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PubMed1994

De novo sulfation of L-tyrosine in HepG2 human hepatoma cells and its possible functional implication.

HepG2 human hepatoma cells, labeled with [35S]sulfate in the presence of 10-30 micrograms/ml of cycloheximide, released up to 64% of the amount of free tyrosine-O-[35S]sulfate produced and released by cells labeled in the absence of cycloheximide. A time-course study revealed that, in cells incubated in medium containing [3H]tyrosine, free [3H]tyrosine-O-sulfate was produced within 5 min of incubation, whereas no [3H]tyrosine-sulfated proteins were detected until 20 min after the incubation had begun. Using 3'-phosphoadenosine, 5'-phospho[35S]sulfate as the sulfate donor, HepG2 cell homogenate was shown to contain enzymic activity catalyzing the sulfation of L-tyrosine with the formation of tyrosine-O-[35S]sulfate. Upon subcellular fractionation, the majority of the enzyme activity was found in the cytosolic fraction. The enzyme, designated tyrosine sulfotransferase, displayed the optimum activity at pH 8.0 in the presence of 10 mM Mn2+. Under optimum conditions, the apparent Km of the enzyme for L-tyrosine, at 4.5-microM concentration of 3'-phosphoadenosine, 5'-phosphosulfate, was determined to be 1.95 mM, while that for 3'-phosphoadenosine, 5'-phosphosulfate, at 1 mM L-tyrosine concentration, was 8.3 microM. The Vmax determined under these conditions was 1.05 pmol.min-1.mg protein-1. A tyrosine-dependence study showed that, for cells labeled with [35S]sulfate, the production and release of free tyrosine-O-[35S]sulfate appeared to proceed actively and increase proportionally to the L-tyrosine concentration when it was raised above a threshold level in the culture medium. These results may imply a possible involvement of sulfation in removing excess intracellular L-tyrosine.

Carcinoma, Hepatocellular↗
PubMed1997

Intimin-dependent binding of enteropathogenic Escherichia coli to host cells triggers novel signaling events, including tyrosine phosphorylation of phospholipase C-gamma1.

Enteropathogenic Escherichia coli (EPEC) interactions with HeLa epithelial cells induced the tyrosine phosphorylation of a host protein of approximately 150 kDa, Hp150. Phosphorylation of this protein band was dependent on the interaction of the EPEC protein intimin with epithelial cell surfaces and was correlated with pedestal formation. Hp150 phosphorylation was specifically inhibited by the addition of cytochalasin D, an inhibitor of actin polymerization, although this appeared to be an indirect effect preventing interaction of intimin with its receptor, tyrosine-phosphorylated Hp90, and thus triggering Hp150 phosphorylation. This suggests the involvement of an actin-based movement of membrane-bound tyrosine-phosphorylated Hp90 to allow its interaction with intimin. Analysis of the tyrosine-phosphorylated Hp150 protein demonstrated that it is heterogeneous in composition, with phospholipase C-gamma1 (PLC-gamma1) being a minor component. Activation of PLC-gamma1 by tyrosine phosphorylation leads to inositol triphosphate and Ca2+ fluxes, events detected following EPEC infection. EPEC also induced tyrosine dephosphorylation of host proteins, including a 240-kDa host protein (Hp240), following EPEC infection. Protein dephosphorylation appears to be a signaling event which occurs independently of intimin. Inhibition of host tyrosine dephosphorylation events by the addition of the tyrosine phosphatase inhibitor sodium vanadate did not prevent actin accumulation beneath the adherent bacteria. We conclude that EPEC induces two sets of signaling events following infection. One set is dependent on EPEC proteins secreted by the type III secretion pathway (EspA and EspB) which induces Hp90 tyrosine phosphorylation and dephosphorylation of host phosphotyrosine proteins. The second set, which is also dependent on the first signaling events, requires intimin interaction with its receptor, tyrosine-phosphorylated Hp90, to trigger Hp150 and PLC-gamma1 tyrosine phosphorylation as well as pedestal formation. Inhibition of pedestal formation by tyrosine kinase inhibitors indicates an important role for tyrosine phosphorylation events during EPEC subversion of host processes.

Adhesins, Bacterial↗
PubMed1995

Importance of the content and localization of tyrosine residues for thyroxine formation within the N-terminal part of human thyroglobulin.

