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PubMed1996

Glucose-induced tyrosine phosphorylation of p125 in beta cells and pancreatic islets. A novel proximal signal in insulin secretion.

In this study, we demonstrate that stimulation of beta cells with carbachol and glucose causes increased tyrosine phosphorylation of a 125-kDa protein concurrently with increased insulin secretion. The effect was observed in two different insulin-secreting cell lines and in rat pancreatic islets. Tyrosine phosphorylation was largely calcium independent and occurred within 2 min after stimulation of beta cells with glucose and the muscarinic agonist carbachol. In islets, the effect of glucose was greatly diminished by the addition of mannoheptulose, a seven-carbon sugar that inhibits glucokinase, suggesting that glucose metabolism is required for tyrosine phosphorylation of the protein to occur. Neither insulin nor insulin-like growth factor I significantly increased tyrosine phosphorylation of the 125-kDa protein, suggesting that it was not an autocrine effect. Depolarization of beta cells with glyburide or 50 m potassium dramatically increased insulin secretion but had no significant effect on tyrosine phosphorylation. Addition of phorbol ester caused a less than 2-fold increase in tyrosine phosphorylation, whereas the calcium ionophore A23187 had no effect. Among the various fuel secretagogues tested, only -glucose stimulated tyrosine phosphorylation, both alone and in combination with carbachol. Finally, the tyrosine kinase inhibitor AG879 inhibited both tyrosine phosphorylation and insulin secretion in a dose-dependent manner. Taken together, these data demonstrate the presence of a novel signaling pathway in glucose-induced insulin secretion: tyrosine phosphorylation of beta cell p125, which is a proximal step in insulin secretion. Our current working hypothesis is that glucose stimulation of beta cell p125 tyrosine phosphorylation is an essential step for insulin secretion.

Animals↗

PubMed1991

Cyclin B targets p34cdc2 for tyrosine phosphorylation.

A universal intracellular factor, the 'M phase-promoting factor' (MPF), triggers the G2/M transition of the cell cycle in all organisms. In late G2, it is present as an inactive complex of tyrosine-phosphorylated p34cdc2 and unphosphorylated cyclin Bcdc13. In M phase, its activation as an active MPF displaying histone H1 kinase (H1K) originates from the concomitant tyrosine dephosphorylation of the p34cdc2 subunit and the phosphorylation of the cylin Bcdc13 subunit. We have investigated the role of cyclin in the formation of this complex and the tyrosine phosphorylation of p34cdc2, using highly synchronous mitotic sea urchin eggs as a model. As cells leave the S phase and enter the G2 phase, a massive tyrosine phosphorylation of p34cdc2 occurs. This large p34cdc2 tyrosine phosphorylation burst does not arise from a massive increase in p34cdc2 concentration. It even appears to affect only a fraction (non-immunoprecipitable by anti-PSTAIR antibodies) of the total p34cdc2 present in the cell. Several observations point to an extremely close association between accumulation of unphosphorylated cyclin and p34cdc2 tyrosine phosphorylation: (i) both events coincide perfectly during the G2 phase; (ii) both tyrosine-phosphorylated p34cdc2 and cyclin are not immunoprecipitated by anti-PSTAIR antibodies; (iii) accumulation of unphosphorylated cyclin by aphidicolin treatment of the cells, triggers a dramatic accumulation of tyrosine-phosphorylated p34cdc2; and (iv) inhibition of cyclin synthesis by emetine inhibits p34cdc2 tyrosine phosphorylation without affecting the p34cdc2 concentration. These results show that, as it is synthesized, cyclin B binds and recruits p34cdc2 for tyrosine phosphorylation; this inactive complex then requires the completion of DNA replication before it can be turned into fully active MPF. These results fully confirm recent data obtained in vitro with exogenous cyclin added to cycloheximide-treated Xenopus egg extracts.

Animals↗

PubMed1995

Fluoride increases tyrosine kinase activity in osteoblast-like cells: regulatory role for the stimulation of cell proliferation and Pi transport across the plasma membrane.

