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PubMed1985

Evidence from two transformed cell lines that the phosphorylations of peptide tyrosine and phosphatidylinositol are catalyzed by different proteins.

Two transformed rodent cell lines (RS-1 and LSTRA) were studied in vitro to determine if their major protein tyrosine kinases catalyzed the phosphorylation of phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P), or diacylglycerol. RS-1 cells, transformed by Rous sarcoma virus, contain high levels of pp60src; LSTRA cells, transformed by Moloney murine leukemia virus, contain a tyrosine kinase (pp56) that is the product of an unknown cellular gene. Rates of phosphorylation of peptide tyrosine were elevated more than 20-fold in RS-1 and LSTRA particulate fractions compared to fractions from suitable control cells (N2 and YAC-1), but there was not a proportional increase in rates of phosphorylation of PtdIns, PtdIns4P, or diacylglycerol. Heat (34 degrees C) completely inactivated the LSTRA tyrosine kinase, while it enhanced the phosphorylation of PtdIns and PtdIns4P and had no effect on the phosphorylation of diacylglycerol. PtdIns4P inhibited the phosphorylation of PtdIns but had no effect on tyrosine kinase activity. An antibody, raised against a peptide with a sequence homologous to the autophosphorylation site of pp60src, immunoprecipitated tyrosine kinase activity from RS-1 and LSTRA extracts but had no effect on PtdIns kinase or PtdIns4P kinase activity. These results provide evidence that the phosphorylations of tyrosine and PtdIns are catalyzed by different proteins. An additional observation was that a monoclonal antibody that binds to pp60src and pp56 removed PtdIns kinase as well as tyrosine kinase activity from RS-1 and LSTRA particulate extracts. This antibody also removed PtdIns kinase from N2 and YAC-1 extracts, in which tyrosine kinase activity was low or undetectable. Thus, the anti-pp60src monoclonal antibody may recognize the PtdIns kinase in addition to pp60src and pp56.

1-Phosphatidylinositol 4-Kinase↗
PubMed1986

Fluorescence polarization decay of tyrosine in lima bean trypsin inhibitor.

The fluorescence of lima bean trypsin inhibitor is due to a single tyrosine residue at position 69. The lifetime of this tyrosine fluorescence is 620 +/- 50 ps (mean +/- SD) and is little affected by addition of 0.88 M citrate, an efficient quencher of tyrosine fluorescence. The steady-state emission intensity is also only weakly reduced by the quencher. The tyrosine is thus not accessible to the citrate and is probably located in the interior of the protein. The high pK of the tyrosine supports this conclusion. The fluorescence anisotropy decay of the inhibitor's tyrosine can be fitted to a double exponential form, with time constants of about 40 ps and greater than or equal to 3 ns. The anisotropy at time zero is 0.19 +/- 0.015 (mean +/- SD), the same as for N-acetyl-L-tyrosinamide in viscous glycerol solution. The nanosecond component of the decay is consistent with rotation of the entire protein molecule. The 40-ps component demonstrates that the tyrosine has considerable freedom to move independently of the protein as a whole. This rotational correlation time is approximately what is observed for free tyrosine in aqueous solution. Since the polypeptide chain near tyrosine-69 is anchored by several disulfide bonds, the data argue that this interior portion of the protein consists of a rigid, immobile backbone embedded in fluid, mobile amino acid side chains.

Fabaceae↗
PubMed1990

Tyrosine phosphorylation coupled to IgE receptor-mediated signal transduction and histamine release.

Antigen-induced cross-linking of IgE bound to its receptors at the surface of basophils or mast cells initiates a number of biochemical events culminating in the release of histamine-containing granules. In the present study, we investigated the possible involvement of tyrosine phosphorylation in signaling by the high-affinity IgE receptor (Fc epsilon RI). Cross-linking of Fc epsilon RI in rat basophilic leukemia cells (RBL-2H3) led to the phosphorylation of several proteins on tyrosine, the most prominent having a mass of 72 kDa. Tyrosine phosphorylation was rapid, detectable 1 min after stimulation, and correlated with both the time course and antigen dose for histamine release. Reversal of Fc epsilon RI cross-linking prevented continuation of the degranulation process and resulted in rapid loss of tyrosine phosphorylation. The receptor-mediated tyrosine phosphorylation was still induced in the absence of calcium in the medium. Depletion of protein kinase C with phorbol 12-myristate 13-acetate did not dramatically affect the tyrosine phosphorylation signal or the release of histamine. In contrast, the calcium ionophore A23187 induced histamine release in the absence of a perceptible increase in protein tyrosine phosphorylation. Thus, tyrosine phosphorylation is an early signal following Fc epsilon RI aggregation, independent of the exocytotic process itself. Taken together, our findings functionally link protein phosphorylation on tyrosine residues to Fc epsilon RI-mediated signal transduction leading to histamine release.