Thyroxine (T4) is formed by coupling of iodinated tyrosine residues within thyroglobulin (TG). In mature TG, some iodinated tyrosine residues are involved preferentially in T4 formation. In order to investigate the specific role of various tyrosine residues in T4 formation, N-terminal TG fragments with mutated tyrosine residues were constructed. An N-terminal TG fragment 198 amino acids in size and containing seven tyrosine residues at amino acid positions 5, 29, 89, 97, 107, 130 and 192 was expressed in a baculovirus system. Using site-directed mutagenesis, eight mutant TG fragments were constructed in which different tyrosine residues were replaced by phenylalanine. In the first four TG mutants, one single tyrosine residue (5, 89, 97 or 130) was mutated. In the mutant Y(5,89,97,130)F all of these four tyrosine residues were replaced. The sixth mutant Y(29,89,107,130,192)F contained only tyrosine residues 5 and 97 and the seventh (Y(29,89,97,192)F) contained only tyrosine residues 5, 107 and 130. A TG fragment (Y(5,29,89,97,107,130,192)F) in which all tyrosine residues were replaced by phenylalanine was used as a negative control. After in vitro iodination with lactoperoxidase, specific T4 formation was established in the non-mutated wild-type N-terminal TG fragment. In general the T4 formation in the mutant TG constructs decreased when the total number of tyrosine residues in the 198 amino acid fragment decreased, except fragment Y(29,89,97,192) containing three tyrosine residues, two of them being 5 and 130. Although the rate of T4 formation in this mutated N-terminal TG fragment was lower, the ultimate T4 generation was the same as in the wild-type fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Tyrosine content, influx and accumulation rate, and catecholamine biosynthesis measured in vivo, in the central nervous system and in peripheral organs of the young rat. Influence of neonatal hypo- and hyperthyroidism.

The influence of neonatal hypo- and hyperthyroidism on different aspects of tyrosine metabolism in the hypothalamus, striatum, brainstem, adrenal glands, heart and brown adipose tissue (BAT) were studied in 14-day old rats. The synthesis rate of catecholamines (CA) was also determined in vivo after the injection of labelled tyrosine. Hypothyroidism increases tyrosinaemia and endogenous tyrosine concentration in the hypothalamus and BAT. Hyperthyroidism decreases tyrosinaemia and endogenous tyrosine levels in the striatum, adrenals and heart. The accumulation rate of tyrosine determined 30 min after an intravenous injection of the labelled amino acid has been determined in the organs, together with the influx of the amino acid, determined within 20s. Hypothyroidism increases tyrosine accumulation rate in all the organs studied, and tyrosine clearance is decreased in the striatum and brainstem; together with an increased tyrosinaemia, this leads to a normal influx. The influx of tyrosine is increased in the hypothalamus. Hyperthyroidism decreases tyrosine accumulation rate in all the organs except the adrenals. These results indicate that the thyroid status of the young rat can influence tyrosine uptake mechanisms, without modifying an organ's tyrosine content. The fact that hypothyroidism increases tyrosine influx in the hypothalamus without modifying it in the brainstem and striatum reflects an heterogeneous reactivity to the lack of thyroid hormones in different brain structures. Neonatal hypothyroidism decreases the CA synthesis rate in the striatum, the heart and the interscapular brown adipose tissue, while synthesis was enhanced in the brainstem and the adrenals. It is likely that these variations in CA synthesis are due to thyroid hormone modulation of tyrosine hydroxylase activity, the enzyme which catalyses the rate limiting step in CA biosynthesis.

Adipose Tissue, Brown↗
PubMed1984

Role of histone tyrosines in nucleosome formation and histone-histone interaction.

We have studied the functional properties of iodinated histones. Isolated, denatured histones were iodinated at trace levels and then renatured together with carrier histones and high molecular weight DNA to form nucleohistone. Nucleosomes were prepared from the reconstitute using micrococcal nuclease, and the relative representations of the individual iodinated tyrosines of the histones in the reconstituted nucleosomes were determined. Our principal findings are 1) that denatured histones can be iodinated at any tyrosine without interfering in subsequent nucleosome reconstitution and 2) that the resulting reconstituted nucleosomes nevertheless possess histone cores of altered stability, being either more or less stable depending on the particular tyrosine which is iodinated. We show that tyrosines 37, 40, and 42 of H2B are protected from iodination in intact core particles, as expected since these tyrosines lie within the H2B-H2A binding site. Yet iodination of these tyrosines in denatured H2B does not interfere with nucleosome assembly. However, the histone cores isolated from these reconstituted nucleosomes are of diminished stability as assayed by Sephadex column chromatography in 2 M salt. In contrast, iodination of tyrosines 83 and 121 of H2B, as well as iodination of the tyrosines of H2A, increases the stability of the histone octamer core. Iodination of H4 tyrosine 72 is without effect on histone octamer stability. Tyrosine iodination constitutes a profound amino acid alteration in the context of the absolute evolutionary conservation of most histone tyrosines. For example, all H2Bs sequenced to date, from fungi to mammals, possess tyrosines at positions 37, 40, and 42. Our results suggest that the immutability of these tyrosines reflects some sophisticated function of the nucleosome histone core beyond the assembly and mere maintenance of a compact structure.

