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PubMed2007

Two axes in platelet-derived growth factor signaling: tyrosine phosphorylation and reactive oxygen species.

The tyrosine phosphorylation cascade is a hallmark of platelet-derived growth factor (PDGF)- induced signal transduction. The amplitude and propagation of the tyrosine phosphorylation signal relies on the balance between tyrosine kinase and tyrosine phosphatase. The tyrosine kinase is latent in the absence of stimulation, whereas the tyrosine phosphatase is highly and constitutively active. Therefore, the kinase activation should be accompanied by temporal and spatial inactivation of tyrosine phosphatase to achieve the robust amplification of tyrosine phosphorylation. For the past decade, reactive oxygen species have been receiving a great deal of attention with regard to their ability to shut down tyrosine phosphatase activities in a reversible manner. In this article, the crosstalk between tyrosine phosphorylation and reactive oxygen species in PDGF signaling is discussed.

Animals↗

PubMed1996

Surgical denervation increases protein tyrosine phosphatase activity in skeletal muscle.

Protein tyrosine phosphorylation, which plays an important role in synapse formation at the neuromuscular junction, appears to be regulated by presynaptic neurons. Innervation increases whereas denervation decreases the phosphotyrosine content at the neuromuscular junction. The innervation-dependent tyrosine phosphorylation may result from elevated activity of protein tyrosine kinases; alternatively innervation may down-regulate the protein tyrosine phosphatase activity in the skeletal muscle. To investigate the possible neuronal control of protein tyrosine phosphatase activity at the neuromuscular junction, we have characterized protein tyrosine phosphatase activity in rat skeletal muscle and studied the effects of surgical denervation on the phosphatase activity. Protein tyrosine phosphatase activity in the skeletal muscle, assayed using src [32P]-phosphorylated myelin basic protein as a substrate, was both time- and protein concentration-dependent and was inhibited by micromolar concentrations of vanadate and zinc ion, both of which are known to inhibit tyrosine phosphatases specifically. It was not affected, however, by chemicals known to inhibit acid and alkaline phosphatases or serine/threonine phosphatases. Surgical denervation caused an increase in protein tyrosine phosphatase activity in rat hindlimb muscles. The increase in phosphatase activity reached a maximum (2-fold above the normal) 4 days post-denervation and maintained a plateau for up to 24 days. The biochemical properties of the phosphatase activity in denervated muscle were similar to those of the phosphatase activity in the innervated muscles. These results demonstrate that protein tyrosine phosphatase activity in skeletal muscle is regulated by motoneurons.

Animals↗

PubMed1995

The regulation of leukotriene D4-induced calcium influx in human epithelial cells involves protein tyrosine phosphorylation.

Leukotriene D4 (LTD4) has been found to induce calcium signalling in the intestinal epithelial cell line Int 407, and this action involves the activation of both different GTP-binding proteins (G-proteins) and phospholipase C of the gamma-subtype (PLC-gamma). With this knowledge as the incentive, we investigated the possible regulatory role of protein tyrosine kinase activities in the calcium signalling system of the LTD4 receptor. The tyrosine kinase inhibitors genistein and herbimycin. A both reduced the LTD4-induced calcium signal by 70% when Int 407 cells were stimulated in the presence of extracellular calcium, but had no effect on the signal when the cells were stimulated in a calcium-free medium. In accordance with these findings, pretreatment with a tyrosine kinase inhibitor also blocked thapsigargin-induced cellular influx of calcium. These inhibitors had no effect on the intracellular mobilisation of calcium, which was supported by the findings that LTD4 was able to induce an increase in the tyrosine phosphorylation of PLC-gamma even when one of the tyrosine kinase inhibitors was present. Of possible interest regarding the effect of genistein on LTD4-induced calcium influx is that two major tyrosine phosphorylated protein bands were detected in immunoprecipitates obtained with PLC-gamma antibodies from LTD4-stimulated cells. These proteins, which associate with PLC-gamma, have estimated molecular weights of 84 and 97 kD. Preincubation with genistein completely abolished the LTD4-induced increase in tyrosine phosphorylation of the major 97 kD band, whereas the 84 kD protein band, like the PLC-gamma band, still exhibited an increased phosphorylation of tyrosine residues in response to LTD4. Neither this effect nor any of the other effects of genistein were induced when cells were preincubated with daidzein, an inactive analogue of genistein. The present results suggest that LTD4-induced calcium signalling in epithelial cells involves not only tyrosine phosphorylation of PLC-gamma, but also a tyrosine kinase-dependent step which occurs downstream of PLC-gamma activation and is directly implicated in the regulation of agonist-mediated calcium influx.

