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Sample records for caga epiya tyrosine

  1. Multiple repeats of Helicobacter pylori CagA EPIYA-C phosphorylation sites predict risk of gastric ulcer in Iran.

    PubMed

    Honarmand-Jahromy, Sahar; Siavoshi, Farideh; Malekzadeh, Reza; Sattari, Taher Nejad; Latifi-Navid, Saeid

    2015-12-01

    Biological activity of Helicobacter pylori oncoprotein CagA is determined by a diversity in the tyrosine phosphorylation motif sites. In the present study, the diversity and the type of the H. pylori CagA EPIYA motifs and their association with gastric ulcer (GU) and duodenal ulcer (DU) in Iranian dyspeptic patients were assessed. PCR amplification, sequencing, and bioinformatic analysis were performed to determine the pattern of CagA EPIYA motifs. Of 168 H. pylori cagA(+) strains, the frequency of ABC was 93.50%, ABCCC 5.40%, ABC + ABCCC 0.6% and ABCC 0.6%. There was no EPIYA-D segment. The ABCCC pattern of EPIYA motif was more frequent in the H. pylori isolates from GU (8/50, 16%) than in those from chronic gastritis (CG) (0/81, 0%) (P = 0). In contrast, The ABC pattern of EPIYA motif was less frequent in the H. pylori isolates from GU (41/50, 82%) than in those from CG (80/81, 98.80%) (Age-sex-adjusted odds ratio (OR) = 0.020, 95% CI = 0.002-0.259; P = 0.003). The distribution of the ABC motif was almost the same in H. pylori isolates from CG (98.80%) and DU diseases (97.30%). There was no significant association between the number of CagA EPIYA-C segment and DU (P > 0.05). We have proposed that CagA from Iranian H. pylori strains were Western type and all strains had active phosphorylation sites. The three EPIYA-C motifs of CagA were more frequently observed in the H. pylori strains from GU; thus it might be an important biomarker for predicting the GU risk in Iran. PMID:26408373

  2. Multiple repeats of Helicobacter pylori CagA EPIYA-C phosphorylation sites predict risk of gastric ulcer in Iran.

    PubMed

    Honarmand-Jahromy, Sahar; Siavoshi, Farideh; Malekzadeh, Reza; Sattari, Taher Nejad; Latifi-Navid, Saeid

    2015-12-01

    Biological activity of Helicobacter pylori oncoprotein CagA is determined by a diversity in the tyrosine phosphorylation motif sites. In the present study, the diversity and the type of the H. pylori CagA EPIYA motifs and their association with gastric ulcer (GU) and duodenal ulcer (DU) in Iranian dyspeptic patients were assessed. PCR amplification, sequencing, and bioinformatic analysis were performed to determine the pattern of CagA EPIYA motifs. Of 168 H. pylori cagA(+) strains, the frequency of ABC was 93.50%, ABCCC 5.40%, ABC + ABCCC 0.6% and ABCC 0.6%. There was no EPIYA-D segment. The ABCCC pattern of EPIYA motif was more frequent in the H. pylori isolates from GU (8/50, 16%) than in those from chronic gastritis (CG) (0/81, 0%) (P = 0). In contrast, The ABC pattern of EPIYA motif was less frequent in the H. pylori isolates from GU (41/50, 82%) than in those from CG (80/81, 98.80%) (Age-sex-adjusted odds ratio (OR) = 0.020, 95% CI = 0.002-0.259; P = 0.003). The distribution of the ABC motif was almost the same in H. pylori isolates from CG (98.80%) and DU diseases (97.30%). There was no significant association between the number of CagA EPIYA-C segment and DU (P > 0.05). We have proposed that CagA from Iranian H. pylori strains were Western type and all strains had active phosphorylation sites. The three EPIYA-C motifs of CagA were more frequently observed in the H. pylori strains from GU; thus it might be an important biomarker for predicting the GU risk in Iran.

  3. Systematic Analysis of Phosphotyrosine Antibodies Recognizing Single Phosphorylated EPIYA-Motifs in CagA of Western-Type Helicobacter pylori Strains

    PubMed Central

    Lind, Judith; Backert, Steffen; Pfleiderer, Klaus; Berg, Douglas E.; Yamaoka, Yoshio; Sticht, Heinrich; Tegtmeyer, Nicole

    2014-01-01

    The clinical outcome of Helicobacter pylori infections is determined by multiple host-pathogen interactions that may develop to chronic gastritis, and sometimes peptic ulcers or gastric cancer. Highly virulent strains encode a type IV secretion system (T4SS) that delivers the effector protein CagA into gastric epithelial cells. Translocated CagA undergoes tyrosine phosphorylation at EPIYA-sequence motifs, called A, B and C in Western-type strains, by members of the oncogenic Src and Abl host kinases. Phosphorylated EPIYA-motifs mediate interactions of CagA with host signaling factors – in particular various SH2-domain containing human proteins – thereby hijacking multiple downstream signaling cascades. Observations of tyrosine-phosphorylated CagA are mainly based on the use of commercial phosphotyrosine antibodies, which originally were selected to detect phosphotyrosines in mammalian proteins. Systematic studies of phosphorylated EPIYA-motif detection by the different antibodies would be very useful, but are not yet available. To address this issue, we synthesized phospho- and non-phosphopeptides representing each predominant Western CagA EPIYA-motif, and determined the recognition patterns of seven different phosphotyrosine antibodies in Western blots, and also performed infection studies with diverse representative Western H. pylori strains. Our results show that a total of 9–11 amino acids containing the phosphorylated EPIYA-motifs are necessary and sufficient for specific detection by these antibodies, but revealed great variability in sequence recognition. Three of the antibodies recognized phosphorylated EPIYA-motifs A, B and C similarly well; whereas preferential binding to phosphorylated motif A and motifs A and C was found with two and one antibodies, respectively, and the seventh anti-phosphotyrosine antibody did not recognize any phosphorylated EPIYA-motif. Controls showed that none of the antibodies recognized the corresponding non-phospho CagA

  4. Dramatic increase in SHP2 binding activity of Helicobacter pylori Western CagA by EPIYA-C duplication: its implications in gastric carcinogenesis

    PubMed Central

    Nagase, Lisa; Hayashi, Takeru; Senda, Toshiya; Hatakeyama, Masanori

    2015-01-01

    Infection with cagA-positive Helicobacter pylori is critically associated with the development of gastric cancer. The cagA-encoded CagA is delivered into gastric epithelial cells via type IV secretion, where it interacts with and thereby deregulates the pro-oncogenic phosphatase SHP2. East Asian CagA and Western CagA are two major CagA species produced by H. pylori circulating in East Asian countries and in the rest of the world, respectively. The SHP2 binding site of Western CagA, termed the EPIYA-C segment, variably duplicates and infection with H. pylori carrying Western CagA with multiple EPIYA-C segments is a distinct risk factor of gastric cancer. Here we show that duplication of EPIYA-C from one to two or more increases SHP2 binding of Western CagA by more than one hundredfold. Based on the decisive difference in SHP2 binding, Western CagA can be divided into two types: type I CagA carrying a single EPIYA-C segment and type II CagA carrying multiple EPIYA-C segments. Gastric epithelial cells expressing type II CagA acquire the ability to invade extracellular matrices, a malignant cellular trait associated with deregulated SHP2. A big leap in SHP2 binding activity may therefore provide molecular basis that makes type II Western CagA a distinct gastric cancer risk. PMID:26507409

  5. Higher number of Helicobacter pylori CagA EPIYA C phosphorylation sites increases the risk of gastric cancer, but not duodenal ulcer

    PubMed Central

    2011-01-01

    Background Helicobacter pylori infection is one of the most common infections worldwide and is associated with gastric cancer and peptic ulcer. Bacterial virulence factors such as CagA have been shown to increase the risk of both diseases. Studies have suggested a causal role for CagA EPIYA polymorphisms in gastric carcinogenesis, and it has been shown to be geographically diverse. We studied associations between H. pylori CagA EPIYA patterns and gastric cancer and duodenal ulcer, in an ethnically admixed Western population from Brazil. CagA EPIYA was determined by PCR and confirmed by sequencing. A total of 436 patients were included, being 188 with gastric cancer, 112 with duodenal ulcer and 136 with gastritis. Results The number of EPIYA C segments was significantly associated with the increased risk of gastric carcinoma (OR = 3.08, 95% CI = 1.74 to 5.45, p < 10-3) even after adjustment for age and gender. Higher number of EPIYA C segments was also associated with gastric atrophy (p = 0.04) and intestinal metaplasia (p = 0.007). Furthermore, patients infected by cagA strains possessing more than one EPIYA C segment showed decreased serum levels of pepsinogen I in comparison with those infected by strains containing one or less EPIYA C repeat. Otherwise, the number of EPIYA C segments did not associate with duodenal ulcer. Conclusions Our results demonstrate that infection with H. pylori strains harbouring more than one CagA EPIYA C motif was clearly associated with gastric cancer, but not with duodenal ulcer. Higher number of EPIYA C segments was also associated with gastric precancerous lesions as demonstrated by histological gastric atrophic and metaplastic changes and decreased serum levels of pepsinogen I. PMID:21435255

  6. A specific A/T polymorphism in Western tyrosine phosphorylation B-motifs regulates Helicobacter pylori CagA epithelial cell interactions.

    PubMed

    Zhang, Xue-Song; Tegtmeyer, Nicole; Traube, Leah; Jindal, Shawn; Perez-Perez, Guillermo; Sticht, Heinrich; Backert, Steffen; Blaser, Martin J

    2015-02-01

    Helicobacter pylori persistently colonizes the human stomach, with mixed roles in human health. The CagA protein, a key host-interaction factor, is translocated by a type IV secretion system into host epithelial cells, where its EPIYA tyrosine phosphorylation motifs (TPMs) are recognized by host cell kinases, leading to multiple host cell signaling cascades. The CagA TPMs have been described as type A, B, C or D, each with a specific conserved amino acid sequence surrounding EPIYA. Database searching revealed strong non-random distribution of the B-motifs (including EPIYA and EPIYT) in Western H. pylori isolates. In silico analysis of Western H. pylori CagA sequences provided evidence that the EPIYT B-TPMs are significantly less associated with gastric cancer than the EPIYA B-TPMs. By generating and using a phosphorylated CagA B-TPM-specific antibody, we demonstrated the phosphorylated state of the CagA B-TPM EPIYT during H. pylori co-culture with host cells. We also showed that within host cells, CagA interaction with phosphoinositol 3-kinase (PI3-kinase) was B-TPM tyrosine-phosphorylation-dependent, and the recombinant CagA with EPIYT B-TPM had higher affinity to PI3-kinase and enhanced induction of AKT than the isogenic CagA with EPIYA B-TPM. Structural modeling of the CagA B-TPM motif bound to PI3-kinase indicated that the threonine residue at the pY+1 position forms a side-chain hydrogen bond to N-417 of PI3-kinase, which cannot be formed by alanine. During co-culture with AGS cells, an H. pylori strain with a CagA EPIYT B-TPM had significantly attenuated induction of interleukin-8 and hummingbird phenotype, compared to the isogenic strain with B-TPM EPIYA. These results suggest that the A/T polymorphisms could regulate CagA activity through interfering with host signaling pathways related to carcinogenesis, thus influencing cancer risk.

  7. A specific A/T polymorphism in Western tyrosine phosphorylation B-motifs regulates Helicobacter pylori CagA epithelial cell interactions.

    PubMed

    Zhang, Xue-Song; Tegtmeyer, Nicole; Traube, Leah; Jindal, Shawn; Perez-Perez, Guillermo; Sticht, Heinrich; Backert, Steffen; Blaser, Martin J

    2015-02-01

    Helicobacter pylori persistently colonizes the human stomach, with mixed roles in human health. The CagA protein, a key host-interaction factor, is translocated by a type IV secretion system into host epithelial cells, where its EPIYA tyrosine phosphorylation motifs (TPMs) are recognized by host cell kinases, leading to multiple host cell signaling cascades. The CagA TPMs have been described as type A, B, C or D, each with a specific conserved amino acid sequence surrounding EPIYA. Database searching revealed strong non-random distribution of the B-motifs (including EPIYA and EPIYT) in Western H. pylori isolates. In silico analysis of Western H. pylori CagA sequences provided evidence that the EPIYT B-TPMs are significantly less associated with gastric cancer than the EPIYA B-TPMs. By generating and using a phosphorylated CagA B-TPM-specific antibody, we demonstrated the phosphorylated state of the CagA B-TPM EPIYT during H. pylori co-culture with host cells. We also showed that within host cells, CagA interaction with phosphoinositol 3-kinase (PI3-kinase) was B-TPM tyrosine-phosphorylation-dependent, and the recombinant CagA with EPIYT B-TPM had higher affinity to PI3-kinase and enhanced induction of AKT than the isogenic CagA with EPIYA B-TPM. Structural modeling of the CagA B-TPM motif bound to PI3-kinase indicated that the threonine residue at the pY+1 position forms a side-chain hydrogen bond to N-417 of PI3-kinase, which cannot be formed by alanine. During co-culture with AGS cells, an H. pylori strain with a CagA EPIYT B-TPM had significantly attenuated induction of interleukin-8 and hummingbird phenotype, compared to the isogenic strain with B-TPM EPIYA. These results suggest that the A/T polymorphisms could regulate CagA activity through interfering with host signaling pathways related to carcinogenesis, thus influencing cancer risk. PMID:25646814

  8. A Specific A/T Polymorphism in Western Tyrosine Phosphorylation B-Motifs Regulates Helicobacter pylori CagA Epithelial Cell Interactions

    PubMed Central

    Zhang, Xue-Song; Tegtmeyer, Nicole; Traube, Leah; Jindal, Shawn; Perez-Perez, Guillermo; Sticht, Heinrich; Backert, Steffen; Blaser, Martin J.

    2015-01-01

    Helicobacter pylori persistently colonizes the human stomach, with mixed roles in human health. The CagA protein, a key host-interaction factor, is translocated by a type IV secretion system into host epithelial cells, where its EPIYA tyrosine phosphorylation motifs (TPMs) are recognized by host cell kinases, leading to multiple host cell signaling cascades. The CagA TPMs have been described as type A, B, C or D, each with a specific conserved amino acid sequence surrounding EPIYA. Database searching revealed strong non-random distribution of the B-motifs (including EPIYA and EPIYT) in Western H. pylori isolates. In silico analysis of Western H. pylori CagA sequences provided evidence that the EPIYT B-TPMs are significantly less associated with gastric cancer than the EPIYA B-TPMs. By generating and using a phosphorylated CagA B-TPM-specific antibody, we demonstrated the phosphorylated state of the CagA B-TPM EPIYT during H. pylori co-culture with host cells. We also showed that within host cells, CagA interaction with phosphoinositol 3-kinase (PI3-kinase) was B-TPM tyrosine-phosphorylation-dependent, and the recombinant CagA with EPIYT B-TPM had higher affinity to PI3-kinase and enhanced induction of AKT than the isogenic CagA with EPIYA B-TPM. Structural modeling of the CagA B-TPM motif bound to PI3-kinase indicated that the threonine residue at the pY+1 position forms a side-chain hydrogen bond to N-417 of PI3-kinase, which cannot be formed by alanine. During co-culture with AGS cells, an H. pylori strain with a CagA EPIYT B-TPM had significantly attenuated induction of interleukin-8 and hummingbird phenotype, compared to the isogenic strain with B-TPM EPIYA. These results suggest that the A/T polymorphisms could regulate CagA activity through interfering with host signaling pathways related to carcinogenesis, thus influencing cancer risk. PMID:25646814

  9. Evaluation of the Pattern of EPIYA Motifs in the Helicobacter pylori cagA Gene of Patients with Gastritis and Gastric Adenocarcinoma from the Brazilian Amazon Region

    PubMed Central

    Vilar e Silva, Adenielson; Junior, Mario Ribeiro da Silva; Vinagre, Ruth Maria Dias Ferreira; Santos, Kemper Nunes; da Costa, Renata Aparecida Andrade; Fecury, Amanda Alves; Quaresma, Juarez Antônio Simões; Martins, Luisa Caricio

    2014-01-01

    The Helicobacter pylori is associated with the development of different diseases. The clinical outcome of infection may be associated with the cagA bacterial genotype. The aim of this study was to determine the EPIYA patterns of strains isolated from patients with gastritis and gastric adenocarcinoma and correlate these patterns with the histopathological features. Gastric biopsy samples were selected from 384 patients infected with H. pylori, including 194 with chronic gastritis and 190 with gastric adenocarcinoma. The presence of the cagA gene and the EPIYA motif was determined by PCR. The cagA gene was more prevalent in patients with gastric cancer and was associated with a higher degree of inflammation, neutrophil activity, and development of intestinal metaplasia. The number of EPIYA-C repeats showed a significant association with an increased risk of gastric carcinoma (OR = 3.79, 95% CI = 1.92–7.46, and P = 0.002). A larger number of EPIYA-C motifs were also associated with intestinal metaplasia. In the present study, infection with H. pylori strains harboring more than one EPIYA-C motif in the cagA gene was associated with the development of intestinal metaplasia and gastric adenocarcinoma but not with neutrophil activity or degree of inflammation. PMID:26904732

  10. Evaluation of the Pattern of EPIYA Motifs in the Helicobacter pylori cagA Gene of Patients with Gastritis and Gastric Adenocarcinoma from the Brazilian Amazon Region.

    PubMed

    Vilar E Silva, Adenielson; Junior, Mario Ribeiro da Silva; Vinagre, Ruth Maria Dias Ferreira; Santos, Kemper Nunes; da Costa, Renata Aparecida Andrade; Fecury, Amanda Alves; Quaresma, Juarez Antônio Simões; Martins, Luisa Caricio

    2014-01-01

    The Helicobacter pylori is associated with the development of different diseases. The clinical outcome of infection may be associated with the cagA bacterial genotype. The aim of this study was to determine the EPIYA patterns of strains isolated from patients with gastritis and gastric adenocarcinoma and correlate these patterns with the histopathological features. Gastric biopsy samples were selected from 384 patients infected with H. pylori, including 194 with chronic gastritis and 190 with gastric adenocarcinoma. The presence of the cagA gene and the EPIYA motif was determined by PCR. The cagA gene was more prevalent in patients with gastric cancer and was associated with a higher degree of inflammation, neutrophil activity, and development of intestinal metaplasia. The number of EPIYA-C repeats showed a significant association with an increased risk of gastric carcinoma (OR = 3.79, 95% CI = 1.92-7.46, and P = 0.002). A larger number of EPIYA-C motifs were also associated with intestinal metaplasia. In the present study, infection with H. pylori strains harboring more than one EPIYA-C motif in the cagA gene was associated with the development of intestinal metaplasia and gastric adenocarcinoma but not with neutrophil activity or degree of inflammation. PMID:26904732

  11. Role of Helicobacter pylori cagA EPIYA motif and vacA genotypes for the development of gastrointestinal diseases in Southeast Asian countries: a meta-analysis

    PubMed Central

    2012-01-01

    Background Infection with cagA-positive, cagA EPIYA motif ABD type, and vacA s1, m1, and i1 genotype strains of Helicobacter pylori is associated with an exacerbated inflammatory response and increased risk of gastroduodenal diseases. However, it is unclear whether the prevalence and virulence factor genotypes found in Southeast Asia are similar to those in Western countries. Here, we examined the cagA status and prevalence of cagA EPIYA motifs and vacA genotypes among H. pylori strains found in Southeast Asia and examined their association with gastroduodenal disease. Methods To determine the cagA status, cagA EPIYA motifs, and vacA genotypes of H. pylori, we conducted meta-analyses of 13 previous reports for 1,281 H. pylori strains detected from several Southeast Asian countries. Results The respective frequencies of cagA-positive and vacA s1, m1, and i1 genotypes among examined subjects were 93% (1,056/1,133), 98% (1,010/1,033), 58% (581/1,009), and 96% (248/259), respectively. Stratification showed significant variation in the frequencies of cagA status and vacA genotypes among countries and the individual races residing within each respective country. The frequency of the vacA m-region genotype in patients infected with East Asian-type strains differed significantly between the northern and southern areas of Vietnam (p < 0.001). Infection with vacA m1 type or cagA-positive strains was associated with an increased risk of peptic ulcer disease (odds ratio: 1.46, 95%CI: 1.01-2.12, p = 0.046 and 2.83, 1.50-5.34, p = 0.001, respectively) in the examined Southeast Asian populations. Conclusions Both Western- and East Asian-type strains of H. pylori are found in Southeast Asia and are predominantly cagA-positive and vacA s1 type. In Southeast Asia, patients infected with vacA m1 type or cagA-positive strains have an increased risk of peptic ulcer disease. Thus, testing for this genotype and the presence of cagA may have clinical usefulness. PMID

  12. CagA phosphorylation EPIYA-C motifs and the vacA i genotype in Helicobacter pylori strains of asymptomatic children from a high-risk gastric cancer area in northeastern Brazil

    PubMed Central

    Braga, Lucia Libanez Bessa Campelo; de Oliveira, Maria Aparecida Alves; Gonçalves, Maria Helane Rocha Batista; Chaves, Fernando Kennedy; Benigno, Tiago Gomes da Silva; Gomes, Adriana Dias; Silva, Cícero Igor Simões Moura; Anacleto, Charles; Batista, Sérgio de Assis; Queiroz, Dulciene Maria Magalhães

    2014-01-01

    Helicobacter pylori infection is one of the most common infections worldwide and is associated with gastric diseases. Virulence factors such as VacA and CagA have been shown to increase the risk of these diseases. Studies have suggested a causal role of CagA EPIYA-C in gastric carcinogenesis and this factor has been shown to be geographically diverse. We investigated the number of CagA EPIYA motifs and the vacA i genotypes in H. pylori strains from asymptomatic children. We included samples from 40 infected children (18 females and 22 males), extracted DNA directly from the gastric mucus/juice (obtained using the string procedure) and analysed the DNA using polymerase chain reaction and DNA sequencing. The vacA i1 genotype was present in 30 (75%) samples, the i2 allele was present in nine (22.5%) samples and both alleles were present in one (2.5%) sample. The cagA-positive samples showed distinct patterns in the 3’ variable region of cagA and 18 of the 30 (60%) strains contained 1 EPIYA-C motif, whereas 12 (40%) strains contained two EPIYA-C motifs. We confirmed that the studied population was colonised early by the most virulent H. pylori strains, as demonstrated by the high frequency of the vacA i1 allele and the high number of EPIYA-C motifs. Therefore, asymptomatic children from an urban community in Fortaleza in northeastern Brazil are frequently colonised with the most virulent H. pylori strains. PMID:25494468

  13. Host SHP1 phosphatase antagonizes Helicobacter pylori CagA and can be downregulated by Epstein-Barr virus.

    PubMed

    Saju, Priya; Murata-Kamiya, Naoko; Hayashi, Takeru; Senda, Yoshie; Nagase, Lisa; Noda, Saori; Matsusaka, Keisuke; Funata, Sayaka; Kunita, Akiko; Urabe, Masayuki; Seto, Yasuyuki; Fukayama, Masashi; Kaneda, Atsushi; Hatakeyama, Masanori

    2016-01-01

    Most if not all gastric cancers are associated with chronic infection of the stomach mucosa with Helicobacter pylori cagA-positive strains(1-4). Approximately 10% of gastric cancers also harbour Epstein-Barr virus (EBV) in the cancer cells(5,6). Following delivery into gastric epithelial cells via type IV secretion(7,8), the cagA-encoded CagA protein undergoes tyrosine phosphorylation on the Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs initially by Src family kinases (SFKs) and then by c-Abl(9,10). Tyrosine-phosphorylated CagA binds to the pro-oncogenic protein tyrosine phosphatase SHP2 and thereby deregulates the phosphatase activity(11,12), which has been considered to play an important role in gastric carcinogenesis(13). Here we show that the SHP2 homologue SHP1 interacts with CagA independently of the EPIYA motif. The interaction potentiates the phosphatase activity of SHP1 that dampens the oncogenic action of CagA by dephosphorylating the CagA EPIYA motifs. In vitro infection of gastric epithelial cells with EBV induces SHP1 promoter hypermethylation, which strengthens phosphorylation-dependent CagA action via epigenetic downregulation of SHP1 expression. Clinical specimens of EBV-positive gastric cancers also exhibit SHP1 hypermethylation with reduced SHP1 expression. The results reveal that SHP1 is the long-sought phosphatase that can antagonize CagA. Augmented H. pylori CagA activity, via SHP1 inhibition, might also contribute to the development of EBV-positive gastric cancer. PMID:27572445

  14. Computational approaches for evaluating the effect of sequence variations and the intrinsically disordered C-terminal region of the Helicobacter pylori CagA protein on the interaction with tyrosine kinase Src.

    PubMed

    Delgado, Paula; Peñaranda, Natalia; Zamora, María Antonia; del Pilar Delgado, María; Bohorquez, Eliana; Castro, Harold; Barrios, Andrés Fernando González; Jaramillo, Carlos

    2014-08-01

    The Helicobacter pylori CagA protein was the first bacterial oncoprotein to be identified as important in the development of human malignancies such as gastric cancer. It is not clear how it is able to deregulate a set of cell control mechanisms to induce carcinogenesis following translocation into human gastric epithelial cells. It is likely, however, that structural variations in the CagA sequence alter its affinity with the host proteins inducing differences in the pathogenicity of different H. pylori strains. Using the recently elucidated N-terminal 3D structure of H. pylori CagA, information on the full cagA gene sequence, and intrinsically disordered protein structure predictions methods we evaluated the interaction of different CagA variants with the kinase Src. An automated docking followed by molecular dynamics simulations were performed to explore CagA interaction modes with Src, one of its cellular partners. The computational approach let us establish that even in the presence of the same number and type of EPIYA motifs, CagA protein can reveal different spatial distributions. Based on the lowest affinity energy and higher number of interactions it was established that the principal forces governing the CagA-Src interaction are electrostatic. Results showed that EPIYA-D models presents higher affinity with some host proteins than EPIYA-C. Thus, we highlight the importance and advantage of the use of computational tools in combining chemical and biological data with bioinformatics for modeling and prediction purposes in some cases where experimental techniques present limitations. PMID:25116152

  15. CagA Phosphorylation in Helicobacter pylori-Infected B Cells Is Mediated by the Nonreceptor Tyrosine Kinases of the Src and Abl Families

    PubMed Central

    Krisch, Linda M.; Posselt, Gernot; Hammerl, Peter

    2016-01-01

    CagA is one of the most important virulence factors of the human pathogen Helicobacter pylori. CagA expression can be associated with the induction of severe gastric disorders such as gastritis, ulceration, gastric cancer, or mucosa-associated lymphoid tissue (MALT) lymphoma. After translocation through a type IV secretion system into epithelial cells, CagA is tyrosine phosphorylated by kinases of the Src and Abl families, leading to drastic cell elongation and motility. While the functional role of CagA in epithelial cells is well investigated, knowledge about CagA phosphorylation and its associated signal transduction pathways in B cells is only marginal. Here, we established the B cell line MEC1 derived from a B cell chronic lymphocytic leukemia (B-CLL) patient as a new infection model to study the signal transduction in B cells controlled by H. pylori. We observed that CagA was rapidly injected, strongly tyrosine phosphorylated, and cleaved into a 100-kDa N-terminal and a 40-kDa C-terminal fragment. To identify upstream signal transduction pathways of CagA phosphorylation in MEC1 cells, pharmacological inhibitors were employed to specifically target Src and Abl kinases. We observed that CagA phosphorylation was strongly inhibited upon treatment with an Src inhibitor and slightly diminished when the Abl kinase inhibitor imatinib mesylate (Gleevec) was applied. The addition of dasatinib to block c-Abl and Src kinases led to a complete loss of CagA phosphorylation. In conclusion, these results demonstrate an important role for Src and Abl tyrosine kinases in CagA phosphorylation in B cells, which represent druggable targets in H. pylori-mediated gastric MALT lymphoma. PMID:27382024

  16. Tyrosine phosphorylation of the Helicobacter pylori CagA antigen after cag-driven host cell translocation.

    PubMed

    Stein, M; Rappuoli, R; Covacci, A

    2000-02-01

    Helicobacter pylori strains associated with severe tissue damage and inflammation possess a unique genetic locus, cag, containing 31 genes originating from a distant event of horizontal transfer and retained as a pathogenicity island. The cag system is an Helicobacter-specific type IV secretion engine involved in cellular responses like induction of pedestals, secretion of IL-8, and phosphorylation of proteic targets. It has previously been reported that cocultivation of epithelial cells with Helicobacter pylori triggers signal transduction and tyrosine phosphorylation of a 145-kDa putative host cell protein. Herein, we demonstrate that this protein is not derived from the host but rather is the bacterial immunodominant antigen CagA, a virulence factor commonly expressed in peptic ulcer disease and thought to be an orphan of a specific biological function. Thus, CagA is delivered into the epithelial cells by the cag type IV secretion system where it is phosphorylated on tyrosine residues by an as yet unidentified host cell kinase and wired to eukaryotic signal transduction pathways and cytoskeletal plasticity. PMID:10655519

  17. Evolution of cagA Oncogene of Helicobacter pylori through Recombination

    PubMed Central

    Furuta, Yoshikazu; Yahara, Koji; Hatakeyama, Masanori; Kobayashi, Ichizo

    2011-01-01

    Helicobacter pylori is a gastric pathogen that infects half the human population and causes gastritis, ulcers, and cancer. The cagA gene product is a major virulence factor associated with gastric cancer. It is injected into epithelial cells, undergoes phosphorylation by host cell kinases, and perturbs host signaling pathways. CagA is known for its geographical, structural, and functional diversity in the C-terminal half, where an EPIYA host-interacting motif is repeated. The Western version of CagA carries the EPIYA segment types A, B, and C, while the East Asian CagA carries types A, B, and D and shows higher virulence. Many structural variants such as duplications and deletions are reported. In this study, we gained insight into the relationships of CagA variants through various modes of recombination, by analyzing all known cagA variants at the DNA sequence level with the single nucleotide resolution. Processes that occurred were: (i) homologous recombination between DNA sequences for CagA multimerization (CM) sequence; (ii) recombination between DNA sequences for the EPIYA motif; and (iii) recombination between short similar DNA sequences. The left half of the EPIYA-D segment characteristic of East Asian CagA was derived from Western type EPIYA, with Amerind type EPIYA as the intermediate, through rearrangements of specific sequences within the gene. Adaptive amino acid changes were detected in the variable region as well as in the conserved region at sites to which no specific function has yet been assigned. Each showed a unique evolutionary distribution. These results clarify recombination-mediated routes of cagA evolution and provide a solid basis for a deeper understanding of its function in pathogenesis. PMID:21853141

  18. In vitro effect of amoxicillin and clarithromycin on the 3’ region of cagA gene in Helicobacter pylori isolates

    PubMed Central

    Bustamante-Rengifo, Javier Andrés; Matta, Andrés Januer; Pazos, Alvaro; Bravo, Luis Eduardo

    2013-01-01

    AIM: To evaluate the in vitro effect of amoxicillin and clarithromycin on the cag pathogenicity island (cag PAI). METHODS: One hundred and forty-nine clinical isolates of Helicobacter pylori (H. pylori) cultured from gastric biopsies from 206 Colombian patients with dyspeptic symptoms from a high-risk area for gastric cancer were included as study material. Antimicrobial susceptibility was determined by the agar dilution method. Resistant isolates at baseline and in amoxicillin and clarithromycin serial dilutions were subjected to genotyping (cagA, vacA alleles s and m), Glu-Pro-Ile-Tyr-Ala (EPIYA) polymerase chain reaction and random amplified polymorphic DNA (RAPD). Images of the RAPD amplicons were analyzed by Gel-Pro Analyzer 4.5 program. Cluster analyses was done using SPSS 15.0 statistical package, where each of the fingerprint bands were denoted as variables. Dendrograms were designed by following Ward’s clustering method and the estimation of distances between each pair of H. pylori isolates was calculated with the squared Euclidean distance. RESULTS: Resistance rates were 4% for amoxicillin and 2.7% for clarithromycin with 2% double resistances. Genotyping evidenced a high prevalence of the genotype cagA-positive/vacA s1m1. The 3’ region of cagA gene was successfully amplified in 92.3% (12/13) of the baseline resistant isolates and in 60% (36/60) of the resistant isolates growing in antibiotic dilutions. Upon observing the distribution of the number of EPIYA repetitions in each dilution with respect to baseline isolates, it was found that in 61.5% (8/13) of the baseline isolates, a change in the number of EPIYA repetitions lowered antibiotic pressure. The gain and loss of EPIYA motifs resulted in a diversity of H. pylori subclones after bacterial adjustment to changing conditions product of antibiotic pressure. RAPD PCR evidenced the close clonal relationship between baseline isolates and isolates growing in antibiotic dilutions. CONCLUSION: Antibiotic

  19. [Production of a recombinant CagA protein for the detection of Helicobacter pylori CagA antibodies].

    PubMed

    Akgüç, Miray; Karatayli, Ersin; Çelik, Esra; Koyuncu, Duygu; Çelik, İnci; Karatayli, Senem Ceren; Özden, Ali; Bozdayi, A Mithat

    2014-07-01

    At present, Helicobacter pylori infections affect approximately 50% of the world population. It is known that H.pylori is related with several gastric diseases including chronic atrophic gastritis, peptic and gastric ulcers as well as gastric carcinomas. CagA (Cytotoxin-associated gene A) protein which is one of the most important virulence factors of H.pylori, is thought to be responsible for the development of gastric cancer. CagA is a 128 kDa hydrophilic protein which binds to the epitelial stomach cells and is known to be phosphorylated on its EPIYA regions. The EPIYA regions are highly variable and carry a higher risk of developing gastric cancer than CagA negative strains. The aim of this study was to construct a prokaryotic expression system expressing a recombinant CagA protein, which can be used for the detection of anti-CagA antibodies. For the isolation of H.pylori genomic DNA, a total of 112 gastric biopsy samples obtained from patients who were previously found positive for rapid urease (CLO) test, were used. H.pylori DNAs were amplified from 57 of those samples by polymerase chain reaction (PCR) and of them 35 were found positive in terms of cagA gene. Different EPIYA motifs were detected in 25 out of 35 cagA positive samples, and one of those samples that contained the highest number of EPIYA motif, was chosen for the cloning procedure. Molecular cloning and expression of the recombinant fragment were performed with Champion Pet151/D expression vector (Invitrogen, USA), the expression of which was induced by the addition of IPTG (Isopropyl-beta-D-thiogalactopyranoside) into the E.coli culture medium. Expression was observed with anti-histidin HRP (Horse Radish Peroxidase) antibodies by SDS-PAGE and Western Blot (WB) analysis. In our study, two clones possessing different fragments from the same H.pylori strain with three different EPIYA motifs were succesfully expressed. Since CagA antigen plays a signicant role in the pathogenesis of H

  20. [Production of a recombinant CagA protein for the detection of Helicobacter pylori CagA antibodies].

