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PubMed1996

Bradykinin-stimulated protein tyrosine phosphorylation promotes endothelial nitric oxide synthase translocation to the cytoskeleton.

Stimulation of bovine aortic endothelial cells (BAEC) with bradykinin produces cycles of tyrosine phosphorylation/dephosphorylation of a 90 kDa endothelial nitric oxide synthase (eNOS)-associated protein which we have termed ENAP-1 (for endothelial nitric oxide synthase-associated protein 1). ENAP-1 interacts specifically and tightly with eNOS in BAEC and is co-immunoprecipitated from cell lysates with anti-eNOS antibodies. In addition, anti-phosphotyrosine antibodies co-precipitate eNOS. Bradykinin-stimulated tyrosine phosphorylation of ENAP-1 is blocked by the tyrosine kinase inhibitor, tyrphostin. Dephosphorylation is blocked by the tyrosine phosphatase inhibitor, orthovanadate. Treatment of BAEC with bradykinin or the tyrosine phosphatase inhibitor, phenylarsine oxide promotes tyrosine phosphorylation of detergent-insoluble, cytoskeletal proteins accompanied by translocation of eNOS to the cytoskeletal subcellular compartment. Translocation is blocked by the tyrosine kinase inhibitor, geldanamycin and does not appear to alter enzyme catalytic activity. Tyrosine phosphorylation-dependent association of eNOS with the cytoskeleton may have a role in targeting NO production to specific subcellular locations.

Animals↗
PubMed1999

Activation of three classes of nonreceptor tyrosine kinases following Fc gamma receptor crosslinking in human monocytes.

Fc gamma receptors on monocytes/macrophages play an important role in both host defense and autoimmune disorders. Fc gamma receptor signaling can lead to such downstream events as phagocytosis and the release of intracellular cytokines and reactive oxygen species. Freshly isolated human monocytes express two major classes of Fc gamma receptor proteins, Fc gamma RI (CD64) and Fc gamma RII (CD32). Crosslinking of Fc gamma RI and Fc gamma RII gives rise to rapid and transient phosphorylation of multiple monocyte intracellular proteins including proteins of 40, 68-72, 75-85, 95, and 115-165 kDa. A 72-kDa protein was earlier identified as the tyrosine kinase Syk. Here we identify one of the proteins in the 115- to 165-kDa cluster as FAK, a protein tyrosine kinase localized to focal adhesions. A 68-kDa phosphoprotein was identified as paxillin, a cytoskeleton associated substrate for tyrosine kinases, and a 95-kDa protein was found to be the proto-oncogene product Vav. The Src family protein tyrosine kinase Fgr (p58) also displayed enhanced tyrosine phosphorylation after Fc gamma RI and Fc gamma RII crosslinking. Although Fc gamma RIIA utilizes tyrosines within its own cytoplasmic domain for signaling while Fc gamma RI utilizes the cytoplasmic tyrosines of its associated gamma subunit, our results indicate sharing of several proteins for signaling in monocytes by these Fc receptors. These molecules include three distinct classes of tyrosine kinases, Syk, FAK, and Fgr, and the functionally diverse proteins Vav and paxillin.

Cell Adhesion Molecules↗
PubMed1992

Tyrosine phosphorylation and its possible role in superoxide production by human neutrophils stimulated with FMLP and IgG.

Superoxide production by human neutrophils stimulated with FMLP and soluble aggregated human IgG were inhibited in a dose dependent manner by two kinds of tyrosine kinase inhibitors, erbstatin and genistein. Superoxide production stimulated with surface bound IgG, however, was scarcely inhibited by either inhibitor. Protein tyrosine phosphorylation studies with immunoblotting revealed specific tyrosine phosphorylation of a 40 Kd protein by soluble aggregated and surface bound IgG, and that of a 39 Kd protein, as well as the 40 Kd protein, by FMLP. These were all inhibited by the tyrosine kinase inhibitors. These data suggest that superoxide production induced by FMLP and soluble aggregated IgG are, at least in part, tyrosine kinase dependent, but the tyrosine kinases and/or substrates of tyrosine kinases involved may be different. In addition, tyrosine kinase independent pathways are also suggested to be involved in superoxide production by stimulation with surface bound IgG.