Animals↗
PubMed1996

Phosphorylation of caveolin by src tyrosine kinases. The alpha-isoform of caveolin is selectively phosphorylated by v-Src in vivo.

Caveolae are flask-shaped plasma membrane specializations that are thought to exist in most cell types. A 22-kDa protein, caveolin, is an integral membrane component of caveolae membranes in vivo. Previous studies have demonstrated that caveolin is phosphorylated on tyrosine by oncogenic viral Src (v-Src) and that caveolin is physically associated as a hetero-oligomeric complex with normal cellular Src (c-Src) and other Src family tyrosine kinases. Caveolin contains eight conserved tyrosine residues that may serve as potential substrates for Src. Here, we have begun to study the phosphorylation of caveolin by Src family tyrosine kinases both in vitro and in vivo. Using purified recombinant components, we first reconstituted the phosphorylation of caveolin by Src kinase in vitro. Microsequencing of Src-phosphorylated caveolin revealed that phosphorylation occurs within the extreme N-terminal region of full-length caveolin between residues 6 and 26. This region contains three tyrosine residues at positions 6, 14, and 25. Deletion mutagenesis demonstrates that caveolin residues 1-21 are sufficient to support this phosphorylation event, implicating tyrosine 6 and/or 14. In vitro phosphorylation of caveolin-derived synthetic peptides and site-directed mutagenesis directly show that tyrosine 14 is the principal substrate for Src kinase. In support of these observations, tyrosine 14 is the only tyrosine residue within caveolin that bears any resemblance to the known recognition motifs for Src family tyrosine kinases. In order to confirm or refute the relevance of these in vitro studies, we next analyzed the tyrosine phosphorylation of endogenous caveolin in v-Src transformed NIH 3T3 cells. In vivo, two isoforms of caveolin are known to exist: alpha-caveolin contains residues 1-178 and beta-caveolin contains residues 32-178. Only alpha-caveolin underwent tyrosine phosphorylation in v-Src transformed NIH 3T3 cells, although beta-caveolin is well expressed in these cells. As beta-caveolin lacks residues 1-31 (and therefore tyrosine 14), these in vivo studies directly demonstrate the validity of our in vitro studies. Because alpha- and beta-caveolin are known to assume a distinct but overlapping subcellular distribution within a single cell, v-Src phosphorylation of alpha-caveolin may only affect a subpopulation of caveolae that contain alpha-caveolin.

3T3 Cells↗
PubMed1999

Possible regulation of epidermal growth factor-receptor tyrosine autophosphorylation by calcium and G proteins in chemically permeabilized rat UMR106 cells.

A model using chemically permeabilized cells was developed to examine mechanisms that regulate protein tyrosine phosphorylation in osteoblastic cells. Using either permeabilized UMR106 osteoblastic or A431 (reference) cells, epidermal growth factor (EGF)-induced cellular tyrosine phosphorylation, and whether there are previously unrecognized interactions between this transduction pathway and Ca2+- or G protein-dependent signalling pathways, were investigated. Both permeabilized cell types, when maintained in non-supplemented cytoplasmic substitution solution (basic CSS), responded to EGF (1-100 ng/ml) with dose-dependent increases in tyrosine phosphorylation. A complex and time-dependent pattern of phosphotyrosine-containing proteins resulted, but the profile of tyrosine phosphorylated proteins was appreciably less complex than in intact cells. Supplementation of basic CSS with MgATP restored the normal complexity of the profiles for EGF-induced tyrosine phosphorylation proteins in both permeabilized cell lines and produced a more sustained accumulation of phosphoprotein products in A431 cells. Adding Ca2+ (< or = 10(-6) M), with or without exogenous MgATP, dose-dependently attenuated EGF-induced tyrosine phosphorylation of EGF receptors (EGFR) and other substrates in UMR106 cells, but was less effective in A431 cells. In both cell types, genistein, an inhibitor of tyrosine kinases, was more effective in attenuating EGF-induced receptor tyrosine phosphorylation in permeabilized cells. Similarly, orthovanadate, an inhibitor of protein tyrosine phosphatases, stimulated the accumulation of phosphoprotein products more effectively in permeabilized cells. Thus, the permeabilization preserves many features of intact cells while facilitating manipulation of intracellular conditions. NaF reproducibly produced a significant vanadate-like action in permeabilized cells that was somewhat stronger than its effect on intact cells. In contrast, the well-known inhibition of tyrosine phosphorylation by phorbol 12-myristate 13-acetate (PMA) was less effective in permeabilized cells than in intact cells; these actions of PMA were Ca2+-dependent. In addition, guanylyl-imidodiphosphate (Gpp(NH)p) attenuated tyrosine phosphorylation in UMR106 cells, and this effect was specifically blocked by guanosine 5'-O-(2-thiodiphosphate) (GDPbetas). These results strongly suggest that there is crosstalk between EGFR-activated tyrosine phosphorylation/dephosphorylation pathways and both Ca2+- and G protein-mediated pathways in UMR106 cells, revealing a previously unrecognized modulation of EGF signalling in osteoblast-like cells that contrasts with the simpler regulatory mechanisms found in A431 cells.

