Stimulation of tyrosine phosphorylation in lectin treated human lymphocytes

Large increases in tyrosine phosphorylation have been detected in subcellular matrixes isolated from lectin treated human lymphocytes. In lectin stimulated cells proteins of molecular weight 105, 75, 58 and 35 kDa contained phosphotyrosine (P-tyr) whereas non-stimulated cells had no 105 and low levels of P-tyr in proteins of 75, 58 and 35 kDa. In stimulated cells increased tyrosine kinase activity was also shown using gastrin as substrate. In both stimulated and non-stimulated cells the 58 kDa phosphoprotein was the most heavily labelled, after partial proteolysis of the 58 kDa different phosphopeptides were generated. A peptide with a sequence analogous to the autophosphorylated tyrosine site of pp60src inhibited tyrosine phosphorylation in stimulated cells. The lymphocyte system provides a useful tool to study normal tyrosine protein kinases and their role in cellular proliferation.     

Incorporation of sulphate into type III procollagen by cultured human fibroblasts. Identification of tyrosine O-sulphate

Confluent cultures of normal human skin fibroblasts were labelled overnight with [35S]sulphate, and the incorporation of the isotope into type III procollagen, secreted into the medium, was verified by radioimmunoassay and immunoprecipitation after removing the heavily sulphated proteoglycans by anion-exchange chromatography. Type III procollagen and its pro and pN alpha chains were visualized in fluorographs of the immunoprecipitates. The labelled procollagen could be isolated by a combination of ion-exchange chromatography and gel filtration and was found to contain tyrosine O-sulphate, which was identified by thin-layer electrophoresis after Ba(OH)2 hydrolysis. The regions sulphated in the type III procollagen molecule were susceptible to pepsin digestion. Digestion with purified bacterial collagenase at +37 degrees C produced a labelled fragment that was recognized by antibodies against the aminoterminal propeptide of type III procollagen, indicating that the sulphated tyrosine residues are located either in this propeptide or in the non-helical telopeptide region of the type III collagen molecule proper. Sulphation of tyrosine residues is a new post-translational modification in procollagen, which could be involved in the regulation of the processing of type III procollagen into collagen and thus affect the formation of collagen fibres.     

The activated human met gene encodes a protein tyrosine kinase

We have raised antibodies against a synthetic dodecapeptide corresponding to the carboxyl terminus of the predicted met gene product. Phosphorylation of 60 kDa and 65 kDa proteins on tyrosine residues was observed when immunoprecipitates of cells containing the activated human met gene were incubated with [gamma-32P]ATP. Phosphoproteins with the same molecular masses could be immunoprecipitated from cells metabolically labelled with [32P]orthophosphate. When considered together, these observations indicate that the activated human met gene encodes 60 kDa and 65 kDa proteins that can catalyse autophosphorylation on tyrosine residues.     

Structural identification of sulfated tyrosine in human urine

A reliable HPLC method was used for the identification of positional isomerism and stereoisomerism of sulfated tyrosine residues in human urine. Upon separation of human urine by ion-pair HPLC on a reverse-phase column, p-tyrosine-O-sulfate (p-TyrS) was identified. Differentiation of the L and D forms was done by using a column with a chiral stationary phase. It was concluded that L-p-tyrosine (L-p-Tyr) which is the predominant tyrosine isomer in the human body, was sulfated and excreted in human urine as a normal constituent. The sulfated forms of D-p-Tyr and m-Tyr could not be detected under these analytical conditions.     

Assignment of the human tyrosine aminotransferase gene to chromosome 16

Summary The liver enzyme tyrosine aminotransferase (TAT; EC 2.6.1.5) catalyzes the rate-limiting step in the catabolic pathway of tyrosine. Deficiency in TAT enzyme activity underlies the autosomally inherited disorder tyrosinemia II (Richner-Hanhart syndrome). Using a human TAT cDNA clone as hybridization probe, we have determined the chromosomal location of the TAT structural gene by Southern blot analysis of DNAs from a series of human x rodent somatic cell hybrids. The results assign the TAT gene to human chromosome 16.     