    PubMed

    Akgüç, Miray; Karatayli, Ersin; Çelik, Esra; Koyuncu, Duygu; Çelik, İnci; Karatayli, Senem Ceren; Özden, Ali; Bozdayi, A Mithat

    2014-07-01

    At present, Helicobacter pylori infections affect approximately 50% of the world population. It is known that H.pylori is related with several gastric diseases including chronic atrophic gastritis, peptic and gastric ulcers as well as gastric carcinomas. CagA (Cytotoxin-associated gene A) protein which is one of the most important virulence factors of H.pylori, is thought to be responsible for the development of gastric cancer. CagA is a 128 kDa hydrophilic protein which binds to the epitelial stomach cells and is known to be phosphorylated on its EPIYA regions. The EPIYA regions are highly variable and carry a higher risk of developing gastric cancer than CagA negative strains. The aim of this study was to construct a prokaryotic expression system expressing a recombinant CagA protein, which can be used for the detection of anti-CagA antibodies. For the isolation of H.pylori genomic DNA, a total of 112 gastric biopsy samples obtained from patients who were previously found positive for rapid urease (CLO) test, were used. H.pylori DNAs were amplified from 57 of those samples by polymerase chain reaction (PCR) and of them 35 were found positive in terms of cagA gene. Different EPIYA motifs were detected in 25 out of 35 cagA positive samples, and one of those samples that contained the highest number of EPIYA motif, was chosen for the cloning procedure. Molecular cloning and expression of the recombinant fragment were performed with Champion Pet151/D expression vector (Invitrogen, USA), the expression of which was induced by the addition of IPTG (Isopropyl-beta-D-thiogalactopyranoside) into the E.coli culture medium. Expression was observed with anti-histidin HRP (Horse Radish Peroxidase) antibodies by SDS-PAGE and Western Blot (WB) analysis. In our study, two clones possessing different fragments from the same H.pylori strain with three different EPIYA motifs were succesfully expressed. Since CagA antigen plays a signicant role in the pathogenesis of H

  1. Infection with CagA-Positive Helicobacter Pylori Strain Containing Three EPIYA C Phosphorylation Sites is Associated with More Severe Gastric Lesions in Experimentally Infected Mongolian Gerbils (Meriones Unguiculatus)

    PubMed Central

    Junior, M. Ferreira; Batista, S.A.; Vidigal, P.V.T; Cordeiro, A.A.C.; Oliveira, F.M.S.; Prata, L.O.; Diniz, A.E.T.; Barral, C.M.; Barbuto, R.C.; Comes, A.D.; Araujo, I.D.; Queiroz, D.M.M.; Caliari, M.V.

    2015-01-01

    Infection with Helicobacter pylori strains containing high number of EPIYA-C phosphorylation sites in the CagA is associated with significant gastritis and increased risk of developing pre-malignant gastric lesions and gastric carcinoma. However, these findings have not been reproduced in animal models yet. Therefore, we investigated the effect on the gastric mucosa of Mongolian gerbil (Meriones unguiculatus) infected with CagA-positive H. pylori strains exhibiting one or three EPIYA-C phosphorilation sites. Mongolian gerbils were inoculated with H. pylori clonal isolates containing one or three EPIYA-C phosphorylation sites. Control group was composed by uninfected animals challenged with Brucella broth alone. Gastric fragments were evaluated by the modified Sydney System and digital morphometry. Clonal relatedness between the isolates was considered by the identical RAPD-PCR profiles and sequencing of five housekeeping genes, vacA i/d region and of oipA. The other virulence markers were present in both isolates (vacA s1i1d1m1, iceA2, and intact dupA). CagA of both isolates was translocated and phosphorylated in AGS cells. After 45 days of infection, there was a significant increase in the number of inflammatory cells and in the area of the lamina propria in the infected animals, notably in those infected by the CagA-positive strain with three EPIYA-C phosphorylation sites. After six months of infection, a high number of EPIYA-C phosphorylation sites was associated with progressive increase in the intensity of gastritis and in the area of the lamina propria. Atrophy, intestinal metaplasia, and dysplasia were also observed more frequently in animals infected with the CagA-positive isolate with three EPIYA-C sites. We conclude that infection with H. pylori strain carrying a high number of CagA EPIYA-C phosphorylation sites is associated with more severe gastric lesions in an animal model of H. pylori infection. PMID:26150158

  2. Translocation of Helicobacter pylori CagA into Gastric Epithelial Cells by Type IV Secretion

    NASA Astrophysics Data System (ADS)

    Odenbreit, Stefan; Püls, Jürgen; Sedlmaier, Bettina; Gerland, Elke; Fischer, Wolfgang; Haas, Rainer

    2000-02-01

    The Gram-negative bacterium Helicobacter pylori is a causative agent of gastritis and peptic ulcer disease in humans. Strains producing the CagA antigen (cagA+) induce strong gastric inflammation and are strongly associated with gastric adenocarcinoma and MALT lymphoma. We show here that such strains translocate the bacterial protein CagA into gastric epithelial cells by a type IV secretion system, encoded by the cag pathogenicity island. CagA is tyrosine-phosphorylated and induces changes in the tyrosine phosphorylation state of distinct cellular proteins. Modulation of host cells by bacterial protein translocation adds a new dimension to the chronic Helicobacter infection with yet unknown consequences.

  3. Fragmentation of CagA Reduces Hummingbird Phenotype Induction by Helicobactor pylori

    PubMed Central

    Chen, Ying-Chieh; Perng, Chin-Lin; Lin, Hwai-Jeng; Ou, Yueh-Hsing

    2016-01-01

    Infection with Helicobacter pylori (H. pylori) has been linked to various gastro-intestinal diseases; nevertheless it remains to be clarified why only a minority of infected individuals develop illness. Studies from the West have indicated that the cagA gene and the associated EPIYA genotype of H. pylori is closely linked to the development of severe gastritis and gastric carcinoma; however, as yet no consistent correlation has been found among the bacteria from East Asia. In addition to genotype variation, the CagA protein undergoes fragmentation; however, the functional significance of fragmentation with respect to H. pylori infection remains unknown. In this study, we isolated 594 H. pylori colonies from 99 patients and examined the fragmentation patterns of CagA protein using immunoblotting. By analyzing the ability of the isolates to induce the host cell morphological transition to the highly invasive hummingbird phenotype, we demonstrated that H. pylori colonies with substantial CagA fragmentation are less potent in terms of causing this morphological transition. Our results uncovered a functional role for CagA fragmentation with respect to H. pylori-induced hummingbird phenotype formation and these findings suggest the possibility that the post-translational processing of CagA may be involved in H. pylori infection pathogenesis. PMID:26934189

  4. Fragmentation of CagA Reduces Hummingbird Phenotype Induction by Helicobactor pylori.

    PubMed

    Chang, Chih-Chi; Kuo, Wein-Shung; Chen, Ying-Chieh; Perng, Chin-Lin; Lin, Hwai-Jeng; Ou, Yueh-Hsing

    2016-01-01

    Infection with Helicobacter pylori (H. pylori) has been linked to various gastro-intestinal diseases; nevertheless it remains to be clarified why only a minority of infected individuals develop illness. Studies from the West have indicated that the cagA gene and the associated EPIYA genotype of H. pylori is closely linked to the development of severe gastritis and gastric carcinoma; however, as yet no consistent correlation has been found among the bacteria from East Asia. In addition to genotype variation, the CagA protein undergoes fragmentation; however, the functional significance of fragmentation with respect to H. pylori infection remains unknown. In this study, we isolated 594 H. pylori colonies from 99 patients and examined the fragmentation patterns of CagA protein using immunoblotting. By analyzing the ability of the isolates to induce the host cell morphological transition to the highly invasive hummingbird phenotype, we demonstrated that H. pylori colonies with substantial CagA fragmentation are less potent in terms of causing this morphological transition. Our results uncovered a functional role for CagA fragmentation with respect to H. pylori-induced hummingbird phenotype formation and these findings suggest the possibility that the post-translational processing of CagA may be involved in H. pylori infection pathogenesis. PMID:26934189

  5. Novel effects of Helicobacter pylori CagA on key genes of gastric cancer signal transduction: a comparative transfection study.

    PubMed

    Vaziri, Farzam; Peerayeh, Shahin N; Alebouyeh, Masoud; Maghsoudi, Nader; Azimzadeh, Pedram; Siadat, Seyed D; Zali, Mohammad R

    2015-04-01

    Helicobacter pylori (H. pylori) infection is now recognized as a worldwide problem. Helicobacter pylori CagA is the first bacterial oncoprotein to be identified in relation to human cancer. Helicobacter pylori CagA is noted for structural diversity in its C-terminal region (contains EPIYA motifs), with which CagA interacts with numerous host cell proteins. Deregulation of host signaling by translocated bacterial proteins provides a new aspect of microbial-host cell interaction. The aim of this study is to compare the cellular effects of two different CagA EPIYA motifs on identified signaling pathways involve in gastric carcinogenesis. To investigate the effects of CagA protein carboxyl region variations on the transcription of genes involved in gastric epithelial carcinogenesis pathways, the eukaryotic vector carrying the cagA gene (ABC and ABCCC types) was transfected into gastric cancer cell line. The 42 identified key genes of signal transduction involved in gastric cancer were analyzed at the transcription level by real-time PCR. The results of real-time PCR provide us important clue that the ABCCC oncoprotein variant can change the fate of the cell completely different from ABC type. In fact, these result proposed that the ABCCC type can induce the intestinal metaplasia, IL-8, perturbation of Crk adaptor proteins, anti-apoptotic effect and carcinogenic effect more significantly than ABC type. These data support our hypothesis of a complex interaction of host cell and these two different H. pylori effector variants that determines host cellular fate.

  6. Analysis of the intactness of Helicobacter pylori cag pathogenicity island in Iranian strains by a new PCR-based strategy and its relationship with virulence genotypes and EPIYA motifs.

    PubMed

    Yadegar, Abbas; Alebouyeh, Masoud; Zali, Mohammad Reza

    2015-10-01

    Variants of the Helicobacter pylori cag pathogenicity island (cagPAI) and certain virulence genotypes have been proposed to be associated with different gastric disorders. In the present study, we designed a new PCR-based strategy to investigate the intactness of cagPAI in Iranian patients using highly specific primer sets spanning the cagPAI region. The possible relationship between the cagPAI status of the strains and clinical outcomes was also determined. We also characterized virulence genotypes (cagL, cagA, vacA, babA2 and sabA) and variants of CagA EPIYA motifs in these strains. H. pylori was detected in 61 out of 126 patients with various gastroduodenal diseases. The cagL, cagA, vacA s1m1, vacA s1m2, vacA s2m2, babA2, and sabA genotypes were detected in 96.7%, 85.2%, 29.5%, 45.9%, 24.6%, 96.7%, and 83.6% of the strains, respectively. Among the 52 cagA-positive strains, EPIYA motifs ABC, ABCC, ABCCC, and mixed types were orderly detected in the 39, 7, 1, and 5 strains. The cagPAI positivity included both intact and partially deleted, with the overall frequencies of 70.5% and 26.2%, respectively. The majority of the strains from patients with PUD (87.5%), gastric erosion (83.3%) and cancer (80%) presented an intact cagPAI, while a lower frequency of cagPAI intactness was detected in gastritis patients (61.1%). However, no significant relationship was found between the possession of intact cagPAI and clinical outcomes. Furthermore, we found that cagA and vacA s1m1 genotypes were significantly correlated with intact cagPAI (P=0.015 and P=0.012). A significant correlation was also found between EPIYA-ABC and intact cagPAI (P=0.010). The proposed PCR-based scheme was found to be useful for determining the intactness of cagPAI. Our findings also indicate that the cagPAI appears to be intact and rather conserved in majority of Iranian strains. Finally, our study proposed that H. pylori strains with partially deleted cagPAI were less likely to cause severe diseases

  7. Heme oxygenase-1 inhibits phosphorylation of the Helicobacter pylori oncoprotein CagA in gastric epithelial cells

    PubMed Central

    Gobert, Alain P.; Verriere, Thomas; de Sablet, Thibaut; Peek, Richard M.; Chaturvedi, Rupesh; Wilson, Keith T.

    2012-01-01

    Summary The cytotoxin-associated gene A protein (CagA) plays a pivotal role in the etiology of Helicobacter (H.) pylori-associated gastric diseases. CagA is injected into the cytoplasm of host cells by a type IV secretion system, and is phosphorylated on tyrosine residues by the host enzyme c-Src. We previously reported that the enzyme heme oxygenase-1 (HO-1) inhibits IL-8 secretion by H. pylori-infected cells. However, the cellular mechanism by which HO-1 regulates the innate immune function of infected cells remains unknown. We now show that nitric oxide and hemin, two inducers of HO-1, decrease the level of phosphorylated CagA (p-CagA) in H. pylori-infected gastric epithelial cells and this is blocked by either pharmacologic inhibition of HO-1 or siRNA knockdown of hmox-1. Moreover, forced expression of HO-1 by transfection of a plasmid expressing hmox-1 also results in a strong attenuation of CagA phosphorylation. This occurs through the inhibition of H. pylori-induced c-Src phosphorylation/activation by HO-1. Consequently, H. pylori-induced cytoskeletal rearrangements and activation of the pro-inflammatory response mediated by p-CagA are inhibited in HO-1-expressing cells. These data highlight a mechanism by which the innate immune response of the host can restrict the pathogenicity of H. pylori by attenuating CagA phosphorylation in gastric epithelial cells. PMID:23051580

  8. Helicobacter pylori cagA Promoter Region Sequences Influence CagA Expression and Interleukin 8 Secretion.

    PubMed

    Ferreira, Rui M; Pinto-Ribeiro, Ines; Wen, Xiaogang; Marcos-Pinto, Ricardo; Dinis-Ribeiro, Mário; Carneiro, Fátima; Figueiredo, Ceu

    2016-02-15

    Heterogeneity at the Helicobacter pylori cagA gene promoter region has been linked to variation in CagA expression and gastric histopathology. Here, we characterized the cagA promoter and expression in 46 H. pylori strains from Portugal. Our results confirm the relationship between cagA promoter region variation and protein expression originally observed in strains from Colombia. We observed that individuals with intestinal metaplasia were all infected with H. pylori strains containing a specific cagA motif. Additionally, we provided novel functional evidence that strain-specific sequences in the cagA promoter region and CagA expression levels influence interleukin 8 secretion by the host gastric epithelial cells.

  9. Construction of prokaryotic expression system of 2 148-bp fragment from cagA gene and detection of cagA gene, CagA protein in Helicobacter pylori isolates and its antibody in sera of patients

    PubMed Central

    Yan, Jie; Wang, Yuan; Shao, Shi-He; Mao, Ya-Fei; Li, Hua-Wen; Luo, Yi-Hui

    2004-01-01

    AIM: To construct a prokaryotic expression system of a Helicobacter pylori (H pylori) cagA gene fragment and establish enzyme-linked immunosorbent assays (ELISA) for detecting CagA and its antibody, so as to understand the manner in which the infection of CagA-expressing H pylori (CagA+ H pylori) isolates cause diseases. METHODS: H pylori strains in gastric biopsy specimens from 156 patients with positive results in rapid urease test were isolated. PCR was used to detect the frequency of cagA gene in the 109 H pylori isolates and to amplify a 2 148-bp fragment (cagA1) of cagA gene from a clinical strain Y06. A prokaryotic expression system of cagA1 gene was constructed, and the expression of the target recombinant protein (rCagA1) was examined by SDS-PAGE. Western blotting and immunodiffusion assay were employed to determine the immunoreactivity and antigenicity of rCagA1, respectively. Two ELISAs were established to detect CagA expression in 109 H pylori isolates and the presence of CagA antibody in the corresponding patients’ sera, and the correlations between infection with CagA+ H pylori and gastritis as well as peptic ulcer were analyzed. RESULTS: Of all the clinical specimens obtained, 80.8% (126/156) were found to have H pylori isolates and 97.2% of the isolates (106/109) were positive for cagA gene. In comparison with the reported data, the cloned cagA1 fragment possessed 94.83% and 93.30% homologies with the nucleotide and putative amino acid sequences, respectively. The output of rCagA1 produced by the constructed recombinant prokaryotic expression system was approximately 30% of the total bacterial protein. rCagA1 was able to bind to the commercial antibody against the whole-cells of H pylori and to induce the immunized rabbits to produce antibody with an immunodiffusion titer of 1:4. A proportion as high as 92.6% of the H pylori isolates (101/109) expressed CagA and 88.1% of the patients’ serum samples (96/109) were CagA antibody-positive. The

  10. Helicobacter pylori CagA Inhibits PAR1-MARK Family Kinases by Mimicking Host Substrates

    SciTech Connect

    Nesic, D.; Miller, M; Quinkert, Z; Stein, M; Chait, B; Stebbins, C

    2010-01-01

    The CagA protein of Helicobacter pylori interacts with numerous cellular factors and is associated with increased virulence and risk of gastric carcinoma. We present here the cocrystal structure of a subdomain of CagA with the human kinase PAR1b/MARK2, revealing that a CagA peptide mimics substrates of this kinase family, resembling eukaryotic protein kinase inhibitors. Mutagenesis of conserved residues central to this interaction renders CagA inactive as an inhibitor of MARK2.

  11. Helicobacter pylori and CagA under conditions of iron deficiency.

    PubMed

    Noto, Jennifer M; Peek, Richard M

    2015-01-01

    Iron deficiency is the most common nutritional deficiency worldwide and compelling evidence has demonstrated that this condition heightens the risk of gastric cancer. Infection with Helicobacter pylori is the strongest known risk factor for the development of gastric adenocarcinoma. Recent work has demonstrated that, under conditions of iron deficiency, H. pylori-induced gastric carcinogenesis is augmented through increased formation of the strain-specific cag type IV secretion system and enhanced delivery of the bacterial oncoprotein CagA into host cells. Although CagA is a potent virulence factor that promotes oncogenic responses, additional studies have now demonstrated that CagA modulates host cell iron homeostasis in vitro and fundamental metabolic functions of the bacterial cell in vivo. Here we discuss these findings and describe working models by which CagA exerts its effects on gastric epithelial cells, with particular emphasis on its potential role in modulation of host iron homeostasis.

  12. The CagA toxin of Helicobacter pylori: abundant production but relatively low amount translocated

    PubMed Central

    Jiménez-Soto, Luisa F.; Haas, Rainer

    2016-01-01

    CagA is one of the most studied pathogenicity factors of the bacterial pathogen Helicobacter pylori. It is injected into host cells via the H. pylori cag-Type IV secretion system. Due to its association with gastric cancer, CagA is classified as oncogenic protein. At the same time CagA represents the 4th most abundant protein produced by H. pylori, suggesting that high amounts of toxin are required to cause the physiological changes or damage observed in cells. We were able to quantify the injection of CagA into gastric AGS epithelial cells in vitro by the adaptation of a novel protease-based approach to remove the tightly adherent extracellular bacteria. After one hour of infection only 1.5% of the total CagA available was injected by the adherent bacteria, whereas after 3 hours 7.5% was found within the host cell. Thus, our data show that only a surprisingly small amount of the CagA available in the infection is finally injected under in vitro infection conditions. PMID:26983895

  13. Mixed Infection with cagA Positive and cagA Negative Strains of Helicobacter pylori Lowers Disease Burden in The Gambia

    PubMed Central

    Secka, Ousman; Antonio, Martin; Berg, Douglas E.; Tapgun, Mary; Bottomley, Christian; Thomas, Vivat; Walton, Robert; Corrah, Tumani; Thomas, Julian E.; Adegbola, Richard A.

    2011-01-01

    Background The prevalence of Helicobacter pylori including strains with putatively virulent genotypes is high, whereas the H. pylori-associated disease burden is low, in Africa compared to developed countries. In this study, we investigated the prevalence of virulence-related H. pylori genotypes and their association with gastroduodenal diseases in The Gambia. Methods and Findings DNA extracted from biopsies and H. pylori cultures from 169 subjects with abdominal pain, dyspepsia or other gastroduodenal diseases were tested by PCR for H. pylori. The H. pylori positive samples were further tested for the cagA oncogene and vacA toxin gene. One hundred and twenty one subjects (71.6%) were H. pylori positive. The cagA gene and more toxigenic s1 and m1 alleles of the vacA gene were found in 61.2%, 76.9% and 45.5% respectively of Gambian patients harbouring H. pylori. There was a high prevalence of cagA positive strains in patients with overt gastric diseases than those with non-ulcerative dyspepsia (NUD) (p = 0.05); however, mixed infection by cagA positive and cagA negative strains was more common in patients with NUD compared to patients with gastric disease (24.5% versus 0%; p = 0.002). Conclusion This study shows that the prevalence of H. pylori is high in dyspeptic patients in The Gambia and that many strains are of the putatively more virulent cagA+, vacAs1 and vacAm1 genotypes. This study has also shown significantly lower disease burden in Gambians infected with a mixture of cag-positive and cag-negative strains, relative to those containing only cag-positive or only cag-negative strains, which suggests that harbouring both cag-positive and cag-negative strains is protective. PMID:22140492

  14. Association between TNF-α promoter polymorphism and Helicobacter pylori cagA subtype infection

    PubMed Central

    Yea, S; Yang, Y; Jang, W; Lee, Y; Bae, H; Paik, K

    2001-01-01

    Aims—To assess the importance of tumour necrosis factor α (TNF-α) promoter polymorphism in relation to infection with the cytotoxin associated gene A (cagA) subtype of Helicobacter pylori within a dyspeptic Korean population. Methods—Eighty three patients with gastric disease and 113 healthy controls were studied. The DNA from gastric biopsy specimens was analysed by H pylori specific and cagA specific polymerase chain reaction (PCR). To characterise TNF-α polymorphism at positions -308 and -238, PCR based restriction fragment length polymorphism analysis was performed. Results—Helicobacter pylori infection was closely correlated with G to A transition at position -308 of the TNF-α promoter when compared with healthy controls (odds ratio (OR), 2.912; 95% confidence interval (CI), 1.082 to 7.836; p = 0.034). Although TNF-α -308 polymorphism in patients with H pylori was not significantly different from that in patients without H pylori, the -308A polymorphism was strongly associated with H pylori cagA subtype infection when compared with the polymorphism in cagA negative H pylori infection (OR, 8.757; 95% CI, 1.413 to 54.262; p = 0.019) and healthy controls (OR, 3.683; 95% CI, 1.343 to 10.101; p = 0.011). G to A genetic change at position -238 of the TNF-α gene was not significantly associated with H pylori cagA subtype infection. In addition, genetic polymorphisms at both sites of the TNF-α promoter in patients with H pylori infection did not correlate with the severity of disease. Conclusion—TNF-α -308A polymorphism was significantly related to infection with the H pylori cagA subtype in Korean patients with gastric disease. Key Words: Helicobacter pylori • cagA • tumour necrosis factor α • polymorphism PMID:11533078

  15. Mutual reinforcement of inflammation and carcinogenesis by the Helicobacter pylori CagA oncoprotein

    PubMed Central

    Suzuki, Nobumi; Murata-Kamiya, Naoko; Yanagiya, Kohei; Suda, Wataru; Hattori, Masahira; Kanda, Hiroaki; Bingo, Atsuhiro; Fujii, Yumiko; Maeda, Shin; Koike, Kazuhiko; Hatakeyama, Masanori

    2015-01-01

    Helicobacter pylori cagA-positive strain delivers the CagA oncoprotein into gastric epithelial cells and at the same time elicits stomach inflammation. To experimentally investigate the pathophysiological interplay between CagA and inflammation, transgenic mice systemically expressing the bacterial cagA gene were treated with a colitis inducer, dextran sulfate sodium (DSS). Compared with control mice, DSS-induced colitis was markedly deteriorated in cagA-transgenic mice. In the colonic epithelia of cagA-transgenic mice, there was a substantial decrease in the level of IκB, which binds and sequesters NF-κB in the cytoplasm. This IκB reduction was due to CagA-mediated inhibition of PAR1, which may stimulate IκB degradation by perturbing microtubule stability. Whereas the CagA-mediated IκB reduction did not automatically activate NF-κB, it lowered the threshold of NF-κB activation by inflammogenic insults, thereby contributing to colitis exacerbation in cagA-transgenic mice. CagA also activates inflammasomes independently of NF-κB signaling, which further potentiates inflammation. The incidence of colonic dysplasia was elevated in DSS-treated cagA-transgenic mice due to a robust increase in the number of pre-cancerous flat-type dysplasias. Thus, CagA deteriorated inflammation, whereas inflammation strengthened the oncogenic potential of CagA. This work revealed that H. pylori CagA and inflammation reinforce each other in creating a downward spiral that instigates neoplastic transformation. PMID:25944120

  16. Helicobacter pylori virulence factor CagA promotes tumorigenesis of gastric cancer via multiple signaling pathways.

    PubMed

    Yong, Xin; Tang, Bo; Li, Bo-Sheng; Xie, Rui; Hu, Chang-Jiang; Luo, Gang; Qin, Yong; Dong, Hui; Yang, Shi-Ming

    2015-01-01

    Helicobacter pylori (H. pylori) infection is strongly associated with the development of gastric diseases but also with several extragastric diseases. The clinical outcomes caused by H. pylori infection are considered to be associated with a complex combination of host susceptibility, environmental factors and bacterial isolates. Infections involving H. pylori strains that possess the virulence factor CagA have a worse clinical outcome than those involving CagA-negative strains. It is remarkable that CagA-positive H. pylori increase the risk for gastric cancer over the risk associated with H. pylori infection alone. CagA behaves as a bacterial oncoprotein playing a key role in H. pylori-induced gastric cancer. Activation of oncogenic signaling pathways and inactivation of tumor suppressor pathways are two crucial events in the development of gastric cancer. CagA shows the ability to affect the expression or function of vital protein in oncogenic or tumor suppressor signaling pathways via several molecular mechanisms, such as direct binding or interaction, phosphorylation of vital signaling proteins and methylation of tumor suppressor genes. As a result, CagA continuously dysregulates of these signaling pathways and promotes tumorigenesis. Recent research has enriched our understanding of how CagA effects on these signaling pathways. This review summarizes the results of the most relevant studies, discusses the complex molecular mechanism involved and attempts to delineate the entire signaling pathway.

  17. NF-κB activation and potentiation of proinflammatory responses by the Helicobacter pylori CagA protein

    PubMed Central

    Brandt, Sabine; Kwok, Terry; Hartig, Roland; König, Wolfgang; Backert, Steffen

    2005-01-01

    The Helicobacter pylori immunodominant protein, CagA, is associated with severe gastritis and carcinoma. Injection of CagA into gastric epithelial cells by type IV secretion leads to actin-cytoskeletal rearrangements and cell scattering. CagA has been reported to have no role in the induction of transcription factor NF-κB and IL-8, which are crucial determinants for chronic inflammation. Here, we provide several lines of evidence showing that CagA is able to induce IL-8 in a time- and strain-dependent manner. We also show that by exchanging specific cagA genes, high IL-8-inducing H. pylori strains could be converted into low inducing strains and vice versa. Our results suggest that IL-8 release induced by CagA occurs via a Ras→Raf→Mek→Erk→NF-κB signaling pathway in a Shp-2- and c-Met-independent manner. Thus, CagA is a multifunctional protein capable of effecting both actin remodeling and potentiation of chemokine release. PMID:15972330

  18. Association of Helicobacter pylori cagA Gene with Gastric Cancer and Peptic Ulcer in Saudi Patients.

    PubMed

    Saber, Taisir; Ghonaim, Mabrouk M; Yousef, Amany R; Khalifa, Amany; Al Qurashi, Hesham; Shaqhan, Mohammad; Samaha, Mohammad

    2015-07-01

    This study was conducted to assess the relationship between occurrence of gastric cancer and peptic ulcer, and the presence of H. pylori cagA gene and anti-CagA IgG, and to estimate the value of these antibodies in detecting infection by cagA gene-positive H. pylori strains in Saudi patients. The study included 180 patients who were subjected to upper gastrointestinal endoscopy in Taif province and Western region of Saudi Arabia (60 gastric cancer, 60 peptic ulcer, and 60 with non-ulcer dyspepsia). Gastric biopsy specimens were obtained and tested for H. pylori infection by rapid urease test and culture. PCR was performed on the isolated strains and biopsy specimens for detection of the cagA gene. Blood samples were collected and tested for CagA IgG by ELISA. H. pylori infection was detected among 72.8% of patients. The cagA gene and anti-CagA IgG were found in 63.4% and 61.8% of H. pylori-infected patients, respectively. They were significantly (p < 0.01) higher in patients with gastric cancer and peptic ulcer compared with those with non-ulcer dyspepsia. Detection of the CagA IgG was 91.6% sensitive, 89.6% specific, and 90.8% accurate compared with detection of the cagA gene. Its positive and negative predictive values were 93.8% and 86%, respectively. The study showed a significant association between the presence of the cagA gene and gastric cancer and peptic ulcer disease, and between anti-CagA IgG and the cagA gene in Saudi patients. However, a further larger study is required to confirm this finding.

  19. H. pylori virulence factor CagA increases intestinal cell proliferation by Wnt pathway activation in a transgenic zebrafish model

    PubMed Central

    Neal, James T.; Peterson, Tracy S.; Kent, Michael L.; Guillemin, Karen

    2013-01-01

    SUMMARY Infection with Helicobacter pylori is a major risk factor for the development of gastric cancer, and infection with strains carrying the virulence factor CagA significantly increases this risk. To investigate the mechanisms by which CagA promotes carcinogenesis, we generated transgenic zebrafish expressing CagA ubiquitously or in the anterior intestine. Transgenic zebrafish expressing either the wild-type or a phosphorylation-resistant form of CagA exhibited significantly increased rates of intestinal epithelial cell proliferation and showed significant upregulation of the Wnt target genes cyclinD1, axin2 and the zebrafish c-myc ortholog myca. Coexpression of CagA with a loss-of-function allele encoding the β-catenin destruction complex protein Axin1 resulted in a further increase in intestinal proliferation. Coexpression of CagA with a null allele of the key β-catenin transcriptional cofactor Tcf4 restored intestinal proliferation to wild-type levels. These results provide in vivo evidence of Wnt pathway activation by CagA downstream of or in parallel to the β-catenin destruction complex and upstream of Tcf4. Long-term transgenic expression of wild-type CagA, but not the phosphorylation-resistant form, resulted in significant hyperplasia of the adult intestinal epithelium. We further utilized this model to demonstrate that oncogenic cooperation between CagA and a loss-of-function allele of p53 is sufficient to induce high rates of intestinal small cell carcinoma and adenocarcinoma, establishing the utility of our transgenic zebrafish model in the study of CagA-associated gastrointestinal cancers. PMID:23471915

  20. CagA, a major virulence factor of Helicobacter pylori, promotes the production and underglycosylation of IgA1 in DAKIKI cells

    SciTech Connect

    Yang, Man; Li, Fu-gang; Xie, Xi-sheng; Wang, Shao-qing; Fan, Jun-ming

    2014-02-07

    Highlights: • CagA stimulated cell proliferation and the production of IgA1 in DAKIKI cells. • CagA promoted the underglycosylation of IgA1 in DAKIKI cells. • CagA decreased the expression of C1GALT1 and its chaperone Cosmc in DAKIKI cells. • Helicobacter pylori infection may participate in the pathogenesis of IgAN via CagA. - Abstract: While Helicobacter pylori (Hp) infection is closely associated with IgA nephropathy (IgAN), the underlying molecular mechanisms remain to be elucidated. This study was to investigate the effect of cytotoxin associated gene A protein (CagA), a major virulence factor of Hp, on the production and underglycosylation of IgA1 in the B cell line DAKIKI cells. Cells were cultured and treated with recombinant CagA protein. We found that CagA stimulated cell proliferation and the production of IgA1 in a dose-dependent and time-dependent manner. Moreover, CagA promoted the underglycosylation of IgA1, which at least partly attributed to the downregulation of β1,3-galactosyltransferase (C1GALT1) and its chaperone Cosmc. In conclusion, we demonstrated that Hp infection, at least via CagA, may participate in the pathogenesis of IgAN by influencing the production and glycosylation of IgA1 in B cells.

  1. Receptor Tyrosine Kinase and Tyrosine Kinase Inhibitors

    PubMed Central

    Mirshafiey, Abbas; Ghalamfarsa, Ghasem; Asghari, Babak

    2014-01-01

    Receptor tyrosine kinases (RTKs) are essential components of signal transduction pathways that mediate cell-to-cell communication and their function as relay points for signaling pathways. They have a key role in numerous processes that control cellular proliferation and differentiation, regulate cell growth and cellular metabolism, and promote cell survival and apoptosis. Recently, the role of RTKs including TCR, FLT-3, c-Kit, c-Fms, PDGFR, ephrin, neurotrophin receptor, and TAM receptor in autoimmune disorder, especially rheumatoid arthritis and multiple sclerosis has been suggested. In multiple sclerosis pathogenesis, RTKs and their tyrosine kinase enzymes are selective important targets for tyrosine kinase inhibitor (TKI) agents. TKIs, compete with the ATP binding site of the catalytic domain of several tyrosine kinases, and act as small molecules that have a favorable safety profile in disease treatment. Up to now, the efficacy of TKIs in numerous animal models of MS has been demonstrated, but application of these drugs in human diseases should be tested in future clinical trials. PMID:25337443

  2. What exists beyond cagA and vacA? Helicobacter pylori genes in gastric diseases.

    PubMed

    da Costa, Débora Menezes; Pereira, Eliane dos Santos; Rabenhorst, Silvia Helena Barem

    2015-10-01

    Helicobacter pylori (H. pylori) infection is present in more than half the world's population and has been associated with several gastric disorders, such as gastritis, peptic ulceration, and gastric adenocarcinoma. The clinical outcome of this infection depends on host and bacterial factors where H. pylori virulence genes seem to play a relevant role. Studies of cagA and vacA genes established that they were determining factors in gastric pathogenesis. However, there are gastric cancer cases that are cagA-negative. Several other virulence genes have been searched for, but these genes remain less well known that cagA and vacA. Thus, this review aimed to establish which genes have been suggested as potentially relevant virulence factors for H. pylori-associated gastrointestinal diseases. We focused on the cag-pathogenicity island, genes with adherence and motility functions, and iceA based on the relevance shown in several studies in the literature.