Fluoride is one of the most effective agents for the treatment of vertebral osteoporosis because of its ability to increase osteoblast proliferation. The present study further investigates the role of protein tyrosine phosphorylation previously suggested to mediate the mitogenic effect of fluoride on bone-forming cells. The activity of the plasma membrane Na-coupled Pi transport system was monitored to assess the relationship between alterations in tyrosine phosphorylation and osteoblast activity induced by fluoride. The results indicate that vanadate, a selective inhibitor of tyrosine phosphatase, mimicked the stimulatory effect of fluoride on Pi transport. The change in Pi transport induced by fluoride was dose dependently inhibited by genistein, a potent inhibitor of tyrosine kinase. Genistein also inhibited the change in cell proliferation induced by fluoride. Associated with these observations, tyrosine phosphorylation activity was significantly increased in subcellular fractions isolated from UMR-106 cells treated with fluoride as compared with those isolated from vehicle-treated cells. This change in tyrosine phosphorylation activity was markedly blunted when genistein was added to the kinase assay buffer. It was not associated with any alteration in specific tyrosine phosphatase activity. There was also no evidence of a direct effect of fluoride on tyrosine phosphatase activity in isolated plasma membrane of UMR-106 cells. In conclusion, the results of the present study suggest that fluoride enhances protein tyrosine phosphorylation in osteoblast-like cells by enhancing tyrosine kinase activity. The results further support the hypothesis that this signal transduction mechanism is involved in the osteogenic effects of fluoride.

Animals↗

PubMed2000

Activation of m3 muscarinic receptors induces rapid tyrosine phosphorylation of p125(FAK), p130(cas), and paxillin in rat pancreatic acini.

Tyrosine phosphorylation plays a key role in transmembrane and cytoplasmic signal transduction mechanisms stimulated by oncogenes, integrins, growth factors, neuropeptides, and bioactive lipids. Moreover, recent studies show that stimulation of odd-numbered muscarinic receptors increases the tyrosine phosphorylation of several proteins in different cellular types. The present study was aimed at examining whether activation of m3 muscarinic receptors in rat pancreatic acini evokes tyrosine phosphorylation of p125(FAK), and its substrates, p130(cas) and paxillin. Results show that stimulation of pancreatic acini with carbachol resulted in a rapid and transient increase in tyrosine phosphorylation of p125(FAK), p130(cas), and paxillin. Tyrosine phosphorylation of these proteins occurred in a time- and concentration-dependent manner. Simultaneous blockage of both PKC activation and increases in [Ca(2+)](i) partially decreased p125(FAK), p130(cas), and paxillin tyrosine phosphorylation stimulated by carbachol. Pretreatment of pancreatic acini with Clostridium botulinum C3 transferase, which specifically inactivates p21(rho), partially inhibited carbachol-induced p125(FAK), p130(cas), and paxillin tyrosine phosphorylation. In contrast, this treatment had no effect on amylase release stimulated by carbachol. Cytochalasin D, which disrupts actin microfilaments network, completely inhibited carbachol stimulated tyrosine phosphorylation of these proteins without having significant effects in carbachol-stimulated amylase secretion. These results dissociate tyrosine phosphorylation of p125(FAK), p130(cas), and paxillin from amylase secretion after m3 muscarinic receptors occupation in rat pancreatic acini. Taken together, these data suggest that (a) activation of m3 muscarinic receptors in rat pancreatic acini increases tyrosine phosphorylation of p125(FAK) and its substrates, p130(cas) and paxillin by diacylglycerol-activated PKC- and calcium- dependent, and independent pathways, (b) these responses require activation of p21(rho) and an intact actin cytoskeleton, and (c) p125(FAK), p130(cas), and paxillin are unlikely related to secretion in rat pancreatic acinar cells.

ADP Ribose Transferases↗

PubMed1998

Effects of modulation of tyrosine phosphorylation on brush border enzyme activity in human Caco-2 intestinal epithelial cells.