Adenosine Triphosphate↗
PubMed1998

Are tyrosine phosphorylation of p125(FAK) and paxillin or the small GTP binding protein, rho, needed for CCK-stimulated pancreatic amylase secretion?

Studies of a possible role of tyrosine phosphorylation in the secretory process in rat pancreatic acinar cells provide conflicting conclusions. Recent studies show that tyrosine phosphorylation of the focal adhesion kinase, p125FAK and the cytoskeletal protein, paxillin, may mediate a number of cellular changes and this phosphorylation is dependent on the activation of the small GTP binding protein, p21Rho (Rho). In this work we have investigated the role of tyrosine phosphorylation of each of these proteins and of the activation of Rho in pancreatic enzyme secretion. Pretreatment with genistein, a tyrosine kinase inhibitor, decreased CCK-8-stimulated tyrosine phosphorylation of p125FAK and paxillin and CCK-8-stimulated amylase secretion by more than 60%, raising the possibility that tyrosine phosphorylation of these two proteins could be important in the ability of CCK-8 to stimulate amylase release. However, genistein did not alter the amylase release stimulated by TPA but inhibited TPA-stimulated p125FAK and paxillin tyrosine phosphorylation by 70%. Pretreatment with C3 transferase, which specifically inactivates Rho, causes a decrease in CCK-8-induced maximal amylase release by 33%. Moreover, C3 transferase pretreatment causes a 48% and a 38% decrease in the tyrosine phosphorylation of p125FAK and paxillin by CCK-8, respectively. Pretreatment with different concentrations of cytochalasin D, an actin cytoskeleton assembly inhibitor, completely inhibited CCK-8-stimulated tyrosine phosphorylation of p125FAK and paxillin without having any effect on either the potency or efficacy of CCK-8 at stimulating amylase release. Furthermore, cytochalasin D completely inhibited TPA-stimulated tyrosine phosphorylation of both proteins without affecting TPA-stimulated amylase release. These results show that tyrosine phosphorylation of p125FAK and paxillin is not required for CCK-8 stimulation of enzyme secretion. However, our results suggest Rho is involved in the CCK-8 stimulation of amylase release by a parallel pathway to its involvement in the CCK-8-stimulated tyrosine phosphorylation of p125FAK and paxillin.

ADP Ribose Transferases↗
PubMed1999

Clustering of integrin alphaIIb-beta3 differently regulates tyrosine phosphorylation of pp72syk, PLCgamma2 and pp125FAK in concanavalin A-stimulated platelets.

Tyrosine phosphorylation of the non-receptor tyrosine kinases pp72syk and pp125FAK and of the gamma2 isoform of phospholipase C (PLCgamma2) in human platelets stimulated with the lectin Concanavalin A was investigated. Concanavalin A induced the rapid tyrosine phosphorylation of pp72syk and PLCgamma2 with a similar kinetics, while tyrosine phosphorylation of pp125FAK occurred in a later phase of platelet activation. When compared with other platelet agonists, Concanavalin A revealed to be at least as potent as collagen in inducing tyrosine phosphorylation of PLCgamma2 and pp125FAK, while tyrosine phosphorylation of pp72syk induced by the lectin was much stronger than that induced by thrombin or collagen. Concanavalin A-induced tyrosine phosphorylation of pp72syk, PLCgamma2 and pp125FAK was not dependent on platelet aggregation as it occurred normally even in the absence of sample stirring and when fibrinogen binding to integrin alphaIIb-beta3 was inhibited by the peptide RGDS. Tyrosine phosphorylation of pp72syk, PLCgamma2 and pp125FAK required the binding of the lectin to the platelet surface, but was not observed in platelets treated with succinyl-Concanavalin A, a derivative of the lectin that interacts with the same receptors but does not promote clustering of membrane glycoproteins. Moreover, the aggregation-independent tyrosine phosphorylation of pp125FAK and pp72syk induced by Concanavalin A required the expression of integrin alphaIIb-beta3 on the platelet surface as it was strongly inhibited in platelets from patients affected by Glanzmann thrombasthenia. By contrast, tyrosine phosphorylation of PLCalpha2 occurred normally also in thrombasthenic platelets stimulated with Concanavalin A. These results demonstrate that, even in the absence of aggregation, the clustering of integrin alphaIIb-beta3 induced by Concanavalin A on the platelet surface directly promotes tyrosine phosphorylation of pp72syk and pp125FAK and provide further evidence that the oligomerization of the fibrinogen receptor promoted by its natural ligand during platelet aggregation may be responsible for the tyrosine phosphorylation of these proteins induced by physiological agonists.