Identification and characterization of tyrosine kinase activity associated with mitochondrial outer membrane in sarcoma 180 cells

Tyrosine protein kinase activity has been detected in the mitochondrial fraction purified from sarcoma 180 tumor cells. Following hypotonic disruption of mitochondria, tyrosine kinase activity appeared to cosediment with monamine oxidase, marker enzyme of mitochondrial outer membrane; meanwhile, serine and threonine kinases were found to be associated with the inner membrane and matrix of mitochondria. Mitochondrial tyrosine kinase(s) showed thermosensitivity and Mn2+ dependence, useful properties for its characterization and separation from tyrosine kinases associated with other particulate fraction and from serine and threonine kinases associated with mitochondria. Following in vitro incubation of mitochondria with labelled ATP as substrate and analysis by PAGE, a complex pattern of phosphotyrosine containing proteins with a major band of 50-55 kilodaltons resulted.     

Regulation of cell motility by tyrosine phosphorylated villin

Temporal and spatial regulation of the actin cytoskeleton is vital for cell migration. Here, we show that an epithelial cell actin-binding protein, villin, plays a crucial role in this process. Overexpression of villin in doxycyline-regulated HeLa cells enhanced cell migration. Villin-induced cell migration was modestly augmented by growth factors. In contrast, tyrosine phosphorylation of villin and villin-induced cell migration was significantly inhibited by the src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) as well as by overexpression of a dominant negative mutant of c-src. These data suggest that phosphorylation of villin by c-src is involved in the actin cytoskeleton remodeling necessary for cell migration. We have previously shown that villin is tyrosine phosphorylated at four major sites. To further investigate the role of tyrosine phosphorylated villin in cell migration, we used phosphorylation site mutants (tyrosine to phenylalanine or tyrosine to glutamic acid) in HeLa cells. We determined that tyrosine phosphorylation at residues 60, 81, and 256 of human villin played an essential role in cell migration as well as in the reorganization of the actin cytoskeleton. Collectively, these studies define how biophysical events such as cell migration are actuated by biochemical signaling pathways involving tyrosine phosphorylation of actin binding proteins, in this case villin.     

Determination of the sites of tyrosine O-sulfation in peptides and proteins

Tyrosine O-sulfation is a key post-translational modification that regulates protein-protein interactions in extracellular space. We describe a subtractive strategy to determine the sites of tyrosine O-sulfation in proteins. Hydroxyl groups on unsulfated tyrosines are blocked by stoichiometric acetylation in a one-step reaction using sulfosuccinimidyl acetate (S-NHSAc) in the presence of imidazole at pH 7.0. The presence of sulfotyrosine is indicated by the detection of free tyrosine after tandem mass spectrometry (MS/MS) analysis under conditions in which the sulfuryl group of sulfotyrosine is labile. Since phosphorylation and sulfation of tyrosine are isobaric, we used alkaline phosphatase treatment to distinguish these two modifications. Using this methodology we identified the sites and the order of sulfation of several peptides mediated by purified human tyrosylprotein sulfotransferases (TPSTs), and unambiguously determined the tyrosine sulfation sites in mouse lumican and human vitronectin.     

Temperature-dependent tyrosine phosphorylation of microtubule-associated protein kinase in epidermal growth factor-stimulated human fibroblasts.

Treatment of normal human fibroblasts with epidermal growth factor (EGF) results in the rapid (0.5 min) and simultaneous tyrosine phosphorylation of the EGF receptor (EGFr) and several other proteins. An exception to this tyrosine phosphorylation wave was a protein (42 kDa) that became phosphorylated on tyrosine only after a short lag time (5 min). We identified this p42 kDa substrate as the microtubule-associated protein (MAP) kinase using a monoclonal antibody to a peptide corresponding to the C-terminus of the predicted protein (Science 249, 64-67, 1990). EGF treatment of human fibroblasts at 37 degrees C for 5 min resulted in the tyrosine phosphorylation of 60-70% of MAP kinase as determined by the percent that was immunoprecipitated with antiphosphotyrosine antibodies. Like other tyrosine kinase growth factor receptors, the EGFr is activated and phosphorylated at 4 degrees C but is not internalized. Whereas most other substrates were readily tyrosine phosphorylated at 4 degrees C, MAP kinase was not. When cells were first stimulated with EGF at 4 degrees C and then warmed to 37 degrees C without EGF, tyrosine phosphorylation of MAP kinase was again observed. Treatment of cells with the protein kinase C activator phorbol myristate acetate (PMA) also resulted in the tyrosine phosphorylation of MAP kinase, and again only at 37 degrees C. Tryptic phosphopeptide maps demonstrated that EGF and PMA both induced the phosphorylation of the same peptide on tyrosine and threonine. This temperature and PMA sensitivity distinguishes MAP kinase from most other tyrosine kinase substrates in activated human fibroblasts.     

