Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “TYROSINE”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 289 records · Page 16Linked to original sources
PubMed2004

Simultaneous determination of 3-nitro tyrosine, o-, m-, and p-tyrosine in urine samples by liquid chromatography-ultraviolet absorbance detection with pre-column cloud point extraction.

Stable 3-nitro tyrosine (3-NO(2)-Tyr), o-, m-, and p-tyrosine isomers induced by oxidation of tyrosine residues in protein were considered important biomarkers for the existence of toxic oxidizing agents peroxynitrite (ONOO(-)) and OH*, which could lead to such diseases as acute lung injury, neurodegenerative disorders, atherosclerosis, cancers and many other diseases. Therefore, development of an accurate, simple and sensitive method to simultaneously detect o-, m-, and p-tyrosine and 3-NO(2)-Tyr is necessary. Fluorescence detection is highly sensitive to o-, m-, and p-tyrosine, but it cannot be used to detect 3-NO(2)-Tyr, due to the strong fluorescence-quenching characteristic of the NO(2) group. In this study, we developed a highly sensitive reversed HPLC-UV method, combined with pre-column cloud point extraction (CPE), to simultaneously determine o-, m-, and p-tyrosine and 3-NO(2)-Tyr. The procedure included derivatization of a sample with 6-aminoquinolyl-N-hydroxy-succinimidyl carbomate (AccQ) at 0.20 mol/l borate buffer (pH 8.80) for 30 min at 70 degrees C, and pre-concentration with surfactant cloud point extraction. The surfactant-rich phase was then diluted with deionized water and injected directly into the to HPLC column for analysis. A C(18) column (3.9 mm i.d. x 300 mm) was used for gradient elution separation at 25 degrees C and the detection wavelength was at 254 nm. Nineteen general amino acids showed no interference. The detection limits of p-, o-, m-Tyr and 3-NO(2)-Tyr were between 5 and 15 nmol/l. The linear range was from 0.05 to approximately 100 micromol/l.

Chromatography, High Pressure Liquid↗
PubMed1988

Isolation and characterization of a bovine liver tyrosine-O-sulfate-binding protein--a putative receptor molecular for tyrosine-sulfated proteins?

Golgi-enriched microsomal membrane fraction was prepared from bovine liver. Sodium choleate extract of this membrane preparation was subjected to fractionation using Sepharose gel covalently bonded with tyrosine-O-sulfate. SDS gel electrophoresis of the fractionated sample revealed the presence of a major protein with an apparent molecular weight of 175,000. The protein appears to be specific for tyrosine-O-sulfate as it binds neither the unmodified tyrosine nor the structurally similar tyrosine-O-phosphate. pH-dependence study showed the binding of the protein to tyrosine-O-sulfate-Sepharose gel to be strong from pH 8.0 down through 6.0. At pH 5.5, the binding affinity became dramatically reduced. A similar tyrosine-O-sulfate-binding protein was also detected in the choleate extracts of the Golgi-enriched microsomal membrane fractions prepared from bovine pancreas and from both liver and pancreas of dog.

Animals↗
PubMed1997

Ovulation rate and the concentrations of LH, FSH, GH, prolactin and insulin in ewes infused with tryptophan, tyrosine or tyrosine plus phenylalanine during the luteal phase of the oestrous cycle.

Dietary amino acid precursors for cathecholamineric and serotonergic neurotransmitters may be important in the mechanism of nutritional effects on ovulation rate. This paper reports the results of three experiments that examined the effect of such amino acids on ovulation rate and the concentrations of FSH and LH in sheep. In three separate experiments, groups of ewes were infused, over Days 9 to 13 of the oestrous cycle, with either tryptophan (n = 11), tyrosine (n = 11) or a mixture of tyrosine and phenylalanine (n = 11). Control ewes (n = 12 in each experiment) were infused with a vehicle over the same period. None of the amino acids infused effected ovulation rate or plasma concentrations of LH, FSH, GH or prolactin. The infusion of a mixture of tyrosine and phenylalanine increased insulin concentrations. The infusion of these amino acids was not associated with changes in gonadotrophin concentrations and therefore the effect of nutrition on ovulation rate in ewes does not seem to involve an increase in the availability of tryptophan, tyrosine or phenylalanine. Increasing the uptake of other amino acids that compete with tryptophan, tyrosine or phenylalanine for the large neutral amino acid transporter may cause a decrease in the availability of tryptophan, tyrosine or phenylalanine thereby eliciting the effects of nutrition on ovulation rate. However, this hypothesis remains to be tested.