  3. Helicobacter pylori induced interleukin-8 expression in gastric epithelial cells is associated with CagA positive phenotype.

    PubMed Central

    Crabtree, J E; Covacci, A; Farmery, S M; Xiang, Z; Tompkins, D S; Perry, S; Lindley, I J; Rappuoli, R

    1995-01-01

    AIMS--To use a range of natural phenotypically variant strains of Helicobacter pylori with disparate CagA and VacA (vacuolating cytotoxin) expression to determine which bacterial factors are more closely associated with epithelial interleukin-8 (IL-8) induction. METHODS--Gastric epithelial cells (AGS and KATO-3) were co-cultured with five H pylori strains which were variously shown to express the cagA gene/CagA protein, VacA and/or to exhibit biological cytotoxicity. Secreted IL-8 was assayed by enzyme leaked immunosorbent assay (ELISA) and IL-8 messenger RNA (mRNA) was assayed using a reverse transcription polymerase chain reaction based technique (RT-PCR). RESULTS--Strains expressing CagA, including a variant strain (D931) which is non-cytotoxic and does not express the VacA protein, were found to upregulate epithelial IL-8 secretion and gene expression. In contrast, strains with no CagA expression, even in the presence of VacA and/or biological cytotoxicity, (G104, BA142), failed to induce IL-8 protein or mRNA above control values. CONCLUSIONS--These results strongly support a role for H pylori CagA or coexpressed factors other than the cytotoxin in upregulation of gastric epithelial IL-8. Increased epithelial IL-8 secretion and concomitant neutrophil chemotaxis and activation in addition to direct cytotoxicity may be an important factor in tissue damage and ulceration. Images PMID:7706517

  4. CagA and VacA Helicobacter pylori antibodies in gastric cancer

    PubMed Central

    Suriani, Renzo; Colozza, Maurilio; Cardesi, Enrico; Mazzucco, Dario; Marino, Maria; Grosso, Silvia; Sanseverinati, Sabina; Venturini, Ivo; Borghi, Athos; Zeneroli, Maria Luisa

    2008-01-01

    BACKGROUND: Infection with different genotypes of virulent Helicobacter pylori strains (cytotoxin-associated gene A [CagA]-and/or vacuolating cytotoxin A [VacA]-positive) can play a role in the development of atrophic gastritis, duodenal ulcer (DU) and gastric cancer (GC). OBJECTIVE: To determine whether patients with GC and H pylori-negative histological staining had previously been infected with H pylori CagA- and/or VacA-positive virulent strains. METHODS: Twenty-three GC patients with a mean (± SD) age of 68.14±9.8 years who tested H pylori-negative on histological staining took part in the study. Three control groups were included. The first group comprised 19 patients with past H pylori infection and DUs eradicated 10 years earlier, with a mean age of 58±18.2 years. H pylori-negative status for this group was determined every year with Giemsa staining, and follow-up testing occured 120±32 months (mean ± SD) after therapy. The subsequent control groups included 20 asymptomatic children, with a mean age of 7±4.47 years, and with H pylori-negative fecal tests; the final group contained 30 patients without clinical symptoms of H pylori infection, with a mean age of 68±11.6 years, who tested H pylori-negative by histological staining. RESULTS: Prevalence of CagA and VacA seropositivity, respectively was 82.6% and 73.91% in GC patients; 84.2% and 84.2% in H pylori-negative DU patients; 25% and 5% in H pylori-negative children; and 36.6% and 16.6% in the patients without clinical symptoms on histological staining. CagA and VacA antibody positivity was not significantly different between GC patients and patients with DUs that had been eradicated 10 years earlier. Significant positivity was found between the children’s group and the H pylori-negative (with past DUs) group (P<0.001). A statistically significant difference was found in age between groups (P<0.03). CONCLUSIONS: Patients with GC, even when H pylori-negative at the time of the present study, may

  5. Helicobacter pylori CagA and IL-1β Promote the Epithelial-to-Mesenchymal Transition in a Nontransformed Epithelial Cell Model

    PubMed Central

    Arévalo-Romero, Haruki; Meza, Isaura; Vallejo-Flores, Gabriela

    2016-01-01

    Gastric cancer is the third cause of cancer death worldwide and infection by Helicobacter pylori (H. pylori) is considered the most important risk factor, mainly by the activity of its virulence factor CagA. H. pylori/CagA-induced chronic inflammation triggers a series of gastric lesions of increased severity, starting with gastritis and ending with cancer. IL-1β has been associated with tumor development and invasiveness in different types of cancer, including gastric cancer. Currently, it is not clear if there is an association between CagA and IL-1β at a cellular level. In this study, we analyzed the effects of IL-1β and CagA on MCF-10A nontransformed cells. We found evidence that both CagA and IL-1β trigger the initiation of the epithelial-to-mesenchymal transition characterized by β-catenin nuclear translocation, increased expression of Snail1 and ZEB1, downregulation of CDH1, and morphological changes during MCF-10A acini formation. However, only CagA induced MMP9 activity and cell invasion. Our data support that IL-1β and CagA target the β-catenin pathway, with CagA leading to acquisition of a stage related to aggressive tumors. PMID:27525003

  6. Helicobacter pylori CagA and IL-1β Promote the Epithelial-to-Mesenchymal Transition in a Nontransformed Epithelial Cell Model.

    PubMed

    Arévalo-Romero, Haruki; Meza, Isaura; Vallejo-Flores, Gabriela; Fuentes-Pananá, Ezequiel M

    2016-01-01

    Gastric cancer is the third cause of cancer death worldwide and infection by Helicobacter pylori (H. pylori) is considered the most important risk factor, mainly by the activity of its virulence factor CagA. H. pylori/CagA-induced chronic inflammation triggers a series of gastric lesions of increased severity, starting with gastritis and ending with cancer. IL-1β has been associated with tumor development and invasiveness in different types of cancer, including gastric cancer. Currently, it is not clear if there is an association between CagA and IL-1β at a cellular level. In this study, we analyzed the effects of IL-1β and CagA on MCF-10A nontransformed cells. We found evidence that both CagA and IL-1β trigger the initiation of the epithelial-to-mesenchymal transition characterized by β-catenin nuclear translocation, increased expression of Snail1 and ZEB1, downregulation of CDH1, and morphological changes during MCF-10A acini formation. However, only CagA induced MMP9 activity and cell invasion. Our data support that IL-1β and CagA target the β-catenin pathway, with CagA leading to acquisition of a stage related to aggressive tumors. PMID:27525003

  7. Impact of structural polymorphism for the Helicobacter pylori CagA oncoprotein on binding to polarity-regulating kinase PAR1b

    PubMed Central

    Nishikawa, Hiroko; Hayashi, Takeru; Arisaka, Fumio; Senda, Toshiya; Hatakeyama, Masanori

    2016-01-01

    Chronic infection with cagA-positive Helicobacter pylori is the strongest risk factor for atrophic gastritis, peptic ulcers, and gastric cancer. CagA, the product of the cagA gene, is a bacterial oncoprotein, which, upon delivery into gastric epithelial cells, binds to and inhibits the polarity-regulating kinase, partitioning-defective 1b (PAR1b) [also known as microtubule affinity-regulating kinase 2 (MARK2)], via its CagA multimerization (CM) motif. The inhibition of PAR1b elicits junctional and polarity defects, rendering cells susceptible to oncogenesis. Notably, the polymorphism in the CM motif has been identified among geographic variants of CagA, differing in either the copy number or the sequence composition. In this study, through quantitative analysis of the complex formation between CagA and PAR1b, we found that several CagA species have acquired elevated PAR1b-binding activity via duplication of the CM motifs, while others have lost their PAR1b-binding activity. We also found that strength of CagA-PAR1b interaction was proportional to the degrees of stress fiber formation and tight junctional disruption by CagA in gastric epithelial cells. These results indicate that the CM polymorphism is a determinant for the magnitude of CagA-mediated deregulation of the cytoskeletal system and thereby possibly affects disease outcome of cagA-positive H. pylori infection, including gastric cancer. PMID:27445265

  8. Linking epithelial polarity and carcinogenesis by multitasking Helicobacter pylori virulence factor CagA.

    PubMed

    Hatakeyama, M

    2008-11-24

    Loss of cell polarity and tissue architecture is a hallmark of carcinomas that arise from epithelial cells. Recent studies on Drosophila tumor suppressors have provided evidence that epithelial polarity and cell proliferation are functionally coupled, suggesting a function for polarity defects in the development of carcinomas. This notion is supported by the findings that mammalian orthologs of these Drosophila tumor suppressors are targeted by a number of viral oncoproteins. Chronic infection with Helicobacter pylori is causally associated with gastric carcinoma. H. pylori virulence factor CagA (cytotoxin-associated gene A), which is delivered into gastric epithelial cells through a bacterial type IV secretion system, has an important function in cell transformation through interacting with and deregulating SHP-2 phosphatase, a bona fide oncoprotein that is associated with human malignancies. Recent studies have further revealed that CagA specifically binds and inhibits PAR1/MARK polarity-regulating kinase, thereby causing junctional and polarity defects in epithelial cells. Thus, the bacterial oncoprotein simultaneously targets the polarity-regulating system and growth-regulatory system. These findings indicate that loss of cell polarity underlies the abnormal proliferation of epithelial cells that directs carcinogenesis.

  9. In Silico Profiling of the Potentiality of Curcumin and Conventional Drugs for CagA Oncoprotein Inactivation.

    PubMed

    Srivastava, Akhileshwar K; Tewari, Mallika; Shukla, Hari S; Roy, Bijoy K

    2015-08-01

    The oncoprotein cytotoxic associated gene A (CagA) of Helicobacter pylori plays a pivotal role in the development of gastric cancer, so it has been an important target for anti-H. pylori drugs. Conventional drugs are currently being implemented against H. pylori. The inhibitory role of plant metabolites like curcumin against H. pylori is still a major scientific challenge. Curcumin may represent a novel promising drug against H. pylori infection without producing side effects. In the present study, a comparative analysis between curcumin and conventional drugs (clarithromycin, amoxicillin, pantoprazole, and metronidazole) was carried out using databases to investigate the potential of curcumin against H. pylori targeting the CagA oncoprotein. Curcumin was filtered using Lipinski's rule of five and the druglikeness property for evaluation of pharmacological properties. Subsequently, molecular docking was employed to determine the binding affinities of curcumin and conventional drugs to the CagA oncoprotein. According to the results obtained from FireDock, the binding energy of curcumin was higher than those of amoxicillin, pantoprazole, and metronidazole, except for clarithromycin, which had the highest binding energy. Accordingly, curcumin may become a promising lead compound against CagA+ H. pylori infection. PMID:25996140

  10. Protein tyrosine nitration

    PubMed Central

    Chaki, Mounira; Leterrier, Marina; Barroso, Juan B

    2009-01-01

    Nitric oxide metabolism in plant cells has a relative short history. Nitration is a chemical process which consists of introducing a nitro group (-NO2) into a chemical compound. in biological systems, this process has been found in different molecules such as proteins, lipids and nucleic acids that can affect its function. This mini-review offers an overview of this process with special emphasis on protein tyrosine nitration in plants and its involvement in the process of nitrosative stress. PMID:19826215

  11. Helicobacter pylori CagA induces tumor suppressor gene hypermethylation by upregulating DNMT1 via AKT-NFκB pathway in gastric cancer development

    PubMed Central

    Wang, He-xiao; Zhao, Wei; Li, Jian-fang; Su, Li-ping; Shao, Zhifeng; Zhao, Xiaodong; Zhu, Zheng-gang; Yan, Min; Liu, Bingya

    2016-01-01

    Methylation of CpG islands in tumor suppressor gene prompter is one of the most characteristic abnormalities in Helicobacter pylori (HP)-associated gastric carcinoma (GC). Here, we investigated the pathogenic and molecular mechanisms underlying hypermethylation of tumor suppressor genes in HP induced GC development. We found that tumor suppressor genes hypermethylation, represented by MGMT, positively correlated with CagA in clinical specimens, gastric tissues from HP infected C57 mice and GC cell lines transfected by CagA or treated by HP infection. CagA enhanced PDK1 and AKT interaction and increased AKT phosphorylation. The P-AKT subsequent activated NFκB, which then bound to DNMT1 promoter and increased its expression. Finally, the upregulated DNMT1 promoted tumor suppressor genes hypermethylation with MGMT as a representative. In conclusion, CagA increased tumor suppressor genes hypermethylation via stimulating DNMT1 expression through the AKT-NFκB pathway. PMID:26848521

  12. Microbial Protein-tyrosine Kinases*

    PubMed Central

    Chao, Joseph D.; Wong, Dennis; Av-Gay, Yossef

    2014-01-01

    Microbial ester kinases identified in the past 3 decades came as a surprise, as protein phosphorylation on Ser, Thr, and Tyr amino acids was thought to be unique to eukaryotes. Current analysis of available microbial genomes reveals that “eukaryote-like” protein kinases are prevalent in prokaryotes and can converge in the same signaling pathway with the classical microbial “two-component” systems. Most microbial tyrosine kinases lack the “eukaryotic” Hanks domain signature and are designated tyrosine kinases based upon their biochemical activity. These include the tyrosine kinases termed bacterial tyrosine kinases (BY-kinases), which are responsible for the majority of known bacterial tyrosine phosphorylation events. Although termed generally as bacterial tyrosine kinases, BY-kinases can be considered as one family belonging to the superfamily of prokaryotic protein-tyrosine kinases in bacteria. Other members of this superfamily include atypical “odd” tyrosine kinases with diverse mechanisms of protein phosphorylation and the “eukaryote-like” Hanks-type tyrosine kinases. Here, we discuss the distribution, phylogeny, and function of the various prokaryotic protein-tyrosine kinases, focusing on the recently discovered Mycobacterium tuberculosis PtkA and its relationship with other members of this diverse family of proteins. PMID:24554699

  13. Characterization of the Translocation-competent Complex between the Helicobacter pylori Oncogenic Protein CagA and the Accessory Protein CagF*

    PubMed Central

    Bonsor, Daniel A.; Weiss, Evelyn; Iosub-Amir, Anat; Reingewertz, Tali H.; Chen, Tiffany W.; Haas, Rainer; Friedler, Assaf; Fischer, Wolfgang; Sundberg, Eric J.

    2013-01-01

    CagA is a virulence factor that Helicobacter pylori inject into gastric epithelial cells through a type IV secretion system where it can cause gastric adenocarcinoma. Translocation is dependent on the presence of secretion signals found in both the N- and C-terminal domains of CagA and an interaction with the accessory protein CagF. However, the molecular basis of this essential protein-protein interaction is not fully understood. Herein we report, using isothermal titration calorimetry, that CagA forms a 1:1 complex with a monomer of CagF with nm affinity. Peptide arrays and isothermal titration calorimetry both show that CagF binds to all five domains of CagA, each with μm affinity. More specifically, a coiled coil domain and a C-terminal helix within CagF contacts domains II-III and domain IV of CagA, respectively. In vivo complementation assays of H. pylori with a double mutant, L36A/I39A, in the coiled coil region of CagF showed a severe weakening of the CagA-CagF interaction to such an extent that it was nearly undetectable. However, it had no apparent effect on CagA translocation. Deletion of the C-terminal helix of CagF also weakened the interaction with CagA but likewise had no effect on translocation. These results indicate that the CagA-CagF interface is distributed broadly across the molecular surfaces of these two proteins to provide maximal protection of the highly labile effector protein CagA. PMID:24072713

  14. Characterization of the translocation-competent complex between the Helicobacter pylori oncogenic protein CagA and the accessory protein CagF.

    PubMed

    Bonsor, Daniel A; Weiss, Evelyn; Iosub-Amir, Anat; Reingewertz, Tali H; Chen, Tiffany W; Haas, Rainer; Friedler, Assaf; Fischer, Wolfgang; Sundberg, Eric J

    2013-11-15

    CagA is a virulence factor that Helicobacter pylori inject into gastric epithelial cells through a type IV secretion system where it can cause gastric adenocarcinoma. Translocation is dependent on the presence of secretion signals found in both the N- and C-terminal domains of CagA and an interaction with the accessory protein CagF. However, the molecular basis of this essential protein-protein interaction is not fully understood. Herein we report, using isothermal titration calorimetry, that CagA forms a 1:1 complex with a monomer of CagF with nM affinity. Peptide arrays and isothermal titration calorimetry both show that CagF binds to all five domains of CagA, each with μM affinity. More specifically, a coiled coil domain and a C-terminal helix within CagF contacts domains II-III and domain IV of CagA, respectively. In vivo complementation assays of H. pylori with a double mutant, L36A/I39A, in the coiled coil region of CagF showed a severe weakening of the CagA-CagF interaction to such an extent that it was nearly undetectable. However, it had no apparent effect on CagA translocation. Deletion of the C-terminal helix of CagF also weakened the interaction with CagA but likewise had no effect on translocation. These results indicate that the CagA-CagF interface is distributed broadly across the molecular surfaces of these two proteins to provide maximal protection of the highly labile effector protein CagA.

  15. H. pylori-encoded CagA disrupts tight junctions and induces invasiveness of AGS gastric carcinoma cells via Cdx2-dependent targeting of Claudin-2.

    PubMed

    Song, Xin; Chen, Hui-Xin; Wang, Xiao-Yan; Deng, Xi-Yun; Xi, Yin-Xue; He, Qing; Peng, Tie-Li; Chen, Jie; Chen, Wei; Wong, Benjamin Chun-Yu; Chen, Min-Hu

    2013-01-01

    Helicobacter pylori encoded CagA is presently the only known virulence factor that is injected into gastric epithelial cells where it destroys apical junctional complexes and induces dedifferentiation of gastric epithelial cells, leading to H. pylori-related gastric carcinogensis. However, little is known about the molecular mechanisms by which CagA mediates these changes. Caudal-related homeobox 2 (Cdx2) is an intestine-specific transcription factor highly expressed in multistage tissues of dysplasia and cancer. One specific target of Cdx2, Claudin-2, is involved in the regulation of tight junction (TJ) permeability. In this study, our findings showed that the activity of Cdx2 binding to Cdx binding sites of CdxA (GTTTATG) and CdxB (TTTTAGG) of probes corresponding to claudin-2 flanking region increased in AGS cells, infected with CagA positive wild-type strain of H. pylori, compared to CagA negative isogenic mutant-type strain. Moreover, Cdx2 upregulated claudin-2 expression at transcriptional level and translational level. In the meantime, we found that TJs of AGS cells, infected with CagA positive wild-type strain of H. pylori, compared to CagA negative isogenic mutant-type strain, were more severely destroyed, leading to wider cell gap, interference of contact, scattering and highly elevated migration of cells. Herein, this study is firstly demonstrated that H. pylori-encoded CagA disrupts TJs and induces invasiveness of AGS gastric carcinoma cells via Cdx2-dependent targeting of Claudin-2. This provides a new mechanism whereby CagA induced dedifferentiation of AGS cells, leading to malignant behavior of biology. PMID:24287273

  16. Diversity of Helicobacter Pylori cagA and vacA Genes and Its Relationship with Clinical Outcomes in Azerbaijan, Iran

    PubMed Central

    Ghotaslou, Reza; Milani, Morteza; Akhi, Mohammad Taghi; Nahaei, Mohammad Reza; Hasani, Alka; Hejazi, Mohammad Saeid; Meshkini, Mohammad

    2013-01-01

    Purpose: The purpose of this research was to analyze cagA and vacA genotypes status in H. pylori isolates and relationship with clinical outcomes. Methods: Gastric biopsy specimens were cultured for H. pylori isolation and cagA and vacA genes were detected in these isolates. Data were collected and the results were analyzed using χ2 and Fishers exact tests by SPSS software version. 16. Results: Of the total 115 H. pylori isolates, 79 (68.7 %) were cagA positive and 82 (71.3%) of isolates contained the s1 allele which 33 (28.7%) were subtype s2. s1m2 was the most frequent vacA allelic combination in the H. pylori isolates examined (63 cases), followed by s2m2 (31 cases), s1m1 (19 cases) and s2m1 (2 case). Strains cagA positive were more frequent in peptic ulcer diseases patients than non ulcer diseases patients, as 47 (59.5%) and 32 (40.5%), while cagA negative were low, as 15 (41.7%) and 21 (58.3%), respectively. Conclusion: We found that the cagA and vacA status were not related to clinical outcomes in this area. Overall, in the present study, vacA s1/m2, cagA-positive strains were predominant irrespective of clinical outcome, but s2/m1 was rare. PMID:24312813

  17. Proteomic Characterization of Helicobacter pylori CagA Antigen Recognized by Child Serum Antibodies and Its Epitope Mapping by Peptide Array

    PubMed Central

    Akada, Junko; Okuda, Masumi; Hiramoto, Narumi; Kitagawa, Takao; Zhang, Xiulian; Kamei, Shuichi; Ito, Akane; Nakamura, Mikiko; Uchida, Tomohisa; Hiwatani, Tomoko; Fukuda, Yoshihiro; Nakazawa, Teruko; Kuramitsu, Yasuhiro; Nakamura, Kazuyuki

    2014-01-01

    Serum antibodies against pathogenic bacteria play immunologically protective roles, and can be utilized as diagnostic markers of infection. This study focused on Japanese child serum antibodies against Helicobacter pylori, a chronically-infected gastric bacterium which causes gastric cancer in adults. Serological diagnosis for H. pylori infection is well established for adults, but it needs to be improved for children. Serum samples from 24 children, 22 H. pylori (Hp)-positive and 2 Hp-negative children, were used to catalogue antigenic proteins of a Japanese strain CPY2052 by two-dimensional electrophoresis followed by immunoblot and LC-MS/MS analysis. In total, 24 proteins were identified as candidate antigen proteins. Among these, the major virulence factor, cytotoxin-associated gene A protein (CagA) was the most reactive antigen recognized by all the Hp-positive sera even from children under the age of 3 years. The major antigenic part of CagA was identified in the middle region, and two peptides containing CagA epitopes were identified using a newly developed peptide/protein-combined array chip method, modified from our previous protein chip method. Each of the epitopes was found to contain amino acid residue(s) unique to East Asian CagA. Epitope analysis of CagA indicated importance of the regional CagA antigens for serodiagnosis of H. pylori infection in children. PMID:25141238

  18. Roles of the tyrosine isomers meta-tyrosine and ortho-tyrosine in oxidative stress.

    PubMed

    Ipson, Brett R; Fisher, Alfred L

    2016-05-01

    The damage to cellular components by reactive oxygen species, termed oxidative stress, both increases with age and likely contributes to age-related diseases including Alzheimer's disease, atherosclerosis, diabetes, and cataract formation. In the setting of oxidative stress, hydroxyl radicals can oxidize the benzyl ring of the amino acid phenylalanine, which then produces the abnormal tyrosine isomers meta-tyrosine or ortho-tyrosine. While elevations in m-tyrosine and o-tyrosine concentrations have been used as a biological marker of oxidative stress, there is emerging evidence from bacterial, plant, and mammalian studies demonstrating that these isomers, particularly m-tyrosine, directly produce adverse effects to cells and tissues. These new findings suggest that the abnormal tyrosine isomers could in fact represent mediators of the effects of oxidative stress. Consequently the accumulation of m- and o-tyrosine may disrupt cellular homeostasis and contribute to disease pathogenesis, and as result, effective defenses against oxidative stress can encompass not only the elimination of reactive oxygen species but also the metabolism and ultimately the removal of the abnormal tyrosine isomers from the cellular amino acid pool. Future research in this area is needed to clarify the biologic mechanisms by which the tyrosine isomers damage cells and disrupt the function of tissues and organs and to identify the metabolic pathways involved in removing the accumulated isomers after exposure to oxidative stress.

  19. High Diversity of vacA and cagA Helicobacter pylori Genotypes in Patients with and without Gastric Cancer

    PubMed Central

    López-Vidal, Yolanda; Ponce-de-León, Sergio; Castillo-Rojas, Gonzalo; Barreto-Zúñiga, Rafael; Torre-Delgadillo, Aldo

    2008-01-01

    Background Helicobacter pylori is associated with chronic gastritis, peptic ulcers, and gastric cancer. The aim of this study was to assess the topographical distribution of H. pylori in the stomach as well as the vacA and cagA genotypes in patients with and without gastric cancer. Methodology/Principal Findings Three gastric biopsies, from predetermined regions, were evaluated in 16 patients with gastric cancer and 14 patients with dyspeptic symptoms. From cancer patients, additional biopsy specimens were obtained from tumor centers and margins; among these samples, the presence of H. pylori vacA and cagA genotypes was evaluated. Positive H. pylori was 38% and 26% in biopsies obtained from the gastric cancer and non-cancer groups, respectively (p = 0.008), and 36% in tumor sites. In cancer patients, we found a preferential distribution of H. pylori in the fundus and corpus, whereas, in the non-cancer group, the distribution was uniform (p = 0.003). A majority of the biopsies were simultaneously cagA gene-positive and -negative. The fundus and corpus demonstrated a higher positivity rate for the cagA gene in the non-cancer group (p = 0.036). A mixture of cagA gene sizes was also significantly more frequent in this group (p = 0.003). Ninety-two percent of all the subjects showed more than one vacA gene genotype; s1b and m1 vacA genotypes were predominantly found in the gastric cancer group. The highest vacA-genotype signal-sequence diversity was found in the corpus and 5 cm from tumor margins. Conclusion/Significance High H. pylori colonization diversity, along with the cagA gene, was found predominantly in the fundus and corpus of patients with gastric cancer. The genotype diversity observed across systematic whole-organ and tumor sampling was remarkable. We find that there is insufficient evidence to support the association of one isolate with a specific disease, due to the multistrain nature of H. pylori infection shown in this work. PMID:19050763

  20. CagA+ Helicobacter pylori infection and gastric cancer risk in the EPIC-EURGAST study.

    PubMed

    Palli, Domenico; Masala, Giovanna; Del Giudice, Giuseppe; Plebani, Mario; Basso, Daniela; Berti, Duccio; Numans, Mattijs E; E Numans, Mattijs; Ceroti, Marco; Peeters, Petra H M; Bueno de Mesquita, H Bas; Buchner, Frederike L; Clavel-Chapelon, Francoise; Boutron-Ruault, Marie-Christine; Krogh, Vittorio; Saieva, Calogero; Vineis, Paolo; Panico, Salvatore; Tumino, Rosario; Nyrén, Olof; Simán, Henrik; Berglund, Goran; Hallmans, Goran; Sanchez, Maria-Jose; Larrãnaga, Nerea; Barricarte, Aurelio; Navarro, Carmen; Quiros, Jose R; Key, Tim; Allen, Naomi; Bingham, Sheila; Khaw, Kay Tee; Boeing, Heiner; Weikert, Cornelia; Linseisen, Jakob; Nagel, Gabriele; Overvad, Kim; Thomsen, Reimar W; Tjonneland, Anne; Olsen, Anja; Trichoupoulou, Antonia; Trichopoulos, Dimitrios; Arvaniti, Athina; Pera, Guillem; Kaaks, Rudolf; Jenab, Mazda; Ferrari, Pietro; Nesi, Gabriella; Carneiro, Fatima; Riboli, Elio; Gonzalez, Carlos A

    2007-02-15

    Helicobacter pylori (H. pylori), atrophic gastritis, dietary and life-style factors have been associated with gastric cancer (GC). These factors have been evaluated in a large case-control study nested in the European Prospective Investigation into Cancer and Nutrition carried out in 9 countries, including the Mediterranean area. Participants, enrolled in 1992-1998, provided life-style and dietary information and a blood sample (360,000; mean follow-up: 6.1 years). For 233 GC cases diagnosed after enrolment and their 910 controls individually-matched by center, gender, age and blood donation date H. pylori antibodies (antilysate and antiCagA) and plasma Pepsinogen A (PGA) were measured by ELISA methods. Severe chronic atrophic gastritis (SCAG) was defined as PGA circulating levels <22 microg/l. Overall, in a conditional logistic regression analysis adjusted for education, smoke, weight and consumption of total vegetables, fruit, red and preserved meat, H. pylori seropositivity was associated with GC risk. Subjects showing only antibodies anti-H. pylori lysate, however, were not at increased risk, while those with antiCagA antibodies had a 3.4-fold increased risk. Overall, the odds ratio associated with SCAG was 3.3 (95% CI 2.2-5.2). According to site, the risk of noncardia GC associated with CagA seropositivity showed a further increase (OR 6.5; 95% CI 3.3-12.6); on the other hand, a ten-fold increased risk of cardia GC was associated with SCAG (OR 11.0; 95% CI 3.0-40.9). These results support the causal relationship between H. pylori CagA+ strains infection, and GC in these European populations even after taking into account dietary habits. This association was limited to distal GC, while serologically defined SCAG was strongly associated with cardia GC, thus suggesting a divergent risk pattern for these 2 sites. PMID:17131317

  1. Hydrogen Metabolism in Helicobacter pylori Plays a Role in Gastric Carcinogenesis through Facilitating CagA Translocation

    PubMed Central

    Wang, Ge; Romero-Gallo, Judith; Benoit, Stéphane L.; Piazuelo, M. Blanca; Dominguez, Ricardo L.; Morgan, Douglas R.; Peek, Richard M.

    2016-01-01

    ABSTRACT A known virulence factor of Helicobacter pylori that augments gastric cancer risk is the CagA cytotoxin. A carcinogenic derivative strain, 7.13, that has a greater ability to translocate CagA exhibits much higher hydrogenase activity than its parent noncarcinogenic strain, B128. A Δhyd mutant strain with deletion of hydrogenase genes was ineffective in CagA translocation into human gastric epithelial AGS cells, while no significant attenuation of cell adhesion was observed. The quinone reductase inhibitor 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO) was used to specifically inhibit the H2-utilizing respiratory chain of outer membrane-permeabilized bacterial cells; that level of inhibitor also greatly attenuated CagA translocation into AGS cells, indicating the H2-generated transmembrane potential is a contributor to toxin translocation. The Δhyd strain showed a decreased frequency of DNA transformation, suggesting that H. pylori hydrogenase is also involved in energizing the DNA uptake apparatus. In a gerbil model of infection, the ability of the Δhyd strain to induce inflammation was significantly attenuated (at 12 weeks postinoculation), while all of the gerbils infected with the parent strain (7.13) exhibited a high level of inflammation. Gastric cancer developed in 50% of gerbils infected with the wild-type strain 7.13 but in none of the animals infected with the Δhyd strain. By examining the hydrogenase activities from well-defined clinical H. pylori isolates, we observed that strains isolated from cancer patients (n = 6) have a significantly higher hydrogenase (H2/O2) activity than the strains isolated from gastritis patients (n = 6), further supporting an association between H. pylori hydrogenase activity and gastric carcinogenesis in humans. PMID:27531909

  2. Increased oxidative and nitrative stress in human stomach associated with cagA+ Helicobacter pylori infection and inflammation.

    PubMed

    Li, C Q; Pignatelli, B; Ohshima, H

    2001-04-01

    In order to study the role of Helicobacter pylori infection in gastric carcinogenesis, we have measured oxidized (carbonyls) and nitrated (nitrotyrosine-containing) proteins as markers for oxidative and nitrative stress in 216 human gastric biopsies using dot and western immunoblots and correlated the results with H. pylori, cagA status, expression of interleukin-8 and inducible nitric oxide synthase (iNOS) mRNAs, and gastric pathology. Higher levels of both oxidized and nitrated proteins were found in patients with either chronic gastritis or duodenal ulcer than in those with normal mucosa. The levels of modified proteins were significantly higher in inflamed samples infected with H. pylori, especially cagA+ strains, and in those with expression of interleukin-8 and iNOS mRNAs than in those negative for these parameters. These results indicate that infection with cagA+ H. pylori induces significant oxidative and nitrative stress in stomach mucosa, contributing to the pathogenesis of H. pylori-associated gastroduodenal diseases.

  3. Immunomodulatory Effect of H. Pylori CagA Genotype and Gastric Hormones On Gastric Versus Inflammatory Cells Fas Gene Expression in Iraqi Patients with Gastroduodenal Disorders

    PubMed Central

    AL-Ezzy, Ali Ibrahim Ali

    2016-01-01

    AIM: To evaluate the Immunomodulatory effects of CagA expression; pepsinogen I, II & gastrin-17 on PMNs and lymphocytes Fas expression in inflammatory and gastric cells; demographic distribution of Fas molecule in gastric tissue and inflammatory cells. METHODS: Gastroduodenal biopsies were taken from 80 patients for histopathology and H. pylori diagnosis. Serum samples were used for evaluation of pepsinogen I (PGI); (PGII); gastrin-17 (G-17). RESULTS: Significant difference (p < 0.001) in lymphocytes & PMNs Fas expression; epithelial & lamina propria Fas localization among H. pylori associated gastric disorders. No correlation between grade of lymphocytes & PMNs Fas expression in gastric epithelia; lamina propria and types of gastric disorder. Significant difference (p < 0.001) in total gastric Fas expression, epithelial Fas; lamina propria and gastric gland Fas expression according to CagA, PGI; PGII; PGI/PGII; Gastrin-17. Total gastric Fas expression has significant correlation with CagA, PGII levels. Gastric epithelial and gastric lamina propria Fas expression have significant correlation with CagA, PGI; PGII levels. Significant difference (p < 0.001) was found in lymphocytes & PMNs Fas expression; epithelial & lamina propria localization of lymphocytes & PMNs Fas expression according to CagA, PGI; PGII; PGI/PGII; Gastrin-17. Lymphocytes Fas expression have correlation with PGI, PGII, PGI/PGII. PMNs Fas expression have correlation with PGI, PGII. CONCLUSION: Fas gene expression and localization on gastric and inflammatory cells affected directly by H. pylori CagA and indirectly by gastric hormones. This contributes to progression of various gastric disorders according to severity of CagA induced gastric pathology and gastric hormones disturbance throughout the course of infection and disease. PMID:27703557

  4. Tyrosine phosphorylation and bacterial virulence

    PubMed Central

    Whitmore, Sarah E; Lamont, Richard J

    2012-01-01

    Protein phosphorylation on tyrosine has emerged as a key device in the control of numerous cellular functions in bacteria. In this article, we review the structure and function of bacterial tyrosine kinases and phosphatases. Phosphorylation is catalyzed by autophosphorylating adenosine triphosphate-dependent enzymes (bacterial tyrosine (BY) kinases) that are characterized by the presence of Walker motifs. The reverse reaction is catalyzed by three classes of enzymes: the eukaryotic-like phosphatases (PTPs) and dual-specific phosphatases; the low molecular weight protein-tyrosine phosphatases (LMW-PTPs); and the polymerase–histidinol phosphatases (PHP). Many BY kinases and tyrosine phosphatases can utilize host cell proteins as substrates, thereby contributing to bacterial pathogenicity. Bacterial tyrosine phosphorylation/dephosphorylation is also involved in biofilm formation and community development. The Porphyromonas gingivalis tyrosine phosphatase Ltp1 is involved in a restraint pathway that regulates heterotypic community development with Streptococcus gordonii. Ltp1 is upregulated by contact with S. gordonii and Ltp1 activity controls adhesin expression and levels of the interspecies signal AI-2. PMID:22388693

  5. CagA, a major virulence factor of Helicobacter pylori, promotes the production and underglycosylation of IgA1 in DAKIKI cells.