Involvement of reactive oxygen species produced via NADPH oxidase in tyrosine phosphorylation in human B- and T-lineage lymphoid cells

In several human B- and T-lymphoid cell lines, reactive oxygen species (ROS) were produced in a time- and dose-dependent manner in response to menadione (vitamin K3) and anti-Fas (CD95/APO-1) mAb when ROS formation was determined by a chemiluminescence-based method. The ROS evoked by menadione and anti-Fas could be first observed as rapidly as within 20 seconds after the stimulation, reaching a maximum within 5-10 min, and declining slowly thereafter. Both menadione and anti-Fas also induced increased tyrosine phosphorylation of multiple cellular proteins whose pattern was similar to that observed upon hydrogen peroxide treatment. For each agent, the kinetics of the increased tyrosine phosphorylation was similar to that of ROS production, and an NADPH oxidase inhibitor, diphenyleneiodonium, prevented both of these two events. Our results suggest a close link between ROS production and tyrosine phosphorylation induced by divergent extracellular stimuli and the possible role of NADPH oxidase or its related enzyme.     

The Raman detection of peptide tyrosine phosphorylation

Drop-coating-deposition-Raman (DCDR) is used to detect spectral changes induced by phosphorylation of tyrosine amino acid residues in peptides. Four peptides are investigated, with sequences derived from the human protein-tyrosine kinase, p60c-src, with Y-216, Y-419, and Y-530 phosphorylation sites. Although the spectra of the four peptides are quite different, tyrosine phosphorylation is found to invariably induce the collapse of a doublet at 820-850cm(-1) and the attenuation of a peak around 1205cm(-1). Moreover, amide III band shifts suggest that tyrosine phosphorylation may promote beta sheet formation, particularly in peptides that lack phenylalanine residues. The degree of tyrosine phosphorylation in peptide mixtures is determined using DCDR combined with partial least squares multivariate calibration with a 2% root mean standard error of prediction.     

Elevated free tyrosine in rhinoceros erythrocytes

Red blood cells of African black rhinoceroses (Diceros bicornis) are highly sensitive to oxidant-induced hemolysis and they possess a number of enzymatic and biochemical features that differ radically from other mammals. Here we show concentrations of free tyrosine in rhinoceros red blood cells which can approach levels as high as 1 mM, 50-fold higher than in human red blood cells. Elevated levels of tyrosine are also observed in red blood cells of other members of the order Perissodactyla such as the horse and zebra. Captive black rhinoceroses have significantly lower levels of red blood cell tyrosine than black rhinoceroses in the wild. Tyrosine transport studies indicate that black rhinoceros red blood cells have lost the ability to transport tyrosine as efficiently as human red blood cells.     

Profiling the global tyrosine phosphorylation state

Protein tyrosine kinases and protein tyrosine phosphatases play a key role in cell signaling, and the recent success of specific tyrosine kinase inhibitors in cancer treatment strongly validates the clinical relevance of basic research on tyrosine phosphorylation. Functional profiling of the tyrosine phosphoproteome is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel molecular diagnostic approaches. The ultimate aim of current mass spectrometry-based phosphoproteomic approaches is the comprehensive characterization of the phosphoproteome. However, current methods are not yet sensitive enough for routine detection of a large percentage of tyrosine-phosphorylated proteins, which are generally of low abundance. In this article, we discuss alternative methods that exploit Src homology 2 (SH2) domains for profiling the tyrosine phosphoproteome. SH2 domains are small protein modules that bind specifically to tyrosine-phosphorylated peptides; there are more than 100 SH2 domains in the human genome, and different SH2 domains bind to different classes of tyrosine-phosphorylated ligands. These domains play a critical role in the propagation of signals in the cell, mediating the relocalization and complex formation of proteins in response to changes in tyrosine phosphorylation. We have developed an SH2 profiling method based on far-Western blotting, in which a battery of SH2 domains is used to probe the global state of tyrosine phosphorylation. Application to the classification of human malignancies suggests that this approach has potential as a molecular diagnostic tool. We also describe ongoing efforts to modify and improve SH2 profiling, including the development of a multiplexed assay system that will allow high-throughput functional profiling of the tyrosine phosphoproteome.     