Humans↗
PubMed1988

Diurnal alterations in retinal tyrosine level and dopamine turnover in diabetic rats.

Retinal tyrosine level is correlated not only to its serum concentration, but also to the serum tyrosine ratio (the ratio of serum tyrosine to the sum of 5 neutral amino acids, leucine, isoleucine, valine, tryptophan and phenylalanine which compete with it for tissue uptake). In control animals, serum tyrosine ratio, retinal tyrosine, dopamine (DA) and its metabolites, and DOPA synthesis exhibit roughly parallel daily rhythms with night-time lows and peak levels occurring in the early portion of the daily light phase (L + 4 HR). In rats made diabetic by streptozotocin injection serum leucine, isoleucine and valine are elevated, lowering the serum tyrosine ratio and retinal tyrosine level by about 50% throughout the daily cycle. Parameters of DA turnover are still highest in the light phase but normal peak levels are delayed by 4 h until the latter half of the light phase (L + 8 HR). In diabetic rats, the normal increase in retinal DA synthesis and release upon exposure to light may be compromised by the decreased availability of its precursor, tyrosine.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1983

Regulation of hepatic tyrosine aminotransferase in genetically obese rats.

The activities of hepatic tyrosine aminotransferase, tryptophan oxygenase and serine dehydratase were increased in obese rats shortly after weaning. Immunotitration experiments showed that the increase in tyrosine aminotransferase activity resulted from an increase in enzyme protein in obese rats. No increase in hepatic tyrosine aminotransferase was observed in suckling pre-obese rats. The post-weaning increase in hepatic tyrosine aminotransferase of obese rats was only observed during the light phase of the diurnal cycle, but was prevented by pair-feeding and by starvation. Tryptophan increased hepatic tyrosine aminotransferase of lean rats to obese levels but had no effect in obese rats until tyrosine aminotransferase levels were reduced by starvation or adrenalectomy. Adrenalectomy abolished the increase in hepatic tyrosine aminotransferase activity in obese rats although serum corticosterone was normal in these animals. Hepatic and brain tyrosine concentrations were decreased in obese rats but normalized after adrenalectomy. The results suggest that the corticosteroid-dependent increase in food and tryptophan intake may be the primary cause of the increased hepatic amino acid catabolism of obese rats.

Adrenalectomy↗
PubMed2000

Tyrosine phosphorylation of the delta-opioid receptor. Evidence for its role in mitogen-activated protein kinase activation and receptor internalization*.

The internalization of G-protein-coupled receptors (GPCRs), including the delta opioid receptor (delta-OR), has been shown to involve the phosphorylation of serine and threonine residues. However, recent studies suggest that these residues may not be the only ones phosphorylated in response to prolonged opioid exposure. Tyrosines also appear important for delta-OR signalling, but it remains unclear whether they undergo phosphorylation. We examined whether the delta-OR, stably expressed in Chinese hamster ovary (CHO-K1) cells, was tyrosine-phosphorylated during prolonged agonist treatment. The epitope-tagged delta-OR was purified by immunoprecipitation, and the presence of phosphorylated tyrosines was detected using anti-phosphotyrosine antibodies. Tyrosine residues in the delta-OR were phosphorylated after exposure to the high-affinity agonist [d-Thr(2)]-Leu-enkephalin-Thr (DTLET) in a time- and concentration-dependent manner. Tyrosine phosphorylation of the delta-OR appeared to require the actions of a Src-like protein tyrosine kinase, since the Src inhibitor 4-amino-5-(4-methylphenyl)-7-(t-butyl)-pyrazolo-[3,4-d]-pyrimidine (PP1) attenuated this response. PP1 also attenuated the DTLET-mediated activation of mitogen-activated protein kinase, as well as rapid delta-OR internalization, but not receptor down-regulation. Finally, only opioid agonists that induce receptor internalization via the clathrin-dependent endosomal pathway stimulated significant tyrosine phosphorylation of the delta-OR protein. Evidence is presented that the delta-OR is tyrosine-phosphorylated, and we suggest how this may have an active role in opioid receptor signalling and regulation.