Blood Platelets↗
PubMed2000

Mechanisms of regulation of tyrosine phosphorylation of NMDA receptor subunit 2B after cerebral ischemia/reperfusion.

AIM: To study the mechanisms of the regulation of the tyrosine phosphorylation of N-methyl-D-aspartate (NMDA) receptor subunit 2B(NR2B) in the gerbil hippocampal synaptosomes following ischemia/reperfusion (I/R). METHODS: Transient (15 min) cerebral ischemia was produced by bilateral carotid artery occlusion procedure. The tyrosine phosphorylation of NR2B was analyzed by immunoprecipitation and immunoblot assay. RESULTS: Transient forebrain ischemia for 15 min caused a marked decrease in the levels of tyrosine phosphorylation of many protein bands including 180 kDa protein. Transient ischemia followed by reperfusion induced rapid (within 15 min of reperfusion), and sustained (for at least 48 h) increase in the tyrosine phosphorylation of many protein bands including 180 kDa protein. Immunoprecipitation and immunoblot confirmed that NR2B is among the phosphorylated 180 kDa protein. Maximal phosphorylation of 180 kDa band corresponding to NR2B (1.8 fold relative to sham-operated controls) was reached at 6 h of reperfusion following 15 min of cerebral ischemia. But the level of protein expression of NR2B did not change. Administration of ketamine (KT), a non-competitive NMDA receptor antagonist, or nifedipine (ND), an L-type voltage gated calcium channel (L-type VGCC) blocker, 20 min before ischemia attenuated stimulation of the tyrosine phosphorylation of NR2B without affecting the level of protein expression of NR2B. Under these conditions, non-NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) had no effect on the level of tyrosine phosphorylation. Protein tyrosine phosphatase (PTP) inhibitor vanadate and protein tyrosine kinase (PTK) inhibitor genestein resulted in the increase and the decrease of the tyrosine phosphorylation of NR2B, respectively. Src coprecipitated with NR2B protein. CONCLUSION: The increase of the tyrosine phosphorylation of NR2B induced by I/R has relation to NR and L-type VGCC; PTK and PTP participate in the regulation of the tyrosine phosphorylation of NR2B during I/R. Src that associates with NR2B may play an important role in the regulation of the tyrosine phosphorylation of NR2B during I/R.

Animals↗
PubMed1990

Protein tyrosine phosphorylation and p56lck modification in IL-2 or phorbol ester-activated human natural killer cells.

Protein tyrosine kinases play fundamental roles in the transduction of signals that regulate cell growth, differentiation, and functional responses to a diversity of external stimuli. It is therefore likely that understanding protein tyrosine kinase activity in NK cells will be crucial in further defining the intracellular regulation of their unique and specialized functions. We investigated the role of protein tyrosine phosphorylation in receptor-mediated signal transduction using stimuli known to play major roles in regulating NK cell activation. Immunoblot analyses with antiphosphotyrosine antibodies demonstrated that IL-2, a potent stimulus for NK cell proliferation and an agent that enhances NK cytotoxic function, induced the tyrosine phosphorylation of at least eight proteins in clonal CD16+/CD3-human NK cells. In contrast, IL-4, which modulates NK cell function without inducing proliferation, had no apparent effect on protein tyrosine phosphorylation. Because protein kinase C (PKC) activation plays a prominent, yet distinct role in NK cell-mediated cytolytic reactions, we next investigated whether PKC activation affects NK cell protein tyrosine phosphorylation. Surprisingly, PKC-activating agents, including the phorbol esters 12-O-tetradecanoylphorbol-13-acetate and 4 beta-phorbol 12, 13-didecanoate, as well as the synthetic diacylglycerol,1-oleoyl-2-acetylglycerol, also induced the tyrosine phosphorylation of a distinct set of proteins. The 4 beta-phorbol 12,13-didecanoate homolog, 4 alpha-phorbol 12,13-didecanoate, which does not activate PKC, also failed to induce protein tyrosine phosphorylation. Further, the PKC inhibitor, 1-O-hexadecyl-2-O-methylglycerol blocked tyrosine phosphorylation induced by 1-oleoyl-2-acetylglycerol. In subsequent studies, both CD8+ and CD8- NK clones were found to express the src-family tyrosine kinase, p56lck, which was detected by immunoblot analysis with anti-p56lck antiserum. In both types of clonal NK cell lines, IL-2 and 12-O-tetradecanoyl-phorbol appeared to stimulate the differential phosphorylation of p56lck as evidenced by the appearance of higher molecular mass isoforms on SDS-polyacrylamide gels. Thus, our results identify and characterize a potential role for tyrosine phosphorylation and for the lymphocyte-specific tyrosine kinase p56lck in the signaling events that regulate NK cell activation.