Animals↗
PubMed2002

Signal transduction by protein tyrosine nitration: competition or cooperation with tyrosine phosphorylation-dependent signaling events?

This review article is an attempt to stimulate a discussion on the significance of protein tyrosine nitration to cellular signaling and its relationships with protein tyrosine phosphorylation. Initially, it provides basic information on growth factor and oxidants as modulators/mediators of tyrosine phosphorylation-dependent signal transduction pathways. The effects of exogenous and endogenous tyrosine nitration on such pathways were examined by reviewing published and unpublished observations. From an initial perspective that tyrosine nitration was a toxic manifestation of nitric oxide, the concept evolved to a protein modification that could also function in cellular signaling events, possibly cooperating with tyrosine phosphorylation.

Animals↗
PubMed2001

Nitration and oxidation of a hydrophobic tyrosine probe by peroxynitrite in membranes: comparison with nitration and oxidation of tyrosine by peroxynitrite in aqueous solution.

It has been reported that peroxynitrite will initiate both oxidation and nitration of tyrosine, forming dityrosine and nitrotyrosine, respectively. We compared peroxynitrite-dependent oxidation and nitration of a hydrophobic tyrosine analogue in membranes and tyrosine in aqueous solution. Reactions were carried out in the presence of either bolus addition or slow infusion of peroxynitrite, and also using the simultaneous generation of superoxide and nitric oxide. Results indicate that the level of nitration of the hydrophobic tyrosyl probe located in a lipid bilayer was significantly greater than its level of oxidation to the corresponding dimer. During slow infusion of peroxynitrite, the level of nitration of the membrane-incorporated tyrosyl probe was greater than that of tyrosine in aqueous solution. Evidence for hydroxyl radical formation from decomposition of peroxynitrite in a dimethylformamide/water mixture was obtained by electron spin resonance spin trapping. Mechanisms for nitration of the tyrosyl probe in the membrane are discussed. We conclude that nitration but not oxidation of a tyrosyl probe by peroxynitrite is a predominant reaction in the membrane. Thus, the local environment of target tyrosine residues is an important factor governing its propensity to undergo nitration in the presence of peroxynitrite. This work provides a new perspective on selective nitration of membrane-incorporated tyrosine analogues.

Bicarbonates↗
PubMed1986

Time-resolved fluorescence and 1H NMR studies of tyrosine and tyrosine analogues: correlation of NMR-determined rotamer populations and fluorescence kinetics.

The time-resolved fluorescence properties of phenol and straight-chained phenol derivatives and tyrosine and simple tyrosine derivatives are reported for the pH range below neutrality. Phenol and straight-chained phenol derivatives exhibit single exponential fluorescence decay kinetics in this pH range unless they have a titratable carboxyl group. If a carboxyl group is present, the data follow a two-state, ground-state, Henderson-Hasselbalch relationship. Tyrosine and its derivatives with a free carboxyl group display complex fluorescence decay behavior as a function of pH. The complex kinetics cannot be fully explained by titration of a carboxyl group; other ground-state processes are evident, especially since tyrosine analogues with a blocked carboxyl group are also multiexponential. The fluorescence kinetics can be explained by a ground-state rotamer model. Comparison of the preexponential weighting factors (amplitudes) of the fluorescence decay constants with the 1H NMR determined phenol side-chain rotamer populations shows that tyrosine derivatives with a blocked or protonated carboxyl group have at least one rotamer exchanging more slowly than the radiative and nonradiative rates, and the fluorescence data are consistent with a slow-exchange model for all three rotamers, the shortest fluorescence decay constant is associated with a rotamer where the carbonyl group can contact the phenol ring, and in the tyrosine zwitterion, either rotamer interconversion is fast and an average lifetime is seen or rotamer interconversion is slow and the individual fluorescence decay constants are similar.