    PubMed

    Yang, Man; Li, Fu-gang; Xie, Xi-sheng; Wang, Shao-qing; Fan, Jun-ming

    2014-02-01

    While Helicobacter pylori (Hp) infection is closely associated with IgA nephropathy (IgAN), the underlying molecular mechanisms remain to be elucidated. This study was to investigate the effect of cytotoxin associated gene A protein (CagA), a major virulence factor of Hp, on the production and underglycosylation of IgA1 in the B cell line DAKIKI cells. Cells were cultured and treated with recombinant CagA protein. We found that CagA stimulated cell proliferation and the production of IgA1 in a dose-dependent and time-dependent manner. Moreover, CagA promoted the underglycosylation of IgA1, which at least partly attributed to the downregulation of β1,3-galactosyltransferase (C1GALT1) and its chaperone Cosmc. In conclusion, we demonstrated that Hp infection, at least via CagA, may participate in the pathogenesis of IgAN by influencing the production and glycosylation of IgA1 in B cells.

  6. UreA and cagA genes of Helicobacter pylori in Egyptian patients with laryngeal squamous cell carcinoma and benign laryngeal polyps: a cohort study.

    PubMed

    Barakat, Ghada; Nabiel, Yasmin; Ali, Omima; El-Nady, Ghada; Musaad, Ahmed; El-Sharkawy, Asser

    2016-10-01

    This work aims to estimate the prevalence of Helicobacter pylori ureA gene and evaluate cagA gene-positive strains in both patients of laryngeal squamous cell carcinoma (LSCC) and those with benign laryngeal polyps. This study included 49 patients confirmed pathologically to have LSCC and 15 patients with benign laryngeal polyps over a period from June 2013 to March 2015. Samples of laryngeal tissue were collected during direct laryngoscope under general anesthesia to be pathologically evaluated followed by analysis for H. pylori detection. Each laryngeal tissue sample was divided into three parts; one for bacteriological examination, the second for pathological examination and the third for PCR to detect both ureA and cagA genes. Out of 49 LSCC samples, 31 (64.6 %) was positive for ureA by PCR. Out of them, 29 samples (93.5 %) were cagA positive. Only three cases (20 %) of the benign laryngeal polyp were ureA positive by PCR and one of them was cagA positive by PCR. By the bacteriological culture, only eight samples (25.8 %) gave growth. All of them were ureA positive and only seven of them were cagA positive. There was a significant association between presence of H. pylori and LSCC as compared to benign laryngeal polyp which may contribute in the pathogenesis of laryngeal carcinoma. These results should be confirmed by further studies over larger number of cases.

  7. Tyrosine phosphorylation of WW proteins

    PubMed Central

    Reuven, Nina; Shanzer, Matan

    2015-01-01

    A number of key regulatory proteins contain one or two copies of the WW domain known to mediate protein–protein interaction via proline-rich motifs, such as PPxY. The Hippo pathway components take advantage of this module to transduce tumor suppressor signaling. It is becoming evident that tyrosine phosphorylation is a critical regulator of the WW proteins. Here, we review the current knowledge on the involved tyrosine kinases and their roles in regulating the WW proteins. PMID:25627656

  8. Risk of advanced gastric precancerous lesions in Helicobacter pylori infected subjects is influenced by ABO blood group and cagA status

    PubMed Central

    Rizzato, Cosmeri; Kato, Ikuko; Plummer, Martyn; Muñoz, Nubia; Stein, Angelika; van Doorn, Leen Jan; Franceschi, Silvia; Canzian, Federico

    2013-01-01

    A higher incidence of stomach cancer in ABO blood type A individuals than in those with blood type O has been known for a long time. We studied this association in relation to Helicobacter pylori (Hp) of different cagA status. For this study we used baseline gastric histopathology data and DNAs from frozen gastric biopsies of 2077 subjects enrolled in a chemoprevention trial for gastric precancerous lesions in Venezuela. We analyzed 6 single nucleotide polymorphisms in the ABO gene and we assessed the presence of the Hp cagA gene. Odds ratios for risk of advanced precancerous gastric lesions were calculated using individuals with normal gastric epithelium or non-atrophic gastritis as a reference. Among individuals carrying a cagA negative Hp infection or no Hp infection, those with blood type A had a lower risk of intestinal metaplasia and dysplasia than those with blood type O (OR=0.60; 95% CI 0.38-0.94). In carriers of cagA positive Hp strains, individuals with blood type A had a higher risk of intestinal metaplasia or dysplasia than those with blood type O (OR=1.42, 95% CI 1.09-1.86) and a higher risk if compared with subjects carrying cagA− strain and non-A blood group (OR=3.82, 95%CI=2.80-5.20). The interaction between Hp cagA status and blood type was statistically significant (P=0.0006). We showed that SNPs in the ABO gene, predictive of ABO blood groups, are associated with risk of advanced precancerous gastric lesions in individuals infected with Hp, but the assessment of the risk is strictly dependent on cagA status. PMID:23319424

  9. Targeted tyrosine iodination in a multi-tyrosine vasopressin analog.

    PubMed

    Durr, Jacques A; Blankenship, Mary; Chauhan, Satendra S; Pennington, Michael W

    2007-11-01

    Iodination of the conserved 2-tyrosine (Tyr(2)) residue in the pressin and tocin rings of arginine- or lysine-vasopressin (AVP or LVP), and oxytocin, respectively, impairs binding to their respective receptors. Synthetic antagonists that have their Tyr(2) either replaced by another amino acid or irreversibly blocked by an O-methyl or O-ethyl ether, but have, instead, an iodinatable phenol moiety outside the pressin/tocin ring, are used for radiolabeling. We explored another approach to avoid iodinating Tyr(2) by capping this residue with a reversible O-acetyl group, incorporated during peptide synthesis. The O-acetyl-Tyr(2) LVP peptide, with a free iodinatable tyrosine attached to the epsilon-amine of 8-lysine, is iodinated at a neutral pH and purified by reverse-phase high-pressure liquid chromatography (HPLC) at an acidic pH, conditions under which the O-acetyl groups are stable. Deacetylation with hydroxylamine is selective, and leaves intact the disulfide bridge. The marked shortening of the HPLC retention time after deblocking produces a chemically homogeneous label, iodinated exclusively on the free tyrosine residue attached to the epsilon-amine of LVP. Hitherto, this (125)I labeled vasopressin agonist could be obtained only in low yield, via conjugation labeling with iodinated N-t-Boc-tyrosine succinimidyl ester. This fully reversible tyrosine protection strategy does not require special equipment, and retains the conserved Tyr(2), typical of vasopressin and oxytocin agonists.

  10. Expression of cagA, virB/D Complex and/or vacA Genes in Helicobacter pylori Strains Originating from Patients with Gastric Diseases

    PubMed Central

    Szkaradkiewicz, Andrzej; Karpiński, Tomasz M.; Linke, Krzysztof; Majewski, Przemysław; Rożkiewicz, Dorota; Goślińska-Kuźniarek, Olga

    2016-01-01

    In order to better understand pathogenicity of Helicobacter pylori, particularly in the context of its carcinogenic activity, we analysed expression of virulence genes: cagA, virB/D complex (virB4, virB7, virB8, virB9, virB10, virB11, virD4) and vacA in strains of the pathogen originating from persons with gastric diseases. The studies were conducted on 42 strains of H. pylori isolated from patients with histological diagnosis of non-atrophic gastritis—NAG (group 1, including subgroup 1 containing cagA+ isolates and subgroup 2 containing cagA- strains), multifocal atrophic gastritis—MAG (group 2) and gastric adenocarcinoma—GC (group 3). Expression of H. pylori genes was studied using microarray technology. In group 1, in all strains of H. pylori cagA+ (subgroup 1) high expression of the gene as well as of virB/D was disclosed, accompanied by moderate expression of vacA. In strains of subgroup 2 a moderate expression of vacA was detected. All strains in groups 2 and 3 carried cagA gene but they differed in its expression: a high expression was detected in isolates of group 2 and its hyperexpression in strains of group 3 (hypervirulent strains). In both groups high expression of virB/D and vacA was disclosed. Our results indicate that chronic active gastritis may be induced by both cagA+ strains of H. pylori, manifesting high expression of virB/D complex but moderate activity of vacA, and cagA- strains with moderate expression of vacA gene. On the other hand, in progression of gastric pathology and carcinogenesis linked to H. pylori a significant role was played by hypervirulent strains, manifesting a very high expression of cagA and high activity of virB/D and vacA genes. PMID:26866365

  11. Tyrosine - Effects on catecholamine release

    NASA Technical Reports Server (NTRS)

    Acworth, Ian N.; During, Matthew J.; Wurtman, Richard J.

    1988-01-01

    Tyrosine administration elevates striatal levels of dopamine metabolites in animals given treatments that accelerate nigrostriatal firing, but not in untreated rats. We examined the possibility that the amino acid might actually enhance dopamine release in untreated animals, but that the technique of measuring striatal dopamine metabolism was too insensitive to demonstrate such an effect. Dopamine release was assessed directly, using brain microdialysis of striatal extracellular fluid. Tyrosine administration (50-200 mg/kg IP) did indeed cause a dose related increase in extracellular fluid dopamine levels with minor elevations in levels of DOPAC and HVA, its major metabolites, which were not dose-related. The rise in dopamine was short-lived, suggesting that receptor-mediated feedback mechanisms responded to the increased dopamine release by diminishing neuronal firing or sensitivity to tyrosine. These observations indicate that measurement of changes in striatal DOPAC and HVA, if negative, need not rule out increases in nigrostriatal dopamine release.

  12. High photoluminescence stability of CaGa4O7:Eu3+ red phosphor in wide excitation intensity interval

    NASA Astrophysics Data System (ADS)

    Leanenia, M. S.; Lutsenko, E. V.; Rzheutski, M. V.; Yablonskii, G. P.; Naghiyev, T. G.; Ganbarova, H. B.; Tagiev, O. B.

    2016-04-01

    The photoluminescence (PL) of CaGa4O7 compound doped with Eu3+ ions is studied. It is shown that room temperature emission spectrum of CaGa4O7 compound consists of groups of lines in the red spectral region, caused by electronic transitions 5D0 → 7Fj (j = 1, 2, 3, 4) in Eu3+ ions with the most intensive lines at 612 nm and 615 nm. It is evaluated that low local symmetry of Eu3+ ion appears in the presence of several lines for each transitions. The PL decay constants were found to be in the range of 1.1-1.3 ms. Non-exponential rise was observed in the beginning of PL kinetics and assumed with energy transfer between Eu3+ ions in different sites in host lattice. Weak thermal quenching is shown in the temperature range of 10-300 K. Extreme stability of PL spectra and efficiency in a wide excitation intensity range of 104-108 W/cm2 were achieved. A slight emission efficiency reversible droop by only 25% is found to be at the excitation intensities from 2 · 107 W/cm2 to 108 W/cm2.

  13. Characterization of virulence genes cagA and vacA in Helicobacter Pylori and their prevalence in gastrointestinal disorders

    PubMed Central

    Cogo, Laura Lúcia; Monteiro, Cristina Leise Bastos; Nogueira, Keite da Silva; Palmeiro, Jussara Kasuko; Ribeiro, Marcelo Lima; de Camargo, Eloá Ramalho; Neves, Daniel Locatelli; do Nascimento, Aguinaldo José; Costa, Libera Maria Dalla

    2011-01-01

    Prevalence of H. pylori infection was determined using cultures of gastric biopsy samples of patients attended at the academic hospital of the Federal University of Paraná, Curitiba, Paraná, Brazil. Molecular methods were used to characterize the cagA and vacA genes from bacterial isolates associated with different diseases presented by patients. Out of a total of 81, forty-two gastric biopsy samples tested were positive for H. pylori, with a prevalence of 51.9%. No significant difference was found with regard to the gender (p=0.793) and age (p=0.183) of the patients. Genotype s1m1 vacA gene was found in 67% of the cases of peptic ulcer investigated (p=1.0), despite the limited number of patients with this disease (n=3). A correlation between the presence of less virulent strains (s2m2) and reflux esophagitis was found in the majority of the cases (45%), but without statistical significance. An association between the prevalence of cagA gene, found in 92% of isolates, and peptic ulcer was not observed (p=1.0), suggesting that this gene cannot be considered a specific marker of severity in our environment. The results reinforce the importance of conducting regional studies and the need to characterize H. pylori virulence genes associated with different diseases. PMID:24031754

  14. Discovering the first tyrosine kinase

    PubMed Central

    Hunter, Tony

    2015-01-01

    In the middle of the 20th century, animal tumor viruses were heralded as possible models for understanding human cancer. By the mid-1970s, the molecular basis by which tumor viruses transform cells into a malignant state was beginning to emerge as the first viral genomic sequences were reported and the proteins encoded by their transforming genes were identified and characterized. This was a time of great excitement and rapid progress. In 1978, prompted by the discovery from Ray Erikson’s group that the Rous sarcoma virus (RSV) v-Src–transforming protein had an associated protein kinase activity specific for threonine, my group at the Salk Institute set out to determine whether the polyomavirus middle T-transforming protein had a similar kinase activity. Here, I describe the experiments that led to the identification of a kinase activity associated with middle T antigen and our serendipitous discovery that this activity was specific for tyrosine in vitro, and how this in turn led to the fortuitous observation that the v-Src–associated kinase activity was also specific for tyrosine. Our finding that v-Src increased the level of phosphotyrosine in cellular proteins in RSV-transformed cells confirmed that v-Src is a tyrosine kinase and transforms cells by phosphorylating proteins on tyrosine. My colleague Bart Sefton and I reported these findings in the March issue of PNAS in 1980. Remarkably, all of the experiments in this paper were accomplished in less than one month. PMID:26130799

  15. 21 CFR 582.5920 - Tyrosine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Tyrosine. 582.5920 Section 582.5920 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS... § 582.5920 Tyrosine. (a) Product. Tyrosine (L- and DL-forms). (b) Conditions of use. This substance...

  16. 21 CFR 582.5920 - Tyrosine.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Tyrosine. 582.5920 Section 582.5920 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS... § 582.5920 Tyrosine. (a) Product. Tyrosine (L- and DL-forms). (b) Conditions of use. This substance...

  17. Prevalence of cagA and vacA among Helicobacter pylori-infected patients in Iran: a systematic review and meta-analysis.

    PubMed

    Sayehmiri, Fatemeh; Kiani, Faezeh; Sayehmiri, Kourosh; Soroush, Setareh; Asadollahi, Khairollah; Alikhani, Mohammad Yousef; Delpisheh, Ali; Emaneini, Mohammad; Bogdanović, Lidija; Varzi, Ali Mohammad; Zarrilli, Raffaele; Taherikalani, Morovat

    2015-07-30

    The varieties of infections caused by Helicobacter pylori may be due to differences in bacterial genotypes and virulence factors as well as environmental and host-related factors. This study aimed to investigate the prevalence of cagA and vacA genes among H. pylori-infected patients in Iran and analyze their relevance to the disease status between two clinical groups via a meta-analysis method. Different databases including PubMed, ISI, Scopus, SID, Magiran, Science Direct, and Medlib were investigated, and 23 relevant articles from the period between 2001 and 2012 were finally analyzed. The relevant data obtained from these papers were analyzed by a random-effects model. Data were analyzed using R software and STATA. The prevalence of cagA and vacA genes among H. pylori-infected patients was 70% (95% CI, 64-75) and 41% (95% CI, 24.3-57.7), respectively. The prevalence of duodenal ulcers, peptic ulcers, and gastritis among cagA+ individuals was 53% (95% CI, 20-86), 65% (95% CI, 34-97), and 71% (95% CI, 59-84), respectively. Odds ratio (OR) between cagA-positive compared with cagA-negative patients showed a 1.89 (95% CI, 1.38-2.57) risk of ulcers. In conclusion, the frequency of cagA gene among H. pylori strains is elevated in Iran and it seems to be more frequently associated with gastritis. Therefore, any information about cagA and vacA prevalence among different H. pylori-infected clinical groups in the country can help public health authorities to plan preventive policies to reduce the prevalence of diseases associated with H. pylori infection.

  18. Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island: essential genes for CagA translocation in host cells and induction of interleukin-8.

    PubMed

    Fischer, W; Püls, J; Buhrdorf, R; Gebert, B; Odenbreit, S; Haas, R

    2001-12-01

    Helicobacter pylori (Hp) carries a type IV secretion system encoded by the cag pathogenicity island (cag-PAI), which is used to: (i) translocate the bacterial effector protein CagA into different types of eukaryotic cells; and (ii) induce the synthesis and secretion of chemokines, such as interleukin-8 (IL-8). The cag-PAI in Hp 26695 consists of 27 putative genes, six of which were identified as homologues to the basic type IV secretion system represented by the Agrobacterium tumefaciens virB operon. To define the role and contribution of each of the 27 genes, we applied a precise deletion/insertion mutagenesis procedure to knock out each individual gene without causing polar effects on the expression of downstream genes. Seventeen out of 27 genes were found to be absolutely essential for translocation of CagA into host cells and 14 out of 27 for the ability of Hp fully to induce transcription of IL-8. The products of hp0524 (virD4 homologue), hp0526 and hp0540 are absolutely essential for the translocation of CagA, but not for the induction of IL-8. In contrast, the products of hp0520, hp0521, hp0534, hp0535, hp0536 and hp0543 are not necessary for either translocation of CagA or for IL-8 induction. Our data argue against a translocated IL-8-inducing effector protein encoded by the cag-PAI. We isolated a variant of Hp 26695, which spontaneously switched off its capacity for IL-8 induction and translocation of CagA, but retained the complete cag-PAI. We identified a point mutation in gene hp0532, causing a premature translational stop in the corresponding polypeptide chain, providing a putative explanation for the defect in the type IV secretion system of the spontaneous mutant. PMID:11886563

  19. Structural basis of tubulin tyrosination by tubulin tyrosine ligase.

    PubMed

    Prota, Andrea E; Magiera, Maria M; Kuijpers, Marijn; Bargsten, Katja; Frey, Daniel; Wieser, Mara; Jaussi, Rolf; Hoogenraad, Casper C; Kammerer, Richard A; Janke, Carsten; Steinmetz, Michel O

    2013-02-01

    Tubulin tyrosine ligase (TTL) catalyzes the post-translational retyrosination of detyrosinated α-tubulin. Despite the indispensable role of TTL in cell and organism development, its molecular mechanism of action is poorly understood. By solving crystal structures of TTL in complex with tubulin, we here demonstrate that TTL binds to the α and β subunits of tubulin and recognizes the curved conformation of the dimer. Biochemical and cellular assays revealed that specific tubulin dimer recognition controls the activity of the enzyme, and as a consequence, neuronal development. The TTL-tubulin structure further illustrates how the enzyme binds the functionally crucial C-terminal tail sequence of α-tubulin and how this interaction catalyzes the tyrosination reaction. It also reveals how TTL discriminates between α- and β-tubulin, and between different post-translationally modified forms of α-tubulin. Together, our data suggest that TTL has specifically evolved to recognize and modify tubulin, thus highlighting a fundamental role of the evolutionary conserved tubulin tyrosination cycle in regulating the microtubule cytoskeleton. PMID:23358242

  20. Receptor Tyrosine Kinases in Drosophila Development

    PubMed Central

    Sopko, Richelle; Perrimon, Norbert

    2013-01-01

    Tyrosine phosphorylation plays a significant role in a wide range of cellular processes. The Drosophila genome encodes more than 20 receptor tyrosine kinases and extensive studies in the past 20 years have illustrated their diverse roles and complex signaling mechanisms. Although some receptor tyrosine kinases have highly specific functions, others strikingly are used in rather ubiquitous manners. Receptor tyrosine kinases regulate a broad expanse of processes, ranging from cell survival and proliferation to differentiation and patterning. Remarkably, different receptor tyrosine kinases share many of the same effectors and their hierarchical organization is retained in disparate biological contexts. In this comprehensive review, we summarize what is known regarding each receptor tyrosine kinase during Drosophila development. Astonishingly, very little is known for approximately half of all Drosophila receptor tyrosine kinases. PMID:23732470

  1. Endocytosis of Receptor Tyrosine Kinases

    PubMed Central

    Goh, Lai Kuan

    2013-01-01

    Endocytosis is the major regulator of signaling from receptor tyrosine kinases (RTKs). The canonical model of RTK endocytosis involves rapid internalization of an RTK activated by ligand binding at the cell surface and subsequent sorting of internalized ligand-RTK complexes to lysosomes for degradation. Activation of the intrinsic tyrosine kinase activity of RTKs results in autophosphorylation, which is mechanistically coupled to the recruitment of adaptor proteins and conjugation of ubiquitin to RTKs. Ubiquitination serves to mediate interactions of RTKs with sorting machineries both at the cell surface and on endosomes. The pathways and kinetics of RTK endocytic trafficking, molecular mechanisms underlying sorting processes, and examples of deviations from the standard trafficking itinerary in the RTK family are discussed in this work. PMID:23637288

  2. Tyrosine Recombinase Retrotransposons and Transposons.

    PubMed

    Poulter, Russell T M; Butler, Margi I

    2015-04-01

    Retrotransposons carrying tyrosine recombinases (YR) are widespread in eukaryotes. The first described tyrosine recombinase mobile element, DIRS1, is a retroelement from the slime mold Dictyostelium discoideum. The YR elements are bordered by terminal repeats related to their replication via free circular dsDNA intermediates. Site-specific recombination is believed to integrate the circle without creating duplications of the target sites. Recently a large number of YR retrotransposons have been described, including elements from fungi (mucorales and basidiomycetes), plants (green algae) and a wide range of animals including nematodes, insects, sea urchins, fish, amphibia and reptiles. YR retrotransposons can be divided into three major groups: the DIRS elements, PAT-like and the Ngaro elements. The three groups form distinct clades on phylogenetic trees based on alignments of reverse transcriptase/ribonuclease H (RT/RH) and YR sequences, and also having some structural distinctions. A group of eukaryote DNA transposons, cryptons, also carry tyrosine recombinases. These DNA transposons do not encode a reverse transcriptase. They have been detected in several pathogenic fungi and oomycetes. Sequence comparisons suggest that the crypton YRs are related to those of the YR retrotransposons. We suggest that the YR retrotransposons arose from the combination of a crypton-like YR DNA transposon and the RT/RH encoding sequence of a retrotransposon. This acquisition must have occurred at a very early point in the evolution of eukaryotes. PMID:26104693

  3. Evaluation of immobilized metal affinity chromatography kits for the purification of histidine-tagged recombinant CagA protein.

    PubMed

    Karakus, Cebrail; Uslu, Merve; Yazici, Duygu; Salih, Barik A

    2016-05-15

    Immobilized metal affinity chromatography (IMAC) technique is used for fast and reliable purification of histidine(His)-tagged recombinant proteins. The technique provides purification under native and denaturing conditions. The aim of this study is to evaluate three commercially available IMAC kits (Thermo Scientific, GE Healthcare and Qiagen) for the purification of a 6xHis-tagged recombinant CagA (cytotoxin-associated gene A) protein from IPTG-induced Escherichia coli BL21(DE3) culture. The kits were tested according to the manufacturer instructions and the protein was purified with only GE Healthcare and Qiagen kits under denaturing conditions. 1% (w/v) SDS was used as denaturing agent in PBS instead of extraction reagent of Thermo Scientific kit to lyse bacterial cells from 100ml culture. The 6xHis-tagged recombinant protein was purified by the three kits equally. PMID:26657801

  4. Cisplatin stimulates protein tyrosine phosphorylation in macrophages.

    PubMed

    Kumar, R; Shrivastava, A; Sodhi, A

    1995-03-01

    Cisplatin [cis-dichlorodiamine platinum (II)], a potent anti-tumor compound, stimulates immune responses by activating monocyte-macrophages and other cells of the immune system. The mechanism by which cisplatin activates these cells is poorly characterized. Since protein tyrosine phosphorylation appears to be a major intracellular signalling event that mediates cellular responses, we examined whether cisplatin alters tyrosine phosphorylation in macrophages. We found that cisplatin increased tyrosine phosphorylation of several proteins in peritoneal macrophages and in P388D1 and IC-21 macrophage cell lines. Treatment of macrophages with tyrosine kinase inhibitors, genestein and lavendustin A, inhibited cisplatin-stimulated protein tyrosine phosphorylation in macrophages. Macrophages treated with cisplatin also exhibit increased fluorescence with anti-phosphotyrosine-FITC antibody. These data indicate that protein tyrosine phosphorylation plays a role in cisplatin-induced activation of macrophages. PMID:7539662

  5. The rotational spectrum of tyrosine.

    PubMed

    Pérez, Cristóbal; Mata, Santiago; Cabezas, Carlos; López, Juan C; Alonso, José L

    2015-04-23

    In this work neutral tyrosine has been generated in the gas phase by laser ablation of solid samples, and its most abundant conformers characterized through their rotational spectra. Their identification has been made by comparison between the experimental and ab initio values of the rotational and quadrupole coupling constants. Both conformers are stabilized by an O-H•••N hydrogen bond established within the amino acid skeleton chain and an additional weak N-H•••π hydrogen bond. The observed conformers differ in the orientation of the phenolic -OH group.

  6. Relationship between Tobacco, cagA and vacA i1 Virulence Factors and Bacterial Load in Patients Infected by Helicobacter pylori

    PubMed Central

    Aguirre, Estefanía; Aragones, Nuria; Saez, Jesús; Galiana, Antonio; Sola-Vera, Javier; Ruiz-García, Montserrat; Paz-Zulueta, María; Sarabia-Lavín, Raquel; Brotons, Alicia; López-Girona, Elena; Pérez, Estefanía; Sillero, Carlos

    2015-01-01

    Background and Aim Several biological and epidemiological studies support a relationship between smoking and Helicobacter pylori (H. pylori) to increase the risk of pathology. However, there have been few studies on the potential synergistic association between specific cagA and vacA virulence factors and smoking in patients infected by Helicobacter pylori. We studied the relationship between smoking and cagA, vacA i1 virulence factors and bacterial load in H. pylori infected patients. Methods Biopsies of the gastric corpus and antrum from 155 consecutive patients in whom there was clinical suspicion of infection by H. pylori were processed. In 106 patients H. pylori infection was detected. Molecular methods were used to quantify the number of microorganisms and presence of cagA and vacA i1 genes. A standardized questionnaire was used to obtain patients’ clinical data and lifestyle variables, including tobacco and alcohol consumption. Adjusted Odds Ratios (ORadjusted) were estimated by unconditional logistic regression. Results cagA was significantly associated with active-smoking at endoscope: ORadjusted 4.52. Evidence of association was found for vacA i1 (ORadjusted 3.15). Bacterial load was higher in active-smokers, although these differences did not yield statistical significance (median of 262.2 versus 79.4 copies of H. pylori per cell). Conclusions The association between smoking and a higher risk of being infected by a virulent bacterial population and with higher bacterial load, support a complex interaction between H. pylori infection and environmental factors. PMID:25794002

  7. A CCD-based reader combined with CdS quantum dot-labeled lateral flow strips for ultrasensitive quantitative detection of CagA

    NASA Astrophysics Data System (ADS)

    Gui, Chen; Wang, Kan; Li, Chao; Dai, Xuan; Cui, Daxiang

    2014-02-01

    Immunochromatographic assays are widely used to detect many analytes. CagA is proved to be associated closely with initiation of gastric carcinoma. Here, we reported that a charge-coupled device (CCD)-based test strip reader combined with CdS quantum dot-labeled lateral flow strips for quantitative detection of CagA was developed, which used 365-nm ultraviolet LED as the excitation light source, and captured the test strip images through an acquisition module. Then, the captured image was transferred to the computer and was processed by a software system. A revised weighted threshold histogram equalization (WTHE) image processing algorithm was applied to analyze the result. CdS quantum dot-labeled lateral flow strips for detection of CagA were prepared. One hundred sera samples from clinical patients with gastric cancer and healthy people were prepared for detection, which demonstrated that the device could realize rapid, stable, and point-of-care detection, with a sensitivity of 20 pg/mL.

  8. A CCD-based reader combined with CdS quantum dot-labeled lateral flow strips for ultrasensitive quantitative detection of CagA

    PubMed Central

    2014-01-01

    Immunochromatographic assays are widely used to detect many analytes. CagA is proved to be associated closely with initiation of gastric carcinoma. Here, we reported that a charge-coupled device (CCD)-based test strip reader combined with CdS quantum dot-labeled lateral flow strips for quantitative detection of CagA was developed, which used 365-nm ultraviolet LED as the excitation light source, and captured the test strip images through an acquisition module. Then, the captured image was transferred to the computer and was processed by a software system. A revised weighted threshold histogram equalization (WTHE) image processing algorithm was applied to analyze the result. CdS quantum dot-labeled lateral flow strips for detection of CagA were prepared. One hundred sera samples from clinical patients with gastric cancer and healthy people were prepared for detection, which demonstrated that the device could realize rapid, stable, and point-of-care detection, with a sensitivity of 20 pg/mL. PMID:24495570

  9. Polyclonal antibody to soman-tyrosine

    PubMed Central

    Li, Bin; Duysen, Ellen G.; Froment, Marie-Thérèse; Masson, Patrick; Nachon, Florian; Jiang, Wei; Schopfer, Lawrence M.; Thiele, Geoffrey M.; Klassen, Lynell W.; Cashman, John; Williams, Gareth R.; Lockridge, Oksana

    2013-01-01

    Soman forms a stable, covalent bond with tyrosine 411 of human albumin, with tyrosines 257 and 593 in human transferrin, and with tyrosine in many other proteins. The pinacolyl group of soman is retained, suggesting that pinacolyl methylphosphonate bound to tyrosine could generate specific antibodies. Tyrosine in the pentapeptide RYGRK was covalently modified with soman simply by adding soman to the peptide. The phosphonylated-peptide was linked to keyhole limpet hemocyanin, and the conjugate was injected into rabbits. The polyclonal antiserum recognized soman-labeled human albumin, soman-mouse albumin, and soman human transferrin, but not non-phosphonylated control proteins. The soman-labeled tyrosines in these proteins are surrounded by different amino acid sequences, suggesting that the polyclonal recognizes soman-tyrosine independent of the amino acid sequence. Antiserum obtained after 4 antigen injections over a period of 18 weeks was tested in a competition ELISA where it had an IC50 of 10−11 M. The limit of detection on Western blots was 0.01 μg (15 picomoles) of soman-labeled albumin. In conclusion, a high-affinity, polyclonal antibody that specifically recognizes soman adducts on tyrosine in a variety of proteins has been produced. Such an antibody could be useful for identifying secondary targets of soman toxicity. PMID:23469927

  10. Tyrosine Kinase Inhibitors and Pregnancy

    PubMed Central

    Abruzzese, Elisabetta; Trawinska, Malgorzata Monika; Perrotti, Alessio Pio; De Fabritiis, Paolo

    2014-01-01

    The management of patients with chronic myeloid leukemia (CML) during pregnancy has become recently a matter of continuous debate. The introduction of the Tyrosine Kinase Inhibitors (TKIs) in clinical practice has dramatically changed the prognosis of CML patients; in fact, patients diagnosed in chronic phase can reasonably expect many years of excellent disease control and good quality of life, as well as a normal life expectancy, including the necessity to address issues relating to fertility and pregnancy. Physicians are frequently being asked for advice regarding the need for, and/or the appropriateness of, stopping treatment in order to conceive. In this report, we will review the data published in terms of fertility, conception, pregnancy, pregnancy outcome and illness control for TKI treated CML patients, as well as how to manage a planned and/or unplanned pregnancy. PMID:24804001

  11. cagA, vacA and iceA virulence genes of Helicobacter pylori isolates of children in Finland.

    PubMed

    Karhukorpi, J; Yan, Y; Kolho, K L; Rautelin, H; Lahti, M; Sirviö, A; Riipinen, K; Lindahl, H; Verkasalo, M; Fagerholm, R; Karttunen, R

    2000-10-01

    cagA, vacA s and m genotypes and iceA alleles were analyzed from Helicobacter pylori strains isolated from 17 Finnish children and 32 children of non-Finnish origin living in Finland. Twelve children in the latter group were eastern European and 15 were of African origin. Only three children of non-Finnish origin were born in Finland. The vacA sla subtype was more prevalent in the isolates from Finnish children than African children (76% vs. 7%, P<0.001); vacA s1b frequencies were 5% and 67%, respectively (P<0.001). The iceA1 allele was significantly more prevalent in African than Finnish isolates (93% vs. 35%, P< 0.01). Considerable variation was noted in the frequency of vacA s1 subtypes and iceA alleles in children originating from different geographic regions, but the geographic variation of s1 subtypes resembled that described in other reports.