Benzoquinones↗

PubMed1990

Activation of the EGF receptor tyrosine kinase by divalent metal ions: comparison of holoreceptor and isolated kinase domain properties.

The activation of the epidermal growth factor (EGF) receptor tyrosine kinase activity is thought to represent a key initial step in EGF-mediated mitogenesis. The mechanisms underlying the regulation of the EGF receptor tyrosine kinase activity were examined through comparisons of the holoreceptor, purified from human placenta, and a soluble 42 kDa tyrosine kinase domain (TKD), generated by the limited trypsin proteolysis of the holoreceptor. The results of these studies highlight the importance of divalent metal ions (Me2+), i.e., Mn2+ and Mg2+, as activators of the tyrosine kinase activity. Manganese is an extremely effective activator of the holoreceptor tyrosine kinase, and under some conditions (low ionic strength) it completely alleviates the need for EGF to stimulate activity. In contrast, Mg2+ only weakly stimulates the holoreceptor tyrosine kinase activity in the absence of EGF, but promotes essentially full activity in the presence of the growth factor. Like the holoreceptor, the soluble TKD is highly active in the presence of Mn2+. However, the isolated TKD is completely inactive in the presence of Mg2+, and, in fact, Mg2+ inhibits the Mn2(+)-stimulated tyrosine kinase activity. The differences in the effects of Mn2+ and Mg2+ on the isolated TKD were further demonstrated by monitoring the effects of Me2+ on the modification of a reactive cysteine residue(s) on the TKD. While Mn2+ potentiates the inhibition by cysteine-directed reagents of the tyrosine kinase activity, Mg2+ has no effect on either the rate or the extent of the inhibition. Both the regulation by Mn2+ of the kinase activity of the TKD and the potentiation by Mn2+ of the cysteine reactivity of the TKD occur over a millimolar concentration range, which implicates a direct binding interaction by the metal ion. Overall, these results demonstrate that there are two key activator sites on the EGF receptor, i.e., the EGF binding site on the extracellular domain and a Me2+ binding site on the cytoplasmic TKD. Me2+ interactions with the cytoplasmic kinase domain apparently result in conformational changes which regulate the levels of tyrosine kinase activity, influence the degree to which this activity is responsive to EGF, and probably account for the effects of Me2+ on the aggregation state of the receptor (Carraway, K.L., III, Koland, J.G. and Cerione, R.A. (1989) J. Biol. Chem. 264, 8699-8707). In general, Mg2(+)-induced conformation changes prime the receptor for activation by EGF, while Mn2+ can fully activate the receptor tyrosine kinase and thereby short-circuit growth factor control.

Enzyme Activation↗

PubMed1993

Four isoforms of tyrosine hydroxylase are expressed in human brain.

In contrast to nonprimate species, the RNA for human tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, can undergo alternative splicing to produce four different types of mRNA. Although types 1 and 2 of these human tyrosine hydroxylase mRNAs have been identified in human brain, whether types 3 and 4 human tyrosine hydroxylase mRNAs are present in the central nervous system remains controversial. Furthermore, little is known about the expression of the protein products of these mRNAs in human brain. In this study we used antibodies raised against different octapeptide sequences from each of the predicted human tyrosine hydroxylase protein forms to determine the presence and distribution of each human tyrosine hydroxylase isoforms in several regions of human brain. Control immunocytochemical and blot immunolabeling experiments demonstrated that each antibody selectively recognized the human tyrosine hydroxylase isoform against which it was directed. In immunocytochemical studies, all four human tyrosine hydroxylase isoforms were clearly detectable in neurons of both the substantia nigra and locus coeruleus. The presence of all four isoforms in these nuclei was confirmed with blot immunolabeling studies. Single-label immunocytochemical studies of adjacent sections as well as dual-label comparisons of immunoreactivity for human tyrosine hydroxylase type 1 with type 2, type 3, or type 4 suggested that at least some neurons in these brain regions contain all four human tyrosine hydroxylase isoforms. In contrast, some neurons of the mesencephalon appeared to be selectively immunoreactive with the antibodies against type 1. In the caudate nucleus and putamen, the terminal zones of the dopaminergic projection from the substantia nigra, all four isoforms were detected, although in immunocytochemical studies type 1 appeared to be the predominant isoform present in axons and terminals. These findings demonstrate that human brain contains four distinct isoforms of human tyrosine hydroxylase and that the presence or relative amount of each isoform may differ among catecholaminergic cell populations and between catecholaminergic neurons and terminal fields. These patterns of expression may have important implications for understanding the regulation of catecholamine biosynthesis in human brain both in normal and pathological states.