Hydrogen-Ion Concentration↗
PubMed2006

Mechanistic studies of peroxynitrite-mediated tyrosine nitration in membranes using the hydrophobic probe N-t-BOC-L-tyrosine tert-butyl ester.

Most of the mechanistic studies of tyrosine nitration have been performed in aqueous solution. However, many protein tyrosine residues shown to be nitrated in vitro and in vivo are associated to nonpolar compartments. In this work, we have used the stable hydrophobic tyrosine analogue N-t-BOC-L-tyrosine tert-butyl ester (BTBE) incorporated into phosphatidylcholine (PC) liposomes to study physicochemical and biochemical factors that control peroxynitrite-dependent tyrosine nitration in phospholipid bilayers. Peroxynitrite leads to maximum 3-nitro-BTBE yields (3%) at pH 7.4. In addition, small amounts of 3,3'-di-BTBE were formed at pH 7.4 (0.02%) which increased over alkaline pH; at pH 6, a hydroxylated derivative of BTBE was identified by HPLC-MS analysis. BTBE nitration yields were similar in dilauroyl- and dimyristoyl-PC and were also significant in the polyunsaturated fatty acid-containing egg PC. *OH and *NO2 scavengers inhibited BTBE nitration. In contrast to tyrosine in the aqueous phase, the presence of CO2 decreased BTBE nitration, indicating that CO3*- cannot permeate to the compartment where BTBE is located. On the other hand, micromolar concentrations of hemin and Mn-tccp strongly enhanced BTBE nitration. Electron spin resonance (ESR) detection of the BTBE phenoxyl radical and kinetic modeling of the pH profiles of BTBE nitration and dimerization were in full agreement with a free radical mechanism of oxidation initiated by ONOOH homolysis in the immediacy of or even inside the bilayer and with a diffusion coefficient of BTBE phenoxyl radical 100 times less than for the aqueous phase tyrosyl radical. BTBE was successfully applied as a hydrophobic probe to study nitration mechanisms and will serve to study factors controlling protein and lipid nitration in biomembranes and lipoproteins.

Carbon Dioxide↗
PubMedDate not supplied

Electropolymerized films formed from the amphiphilic decyl esters of D- and L-tyrosine compared to L-tyrosine using the electrochemical quartz crystal microbalance.

Using the electrochemical quartz crystal microbalance (EQCM), we compared thin films formed on Pt by electropolymerization of l-tyrosine to that of the amphiphilic monomers, decyl esters of d- and l-tyrosine (DEDT and DELT). Mass build-up and film properties were determined as a function of monomer concentration via frequency, f, motional resistance, R, and charge passage, Q, measurements. Films were found to occur by a combination of monomer electropolymerization and adsorption for DEDT and DELT, but only by electropolymerization for l-tyrosine. This difference in film formation process for the monomers is reflected in the net mass build-up for each film, as represented by calculated df/dQ values. For the adsorbing monomers DEDT and DELT, films possessed concentration dependent df/dQ values, more than 100-fold greater than that for l-tyrosine film formation under equivalent electropolymerization conditions. During the entire film growth process, all three films exhibited no significant energy dissipation properties (DeltaR invariant). Concentration dependent adsorption of significant levels of unpolymerized but self-assembled DEDT and DELT monomers account for the subsequent time dependent mass loss observed from the films maintained in buffer in the absence of monomer. Contact angle measurements demonstrated a pH dependent increase in the surface hydrophilicity of films electropolymerized from the DEDT, DELT, and l-tyrosine monomers but not films formed from phenol and 3-nitrophenol monomers. This behavior is consistent with the monomers' known changes in titration/charge state properties with increasing pH. This study provided insight into the film formation, stability, and surface hydrophilicity resulting from electropolymerization of these related tyrosine based monomers. This information is critical to assessing the utility these films may have in the development of new biomaterials and as biological macromolecule or cell immobilization strategies in biosensors.

Electrochemistry↗
PubMed2003

Mass mapping sites of nitration in tyrosine hydroxylase: random vs selective nitration of three tyrosine residues.