Animals↗
PubMed1998

Activation of protein tyrosine kinase PYK2 by the m1 muscarinic acetylcholine receptor.

Several G protein-coupled receptors are known to direct the tyrosine phosphorylation, and in some cases the activation, of diverse tyrosine kinases. Using a stable cell line approach, we characterize the activation of PYK2, a tyrosine kinase structurally related to focal adhesion kinase, by the G protein-coupled m1 muscarinic acetylcholine receptor. We find that PYK2 tyrosine kinase activity is critical for the m1 receptor-stimulated tyrosine phosphorylation of PYK2. Furthermore, we identify two tyrosine residues that are subject to phosphorylation in response to muscarinic signaling and show that this phosphorylation induces two cytosolic proteins, c-Src and Grb2, to bind to PYK2. This is the first demonstration of the significance played by distinct PYK2 tyrosine residues in G protein-coupled signaling to this kinase. By comparison, though m1 receptors induce the tyrosine phosphorylation of the cytoskeletal protein paxillin, the association of paxillin with PYK2 is unaffected by muscarinic signaling. We also provide evidence that PYK2 specifically phosphorylates the carboxyl-terminal cytosolic portion of the potassium channel Kv1.2 in a manner regulated by the m1 receptor. These results delineate molecular events attending the m1 muscarinic receptor stimulation of this tyrosine kinase and establish PYK2 as an effector of the m1 muscarinic receptor in the regulation of multiple cell functions.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Tyrosine phosphorylation and enhanced expression of paxillin during neuronal differentiation in vitro.

Tyrosine phosphorylation has been implicated as a means by which neurite outgrowth is regulated. Because paxillin is a tyrosine-phosphorylated protein that may play a role in regulating cell morphology, we examined its expression in neuronal cells and how its tyrosine phosphorylation is related to neurite outgrowth. Paxillin was identified in several neuronal cell lines with an increased level upon differentiation. In SH-SY5Y cells, paxillin was localized along with actin filaments where processes extended from the cell body and in neuritic growth cones. Furthermore, paxillin was tyrosine-phosphorylated in SH-SY5Y cells upon adhesion to laminin. Paxillin tyrosine phosphorylation paralleled that of focal adhesion kinase and occurred as cell spreading, and neurite formation was initiated. Colchicine blocked neurite outgrowth but had no effect on cell spreading or on paxillin or focal adhesion kinase tyrosine phosphorylation. In contrast, cytochalasin D eliminated neurite outgrowth, cell spreading, and the tyrosine phosphorylation of paxillin and focal adhesion kinase. These results show that paxillin is tyrosine-phosphorylated upon integrin ligand binding in neuronal cells. Our findings suggest that paxillin tyrosine phosphorylation is linked to a remodeling of the actin cytoskeleton that leads to cell spreading and neurite formation and thus a differentiated neuronal phenotype.

Actins↗
PubMed1996

Identification of tyrosine residues in the intracellular domain of the growth hormone receptor required for transcriptional signaling and Stat5 activation.

The binding of growth hormone (GH) to its receptor results in its dimerization followed by activation of Jak2 kinase and tyrosine phosphorylation of the GH receptor itself, as well as Jak2 and the transcription factors Stat1, -3, and -5. In order to study the role of GH receptor tyrosine phosphorylation in intracellular signaling, we constructed GH receptors in which combinations of tyrosines were mutated to phenylalanines. We identified three tyrosine residues at positions 534, 566, and 627 that were required for activation of GH-stimulated transcription of the serine protease inhibitor (Spi) 2.1 promoter. Any of these three tyrosines is able to independently mediate GH-induced transcription, indicating redundancy in this part of the GH receptor. Tyrosine phosphorylation was not required for GH stimulation of mitogen-activated protein (MAP) kinase activity or for GH-stimulated Ca2+ channel activation since these pathways were normal in cells expressing a GH receptor in which all eight intracellular tyrosines were mutated to phenylalanines. Activation of Stat5 by GH was, however, abolished in cells expressing the GH receptor lacking intracellular tyrosines. This study demonstrates that specific tyrosines in the GH receptor are required for transcriptional signaling possibly by their role in the activation of transcription factor Stat5.