Intestinal epithelial cell differentiation is closely regulated during normal cell renewal, maturation, and malignant transformation. Since tyrosine phosphorylation influences differentiation in other cell types and has been reported to vary between crypt cells to differentiated villus tip cells, we investigated the influence of tyrosine phosphorylation in colonocyte differentiation, by using human colonic Caco-2 cells as a model and expression of the brush border enzymes alkaline phosphatase (AKP) and dipeptidyl peptidase (DPDD) as differentiation markers. We studied three tyrosine kinase inhibitors with different modes of action and specificities, viz., genistein, erbstatin analog (EA), and tyrphostin, and the tyrosine phosphatase inhibitor sodium orthovanadate. AKP- and DPDD-specific activities were assayed in protein-matched cell lysates by synthetic substrate digestion. We also correlated the effects of these agents on brush border enzyme activity with tyrosine phosphorylation of phosphoproteins by Western blotting. Genistein (5-75 mg/ml) dose-dependently stimulated AKP and DPDD with a maximal stimulation at 75 mg/ml by 158.6+/- 17.5% and 228.6+/-37.1% of control values, respectively (n=12, P<0.001). The inactive analog genistin had no effect. Tyrphostin (25 mM) similarly stimulated AKP and DPDD by 138. 6+/-6.6% and 131.8+/-1.5% of control values (n=12, P<0.001). Unexpectedly, EA (0.1-10 mM) had the opposite effect, inhibiting AKP- and DPDD-specific activity significantly at 10 mM with a maximal 14.8+/-6.4% and 26.5+/-2.5% of control values (n=12, each P<0.001). Sodium orthovanadate had a discordant effect on these two differentiation markers. Orthovanadate dose-dependently increased AKP to a maximal 188.5+/-16.1% of basal activity at 1.5 mM but decreased DPDD activity at 1.5 mM to 47.2+/-3.8% (n=9, P<0.001 each). The effects of each agent were preserved when proliferation was blocked with mitomycin C, suggesting that the modulation of phenotype by these agents was independent of any effects of proliferation. The tyrosine phosphorylation of several phosphoprotein bands was affected differently by these agents. In particular, the tyrosine phosphorylation of one 70-kDa to 71-kDa band was increased by genistein and tyrophostin but deceased by EA. The different effects of these modulators of tyrosine kinase activity raise the possibility that at least two independent enzymes or pathways regulating tyrosine phosphorylation modulate intestinal epithelial differentiation. Furthermore, tyrosine phosphorylation of the 70-kDa to 71-kDa phosphoprotein may be important in the intracellular signaling by which intestinal epithelial cell differentiation is controlled.

Alkaline Phosphatase↗

PubMed1986

Properties of a membrane-bound tyrosine kinase phosphorylating the cytosolic fragment of the red cell membrane band 3 protein.

Band 3 protein of human erythrocyte membrane is phosphorylated on a tyrosine residue located near the NH2 terminal by an endogenous tyrosine kinase activity (Dekowski, S., Rybicki, A. and Drickamer, K. (1983) J. Biol. Chem. 258, 2750-2753). A tyrosine kinase phosphorylating the band 3 protein in situ has been extracted from ghosts by non-ionic detergent and partially characterized (Phan-Dinh-Tuy, F., Henry, J. and Kahn, A. (1985) Biochem. Biophys. Res. Commun. 126, 304-312). We have studied the properties of the tyrosine kinase activity which remains bound to the ghosts after detergent extraction using the 43 kDa fragment of protein 3 as substrate. This activity, solubilized from the detergent-resistant material at 0.25 M NaCl and concentrated by phosphocellulose and tyrosine-agarose chromatographies, remains linked to high molecular weight complexes. It is specific for tyrosine. Assayed with the purified 43 kDa fragment it requires the presence of Mn2+ which cannot be replaced by Mg2+. Its affinity for 43 kDa fragment is very high with a Km of 3.3 microM. ATP acts as a phosphoryl donor with a Km of 0.55 microM. The tyrosine kinase activity was not modified by insulin, DMSO, phorbol ester and epidermal growth factor, vanadate and xanthine derivatives. Polyamines spermidine and the polylysine are inhibitors in the presence of Mn2+ but not in the presence of Mg2+. Heparin is a competitive inhibitor of ATP. 2,3-Diphosphoglycerate is an inhibitor at physiological concentrations (Ki = 2 mM). Purified red cell actin is not phosphorylated by the tyrosine kinase. These properties distinguish the red cell membrane-bound tyrosine kinase from other tyrosine kinases extracted from normal cells.