Adrenal Medulla↗

PubMed1994

Postnatal development of the suprachiasmatic nucleus in the rat. Morpho-functional characteristics and time course of tyrosine hydroxylase immunopositive fibers.

According to earlier data, the suprachiasmatic nucleus of neonatal rats is highly innervated by serotonin- and tyrosine hydroxylase-immunopositive fibers [Ugrumov M. V. (1992) Zool. Sci. (Tokyo) 9, 37-45], while the latter were no longer observed in adults. This study has attempted to evaluate the timing of the innervation of the rat suprachiasmatic nucleus by tyrosine hydroxylase-immunopositive fibers, as well as to specify some morpho-functional characteristics of these fibers. According to our semi-quantitative light microscopic immunocytochemical data, few tyrosine hydroxylase-immunopositive fibers were observed in the suprachiasmatic nucleus as early as the second postnatal day. They highly increased in number neonatally reaching a maximum at the 10th postnatal day, and then decreased dramatically in adulthood. These data suggest either the provisional character of the tyrosine hydroxylase-immunopositive fibers themselves or the transient expression of tyrosine hydroxylase within permanent fibers. The tyrosine hydroxylase immunopositivity in the fibers points to their catecholaminergic nature, while the overlapping in the distribution of tyrosine hydroxylase- and serotonin-immunopositive fibers might also suggest the transient expression of tyrosine hydroxylase in serotoninergic neurons. In order to check this hypothesis, the neurotoxins of catecholamine- and serotoninergic neurons, 6-hydroxydopamine and 5,7-dihydroxytryptamine, were intraventricularly injected at the second postnatal day, while their effects were specified by the semi-quantitative immunocytochemistry eight days later. 6-Hydroxydopamine did not modify the content of tyrosine hydroxylase-immunopositive fibers in the suprachiasmatic nucleus. Conversely, the treatment with 5,7-dihydroxytryptamine resulted in a significant increase in the number of the tyrosine hydroxylase-immunopositive fibers, while reducing the amount of the serotoninergic ones.(ABSTRACT TRUNCATED AT 250 WORDS)

5,7-Dihydroxytryptamine↗

PubMed2005

Tyrosine 769 of the keratinocyte growth factor receptor is required for receptor signaling but not endocytosis.

Keratinocyte growth factor receptor (KGFR) is a receptor tyrosine kinase expressed on epithelial cells which belongs to the family of fibroblast growth factor receptors (FGFRs). Following ligand binding, KGFR is rapidly autophosphorylated on specific tyrosine residues in the intracellular domain, recruits substrate proteins, and is rapidly internalized by clathrin-mediated endocytosis. The role of different autophosphorylation sites in FGFRs, and in particular the role of the tyrosine 766 in FGFR1, first identified as PLCgamma binding site, has been extensively studied. We analyzed here the possible role of the tyrosine 769 in KGFR, corresponding to tyrosine 766 in FGFR1, in the regulation of KGFR signal transduction and MAPK activation as well as in the control of the endocytic process of KGFR. A mutant KGFR in which tyrosine 769 was substituted by phenylalanine was generated and transfected in NIH3T3 and HeLa cells. Our results indicate that tyrosine 769 is required for the binding to KGFR and tyrosine phosphorylation of PLCgamma as well as for the full activation of MAPKs and for cell proliferation through the regulation of FRS2 tyrosine phosphorylation, suggesting that this residue represents a key regulator of KGFR signal transduction. Our data also show that tyrosine 769 is not involved in the regulation of the endocytic process of KGFR.

Animals↗

PubMed2005

Tumor necrosis factor-alpha mediates pancreatitis responses in acinar cells via protein kinase C and proline-rich tyrosine kinase 2.