Animals↗
PubMed1996

Identification of tyrosine 187 as a protein kinase C-delta phosphorylation site.

Protein kinase C-delta (PKC-delta) has been demonstrated to be phosphorylated on tyrosine residue(s) in many different biological systems (Li, W., Yu, J.-C., Michieli, P., Beeler, J. F., Ellmore, N., Heidaran, M. A., and Pierce, J. H. (1994) Mol. Cell. Biol. 14, 6727-6735; Li, W., Mischak, H., Yu, J.-C., Wang, L.-M., Mushinski, J. F., Heidaran, M. A., and Pierce, J. H. (1994) J. Biol. Chem. 269, 2349-2352; Denning, M. F., Dlugosz, A. A., Howett, M. A., and Yuspa, S. H. (1993) J. Biol. Chem. 268, 26079-26081). Tyrosine phosphorylation of PKC-delta has also been shown to occur in vitro when purified PKC-delta is coincubated with different tyrosine kinase sources. However, the tyrosine phosphorylation site(s) is currently unknown and the exact effect of this phosphorylation on its serine/threonine kinase activity and biological functions is still controversial. To directly investigate the potential role of PKC-delta tyrosine phosphorylation, tyrosine 187 was converted to phenylalanine (PKC-deltaY187F) by site-directed mutagenesis, and expression vectors containing PKC-deltaY187F cDNAs were transfected into both 32D myeloid progenitor cells and NIH 3T3 fibroblasts. The results showed that tyrosine 187 of PKC-delta became phosphorylated in vivo in response to 12-O-tetradecanoylphorbol-13-acetate stimulation or platelet-derived growth factor receptor activation. In vivo labeling and subsequent two-dimensional phosphopeptide analysis demonstrated that one phosphopeptide was absent in PKC-deltaY187F when compared to wild type PKC-delta, further substantiating that tyrosine 187 of PKC-delta is phosphorylated in vivo. Although the phosphotyrosine content of PKC-deltaY187F was reduced compared with PKC-deltaWT, the kinase activity of PKC-deltaY187F toward a PKC-delta substrate was not altered. Moreover, 12-O-tetradecanoylphorbol-13-acetate-mediated monocytic differentiation of 32D cells was not affected by expression of the PKC-deltaY187F mutant. Taken together, these results suggest that tyrosine phosphorylation of PKC-delta on 187 may not influence PKC-delta activation and known functions.

3T3 Cells↗
PubMed1999

Inhibition of hepatoma cell growth in vitro by arylating and non-arylating K vitamin analogs. Significance of protein tyrosine phosphatase inhibition.

We recently found that a thioether analog of K vitamin (Cpd 5) inhibited the activity of protein-tyrosine phosphatases (PTPases) and induced protein-tyrosine phosphorylation in a human hepatoma cell line (Hep3B). We have now examined the structural requirements for induction of protein-tyrosine phosphorylation and PTPase inhibition by several K vitamin analogs. Thioether analogs with sulfhydryl arylation capacity, especially those with a hydroxy (Cpd 5) or a methoxy group at the end of the side chain, induced protein-tyrosine phosphorylation, but non-arylating analogs, such as those with an all-carbon or O-ether side chain, did not. Among the receptor-tyrosine kinases, epidermal growth factor receptors were tyrosine-phosphorylated by treatment with thioether analogs, whereas insulin and hepatocyte growth factor receptors were not. An increase in tyrosine-phosphorylated ERK2 mitogen-activated protein kinase was also observed. The activity of purified T cell PTPase was inhibited only by the thioether analogs, but not by non-arylating analogs. Furthermore, the epidermal growth factor receptor dephosphorylation activity of Hep3B cell lysates was inhibited by Cpd 5 treatment. A similar induction of protein-tyrosine phosphorylation by Cpd 5 was seen in other human hepatoma cell lines together with growth inhibition. However, one cell line (HepG2), which was relatively resistant to growth inhibition by Cpd 5, did not increase its phosphorylation levels upon Cpd 5 treatment. These results suggest that cell growth inhibition by thioether analogs is closely associated with inhibition of PTPases by sulfhydryl arylation and with tyrosine phosphorylation of selected proteins.