Animals↗
PubMed2001

Inhibition of tyrosine kinase activity induces caspase-dependent apoptosis in anaplastic large cell lymphoma with NPM-ALK (p80) fusion protein.

OBJECTIVE: The t(2;5)(p23;q35) translocation creates a fusion gene NPM-ALK (p80) that encodes a product with tyrosine kinase activity believed to play an important role in development of anaplastic large cell lymphoma (ALCL). Our study was aimed to analyze tyrosine kinase activity and phosphotyrosine in ALCLs. We were also interested in determining the effect of tyrosine kinase inhibitors on survival of ALCL. METHODS: Eleven cases of ALCL and three ALCL cell lines with t(2;5)(Karpas-299, SUPM2, SU-DHL-1) and 10 Hodgkin's disease (HD) samples were stained with anti-phosphotyrosine antibody. The tyrosine kinase activity, p80 phosphorylation, and the apoptotic effects of two tyrosine kinase inhibitors, herbimycin A and STI-571, were determined on ALCL cell lines. RESULTS: Herbimycin A had showed both a time- and dose-dependent apoptotic effect on all three cell lines, while STI-571 demonstrated a minimal effect. Following herbimycin A treatment, a decrease in tyrosine kinase activity in the ALCL cell lines and inhibition in NPM-ALK (p80) autophosphorylation was demonstrated by immunoprecipitation and Western blotting. Herbimycin A-induced apoptosis was accompanied by caspase-3 activation. Furthermore, apoptosis induced by herbimycin A was blocked by both z-VAD-FMK and z-DEVD-FMK, suggesting a critical role of caspases. CONCLUSIONS: These findings indicate that tyrosine kinase activity is a common characteristic of ALCLs and necessary for ALCL cell survival. These findings further suggest that therapies targeting tyrosine kinases, including p80, may have clinical utility.

Anaplastic Lymphoma Kinase↗
PubMed2000

Oxidative stress and vanadate induce tyrosine phosphorylation of phosphoinositide-dependent kinase 1 (PDK1).

Phosphoinositide-dependent kinase (PDK1) regulates a number of pathways involved in responses to stress and in growth factor signaling; however, little is known concerning the mechanisms governing the activity of PDK1. In this report, we find that oxidative stress (H(2)O(2)) and vanadate induce tyrosine phosphorylation of PDK1. These effects of H(2)O(2) and vanadate were found in 293T cells and CH310T1/2 cells expressing exogenous PDK1 and in A20 lymphoma cells expressing endogenous PDK1. Exogenously expressed PDK1 was also tyrosine-phosphorylated in response to NGF treatment of 293T expressing TrkA. H(2)O(2) induced a more rapid tyrosine phosphorylation of PDK1 relative to vanadate, and only vanadate-induced tyrosine phosphorylation of PDK1 was sensitive to pretreatment of cells with wortmannin. In vitro, PDK1 could be tyrosine-phosphorylated by both the c-Src and Abl tyrosine kinases. Both H(2)O(2) and vanadate treatments increased the activity of PDK1 when the serum/glucocorticoid regulated kinase (SGK) was used as substrate. Vanadate treatment appeared to bypass the requirement for phosphatidylinositol 3,4,5-trisphosphate when Akt was used as substrate for PDK1. Tyrosine phosphorylation of PDK1 by the Abl tyrosine kinase also increased the activity of PDK1 toward SGK and Akt. These data suggest a novel mechanism through which PDK1 activity may be regulated.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed1998

Constitutively tyrosine phosphorylated p52 Shc in breast cancer cells: correlation with ErbB2 and p66 Shc expression.