BACKGROUND & AIMS: Although tumor necrosis factor alpha is implicated as an important mediator of the inflammatory response in acute pancreatitis, its role in other pathologic features of the disease remains unknown. We investigated the role for tumor necrosis factor alpha in cytoskeletal responses and the underlying signaling mechanisms in pancreatic acinar cells. METHODS: In isolated rat pancreatic acini and AR42J cells, we determined the effect of tumor necrosis factor alpha on the actin cytoskeleton by rhodamine-phalloidin. Using pharmacological and molecular approaches, we assessed the involvement of protein kinase C, Src kinases, and proline-rich tyrosine kinase 2 in the process. We also studied the involvement of these signaling pathways in tumor necrosis factor-alpha-induced nuclear factor-kappaB activation and apoptosis. RESULTS: Tumor necrosis factor-alpha increased the tyrosine phosphorylation of proline-rich tyrosine kinase 2 in acinar cells. The broad-spectrum protein kinase C inhibitor and the Src kinase inhibitor both inhibited tumor necrosis factor-alpha-induced proline-rich tyrosine kinase 2 phosphorylation, but at different tyrosine residues. Using protein kinase C isoform-specific inhibitors and the antisense approach, we showed that protein kinase C delta and mediate proline-rich tyrosine kinase 2 tyrosine phosphorylation. Tumor necrosis factor-alpha caused disorganization of the actin cytoskeleton by a mechanism dependent on protein kinase C, Src kinases, and proline-rich tyrosine kinase 2. Inhibition of protein kinase C, but not Src kinases, decreased tumor necrosis factor-alpha-induced apoptosis. Furthermore, with antisense transfections, we showed that protein kinase C delta and , but not proline-rich tyrosine kinase 2, mediate tumor necrosis factor alpha-induced nuclear factor-kappaB activation. CONCLUSIONS: Tumor necrosis factor-alpha activates proline-rich tyrosine kinase 2 to cause cytoskeletal disorganization and nuclear factor-kappaB to cause inflammatory response, and it triggers cell death signaling through divergent mechanisms mediated by protein kinase C. The results provide insights into the mechanisms in pancreatic acinar cells that link tumor necrosis factor alpha to critical processes in acute pancreatitis.

Acute Disease↗

PubMed2003

Nitric oxide and cGMP activate the Ras-MAP kinase pathway-stimulating protein tyrosine phosphorylation in rabbit aortic endothelial cells.

The free radical nitric oxide is a very effective signal transducer, stimulating the enzyme guanylyl cyclase, the oncoprotein p21Ras, and protein tyrosine phosphorylation. In the present study using rabbit aortic endothelial cells (RAEC), it is demonstrated that the nitric-oxide-generating substances sodium nitroprusside and S-nitroso-N-acetylpenicillamine, and a stable analog of cyclic GMP, 8BrcGMP stimulate p21Ras activity. Tyrosine phosphorylation of cytosolic proteins was stimulated and intracellular production of cGMP was increased, indicating that the NO/cGMP-stimulated tyrosine phosphorylation-dependent signaling pathway is most likely associated with the activation of p21Ras. NO and cGMP-dependent activation of p21Ras result in binding of the oncoprotein to the Ras-binding domain of Raf-1 kinase. Incubation of RAEC with FPT II, a potent and selective inhibitor of p21Ras, prevented NO-dependent tyrosine phosphorylation. ODQ, a potent inhibitor of the soluble form of guanylyl cyclase, inhibited the signal as well. Conversely, the use of KT5823, a cGMP-dependent protein kinase (PKG) blocker, showed no effect on protein tyrosine phosphorylation. To further establish a role for p21Ras on the NO-stimulated tyrosine phosphorylation-signaling pathway, RAEC were constitutively transfected with a dominant negative mutant of p21Ras, N17Ras. NO and cGMP-stimulated tyrosine phosphorylation were prevented in N17Ras-expressing RAEC exposed to NO donors and 8BrcGMP. The above findings indicate that NO and cGMP stimulation of protein tyrosine phosphorylation requires the participation of fully functional p21Ras. ERK1/2 MAP kinases and their subsequent targets, the transcription factors, lie downstream to Ras, Raf-1 kinase, and MEK. Treatment of both RAEC and mock-transfected RAEC with NO resulted in phosphorylation and activation of ERK1/2. On the other hand, NO did not stimulate phosphorylation of ERK1/2 in N17Ras-expressing RAEC. In addition, PD98059, a MEK inhibitor, prevented overall tyrosine phosphorylation and phosphorylation of ERK1/2. Upstream to Ras ERK1/2 MAP kinases target the EGF receptor. Incubation of RAEC or mock-transfected RAEC with NO donors resulted in activation of the EGF receptor autophosphorylation. PD98059 effectively blocked this activation. EGF receptor autophosphorylation was insensitive to NO stimulation in N17Ras-expressing RAEC. It is concluded that NO and cGMP stimulate a signaling pathway involving p21Ras-Raf-1 kinase-MEK-ERK1/2. Activation of this signaling pathway is connected to NO-stimulated overall tyrosine phosphorylation that also involves the transactivation of the EGF receptor mediated by ERK1/2.