Antigens, Differentiation↗
PubMed1993

Inhibitors of tyrosine phosphorylation induce apoptosis in human leukemic cell lines.

Experimental evidence suggests that hematopoietic growth factors promote cell survival by suppressing apoptosis or programmed cell death. Since interleukin 3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) induce tyrosine phosphorylation of a common set of proteins in the factor-dependent cell line M07e, we have investigated whether growth-factor-induced tyrosine phosphorylation is involved in the promotion of cell survival and suppression of apoptosis. Experiments were carried out with the leukemic cell lines HL-60 and M07e and the tyrosine kinase inhibitors genistein and tyrphostin AG82. Both the tyrosine kinase inhibitors induced apoptosis of HL-60 and M07e cells. This was indicated by the appearance of DNA degradation and morphologic evidence of nuclear condensation and fragmentation. It was also confirmed by flow cytometry of DNA, which showed apoptotic cells as a fraction of cells characterized by a diminished DNA stainability, represented on the DNA frequency histograms as a distinct peak below the G0/G1 population. Kinase inhibitors also reduced the fraction of cells in the S phase of the cell cycle. That tyrphostin specifically inhibited tyrosine kinases was further suggested by the prevention of its effects by the tyrosine phosphatase inhibitor sodium orthovanadate (vanadate), at least during the first 18-24 h of treatment. The incomplete prevention of genistein effects by vanadate suggests that genistein is a less specific inhibitor of tyrosine kinases than tyrphostin, and may also act as an inhibitor of topoisomerase II. Vanadate also prevented apoptosis and reduction of the S phase in M07e cells cultured for 24 h in the absence of growth factors. These results suggest that tyrosine phosphorylation is an essential step in IL-3 and GM-CSF signal transduction. Since in our experimental model the effects of tyrosine kinase inhibition and growth factor deprivation could be reversed by concomitant inhibition of tyrosine phosphatases, it is suggested that a balance between tyrosine kinases and tyrosine phosphatases establishes whether a cell will survive or undergo apoptosis.

Apoptosis↗
PubMed1997

Protein tyrosine phosphatase activity modulation by endothelin-1 in rabbit platelets.

Protein tyrosine phosphorylation, modulated by the rate of both protein tyrosine kinase and protein tyrosine phosphatase activities, is critical for cellular signal transduction cascades. We report that endothelin-1 stimulation of rabbit platelets resulted in a dose- and time-dependent tyrosine phosphorylation of four groups of proteins in the molecular mass ranges of 50, 60, 70-100 and 100-200 kDa and that one of these corresponds to focal adhesion kinase. This effect is also related to the approximately 60% decrease in protein tyrosine phosphatase activity. Moreover, this inhibited activity was less sensitive to orthovanadate. In the presence of forskolin that increases the cAMP level a dose-dependent inhibition of the endothelin-stimulated tyrosine phosphorylation of different protein substrates and a correlation with an increase in the protein tyrosine phosphatase activity (11.6-fold compared to control) have been found. Further studies by immunoblotting of immunoprecipitated soluble fraction with anti-protein tyrosine phosphatase-1C from endothelin-stimulated platelets have demonstrated that the tyrosine phosphorylation of platelet protein tyrosine phosphatase-1C is correlated with the decrease in its phosphatase activity. As a consequence, modulation and regulation by endothelin-1 in rabbit platelets can be proposed through a cAMP-dependent pathway and a tyrosine phosphorylation process that may affect some relevant proteins such as focal adhesion kinase.

Animals↗
PubMed1997

Signaling functions of the tyrosine residues in the betac chain of the granulocyte-macrophage colony-stimulating factor receptor.

The granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) is a heterodimeric receptor expressed by myeloid lineage cells. Binding of GM-CSF activates at least one receptor-associated tyrosine kinase, JAK2, and rapidly induces tyrosine phosphorylation of the GMR betac-chain (GMRbeta), but not the GMR alpha-chain (GMRalpha). To examine the role of GMRbeta tyrosine phosphorylaiton, each of the 8 tyrosine residues in the cytoplasmic domain of the human GMRbeta was mutated to phenylalanine (GMRbeta-F8), and this mutant receptor was expressed with wild-type GMRalpha in the interleukin-3-dependent murine hematopoietic cell line, Ba/F3. GM-CSF induced tyrosine phosphorylation of multiple cellular proteins in cells expressing GMRbeta-F8 , including JAK2 and STAT5. However, GM-CSF-induced tyrosine phosphorylation of both SHP2 and SHC was reduced or absent compared with wild-type. Next, a series of 8 receptors were generated, each containing only a single, restored, tyrosine residue. Tyrosine 577 was found to be sufficient to regenerate GM-CSF-dependent phosphorylation of SHC, and any of Y577, Y612, or Y695 was sufficient to regenerate GM-CSF-inducible phosphorylation of SHP2. Despite the signaling defect to SHC and SHP2, Ba/F3 cells expressing GMRbeta-F8 were still able to proliferate in response to 10 ng/mL of human GM-CSF, although mitogenesis was impaired compared with wild-type GMRbeta, and this effect was even more prominent at lower concentrations of GM-CSF (1 ng/mL). Overall, these results indicate that GMRbeta tyrosine residues are not necessary for activation of the JAK/STAT pathway or for proliferation, viability, or adhesion signaling in Ba/F3 cells, although tyrosine residues significantly affect the magnitude of the response. However, specific tyrosine residues are needed for activation of SHC and SHP2.