Previous work has shown that tyrosine residues Y423, Y428, and Y432 are potential targets for nitration when tyrosine hydroxylase is exposed to peroxynitrite. For any given protein molecule, up to three nitration events involving these residues may occur. In an effort to determine whether this nitration is directed toward a particular tyrosine residue or randomly distributed among all three tyrosine residues, the goal of this study was to determine whether a singly nitrated molecule was always nitrated at a particular tyrosine residue or whether the initial nitration event was randomly distributed among all three tyrosine residues. Isolation of the singly nitrated peptide V410-E436, followed by subsequent cleavage at D425 and analysis of the resulting peptide fragments by matrix-assisted laser desorption ionization mass spectrometry and LC/MS/MS revealed that the first nitration event was randomly distributed among Y423, Y428, and Y432.

Binding Sites↗
PubMed1987

Tyrosine and tyrosinate fluorescence of pig intestinal Ca2+-binding protein.

The single tyrosine residue in both pig and cow intestinal Ca2+-binding proteins fluoresces at 303 nm although the crystal structure of the cow protein shows a hydrogen bond between the hydroxy group of the tyrosine and glutamate-38 [Szebenyi & Moffat (1986) J. Biol. Chem. 261, 8761-8777]. The latter interaction suggests that tyrosinate fluorescence should dominate the emission spectra of these proteins. A fluorescence difference spectrum, produced by subtracting the spectrum of free tyrosine from the spectrum of the protein, gives a peak at 334 nm due to ionized tyrosine. That this component of the emission spectrum is not due to a tryptophan-containing contaminant is shown by its elimination when the protein is denatured by guanidine and when glutamate-38 is protonated. We conclude that, in solution, the tyrosine residue in this protein interacts occasionally with glutamate-38 but that a permanent hydrogen bond is not formed.

Animals↗
PubMed1992

Fc epsilon RI-mediated tyrosine phosphorylation and activation of the 72-kDa protein-tyrosine kinase, PTK72, in RBL-2H3 rat tumor mast cells.

In RBL-2H3 rat tumor mast cells, cross-linking the high-affinity IgE receptor Fc epsilon RI causes tyrosine phosphorylation of multiple proteins. These phosphoproteins include phospholipase C gamma 1, the beta and gamma subunits of the Fc epsilon RI, the Src family protein-tyrosine kinase Lyn, and a 72-kDa protein that coimmunoprecipitates from lysates of antigen-stimulated cells with antibody to the receptor beta subunit. We now present evidence that the 72-kDa Fc epsilon RI-associated protein is the protein-tyrosine kinase PTK72 that forms part of the antigen receptor complex in B lymphocytes. The identification is based on immunoreactivity with anti-PTK72 antiserum, chromatographic profiles on the affinity resin heparin/agarose, and one-dimensional phosphopeptide mapping studies. Enzymatic activity of the kinase is increased in anti-PTK72 immune complexes prepared from lysates of antigen-activated RBL-2H3 cells. The 72-kDa protein-tyrosine kinase is the principal substrate for in vitro tyrosine phosphorylation in anti-phosphotyrosine immunoprecipitates of RBL-2H3 cells. The discovery that RBL-2H3 mast cells share a receptor-activated protein-tyrosine kinase, PTK72, with B lymphocytes provides additional support for the existence of common signaling pathways initiated by multichain immune recognition receptors.

Animals↗
PubMed1997

The related adhesion focal tyrosine kinase is tyrosine-phosphorylated after beta1-integrin stimulation in B cells and binds to p130cas.

