may not be the primary route by which these agents act. At least one of these pathways is shared with the growth factor EGF, suggesting not only common intermediates in the signaling pathways for growth factors and vasoactive peptides but also perhaps common cellular tyrosine kinases which phosphorylate these intermediates.

Alkaloids↗

Tyrosine residues of bovine thyrotropin. Accessibility to iodination in the intact hormone and isolated subunits.

The susceptibility of the tyrosines of bovine thyrotropin (bTSH) and of its isolated alpha and beta subunits to lactoperoxidase catalyzed iodination was examined as a probe of the reactivity of these residues. In isolated TSH alpha, tyrosines at positions 21, 41, 92 and 93 were labeled with radioactive iodine to a nearly equivalent extent while residue 30 did not incorporate iodine. In intact TSH tyrosine 41, as well as 30, was not modified showing that, upon association with beta subunit, residue 41 becomes substantially less reactive. The other three tyrosines of the alpha subunit retained reactivity; residue 21 incorporated relatively more label than residues 92 and 93. The pattern of reactivity of the TSH alpha tyrosines in intact TSH parallels previous studies on the alpha subunits of lutropin (LH) and human choriogonadotropin (hCG). Thus the different primary sequences of the beta subunits do not influence environments around tyrosines at positions which are spaced throughout the alpha subunit structure. Most of the 11 tyrosine residues of TSH beta incorporate significant amounts of iodine. In intact TSH, beta tyrosines 25, 37, and 119 are most readily iodinated and tyrosines 14, 25 and 37 are the most reactive in the isolated subunit. Of particular interest in intact TSH is that tyrosine 37 and to a lesser extent 61 are modified. These tyrosines are found in analogous and highly conserved domains in the beta subunits of all glycoprotein hormones whose sequences have been determined, but in LH and hCG they can only be iodinated in isolated beta subunits.

Animals↗

Expression of human tyrosine kinase-negative epidermal growth factor receptor amplifies signaling through endogenous murine epidermal growth factor receptor.

Recent findings have suggested that certain ligand-dependent responses to EGF may be propagated in a manner that is not dependent on the intrinsic tyrosine kinase activity of the epidermal growth factor receptor (EGF-R, Campos-Gonzalez, R., and Glenney, J. R., Jr. (1992) J. Biol. Chem. 267, 14535-14538) or, alternatively, that these responses may occur through the interaction of the human tyrosine kinase-deficient EGF-R with an as yet unidentified kinase (Selva, E., Raden, D. L., and Davis, R. J. (1993) J. Biol. Chem. 268, 2250-2254). These conclusions represent a significant departure from our current understanding of signal transduction by receptor tyrosine kinases. Therefore we examined the effect of expression of tyrosine kinase-negative human EGF receptor in murine NIH-3T3-2.2 cells on the EGF-dependent phosphorylation of mitogen-activated protein (MAP-2) kinase. In parental cells (NIH-3T3-2.2) that express low levels of endogenous murine EGF-R, there was no demonstrable EGF-dependent coupling to MAP-2 kinase. In NIH-3T3-2.2 cells transfected with tyrosine kinase-negative human EGF-R, there was unexpected EGF-dependent phosphorylation of MAP-2 kinase. Analysis of the tyrosine kinase-negative human EGF-R in these cells revealed significant tyrosine phosphorylation of the EGF-R. A low level of endogenous murine EGF-R present in these cells were also phosphorylated on tyrosine residues and displayed autokinase activity. Similar results were obtained using an unrelated cell line (B82L cells), in which EGF-dependent phosphorylation of MAP-2 kinase was previously attributed to signal propagation through a tyrosine kinase-negative human EGF-R (Campos-Gonzalez, R., and Glenney, J. R., Jr. (1992) J. Biol. Chem. 267, 14535-14538). Taken together, these results suggest that the tyrosine kinase-negative human EGF-R are able to amplify the response to activation of low levels of endogenous murine EGF-R, thus leading to EGF-dependent phosphorylation of MAP-2 kinase in cells expressing tyrosine kinase-negative human EGF-R.

3T3 Cells↗

Angiotensin II-induced tyrosine phosphorylation in mesangial and vascular smooth muscle cells.

1. Angiotensin II (AngII)-induced, activation of phospholipase C (PLC) and Ca2+-dependent Cl- channels is an important signal transduction pathway for the regulation of vascular smooth muscle cell (VSMC) and glomerular mesangial cell contraction and growth. While AT receptors are traditionally thought to be G-protein coupled to the beta isoform of PLC, recent evidence suggests that in some tissues AT receptors may also activate the PLC-gamma isoform via tyrosine phosphorylation. 2. By western analysis, we identified PLC-gamma1 in the above cell types. We found that within 3 min of exposure to 10(-7) mol/L AngII, tyrosine phosphorylation of PLC-gamma1 was observed; however, peak response (>3-fold increase) occurred within 0.5 min. In addition, pre-incubation of these cells with the tyrosine kinase inhibitor genistein blocked the tyrosine phosphorylation of PLC-gamma1 by AngII. In contrast, preincubation with the tyrosine phosphatase inhibitor sodium vanadate increased the levels of tyrosine phosphorylation of PLC-gamma1. Similar results were found when intracellular levels of 1,4,5-IP3 were measured after AngII exposure. 3. By using patch clamp techniques on cultured rat mesangial cells exposed to AngII, we found that the release of 1,4,5-IP3-sensitive intracellular Ca2+ stores stimulated low conductance Cl- channels. Preincubation with genistein, abolished the usual 10-fold increase in Cl- channel activity observed with AngII. 4. Therefore, we conclude that in VSMC and glomerular mesangial cells (i) AngII transiently stimulates PLC activity via tyrosine phosphorylation of the gamma1 isoenzyme, (ii) tyrosine phosphorylation of PLC-gamma1 and production of 1,4,5-IP3 in response to AngII is dramatically inhibited by tyrosine kinase inhibition and stimulated by tyrosine phosphatase inhibition, (iii) activation of Ca2+-dependent Cl- channels by AngII-induced release of 1,4,5-IP3-dependent intracellular Ca2+ stores is also abolished by tyrosine kinase inhibition. In summary, this AngII-induced signal transduction cascade provides a possible mechanism for both the contractile and growth-stimulating effects of AngII on VSMC and glomerular mesangial cells.

Angiotensin II↗