2,3-Diphosphoglycerate↗

PubMed1989

Tyrosine phosphorylation in human neutrophil.

Protein tyrosine phosphorylation in human neutrophils was examined by immunoblotting with antibodies specific for phosphotyrosine. The addition of the human hormone granulocyte-macrophage colony stimulating factor to human neutrophils caused an increase in the tyrosine phosphorylation levels of several proteins. The increases in at least two of these proteins having molecular masses of 40 kDa (p40) and 54 kDa (p54) were rapid and were inhibited in pertussis toxin treated cells. The newly synthesized tyrosine kinase inhibitor ST 638 inhibited the increases in the levels of the tyrosine phosphorylation in p92, p78, p54 and p40 proteins. The epidermal growth factor receptor tyrosine kinase inhibitors were less effective. The addition of the chemotactic factor fMet-Leu-Phe to human neutrophils also caused an increase in tyrosine phosphorylation in some of these proteins. The pattern of the fMet-Leu-Phe-induced tyrosine phosphorylation was different from that produced by GM-CSF. The increases were also inhibited by ST 638. In addition, ST 638 inhibited superoxide production but not actin polymerization in control and GM-CSF-treated cells stimulated with fMet-Leu-Phe. Moreover, the active but not inactive phorbol esters increase the tyrosine phosphorylation only in the 40 kDa protein. These results suggest several points: (a) some of the responses produced by GM-CSF and fMet-Leu-Phe are mediated through tyrosine phosphorylation, (b) the GM-CSF receptor is coupled to a pertussis toxin sensitive G-protein, (c) the 40 kDa protein is probably the Gi alpha 2, and (d) the 78 or the 92 kDa protein is most likely the receptor for GM-CSF, which indicates that the receptor may have a tyrosine kinase domain.

Cinnamates↗

PubMed1983

Role of tyrosine in the acute effects of ethanol on rat brain catecholamine synthesis.

Acute ethanol administration exerts multiple effects on rat brain catecholamine synthesis, associated with corresponding changes in cerebral tyrosine concentration. Catecholamine synthesis is enhanced at 1 hr by an increased availability of circulating tyrosine to the brain after inhibition of liver tyrosine aminotransferase activity. Tyrosine hydroxylation in vivo and tyrosine hydroxylase activity measured in vitro are also enhanced at 1 hr. Catecholamine synthesis is inhibited at 2-4 hr when tyrosine availability to the brain is decreased because of an enhancement of liver tyrosine aminotransferase activity. Serum neutral amino acid concentrations are decreased at 5 hr. This is followed 1 hr later by normalization of cerebral catecholamine synthesis. By 8 hr after ethanol administration, the latter becomes enhanced because of increased cerebral uptake of tyrosine. Catecholamine synthesis is inhibited at 12 hr because of enhanced transamination of brain tyrosine. Tyrosine metabolism finally returns to normal at 16 hr after ethanol administration. These results are discussed in relation to previous work with ethanol, and to central and peripheral mechanisms of regulation of brain catecholamine synthesis.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1992

A possible mechanism for the increase in brain tyrosine levels induced by cyanide in mice.

Subcutaneous administration of cyanide significantly increased blood tyrosine levels of mice in a dose dependent manner. Tyrosine aminotransferase activity in liver of mice was significantly decreased in the presence of cyanide (8, 10, 20, 40, 50, 65, 80 and 100 microM), also in a concentration-dependent manner, with a positive correlation between the percentage increase of blood tyrosine levels and the percentage decrease of hepatic tyrosine aminotransferase activity. These results suggest that the increased tyrosine levels induced in blood by cyanide may be related to its inhibition of tyrosine aminotransferase activity in the liver. Cyanide decreased hepatic ATP content and increased blood ammonia levels and brain tyrosine in a dose-dependent manner. As it is known that hyperammonaemia increases the uptake of neutral amino acids such as tyrosine into the brain from blood, the mechanism by which tyrosine levels increase in the brain may be based on increases of both tyrosine and ammonia levels in blood.