  12. Ocular Toxicity of Tyrosine Kinase Inhibitors

    PubMed Central

    Davis, Mary Elizabeth

    2016-01-01

    Purpose/Objectives To review common tyrosine kinase inhibitors, as well as their ocular side effects and management. Data Sources A comprehensive literature search was conducted using cINahl®, Pubmed, and cochrane databases for articles published since 2004 with the following search terms: ocular toxicities, tyrosine kinase inhibitors, ophthalmology, adverse events, eye, and vision. Data Synthesis Tyrosine kinase inhibitors can cause significant eye toxicity. Conclusions Given the prevalence of new tyrosine kinase inhibitor therapies and the complexity of possible pathogenesis of ocular pathology, oncology nurses can appreciate the occurrence of ocular toxicities and the role of nursing in the management of these problems. Implications for Nursing Knowledge of the risk factors and etiology of ocular toxicity of targeted cancer therapies can guide nursing assessment, enhance patient education, and improve care management. Including a review of eye symptoms and vision issues in nursing assessment can enhance early detection and treatment of ocular toxicity. PMID:26906134

  13. The Tyrosine Aminomutase TAM1 Is Required for β-Tyrosine Biosynthesis in Rice

    PubMed Central

    Yan, Jian; Aboshi, Takako; Teraishi, Masayoshi; Strickler, Susan R.; Spindel, Jennifer E.; Tung, Chih-Wei; Takata, Ryo; Matsumoto, Fuka; Maesaka, Yoshihiro; McCouch, Susan R.; Okumoto, Yutaka; Mori, Naoki; Jander, Georg

    2015-01-01

    Non-protein amino acids, often isomers of the standard 20 protein amino acids, have defense-related functions in many plant species. A targeted search for jasmonate-induced metabolites in cultivated rice (Oryza sativa) identified (R)-β-tyrosine, an isomer of the common amino acid (S)-α-tyrosine in the seeds, leaves, roots, and root exudates of the Nipponbare cultivar. Assays with 119 diverse cultivars showed a distinct presence/absence polymorphism, with β-tyrosine being most prevalent in temperate japonica cultivars. Genetic mapping identified a candidate gene on chromosome 12, which was confirmed to encode a tyrosine aminomutase (TAM1) by transient expression in Nicotiana benthamiana and in vitro enzyme assays. A point mutation in TAM1 eliminated β-tyrosine production in Nipponbare. Rice cultivars that do not produce β-tyrosine have a chromosome 12 deletion that encompasses TAM1. Although β-tyrosine accumulation was induced by the plant defense signaling molecule jasmonic acid, bioassays with hemipteran and lepidopteran herbivores showed no negative effects at physiologically relevant β-tyrosine concentrations. In contrast, root growth of Arabidopsis thaliana and other tested dicot plants was inhibited by concentrations as low as 1 μM. As β-tyrosine is exuded into hydroponic medium at higher concentrations, it may contribute to the allelopathic potential of rice. PMID:25901084

  14. Protein tyrosine nitration in the cell cycle

    SciTech Connect

    Jia, Min; Mateoiu, Claudia; Souchelnytskyi, Serhiy

    2011-09-23

    Highlights: {yields} Enrichment of 3-nitrotyrosine containing proteins from cells synchronized in different phases of the cell cycle. {yields} Identification of 76 tyrosine nitrated proteins that change expression during the cell cycle. {yields} Nineteen identified proteins were previously described as regulators of cell proliferation. -- Abstract: Nitration of tyrosine residues in proteins is associated with cell response to oxidative/nitrosative stress. Tyrosine nitration is relatively low abundant post-translational modification that may affect protein functions. Little is known about the extent of protein tyrosine nitration in cells during progression through the cell cycle. Here we report identification of proteins enriched for tyrosine nitration in cells synchronized in G0/G1, S or G2/M phases of the cell cycle. We identified 27 proteins in cells synchronized in G0/G1 phase, 37 proteins in S phase synchronized cells, and 12 proteins related to G2/M phase. Nineteen of the identified proteins were previously described as regulators of cell proliferation. Thus, our data indicate which tyrosine nitrated proteins may affect regulation of the cell cycle.

  15. An RGD Helper Sequence in CagL of Helicobacter pylori Assists in Interactions with Integrins and Injection of CagA

    PubMed Central

    Conradi, Jens; Tegtmeyer, Nicole; Woźna, Marta; Wissbrock, Marco; Michalek, Carmela; Gagell, Corinna; Cover, Timothy L.; Frank, Ronald; Sewald, Norbert; Backert, Steffen

    2012-01-01

    Helicobacter pylori is a specific gastric pathogen that colonizes the stomach in more than 50% of the world’s human population. Infection with this bacterium can induce several types of gastric pathology, ranging from chronic gastritis to peptic ulcers and even adenocarcinoma. Virulent H. pylori isolates encode components of a type IV secretion system (T4SS), which form a pilus for the injection of virulence proteins such as CagA into host target cells. This is accomplished by a specialized adhesin on the pilus surface, the protein CagL, a putative VirB5 ortholog, which binds to host cell β1 integrin, triggering subsequent delivery of CagA across the host cell membrane. Like the human extracellular matrix protein fibronectin, CagL contains an RGD (Arg-Gly-Asp) motif and is able to trigger intracellular signaling pathways by RGD-dependent binding to integrins. While CagL binding to host cells is mediated primarily by the RGD motif, we identified an auxiliary binding motif for CagL–integrin interaction. Here, we report on a surface exposed FEANE (Phe-Glu-Ala-Asn-Glu) interaction motif in spatial proximity to the RGD sequence, which enhances the interactions of CagL with integrins. It will be referred to as RGD helper sequence (RHS). Competitive cell adhesion assays with recombinant wild type CagL and point mutants, competition experiments with synthetic cyclic and linear peptides, and peptide array experiments revealed amino acids essential for the interaction of the RHS motif with integrins. Infection experiments indicate that the RHS motif plays a role in the early interaction of H. pylori T4SS with integrin, to trigger signaling and to inject CagA into host cells. We thus postulate that CagL is a versatile T4SS surface protein equipped with at least two motifs to promote binding to integrins, thereby causing aberrant signaling within host cells and facilitating translocation of CagA into host cells, thus contributing directly to H. pylori pathogenesis. PMID

  16. Capsaicin consumption, Helicobacter pylori CagA status and IL1B-31C>T genotypes: a host and environment interaction in gastric cancer.

    PubMed

    López-Carrillo, Lizbeth; Camargo, M Constanza; Schneider, Barbara G; Sicinschi, Liviu A; Hernández-Ramírez, Raúl U; Correa, Pelayo; Cebrian, Mariano E

    2012-06-01

    Gastric cancer (GC) has been associated with a complex combination of genetic and environmental factors. In contrast to most countries, available information on GC mortality trends showed a gradual increase in Mexico. Our aim was to explore potential interactions among dietary (chili pepper consumption), infectious (Helicobacter pylori) and genetic factors (IL1B-31 genotypes) on GC risk. The study was performed in three areas of Mexico, with different GC mortality rates. We included 158 GC patients and 317 clinical controls. Consumption of capsaicin (Cap), the pungent active substance of chili peppers, was estimated by food frequency questionnaire. H. pylori CagA status was assessed by ELISA, and IL1B-31 genotypes were determined by TaqMan assays and Pyrosequencing in DNA samples. Multivariate unconditional logistic regression was used to estimate potential interactions. Moderate to high Cap consumption synergistically increased GC risk in genetically susceptible individuals (IL1B-31C allele carriers) infected with the more virulent H. pylori (CagA+) strains. The combined presence of these factors might explain the absence of a decreasing trend for GC in Mexico. However, further research on gene-environment interactions is required to fully understand the factors determining GC patterns in susceptible populations, with the aim of recommending preventive measures for high risk individuals. PMID:22414649

  17. Helicobacter pylori CagA Induces AGS Cell Elongation through a Cell Retraction Defect That Is Independent of Cdc42, Rac1, and Arp2/3▿ †

    PubMed Central

    Bourzac, Kevin M.; Botham, Crystal M.; Guillemin, Karen

    2007-01-01

    Helicobacter pylori, which infects over one-half the world's population, is a significant risk factor in a spectrum of gastric diseases, including peptic ulcers and gastric cancer. Strains of H. pylori that deliver the effector molecule CagA into host cells via a type IV secretion system are associated with more severe disease outcomes. In a tissue culture model of infection, CagA delivery results in a dramatic cellular elongation referred to as the “hummingbird” phenotype, which is characterized by long, thin cellular extensions. These actin-based cytoskeletal rearrangements are reminiscent of structures that are regulated by Rho GTPases and the Arp2/3 complex. We tested whether these signaling pathways were important in the H. pylori-induced cell elongation phenotype. Contrary to our expectations, we found that these molecules are dispensable for cell elongation. Instead, time-lapse video microscopy revealed that cells infected by cagA+ H. pylori become elongated because they fail to release their back ends during cell locomotion. Consistent with a model in which CagA causes cell elongation by inhibiting the disassembly of adhesive cell contacts at migrating cells' lagging ends, immunohistochemical analysis revealed that focal adhesion complexes persist at the distal tips of elongated cell projections. Thus, our data implicate a set of signaling molecules in the hummingbird phenotype that are different than the molecules previously suspected. PMID:17194805

  18. Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase.

    PubMed Central

    Mahajan, S; Fargnoli, J; Burkhardt, A L; Kut, S A; Saouaf, S J; Bolen, J B

    1995-01-01

    Bruton's tyrosine kinase (Btk) is tyrosine phosphorylated and enzymatically activated following ligation of the B-cell antigen receptor. These events are temporally regulated, and Btk activation follows that of various members of the Src family of protein tyrosine kinases, thus raising the possibility that Src kinases participate in the Btk activation process. We have evaluated the mechanism underlying Btk enzyme activation and have explored the potential regulatory relationship between Btk and Src protein kinases. We demonstrate in COS transient-expression assays that Btk can be activated through intramolecular autophosphorylation at tyrosine 551 and that Btk autophosphorylation is required for Btk catalytic functions. Coexpression of Btk with members of the Src family of protein tyrosine kinases, but not Syk, led to Btk tyrosine phosphorylation and activation. Using a series of point mutations in Blk (a representative Src protein kinase) and Btk, we show that Src kinases activate Btk through an indirect mechanism that requires membrane association of the Src enzymes as well as functional Btk SH3 and SH2 domains. Our results are compatible with the idea that Src protein tyrosine kinases contribute to Btk activation by indirectly stimulating Btk intramolecular autophosphorylation. PMID:7565679

  19. Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase.

    PubMed

    Mahajan, S; Fargnoli, J; Burkhardt, A L; Kut, S A; Saouaf, S J; Bolen, J B

    1995-10-01

    Bruton's tyrosine kinase (Btk) is tyrosine phosphorylated and enzymatically activated following ligation of the B-cell antigen receptor. These events are temporally regulated, and Btk activation follows that of various members of the Src family of protein tyrosine kinases, thus raising the possibility that Src kinases participate in the Btk activation process. We have evaluated the mechanism underlying Btk enzyme activation and have explored the potential regulatory relationship between Btk and Src protein kinases. We demonstrate in COS transient-expression assays that Btk can be activated through intramolecular autophosphorylation at tyrosine 551 and that Btk autophosphorylation is required for Btk catalytic functions. Coexpression of Btk with members of the Src family of protein tyrosine kinases, but not Syk, led to Btk tyrosine phosphorylation and activation. Using a series of point mutations in Blk (a representative Src protein kinase) and Btk, we show that Src kinases activate Btk through an indirect mechanism that requires membrane association of the Src enzymes as well as functional Btk SH3 and SH2 domains. Our results are compatible with the idea that Src protein tyrosine kinases contribute to Btk activation by indirectly stimulating Btk intramolecular autophosphorylation. PMID:7565679

  20. Cell signaling by receptor-tyrosine kinases

    PubMed Central

    Lemmon, Mark A.; Schlessinger, Joseph

    2010-01-01

    Recent structural studies of receptor tyrosine kinases (RTKs) have revealed unexpected diversity in the mechanisms of their activation by growth factor ligands. Strategies for inducing dimerization by ligand binding are surprisingly diverse, as are mechanisms that couple this event to activation of the intracellular tyrosine kinase domains. As our understanding of these details becomes increasingly sophisticated, it provides an important context for therapeutically countering the effects of pathogenic RTK mutations in cancer and other diseases. Much remains to be learned, however, about the complex signaling networks downstream from RTKs and how alterations in these networks are translated into cellular responses. PMID:20602996

  1. 21 CFR 582.5920 - Tyrosine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Tyrosine. 582.5920 Section 582.5920 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  2. 21 CFR 582.5920 - Tyrosine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Tyrosine. 582.5920 Section 582.5920 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  3. 21 CFR 582.5920 - Tyrosine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Tyrosine. 582.5920 Section 582.5920 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  4. Multiple tyrosine metabolites are GPR35 agonists

    PubMed Central

    Deng, Huayun; Hu, Haibei; Fang, Ye

    2012-01-01

    Both kynurenic acid and 2-acyl lysophosphatidic acid have been postulated to be the endogenous agonists of GPR35. However, controversy remains whether alternative endogenous agonists exist. The molecular targets accounted for many nongenomic actions of thyroid hormones are mostly unknown. Here we report the agonist activity of multiple tyrosine metabolites at the GPR35. Tyrosine metabolism intermediates that contain carboxylic acid and/or catechol functional groups were first selected. Whole cell dynamic mass redistribution (DMR) assays enabled by label-free optical biosensor were then used to characterize their agonist activity in native HT-29. Molecular assays including β-arrestin translocation, ERK phosphorylation and receptor internalization confirmed that GPR35 functions as a receptor for 5,6-dihydroxyindole-2-carboxylic acid, 3,3′,5′-triiodothyronine, 3,3′,5-triiodothyronine, gentisate, rosmarinate, and 3-nitrotyrosine. These results suggest that multiple tyrosine metabolites are alternative endogenous ligands of GPR35, and GPR35 may represent a druggable target for treating certain diseases associated with abnormality of tyrosine metabolism. PMID:22523636

  5. RTKdb: database of Receptor Tyrosine Kinase.

    PubMed

    Grassot, Julien; Mouchiroud, Guy; Perrière, Guy

    2003-01-01

    Receptor Tyrosine Kinases (RTK) are transmembrane receptors specifically found in metazoans. They represent an excellent model for studying evolution of cellular processes in metazoans because they encompass large families of modular proteins and belong to a major family of contingency generating molecules in eukaryotic cells: the protein kinases. Because tyrosine kinases have been under close scrutiny for many years in various species, they are associated with a wealth of information, mainly in mammals. Presently, most categories of RTK were identified in mammals, but in a near future other model species will be sequenced, and will bring us RTKs from other metazoan clades. Thus, collecting RTK sequences would provide a good starting point as a new model for comparative and evolutionary studies applying to multigene families. In this context, we are developing the Receptor Tyrosine Kinase database (RTKdb), which is the only database on tyrosine kinase receptors presently available. In this database, protein sequences from eight model metazoan species are organized under the format previously used for the HOVERGEN, HOBACGEN and NUREBASE systems. RTKdb can be accessed through the PBIL (Pôle Bioinformatique Lyonnais) World Wide Web server at http://pbil.univ-lyon1.fr/RTKdb/, or through the FamFetch graphical user interface available at the same address.

  6. Brain catechol synthesis - Control by brain tyrosine concentration

    NASA Technical Reports Server (NTRS)

    Wurtman, R. J.; Larin, F.; Mostafapour, S.; Fernstrom, J. D.

    1974-01-01

    Brain catechol synthesis was estimated by measuring the rate at which brain dopa levels rose following decarboxylase inhibition. Dopa accumulation was accelerated by tyrosine administration, and decreased by treatments that lowered brain tyrosine concentrations (for example, intraperitoneal tryptophan, leucine, or parachlorophenylalanine). A low dose of phenylalanine elevated brain tyrosine without accelerating dopa synthesis. Our findings raise the possibility that nutritional and endocrine factors might influence brain catecholamine synthesis by controlling the availability of tyrosine.

  7. Conservation and early expression of zebrafish tyrosine kinases support the utility of zebrafish as a model for tyrosine kinase biology.

    PubMed

    Challa, Anil Kumar; Chatti, Kiranam

    2013-09-01

    Tyrosine kinases have significant roles in cell growth, apoptosis, development, and disease. To explore the use of zebrafish as a vertebrate model for tyrosine kinase signaling and to better understand their roles, we have identified all of the tyrosine kinases encoded in the zebrafish genome and quantified RNA expression of selected tyrosine kinases during early development. Using profile hidden Markov model analysis, we identified 122 zebrafish tyrosine kinase genes and proposed unambiguous gene names where needed. We found them to be organized into 39 nonreceptor and 83 receptor type, and 30 families consistent with human tyrosine kinase family assignments. We found five human tyrosine kinase genes (epha1, bmx, fgr, srm, and insrr) with no identifiable zebrafish ortholog, and one zebrafish gene (yrk) with no identifiable human ortholog. We also found that receptor tyrosine kinase genes were duplicated more often than nonreceptor tyrosine kinase genes in zebrafish. We profiled expression levels of 30 tyrosine kinases representing all families using direct digital detection at different stages during the first 24 hours of development. The profiling experiments clearly indicate regulated expression of tyrosine kinases in the zebrafish, suggesting their role during early embryonic development. In summary, our study has resulted in the first comprehensive description of the zebrafish tyrosine kinome. PMID:23234507

  8. Conservation and early expression of zebrafish tyrosine kinases support the utility of zebrafish as a model for tyrosine kinase biology.

    PubMed

    Challa, Anil Kumar; Chatti, Kiranam

    2013-09-01

    Tyrosine kinases have significant roles in cell growth, apoptosis, development, and disease. To explore the use of zebrafish as a vertebrate model for tyrosine kinase signaling and to better understand their roles, we have identified all of the tyrosine kinases encoded in the zebrafish genome and quantified RNA expression of selected tyrosine kinases during early development. Using profile hidden Markov model analysis, we identified 122 zebrafish tyrosine kinase genes and proposed unambiguous gene names where needed. We found them to be organized into 39 nonreceptor and 83 receptor type, and 30 families consistent with human tyrosine kinase family assignments. We found five human tyrosine kinase genes (epha1, bmx, fgr, srm, and insrr) with no identifiable zebrafish ortholog, and one zebrafish gene (yrk) with no identifiable human ortholog. We also found that receptor tyrosine kinase genes were duplicated more often than nonreceptor tyrosine kinase genes in zebrafish. We profiled expression levels of 30 tyrosine kinases representing all families using direct digital detection at different stages during the first 24 hours of development. The profiling experiments clearly indicate regulated expression of tyrosine kinases in the zebrafish, suggesting their role during early embryonic development. In summary, our study has resulted in the first comprehensive description of the zebrafish tyrosine kinome.

  9. No effect of oral tyrosine on total tyrosine levels in breast milk: implications for dietary supplementation in early postpartum.

    PubMed

    Dowlati, Yekta; Ravindran, Arun V; Maheux, Maxim; Steiner, Meir; Stewart, Donna E; Meyer, Jeffrey H

    2014-12-01

    Postpartum depression (PPD) is the most common complication of childbearing with a 13 % prevalence rate, and there is no widespread approach for prevention. There is an appealing theoretical rationale for oral tyrosine to help prevent PPD. However, the effect of oral tyrosine on its total and free concentrations in breast milk and plasma of breastfeeding mothers is not known. Twenty-four healthy breastfeeding women were randomly assigned to 0, 2, 5, or 10 g of oral tyrosine. Free and total tyrosine in breast milk and free tyrosine in plasma were measured. Free tyrosine was also measured in 12 different infant formulas. Total tyrosine in breast milk did not rise, but there was a slight tendency towards a reduction (up to −12 %; repeated measures ANOVA (RMANOVA): p = 0.074). Maternal plasma tyrosine rose (RMANOVA: p < 0.005). In breast milk, 98 % of tyrosine was in proteins or peptides and 2 % was free. Free tyrosine levels in breast milk rose in each group (RMANOVA: p < 0.005), but levels were within the range found in common infant formulas. The negligible effect of oral tyrosine on its concentration in breast milk supports further development of oral tyrosine as part of a prevention strategy for PPD. PMID:25015680

  10. Therapeutic efficacy of oral immunization with attenuated Salmonella typhimurium expressing Helicobacter pylori CagA, VacA and UreB fusion proteins in mice model.

    PubMed

    Liu, Kai-Yun; Shi, Yun; Luo, Ping; Yu, Shu; Chen, Li; Zhao, Zhuo; Mao, Xu-Hu; Guo, Gang; Wu, Chao; Zou, Quan-Ming

    2011-09-01

    Therapeutic vaccination is a desirable alternative for controlling Helicobacter pylori (H. pylori) infection. In the present study, attenuated Salmonella vector vaccines were constructed that expressed fusion proteins complexed with H. pylori CagA, VacA and UreB in different arrangements, and their therapeutic efficacy was evaluated in H. pylori-infected mice. Oral therapeutic immunization with attenuated Salmonella, which expressed the fused protein CVU, significantly decreased H. pylori colonization in the stomach; protection was related to specific CD4(+) T cell Th1 type responses and serum IgG and mucosal sIgA antibody responses. These findings suggested that therapeutic efficacy was related to the arrangement of the fusion protein. It is possible that arrangement decides the expression of recombinant antigen in mice, and the latter results in different therapeutic efficacy. The attenuated Salmonella vector vaccine, which expressed the fused protein arrangement CVU, is superior to others, and could be a candidate vaccine against H. pylori.

  11. Helicobacter pylori Genotyping from American Indigenous Groups Shows Novel Amerindian vacA and cagA Alleles and Asian, African and European Admixture

    PubMed Central

    Camorlinga-Ponce, Margarita; Perez-Perez, Guillermo; Gonzalez-Valencia, Gerardo; Mendoza, Irma; Peñaloza-Espinosa, Rosenda; Ramos, Irma; Kersulyte, Dangeruta; Reyes-Leon, Adriana; Romo, Carolina; Granados, Julio; Muñoz, Leopoldo; Berg, Douglas E.; Torres, Javier

    2011-01-01

    It is valuable to extend genotyping studies of Helicobacter pylori to strains from indigenous communities across the world to better define adaption, evolution, and associated diseases. We aimed to genetically characterize both human individuals and their infecting H. pylori from indigenous communities of Mexico, and to compare them with those from other human groups. We studied individuals from three indigenous groups, Tarahumaras from the North, Huichols from the West and Nahuas from the center of Mexico. Volunteers were sampled at their community site, DNA was isolated from white blood cells and mtDNA, Y-chromosome, and STR alleles were studied. H. pylori was cultured from gastric juice, and DNA extracted for genotyping of virulence and housekeeping genes. We found Amerindian mtDNA haplogroups (A, B, C, and D), Y-chromosome DYS19T, and Amerindian STRs alleles frequent in the three groups, confirming Amerindian ancestry in these Mexican groups. Concerning H.pylori cagA phylogenetic analyses, although most isolates were of the Western type, a new Amerindian cluster neither Western nor Asian, was formed by some indigenous Mexican, Colombian, Peruvian and Venezuelan isolates. Similarly, vacA phylogenetic analyses showed the existence of a novel Amerindian type in isolates from Alaska, Mexico and Colombia. With hspA strains from Mexico and other American groups clustered within the three major groups, Asian, African or European. Genotyping of housekeeping genes confirmed that Mexican strains formed a novel Asian-related Amerindian group together with strains from remote Amazon Aborigines. This study shows that Mexican indigenous people with Amerindian markers are colonized with H. pylori showing admixture of Asian, European and African strains in genes known to interact with the gastric mucosa. We present evidence of novel Amerindian cagA and vacA alleles in indigenous groups of North and South America. PMID:22073291

  12. No Association between CagA- and VacA-Positive Strains of Helicobacter pylori and Primary Open-Angle Glaucoma: A Case–Control Study

    PubMed Central

    Noche, C. Domngang; Njajou, O.; Etoa, F. X.

    2016-01-01

    INTRODUCTION Glaucoma is a public health issue worldwide, particularly in Africa. In Cameroon, the prevalence rate of primary open-angle glaucoma (POAG) ranges between 4.5% and 8.2%. Helicobacter pylori (HP) has been implicated in digestive and extra-digestive diseases, including glaucoma. The objective of this work was to evaluate the implication of CagA- and VacA-positive strains of HP in POAG using a case–control design. METHODS An analytical study was conducted from October 2013 to December 2013. Participants were recruited in eye care centers in Yaoundé. Enzyme-linked immunosorbent assays (ELISAs) were carried out in the La Grace Laboratory in Yaoundé. RESULTS The total sample consisted of 50 POAG patients and 31 controls with a mean age of 58.5 ± 12.2 years and 45.5 ± 14.6 years, respectively. The prevalence rates of HP in the POAG and control groups were 74% (37/50) and 87% (27/31), respectively (P = 0.125). The prevalence rates of CagA-positive HP seropositivity in the POAG and control groups were 26% and 22.58%, respectively (P = 0.47), and the prevalence rates of VacA-positive HP participants were 6% and 0%, respectively (P = 0.22). CONCLUSION The HP prevalence rates among POAG patients and controls were 74% and 87%, respectively. There was no significant difference between prevalence rates of HP in the POAG and control groups. There was no association between POAG and CagA- or VacA-positive HP infection. PMID:26917977

  13. Helicobacter pylori genotyping from American indigenous groups shows novel Amerindian vacA and cagA alleles and Asian, African and European admixture.

    PubMed

    Camorlinga-Ponce, Margarita; Perez-Perez, Guillermo; Gonzalez-Valencia, Gerardo; Mendoza, Irma; Peñaloza-Espinosa, Rosenda; Ramos, Irma; Kersulyte, Dangeruta; Reyes-Leon, Adriana; Romo, Carolina; Granados, Julio; Muñoz, Leopoldo; Berg, Douglas E; Torres, Javier

    2011-01-01

    It is valuable to extend genotyping studies of Helicobacter pylori to strains from indigenous communities across the world to better define adaption, evolution, and associated diseases. We aimed to genetically characterize both human individuals and their infecting H. pylori from indigenous communities of Mexico, and to compare them with those from other human groups. We studied individuals from three indigenous groups, Tarahumaras from the North, Huichols from the West and Nahuas from the center of Mexico. Volunteers were sampled at their community site, DNA was isolated from white blood cells and mtDNA, Y-chromosome, and STR alleles were studied. H. pylori was cultured from gastric juice, and DNA extracted for genotyping of virulence and housekeeping genes. We found Amerindian mtDNA haplogroups (A, B, C, and D), Y-chromosome DYS19T, and Amerindian STRs alleles frequent in the three groups, confirming Amerindian ancestry in these Mexican groups. Concerning H.pylori cagA phylogenetic analyses, although most isolates were of the Western type, a new Amerindian cluster neither Western nor Asian, was formed by some indigenous Mexican, Colombian, Peruvian and Venezuelan isolates. Similarly, vacA phylogenetic analyses showed the existence of a novel Amerindian type in isolates from Alaska, Mexico and Colombia. With hspA strains from Mexico and other American groups clustered within the three major groups, Asian, African or European. Genotyping of housekeeping genes confirmed that Mexican strains formed a novel Asian-related Amerindian group together with strains from remote Amazon Aborigines. This study shows that Mexican indigenous people with Amerindian markers are colonized with H. pylori showing admixture of Asian, European and African strains in genes known to interact with the gastric mucosa. We present evidence of novel Amerindian cagA and vacA alleles in indigenous groups of North and South America.

  14. Mechanosensing Controlled Directly by Tyrosine Kinases.

    PubMed

    Yang, Bo; Lieu, Zi Zhao; Wolfenson, Haguy; Hameed, Feroz M; Bershadsky, Alexander D; Sheetz, Michael P

    2016-09-14

    To understand how cells form tissues, we need to understand how the tyrosine kinases are involved in controlling cell mechanics, whether they act directly as parts of mechanosensing machines or indirectly. Cells test the critical parameter of matrix rigidity by locally contracting ("pinching") matrices and measuring forces, and the depletion of contractile units causes transformation. We report here that knocking down the receptor tyrosine kinases (RTKs), AXL, and ROR2, alters rigidity sensing and increases the magnitude or duration of local contraction events, respectively. Phospho-AXL and ROR2 localize to contraction units and bind major contractile components, tropomyosin 2.1 (AXL), myosin IIA (AXL), and filamin A (ROR2). At a molecular level, phosphorylated AXL localizes to active myosin filaments and phosphorylates tropomyosin at a tyrosine critical for adhesion formation. ROR2 binding of ligand is unnecessary, but binding filamin A helps function. Thus, AXL and ROR2 alter rigidity sensing and consequently morphogenic processes by directly controlling local mechanosensory contractions without ligands. PMID:27559755

  15. Protein tyrosine adduct in humans self-poisoned by chlorpyrifos

    SciTech Connect

    Li, Bin; Eyer, Peter; Eddleston, Michael; Jiang, Wei; Schopfer, Lawrence M.; Lockridge, Oksana

    2013-06-15

    Studies of human cases of self-inflicted poisoning suggest that chlorpyrifos oxon reacts not only with acetylcholinesterase and butyrylcholinesterase but also with other blood proteins. A favored candidate is albumin because in vitro and animal studies have identified tyrosine 411 of albumin as a site covalently modified by organophosphorus poisons. Our goal was to test this proposal in humans by determining whether plasma from humans poisoned by chlorpyrifos has adducts on tyrosine. Plasma samples from 5 self-poisoned humans were drawn at various time intervals after ingestion of chlorpyrifos for a total of 34 samples. All 34 samples were analyzed for plasma levels of chlorpyrifos and chlorpyrifos oxon (CPO) as a function of time post-ingestion. Eleven samples were analyzed for the presence of diethoxyphosphorylated tyrosine by mass spectrometry. Six samples yielded diethoxyphosphorylated tyrosine in pronase digests. Blood collected as late as 5 days after chlorpyrifos ingestion was positive for CPO-tyrosine, consistent with the 20-day half-life of albumin. High plasma CPO levels did not predict detectable levels of CPO-tyrosine. CPO-tyrosine was identified in pralidoxime treated patients as well as in patients not treated with pralidoxime, indicating that pralidoxime does not reverse CPO binding to tyrosine in humans. Plasma butyrylcholinesterase was a more sensitive biomarker of exposure than adducts on tyrosine. In conclusion, chlorpyrifos oxon makes a stable covalent adduct on the tyrosine residue of blood proteins in humans who ingested chlorpyrifos. - Highlights: • Chlorpyrifos-poisoned patients have adducts on protein tyrosine. • Diethoxyphosphate-tyrosine does not lose an alkyl group. • Proteins in addition to AChE and BChE are modified by organophosphates.

  16. Tyrosine metabolic enzymes from insects and mammals: a comparative perspective.

    PubMed

    Vavricka, Christopher John; Han, Qian; Mehere, Prajwalini; Ding, Haizhen; Christensen, Bruce M; Li, Jianyong

    2014-02-01

    Differences in the metabolism of tyrosine between insects and mammals present an interesting example of molecular evolution. Both insects and mammals possess fine-tuned systems of enzymes to meet their specific demands for tyrosine metabolites; however, more homologous enzymes involved in tyrosine metabolism have emerged in many insect species. Without knowledge of modern genomics, one might suppose that mammals, which are generally more complex than insects and require tyrosine as a precursor for important catecholamine neurotransmitters and for melanin, should possess more enzymes to control tyrosine metabolism. Therefore, the question of why insects actually possess more tyrosine metabolic enzymes is quite interesting. It has long been known that insects rely heavily on tyrosine metabolism for cuticle hardening and for innate immune responses, and these evolutionary constraints are likely the key answers to this question. In terms of melanogenesis, mammals also possess a high level of regulation; yet mammalian systems possess more mechanisms for detoxification whereas insects accelerate pathways like melanogenesis and therefore must bear increased oxidative pressure. Our research group has had the opportunity to characterize the structure and function of many key proteins involved in tyrosine metabolism from both insects and mammals. In this mini review we will give a brief overview of our research on tyrosine metabolic enzymes in the scope of an evolutionary perspective of mammals in comparison to insects.

  17. The Tyrosine Kinome Dictates Breast Cancer Heterogeneity and Therapeutic Responsiveness.

    PubMed

    Ha, Jacqueline R; Siegel, Peter M; Ursini-Siegel, Josie

    2016-09-01

    Phospho-tyrosine signaling networks control numerous biological processes including cellular differentiation, cell growth and survival, motility, and invasion. Aberrant regulation of the tyrosine kinome is a hallmark of malignancy and influences all stages of breast cancer progression, from initiation to the development of metastatic disease. The success of specific tyrosine kinase inhibitors strongly validates the clinical relevance of tyrosine phosphorylation networks in breast cancer pathology. However, a significant degree of redundancy exists within the tyrosine kinome. Numerous receptor and cytoplasmic tyrosine kinases converge on a core set of signaling regulators, including adaptor proteins and tyrosine phosphatases, to amplify pro-tumorigenic signal transduction pathways. Mutational activation, amplification, or overexpression of one or more components of the tyrosine kinome represents key contributing events responsible for the tumor heterogeneity that is observed in breast cancers. It is this molecular heterogeneity that has become the most significant barrier to durable clinical responses due to the development of therapeutic resistance. This review focuses on recent literature that supports a prominent role for specific components of the tyrosine kinome in the emergence of unique breast cancer subtypes and in shaping breast cancer plasticity, sensitivity to targeted therapies, and the eventual emergence of acquired resistance. J. Cell. Biochem. 117: 1971-1990, 2016. © 2016 Wiley Periodicals, Inc.

  18. The Tyrosine Kinome Dictates Breast Cancer Heterogeneity and Therapeutic Responsiveness.

    PubMed

    Ha, Jacqueline R; Siegel, Peter M; Ursini-Siegel, Josie

    2016-09-01

    Phospho-tyrosine signaling networks control numerous biological processes including cellular differentiation, cell growth and survival, motility, and invasion. Aberrant regulation of the tyrosine kinome is a hallmark of malignancy and influences all stages of breast cancer progression, from initiation to the development of metastatic disease. The success of specific tyrosine kinase inhibitors strongly validates the clinical relevance of tyrosine phosphorylation networks in breast cancer pathology. However, a significant degree of redundancy exists within the tyrosine kinome. Numerous receptor and cytoplasmic tyrosine kinases converge on a core set of signaling regulators, including adaptor proteins and tyrosine phosphatases, to amplify pro-tumorigenic signal transduction pathways. Mutational activation, amplification, or overexpression of one or more components of the tyrosine kinome represents key contributing events responsible for the tumor heterogeneity that is observed in breast cancers. It is this molecular heterogeneity that has become the most significant barrier to durable clinical responses due to the development of therapeutic resistance. This review focuses on recent literature that supports a prominent role for specific components of the tyrosine kinome in the emergence of unique breast cancer subtypes and in shaping breast cancer plasticity, sensitivity to targeted therapies, and the eventual emergence of acquired resistance. J. Cell. Biochem. 117: 1971-1990, 2016. © 2016 Wiley Periodicals, Inc. PMID:27392311

  19. Tyrosine Kinase Inhibition: An Approach to Drug Development

    NASA Astrophysics Data System (ADS)

    Levitzki, Alexander; Gazit, Aviv

    1995-03-01

    Protein tyrosine kinases (PTKs) regulate cell proliferation, cell differentiation, and signaling processes in the cells of the immune system. Uncontrolled signaling from receptor tyrosine kinases and intracellular tyrosine kinases can lead to inflammatory responses and to diseases such as cancer, atherosclerosis, and psoriasis. Thus, inhibitors that block the activity of tyrosine kinases and the signaling pathways they activate may provide a useful basis for drug development. This article summarizes recent progress in the development of PTK inhibitors and demonstrates their potential use in the treatment of disease.