Animals↗

PubMed1992

Identification and functional importance of tyrosine sulfate residues within recombinant factor VIII.

Sulfated tyrosine residues within recombinant human factor VIII were identified by [35S]sulfate biosynthetic labeling of Chinese hamster ovary cells which express human recombinant factor VIII. Alkaline hydrolysis of purified [35S]sulfate-labeled factor VIII showed that greater than 95% of the [35S]sulfate was incorporated into tyrosine. [3H]Tyrosine and [35S]sulfate double labeling was used to quantify the presence of 6 mol of tyrosine sulfate per mole of factor VIII. Amino acid sequence analysis of thrombin and tryptic peptides isolated from [35S]sulfate-labeled factor VIII demonstrated tyrosine sulfate at residue 346 in the factor VIII heavy chain and at residues 1664 and 1680 in the factor VIII light chain. In addition, the carboxyl-terminal half of the A2 domain contained three tyrosine sulfate residues, likely at positions 718, 719, and 723. Interestingly, all sites of tyrosine sulfation border thrombin cleavage sites. The functional importance of tyrosine sulfation was examined by treatment of cells expressing factor VIII with sodium chlorate, a potent inhibitor of tyrosine sulfation. Increasing concentrations of sodium chlorate inhibited sulfate incorporation into factor VIII without affecting its synthesis and/or secretion. However, factor VIII secreted in the presence of sodium chlorate exhibited a 5-fold reduction in procoagulant activity, although the protein was susceptible to thrombin cleavage. These results suggest that tyrosine sulfation is required for full factor VIII activity and may affect the interaction of factor VIII with other components of the coagulation cascade.

Amino Acid Sequence↗

PubMed1996

Mechanism of carbon dioxide-catalyzed oxidation of tyrosine by peroxynitrite.

Peroxynitrite ion (ONO2-) reacted rapidly with CO2 to form a short-lived intermediate provisionally identified as the ONO2CO2- adduct. This adduct was more reactive in tyrosine oxidation than ONO2- itself and produced 3-nitrotyrosine and 3,3'-dityrosine as the major oxidation products. With tyrosine in excess, the rate of 3-nitrotyrosine formation was independent of the tyrosine concentration and was determined by the rate of formation of the ONO2CO2- adduct. The overall yield of oxidation products was also independent of the concentration of tyrosine and medium acidity; approximately 19% of the added ONO2- was converted to products under all reaction conditions. However, the 3-nitrotyrosine/3,3'-dityrosine product ratio depended upon the pH, tyrosine concentration, and absolute reaction rate. These data are in quantitative agreement with a reaction mechanism in which the one-electron oxidation of tyrosine by ONO2CO2- generates tyrosyl and NO2 radicals as intermediary species, but are inconsistent with mechanisms that invoke direct electrophilic attack on the tyrosine aromatic ring by the adduct. Based upon its reactivity characteristics, ONO2CO2- has a lifetime shorter than 3 ms and a redox potential in excess of 1 V, and oxidizes tyrosine with a bimolecular rate constant greater than 2 x 10(5) M-1 s-1. In comparison, in CO2-free solutions, oxidation of tyrosine by peroxynitrite was much slower and gave significantly lower yields (approximately 8%) of the same products. When tyrosine was the limiting reactant, 3,5-dinitrotyrosine was found among the reaction products of the CO2-catalyzed reaction, but this compound was not detected in the uncatalyzed reaction.

Carbon Dioxide↗

PubMed1997

Structural coupling between the oxygen-evolving Mn cluster and a tyrosine residue in photosystem II as revealed by Fourier transform infrared spectroscopy.