Breast cancer cell lines display a wide variety of growth factor receptors, and considerable evidence implicates signaling from these receptors, especially ErbB2, in the important early stages of this disease, contributing to malignant progression. If this is true, then we would hypothesize that a useful prognostic indicator would be the level of activity of a second messenger protein used in common by these receptors. One such second messenger is the Shc adapter protein, which is activated when tyrosine phosphorylated by receptors. Therefore, one prediction from the hypothesis is that the level of tyrosine-phosphorylated Shc (PY-Shc) in breast cancer cell lines would correlate with total receptor tyrosine kinase activity. To begin to test this prediction, we examined Shc tyrosine phosphorylation in a diverse group of breast cancer cell lines that display varied levels of ErbB2. Using Shc immunoprecipitation and anti-phosphotyrosine immunoblotting analysis, we found a strong correlation between the level of ErbB2 overexpression (r = 0.91, p < 0.0002) and PY-ErbB2 levels (r = 0.89, p = 0.0005) compared with the level of tyrosine phosphorylation of the p52 and p46 Shc isoforms. Consistent with Shc tyrosine phosphorylation being driven by ErbB2, an ErbB2-specific tyrosine kinase inhibitor markedly reduced Shc tyrosine phosphorylation. Unexpectedly, although all cell lines had comparable total amounts of p52 and p46 Shc, the amount of an inhibitory Shc isoform, p66, was inversely related to the level of ErbB2 expression (r = -0.86, p = 0.0013). This suggests that reduced p66 Shc expression may play a role in ErbB2-positive breast cancer. In summary, these data are consistent with our prediction that the cellular level of PY-Shc would correlate with the levels of activated ErbB2 displayed by cell lines derived from breast cancers.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Catalytic specificity of protein-tyrosine kinases is critical for selective signalling.

How do distinct protein-tyrosine kinases activate specific down-stream events? Src-homology-2 (SH2) domains on tyrosine kinases or targets of tyrosine kinases recognize phosphotyrosine in a specific sequence context and thereby provide some specificity. The role of the catalytic site of tyrosine kinases in determining target specificity has not been fully investigated. Here we use a degenerate peptide library to show that each of nine tyrosine kinases investigated has a unique optimal peptide substrate. We find that the cytosolic tyrosine kinases preferentially phosphorylate peptides recognized by their own SH2 domains or closely related SH2 domains (group I; ref. 3), whereas receptor tyrosine kinases preferentially phosphorylate peptides recognized by subsets of group III SH2 domains. The importance of these findings for human disease is underscored by our observation that a point mutation in the RET receptor-type tyrosine kinase, which causes multiple endocrine neoplasia type 2B, results in a shift in peptide substrate specificity.

Amino Acid Sequence↗
PubMed1997

Tyrosine phosphorylation in DNA damage and cell death in hypoxic injury to LLC-PK1 cells.

Hypoxia is classically considered to result in a necrotic form of cell injury. We have recently demonstrated a role of endonuclease activation, considered a feature of apoptosis, in DNA damage and cell death in chemical hypoxic injury to renal tubular epithelial cells (LLC-PK1 cells). Tyrosine phosphorylation has been implicated to be involved in cell signaling pathway leading to cell growth, proliferation, and apoptotic death. However, a role of tyrosine phosphorylation as a signal transduction pathway involved in DNA damage and cell death has not been previously examined in hypoxic injury in any tissue. In the present study, we have demonstrated that chemical hypoxia with a combination of antimycin A, a mitochondrial respiration inhibitor, and substrate deprivation resulted in rapid increase in protein tyrosine kinases activity and protein tyrosine phosphorylation prior to any evidence of cell death in LLC-PK1 cells. The inhibitors of protein tyrosine kinases, genistein, lavendustin A, tyrphostin, and herbimycin A provided a marked protection against chemical hypoxia-induced DNA damage (as measured by alkaline unwinding assay) and cell death (as measured by trypan blue exclusion assay). In a separate study, we confirmed the ability of the inhibitors, lavendustin A and herbimycin A to prevent chemical hypoxia-induced increase in protein tyrosine kinases activity and protein tyrosine phosphorylation. In addition, the inhibitors used did not affect ATP depletion induced by antimycin A, suggesting that the inhibitors do not alter cellular uptake of antimycin A. Taken together, our data provide a strong evidence that tyrosine phosphorylation plays as important role in DNA damage and cell death in chemical hypoxic injury to renal tubular epithelial cells.