Integrin ligation initiates intracellular signaling events, among which are the activation of protein tyrosine kinases. The related adhesion focal tyrosine kinase (RAFTK), also known as PYK2 and CAKbeta, is a tyrosine kinase that is homologous to the focal adhesion kinase (FAK) p125FAK. The structure of RAFTK is similar to p125FAK in that it lacks a transmembrane region, does not contain Src homology 2 or 3 domains, and has a proline-rich region in its C terminus. Here we report that RAFTK is a target for beta1-integrin-mediated tyrosine phosphorylation in both transformed and normal human B cells. Ligation of the B cell antigen receptor also induced tyrosine phosphorylation of RAFTK. Phosphorylation of RAFTK following integrin- or B cell antigen receptor-mediated stimulation was decreased by prior treatment of cells with cytochalasin B, indicating that this process was at least partially cytoskeleton-dependent. One of the tyrosine-phosphorylated substrates after integrin stimulation in fibroblasts is p130cas, which can associate with p125FAK. RAFTK also interacted constitutively with p130cas in B cells, since p130cas was detected in RAFTK immunoprecipitates. Although the function of RAFTK remains unknown, these data suggest that RAFTK may have a significant function in integrin-mediated signaling pathways in B cells.

B-Lymphocytes↗
PubMed1997

Tyrosine phosphorylation of the novel protein-tyrosine kinase RAFTK during an early phase of platelet activation by an integrin glycoprotein IIb-IIIa-independent mechanism.

A key regulatory event controlling platelet activation is mediated through the phosphorylation of several cellular proteins by protein-tyrosine kinases. The related adhesion focal tyrosine kinase (RAFTK) is a novel cytoplasmic tyrosine kinase and a member of the focal adhesion kinase (FAK) gene family. FAK phosphorylation in platelets is integrin-dependent, occurs in a late stage of platelet activation, and is dependent on platelet aggregation. In this study, we have investigated the involvement of RAFTK phosphorylation during different stages of platelet activation. Treatment of platelets with thrombin induced, in as early as 10 s, a rapid tyrosine phosphorylation of RAFTK in a time- and concentration-dependent manner. Treatment of platelets with thrombin in the absence of stirring or pretreatment of platelets with RGDS peptide prevented platelet aggregation, but not RAFTK phosphorylation. Furthermore, phosphorylation of RAFTK did not require integrin engagement since platelets treated with the 7E3 inhibitory antibodies that block fibrinogen binding to glycoprotein IIb-IIIa did not inhibit RAFTK phosphorylation. Similarly, platelets treated with LIBS6 antibodies, which specifically activate glycoprotein IIb-IIIa, did not induce RAFTK phosphorylation. Stimulation of platelets by several agonists such as collagen, ADP, epinephrine, and calcium ionophore A23187 induced RAFTK phosphorylation. Tyrosine phosphorylation of RAFTK in platelets is regulated by calcium and is mediated through the protein kinase C pathway. Phosphorylation of RAFTK is dependent upon the formation of actin cytoskeleton as disruption of actin polymerization by cytochalasin D significantly inhibited this phosphorylation. The RAFTK protein appears to be proteolytically cleaved by calpain in an aggregation dependent manner upon thrombin stimulation. These results demonstrate that RAFTK is tyrosine-phosphorylated during an early phase of platelet activation by an integrin- independent mechanism and is not dependent on platelet aggregation, suggesting different mechanisms of regulation for FAK and RAFTK phosphorylation during platelet activation.

Adenosine Diphosphate↗
PubMed1999

Evidence for a calpeptin-sensitive protein-tyrosine phosphatase upstream of the small GTPase Rho. A novel role for the calpain inhibitor calpeptin in the inhibition of protein-tyrosine phosphatases.

Activation of the thiol protease calpain results in proteolysis of focal adhesion-associated proteins and severing of cytoskeletal-integrin links. We employed a commonly used inhibitor of calpain, calpeptin, to examine a role for this protease in the reorganization of the cytoskeleton under a variety of conditions. Calpeptin induced stress fiber formation in both forskolin-treated REF-52 fibroblasts and serum-starved Swiss 3T3 fibroblasts. Surprisingly, calpeptin was the only calpain inhibitor of several tested with the ability to induce these effects, suggesting that calpeptin may act on targets besides calpain. Here we show that calpeptin inhibits tyrosine phosphatases, enhancing tyrosine phosphorylation particularly of paxillin. Calpeptin preferentially inhibits membrane-associated phosphatase activity. Consistent with this observation, in vitro phosphatase assays using purified glutathione S-transferase fusion proteins demonstrated a preference for the transmembrane protein-tyrosine phosphatase-alpha over the cytosolic protein-tyrosine phosphatase-1B. Furthermore, unlike wide spectrum inhibitors of tyrosine phosphatases such as pervanadate, calpeptin appeared to inhibit a subset of phosphatases. Calpeptin-induced assembly of stress fibers was inhibited by botulinum toxin C3, indicating that calpeptin is acting on a phosphatase upstream of the small GTPase Rho, a protein that controls stress fiber and focal adhesion assembly. Not only does this work reveal that calpeptin is an inhibitor of protein-tyrosine phosphatases, but it suggests that calpeptin will be a valuable tool to identify the phosphatase activity upstream of Rho.