Relationship between RNA lariat debranching and Ty1 element retrotransposition

The Saccharomyces cerevisiae DBR1 gene encodes a 2'-5' phosphodiesterase that debranches intron RNA lariats following splicing. Yeast dbr1 mutants accumulate intron lariats and are also defective for mobility of the retrotransposons Ty1 and Ty3. We used a mutagenic PCR method to generate a collection of dbr1 mutant alleles to explore the relationship between the roles of DBR1 in transposition and debranching. Eight mutants defective for Ty1 transposition contained single amino acid changes in Dbr1p. Two mutations, G84A and N85D, are in a conserved phosphoesterase motif that is believed to be part of the active site of the enzyme, supporting a connection between enzymatic activity and Ty1 transposition. Two other mutations, Y68F and Y68D, occur at a potential phosphorylation site, and we have shown that Dbr1p is phosphorylated on tyrosine. We have developed an RNase protection assay to quantitate intron RNA accumulation in cells. The assay uses RNA probes that hybridize to ACT1 intron RNA. Protection patterns confirm that sequences from the 5' end of the intron to the lariat branch point accumulate in dbr1 mutants in a branched (lariat) conformation. RNase protection assays indicate that all of the newly generated dbr1 mutant alleles are also deficient for debranching, further supporting a role for 2'-5' phosphodiesterase activity in Ty1 transposition. A Ty1 element lacking most of its internal sequences transposes independently of DBR1. The existence of Dbr1p-dependent Ty1 sequences raises the possibility that Dbr1p acts on Ty1 RNA.     

Protein tyrosine phosphatases in disease processes

Given the importance of tyrosine phosphorylation of proteins in signalling pathways, it is perhaps not surprising that protein tyrosine phosphatases (PTPs) are involved in the pathogenesis of certain human diseases. A PTP produced by the Yersinia bacteria (which can cause bubonic plague, septicemia and enteric diseases) is thought to be used as a ‘weapon’ against host cell functions. In addition, dysfunction of cells' endogenous PTPs may contribute to defective immune function, to cancer and to diabetes.     

Oxygen dependence of tyrosine hydroxylase

Summary. The effects of dioxygen on tyrosine hydroxylase (TH) activity was studied, measuring the formation of DOPA from tyrosine, ³H₂O from 3,5-³H-tyrosine, or by direct oxygraphic determination of oxygen consumption. A high enzyme activity was observed during the initial 1–2 min of the reactions, followed by a decline in activity, possibly related to a turnover dependent substoichiometrical oxidation of enzyme bound Fe(II) to the inactive Fe(III) state. During the initial reaction phase, apparent K _m-values of 29–45 μM for dioxygen were determined for all human TH isoforms, i.e. 2–40 times higher than previously reported for TH isolated from animal tissues. After 8 min incubation, the K _m (O₂)-values had declined to an average of 20 ± 4 μM. Thus, TH activity may be severely limited by oxygen availability even at moderate hypoxic conditions, and the enzyme is rapidly and turnover dependent inactivated at the experimental conditions commonly employed to measure in vitro activities. Authors’ address: Jan Haavik, Department of Biomedicine, University of Bergen, 5009 Bergen, Norway     

Analysis of the immunoadjuvant octadecyl tyrosine hydrochloride

A simple, rapid, high performance liquid chromatographic (HPLC) method for the analysis of the immunoadjuvant octadecyl tyrosine hydrochloride is described. The HPLC procedure can be applied to the direct determination of amino acid reactants present as contaminants in the adjuvant (tyrosine, ethyl tyrosine) and from this information the content of octadecanol reactant can be estimated. Further, these same determinations provide a means of monitoring immunoadjuvant stability in any vaccine preparation.     

Spectrophotometric resolution of ternary mixtures of tryptophan, tyrosine, and histidine with the aid of principal component-artificial neural network models

A simple and sensitive spectrophotometric method to resolve ternary mixtures of tryptophan (Trp), tyrosine (Tyr), and histidine (His) in synthetic and water samples is described. It relies on the different kinetic rates of the analytes in their oxidative reaction with potassium ferricyanide (K(3)Fe(CN)(6)) in alkaline medium. The absorbance data were monitored on the analytical wavelength (420 nm) of K(3)Fe(CN)(6) spectrum. Synthetic mixtures of the three amino acids were analyzed, and the data obtained were processed by principal component-artificial neural network (PC-ANN) models. After reducing the number of spectral data using principal component analysis, an artificial neural network consisting of three layers of nodes was trained by applying a back-propagation learning rule. Tangent and sigmoidal transfer function were used in the hidden and output layers, respectively. The analytical performance of this method was characterized by relative standard error. The method allowed the determination of Trp, Tyr, and His at concentrations between 10 and 55, 10 and 60, and 10 and 40 microg ml(-1), respectively. The results show that the PC-ANN is an efficient method for prediction of the three analytes.