Adenosine Triphosphate↗

PubMed2000

Biochemical and cellular effects of c-Src kinase-selective pyrido[2, 3-d]pyrimidine tyrosine kinase inhibitors.

Increased expression or activity of c-Src tyrosine kinase has been associated with the transformed phenotype in tumor cells and with progression of neoplastic disease. A number of pyrido[2, 3-d]pyrimidines have been characterized biochemically and in cells as part of an assessment of their potential as anti-tumor agents. The compounds were ATP-competitive inhibitors of c-Src kinase with IC(50) values < 10 nM and from 6 to >100-fold selectivity for c-Src tyrosine kinase relative to basic fibroblast growth factor receptor (bFGFr) tyrosine kinase, platelet-derived growth factor receptor (PDGFr) tyrosine kinase, and epidermal growth factor receptor (EGFr) tyrosine kinase. The compounds yielded IC(50) values < 5 nM against Lck. Human colon tumor cell growth in culture was inhibited, as was colony formation in soft agar at concentrations < 1 microM. Phosphorylation of the c-Src cellular substrates paxillin, p130(cas), and Stat3 was also inhibited at concentrations < 1 microM. Autophosphorylation of EGFr tyrosine kinase or PDGFr tyrosine kinase was not inhibited by c-Src inhibitors, thus showing the selective nature of the compounds in cells. In a mitogenesis assay measuring thymidine incorporation stimulated by specific mitogens, the c-Src tyrosine kinase inhibitors reduced incorporated thymidine in a manner consistent with previously reported roles of c-Src in mitogenic signaling. Progression through the cell cycle was inhibited at G(2)/M in human colon tumor cells treated with two of the c-Src-selective compounds, which is also consistent with earlier reports describing a requirement for active c-Src tyrosine kinase for G(2) to M phase progression. The compounds described here are selective inhibitors of c-Src tyrosine kinase and have antiproliferative effects in tumor cells consistent with inhibition of c-Src.

CSK Tyrosine-Protein Kinase↗

PubMed1999

Contribution of kinases and the CD45 phosphatase to the generation of tyrosine phosphorylation patterns in the T-cell receptor complex zeta chain.

The zeta subunit of the T-cell receptor complex plays a crucial role in coupling the antigen binding alphabeta and gammadelta heterodimers to the downstream activation pathways. Three tandem amino acid sequence motifs containing pairs of exactly spaced Tyr-X-X-Leu/Ile sequences, designated as Immunoreceptor Tyrosine-based Activation Motifs (ITAMs), control this function. The phosphorylated forms of ITAMs serve as docking sites for several src homology 2 (SH2) domain containing signaling proteins. The composition of the assembled signaling complex and the outcome of cell activation depends on the tyrosine phosphorylation pattern of the zeta polypeptide. The mechanism that conducts the generation of various phosphorylated forms has not yet been well established. In this study we have analyzed the ability of src family tyrosine kinases and the CD45 tyrosine phosphatase in determining the phosphorylation state of the different ITAMs and the individual tyrosine residues of the TCR zeta chain. The intracellular part of the zeta chain was phosphorylated by src family tyrosine kinases, p56lck and p59fyn in vitro. Synthetic oligopeptides representing full-length or half-sized ITAMs with a single tyrosine residue were also phosphorylated by both p56lck and p59fyn. In contrast, an additional membrane proximal tyrosine residue in the human zeta chain, located outside of the ITAMs, was not phosphorylated. We also examined the activity of the CD45 phosphatase, using a panel of ITAM derivatives, in which one or both tyrosines were phosphorylated. The efficiency of ITAM dephosphorylation by CD45 was dependent on the primary sequence of the oligopeptides and the position of the phosphotyrosine residues. The in vitro data suggest that the CD45 phosphatase rather than the tyrosine kinase(s) may control the generation of specific phosphorylation patterns of the zeta chain during cell activation.

Cell Line↗

PubMed1994

NK-LAK induction with IL-2 is regulated by tyrosine phosphorylation of a 105-110 kDa protein.