The flash-induced Fourier transform infrared (FTIR) difference spectrum of the oxygen-evolving Mn cluster upon S1-to-S2 transition (S2/S1 spectrum) was measured using photosystem II (PS II) core complexes of Synechocystis 6803 in which tyrosine residues were specifically labeled with 13C at the ring-4 position. The double-difference spectrum between the unlabeled and labeled S2/S1 spectra showed that the bands at 1254 and 1521 cm-1 downshifted by 25 and 15 cm-1, respectively, upon ring-4-13C-Tyr labeling. This observation indicates that there is a tyrosine residue coupled to the Mn cluster, and the vibrational modes of this tyrosine are affected upon S2 formation. From a comparison of the above band positions and isotopic shifts in the S2/S1 spectrum with those of the FTIR spectra of tyrosine in aqueous solution at pH 0.6 (Tyr-OH) and pH 13.4 (Tyr-O-) and of the YD./YD FTIR difference spectrum, the 1254 and 1521 cm-1 bands were assigned to the CO stretching and ring CC stretching modes of tyrosine, respectively, and this tyrosine was suggested to be protonated in PS II. The observation that the effect of the S2 formation on the tyrosine bands appeared as a decrease in intensity with little frequency change could not be explained by a simple electrostatic effect by Mn oxidation, suggesting that the Mn cluster and a tyrosine are linked via chemical and/or hydrogen bonds and the structural changes of the Mn cluster are transmitted to the tyrosine through these bonds. On the basis of previous EPR studies that showed close proximity of YZ to the Mn cluster, YZ was proposed as the most probable candidate for the above tyrosine. This is the first demonstration of the structural coupling between YZ and the Mn cluster in an intact oxygen-evolving complex. This structural coupling may facilitate electron transfer from the Mn cluster to YZ. Our observation also provides an experimental support in favor of the proton or hydrogen atom abstraction model for the YZ function.

Bacterial Proteins↗

PubMed2002

Phosphorylated tyrosine-containing proteins in primary lung cancer correlates with proliferation and prognosis.

To determine the usefulness of tyrosine phosphorylation in evaluating biological characteristics, we attempted to evaluate the relationship between the amount of phosphorylated tyrosine-containing proteins and clinicopathological factors, cell proliferation and outcome in non-small cell lung cancer. To evaluate phosphorylated tyrosine-containing proteins we used 96 surgically resected materials of non-small cell lung cancer and normal peripheral lung, while immunohistochemical evaluation was performed. Cell proliferating ability was evaluated using the labelling index of proliferating cell nuclear antigen-positive nuclear staining cells. There were statistically significant differences between the expression levels of phosphorylated tyrosine-containing proteins of normal and cancerous tissues (P<0.0001). Evaluations based on clinicopathological factors apart from histopathological differentiation, showed no statistically significant differences of phosphorylated tyrosine-containing proteins expression. However, phosphorylated tyrosine-containing proteins correlated with cell proliferation activity evaluated (P((Low, High))<0.0001; P((Low, Int)) <0.0001; P((Int, High))<0.0001). Furthermore, non-small cell lung cancer cases with high expression and intermediate expression of phosphorylated tyrosine-containing proteins had a significantly shorter disease-free postoperative survival than those with low expression of phosphorylated tyrosine-containing proteins using log-rank analysis (P((Low, Int)) <0.0028; P((Low, High))=0.0002). Furthermore, phosphorylated tyrosine-containing proteins expression level statistically contributed to disease-free survival in Cox's proportional hazard model. Therefore, phosphorylated tyrosine-containing proteins in non-small cell lung cancer tissues seem to reflect its biological malignancy, and this evaluation may be valuable for constructing the most appropriate therapeutic strategy.

Aged↗

PubMed1994

Tyrosine phosphorylation and activation of NADPH oxidase in human neutrophils: a possible role for MAP kinases and for a 75 kDa protein.