Animals↗
PubMed1998

Effect of inhibition of tyrosine phosphatases on voltage-operated calcium channel currents in rabbit isolated ear artery cells.

1. The effect of increasing cellular tyrosine phosphorylation by inhibiting endogenous tyrosine phosphatases was examined on voltage-operated calcium channel currents in vascular smooth muscle cells. 2. In single ear artery smooth muscle cells of the rabbit, studied by the whole cell voltage clamp technique, intracellular application of the tyrosine phosphatase inhibitors, sodium orthovanadate (100 microM) and peroxyvanadate (100 microM orthovanadate + 1 mM H2O2) increased voltage-operated calcium channel currents by 56% and 83%, respectively. 3. Bath application of two other membrane permeant tyrosine phosphatase inhibitors, phenylarsine oxide (100 microM) and dephostatin (50 microM) also increased voltage-operated calcium channel currents by 48% and 52%, respectively. 4. The selective tyrosine kinase inhibitor, tyrphostin-23 (100 microM) reduced calcium channel currents by 41%. Pre-incubation with tyrphostin-23 abolished the effects of peroxyvanadate, phenylarsine oxide and dephostatin on calcium channels. 5. Western blot analysis of rabbit ear artery cell lysates showed increased tyrosine phosphorylation of several endogenous proteins following treatment with peroxyvanadate. 6. These results indicate that a number of structurally dissimilar inhibitors of tyrosine phosphatases increase voltage-operated calcium channel currents in arterial smooth muscle cells presumably due to increased tyrosine phosphorylation.

Animals↗
PubMed2004

Actin depolymerization-induced tyrosine phosphorylation of cortactin: the role of Fer kinase.

The F-actin-binding protein cortactin is an important regulator of cytoskeletal dynamics, and a prominent target of various tyrosine kinases. Tyrosine phosphorylation of cortactin has been suggested to reduce its F-actin cross-linking capability. In the present study, we investigated whether a reciprocal relationship exists, i.e. whether the polymerization state of actin impacts on the cortactin tyrosine phosphorylation. Actin depolymerization by LB (latrunculin B) induced robust phosphorylation of C-terminal tyrosine residues of cortactin. In contrast, F-actin stabilization by jasplakinolide, which redistributed cortactin to F-actin-containing patches, prevented cortactin phosphorylation triggered by hypertonic stress or LB. Using cell lines deficient in candidate tyrosine kinases, we found that the F-actin depolymerization-induced cortactin phosphorylation was mediated by the Fyn/Fer kinase pathway, independent of Src and c-Abl. LB caused modest Fer activation and strongly facilitated the association between Fer and cortactin. Interestingly, the F-actin-binding region within the cortactin N-terminus was essential for the efficient phosphorylation of C-terminal tyrosine residues. Investigating the structural requirements for the Fer-cortactin association, we found that (i) phosphorylation-incompetent cortactin still bound to Fer; (ii) the isolated N-terminus associated with Fer; and (iii) the C-terminus alone was insufficient for binding. Thus the cortactin N-terminus participates in the Fer-cortactin interaction, which cannot be fully due to the binding of the Fer Src homology 2 domain to C-terminal tyrosine residues of cortactin. Taken together, F-actin stabilization prevents cortactin tyrosine phosphorylation, whereas depolymerization promotes it. Depolymerization-induced phosphorylation is mediated by Fer, and requires the actin-binding domain of cortactin. These results define a novel F-actin-dependent pathway that may serve as a feedback mechanism during cytoskeleton remodelling.

Actins↗
PubMed1993

Thrombin Ca(2+)-dependently stimulates protein tyrosine phosphorylation in BC3H1 muscle cells.