  20. Euglena mitochondria and chloroplasts form tyrosine-O-sulfate

    SciTech Connect

    Saidha, T.; Hanfstingl, U.; Schiff, J.A. )

    1989-04-01

    Mitochondria from light-grown wild-type Euglena gracilis var. bacillaris Cori or dark-grown mutant W{sub 10}BSmL incubated with {sup 35}SO{sub 4}{sup 2{minus}} and ATP, or with {sup 14}C-tyrosine, non-radioactive sulfate and ATP accumulate a labeled compound in the medium. Since this compound shows exact coelectrophoresis with tyrosine-O-sulfate (TOS) at pH 2.0, 5.8 or 8.0., yields sulfate and tyrosine on acid hydrolysis, and treatment with aryl sulfatase from Aerobacter aerogenes yields sulfate and tyrosine but no tyrosine methyl ester, it is identified as TOS. No TOS is found outside purified developing chloroplasts incubated with {sup 35}SO{sub 4}{sup 2{minus}} and ATP, but both chloroplasts and mitochondria form to {sup 35}S externally when incubated with adenosine 3{prime} phosphate 5{prime}phospho({sup 35}S) sulfate (PAP{sup 35}S). Since no tyrosine need be added, tyrosine is provided from endogenous sources. Although TOS is found in the free pool of Euglena cells it cannot be detected in proteins of cells or mucus ruling our sulfation of tyrosine of protein or incorporation of TOS into proteins. The system forming TOS is membrane-bound and may be involved in tyrosine transport.

  1. Receptor Tyrosine Kinase and Tyrosine Kinase Inhibitors: New Hope for Success in Multiple Sclerosis Therapy.

    PubMed

    Mirshafiey, Abbas; Ghalamfarsa, Ghasem; Asghari, Babak; Azizi, Gholamreza

    2014-07-01

    Receptor tyrosine kinases (RTKs) are essential components of signal transduction pathways that mediate cell-to-cell communication and their function as relay points for signaling pathways. They have a key role in numerous processes that control cellular proliferation and differentiation, regulate cell growth and cellular metabolism, and promote cell survival and apoptosis. Recently, the role of RTKs including TCR, FLT-3, c-Kit, c-Fms, PDGFR, ephrin, neurotrophin receptor, and TAM receptor in autoimmune disorder, especially rheumatoid arthritis and multiple sclerosis has been suggested. In multiple sclerosis pathogenesis, RTKs and their tyrosine kinase enzymes are selective important targets for tyrosine kinase inhibitor (TKI) agents. TKIs, compete with the ATP binding site of the catalytic domain of several tyrosine kinases, and act as small molecules that have a favorable safety profile in disease treatment. Up to now, the efficacy of TKIs in numerous animal models of MS has been demonstrated, but application of these drugs in human diseases should be tested in future clinical trials.

  2. Tyrosine kinase BMX phosphorylates phosphotyrosine-primed motif mediating the activation of multiple receptor tyrosine kinases.

    PubMed

    Chen, Sen; Jiang, Xinnong; Gewinner, Christina A; Asara, John M; Simon, Nicholas I; Cai, Changmeng; Cantley, Lewis C; Balk, Steven P

    2013-05-28

    The nonreceptor tyrosine kinase BMX (bone marrow tyrosine kinase gene on chromosome X) is abundant in various cell types and activated downstream of phosphatidylinositol-3 kinase (PI3K) and the kinase Src, but its substrates are unknown. Positional scanning peptide library screening revealed a marked preference for a priming phosphorylated tyrosine (pY) in the -1 position, indicating that BMX substrates may include multiple tyrosine kinases that are fully activated by pYpY sites in the kinase domain. BMX phosphorylated focal adhesion kinase (FAK) at Tyr⁵⁷⁷ subsequent to its Src-mediated phosphorylation at Tyr⁵⁷⁶. Loss of BMX by RNA interference or by genetic deletion in mouse embryonic fibroblasts (MEFs) markedly impaired FAK activity. Phosphorylation of the insulin receptor in the kinase domain at Tyr¹¹⁸⁹ and Tyr¹¹⁹⁰, as well as Tyr¹¹⁸⁵, and downstream phosphorylation of the kinase AKT at Thr³⁰⁸ were similarly impaired by BMX deficiency. However, insulin-induced phosphorylation of AKT at Ser⁴⁷³ was not impaired in Bmx knockout MEFs or liver tissue from Bmx knockout mice, which also showed increased insulin-stimulated glucose uptake, possibly because of decreased abundance of the phosphatase PHLPP (PH domain leucine-rich repeat protein phosphatase). Thus, by identifying the pYpY motif as a substrate for BMX, our findings suggest that BMX functions as a central regulator among multiple signaling pathways mediated by tyrosine kinases. PMID:23716717

  3. The Extended Family of Protein Tyrosine Phosphatases.

    PubMed

    Alonso, Andrés; Nunes-Xavier, Caroline E; Bayón, Yolanda; Pulido, Rafael

    2016-01-01

    In higher eukaryotes, the Tyr phosphorylation status of cellular proteins results from the coordinated action of Protein Tyrosine Kinases (PTKs) and Protein Tyrosine Phosphatases (PTPs). PTPs have emerged as highly regulated enzymes with diverse substrate specificity, and proteins with Tyr-dephosphorylation or Tyr-dephosphorylation-like properties can be clustered as the PTPome. This includes proteins from the PTP superfamily, which display a Cys-based catalytic mechanism, as well as enzymes from other gene families (Asp-based phosphatases, His-based phosphatases) that have converged in protein Tyr-dephosphorylation-related functions by using non-Cys-based catalytic mechanisms. Within the Cys-based members of the PTPome, classical PTPs dephosphorylate specific phosphoTyr (pTyr) residues from protein substrates, whereas VH1-like dual-specificity PTPs dephosphorylate pTyr, pSer, and pThr residues, as well as nonproteinaceous substrates, including phosphoinositides and phosphorylated carbohydrates. In addition, several PTPs have impaired catalytic activity as a result of amino acid substitutions at their active sites, but retain regulatory functions related with pTyr signaling. As a result of their relevant biological activity, many PTPs are linked to human disease, including cancer, neurodevelopmental, and metabolic diseases, making these proteins important drug targets and molecular markers in the clinic. Here, a brief overview on the biochemistry and physiology of the different groups of proteins that belong to the mammalian PTPome is presented. PMID:27514797

  4. Targeting receptor tyrosine kinases in gastric cancer

    PubMed Central

    Morishita, Asahiro; Gong, Jian; Masaki, Tsutomu

    2014-01-01

    Molecularly targeted therapeutic agents are constantly being developed and have been shown to be effective in various clinical trials. One group of representative targeted oncogenic kinases, the receptor tyrosine kinases (RTKs), has been associated with gastric cancer development. Trastuzumab, an inhibitor of ERBB2, has been approved for the treatment of gastric cancer, although other receptor tyrosine kinases, such as epidermal growth factor receptor, vascular endothelial growth factor, platelet-derived growth factor receptor, c-Met, IGF-1R and fibroblast growth factor receptor 2, are also activated in gastric cancer. The promising results of the trastuzumab clinical trial for gastric cancer resulted in the approval of trastuzumab-based therapy as a first-line treatment for human epidermal growth factor receptor 2-positive patients. On the other hand, the trial examining bevacizumab in combination with conventional chemotherapy did not meet its primary goal of increasing the overall survival time of gastric cancer patients; however, a significantly higher response rate and a longer progression-free survival were observed in the bevacizumab arm of the trial. Other clinical trials, especially phase III trials that have tested drugs targeting RTKs, such as cetuximab, panitumumab, gefitinib, erlotinib, figitumumab, sorafenib, sunitinib and lapatinib, have shown that these drugs have modest effects against gastric cancer. This review summarizes the recent results from the clinical trials of molecularly targeted drugs and suggests that further improvements in the treatment of advanced gastric cancer can be achieved through the combination of conventional drugs with the new molecularly targeted therapies. PMID:24782606

  5. Role of Protein Tyrosine Phosphatases in Plants

    PubMed Central

    Shankar, Alka; Agrawal, Nisha; Sharma, Manisha; Pandey, Amita; Pandey, Girdhar K.

    2015-01-01

    Reversible protein phosphorylation is a crucial regulatory mechanism that controls many biological processes in eukaryotes. In plants, phosphorylation events primarily occur on serine (Ser) and threonine (Thr) residues, while in certain cases, it was also discovered on tyrosine (Tyr) residues. In contrary to plants, extensive reports on Tyr phosphorylation regulating a large numbers of biological processes exist in animals. Despite of such prodigious function in animals, Tyr phosphorylation is a least studied mechanism of protein regulation in plants. Recently, various chemical analytical procedures have strengthened the view that Tyr phosphorylation is equally prevalent in plants as in animals. However, regardless of Tyr phosphorylation events occuring in plants, no evidence could be found for the existence of gene encoding for Tyr phosphorylation i.e. the typical Tyr kinases. Various methodologies have suggested that plant responses to stress signals and developmental processes involved modifications in protein Tyr phosphorylation. Correspondingly, various reports have established the role of PTPs (Protein Tyrosine Phosphatases) in the dephosphorylation and inactivation of mitogen activated protein kinases (MAPKs) hence, in the regulation of MAPK signaling cascade. Besides this, many dual specificity protein phosphatases (DSPs) are also known to bind starch and regulate starch metabolism through reversible phosphorylation. Here, we are emphasizing the significant progress on protein Tyr phosphatases to understand the role of these enzymes in the regulation of post-translational modification in plant physiology and development. PMID:26962298

  6. Tyrosine can protect against oxidative stress through ferryl hemoglobin reduction.

    PubMed

    Lu, Naihao; He, Yingjie; Chen, Chao; Tian, Rong; Xiao, Qiang; Peng, Yi-Yuan

    2014-08-01

    The toxic mechanism of hemoglobin (Hb) under oxidative stress is linked to the formations of highly cytotoxic ferryl species and subsequently heme-to-protein cross-linked derivative of Hb (Hb-X). In this study, we have examined the effects of free tyrosine and its analogues (3-chlorotyrosine, phenylalanine) on the stability of ferryl hemoglobin and the formation of Hb-X. The results showed that free tyrosine (not phenylalanine, 10-500 μM) was an efficient reducing agent of ferryl species and also effective at preventing the formation of cytotoxic Hb-X. Meanwhile, the dimeric tyrosine was formed as the oxidation product of tyrosine during Hb redox reaction. Compared with free tyrosine, 3-chlorotyrosine, an oxidation product of tyrosine and a proposed biomarker for hypochlorous acid (HOCl) in vivo, exhibited stronger antioxidant properties in Hb-induced oxidative stress, which was consistent with its more efficient ability in the reduction of ferryl species. These results showed that the presence of tyrosine and its derivative in vivo and vitro could ameliorate oxidative damage through ferryl heme reduction. The antioxidant ability, therefore, may provide new insights into the nutritional and physiological significance of free tyrosine with redox active heme proteins-related oxidative stress.

  7. Tyrosine improves working memory in a multitasking environment.

    PubMed

    Thomas, J R; Lockwood, P A; Singh, A; Deuster, P A

    1999-11-01

    Previous studies indicate that tyrosine may prove useful in promoting improved performance in situations in which performance is compromised by stress. To extend the generality of previous tyrosine findings, the present study examined the effects of tyrosine ingestion on performance during both a Multiple Task and a Simple Task battery. The multiple task battery was designed to measure working memory, arithmetic skills, and visual and auditory monitoring simultaneously, whereas the simple task battery measured only working memory and visual monitoring. Ten men and 10 women subjects underwent these batteries 1 h after ingesting 150 mg/kg of l-tyrosine or placebo. Administration of tyrosine significantly enhanced accuracy and decreased frequency of list retrieval on the working memory task during the multiple task battery compared with placebo. However, tyrosine induced no significant changes in performance on the arithmetic, visual, or auditory tasks during the Multiple Task, or modified any performance measures during the Simple Task battery. Blood levels of ACTH and cortisol were not, but heart rate and blood pressure were significantly increased during the performance tasks. The present results indicate that tyrosine may sustain working memory when competing requirements to perform other tasks simultaneously degrade performance, and that supplemental tyrosine may be appropriate for maintaining performance when mild to severe decrements are anticipated.

  8. Toxicological disruption of signaling homeostasis: Tyrosine phosphatses as targets

    EPA Science Inventory

    The protein tyrosine phosphatases (PTP) comprised a diverse group of enzymes whose activity opposes that of the tyrosine kinases. As such, the PTP have critical roles in maintaining signaling quiescence in resting cells and in restoring homeostasis by effecting signal termination...

  9. Pleiotrophin stimulates tyrosine phosphorylation of beta-adducin through inactivation of the transmembrane receptor protein tyrosine phosphatase beta/zeta.

    PubMed

    Pariser, Harold; Perez-Pinera, Pablo; Ezquerra, Laura; Herradon, Gonzalo; Deuel, Thomas F

    2005-09-16

    Pleiotrophin (PTN the protein, Ptn the gene) signals through a unique mechanism; it inactivates the tyrosine phosphatase activity of its receptor, the transmembrane receptor protein tyrosine phosphatase (RPTP)beta/zeta, and increases tyrosine phosphorylation of the substrates of RPTPbeta/zeta through the continued activity of a yet to be described protein tyrosine kinase(s) in PTN-stimulated cells. We have now found that the cytoskeletal protein beta-adducin interacts with the intracellular domain of RPTPbeta/zeta in a yeast two-hybrid system, that beta-adducin is a substrate of RPTPbeta/zeta, that beta-adducin is phosphorylated in tyrosine in cells not stimulated by PTN, and that tyrosine phosphorylation of beta-adducin is sharply increased in PTN-stimulated cells, suggesting that beta-adducin is a downstream target of and regulated by the PTN/RPTPbeta/zeta signaling pathway. beta-Catenin was the first downstream target of the PTN/RPTPbeta/zeta signaling pathway to be identified; these data thus also suggest that PTN coordinately regulates steady state levels of tyrosine phosphorylation of the important cytoskeletal proteins beta-adducin and beta-catenin and, through PTN-stimulated tyrosine phosphorylation, beta-adducin may contribute to the disruption of cytoskeletal structure, increased plasticity, and loss of homophilic cell-cell adhesion that are the consequences of PTN stimulation of cells and a characteristic feature of different malignant cells with mutations that activate constitutive expression of the endogenous Ptn gene.

  10. Mutation of tyrosine-141 inhibits insulin-promoted tyrosine phosphorylation and increased responsiveness of the human beta 2-adrenergic receptor.

    PubMed Central

    Valiquette, M; Parent, S; Loisel, T P; Bouvier, M

    1995-01-01

    The ability of insulin to promote phosphorylation of the human beta 2-adrenergic receptor (beta 2AR) was assessed in Chinese hamster fibroblasts transfected with beta 2AR cDNA. Phosphotyrosine residues were detected in purified beta 2AR using a polyclonal anti-phosphotyrosine antibody and by phosphoamino acid analysis following metabolic labelling with inorganic 32P. Treatment of the cells with insulin induced a 2.4-fold increase in the phosphotyrosine content of the receptor. The insulin-promoted phosphorylation of the beta 2AR was accompanied by an increase in the beta-adrenergic-stimulated adenyl cyclase activity. Substitution of a phenylalanine residue for tyrosine-141 completely prevented both the increased tyrosine phosphorylation and the enhanced responsiveness of the beta 2AR promoted by insulin treatment. Mutation of three other tyrosines located in the cytoplasmic domain of the receptor, tyrosine-366, tyrosine-350 and tyrosine-354, did not abolish the insulin-promoted tyrosine phosphorylation. Taken together, these results suggest that insulin promotes phosphorylation of the beta 2AR on tyrosine-141 and that such phosphorylation leads to a supersensitization of the receptor. Images PMID:8521811

  11. Receptor tyrosine kinase targeting in multicellular spheroids.

    PubMed

    Breslin, Susan; O'Driscoll, Lorraine

    2015-01-01

    While growing cells as a monolayer is the traditional method for cell culture, the incorporation of multicellular spheroids into experimental design is becoming increasingly popular. This is due to the understanding that cells grown as spheroids tend to replicate the in vivo situation more reliably than monolayer cells. Thus, the use of multicellular spheroids may be more clinically relevant than monolayer cell cultures. Here, we describe methods for multicellular 3D spheroid generation that may be used to provide samples for receptor tyrosine kinase (and other protein) detection. Methods described include the forced-floating poly-HEMA method, the hanging-drop method, and the use of ECM to form multicellular 3D spheroids. PMID:25319898

  12. Complexity of Receptor Tyrosine Kinase Signal Processing

    PubMed Central

    Volinsky, Natalia; Kholodenko, Boris N.

    2013-01-01

    Our knowledge of molecular mechanisms of receptor tyrosine kinase (RTK) signaling advances with ever-increasing pace. Yet our understanding of how the spatiotemporal dynamics of RTK signaling control specific cellular outcomes has lagged behind. Systems-centered experimental and computational approaches can help reveal how overlapping networks of signal transducers downstream of RTKs orchestrate specific cell-fate decisions. We discuss how RTK network regulatory structures, which involve the immediate posttranslational and delayed transcriptional controls by multiple feed forward and feedback loops together with pathway cross talk, adapt cells to the combinatorial variety of external cues and conditions. This intricate network circuitry endows cells with emerging capabilities for RTK signal processing and decoding. We illustrate how mathematical modeling facilitates our understanding of RTK network behaviors by unraveling specific systems properties, including bistability, oscillations, excitable responses, and generation of intricate landscapes of signaling activities. PMID:23906711

  13. Antibodies directed against receptor tyrosine kinases

    PubMed Central

    FAUVEL, Bénédicte; Yasri, Aziz

    2014-01-01

    Approximately 30 therapeutic monoclonal antibodies have already been approved for cancers and inflammatory diseases, and monoclonal antibodies continue to be one of the fastest growing classes of therapeutic molecules. Because aberrant signaling by receptor tyrosine kinases (RTKs) is a commonly observed factor in cancer, most of the subclasses of RTKs are being extensively studied as potential targets for treating malignancies. The first two RTKs that have been targeted by antibody therapy, with five currently marketed antibodies, are the growth factor receptors EGFR and HER2. However, due to systemic side effects, refractory patients and the development of drug resistance, these treatments are being challenged by emerging therapeutics. This review examines current monoclonal antibody therapies against RTKs. After an analysis of agents that have already been approved, we present an analysis of antibodies in clinical development that target RTKs. Finally, we highlight promising RTKs that are emerging as new oncological targets for antibody-based therapy. PMID:24859229

  14. NLRP3 tyrosine phosphorylation is controlled by protein tyrosine phosphatase PTPN22

    PubMed Central

    Spalinger, Marianne R.; Kasper, Stephanie; Gottier, Claudia; Lang, Silvia; Atrott, Kirstin; Vavricka, Stephan R.; Scharl, Sylvie; Gutte, Petrus M.; Grütter, Markus G.; Beer, Hans-Dietmar; Contassot, Emmanuel; Chan, Andrew C.; Dai, Xuezhi; Rawlings, David J.; Mair, Florian; Becher, Burkhard; Falk, Werner; Fried, Michael; Rogler, Gerhard

    2016-01-01

    Inflammasomes form as the result of the intracellular presence of danger-associated molecular patterns and mediate the release of active IL-1β, which influences a variety of inflammatory responses. Excessive inflammasome activation results in severe inflammatory conditions, but physiological IL-1β secretion is necessary for intestinal homeostasis. Here, we have described a mechanism of NLRP3 inflammasome regulation by tyrosine phosphorylation of NLRP3 at Tyr861. We demonstrated that protein tyrosine phosphatase non-receptor 22 (PTPN22), variants in which are associated with chronic inflammatory disorders, dephosphorylates NLRP3 upon inflammasome induction, allowing efficient NLRP3 activation and subsequent IL-1β release. In murine models, PTPN22 deficiency resulted in pronounced colitis, increased NLRP3 phosphorylation, but reduced levels of mature IL-1β. Conversely, patients with inflammatory bowel disease (IBD) that carried an autoimmunity-associated PTPN22 variant had increased IL-1β levels. Together, our results identify tyrosine phosphorylation as an important regulatory mechanism for NLRP3 that prevents aberrant inflammasome activation. PMID:27043286

  15. Tyrosine promotes oxidative stress in cerebral cortex of young rats.

    PubMed

    Sgaravatti, Angela M; Vargas, Bethânia A; Zandoná, Bernardo R; Deckmann, Kátia B; Rockenbach, Francieli J; Moraes, Tarsila B; Monserrat, José M; Sgarbi, Mirian B; Pederzolli, Carolina D; Wyse, Angela T S; Wannmacher, Clóvis M D; Wajner, Moacir; Dutra-Filho, Carlos Severo

    2008-10-01

    Tyrosine accumulates in inborn errors of tyrosine catabolism, especially in tyrosinemia type II, where tyrosine levels are highly elevated in tissues and physiological fluids of affected patients. In tyrosinemia type II, high levels of tyrosine are correlated with eyes, skin and central nervous system disturbances. Considering that the mechanisms of brain damage in these disorders are poorly known, in the present study, we investigated whether oxidative stress is elicited by l-tyrosine in cerebral cortex homogenates of 14-day-old Wistar rats. The in vitro effect of 0.1-4.0mM l-tyrosine was studied on the following oxidative stress parameters: total radical-trapping antioxidant potential (TRAP), total antioxidant reactivity (TAR), ascorbic acid content, reduced glutathione (GSH) content, spontaneous chemiluminescence, thiobarbituric acid-reactive substances (TBA-RS), thiol-disulfide redox state (SH/SS ratio), protein carbonyl content, formation of DNA-protein cross-links, and the activities of the enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glucose-6-phosphate dehydrogenase (G6PDH). TRAP, TAR, ascorbic acid content, SH/SS ratio and CAT activity were significantly diminished, while formation of DNA-protein cross-link was significantly enhanced by l-tyrosine in vitro. In contrast, l-tyrosine did not affect the other parameters of oxidative stress evaluated. These results indicate that l-tyrosine decreases enzymatic and non-enzymatic antioxidant defenses, changes the redox state and stimulates DNA damage in cerebral cortex of young rats in vitro. This suggests that oxidative stress may represent a pathophysiological mechanism in tyrosinemic patients, in which this amino acid accumulates.

  16. Dopamine release in rat striatum - Physiological coupling to tyrosine supply

    NASA Technical Reports Server (NTRS)

    During, Matthew J.; Acworth, Ian N.; Wurtman, Richard J.

    1989-01-01

    Intracerebral microdialysis was used to monitor dopamine release in rat striatal extracellular fluid following the intraperitoneal administration of dopamine's precursor amino acid, L-tyrosine. Dopamine concentrations in dialysates increased transiently after tyrosine (50-100 mg/kg) administration. Pretreatment with haloperidol or the partial lesioning of nigrostriatal neurons enhanced the effect of tyrosine on dopamine release, and haloperidol also prolonged this effect. These data suggest that nigrostriatal dopaminergic neurons are responsive to changes in precursor availability under basal conditions, but that receptor-mediated feedback mechanisms limit the magnitude and duration of this effect.

  17. Genetic alterations of protein tyrosine phosphatases in human cancers

    PubMed Central

    Zhao, Shuliang; Sedwick, David; Wang, Zhenghe

    2014-01-01

    Protein tyrosine phosphatases (PTPs) are enzymes that remove phosphate from tyrosine residues in proteins. Recent whole-exome sequencing of human cancer genomes reveals that many PTPs are frequently mutated in a variety of cancers. Among these mutated PTPs, protein tyrosine phosphatase T (PTPRT) appears to be the most frequently mutated PTP in human cancers. Beside PTPN11 which functions as an oncogene in leukemia, genetic and functional studies indicate that most of mutant PTPs are tumor suppressor genes. Identification of the substrates and corresponding kinases of the mutant PTPs may provide novel therapeutic targets for cancers harboring these mutant PTPs. PMID:25263441

  18. Statins Attenuate Helicobacter pylori CagA Translocation and Reduce Incidence of Gastric Cancer: In Vitro and Population-Based Case-Control Studies.

    PubMed

    Lin, Chun-Jung; Liao, Wei-Chih; Lin, Hwai-Jeng; Hsu, Yuan-Man; Lin, Cheng-Li; Chen, Yu-An; Feng, Chun-Lung; Chen, Chih-Jung; Kao, Min-Chuan; Lai, Chih-Ho; Kao, Chia-Hung

    2016-01-01

    Gastric cancer is the second leading cause of cancer-related death worldwide. The correlation of Helicobacter pylori and the etiology of gastric cancer was substantially certain. Cholesterol-rich microdomains (also called lipid rafts), which provide platforms for signaling, are associated with H. pylori-induced pathogenesis leading to gastric cancer. Patients who have been prescribed statins, inhibitors of 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase, have exhibited a reduced risk of several types of cancer. However, no studies have addressed the effect of statins on H. pylori-associated gastric cancer from the antineoplastic perspective. In this study, we showed that treatment of gastric epithelial cells with simvastatin reduced the level of cellular cholesterol and led to attenuation of translocation and phosphorylation of H. pylori cytotoxin-associated gene A (CagA), which is recognized as a major determinant of gastric cancer development. Additionally, a nationwide case-control study based on data from the Taiwanese National Health Insurance Research Database (NHIRD) was conducted. A population-based case-control study revealed that patients who used simvastatin exhibited a significantly reduced risk of gastric cancer (adjusted odds ratio (OR) = 0.76, 95% confidence interval (CI) = 0.70-0.83). In patients exhibiting H. pylori infection who were prescribed simvastatin, the adjusted OR for gastric cancer was 0.25 (95% CI = 0.12-0.50). Our results combined an in vitro study with a nationwide population analysis reveal that statin use might be a feasible approach to prevent H. pylori-associated gastric cancer.

  19. Statins Attenuate Helicobacter pylori CagA Translocation and Reduce Incidence of Gastric Cancer: In Vitro and Population-Based Case-Control Studies

    PubMed Central

    Hsu, Yuan-Man; Lin, Cheng-Li; Chen, Yu-An; Feng, Chun-Lung; Chen, Chih-Jung; Kao, Min-Chuan; Lai, Chih-Ho; Kao, Chia-Hung

    2016-01-01

    Gastric cancer is the second leading cause of cancer-related death worldwide. The correlation of Helicobacter pylori and the etiology of gastric cancer was substantially certain. Cholesterol-rich microdomains (also called lipid rafts), which provide platforms for signaling, are associated with H. pylori-induced pathogenesis leading to gastric cancer. Patients who have been prescribed statins, inhibitors of 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase, have exhibited a reduced risk of several types of cancer. However, no studies have addressed the effect of statins on H. pylori-associated gastric cancer from the antineoplastic perspective. In this study, we showed that treatment of gastric epithelial cells with simvastatin reduced the level of cellular cholesterol and led to attenuation of translocation and phosphorylation of H. pylori cytotoxin-associated gene A (CagA), which is recognized as a major determinant of gastric cancer development. Additionally, a nationwide case-control study based on data from the Taiwanese National Health Insurance Research Database (NHIRD) was conducted. A population-based case-control study revealed that patients who used simvastatin exhibited a significantly reduced risk of gastric cancer (adjusted odds ratio (OR) = 0.76, 95% confidence interval (CI) = 0.70–0.83). In patients exhibiting H. pylori infection who were prescribed simvastatin, the adjusted OR for gastric cancer was 0.25 (95% CI = 0.12–0.50). Our results combined an in vitro study with a nationwide population analysis reveal that statin use might be a feasible approach to prevent H. pylori-associated gastric cancer. PMID:26730715

  20. Studying Protein-Tyrosine Phosphatases in Zebrafish.

    PubMed

    Hale, Alexander James; den Hertog, Jeroen

    2016-01-01

    Protein-tyrosine phosphatases (PTPs) are a large family of signal transduction regulators that have an essential role in normal development and physiology. Aberrant activation or inactivation of PTPs is at the basis of many human diseases. The zebrafish, Danio rerio, is being used extensively to model major aspects of development and disease as well as the mechanism of regeneration of limbs and vital organs, and most classical PTPs have been identified in zebrafish. Zebrafish is an excellent model system for biomedical research because the genome is sequenced, zebrafish produce a large number of offspring, the eggs develop outside the mother and are transparent, facilitating intravital imaging, and transgenesis and (site-directed) mutagenesis are feasible. Together, these traits make zebrafish amenable for the analysis of gene and protein function. In this chapter we cover three manipulations of zebrafish embryos that we have used to study the effects of PTPs in development, regeneration, and biochemistry. Microinjection at the one-cell stage is at the basis of many zebrafish experiments and is described first. This is followed by a description for measuring regeneration of the embryonic caudal fin, a powerful and robust physiological assay. Finally, the considerable but manageable troubleshooting of several complications associated with preparing zebrafish embryos for immunoblotting is explained. Overall, this chapter provides detailed protocols for manipulating zebrafish embryo samples with a compilation of tips collected through extensive experience from the zebrafish research community. PMID:27514815

  1. Therapeutic drug monitoring and tyrosine kinase inhibitors

    PubMed Central

    Herviou, Pauline; Thivat, Emilie; Richard, Damien; Roche, Lucie; Dohou, Joyce; Pouget, Mélanie; Eschalier, Alain; Durando, Xavier; Authier, Nicolas

    2016-01-01

    The therapeutic activity of drugs can be optimized by establishing an individualized dosage, based on the measurement of the drug concentration in the serum, particularly if the drugs are characterized by an inter-individual variation in pharmacokinetics that results in an under- or overexposure to treatment. In recent years, several tyrosine kinase inhibitors (TKIs) have been developed to block intracellular signaling pathways in tumor cells. These oral drugs are candidates for therapeutic drug monitoring (TDM) due to their high inter-individual variability for therapeutic and toxic effects. Following a literature search on PubMed, studies on TKIs and their pharmacokinetic characteristics, plasma quantification and inter-individual variability was studied. TDM is commonly used in various medical fields, including cardiology and psychiatry, but is not often applied in oncology. Plasma concentration monitoring has been thoroughly studied for imatinib, in order to evaluate the usefulness of TDM. The measurement of plasma concentration can be performed by various analytical techniques, with liquid chromatography-mass spectrometry being the reference method. This method is currently used to monitor the efficacy and tolerability of imatinib treatments. Although TDM is already being used for imatinib, additional studies are required in order to improve this practice with the inclusion of other TKIs. PMID:27446421

  2. Transcriptional regulation through glutamate receptors: Involvement of tyrosine kinases.

    PubMed

    López-Bayghen, Esther; Aguirre, Adán; Ortega, Arturo

    2003-12-01

    Glutamate receptors play a key role in neuronal plasticity, learning and memory, and in several neuropathologies. Short-term and long-term changes in synaptic efficacy are triggered by glutamate. Although an enhanced glutamate-dependent tyrosine phosphorylation has been described in several systems, its role in membrane-to-nuclei signaling is unclear. Taking advantage of the fact that the gene encoding the chick kainate-binding protein undergoes a glutamate-dependent transcriptional regulation via an activator protein-1 (AP-1) site, we evaluated the involvement of tyrosine kinases in this process. We describe here the participation of receptor and non-receptor tyrosine kinases in the signaling cascade triggered by glutamate. Our results suggest that in Bergmann glia cells, glutamate receptors transactivate receptor tyrosine kinases, favoring the idea of a complex network of signals activated by this excitatory neurotransmitter that results in regulation of gene expression.

  3. Tyrosine kinase signaling and the emergence of multicellularity.

    PubMed

    Miller, W Todd

    2012-06-01

    Tyrosine phosphorylation is an essential element of signal transduction in multicellular animals. Although tyrosine kinases were originally regarded as specific to the metazoan lineage, it is now clear that they evolved prior to the split between unicellular and multicellular eukaryotes (≈600million years ago). Genome analyses of choanoflagellates and other protists show an abundance of tyrosine kinases that rivals the most complex animals. Some of these kinases are orthologs of metazoan enzymes (e.g., Src), but others display unique domain compositions not seen in any metazoan. Biochemical experiments have highlighted similarities and differences between the unicellular and multicellular tyrosine kinases. In particular, it appears that the complex systems of kinase autoregulation may have evolved later in the metazoan lineage. PMID:22480439

  4. Skin problems and EGFR-tyrosine kinase inhibitor.

    PubMed

    Kozuki, Toshiyuki

    2016-04-01

    Epidermal growth factor receptor inhibition is a good target for the treatment of lung, colon, pancreatic and head and neck cancers. Epidermal growth factor receptor-tyrosine kinase inhibitor was first approved for the treatment of advanced lung cancer in 2002. Epidermal growth factor receptor-tyrosine kinase inhibitor plays an essential role in the treatment of cancer, especially for patients harbouring epidermal growth factor receptor activating mutation. Hence, skin toxicity is the most concerning issue for the epidermal growth factor receptor-tyrosine kinase inhibitor treatment. Skin toxicity is bothersome and sometimes affects the quality of life and treatment compliance. Thus, it is important for physicians to understand the background and how to manage epidermal growth factor receptor-tyrosine kinase inhibitor-associated skin toxicity. Here, the author reviewed the mechanism and upfront preventive and reactive treatments for epidermal growth factor receptor inhibitor-associated skin toxicities.

  5. A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation.

    PubMed

    Macho, Alberto P; Schwessinger, Benjamin; Ntoukakis, Vardis; Brutus, Alexandre; Segonzac, Cécile; Roy, Sonali; Kadota, Yasuhiro; Oh, Man-Ho; Sklenar, Jan; Derbyshire, Paul; Lozano-Durán, Rosa; Malinovsky, Frederikke Gro; Monaghan, Jacqueline; Menke, Frank L; Huber, Steven C; He, Sheng Yang; Zipfel, Cyril

    2014-03-28

    Innate immunity relies on the perception of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) located on the host cell's surface. Many plant PRRs are kinases. Here, we report that the Arabidopsis receptor kinase EF-TU RECEPTOR (EFR), which perceives the elf18 peptide derived from bacterial elongation factor Tu, is activated upon ligand binding by phosphorylation on its tyrosine residues. Phosphorylation of a single tyrosine residue, Y836, is required for activation of EFR and downstream immunity to the phytopathogenic bacterium Pseudomonas syringae. A tyrosine phosphatase, HopAO1, secreted by P. syringae, reduces EFR phosphorylation and prevents subsequent immune responses. Thus, host and pathogen compete to take control of PRR tyrosine phosphorylation used to initiate antibacterial immunity.