3T3 Cells↗
PubMed2000

Dityrosine formation outcompetes tyrosine nitration at low steady-state concentrations of peroxynitrite. Implications for tyrosine modification by nitric oxide/superoxide in vivo.

Formation of peroxynitrite from NO and O-(*2) is considered an important trigger for cellular tyrosine nitration under pathophysiological conditions. However, this view has been questioned by a recent report indicating that NO and O-(*2) generated simultaneously from (Z)-1-(N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino) diazen-1-ium-1,2-diolate] (SPER/NO) and hypoxanthine/xanthine oxidase, respectively, exhibit much lower nitrating efficiency than authentic peroxynitrite (Pfeiffer, S. and Mayer, B. (1998) J. Biol. Chem. 273, 27280-27285). The present study extends those earlier findings to several alternative NO/O-(*2)-generating systems and provides evidence that the apparent lack of tyrosine nitration by NO/O-(*2) is due to a pronounced decrease of nitration efficiency at low steady-state concentrations of authentic peroxynitrite. The decrease in the yields of 3-nitrotyrosine was accompanied by an increase in the recovery of dityrosine, showing that dimerization of tyrosine radicals outcompetes the nitration reaction at low peroxynitrite concentrations. The observed inverse dependence on peroxynitrite concentration of dityrosine formation and tyrosine nitration is predicted by a kinetic model assuming that radical formation by peroxynitrous acid homolysis results in the generation of tyrosyl radicals that either dimerize to yield dityrosine or combine with (*)NO(2) radical to form 3-nitrotyrosine. The present results demonstrate that very high fluxes (>2 microM/s) of NO/O-(*2) are required to render peroxynitrite an efficient trigger of tyrosine nitration and that dityrosine is a major product of tyrosine modification caused by low steady-state concentrations of peroxynitrite.

Dimerization↗
PubMed2002

STAT3 activation by type I interferons is dependent on specific tyrosines located in the cytoplasmic domain of interferon receptor chain 2c. Activation of multiple STATS proceeds through the redundant usage of two tyrosine residues.

Human type I interferons (IFNs) play an important role in the regulation of antiviral defense mechanisms, immunomodulatory activities, and growth control. Recent efforts have demonstrated the importance of IFNs in the activation of signal transducers and activators of transcription (STATs). The role of STAT1 and STAT2 in IFN-dependent JAK-STAT signaling is well established; however, the role of STAT3 and its activation by IFNs remains unclear. Understanding the IFN-dependent regulation of STAT3 is of increasing interest because recent studies have demonstrated that STAT3 may play a role in cancer. Studies have revealed that STAT3 is constitutively active in a number of cancer cell lines and that overexpression of an active form of STAT3 transforms normal fibroblasts. Therefore, STAT3 exhibits properties indicative of known oncogenes. In this report, we define the role of the type I IFN receptor in STAT3 activation and identify for the first time tyrosine residues present in the cytoplasmic domain of IFNAR2c that are critical for STAT3 activation. The regulation of STAT3 activation by IFNs was measured in a human lung fibrosarcoma cell line lacking IFNAR2c but stably expressing various IFNAR2c tyrosine mutants. We show here that in addition to IFN-dependent tyrosine phosphorylation of STAT3, activation using a STAT3-dependent electrophoretic mobility shift assay and a STAT3-specific reporter can also be demonstrated. Furthermore, we demonstrate that type I IFN-dependent activation of STAT3 proceeds through a novel mechanism that is dependent on two tyrosines, Tyr(337) and Tyr(512), present in IFNAR2c and contained within a conserved six-amino acid residue motif, GxGYxM. Surprisingly, both tyrosines were previously shown to be required for type I IFN-dependent STAT1 and STAT2 activation. Our results reveal that type I IFNs activate multiple STATs via the overlapping usage of two tyrosine residues located in the cytoplasmic domain of IFNAR2c.