The proteinase thrombin, known to act via heptahelical G-protein-coupled receptors, is a mitogenic agent for different cell types, including the mouse muscle cell line BC3H1. In this study, the effect of thrombin on tyrosine phosphorylation was examined using anti-phosphotyrosine antibodies. Thrombin was found to induce phosphorylation of 65-70 and 110-120 kDa proteins in BC3H1 cells. The effect of thrombin was concentration-dependent, being half-maximal and maximal at concentrations of 0.03 and 1 unit/ml respectively. The thrombin-induced increase in phosphorylation was rapid (< or = 10 s) and transient, with a peak response after about 1-2 min. The effect of thrombin could be mimicked by the thrombin receptor agonist peptide SFLLRN-NH2. Preincubation of cells with pertussis toxin (PT) had no effect on thrombin-induced tyrosine phosphorylation. Epidermal growth factor, platelet-derived growth factor and insulin stimulated tyrosine phosphorylation of different proteins, among which were 65-70 and 110-120 kDa proteins. The phorbol ester 12-myristate 13-acetate (PMA) as well as the Ca2+ ionophore A23187 both stimulated tyrosine phosphorylation of proteins identical to those phosphorylated by thrombin, suggesting that activation of protein kinase C (PKC) and elevation of the cytosolic Ca2+ concentration alone are sufficient to induce tyrosine phosphorylation. However, calphostin C and other PKC inhibitors, which completely inhibited tyrosine phosphorylation induced by PMA, had no influence on the effect of thrombin, whereas loading of cells with the intracellular Ca2+ chelator bis-(O-aminophenoxy)ethane-NNN'N'-tetra-acetic acid totally blocked thrombin-stimulated tyrosine phosphorylation. Thus tyrosine phosphorylation stimulated by thrombin is an early PT-insensitive cellular response which is either directly mediated by elevation of cytosolic Ca2+ concentration or by a presently unknown mechanism that requires an elevated cytosolic Ca2+ concentration.

Animals↗
PubMed1996

Pervanadate activation of intracellular kinases leads to tyrosine phosphorylation and shedding of syndecan-1.

Syndecan-1 is a transmembrane haparan sulphate proteoglycan that binds extracellular matrices and growth factors, making it a candidate to act between these regulatory molecules and intracellular signalling pathways. It has a highly conserved transmembrane/cytoplasmic domain that contains four conserved tyrosines. One of these is in a consensus sequence for tyrosine kinase phosphorylation. As an initial step to investigating whether or not phosphorylation of these tyrosines is part of a signal-transduction pathway, we have monitored the tyrosine phosphorylation of syndecan-1 by cytoplasmic tyrosine kinases in intact cells. Tyrosine phosphorylation of syndecan-1 is observed when NMuMG cells are treated with sodium orthovanadate or pervanadate, which have been shown to activate intracellular tyrosine kinases. Initial studies with sodium orthovanadate demonstrate a slow accumulation of phosphotyrosine on syndecan-1 over the course of several hours. Pervanadate, a more effective inhibitor of phosphatases, allows detection of phosphotyrosine on syndecan-1 within 5 min, with peak phosphorylation seen by 15 min. Concurrently, in a second process activated by pervanadate, syndecan-1 ectodomain is cleaved and released into the culture medium. Two phosphorylated fragments of syndecan-1 of apparent sizes 6 and 8 kDa remain with the cell after shedding of the ectodomain. The 8 kDa size class appears to be a highly phosphorylated form of the 6 kDa product, as it disappears if samples are dephosphorylated. These fragments contain the C-terminus of syndecan-1 and also retain at least a portion of the transmembrane domain, suggesting that they are produced by a cell surface cleavage event. Thus pervanadate treatment of cells results in two effects of syndecan-1: (i) phosphorylation of one or more of its tyrosines via the action of a cytoplasmic kinase(s) and (ii) cleavage and release of the ectodomain into the medium, producing a C-terminal fragment containing the transmembrane/cytoplasmic domain.

Animals↗
PubMed1996

Oxidative stress triggers tyrosine phosphorylation in B cells through a redox- and inflammatory cytokine-sensitive mechanism.