Challenge of neutrophils with concanavalin A (ConA), formyl-methionyl-leucyl-phenylalanine (FMLP), and phorbol 12-myristate 13-acetate (PMA) induced the tyrosine phosphorylation of several proteins. Among these proteins we have identified two mitogen-activated protein kinase (MAPK) isoforms of 43 kDa (p43 MAPK) and 45 kDa (p45 MAPK) molecular mass. Moreover here we show that: (1) FMLP induced the tyrosine phosphorylation of the p43 MAPK, and ConA that of p45 MAPK, while PMA induced the tyrosine phosphorylation of both p43 and p45 MAPK; all these agonists induced the tyrosine phosphorylation of a 75 kDa protein (p75). (2) With FMLP or ConA as agonists, tyrosine phosphorylations of MAPK and p75 can be involved in the process of NADPH oxidase activation. On the contrary, PMA can activate the respiratory burst independently of these phosphorylations. (3) In Ca(2+)-depleted neutrophils, where phospholipid hydrolysis did not take place, ConA or FMLP did not activate the respiratory burst, but while ConA induced the tyrosine phosphorylation of p45 MAPK and p75, FMLP was not able to phosphorylate p43 MAPK and p75. (4) As previously observed in our laboratory, a double stimulation of Ca(2+)-depleted neutrophils with ConA plus FMLP induced a respiratory burst in the absence of activation of second messengers derived from phospholipase C, D and A2 activity. This respiratory burst was accompanied by tyrosine phosphorylation of both p43 and p45 MAPKs. These results indicate that when FMLP is the agonist, both the tyrosine phosphorylation of p43 MAPK and p75, and the activation of NADPH oxidase, are coupled to Ca(2+)-dependent mechanisms. On the contrary, ConA can induce the tyrosine phosphorylation of p45 MAPK and p75 independently of calcium, but an unknown Ca(2+)-dependent mechanism is necessary for the activation of NADPH oxidase by this agonist. This mechanism could be substituted by the induction of tyrosine phosphorylation of both p43 MAPK and p45 MAPK when Ca(2+)-depleted neutrophils are stimulated with ConA plus FMLP.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed2001

Tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) by oxidant stress in cerebellar granule neurons: modulation by N-methyl-D-aspartate through calcineurin activity.

Insulin receptor-substrate-1 (IRS-1) is a docking protein for several tyrosine kinase receptors. Upon tyrosine phosphorylation, IRS-1 binds to signaling molecules that express Src homology 2 (SH-2) binding domains, including phosphatidylinositol 3-kinase (PI 3-kinase), phosphotyrosine phosphatase SHP-2 (Syp), Nck, Crk and Grb-2. Hydrogen peroxide (H(2)O(2)) induces tyrosine phosphorylation of key signaling mediators presumably by inhibition of tyrosine phosphatases. In many cell types, the activation of extracellular signal-related kinases (e.g. MAPK) and other protein kinases by H(2)O(2) leads to transcriptional activation. In the current study, we examined the effect of H(2)O(2) on IRS-1 tyrosine phosphorylation in primary cultured rat cerebellar granule neurons. H(2)O(2) stimulated the rapid tyrosine phosphorylation of IRS-1 and p42/p44 MAP kinase, and induced its association with PI 3-kinase. H(2)O(2)-induced IRS-1 phosphorylation was rapidly reversible (5 min) whereas MAPK phosphorylation persisted for up to 1 h. NMDA reversed H(2)O(2)-mediated tyrosine phosphorylation of IRS-1 and its association with PI 3-kinase. The dephosphorylation of IRS-1 by NMDA was calcium-dependent and was inhibited by the calcineurin inhibitor cyclosporine. Calmodulin-dependent tyrosine phosphatase activity of calcineurin was observed in vitro using both immunoprecipitated and recombinant tyrosine-phosphorylated IRS-1 as substrates. These data highlight the role of multiple phosphatases in the regulation of IRS-1 tyrosine phosphorylation and identify a novel functional property of calcineurin.

Animals↗

PubMed1986

Protein tyrosine phosphorylation in the cell cycle of BALB/c 3T3 fibroblasts.

Cell cycle-dependent regulation of protein tyrosine phosphorylation in normal BALB/c 3T3 fibroblasts was examined by immunoblotting with a high-affinity antibody specific for phosphotyrosine. At least 15 different tyrosine-phosphorylated proteins are found in normal 3T3 cells. The level of tyrosine phosphorylation is higher in growing cells than in quiescent cells. However, a prominent tyrosine-phosphorylated protein of Mr 150,000 is present in quiescent cells, and its level is inversely proportional to the growth rate of these fibroblasts. Stimulation of quiescent cells with serum causes a major, yet transient, increase in tyrosine phosphorylation. The immediate tyrosine phosphorylation reactions in response to serum stimulation are independent of protein synthesis, but tyrosine phosphorylation reactions occurring later in the G1 phase of the cell cycle are inhibited by cycloheximide. Thus, tyrosine phosphorylation of proteins in normal 3T3 cells occurs predominantly at the G0 to G1 transition of the cell cycle. Maintenance of steady-state tyrosine phosphorylation is dependent on the presence of serum, but at least one tyrosine phosphorylation reaction occurs in the absence of cell growth.