Base Sequence↗
PubMed2004

A novel proteomic approach for specific identification of tyrosine kinase substrates using [13C]tyrosine.

Proteomic studies to find substrates of tyrosine kinases generally rely on identification of protein bands that are "pulled down" by antiphosphotyrosine antibodies from ligand-stimulated samples. One can obtain erroneous results from such experiments because of two major reasons. First, some proteins might be basally phosphorylated on tyrosine residues in the absence of ligand stimulation. Second, proteins can bind non-specifically to the antibodies or the affinity matrix. Induction of phosphorylation of proteins by ligand must therefore be confirmed by a different approach, which is not always feasible. We have developed a novel proteomic approach to identify substrates of tyrosine kinases in signaling pathways studies based on in vivo labeling of proteins with "light" (12C-labeled) or "heavy" (13C-labeled) tyrosine. This stable isotope labeling in cell culture method enables the unequivocal identification of tyrosine kinase substrates, as peptides derived from true substrates give rise to a unique signature in a mass spectrometry experiment. By using this approach, from a single experiment, we have successfully identified several known substrates of insulin signaling pathway and a novel substrate, polymerase I and transcript release factor, a protein that is implicated in the control of RNA metabolism and regulation of type I collagen promoters. This approach is amenable to high throughput global studies as it simplifies the specific identification of substrates of tyrosine kinases as well as serine/threonine kinases using mass spectrometry.

3T3-L1 Cells↗
PubMed1994

Induction of tyrosine phosphorylation during ICAM-3 and LFA-1-mediated intercellular adhesion, and its regulation by the CD45 tyrosine phosphatase.

Intercellular adhesion molecule (ICAM)-3, a recently described counter-receptor for the lymphocyte function-associated antigen (LFA)-1 integrin, appears to play an important role in the initial phase of immune response. We have previously described the involvement of ICAM-3 in the regulation of LFA-1/ICAM-1-dependent cell-cell interaction of T lymphoblasts. In this study, we further investigated the functional role of ICAM-3 in other leukocyte cell-cell interactions as well as the molecular mechanisms regulating these processes. We have found that ICAM-3 is also able to mediate LFA-1/ICAM-1-independent cell aggregation of the leukemic JM T cell line and the LFA-1/CD18-deficient HAFSA B cell line. The ICAM-3-induced cell aggregation of JM and HAFSA cells was not affected by the addition of blocking mAb specific for a number of cell adhesion molecules such as CD1 1a/CD18, ICAM-1 (CD54), CD2, LFA-3 (CD58), very late antigen alpha 4 (CD49d), and very late antigen beta 1 (CD29). Interestingly, some mAb against the leukocyte tyrosine phosphatase CD45 were able to inhibit this interaction. Moreover, they also prevented the aggregation induced on JM T cells by the proaggregatory anti-LFA-1 alpha NKI-L16 mAb. In addition, inhibitors of tyrosine kinase activity also abolished ICAM-3 and LFA-1-mediated cell aggregation. The induction of tyrosine phosphorylation through ICAM-3 and LFA-1 antigens was studied by immunofluorescence, and it was found that tyrosine-phosphorylated proteins were preferentially located at intercellular boundaries upon the induction of cell aggregation by either anti-ICAM-3 or anti-LFA-1 alpha mAb. Western blot analysis revealed that the engagement of ICAM-3 or LFA-1 with activating mAb enhanced tyrosine phosphorylation of polypeptides of 125, 70, and 38 kD on JM cells. This phenomenon was inhibited by preincubation of JM cells with those anti-CD45 mAb that prevented cell aggregation. Altogether these results indicate that CD45 tyrosine phosphatase plays a relevant role in the regulation of both intracellular signaling and cell adhesion induced through ICAM-3 and beta 2 integrins.

Antibodies, Monoclonal↗