Tyrosine phosphorylation of the cellular proteins of IL-2-stimulated NK cells was determined by anti-phosphotyrosine immunoblotting. IL-2 induced tyrosine phosphorylation of a 105-110 kDa protein in a dose-dependent manner. The tyrosine phosphorylation took place within 5 min after the addition of IL-2 to NK cells, and reached a maximal level in 15 min. The degree of the tyrosine phosphorylation correlated with IL-2-induced LAK activity. Staurosporine and pertussis toxin, which slightly suppressed LAK induction, did not inhibit tyrosine phosphorylation of the 105-110 kDa protein. Genistein, TMB-8 and EGTA completely inhibited LAK induction; however, the calcium channel blocker and chelator did not prevent the protein tyrosine phosphorylation. Anti-IL-2R beta mAb almost completely suppressed tyrosine phosphorylation of the 105-110 kDa protein, but anti-IL-2R alpha mAb only slightly suppressed it; this result correlated with that of the suppression of LAK activity. No further suppression of the tyrosine phosphorylation was induced even when both mAbs were added. Western blotting of the immunoprecipitates revealed no association of PLC-gamma 1 or IL-2R beta with the 105-110 kDa protein. These results suggest that both tyrosine phosphorylation of the 105-110 kDa protein and translocation of [Ca++]i are essential for NK-LAK induction, and the tyrosine phosphorylation plays a critical role in the early stage of IL-2 signalling from the IL-2R beta chain.

Blotting, Western↗

PubMed2001

Phosphorylation and activation of protein tyrosine phosphatase (PTP) 1B by insulin receptor.

We have previously reported a direct in vivo interaction between the activated insulin receptor and protein-tyrosine phosphatase-1B (PTP1B), which leads to an increase in PTP1B tyrosine phosphorylation. In order to determine if PTP1B is a substrate for the insulin receptor tyrosine kinase, the phosphorylation of the Cys 215 Ser, catalytically inactive mutant PTP1B (CS-PTP1B) was measured in the presence of partially purified and activated insulin receptor. In vitro, the insulin receptor tyrosine kinase catalyzed the tyrosine phosphorylation of PTP1B. 53% of the total cellular PTP1B became tyrosine phosphorylated in response to insulin in vivo. Tyrosine phosphorylation of PTP1B by the insulin receptor was absolutely dependent upon insulin-stimulated receptor autophosphorylation and required an intact kinase domain, containing insulin receptor tyrosines 1146, 1150 and 1151. Tyrosine phosphorylation of wild type PTP1B by the insulin receptor kinase increased phosphatase activity of the protein. Intermolecular transdephosphorylation was demonstrated both in vitro and in vivo, by dephosphorylation of phosphorylated CS-PTP1B by the active wild type enzyme either in a cell-free system or via expression of the wild type PTP1B into Hirc-M cell line, which constitutively overexpress the human insulin receptor and CS-PTP1B. These results suggest that PTP1B is a target protein for the insulin receptor tyrosine kinase and PTP1B can regulate its own phosphatase activity by maintaining the balance between its phosphorylated (the active form) and dephosphorylated (the inactive form) state.

Animals↗

PubMed1997

Bcr phosphorylated on tyrosine 177 binds Grb2.

We and others have shown that the Bcr-Abl oncoprotein binds activators of the Ras pathway such as Grb2 and Shc. Grb2 binding is mediated through a phosphorylated tyrosine residue (Y177) located within a consensus Grb2 binding site encoded by the first exon of the BCR gene. Our results indicate that P160 BCR is tyrosine phosphorylated at the same site by Bcr-Abl in kinase assays (Puil et al., 1994). We performed experiments to determine whether Bcr, which was tyrosine phosphorylated within cells by activated c-Abl, could also bind Grb2, and whether phosphotyrosine 177 was the major binding site. Complexes between Bcr and Abl were detected in a hemopoietic cell line lacking Bcr-Abl and in COS1 cells coexpressing both Bcr and Abl proteins. P160 BCR was tyrosine phosphorylated in COS1 cells coexpressing Abl and Bcr proteins. Similarly, various deletion mutants of Bcr including BCRN553, BCRN413 and BCRN221 were tyrosine phosphorylated by activated c-Abl whereas BCRN159 was not. Wild-type Bcr and Bcr Y177F were examined under these conditions for their ability to co-precipitate with Grb2. The results showed that while wild-type tyrosine phosphorylated Bcr efficiently bound Grb2, tyrosine phosphorylated Bcr Y177F had greatly reduced Grb2-binding ability. Studies with GST-SH2 (Grb2) revealed that tyrosine phosphorylated Bcr was able to bind to GST SH2 (Grb2) but tyrosine phosphorylated Bcr Y177F was deficient in binding. These results indicate that the Bcr protein when phosphorylated at tyrosine 177 binds Grb2, thereby implicating Bcr as a potantial activator of the Ras pathway.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Complex formation between EphB2 and Src requires phosphorylation of tyrosine 611 in the EphB2 juxtamembrane region.

