A quantitative assay for tyrosine sulfation and tyrosine phosphorylation in peptides.

A method was developed to measure sulfation and phosphorylation of tyrosine in proteins after alkaline hydrolysis, ion-exchange chromatography, reaction with [3H]dinitrofluorobenzene and subsequent thin-layer chromatography. The method allows the detection of 10-20 pmol of modified tyrosine and was applied to determine the content of tyrosine-phosphate and -sulfate in fibrinogens, thyroglobulin, alpha-casein, cytochrome c and glyceraldehyde dehydrogenase.     

A tumor promoter stimulates phosphorylation on tyrosine

The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate is mitogenic for normal chicken embryo fibroblasts and also causes these cells to express transiently many properties of cells transformed by Rous sarcoma virus. Since some mitogenic hormones stimulate a tyrosine-specific protein kinase activity, and since the transforming protein of RSV is a tyrosine-specific protein kinase, we have examined whether TPA also stimulates protein phosphorylation on tyrosine. We report here that TPA treatment of normal cells resulted in a very rapid phosphorylation on tyrosine of a protein peak of Mr 40 to 43 kilodaltons. Thus, a similar biochemical activity (tyrosine phosphorylation) is associated with the action of polypeptide mitogenic hormones, Rous sarcoma virus and a tumor promoter. In addition, TPA treatment resulted in rapid changes in phosphorylation of proteins on serine and threonine.     

A genome-wide survey of human tyrosine phosphatases

Tyrosine phosphatases play an important role in cellular signalling and networking that is antagonistic to the kinases. Near completion of the human genome- sequencing project permits us to review the distribution of this family and study its involvement in different pathways. Ninety-six homologues of the classical and dual- specific tyrosine phosphatases (DuSPs) were identified in the human genome using sensitive sequence search techniques. Uncommon domain architectures were encountered, including an example where a kinase and a phosphatase domain are found to co-exist in a single polypeptide. The evolutionary rate is higher for the DuSP compared with the classical tyrosine phosphatases. Orthologues of the 96 putative human tyrosine phosphatases were identified in four model organisms to study the conservation of the family members. Three nuclear localized tyrosine phosphatases retain an orthologous relationship with all model systems considered but still differ in their domain architectures. The diversity in the multi-domain members of the superfamily occurs mainly through domain recruitment, especially in receptor tyrosine phosphatases. The curation of human tyrosine phosphatases provides a convenient framework for characterizing and analysing the functional and structural properties of this diverse family of proteins.     

Dual-specificity tyrosine phosphorylation-regulated kinase 1A does not require tyrosine phosphorylation for activity in vitro

The dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) gene is localized in human chromosome 21, and its overexpression has been associated with the learning and memory deficits of Down syndrome. DYRK1A contains a Y319XY321 motif shared by all members of the DYRK protein kinase family. Residue Y321 in the motif is phosphorylated in DYRK1A prepared from Escherichia coli and from eukaryotic cells. It has been proposed that the YXY motif is an equivalent of the TXY motif, the activation loop, of mitogen-activated protein kinase and that phosphorylation at the motif is required for DYRK activity. In this study, the role of tyrosine phosphorylation in the activity of DYRK1A was investigated in detail. Wild-type DYRK1A with a reduced level of phosphotyrosine (pY) was prepared by treating E. coli-produced DYRK1A with two different protein tyrosine phosphatases. The resulting pY-depleted DYRK1A could not regain pY during autophosphorylation but was as active as the untreated control. These findings were further supported by the observation that DYRK1A retained significant enzymatic activity when both tyrosine residues in the YXY motif were replaced with either histidine or glutamine. Together, we conclude that tyrosine phosphorylation and tyrosine residues in the YXY motif are not directly involved in DYRK1A enzymatic activity in vitro.     