Carcinoma, Hepatocellular↗
PubMed2001

Tyrosine phosphorylation of villin regulates the organization of the actin cytoskeleton.

We have previously shown that tyrosine phosphorylation of the actin-regulatory protein villin is accompanied by the redistribution of phosphorylated villin and a concomitant decrease in the F-actin content of intestinal epithelial cells. The temporal and spatial correlation of these two events suggested that tyrosine phosphorylation of villin may be involved in the rearrangement of the microvillar cytoskeleton. This hypothesis was investigated by analyzing the effects of tyrosine phosphorylation of villin on the kinetics of actin polymerization by reconstituting in vitro the tyrosine phosphorylation of villin and its association with actin. Full-length recombinant human villin was phosphorylated in vitro by expression in the TKX1-competent cells that carry an inducible tyrosine kinase gene. The actin-binding properties of villin were examined using a co-sedimentation assay. Phosphorylation of villin did not change the stoichiometry (1:2) but decreased the binding affinity (4.4 microm for unphosphorylated versus 0.6 microm for phosphorylated) of villin for actin. Using a pyrene-actin-based fluorescence assay, we demonstrated that tyrosine phosphorylation had a negative effect on actin nucleation by villin. In contrast, tyrosine phosphorylation enhanced actin severing by villin. Electron microscopic analysis showed complementary morphological changes. Phosphorylation inhibited the actin bundling and enhanced the actin severing functions of villin. Taken together our data show that tyrosine phosphorylation of villin decreases the amount of villin bound to actin filaments, inhibits the actin-polymerizing properties of villin, and promotes the actin-depolymerizing functions instead. These observations suggest a role for tyrosine phosphorylation in modulating the microvillar cytoskeleton in vivo by villin in response to specific physiological stimuli.

Actins↗
PubMed1991

Tissue-dependent regulation of protein tyrosine kinase activity during embryonic development.

Protein tyrosine kinase activity was assayed in a variety of chicken tissues during embryonic development and in the adult. In some tissues protein tyrosine kinase activity decreased during embryonic development; however, in other tissues it remained high throughout development, it contrast to the level of protein tyrosine phosphorylation, which decreased during development. The highest levels of tyrosine kinase activity were detected in 17-d embryonic brain although only low levels of protein tyrosine phosphorylation were observed in this tissue. Several alternatives were examined in an effort to determine the mechanism responsible for the low levels of tyrosine phosphorylated proteins in most older embryonic and adult chicken tissues despite the presence of highly active tyrosine kinases. The results show that the regulation of protein tyrosine phosphorylation during embryonic development is complex and varies from tissue to tissue. Furthermore, the results suggest that protein tyrosine phosphatases play an important role in regulating the level of phosphotyrosine in proteins of many older embryonic and adult tissues.

Animals↗
PubMed1992

Cadherin-mediated cell-cell adhesion is perturbed by v-src tyrosine phosphorylation in metastatic fibroblasts.

Rat 3Y1 cells acquire metastatic potential when transformed with v-src, and this potential is enhanced by double transformation with v-src and v-fos (Taniguchi, S., T. Kawano, T. Mitsudomi, G. Kimura, and T. Baba. 1986. Jpn. J. Cancer Res. 77:1193-1197). We compared the activity of cadherin cell adhesion molecules of normal 3Y1 cells with that of v-src transformed (SR3Y1) and v-src and v-fos double transformed (fosSR3Y1) 3Y1 cells. These cells expressed similar amounts of P-cadherin, and showed similar rates of cadherin-mediated aggregation under suspended conditions. However, the aggregates or colonies of these cells were morphologically distinct. Normal 3Y1 cells formed compacted aggregates in which cells are firmly connected with each other, whereas the transformed cells were more loosely associated, and could freely migrate out of the colonies. Overexpression of exogenous E-cadherin in these transformed cells had no significant effect on their adhesive properties. We then found that herbimycin A, a tyrosine kinase inhibitor, induced tighter cell-cell associations in the aggregates of the transformed cells. In contrast, vanadate, a tyrosine phosphatase inhibitor, inhibited the cadherin-mediated aggregation of SR3Y1 and fosSR3Y1 cells but had little effect on that of normal 3Y1 cells. These results suggest that v-src-mediated tyrosine phosphorylation perturbs cadherin function directly or indirectly, and the inhibition of tyrosine phosphorylation restores cadherin action to the normal state. We next studied tyrosine phosphorylation on cadherins and the cadherin-associated proteins, catenins. While similar amounts of catenins were expressed in all of these cells, the 98-kD catenin was strongly tyrosine phosphorylated only in SR3Y1 and fosSR3Y1 cells. Cadherins were also weakly tyrosine phosphorylated only in the transformed cells. The tyrosine phosphorylation of these proteins was enhanced by vanadate, and inhibited by herbimycin A. Thus, the tyrosine phosphorylation of the cadherin-catenin system itself might affect its function, causing instable cell-cell adhesion.