  6. Tyrosine kinase signaling and the emergence of multicellularity.

    PubMed

    Miller, W Todd

    2012-06-01

    Tyrosine phosphorylation is an essential element of signal transduction in multicellular animals. Although tyrosine kinases were originally regarded as specific to the metazoan lineage, it is now clear that they evolved prior to the split between unicellular and multicellular eukaryotes (≈600million years ago). Genome analyses of choanoflagellates and other protists show an abundance of tyrosine kinases that rivals the most complex animals. Some of these kinases are orthologs of metazoan enzymes (e.g., Src), but others display unique domain compositions not seen in any metazoan. Biochemical experiments have highlighted similarities and differences between the unicellular and multicellular tyrosine kinases. In particular, it appears that the complex systems of kinase autoregulation may have evolved later in the metazoan lineage.

  7. Targeting Tyrosine Kinases and Autophagy in Prostate Cancer

    PubMed Central

    2010-01-01

    Tyrosine kinases play significant roles in tumor progression and therapy resistance. Inhibitors of tyrosine kinases are on the forefront of targeted therapy. For prostate cancer, tyrosine kinases play an additional role in the development of castration-resistant disease state, the most troubling aspect of prostate cancinogenesis which presently defies any effective treatment. Among the 30 or so tyrosine kinases expressed in a typical prostate cancer cell, nearly one third of them have been implicated in prostate carcinogenesis. Interestingly, most of them channel signals through a trio of non-receptor tyrosine kinases, Src/Etk/FAK, referred here as Src tyrosine kinase complex. This complex has been shown to play a significant role in the aberrant activation of androgen receptor (AR) mediated by growth factors (e.g., epidermal growth factor (EGF)), cytokines (interleukin (IL)-6), chemokines (IL-8), and neurokines (gastrin-releasing peptide). These factors are induced and released from the prostate cancer to the stromal cells upon androgen withdrawal. The Src kinase complex has the ability to phosphorylate androgen receptor, resulting in the nuclear translocation and stabilization of un-liganded androgen receptor. Indeed, tyrosine kinase inhibitors targeting Src can inhibit androgen-independent growth of prostate cancer cells in vitro and in preclinical xenograft model. While effective in inducing growth arrest and inhibiting metastasis of castration-resistant tumors, Src inhibitors rarely induce a significant level of apoptosis. This is also reflected by the general ineffectiveness of tyrosine kinase inhibitors as monotherapy in clinical trials. One of the underlying causes of apoptosis resistance is “autophagy,” which is induced by tyrosine kinase inhibitors and by androgen withdrawal. Autophagy is a self-digesting process to regenerate energy by removal of long-lived proteins and retired organelles to provide a survival mechanism to cells encountering stresses

  8. Method of making L-dopa from L-tyrosine

    DOEpatents

    Xun, L.; Lee, J.Y.

    1998-11-17

    The invention is a method of making a L-dopa from L-tyrosine in the presence of an enzyme catalyst and oxygen. By starting with L-tyrosine, no variant of the L-dopa is produced and the L-dopa is stable in the presence of the enzyme catalyst. In other words, the reaction favors the L-dopa and is not reversible. 3 figs.

  9. Method of making L-dopa from L-tyrosine

    DOEpatents

    Xun, Luying; Lee, Jang Young

    1998-01-01

    The invention is a method of making a L-dopa from L-tyrosine in the presence of an enzyme catalyst and oxygen. By starting with L-tyrosine, no variant of the L-dopa is produced and the L-dopa is stable in the presence of the enzyme catalyst. In other words, the reaction favors the L-dopa and is not reversible.

  10. Generation Mechanism of Deferoxamine Radical by Tyrosine-Tyrosinase Reaction.

    PubMed

    Tada, Mika; Niwano, Yoshimi; Kohno, Masahiro

    2015-01-01

    Nitroxide radical formations of deferoxamine mesylate (DFX) that is used clinically to treat iron-overload patients was examined by a tyrosine-tyrosinase reaction system as models of the H-atom transfer or proton-coupled electron transfer. When DFX was exposed to the tyrosine-tyrosinase reaction, nine-line ESR spectrum (g = 2.0063, hfcc; aN = 0.78 mT, aH(2) = 0.63 mT) was detected, indicating that the oxidation of DFX leads to a nitroxide radical. The signal intensity of the DFX radical increased dependently on the concentrations of tyrosine and tyrosinase. The amounts of DMPO-OH spin adducts via the tyrosine-tyrosinase reaction declined with DFX. Furthermore, mass spectra of an extra removed from the tyrosine-tyrosinase reaction mixture showed that the enzyme reactions might not be degradations of DFX. Therefore, there might be two types of DFX reaction passways, which could be through an internal electron transfer from tyrosine and hydrogen absorptions by ·OH directly.

  11. Beta 2 integrin-dependent protein tyrosine phosphorylation and activation of the FGR protein tyrosine kinase in human neutrophils

    PubMed Central

    1994-01-01

    Stimulation of adherent human neutrophils (PMN) with tumor necrosis factor (TNF) triggers protein tyrosine phosphorylation (Fuortes, M., W. W. Jin, and C. Nathan. 1993. J. Cell Biol. 120:777-784). We investigated the dependence of this response on beta 2 integrins by using PMN isolated from a leukocyte adhesion deficiency (LAD) patient, which do not express beta 2 integrins, and by plating PMN on surface bound anti-beta 2 (CD18) antibodies. Protein tyrosine phosphorylation increased in PMN plated on fibrinogen and this phosphorylation was enhanced by TNF. Triggering of protein tyrosine phosphorylation did not occur in LAD PMN plated on fibrinogen either in the absence or the presence of TNF. Surface bound anti-CD18, but not isotype-matched anti- Class I major histocompatibility complex (MHC) antigens, antibodies triggered tyrosine phosphorylation in normal, but not in LAD PMN. As the major tyrosine phosphorylated proteins we found in our assay conditions migrated with an apparent molecular mass of 56-60 kD, we investigated whether beta 2 integrins are implicated in activation of members of the src family of intracellular protein-tyrosine kinases. We found that the fgr protein-tyrosine kinase (p58fgr) activity, and its extent of phosphorylation in tyrosine, in PMN adherent to fibrinogen, was enhanced by TNF. Activation of p58fgr in response to TNF was evident within 10 min of treatment and increased with times up to 30 min. Also other activators of beta 2 integrins such as phorbol-12- myristate 13-acetate (PMA), and formyl methionyl-leucyl-phenylalanine (FMLP), induced activation of p58fgr kinase activity. Activation of p58fgr kinase activity, and phosphorylation in tyrosine, did not occur in PMN of a LAD patient in response to TNF. Soluble anti-CD18, but not anti-Class I MHC antigens, antibodies inhibited activation of p58fgr kinase activity in PMN adherent to fibrinogen in response to TNF, PMA, and FMLP. These findings demonstrate that, in PMN, beta 2 integrins

  12. SHP1 tyrosine phosphatase negatively regulates NPM-ALK tyrosine kinase signaling.

    PubMed

    Honorat, Jean-François; Ragab, Ashraf; Lamant, Laurence; Delsol, Georges; Ragab-Thomas, Jeannie

    2006-05-15

    Anaplastic large-cell lymphoma (ALCL) is frequently associated with the 2;5 translocation and expresses the NPM-ALK fusion protein, which possesses a constitutive tyrosine kinase activity. We analyzed SHP1 tyrosine phosphatase expression and activity in 3 ALK-positive ALCL cell lines (Karpas 299, Cost, and SU-DHL1) and in lymph node biopsies (n = 40). We found an inverse correlation between the level of NPM-ALK phosphorylation and SHP1 phosphatase activity. Pull-down and coimmunoprecipitation experiments demonstrated a SHP1/NPM-ALK association. Furthermore, confocal microscopy performed on ALCL cell lines and biopsy specimens showed the colocalization of the 2 proteins in cytoplasmic bodies containing Y664-phosphorylated NPM-ALK. Dephosphorylation of NPM-ALK by SHP1 demonstrated that NPM-ALK was a SHP1 substrate. Downregulation of SHP1 expression by RNAi in Karpas cells led to hyperphosphorylation of NPM-ALK, STAT3 activation, and increase in cell proliferation. Furthermore, SHP1 overexpression in 3T3 fibroblasts stably expressing NPM-ALK led to the decrease of NPM-ALK phosphorylation, lower cell proliferation, and tumor progression in nude mice. These findings show that SHP1 is a negative regulator of NPM-ALK signaling. The use of tissue microarrays revealed that 50% of ALK-positive ALCLs were positive for SHP1. Our results suggest that SHP1 could be a critical enzyme in ALCL biology and a potential therapeutic target.

  13. Are striatal tyrosine hydroxylase interneurons dopaminergic?

    PubMed

    Xenias, Harry S; Ibáñez-Sandoval, Osvaldo; Koós, Tibor; Tepper, James M

    2015-04-22

    Striatal GABAergic interneurons that express the gene for tyrosine hydroxylase (TH) have been identified previously by several methods. Although generally assumed to be dopaminergic, possibly serving as a compensatory source of dopamine (DA) in Parkinson's disease, this assumption has never been tested directly. In TH-Cre mice whose nigrostriatal pathway had been eliminated unilaterally with 6-hydroxydopamine, we injected a Cre-dependent virus coding for channelrhodopsin-2 and enhanced yellow fluorescent protein unilaterally into the unlesioned midbrain or bilaterally into the striatum. Fast-scan cyclic voltammetry in striatal slices revealed that both optical and electrical stimulation readily elicited DA release in control striata but not from contralateral striata when nigrostriatal neurons were transduced. In contrast, neither optical nor electrical stimulation could elicit striatal DA release in either the control or lesioned striata when the virus was injected directly into the striatum transducing only striatal TH interneurons. This demonstrates that striatal TH interneurons do not release DA. Fluorescence immunocytochemistry in enhanced green fluorescent protein (EGFP)-TH mice revealed colocalization of DA, l-amino acid decarboxylase, the DA transporter, and vesicular monoamine transporter-2 with EGFP in midbrain dopaminergic neurons but not in any of the striatal EGFP-TH interneurons. Optogenetic activation of striatal EGFP-TH interneurons produced strong GABAergic inhibition in all spiny neurons tested. These results indicate that striatal TH interneurons are not dopaminergic but rather are a type of GABAergic interneuron that expresses TH but none of the other enzymes or transporters necessary to operate as dopaminergic neurons and exert widespread GABAergic inhibition onto direct and indirect spiny neurons. PMID:25904808

  14. Are striatal tyrosine hydroxylase interneurons dopaminergic?

    PubMed

    Xenias, Harry S; Ibáñez-Sandoval, Osvaldo; Koós, Tibor; Tepper, James M

    2015-04-22

    Striatal GABAergic interneurons that express the gene for tyrosine hydroxylase (TH) have been identified previously by several methods. Although generally assumed to be dopaminergic, possibly serving as a compensatory source of dopamine (DA) in Parkinson's disease, this assumption has never been tested directly. In TH-Cre mice whose nigrostriatal pathway had been eliminated unilaterally with 6-hydroxydopamine, we injected a Cre-dependent virus coding for channelrhodopsin-2 and enhanced yellow fluorescent protein unilaterally into the unlesioned midbrain or bilaterally into the striatum. Fast-scan cyclic voltammetry in striatal slices revealed that both optical and electrical stimulation readily elicited DA release in control striata but not from contralateral striata when nigrostriatal neurons were transduced. In contrast, neither optical nor electrical stimulation could elicit striatal DA release in either the control or lesioned striata when the virus was injected directly into the striatum transducing only striatal TH interneurons. This demonstrates that striatal TH interneurons do not release DA. Fluorescence immunocytochemistry in enhanced green fluorescent protein (EGFP)-TH mice revealed colocalization of DA, l-amino acid decarboxylase, the DA transporter, and vesicular monoamine transporter-2 with EGFP in midbrain dopaminergic neurons but not in any of the striatal EGFP-TH interneurons. Optogenetic activation of striatal EGFP-TH interneurons produced strong GABAergic inhibition in all spiny neurons tested. These results indicate that striatal TH interneurons are not dopaminergic but rather are a type of GABAergic interneuron that expresses TH but none of the other enzymes or transporters necessary to operate as dopaminergic neurons and exert widespread GABAergic inhibition onto direct and indirect spiny neurons.

  15. Role of Tyrosine Isomers in Acute and Chronic Diseases Leading to Oxidative Stress - A Review

    PubMed Central

    Molnár, Gergő A.; Kun, Szilárd; Sélley, Eszter; Kertész, Melinda; Szélig, Lívia; Csontos, Csaba; Böddi, Katalin; Bogár, Lajos; Miseta, Attila; Wittmann, István

    2016-01-01

    Oxidative stress plays a major role in the pathogenesis of a variety of acute and chronic diseases. Measurement of the oxidative stress-related end products may be performed, e.g. that of structural isomers of the physiological para-tyrosine, namely meta- and ortho-tyrosine, that are oxidized derivatives of phenylalanine. Recent data suggest that in sepsis, serum level of meta-tyrosine increases, which peaks on the 2nd and 3rd days (p<0.05 vs. controls), and the kinetics follows the intensity of the systemic inflammation correlating with serum procalcitonin levels. In a similar study subset, urinary meta-tyrosine excretion correlated with both need of daily insulin dose and the insulin-glucose product in non-diabetic septic cases (p<0.01 for both). Using linear regression model, meta-tyrosine excretion, urinary meta-tyrosine/para-tyrosine, urinary ortho-tyrosine/para-tyrosine and urinary (meta- + ortho-tyrosine)/para-tyrosine proved to be markers of carbohydrate homeostasis. In a chronic rodent model, we tried to compensate the abnormal tyrosine isomers using para-tyrosine, the physiological amino acid. Rats were fed a standard high cholesterol-diet, and were given para-tyrosine or vehicle orally. High-cholesterol feeding lead to a significant increase in aortic wall meta-tyrosine content and a decreased vasorelaxation of the aorta to insulin and the glucagon-like peptide-1 analogue, liraglutide, that both could be prevented by administration of para-tyrosine. Concluding, these data suggest that meta- and ortho-tyrosine are potential markers of oxidative stress in acute diseases related to oxidative stress, and may also interfere with insulin action in septic humans. Competition of meta- and ortho-tyrosine by supplementation of para-tyrosine may exert a protective role in oxidative stress-related diseases. PMID:26785996

  16. An Overview of Phenylalanine and Tyrosine Kinetics in Humans12

    PubMed Central

    Matthews, Dwight E.

    2008-01-01

    The initial use of a tracer of phenylalanine was by Moss and Schoenheimer in rats in 1940 to determine that phenylalanine was hydroxylated to tyrosine, defining for the first time the primacy of this pathway. Phenylalanine and tyrosine kinetics were not measured in humans until the 1970–80s. The first application was for determination of the degree of blockage of phenylalanine hydroxylation in patients with hyperphenylalanemia and phenylketonuria, but this approach was expanded to determination of phenylalanine hydroxylation in normal subjects. Far more uses have been demonstrated for measuring rates of phenylalanine disposal and tyrosine production in relatively normal subjects than in patients with in-born errors of metabolism. Key to use of tracers to determine phenylalanine and tyrosine metabolic rates has been development of appropriate tracer models. Most applications have used relatively simple models ignoring the intracellular hydroxylation rate component. Because the liver is the primary site of hydroxylation in the body, the intracellular enrichment at the site of hydroxylation can be assessed from the tracer enrichments at isotopic steady state in rapid-turnover plasma proteins, such as Apo-B, made and secreted by the liver. Although there are potential problems with use of deuterated tracers of phenylalanine, suitable tracers are available and have been demonstrated for general measurement of phenylalanine and tyrosine kinetics in humans. PMID:17513423

  17. A novel nonreceptor tyrosine kinase, Srm: cloning and targeted disruption.

    PubMed Central

    Kohmura, N; Yagi, T; Tomooka, Y; Oyanagi, M; Kominami, R; Takeda, N; Chiba, J; Ikawa, Y; Aizawa, S

    1994-01-01

    We have isolated a novel nonreceptor tyrosine kinase, Srm, that maps to the distal end of chromosome 2. It has SH2, SH2', and SH3 domains and a tyrosine residue for autophosphorylation in the kinase domain but lacks an N-terminal glycine for myristylation and a C-terminal tyrosine which, when phosphorylated, suppresses kinase activity. These are structural features of the recently identified Tec family of nonreceptor tyrosine kinases. The Srm N-terminal unique domain, however, lacks the structural characteristics of the Tec family kinases, and the sequence similarity is highest to Src in the SH region. The expression of two transcripts is rather ubiquitous and changes according to tissue and developmental stage. Mutant mice were generated by gene targeting in embryonic stem cells but displayed no apparent phenotype as in mutant mice expressing Src family kinases. These results suggest that Srm constitutes a new family of nonreceptor tyrosine kinases that may be redundant in function. Images PMID:7935409

  18. Tyrosine phosphorylation of WASP promotes calpain-mediated podosome disassembly

    PubMed Central

    Macpherson, Lee; Monypenny, James; Blundell, Michael P.; Cory, Giles O.; Tomé-García, Jessica; Thrasher, Adrian J.; Jones, Gareth E.; Calle, Yolanda

    2012-01-01

    Podosomes are actin-based adhesions involved in migration of cells that have to cross tissue boundaries such as myeloid cells. The Wiskott Aldrich Syndrome Protein regulates de novo actin polymerization during podosome formation and it is cleaved by the protease calpain during podosome disassembly. The mechanisms that may induce the Wiskott Aldrich Syndrome Protein cleavage by calpain remain undetermined. We now report that in myeloid cells, tyrosine phosphorylation of the Wiskott Aldrich Syndrome Protein-tyrosine291 (Human)/tyrosine293 (mouse) not only enhances Wiskott Aldrich Syndrome Protein-mediated actin polymerization but also promotes its calpain-dependent degradation during podosome disassembly. We also show that activation of the Wiskott Aldrich Syndrome Protein leading to podosome formation occurs independently of tyrosine phosphorylation in spleen-derived dendritic cells. We conclude that tyrosine phosphorylation of the Wiskott Aldrich Syndrome Protein integrates dynamics of actin and cell adhesion proteins during podosome disassembly required for mobilization of myeloid cells during the immune response. PMID:22133775

  19. Tubulin tyrosine nitration regulates microtubule organization in plant cells

    PubMed Central

    Blume, Yaroslav B.; Krasylenko, Yuliya A.; Demchuk, Oleh M.; Yemets, Alla I.

    2013-01-01

    During last years, selective tyrosine nitration of plant proteins gains importance as well-recognized pathway of direct nitric oxide (NO) signal transduction. Plant microtubules are one of the intracellular signaling targets for NO, however, the molecular mechanisms of NO signal transduction with the involvement of cytoskeletal proteins remain to be elucidated. Since biochemical evidence of plant α-tubulin tyrosine nitration has been obtained recently, potential role of this posttranslational modification in regulation of microtubules organization in plant cell is estimated in current paper. It was shown that 3-nitrotyrosine (3-NO2-Tyr) induced partially reversible Arabidopsis primary root growth inhibition, alterations of root hairs morphology and organization of microtubules in root cells. It was also revealed that 3-NO2-Tyr intensively decorates such highly dynamic microtubular arrays as preprophase bands, mitotic spindles and phragmoplasts of Nicotiana tabacum Bright Yellow-2 (BY-2) cells under physiological conditions. Moreover, 3D models of the mitotic kinesin-8 complexes with the tail of detyrosinated, tyrosinated and tyrosine nitrated α-tubulin (on C-terminal Tyr 450 residue) from Arabidopsis were reconstructed in silico to investigate the potential influence of tubulin nitrotyrosination on the molecular dynamics of α-tubulin and kinesin-8 interaction. Generally, presented data suggest that plant α-tubulin tyrosine nitration can be considered as its common posttranslational modification, the direct mechanism of NO signal transduction with the participation of microtubules under physiological conditions and one of the hallmarks of the increased microtubule dynamics. PMID:24421781

  20. Regulation of mouse gamete interaction by a sperm tyrosine kinase.

    PubMed

    Leyton, L; LeGuen, P; Bunch, D; Saling, P M

    1992-12-15

    A 95-kDa mouse sperm protein has been previously identified as a putative receptor involved in the sperm-egg interactions that lead to fertilization. The ligand for this receptor is the zona pellucida glycoprotein ZP3. This constituent of the oocyte-specific extracellular matrix mediates not only sperm binding to the zona but also triggers acrosomal exocytosis. The latter, also termed the acrosome reaction, is a key regulatory event upon which fertilization is absolutely dependent. Previously, we showed that the 95-kDa protein that binds ZP3 is a substrate for tyrosine kinase, and its phosphotyrosine content increases after sperm-zona pellucida binding. Here, we show the presence of protein tyrosine kinase activity in sperm plasma membranes and in electroeluted 95-kDa protein. The tyrosine kinase activity of the isolated protein is stimulated by solubilized zona pellucida and inhibited by tyrphostin RG-50864, a membrane-permeable tyrosine kinase inhibitor. Furthermore, tyrphostin inhibits zona-triggered acrosomal exocytosis in a dose-dependent manner. These findings indicate that the 95-kDa protein participates in a critical regulatory event of gamete interaction; moreover, our experiments suggest that sperm protein tyrosine kinase may be an excellent target for the control of fertility.

  1. Bacterial tyrosine kinases: evolution, biological function and structural insights

    PubMed Central

    Grangeasse, Christophe; Nessler, Sylvie; Mijakovic, Ivan

    2012-01-01

    Reversible protein phosphorylation is a major mechanism in the regulation of fundamental signalling events in all living organisms. Bacteria have been shown to possess a versatile repertoire of protein kinases, including histidine and aspartic acid kinases, serine/threonine kinases, and more recently tyrosine and arginine kinases. Tyrosine phosphorylation is today recognized as a key regulatory device of bacterial physiology, linked to exopolysaccharide production, virulence, stress response and DNA metabolism. However, bacteria have evolved tyrosine kinases that share no resemblance with their eukaryotic counterparts and are unique in exploiting the ATP/GTP-binding Walker motif to catalyse autophosphorylation and substrate phosphorylation on tyrosine. These enzymes, named BY-kinases (for Bacterial tYrosine kinases), have been identified in a majority of sequenced bacterial genomes, and to date no orthologues have been found in Eukarya. The aim of this review was to present the most recent knowledge about BY-kinases by focusing primarily on their evolutionary origin, structural and functional aspects, and emerging regulatory potential based on recent bacterial phosphoproteomic studies. PMID:22889913

  2. Mechanism of fluorescence quenching of tyrosine derivatives by amide group

    NASA Astrophysics Data System (ADS)

    Wiczk, Wiesław; Rzeska, Alicja; Łukomska, Joanna; Stachowiak, Krystyna; Karolczak, Jerzy; Malicka, Joanna; Łankiewicz, Leszek

    2001-06-01

    The difference between fluorescence lifetimes of the following amino acids: phenylalanine (Phe), tyrosine (Tyr), ( O-methyl)tyrosine (Tyr(Me)), (3-hydroxy)tyrosine (Dopa), (3,4-dimethoxy)phenylalanine (Dopa(Me) 2) and their amides was used to testify the mechanism of fluorescence quenching of aromatic amino acids by the amide group. On the basis of the Marcus theory of photoinduced electron transfer parabolic relationships between ln kET and ionization potentials reduced by energy of excitation ( IP-E ∗0,0) for the above-mentioned amino acids were obtained. This finding indicates the occurrence of photoinduced electron transfer from the excited chromophore group to the amide group.

  3. Tyrosine aminotransferase activity in the benzene intoxicated rat

    SciTech Connect

    Bong, M.; Michalska, A.; Laskowska-Klita, T.; Szymczyk, T.

    1985-01-01

    The toxic effect of hydrocarbon solvents on hepatic metabolism manifests itself by changes in the enzymatic pattern of blood serum. Changes in the activity of phosphatases as well as leucine aminopeptidase, glutamine aminotransferase, sorbitol dehydrogenase and ..gamma..-glutamyltransferase were observed in rats intoxicated with different fractions of benzene. Therefore it seemed reasonable to investigate the effect of benzene fraction of petroleum on cellular metabolism. The results of the present work concern the activity of tyrosine aminotransferase, the enzyme involved in catabolism of aromatic amino acid which is known to be under both hormonal and stress dependent control. Changes in tyrosine aminotransferase activity effect the level of tyrosine oxidation as well as the metabolic conversion of this amino acid into tyramine, tyroxin, adrenaline and noradrenaline.

  4. Tyrosine phosphorylation of clathrin heavy chain under oxidative stress.

    PubMed

    Ihara, Yoshito; Yasuoka, Chie; Kageyama, Kan; Wada, Yoshinao; Kondo, Takahito

    2002-09-20

    In mouse pancreatic insulin-producing betaTC cells, oxidative stress due to H(2)O(2) causes tyrosine phosphorylation in various proteins. To identify proteins bearing phosphotyrosine under stress, the proteins were affinity purified using an anti-phosphotyrosine antibody-conjugated agarose column. A protein of 180kDa was identified as clathrin heavy chain (CHC) by electrophoresis and mass spectrometry. Immunoprecipitated CHC showed tyrosine phosphorylation upon H(2)O(2) treatment and the phosphorylation was suppressed by the Src kinase inhibitor, PP2. The phosphorylation status of CHC affected the intracellular localization of CHC and the clathrin-dependent endocytosis of transferrin under oxidative stress. In conclusion, CHC is a protein that is phosphorylated at tyrosine by H(2)O(2) and this phosphorylation status is implicated in the intracellular localization and functions of CHC under oxidative stress. The present study demonstrates that oxidative stress affects intracellular vesicular trafficking via the alteration of clathrin-dependent vesicular trafficking.

  5. Phosphorylation of two regulatory tyrosine residues in the activation of Bruton’s tyrosine kinase via alternative receptors

    PubMed Central

    Wahl, Matthew I.; Fluckiger, Anne-Catherine; Kato, Roberta M.; Park, Hyunsun; Witte, Owen N.; Rawlings, David J.

    1997-01-01

    Mutation of Bruton’s tyrosine kinase (Btk) impairs B cell maturation and function and results in a clinical phenotype of X-linked agammaglobulinemia. Activation of Btk correlates with an increase in the phosphorylation of two regulatory Btk tyrosine residues. Y551 (site 1) within the Src homology type 1 (SH1) domain is transphosphorylated by the Src family tyrosine kinases. Y223 (site 2) is an autophosphorylation site within the Btk SH3 domain. Polyclonal, phosphopeptide-specific antibodies were developed to evaluate the phosphorylation of Btk sites 1 and 2. Crosslinking of the B cell antigen receptor (BCR) or the mast cell Fcɛ receptor, or interleukin 5 receptor stimulation each induced rapid phosphorylation at Btk sites 1 and 2 in a tightly coupled manner. Btk molecules were singly and doubly tyrosine-phosphorylated. Phosphorylated Btk comprised only a small fraction (≤5%) of the total pool of Btk molecules in the BCR-activated B cells. Increased dosage of Lyn in B cells augmented BCR-induced phosphorylation at both sites. Kinetic analysis supports a sequential activation mechanism in which individual Btk molecules undergo serial transphosphorylation (site 1) then autophosphorylation (site 2), followed by successive dephosphorylation of site 1 then site 2. The phosphorylation of conserved tyrosine residues within structurally related Tec family kinases is likely to regulate their activation. PMID:9326643

  6. Fluorination of (E)-{beta}-(fluoromethylene)-m-tyrosine: Regioslective synthesis of 4-fluoro-(E)-{beta}-(fluoromethylene)-m-tyrosine

    SciTech Connect

    Lacan, G.; Satyamurthy, N.; Barrio, J.R.

    1995-01-13

    Fluorination of (R)- and (S)-(E)-{beta}-(fluoromethylene)-m-tyrosine (1) by acetyl hypofluorite yielded a mixture of the corresponding 2-fluoro- (2a), 6-fluoro- (2b), 4-fluoro- (2c), and 2,6-difluoro- (2d) derivatives along with a pair of diastereomeric products of addition across the vinylic double bond. A regioselective synthesis of 4-fluoro-(E)-{beta}-(fluoromethylene)-m-tyrosine has also been developed based on a fluorodestannylation reaction with elemental fluorine followed by acid hydrolysis. This reaction sequence yielded minor byproducts, namely, 4-fluoro-(Z)-{beta}(fluoromethylene)-m-tyrosine (11), 4,6-difluoro- and 2,4-difluoro-(E)-{beta}-(fluoromethylene)-m-tyrosines (12a and 12b). All these products were completely separated by semipreparative HPLC and fully characterized by NMR and mass spectroscopy. Single crystal X-ray crystallographic analyses of 2-fluoro-, 4-fluoro-, and 6-fluoro-(E)-{beta}-(fluoromethylene)-m-tyrosines unequivocally established the structures of these amino acids. 1 fig., 3 tabs.

  7. Tryptophan prenyltransferases showing higher catalytic activities for Friedel-Crafts alkylation of o- and m-tyrosines than tyrosine prenyltransferases.

    PubMed

    Fan, Aili; Xie, Xiulan; Li, Shu-Ming

    2015-07-21

    Tryptophan prenyltransferases FgaPT2, 5-DMATS, 6-DMATSSv and 7-DMATS catalyse regiospecific C-prenylations on the indole ring, while tyrosine prenyltransferases SirD and TyrPT catalyse the O-prenylation of the phenolic hydroxyl group. In this study, we report the Friedel-Crafts alkylation of L-o-tyrosine by these enzymes. Surprisingly, no conversion was detected with SirD and three tryptophan prenyltransferases showed significantly higher activity than another tyrosine prenyltransferase TyrPT. C5-prenylated L-o-tyrosine was identified as a unique product of these enzymes. Using L-m-tyrosine as the prenylation substrate, product formation was only observed with the tryptophan prenyltransferases FgaPT2 and 7-DMATS. C4- and C6-prenylated derivatives were identified in the reaction mixture of FgaPT2. These results provided additional evidence for the similarities and differences between these two subgroups within the DMATS superfamily in their catalytic behaviours.

  8. Tryptophan prenyltransferases showing higher catalytic activities for Friedel-Crafts alkylation of o- and m-tyrosines than tyrosine prenyltransferases.

    PubMed

    Fan, Aili; Xie, Xiulan; Li, Shu-Ming

    2015-07-21

    Tryptophan prenyltransferases FgaPT2, 5-DMATS, 6-DMATSSv and 7-DMATS catalyse regiospecific C-prenylations on the indole ring, while tyrosine prenyltransferases SirD and TyrPT catalyse the O-prenylation of the phenolic hydroxyl group. In this study, we report the Friedel-Crafts alkylation of L-o-tyrosine by these enzymes. Surprisingly, no conversion was detected with SirD and three tryptophan prenyltransferases showed significantly higher activity than another tyrosine prenyltransferase TyrPT. C5-prenylated L-o-tyrosine was identified as a unique product of these enzymes. Using L-m-tyrosine as the prenylation substrate, product formation was only observed with the tryptophan prenyltransferases FgaPT2 and 7-DMATS. C4- and C6-prenylated derivatives were identified in the reaction mixture of FgaPT2. These results provided additional evidence for the similarities and differences between these two subgroups within the DMATS superfamily in their catalytic behaviours. PMID:26077893

  9. L-tyrosine administration increases acetylcholinesterase activity in rats.

    PubMed

    Ferreira, Gabriela K; Carvalho-Silva, Milena; Gonçalves, Cinara L; Vieira, Júlia S; Scaini, Giselli; Ghedim, Fernando V; Deroza, Pedro F; Zugno, Alexandra I; Pereira, Talita C B; Oliveira, Giovanna M T; Kist, Luiza W; Bogo, Maurício R; Schuck, Patrícia F; Ferreira, Gustavo C; Streck, Emilio L

    2012-12-01

    Tyrosinemia is a rare genetic disease caused by mutations on genes that codify enzymes responsible for tyrosine metabolism. Considering that tyrosinemics patients usually present symptoms associated with central nervous system alterations that ranges from slight decreases in intelligence to severe mental retardation, we decided to investigate whether acute and chronic administration of L-tyrosine in rats would affect acetylcholinesterase mRNA expression and enzymatic activity during their development. In our acute protocol, Wistar rats (10 and 30 days old) were killed one hour after a single intraperitoneal L-tyrosine injection (500 mg/kg) or saline. Chronic administration consisted of L-tyrosine (500 mg/kg) or saline injections 12 h apart for 24 days in Wistar rats (7 days old) and rats were killed 12 h after last injection. Acetylcholinesterase activity was measured by Ellman's method and acetylcholinesterase expression was carried out by a semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) assay. We observed that acute (10 and 30 days old rats) and chronic L-tyrosine administration increased acetylcholinesterase activity in serum and all tested brain areas (hippocampus, striatum and cerebral cortex) when compared to control group. Moreover, there was a significant decrease in mRNA levels of acetylcholinesterase in hippocampus was observed after acute protocol (10 and 30 days old rats) and in striatum after chronic protocol. In case these alterations also occur in the brain of the patients, our results may explain, at least in part, the neurological sequelae associated with high plasma concentrations of tyrosine seen in patients affected by tyrosinemia type II. PMID:23046746

  10. Metal-catalyzed protein tyrosine nitration in biological systems.

    PubMed

    Campolo, Nicolás; Bartesaghi, Silvina; Radi, Rafael

    2014-11-01

    Protein tyrosine nitration is an oxidative postranslational modification that can affect protein structure and function. It is mediated in vivo by the production of nitric oxide-derived reactive nitrogen species (RNS), including peroxynitrite (ONOO(-)) and nitrogen dioxide ((•)NO₂). Redox-active transition metals such as iron (Fe), copper (Cu), and manganese (Mn) can actively participate in the processes of tyrosine nitration in biological systems, as they catalyze the production of both reactive oxygen species and RNS, enhance nitration yields and provide site-specificity to this process. Early after the discovery that protein tyrosine nitration can occur under biologically relevant conditions, it was shown that some low molecular weight transition-metal centers and metalloproteins could promote peroxynitrite-dependent nitration. Later studies showed that nitration could be achieved by peroxynitrite-independent routes as well, depending on the transition metal-catalyzed oxidation of nitrite (NO₂(-)) to (•)NO₂ in the presence of hydrogen peroxide. Processes like these can be achieved either by hemeperoxidase-dependent reactions or by ferrous and cuprous ions through Fenton-type chemistry. Besides the in vitro evidence, there are now several in vivo studies that support the close relationship between transition metal levels and protein tyrosine nitration. So, the contribution of transition metals to the levels of tyrosine nitrated proteins observed under basal conditions and, specially, in disease states related with high levels of these metal ions, seems to be quite clear. Altogether, current evidence unambiguously supports a central role of transition metals in determining the extent and selectivity of protein tyrosine nitration mediated both by peroxynitrite-dependent and independent mechanisms.