Exposure to oxidants such as hydrogen peroxide (H2O2) and gamma-ray irradiation has been recently shown to trigger tyrosine phosphorylation in B cells as does cross-linking surface immunoglobulin (sIg) by antigens or anti-immunoglobulins. We studied the mechanism by which H2O2 induced tyrosine phosphorylation in B cells and compared it with the mechanism utilized by sIg. Both anti-immunoglobulin M (anti-IgM) and H2O2 induced tyrosine phosphorylation through protein tyrosine kinase (PTK) activation. However, the tyrosine phosphorylation caused by H2O2 but not that induced by anti-IgM, was modulated by agents affecting cellular thiols and glutathione contents including dithiothreitol, 2-mercaptoethanol, and buthionine sulphoximine. Moreover, the tyrosine phosphorylation caused by the oxidant but not that induced by anti-IgM was markedly augmented by two inflammatory cytokines, tumour necrosis factor-alpha and interleukin-1 alpha, although these agents by themselves did not stimulate PTK activity nor induce tyrosine phosphorylation. These findings demonstrate that oxidative stress but not surface IgM (sIgM) ligation triggers tyrosine phosphorylation through a mechanism that is sensitive to cellular thiols and these inflammatory cytokines.

Antibodies, Anti-Idiotypic↗
PubMed1996

Tyrosine kinases are required for catecholamine secretion and mitogen-activated protein kinase activation in bovine adrenal chromaffin cells.

Nicotine-induced catecholamine secretion in bovine adrenomedullary chromaffin cells is accompanied by rapid tyrosine phosphorylation of multiple cellular proteins, most notably the mitogen-activated protein kinases (MAPKs). The requirement for activation of tyrosine kinases and MAPKs in chromaffin cell exocytosis was investigated using a panel of tyrosine kinase inhibitors. Genistein and tyrphostin 23, two compounds that inhibit tyrosine kinases by distinct mechanisms, were found to inhibit secretion by > 90% in cells stimulated by nicotine, 55 mM KCl, or the Ca2+ ionophore A23187. Inhibition of secretion induced by all three secretagogues correlated with a block in both protein tyrosine phosphorylation and activation of the MAPKs and their activators (MEKs) in situ. However, neither genistein nor tyrphostin 23 inhibited the activities of the MAPKs or MEKs in vitro. These results indicate that the target(s) of inhibition lie downstream of Ca2+ influx and upstream of MEK activation. This Ca(2+)-activated tyrosine kinase activity could not be accounted for entirely by c-Src or Fyn (two nonreceptor tyrosine kinases that are expressed abundantly in chromaffin cells), because their in vitro kinase activities were not inhibited by tyrphostin 23 and only partially inhibited by genistein. These results demonstrate that an unidentified Ca(2+)-activated tyrosine kinase(s) is required for MAPK activation and exocytosis in chromaffin cells and suggest that MAPK participates in the regulation of secretion.

Adrenal Glands↗
PubMed1994

CD28 is associated with and induces the immediate tyrosine phosphorylation and activation of the Tec family kinase ITK/EMT in the human Jurkat leukemic T-cell line.

T lymphocytes require two signals to be activated. The antigen-specific T-cell receptor can deliver the first signal, while ligation of the T-cell surface molecule CD28 by antibodies or its cognate ligands B7-1 (CD80) or B7-2 has been demonstrated to be sufficient for the delivery of the second signal. Signaling via CD28 and the T-cell receptor results (i) in their costimulation of T cells to produce numerous lymphokines including interleukin 2 and (ii) in the prevention of anergy induction. Little is known about the pathway by which CD28 mediates its signals except that protein-tyrosine phosphorylation is involved. We show here in human Jurkat cells that the Tec-family protein-tyrosine kinase ITK/EMT (p72ITK/EMT) is associated with CD28 and becomes tyrosine-phosphorylated and activated within seconds of CD28 ligation. This tyrosine phosphorylation of p72ITK/EMT is rapid (within 30 sec), occurs in the absence of LCK activation, and precedes tyrosine phosphorylation of the guanine nucleotide exchange factor VAV. Secondary crosslinking of CD28 is unnecessary for the induced tyrosine phosphorylation of p72ITK/EMT. Thus, tyrosine phosphorylation of p72ITK/EMT may represent one of the earliest events in CD28 signaling. This demonstrates that a member of the Tec family of protein tyrosine kinases, similar to members of the Src and Syk families, plays a role in the activation of T cells. Furthermore, the data demonstrate that p72ITK/EMT, and by analogy other members of the Tec family, responds to extracellularly generated signals.

CD28 Antigens↗