Animals↗
PubMed1993

Adhesion-dependent protein tyrosine phosphorylation in neutrophils treated with tumor necrosis factor.

Human neutrophils (PMN) respond to tumor necrosis factor (TNF) by releasing their granules, reorganizing their cytoskeleton, and massively secreting hydrogen peroxide. This response is dependent on adhesion to extracellular matrix proteins and expression of CD11b/CD18 integrins (Nathan, C., S. Srimal, C. Farber, E. Sanchez, L. Kabbash, A. Asch, J. Gailit, and S. D. Wright. 1989. J. Cell Biol. 109:1341-1349). We investigated the role of tyrosine phosphorylation in the response of PMN to TNF. PMN adherent to protein-coated surfaces but not suspended PMN showed tyrosine phosphorylation of several proteins (approximately 150, approximately 115, approximately 75, and approximately 65 kD) in response to TNF. Tyrosine phosphorylation was evident 5 min after addition of TNF and lasted at least 2 h. The tyrosine kinase inhibitors K252a, genistein and ST638 suppressed tyrosine phosphorylation and blocked hydrogen peroxide production in a reversible manner at low concentrations. Tyrosine kinase inhibitors also blocked the spreading of PMN in response to TNF. Dihydrocytochalasin B did not inhibit tyrosine phosphorylation, but in its presence phosphorylation was rapidly reversed. By immunocytochemistry, the majority of tyrosine phosphoproteins were localized to focal adhesions. Thus TNF-induced tyrosine phosphorylation depends on adhesion of PMN to extracellular matrix proteins, and participates in the transduction of the signals that direct the cells to spread on a biological surface and undergo a respiratory burst.

Carbazoles↗
PubMed1993

Tyrosine phosphorylation and association with phospholipase C gamma-1 of the GAP-associated 62-kD protein after CD2 stimulation of Jurkat T cell.

Numerous substrates are tyrosine phosphorylated upon CD2 stimulation of human Jurkat T cells using a mitogenic pair of CD2 monoclonal antibodies, including the phospholipase C (PLC)gamma-1-p35/36 complex. Most of these substrates are identically tyrosine phosphorylated after CD3 ligation, suggesting that both stimuli share the same biochemical pathway. We show, however, in this report that a 63-kD protein is specifically phosphorylated on tyrosine residues after ligation of the CD2 molecule. The tyrosine phosphorylation of p63 can be induced independently of other substrates when using a single CD2 mAb recognizing the D66 epitope of the molecule. Importantly, this CD2-induced tyrosine phosphorylation of p63 can also occur in the absence of the CD3 zeta chain membrane expression, and is also distinct from the protein tyrosine kinases p56lck and p59fyn. We demonstrate, moreover, that p63 is physically linked with PLC gamma-1 and p35/36 upon CD2 stimulation. Finally, we also show that a 62-kD protein coimmunoprecipitating with the p21ras GTPase activating protein (GAP) is heavily tyrosine phosphorylated only after CD2 stimulation. This ultimately suggests that p63 may represent in fact the 62-kD protein that associates with GAP after tyrosine phosphorylation. Taken together, these results demonstrate the occurrence in Jurkat cells of a tyrosine kinase pathway specifically coupled to the CD2 molecule. They also suggest a function of the p62-GAP-associated protein as a link between PLC gamma-1 and p21ras activation pathways after CD2 activation.

Antibodies, Monoclonal↗
PubMed1998

Twenty-four-hour L-[1-(13)C]tyrosine and L-[3,3-(2)H2]phenylalanine oral tracer studies at generous, intermediate, and low phenylalanine intakes to estimate aromatic amino acid requirements in adults.