The cellular components of the neuronal signaling pathways of Eph receptor tyrosine kinases are only beginning to be elucidated. Here we show that in vivo tyrosine phosphorylation sites of the Eph receptors EphA3, EphA4, and EphB2 in embryonic retina serve as binding sites for the Src-homology 2 (SH2) domain of Src kinase. Furthermore, tyrosine-phosphorylated EphB2 was detected in Src immunoprecipitates from transfected Cos cells, indicating that EphB2 and Src can physically associate. Interestingly, a form of Src with reduced electrophoretic mobility and increased tyrosine phosphorylation was detected in Cos cells expressing tyrosine-phosphorylated EphB2, suggesting a functional interaction between EphB2 and Src. Yeast two-hybrid analysis in conjunction with site-directed mutagenesis demonstrated that phosphorylated tyrosine 611 in the juxtamembrane region of EphB2 is crucial for the interaction with the SH2 domain of Src. In contrast, binding of the carboxy-terminal SH2 domain of phospholipase Cgamma was not abolished upon mutation of tyrosine 611 in EphB2. Phosphopeptide mapping of autophosphorylated full-length EphB2, and wild-type and tyrosine to phenylalanine mutants of the EphB2 cytoplasmic domain fused to LexA, showed tyrosine 611 in the sequence motif YEDP as a major site of autophosphorylation in EphB2. Our mutational analysis also indicated that tyrosines 605 and 611 are important for EphB2 kinase activity. We propose Src kinase as a downstream effector that mediates the neuron's response to Eph receptor activation.

Animals↗

PubMed1991

Bacterial lipopolysaccharide stimulates protein tyrosine phosphorylation in macrophages.

Lipopolysaccharide (LPS), a membrane component of Gram-negative bacteria, stimulates immune responses by activating macrophages, B lymphocytes, and other cells of the immune system. The mechanisms by which LPS activates these cells are poorly characterized. Since protein tyrosine phosphorylation appears to be a major intracellular signaling event that mediates cellular responses, we examined whether LPS alters tyrosine phosphorylation in macrophages. We found that Escherichia coli K235 LPS increased tyrosine phosphorylation of several proteins in the RAW 264.7 murine macrophage cell line and in resident peritoneal macrophages from C3H/HeSNJ mice. Changes in tyrosine phosphorylation were detectable by 4-5 min, reached a maximum by 15 min, and declined after 30-60 min. Protein tyrosine phosphorylation increased following stimulation with LPS at 100 pg/ml and was maximal with 10 ng/ml. Similar changes in tyrosine phosphorylation were induced by Salmonella minnesota R595 LPS and by the biologically active domain of LPS, lipid A, but not by the inactive lipid A derivative N2-monoacylglucosamine 1-phosphate. Phorbol 12-myristate 13-acetate also stimulated protein tyrosine phosphorylation, but some of the modulated proteins were different than those phosphorylated by LPS. Treatment of RAW 264.7 cells with a tyrosine kinase inhibitor, herbimycin A, inhibited both LPS-stimulated tyrosine phosphorylation and LPS-stimulated release of arachidonic acid metabolites. Thus, increased protein tyrosine phosphorylation is a rapid LPS-activated signaling event that may mediate release of arachidonic acid metabolites in RAW 264.7 cells.

Animals↗

PubMed1991

Nerve growth factor rapidly stimulates tyrosine phosphorylation of phospholipase C-gamma 1 by a kinase activity associated with the product of the trk protooncogene.