Animals↗
PubMed1997

Neuromedin B receptor activation causes tyrosine phosphorylation of p125FAK by a phospholipase C independent mechanism which requires p21rho and integrity of the actin cytoskeleton.

Recent studies show that tyrosine phosphorylation by a number of neuropeptides may be an important intracellular pathway in mediating changes in cell function, particularly related to growth. Neuromedin B (NMB), a mammalian bombesin related peptide, functions through a distinct receptor, the neuromedin B receptor (NMB-R), of which little is known about its cellular basis of action. In the present study we explored the ability of NMB-R activation to cause tyrosine phosphorylation of focal adhesion kinase (p125(FAK)), an important substrate for tyrosine phosphorylation by other neuropeptides. NMB caused rapid increases in p125(FAK) phosphorylation which reached maximum at 2 min in both rat C6 glioblastoma cells which possess native NMB-Rs and rat neuromedin B receptor (rNMR-R) transfected BALB 3T3 cells. NMB had a half-maximal effect was at 0.4 nM and was 30-fold more potent than gastrin-releasing peptide (GRP). The stoichiometric relationships between increased p125(FAK) tyrosine phosphorylation and other cellular processes was similar in both C6 cells and rNMB-R transfected cells. TPA (1 microM) caused 45% and the calcium ionophore, A23187, 11% of maximal tyrosine phosphorylation of p125(FAK) seen with NMB. A23187 potentiated the effect of TPA. Pretreatment with the selective PKC inhibitor, GF109203X, inhibited TPA-induced p125(FAK) tyrosine phosphorylation, but it had no effect on the NMB stimulation. Pretreatment with thapsigargin completely inhibited NMB-stimulated increases in [Ca2+]i, but had no effect on NMB-stimulation of p125(FAK) phosphorylation either alone or with GF109203X. The tyrosine kinase inhibitor, tyrphostin A25, inhibited NMB-induced phosphorylation of p125(FAK) by 52%. However, tyrphostin A25 did not inhibit NMB-stimulated increases in [3H]inositol phosphates. Cytochalasin D, an agent which disrupts actin microfilaments, inhibited BN- and TPA-induced tyrosine phosphorylation of p125(FAK) completely. In contrast, colchicine, an agent which disrupts microtubules, had no effect. Pretreatment with Clostridium botulinum C3 exoenzyme which inactivates the small GTP-binding protein rho p21, also inhibited tyrosine phosphorylation of p125(FAK) by 55%. These results demonstrate that activation of NMB-R can cause rapid tyrosine phosphorylation of p125(FAK). NMB-induced tyrosine phosphorylation of p125(FAK) is independent of NMB-induced changes in [Ca2+]i or PKC. The integrity of the actin cytoskeleton but not of microtubules is necessary for NMB-stimulated phosphorylation of p125(FAK). The ras-related small GTP-binding protein rho p21 is at least partially involved in mediating NMB-induced tyrosine phosphorylation of p125(FAK). These results suggest that similar to some other neuropeptides, activation of this pathway may be an important mechanism in mediating cellular changes by this receptor such as growth.

ADP Ribose Transferases↗
PubMed1995

Sequence requirements for binding of Src family tyrosine kinases to activated growth factor receptors.

Activation of growth factor receptor protein tyrosine kinases frequently results in the binding of numerous proteins to their tyrosine-phosphorylated cytoplasmic domains. These interactions involve the SH2 domains of the binding proteins and phosphorylated tyrosines on the receptor molecules, with the specificity of interaction dictated by the amino acid composition surrounding the phosphorylated tyrosine. In the case of the platelet-derived growth factor (PDGF) receptor, the major binding site for Src family tyrosine kinases is in the juxtamembrane domain and includes tyrosine 579 (Mori, S., Rönnstrand, L., Yokote, K., Engström, A., Courtneidge, S. A., Claesson-Welsh, L., and Heldin, C-H. (1993) EMBO J. 12, 2257-2264). To analyze in more detail which amino acids surrounding the phosphorylated tyrosine at position 579 were important for high affinity interaction with Src family kinases, we synthesized a series of phosphopeptides corresponding to this binding site in which single amino acids were individually changed and tested their ability to compete with the PDGF receptor for binding of Fyn. We found that not only the three residues carboxyl-terminal to the phosphorylated tyrosine were important but that also residues at positions -1 and +4 relative to the tyrosine were required. Phosphorylation of both tyrosines 579 and 581 significantly increased competition efficiency. The activated colony stimulating factor-1 (CSF-1) receptor, which is known to associate with Src family kinases, has a sequence in its juxtamembrane region similar to that surrounding Tyr-579 of the PDGF receptor, and a phosphopeptide modeled on this sequence competed the association of Fyn with the receptor in vitro. Furthermore, mutational analysis demonstrated that these sequences were required for the efficient association of Src family kinases with the activated CSF-1 receptor in vivo. Phosphopeptides corresponding to the Src family binding sites of both PDGF and CSF-1 receptors activated Src kinase activity in vitro. These observations support a model in which the enzymatic activity of Src family tyrosine kinases is controlled by intra- and intermolecular interactions of tyrosine-phosphorylated peptides with the SH2 domain of the kinases.