Daily pattern and rates of whole-body tyrosine oxidation and phenylalanine hydroxylation were determined in young adults (15 men, 1 woman) receiving [13C]tyrosine and [(2)H2]phenylalanine via primed, constant oral infusion and [(2)H4]tyrosine by vein (five subjects also received [(2)H3]leucine simultaneously by vein) continuously for 24 h (12 h fast then 12 h fed). Subjects were given a diet supplying 96.6 (n = 5), 35.6 (the proposed requirement; n = 5), and 18.5 mg phenylalanine x kg(-1) x d(-1) (n = 6) based on an otherwise adequate L-amino acid mixture for 6 d before the 24-h tracer study began. [Each diet was low in tyrosine: 6.79 mg x kg(-1) x d(-1).] Our hypothesis was that subjects would be in tyrosine equilibrium, positive balance, or both, at the 96.6- and 35.6-mg intakes and in distinctly negative balance at the 18.5-mg intake. The diurnal pattern in phenylalanine and tyrosine kinetics was dependent on the intake and, presumably, on the adequacy of dietary phenylalanine. Wholebody tyrosine balances, determined from rates of phenylalanine hydroxylation and tyrosine input and oxidation were negative (0.05 < P < 0.1 from zero balance) with the low (18.5 mg) phenylalanine intake [total aromatic amino acid (AAA) intake: 25.3 mg x kg(-1) x d(-1)] but at equilibrium (P > 0.05 from zero balance) with the two higher phenylalanine intakes. Whole-body AAA balance (AAA intake - tyrosine oxidation) was negative (P < 0.05 from zero balance) with the low intake, at equilibrium with the intermediate intake, and apparently distinctly positive (P < 0.05) with the generous intake. Despite model limitations, as discussed, these findings lend further support for a proposed, tentative value for a total mean requirement of 39 mg AAA x kg(-1) x d(-1).

Adolescent↗
PubMed1975

Protein synthesis in liver, muscle, and brain of rats fed a high tyrosine-low protein diet.

Effects of feeding rats a high tyrosine-low protein diet on protein synthesis in liver, muscle, and brain were investigated both in vitro and in vivo. Tissue tyrosine concentrations of rats consuming the high tyrosine diet for 7 days were substantially elevated; this was accompanied by severe growth retardation. In the livers of rats fed the high tyrosine diet for 6 days, polysomal profiles showed a shift toward heavier ribosomal aggregates, while in muscle and brain, extensive disaggregation of polysomes occurred. In an in vitro amino acid incorporating system, the activities of both the microsomal and pH 5 enzyme fractions isolated from the livers of the high tyrosine animals that had been fed the diet for 6 weeks were elevated. On the other hand, the capacity of muscle or brain ribosomal preparations to incorporate [14-C] leucine was much reduced. Similar results were obtained in a study of [14-C] leucine incorporation in vivo in which rats were force-fed two meals and killed at various times after the last feeding. In rats fed the high tyrosine diet, incorporation of leucine into liver increased progressively; this was accompanied by a gradual decrease in leucine incorporation into muscle. In contrast, leucine incorporation into brain was immediately suppressed. In view of the apparently paradoxical effect of a high tyrosine load on protein synthesis in the liver, rates of the anabolic and catabolic phases of protein turnover in animals fed a high tyrosine diet were determined from radioactivity measurements made after pulse labeling them with [14-C] bicarbonate. Results indicated that the rates of both synthesis and degradation of liver proteins were elevated over control values. Differences in the effects of a toxic load of tyrosine on protein synthesis in the tissues examined could be the consequence of altered metabolic or hormonal balance as a result of nutritional stress.

Amino Acids↗
PubMed1999

Regulation of human sperm capacitation by a cholesterol efflux-stimulated signal transduction pathway leading to protein kinase A-mediated up-regulation of protein tyrosine phosphorylation.

Protein tyrosine phosphorylation is an important intracellular event accompanying the in-vitro capacitation of mouse, bovine and human spermatozoa. Here, we demonstrate that bovine serum albumin (BSA) and NaHCO(3) are required for protein tyrosine phosphorylation in ejaculated human spermatozoa. The absence of protein tyrosine phosphorylation in media minus these two constituents could be recovered by addition to the media of cAMP analogues and/or phosphodiesterase inhibitors. Since BSA is postulated to modulate capacitation by removal of cholesterol from the sperm plasma membrane, we determined whether cholesterol release leads to changes in protein tyrosine phosphorylation. Incubation of spermatozoa in media containing BSA resulted in the release of significant amounts of cholesterol when compared with media devoid of BSA. Preloading BSA with cholesterol-SO(4) inhibited protein tyrosine phosphorylation, as well as capacitation, and this inhibitory effect was overcome by the addition of dibutyryl cAMP plus isobutylmethylxanthine (IBMX). The functional significance of BSA-mediated cholesterol release, protein tyrosine phosphorylation and capacitation was confirmed by examining the effects of the cholesterol-binding heptasaccharides, methyl-beta-cyclodextrin or OH-propyl-beta-cyclodextrin. Both cyclodextrins caused cholesterol efflux from the spermatozoa, increased protein tyrosine phosphorylation, and stimulated capacitation. Therefore, cholesterol release is associated with the activation of a signal transduction pathway involving protein kinase A and tyrosine kinase second messenger systems, and resulting in protein tyrosine phosphorylation and capacitation.