A recombinant tyrosine aminotransferase from Trypanosoma cruzi has both tyrosine aminotransferase and alanine aminotransferase activities

Abstract Tyrosine aminotransferase purified from epimastigotes of Trypanosoma cruzi displays an additional activity of alanine aminotransferase, absent in all other tyrosine aminotransferases characterized so far. Since the parasite's genome contains a high number of copies of the tyrosine aminotransferase gene, we could not rule out the possibility that two very similar proteins, with changed specificity due to a few amino acid substitutions, might be responsible for the two activities. We have now expressed in Escherichia coli a recombinant tyrosine aminotransferase as a fusion protein with glutathione S-trans-ferase. The purified fusion protein, intact or after thrombin cleavage, displays tyrosine aminotransferase and alanine aminotransferase activities with apparent K m values similar to those for the natural enzyme, thus proving that they belong to the same protein.     

Paxillin: A cytoskeletal target for tyrosine kinases

Paxillin is a recently identified member of the complex of cytoskeletal proteins that is found concentrated in cultured cells and in vivo at the cytoplasmic face of regions of cell attachment to the extracellular matrix. These sites, in view of their close proximity to the extracellular matrix, are well positioned to act as signal-transducing centers to ‘report on’ changes in the cells, immediate environment. Recent findings indicate that such signals are in part mediated through the activation of tyrosine kinases concentrated at the sites of adhesion. Changes in the phosphotyrosine content of paxillin accompanying this elevation in kinase activity suggest that paxillin may be an important intermediary in these pathways.     

A strategy for identification of protein tyrosine phosphorylation

To develop methods for studying phosphorylation of protein tyrosine residues is an important task since this protein modification regulates many cellular functions and often is involved in oncogenesis. An optimal protocol includes enrichment of tyrosine phosphorylated (pTyr) peptides or proteins, followed by a high resolving analytical method for identification of the enriched components. In this Methods paper, we describe a working strategy on how immunoaffinity enrichments, using anti-pTyr antibodies, combined with mass spectrometric (MS) analysis can be used to study the pTyr proteome. We describe in detail how our procedure was used to characterize the pTyr proteome of K562 leukemia cells. Important questions concerning the use of different anti-pTyr antibodies, enrichments performed at the peptide and/or the protein level, pooling of enrichments and requirements for the MS characterization are discussed.     

A vesicle surface tyrosine kinase regulates phagosome maturation

Phagocytosis is crucial for host defense against microbial pathogens and for obtaining nutrients in Dictyostelium discoideum. Phagocytosed particles are delivered via a complex route from phagosomes to lysosomes for degradation, but the molecular mechanisms involved in the phagosome maturation process are not well understood. Here, we identify a novel vesicle-associated receptor tyrosine kinase-like protein, VSK3, in D. discoideum. We demonstrate how VSK3 is involved in phagosome maturation. VSK3 resides on the membrane of late endosomes/lysosomes with its C-terminal kinase domain facing the cytoplasm. Inactivation of VSK3 by gene disruption reduces the rate of phagocytosis in cells, which is rescued by re-expression of VSK3. We found that the in vivo function of VSK3 depends on the presence of the kinase domain and vesicle localization. Furthermore, VSK3 is not essential for engulfment, but instead, is required for the fusion of phagosomes with late endosomes/lysosomes. Our findings suggest that localized tyrosine kinase signaling on the surface of endosome/lysosomes represents a control mechanism for phagosome maturation.     

A tyrosine aminotransferase involved in tocopherol synthesis in Arabidopsis

The metabolic function of the predicted Arabidopsis tyrosine aminotransferase (TAT) encoded by the At5g53970 gene was studied using two independent knock-out mutants. Gas chromatography-mass spectrometry based metabolic profiling revealed a specific increase in tyrosine levels, supporting the proposed function of At5g53970 as a tyrosine-specific aminotransferase not involved in tyrosine biosynthesis, but rather in utilization of tyrosine for other metabolic pathways. The TAT activity of the At5g53970-encoded protein was verified by complementation of the Escherichia coli tyrosine auxotrophic mutant DL39, and in vitro activity of recombinantly expressed and purified At5g53970 was found to be specific for tyrosine. To investigate the physiological role of At5g53970, the consequences of reduction in tyrosine utilization on metabolic pathways having tyrosine as a substrate were analysed. We found that tocopherols were substantially reduced in the mutants and we conclude that At5g53970 encodes a TAT important for the synthesis of tocopherols in Arabidopsis.