Animals↗

PubMed1992

Ionizing radiation stimulates unidentified tyrosine-specific protein kinases in human B-lymphocyte precursors, triggering apoptosis and clonogenic cell death.

Very little is known regarding the effects of ionizing radiation on cytoplasmic signal transduction pathways. Here, we show that ionizing radiation induces enhanced tyrosine phosphorylation of multiple substrates in human B-lymphocyte precursors. This response to ionizing radiation was also observed in cells pretreated with vanadate, a potent protein-tyrosine-phosphatase (PTPase) inhibitor, and phosphotyrosyl [Val5]angiotensin II phosphatase assays showed no decreased PTPase activity in irradiated cells. Thus, enhanced tyrosine phosphorylation in irradiated B-lymphocyte precursors is not triggered by inhibition of total cellular PTPase activity. Immune-complex kinase assays using anti-phosphotyrosine antibodies demonstrated enhanced protein-tyrosine kinase (PTK) activity in the immunoprecipitates from irradiated cells, and the PTK inhibitors genistein and herbimycin effectively prevented radiation-induced tyrosine phosphorylation. Immune-complex kinase assays on irradiated and unirradiated B-lymphocyte precursors using antibodies prepared against unique amino acid sequences of p59fyn, p56/p53lyn, p55blk, and p56lck demonstrated that these Src-family tyrosine kinases were not the primary PTKs responsible for enhanced tyrosine kinase activity in the anti-phosphotyrosine antibody immunoprecipitates or for enhanced tyrosine phosphorylation of multiple substrates. Thus, our findings favor the hypothesis that ionizing radiation induces enhanced tyrosine phosphorylation in B-lymphocyte precursors by stimulation of as yet unidentified PTKs. Tyrosine phosphorylation appears to be an important proximal step in radiation-induced apoptosis and clonogenic cell death because inhibition of PTK prevents DNA fragmentation and loss of clonogenicity of irradiated B-lymphocyte precursors. Since PTKs play myriad roles in the regulation of cell function and proliferation, the activation of a PTK cascade, as detailed in this report, may explain some of the pleiotropic effects of ionizing radiation on cellular functions of B-lymphocytes and their precursors.

Apoptosis↗

PubMed1995

The role of the tyrosine kinase domain of the insulin-like growth factor-I receptor in intracellular signaling, cellular proliferation, and tumorigenesis.

Insulin and insulin-like growth factor (IGF-I) receptors are heterotetrameric proteins consisting of two alpha-and two beta-subunits and members of the transmembrane tyrosine kinase receptors. Specific ligand binding to the receptor triggers a cascade of intracellular events, which begins with autophosphorylation of several tyrosine residues of the beta-subunit of the receptor. The triple cluster in the tyrosine kinase domain of the beta-subunit is the earliest and major autophosphorylation site. Previous studies have shown that substitutions of these three tyrosines by phenylalanines of both insulin and IGF-I receptors practically abolish any activation of cellular signaling pathways. We have studied the effect of double tyrosine mutations on IGF-I induced receptor autophosphorylation, activation of Shc and IRS-1 pathways, and cell proliferation and tumorigenicity. Substitution of tyrosines 1131/1135 blocks any detectable autophosphorylation, whereas substitution of tyrosines 1131/1136 or 1135/1136 only reduces autophosphorylation levels in some clones by approximately 50%. Nevertheless, all the cells expressing IGF-I receptors with double tyrosine substitutions demonstrated markedly reduced signaling through Shc and IRS-1 pathways. In addition, they were unable to respond to IGF-I-stimulated cell growth in culture, and tumor formation in nude mice was abrogated. These data suggest that the presence of tyrosine 1131 or 1135 essential for receptor autophosphorylation, whereas the presence of each of these tyrosines is necessary for a fully functional receptor.

3T3 Cells↗