Biological Transport↗
PubMed1982

Affinity chromatography of thyroid peroxidase using tyrosine coupled to Agarose.

A selective adsorbent for thyroid peroxidase was prepared by attaching tyrosine, a possible substrate of peroxidase, to agarose beads. When partially purified calf thyroid peroxidase was passed through a column containing this adsorbent, the peroxidase activity present was bound to the agarose. The binding of thyroid peroxidase on the adsorbent was inhibited by tyrosine and iodotyrosines. Quantitative elution was readily achieved by modifying the pH of eluting buffers. The peroxidase eluted at pH 8.5 or pH 9.8 was slowly inactivated. During this inactivation, enzyme activity assayed by triiodide formation was not affected, while peroxidase activity assayed by guaiacol oxidation and tyrosine iodination were slowly reduced. Enzyme activity was protected by elution under a partially anaerobic state. Iodide and guaiacol did not interfere with the adsorption of thyroid peroxidase by tyrosine residues on agarose. These data indicate the following characteristics of thyroid peroxidase. 1. Tyrosine and iodotyrosines are the substrates of thyroid peroxidase. 2. Thyroid peroxidase has specific active site(s) for tyrosine and iodide which are independent of each other. 3. The active site(s) for tyrosine and iodotyrosines are common. 4. The active site(s) for tyrosine and guaiacol are similar but are not identical. 5. Thyroid peroxidase is able to bind tyrosine before it is activated to "Complex I" by hydrogen peroxide.

Animals↗
PubMed2003

Hyperosmolarity and CD95L trigger CD95/EGF receptor association and tyrosine phosphorylation of CD95 as prerequisites for CD95 membrane trafficking and DISC formation.

The mechanisms underlying CD95 ligand (CD95L)- and hyperosmolarity-induced activation of the CD95 system [Reinehr, R., Graf, D., Fischer, R., Schliess, F., and Haussinger, D. (2002) Hepatology 36, 602-614] as initial steps of apoptosis were studied. Hyperosmotic exposure (405 mosmol/l) of rat hepatocytes induced within 1 min oxidative stress and antioxidant-sensitive activation of the epidermal growth factor receptor (EGFR) and c-Jun-N-terminal-kinase (JNK). After 30 min of hyperosmotic exposure EGFR associated with CD95 and CD95 became tyrosine phosphorylated. Inhibition of JNK or protein kinase C (PKC) had no effect on EGFR phosphorylation but abolished CD95/EGFR association, CD95-tyrosine phosphorylation, membrane targeting, and Fas-associated death domain/caspase 8 recruitment to CD95 [death-inducing signaling complex (DISC) formation]. Inhibition of EGFR tyrosine kinase activity prevented CD95 tyrosine phosphorylation and DISC formation but not hyperosmolarity-induced EGFR phosphorylation and EGFR association with CD95. Tyrosine-phosphorylated CD95 was enriched in the plasma membrane. All maneuvers preventing CD95 tyrosine phosphorylation inhibited CD95 membrane trafficking and DISC formation. Stimulation of EGFR by EGF induced EGFR phosphorylation but no association with CD95 or CD95 phosphorylation. Addition of CD95L also induced EGFR and JNK activation, EGFR/CD95 association, CD95 tyrosine phosphorylation, DISC formation, and CD95 membrane targeting with an inhibitor sensitivity profile similar to that of hyperosmotic CD95 activation, except that inhibition of PKC was ineffective. The data suggest that moderate hyperosmolarity or CD95L trigger oxidative stress and EGFR activation followed by a JNK-dependent EGFR/CD95association and CD95 tyrosine phosphorylation, probably through EGFR tyrosine kinase activity. This provides a signal for CD95 membrane trafficking and DISC formation.

Animals↗