Nerve growth factor (NGF) promotes the survival and differentiation of specific populations of neurons. The molecular mechanisms by which cells respond to NGF are poorly understood, but two clues have emerged recently. First, NGF rapidly stimulates tyrosine phosphorylation of several unidentified proteins in the NGF-responsive pheochromocytoma cell line PC12 [Maher, P. (1988) Proc. Natl. Acad. Sci. USA 85, 6788-6791]. Second, the protein-tyrosine kinase encoded by the protooncogene trk (p140trk), a member of the receptor class of tyrosine kinases, becomes activated and phosphorylated on tyrosine after NGF treatment of PC12 cells [Kaplan, D. R., Martin-Zanca, D. & Parada, L. F. (1991) Nature (London) 350, 158-160]. We now report that NGF rapidly induces tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1), and we present evidence that the responsible tyrosine kinase is either p140trk or a closely associated protein. Treatment of responsive cells with NGF elicited phosphorylation of PLC-gamma 1 on tyrosine and serine. PLC-gamma 1 immunoprecipitated from NGF-stimulated cells was phosphorylated in vitro by coprecipitating protein kinase activity, and the phosphorylations occurred principally on tyrosine. The responsible kinase could be depleted from cellular lysates by antibodies specific for p140trk. This procedure also depleted a 140-kDa protein that normally coprecipitated with PLC-gamma 1 and became phosphorylated on tyrosine in vivo in response to NGF. Analysis of tryptic peptides from PLC-gamma 1 indicated that the residues phosphorylated in vitro by p140trk-associated kinase activity were largely congruent with those phosphorylated in vivo after NGF treatment. Our findings identify PLC-gamma 1 as a likely substrate for the trk-encoded tyrosine kinase, and they provide a link between NGF-dependent activation of p140trk and the stimulation of intracellular second messenger pathways.

Adrenal Gland Neoplasms↗

PubMed1995

Differential tyrosine phosphorylation of N-methyl-D-aspartate receptor subunits.

Protein-tyrosine phosphorylation has recently been suggested to play an important role in synaptic transmission at the neuromuscular junction. The role of tyrosine phosphorylation in the modulation of synaptic function in the central nervous system, however, is not clear. In this study, immunocytochemical staining with an anti-phosphotyrosine antibody demonstrates that there are high levels of phosphotyrosine, which co-localizes with glutamate receptors at excitatory synapses on cultured hippocampal neurons. In addition, the tyrosine phosphorylation of various subtypes of glutamate receptors were examined using subunit-specific antibodies. Glutamate receptors are the major excitatory neurotransmitter receptors in the central nervous system and are classified into three major classes: alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate, kainate, and N-methyl-D-aspartate (NMDA) receptors, based on their electrophysiological and pharmacological properties. NMDA receptors play a central role in synaptic plasticity, synaptogenesis, and excitotoxicity and are thought to be heteromeric complexes of the two types of subunits: NR1 and NR2(A-D) subunits. Immunoaffinity chromatography of detergent extracts of rat synaptic plasma membranes on anti-phosphotyrosine antibody-agarose showed that the NR2A and NR2B subunits but not the NR1 subunit are tyrosine-phosphorylated. Conversely, immunoprecipitation of the NR1, NR2A, and NR2B subunits with subunit specific antibodies followed by immunoblotting with anti-phosphotyrosine antibodies confirmed that the NR2A and NR2B subunits but not the NR1 subunit were phosphorylated on tyrosine residues. No tyrosine phosphorylation of the AMPA (GluR1-4) and kainate (GluR6/7, KA2) receptor subunits was detected. It was estimated that 2.1 +/- 1.3% of the NR2A subunits and 3.6 +/- 2.4% of the NR2B subunits were tyrosine-phosphorylated in vivo. In addition, endogenous protein-tyrosine kinases in synaptic plasma membranes phosphorylated the NR2A subunit in vitro, increasing its phosphorylation 6-8-fold but did not phosphorylate NR1 or NR2B. These studies demonstrate that NMDA receptor subunits are differentially tyrosine-phosphorylated and suggest that tyrosine phosphorylation of the NR2 subunits may be important for regulating NMDA receptor function.

Animals↗