3T3 Cells↗
PubMed2004

The receptor tyrosine kinase Ror2 associates with and is activated by casein kinase Iepsilon.

Ror2, a member of the mammalian Ror family of receptor tyrosine kinases, plays important roles in developmental morphogenesis, although the mechanism underlying activation of Ror2 remains largely elusive. We show that when expressed in mammalian cells, Ror2 associates with casein kinase Iepsilon (CKIepsilon), a crucial regulator of Wnt signaling. This association occurs primarily via the cytoplasmic C-terminal proline-rich domain of Ror2. We also show that Ror2 is phosphorylated by CKIepsilon on serine/threonine residues, in its C-terminal serine/threonine-rich 2 domain, resulting in autophosphorylation of Ror2 on tyrosine residues. Furthermore, it was found that association of Ror2 with CKIepsilon is required for its serine/threonine phosphorylation by CKIepsilon. Site-directed mutagenesis of tyrosine residues in Ror2 reveals that the sites of phosphorylation are contained among the five tyrosine residues in the proline-rich domain but not among the four tyrosine residues in the tyrosine kinase domain. Moreover, we show that in mammalian cells, CKIepsilon-mediated phosphorylation of Ror2 on serine/threonine and tyrosine residues is followed by the tyrosine phosphorylation of G protein-coupled receptor kinase 2, a kinase with a developmental expression pattern that is remarkably similar to that of Ror2. Intriguingly, a mutant of Ror2 lacking five tyrosine residues, including the autophosphorylation sites, fails to tyrosine phosphorylate G protein-coupled receptor kinase 2. This indicates that autophosphorylation of Ror2 is required for full activation of its tyrosine kinase activity. These findings demonstrate a novel role for CKIepsilon in the regulation of Ror2 tyrosine kinase.

3T3 Cells↗
PubMed1997

Fc epsilon RI-ligation induces association of tyrosine phosphorylated proteins with Src homology 2 domains of phospholipase C gamma 1 in RBL-2H3 rat basophilic leukemia cells.

Stimulation of the IgE receptors on mast cells and basophils activates protein tyrosine kinases and phospholipases leading to histamine release. However, the mechanism by which protein tyrosine kinases regulate the phospholipases is not clearly defined yet. In this study, we examined the possibility that phospholipase C gamma 1 associates with protein tyrosine kinases and tyrosine phosphorylated molecules as a result of activation of RBL-2H3 cells, and that this association involves the Src homology 2 domains of phospholipase C gamma 1. An increase in cytoplasmic Ca2+ level and tyrosine phosphorylations of proteins, including 72 and 40 kDa proteins, were observed after the cross-linking of the IgE receptors on RBL-2H3 rat basophilic cells by dinitrophenyl-specific IgE and dinitrophenyl-conjugated human serum albumin. Immunoprecipitation and coprecipitation experiments were performed to determine if the activation of protein tyrosine kinases is linked to the activation of phospholipase C gamma 1 via its SH2 domains. Tyrosine phosphorylation of phospholipase C gamma 1 was observed in 1 min following IgE receptor stimulation. several proteins (72, 50, 40, and 33 kDa) were identified to be tyrosine phosphorylated and specifically associated with phospholipase C gamma 1 by its Src homology 2 domains. In addition, the coprecipitated complex contains the tyrosine kinase activity which phosphorylates 72, 40, and 33 kDa proteins in the complex. In conclusion, these studies establish that tyrosine-phosphorylated proteins of 72, 40, and 33 kDa associate with phospholipase C gamma 1 via its SH2 domains following IgE receptor stimulation of RBL-2H3 basophilic cells, implying that protein tyrosine kinases may tyrosine-phosphorylate and recruit signaling proteins around the phospholipase C gamma 1 and that phospholipase C gamma 1 activation induces calcium mobilization, PKC activation and degranulation in mast cells or basophils.

Animals↗