  11. L-tyrosine administration increases acetylcholinesterase activity in rats.

    PubMed

    Ferreira, Gabriela K; Carvalho-Silva, Milena; Gonçalves, Cinara L; Vieira, Júlia S; Scaini, Giselli; Ghedim, Fernando V; Deroza, Pedro F; Zugno, Alexandra I; Pereira, Talita C B; Oliveira, Giovanna M T; Kist, Luiza W; Bogo, Maurício R; Schuck, Patrícia F; Ferreira, Gustavo C; Streck, Emilio L

    2012-12-01

    Tyrosinemia is a rare genetic disease caused by mutations on genes that codify enzymes responsible for tyrosine metabolism. Considering that tyrosinemics patients usually present symptoms associated with central nervous system alterations that ranges from slight decreases in intelligence to severe mental retardation, we decided to investigate whether acute and chronic administration of L-tyrosine in rats would affect acetylcholinesterase mRNA expression and enzymatic activity during their development. In our acute protocol, Wistar rats (10 and 30 days old) were killed one hour after a single intraperitoneal L-tyrosine injection (500 mg/kg) or saline. Chronic administration consisted of L-tyrosine (500 mg/kg) or saline injections 12 h apart for 24 days in Wistar rats (7 days old) and rats were killed 12 h after last injection. Acetylcholinesterase activity was measured by Ellman's method and acetylcholinesterase expression was carried out by a semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) assay. We observed that acute (10 and 30 days old rats) and chronic L-tyrosine administration increased acetylcholinesterase activity in serum and all tested brain areas (hippocampus, striatum and cerebral cortex) when compared to control group. Moreover, there was a significant decrease in mRNA levels of acetylcholinesterase in hippocampus was observed after acute protocol (10 and 30 days old rats) and in striatum after chronic protocol. In case these alterations also occur in the brain of the patients, our results may explain, at least in part, the neurological sequelae associated with high plasma concentrations of tyrosine seen in patients affected by tyrosinemia type II.

  12. Old Tyrosine Kinase Inhibitors and Newcomers in Gastrointestinal Cancer Treatment.

    PubMed

    Erika, Giordani; Federica, Zoratto; Martina, Strudel; Anselmo, Papa; Luigi, Rossi; Marina, Minozzi; Davide, Caruso; Eleonora, Zaccarelli; Monica, Verrico; Silverio, Tomao

    2016-01-01

    Gastrointestinal cancer treatment is based more on molecular biology that has provided increasing knowledge about cancer pathogenesis on which targeted therapy is being developed. Precisely, targeted therapy is defined as a "type of treatment that uses drugs, such as monoclonal antibodies or tyrosine kinase inhibitors, to identify and attack specific cancer cells". Nowadays, the United States Food and Drug Administration has approved many targeted therapies for gastrointestinal cancer treatment, as many are in various phases of development as well. In a previous review we discussed the main monoclonal antibodies used and studied in gastrointestinal cancer. In addition to monoclonal antibodies, tyrosine kinase inhibitors represent another class of targeted therapy and following the approval of imatinib for gastrointestinal stromal tumours, other tyrosine kinase inhibitors have been approved for gastrointestinal cancers treatment such as sunitinib, regoragenib, sorafenib and erlotinib. Moving forward, the purpose of this review is to focus on the efficacy data of main tyrosine kinase inhibitors commonly used in the personalized treatment of each gastrointestinal tumour and to provide a comprehensive overview about experimental targeted therapies ongoing in this setting. PMID:26278713

  13. Interference of peptone and tyrosine with the lignin peroxidase assay.

    PubMed Central

    ten Have, R; Hartmans, S; Field, J A

    1997-01-01

    The N-unregulated white rot fungus Bjerkandera sp. strain BOS55 was cultured in 1 liter of peptone-yeast extract medium to produce lignin peroxidase (LiP). During the LiP assay, the oxidation of veratryl alcohol to veratraldehyde was inhibited due to tyrosine present in the peptone and the yeast extract. PMID:9251220

  14. 21 CFR 862.1730 - Free tyrosine test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Free tyrosine test system. 862.1730 Section 862.1730 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... phenylketonuria (a disease that can cause brain damage). (b) Classification. Class I....

  15. 21 CFR 862.1730 - Free tyrosine test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Free tyrosine test system. 862.1730 Section 862.1730 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... phenylketonuria (a disease that can cause brain damage). (b) Classification. Class I....

  16. 21 CFR 862.1730 - Free tyrosine test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Free tyrosine test system. 862.1730 Section 862.1730 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... phenylketonuria (a disease that can cause brain damage). (b) Classification. Class I....

  17. Prokaryotic expression cloning of a novel human tyrosine kinase

    SciTech Connect

    Beeler, J.F.; LaRochelle, W.J.; Chedid, M.

    1994-02-01

    Screening of a human embryonic lung fibroblast cDNA expression library with antiphosphotyrosine antibodies led to isolation of a novel protein kinase. A clone, designated A6, contained a 3-kb cDNA insert with a predicted open reading frame of 350 amino acids. DNA sequence analysis failed to reveal any detectable similarity with previously known genes, and the predicted A6 protein lacked any of the motifs commonly conserved in the catalytic domains of protein kinases. However, the bacterially expressed {beta}-galactosidase-A6 fusion protein demonstrated both tyrosine and serine phosphorylation in an in vitro kinase assay and phosphorylated exogenous substrates including myelin basic protein specifically on tyrosine residues. The enzyme also displayed biochemical properties analogous to those of other protein tyrosine kinases. The A6 gene was found to be expressed widely at the transcript level in normal tissues and was evolutionarily conserved. Thus, A6 represents a novel tyrosine kinase which is highly divergent from previously described members of this important class of regulatory molecules. 29 refs., 8 figs., 1 tab.

  18. Enhanced photoluminescence by tyrosine-containing bolaamphiphile self-assembly.

    PubMed

    Kwak, Jinyoung; Lee, Sang-Yup

    2013-04-01

    Photoluminescent spherical nanostructures were prepared through the self-assembly of a tyrosine-containing bolaamphiphilic molecule, and their antenna effect was examined. The photoluminescent spherical nanostructures were simply prepared by self-assembly of bolaamphiphile molecules in an aqueous solution in which water-soluble photosensitizers and lanthanide ions were dissolved. The photosensitizers and lanthanide cations were incorporated with the phenol group and the carboxyl end of the tyrosine moiety, respectively. Through fluorescence microscopy and photoluminescence spectroscopy analyses, the various combinations of two lanthanide ions (Eu and Tb) and four photosensitizers were screened for synergetic photoluminescence with bolaamphiphile self-assembly. The bolaamphiphile assembly enhanced the photoluminescence intensity by a factor of around 2 when it was associated with Tb and salicylic acid. This enhancement is driven by the phosphorescence enhancement of the photosensitizer induced by the π-π interactions with the phenol group in tyrosine. These results indicate that the tyrosine-containing bolaamphiphile is a promising molecule that can easily produce a soft nanoscaled host matrix with an antenna effect for photoluminescence.

  19. Old Tyrosine Kinase Inhibitors and Newcomers in Gastrointestinal Cancer Treatment.

    PubMed

    Giordani, Erika; Zoratto, Federica; Strudel, Martina; Papa, Anselmo; Rossi, Luigi; Minozzi, Marina; Caruso, Davide; Zaccarelli, Eleonora; Verrico, Monica; Tomao, Silverio

    2016-01-01

    Gastrointestinal cancer treatment is based more on molecular biology that has provided increasing knowledge about cancer pathogenesis on which targeted therapy is being developed. Precisely, targeted therapy is defined as a "type of treatment that uses drugs, such as monoclonal antibodies or tyrosine kinase inhibitors, to identify and attack specific cancer cells". Nowadays, the United States Food and Drug Administration has approved many targeted therapies for gastrointestinal cancer treatment, as many are in various phases of development as well. In a previous review we discussed the main monoclonal antibodies used and studied in gastrointestinal cancer. In addition to monoclonal antibodies, tyrosine kinase inhibitors represent another class of targeted therapy and following the approval of imatinib for gastrointestinal stromal tumours, other tyrosine kinase inhibitors have been approved for gastrointestinal cancers treatment such as sunitinib, regoragenib, sorafenib and erlotinib. Moving forward, the purpose of this review is to focus on the efficacy data of main tyrosine kinase inhibitors commonly used in the personalized treatment of each gastrointestinal tumour and to provide a comprehensive overview about experimental targeted therapies ongoing in this setting.

  20. Specific dephosphorylation of Janus Kinase 2 by protein tyrosine phosphatases.

    PubMed

    Li, Jianzhuo; Liu, Xidong; Chu, Huiying; Fu, Xueqi; Li, Tianbao; Hu, Lianghai; Xing, Shu; Li, Guohui; Gu, Jingkai; Zhao, Zhizhuang Joe

    2015-01-01

    Many protein kinases are activated through phosphorylation of an activation loop thereby turning on downstream signaling pathways. Activation of JAK2, a nonreceptor tyrosine kinase with an important role in growth factor and cytokine signaling, requires phosphorylation of the 1007 and 1008 tyrosyl residues. Dephosphorylation of these two sites by phosphatases presumably inactivates the enzyme, but the underlying mechanism is not known. In this study, we employed MALDI-TOF/TOF and triple quadrupole mass spectrometers to analyze qualitatively and quantitatively the dephosphorylation process by using synthetic peptides derived from the tandem autophosphorylation sites (Y1007 and Y1008) of human JAK2. We found that tyrosine phosphatases catalyzed the dephosphorylation reaction sequentially, but different enzymes exhibited different selectivity. Protein tyrosine phosphatase 1B caused rapid dephosphorylation of Y1008 followed by Y1007, while SHP1 and SHP2 selectively dephosphorylated Y1008 only, and yet HePTP randomly removed a single phosphate from either Y1007 or Y1008, leaving behind mono-phosphorylated peptides. The specificity of dephosphorylation was further confirmed by molecular modeling. The data reveal multiple modes of JAK2 regulation by tyrosine phosphatases, reflecting a complex, and intricate interplay between protein phosphorylation and dephosphorylation.

  1. 21 CFR 862.1730 - Free tyrosine test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Free tyrosine test system. 862.1730 Section 862.1730 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... phenylketonuria (a disease that can cause brain damage). (b) Classification. Class I....

  2. 21 CFR 862.1730 - Free tyrosine test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Free tyrosine test system. 862.1730 Section 862.1730 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... phenylketonuria (a disease that can cause brain damage). (b) Classification. Class I....

  3. Effects of cytotoxin-associated gene A (CagA) positive Helicobacter pylori infection on anti-platelet glycoprotein antibody producing B cells in patients with primary idiopathic thrombocytopenic purpura (ITP)

    PubMed Central

    Cheng, Yuan-Shan; Kuang, Li-Ping; Zhuang, Chun-Lan; Jiang, Jia-Dian; Shi, Man

    2015-01-01

    Objective: To explore the effects of cytotoxin-associated gene A (CagA) positive Helicobacter pylori (H. pylori or HP) infection on circulating B cells producing specific platelet glycoprotein antibodies and the association between therapeutic outcomes in primary idiopathic thrombocytopenic purpura (ITP) patients. Methods: A total of 76 newly diagnosed primary ITP patients were included in the study which was conducted at the first affiliated hospital of Shantou University Medical college, in Shantou city China, between January 2013 and January 2014. These patients were tested for H. pylori infection by 13C urea breath test and for anti-CagA antibody in H. pylori positive cases by enzyme-linked immunosorbent assay (ELISA) method. Anti-GPIb and anti-GPIIb/IIIa antibody-producing B cells were measured using an enzyme-linked immunospot (ELISPOT) assay in all ITP patients and 30 controls. Anti-nuclear antibody (ANA) was also detected in ITP patients. Results: The numbers of anti-GPIIb/IIIa antibody-producing B cells in HP+CagA+ patients were higher than in HP+CagA- or HP- patients. However, anti-GPIb antibody-producing B cells were found higher in HP- patients. Analysis of treatment outcomes showed that a therapeutic response was more likely in patients presenting anti-GPIIb/IIIa B cells, but the poor response was found to be associated with anti-GPIb B cells and ANA presences. Conclusion: CagA antigen of H. pylori may induce anti-GPIIb/IIIa antibodies production by a molecular mimicry mechanism. Anti-GPIIb/IIIa and anti-GPIb antibody producing B Cells detection is useful for predicting treatment effects of primary ITP. PMID:25878627

  4. Helicobacter pylori isolated from Iranian drinking water: vacA, cagA, iceA, oipA and babA2 genotype status and antimicrobial resistance properties.

    PubMed

    Ranjbar, Reza; Khamesipour, Faham; Jonaidi-Jafari, Nematollah; Rahimi, Ebrahim

    2016-05-01

    Despite the clinical importance of Helicobacter pylori in human gastric disorders, its exact route of transmission is still uncertain. Based on the contentious hypothesis and findings of previous investigations, water may play an important role in the transmission of H. pylori to humans. This study was carried out to investigate the vacA, cagA, oipA, iceA and babA2 genotype status and antimicrobial resistance properties of H. pylori strains isolated from the drinking water samples of four major provinces in Iran. A total of 400 drinking water samples were cultured and tested. H. pylori-positive strains were analyzed for the presence of various genotypes and antimicrobial resistance. Twelve of 400 (3%) water samples were positive for H. pylori. Samples from Isfahan province had the highest, while those from Shiraz had the lowest prevalence of H. pylori. The seasonal distribution was also determined, with the highest prevalence of bacteria in the summer season (7.36%). H. pylori strains harbored the highest levels of resistance against ampicillin (100%), erythromycin (75%), clarithromycin (75%), and trimethoprim (58.3%). The most commonly detected genotypes were vacAs1a (83.3%), vacAm1a (66.6%), vacAs2 (50%) and cagA (50%). The presence of similar genotypes in the H. pylori strains of drinking water and those of human clinical samples suggest that contaminated water maybe the sources of bacteria. Spiramycin and furazolidone are suggested for the treatment of cases of H. pylori infection.

  5. Helicobacter pylori isolated from Iranian drinking water: vacA, cagA, iceA, oipA and babA2 genotype status and antimicrobial resistance properties.

    PubMed

    Ranjbar, Reza; Khamesipour, Faham; Jonaidi-Jafari, Nematollah; Rahimi, Ebrahim

    2016-05-01

    Despite the clinical importance of Helicobacter pylori in human gastric disorders, its exact route of transmission is still uncertain. Based on the contentious hypothesis and findings of previous investigations, water may play an important role in the transmission of H. pylori to humans. This study was carried out to investigate the vacA, cagA, oipA, iceA and babA2 genotype status and antimicrobial resistance properties of H. pylori strains isolated from the drinking water samples of four major provinces in Iran. A total of 400 drinking water samples were cultured and tested. H. pylori-positive strains were analyzed for the presence of various genotypes and antimicrobial resistance. Twelve of 400 (3%) water samples were positive for H. pylori. Samples from Isfahan province had the highest, while those from Shiraz had the lowest prevalence of H. pylori. The seasonal distribution was also determined, with the highest prevalence of bacteria in the summer season (7.36%). H. pylori strains harbored the highest levels of resistance against ampicillin (100%), erythromycin (75%), clarithromycin (75%), and trimethoprim (58.3%). The most commonly detected genotypes were vacAs1a (83.3%), vacAm1a (66.6%), vacAs2 (50%) and cagA (50%). The presence of similar genotypes in the H. pylori strains of drinking water and those of human clinical samples suggest that contaminated water maybe the sources of bacteria. Spiramycin and furazolidone are suggested for the treatment of cases of H. pylori infection. PMID:27419049

  6. Asymmetric Tyrosine Kinase Arrangements in Activation or Autophosphorylation of Receptor Tyrosine Kinases

    SciTech Connect

    J Bae; J Schlessinger

    2011-12-31

    Receptor tyrosine kinases (RTKs) play important roles in the control of many cellular processes including cell proliferation, cell adhesion, angiogenesis, and apoptosis. Ligand-induced dimerization of RTKs leads to autophosphorylation and activation of RTKs. Structural studies have shown that while isolated ectodomains of several RTKs form symmetric dimers the isolated cytoplasmic kinase domains of epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR) form asymmetric dimers during their activation. Binding of one kinase molecule of EGFR to a second kinase molecule asymmetrically leads to stimulation of kinase activity and enhanced autophosphorylation. Furthermore, the structures of the kinase domain of FGFR1 and FGFR2 reveal the formation of asymmetric interfaces in the processes of autophosphorylation at their specific phosphotyrosine (pY) sites. Disruption of asymmetric dimer interface of EGFR leads to reduction in enzymatic activity and drastic reduction of autophosphorylation of FGFRs in ligandstimulated live cells. These studies demonstrate that asymmetric dimer formation is as a common phenomenon critical for activation and autophosphorylation of RTKs.

  7. Effects of tyrosine-26 and tyrosine-64 nitration on the photoreactions of bacteriorhodopsin

    NASA Technical Reports Server (NTRS)

    Scherrer, P.; Stoeckenius, W.

    1985-01-01

    The photoreactions of nitrated bacteriorhodopsin (bR) are examined. Flash-induced difference spectra of bR, bR with aminotyrosine in position 26 (bR-N26R) and bR with aminotyrosine in position 64 are analyzed. It is observed that changes in the actinic wavelength (from 520 to 500 or 580 nm) have no affect on the shape of the spectra and the formation and decay kinetics of the O and M intermediates. Nitration of tyrosine-64 decreases the chromophore absorbance, shifts the absorption maximum to 535 nm, and affects photocycle kinetics independent of the pK of its phenolic group. Light-dark adaptation spectra for bR are studied. The kinetics of the M and O intermediates in bR with nitrotyrosine in position 64 (bR-N64) and bR with aminotyrosine in position 64 and bR with nitrotyrosine in position 26 and bR-N26R are described and compared to bR; the pH dependence and M and O decay rates are considered. The deprotonation of bR-N64 during the photoreaction cycle and the effects of nitration on the activity of proton pumping are investigated.

  8. Protein (tyrosine)-chromophore (protonated Schiff base) coupling in bacteriorhodopsin

    SciTech Connect

    Hanamoto, J.H.; Dupuis, P.; El-Sayed, M.A.

    1984-11-01

    The kinetics of formation of both the tyrosinate ion (from its absorption at 296 nm) and the deprotonated Schiff base (M/sub 412/) (from its absorption at 404 nm) are studied simultaneously at different pH values (7-11) and temperatures (5-25/sup 0/C). Two formation rates are observed for M/sub 412/ in agreement with previous observations. The slow one is dominant under physiological conditions and is found to be slightly faster than that for the tyrosinate formation. This is in disagreement with the proposal that the tyrosinate formation is a prerequisite to the deprotonation of the Schiff base (M/sub 412/). The ratio of the amplitudes of the fast and slow components is found to be sensitive to pH and, at any pH, it can be used to calculate an amino acid pK/sub a/ value of 9.6. This is explained by proposing the existence of two sites for the protonated Schiff base within the protein. In one site, the Schiff base is near the neutral form of an amino acid residue with a pK/sub a/ value of 9.6 (giving rise to the slow component), while in the other, it is near its conjugate base. The formation of the tyrosinate ion as well as the formation of the slow and fast components of M/sub 412/ all have activation energies that are comparable to H-bond energies. A model is suggested to account for this and the comparable deprotonation rates of tyrosine and the slow component of the protonated Schiff base. It involves the reduction of their pK/sub a/ by their exposure to a positively charged species. 43 references, 2 figures, 2 tables.

  9. Biochemistry of primary headaches: role of tyrosine and tryptophan metabolism.

    PubMed

    D'Andrea, G; Cevoli, S; Colavito, D; Leon, A

    2015-05-01

    The pathogenesis of migraine as well as cluster headache (CH) is yet a debated question. In this review, we discuss the possible role of the of tyrosine and tryptophan metabolism in the pathogenesis of these primary headaches. These include the abnormalities in the synthesis of neurotransmitters: high level of DA, low level of NE and very elevated levels of octopamine and synephrine (neuromodulators) in plasma of episodic migraine without aura and CH patients. We hypothesize that the imbalance between the levels of neurotransmitters and elusive amines synthesis is due to a metabolic shift directing tyrosine toward an increased decarboxylase and reduced hydroxylase enzyme activities. The metabolic shift of the tyrosine is favored by a state of neuronal hyperexcitability and a reduced mitochondrial activity present in migraine. In addition we present biochemical studies performed in chronic migraine and chronic tension-type headache patients to verify if the same anomalies of the tyrosine and tryptophan metabolism are present in these primary headaches and, if so, their possible role in the chronicity process of CM and CTTH. The results show that important abnormalities of tyrosine metabolism are present only in CM patients (very high plasma levels of DA, NE and tryptamine). Tryptamine plasma levels were found significantly lower in both CM and CTTH patients. In view of this, we propose that migraine and, possibly, CH attacks derive from neurotransmitter and neuromodulator metabolic abnormalities in a hyperexcitable and hypoenergetic brain that spread from the frontal lobe, downstream, resulting in abnormally activated nuclei of the pain matrix. The low tryptamine plasma levels found in CM and CTTH patients suggest that these two primary chronic headaches are characterized by a common insufficient serotoninergic control of the pain threshold.

  10. Protein tyrosine phosphatase regulation of endothelial cell apoptosis and differentiation.

    PubMed

    Yang, C; Chang, J; Gorospe, M; Passaniti, A

    1996-02-01

    Apoptosis, or programmed cell death, occurs during development and may also be an important factor in many diseases. However, little is known about the signal transduction pathways regulating apoptosis. In these studies, loss of endothelial cell-substrate attachment and apoptosis after removal of growth factors was associated with dephosphorylation of tyrosine residues at the cell periphery. Dephosphorylation of total cellular proteins accompanied apoptosis and was reduced by orthovanadate, an inhibitor of protein tyrosine phosphatases. Orthovanadate blocked the fragmentation of nuclear DNA, inhibited DNA laddering, and suppressed the expression of TRPM-2, an apoptosis-associated gene. The tyrosine phosphorylation levels of FAK125, erk1 (mitogen-activated kinase kinase), and cdc-2 were reduced during apoptosis. FAK125 dephosphorylation was inhibited by orthovanadate, but premature activation (tyrosine dephosphorylation) of cdc-2 was not. Orthovanadate was as effective as basic fibroblast growth factor in activating erk1 without increasing cell proliferation and in preventing the apoptosis of endothelial cells after treatment with tumor necrosis factor alpha. Endothelial cell differentiation on extracellular matrix (Matrigel) was also stimulated by orthovanadate in the absence of basic fibroblast growth factor without affecting growth arrest and inhibition of DNA synthesis. Expression of the cyclin-dependent kinase inhibitor p21 (Waf1/Cip1/Sdi1) was down-regulated during the early stages of differentiation, remained low for at least 6 hours as differentiation proceeded, and increased upon completion of differentiation. Cells that failed to down-regulate p21 mRNA on Matrigel in the absence of angiogenic factors underwent apoptosis. These results suggest that protein tyrosine phosphatases are actively involved in signal transduction during apoptosis and may regulate p21 expression to inhibit endothelial cell differentiation.

  11. Integrin Ligation Results in Nephrin Tyrosine Phosphorylation In Vitro

    PubMed Central

    Verma, Rakesh; Venkatareddy, Madhusudan; Kalinowski, Anne; Patel, Sanjeevkumar R.; Garg, Puneet

    2016-01-01

    Nephrin is expressed at the basolateral aspect of podocytes and is an important signaling protein at the glomerular slit diaphragm. In vitro studies have demonstrated that Nephrin phosphorylation-dependent signaling is able to assemble a protein complex that is able to polymerize actin. However, proximal signaling events that result in nephrin tyrosine phosphorylation are not well understood. Nephrin deletion in mice and human nephrin mutations result in developmental failure of the podocyte intercellular junction resutling in proteinuria. This has been presumed to be due to a failure to respond to an external polarized cue in the absence of nephrin or a failure to transduce an outside-in signal in patients with nephrin mutations. The nephrin extracellular domain binds to itself or neph1 across the foot process intercellular junction. Nephrin is tyrosine phosphorylation-silent in healthy glomeruli when presumably the nephrin extracellular domain is in an engaged state. These observations raise the possibility of an alternate proximal signaling mechanism that might be responsible for nephrin tyrosine phosphorylation. Here we present data showing that integrin engagement at the basal aspect of cultured podocytes results in nephrin tyrosine phosphorylation. This is abrogated by incubating podocytes with an antibody that prevents integrin β1 ligation and activation in response to binding to extracellular matrix. Furthermore, nephrin tyrosine phosphorylation was observed in podocytes expressing a membrane-targeted nephrin construct that lacks the extracellular domain. We propose, integrin-activation based signaling might be responsible for nephrin phosphorylation rather than engagment of the nephrin extracellular domain by a ligand. PMID:26848974

  12. Regulation and function of syk tyrosine kinase in mast cell signaling and beyond.

    PubMed

    de Castro, Rodrigo Orlandini

    2011-01-01

    The protein tyrosine kinase Syk plays a critical role in FcεRI signaling in mast cells. Binding of Syk to phosphorylated immunoreceptor tyrosine-based activation motifs (p-ITAM) of the receptor subunits results in conformational changes and tyrosine phosphorylation at multiple sites that leads to activation of Syk. The phosphorylated tyrosines throughout the molecule play an important role in the regulation of Syk-mediated signaling. Reconstitution of receptor-mediated signaling in Syk(-/-) cells by wild-type Syk or mutants which have substitution of these tyrosines with phenylalanine together with in vitro assays has been useful strategies to understand the regulation and function of Syk.

  13. Subcutaneous L-tyrosine elicits cutaneous analgesia in response to local skin pinprick in rats.

    PubMed

    Hung, Ching-Hsia; Chiu, Chong-Chi; Liu, Kuo-Sheng; Chen, Yu-Wen; Wang, Jhi-Joung

    2015-10-15

    The purpose of the study was to estimate the ability of L-tyrosine to induce cutaneous analgesia and to investigate the interaction between L-tyrosine and the local anesthetic lidocaine. After subcutaneously injecting the rats with L-tyrosine and lidocaine in a dose-dependent manner, cutaneous analgesia (by blocking the cutaneous trunci muscle reflex-CTMR) was evaluated in response to the local pinprick. The drug-drug interaction was analyzed by using an isobolographic method. We showed that both L-tyrosine and lidocaine produced dose-dependent cutaneous analgesia. On the 50% effective dose (ED50) basis, the rank of drug potency was lidocaine (5.09 [4.88-5.38] μmol)>L-tyrosine (39.1 [36.5-41.8] μmol) (P<0.05). At the equipotent doses (ED25, ED50, and ED75), the duration of cutaneous analgesia caused by L-tyrosine lasted longer than that caused by lidocaine (P<0.01). Lidocaine co-administered with L-tyrosine exhibited an additive effect on infiltrative cutaneous analgesia. Our pre-clinical study demonstrated that L-tyrosine elicits the local/cutaneous analgesia, and the interaction between L-tyrosine and lidocaine is additive. L-tyrosine has a lower potency but much greater duration of cutaneous analgesia than lidocaine. Adding L-tyrosine to lidocaine preparations showed greater duration of cutaneous analgesia compared with lidocaine alone. PMID:26376025

  14. Unexpected differences between D- and L- tyrosine lead to chiral enhancement in racemic mixtures.

    PubMed

    Shinitzky, Meir; Nudelman, Fabio; Barda, Yaniv; Haimovitz, Rachel; Chen, Effie; Deamer, David W

    2002-08-01

    We report here an unexpected difference in the solubilities of D- and L-tyrosine in water, which could be discerned by their rate of crystallization and the resulting concentrations of their saturated solutions. A supersaturated solution of 10 mM L-tyrosine at 20 degrees C crystallized much more slowly than that of D-tyrosine under the same conditions, and the saturated solution of L-tyrosine was more concentrated than that of D-tyrosine. Supersaturated solutions of 10 mM DL-tyrosine in water formed precipitates of predominantly D-tyrosine and DL-tyrosine, resulting in an excess of L-tyrosine in the saturated solution. The experimental setups were monitored independently by UV-absorption, radioactivity tracing, optical rotation and X-ray diffraction. The process of nucleation and crystallization of D- and L-tyrosine is characterized by an exceptionally high cooperativity. It is possible that minute energy differences between D- and L-tyrosine, originating from parity violation or other non-conservative chiral discriminatory rules, could account for the observations. The physical process that initiated chiral selection in biological systems remains a challenging problem in understanding the origin of life, and it is possible that chiral compounds were concentrated from supersaturated racemic mixtures by preferential crystallization.

  15. Behavioral and cognitive effects of tyrosine intake in healthy human adults.

    PubMed

    Hase, Adrian; Jung, Sophie E; aan het Rot, Marije

    2015-06-01

    The amino acid tyrosine is the precursor to the catecholamine neurotransmitters dopamine and norepinephrine. Increasing tyrosine uptake may positively influence catecholamine-related psychological functioning. We conducted a systematic review to examine the effects of tyrosine on behavior and cognition. Fifteen studies were reviewed. All studies except one involved tyrosine loading during a single test session. In most behavioral studies, there were no significant effects of tyrosine on exercise performance. In contrast, cognitive studies employing neuropsychological measures found that tyrosine loading acutely counteracts decrements in working memory and information processing that are induced by demanding situational conditions such as extreme weather or cognitive load. The buffering effects of tyrosine on cognition may be explained by tyrosine's ability to neutralize depleted brain catecholamine levels. There is evidence that tyrosine may benefit healthy individuals exposed to demanding situational conditions. For future research we recommend moving from studying the acute effects of a single tyrosine load in small samples to studying the behavioral and cognitive effects of tyrosine in larger groups over multiple weeks.

  16. meta-Tyrosine in Festuca rubra ssp. commutata (Chewings fescue) is synthesized by hydroxylation of phenylalanine.

    PubMed

    Huang, Tengfang; Rehak, Ludmila; Jander, Georg

    2012-03-01

    m-Tyrosine is a non-protein amino acid that is structurally similar to the common protein amino acids p-tyrosine and phenylalanine. Copious amounts of m-tyrosine can be found in root exudates of the fine fescue cultivar, Festuca rubra L. ssp. commutata (Chewings fescue). The phytotoxicity of m-tyrosine may contribute to the allelopathic potential of F. rubra. m-Tyrosine in Euphorbia myrsinites (donkey-tail spurge), was previously shown to be synthesized via transamination of m-hydroxyphenylpyruvate. Here we show that m-tyrosine biosynthesis in F. rubra occurs through direct hydroxylation of phenylalanine in the root tips, perhaps through the activity of a cytochrome P450 enzyme. Hence, E. myrsinites and F. rubra, the only two plant species known to produce m-tyrosine, use distinct biosynthetic pathways that likely arose independently in evolutionary history.

  17. Purification and characterization of tyrosine phenol lyase from Citrobacter freundii.

    PubMed

    Chandel, Meenakshi; Azmi, Wamik

    2013-12-01

    The purification and characterization of intracellular tyrosine phenol lyase from Citrobacter freundii has been carried out. The enzyme was purified 35-fold to homogeneity by ammonium sulphate precipitation and hydrophobic interaction chromatography. Its subunit molecular weight was found to be 52 kDa on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The purified tyrosine phenol lyase showed maximum activity in borate buffer (0.05 M at pH 8.5) at 45 °C after 20 min of incubation. The Km and Vmax values of purified enzyme were found to be 0.446 mm and 0.342 mM/min/mg. This enzyme exhibits t1/2 of 10, 52 and 130 min at 55, 45 and 35 °C, respectively. The N-terminal amino acid sequence was determined as MET-ASN-TYR-PRO-ALA-GLU-PRO-PHE-ARG-ILETRP- TRP-VAL-GLY.

  18. Tailoring Tyrosine Kinase Inhibitors to Fit the Lung Cancer Genome

    PubMed Central

    Looyenga, Brendan D; Cherni, Irene; MacKeigan, Jeffrey P; Weiss, Glen J

    2011-01-01

    Tyrosine kinase inhibitors (TKIs) have been in use as cancer therapeutics for nearly a decade, and their utility in targeting specific malignancies with defined genetic lesions has proven to be remarkably effective. Recent efforts to characterize the spectrum of genetic lesions found in non-small cell lung carcinoma (NSCLC) have provided important insights into the molecular basis of this disease and have also revealed a wide array of tyrosine kinases that might be effectively targeted for rationally designed therapies. The findings of these studies, however, also provide a cautionary tale about the limitations of single-agent therapies, which fail to account for the genetic heterogeneity and pathway redundancy that characterize advanced NSCLC. Emergence of drug resistance mechanisms to specific TKIs, such as gefitinib and erlotinib, suggests that more sophisticated chemotherapeutic paradigms that target multiple pathways at the same time will be required to effectively treat this disease. PMID:21461169

  19. Oxidation of Tyrosine-Phosphopeptides by Titanium Dioxide Photocatalysis.

    PubMed

    Ruokolainen, Miina; Ollikainen, Elisa; Sikanen, Tiina; Kotiaho, Tapio; Kostiainen, Risto

    2016-06-22

    Protein phosphorylation has a key role in cell regulation. Oxidation of proteins, in turn, is related to many diseases and to aging, but the effects of phosphorylation on the oxidation of proteins and peptides have been rarely studied. The aim of this study was to examine the mechanistic effect of phosphorylation on peptide oxidation induced by titanium dioxide photocatalysis. The effect of phosphorylation was compared between nonphosphorylated and tyrosine phosphorylated peptides using electrospray tandem mass spectrometry. We observed that tyrosine was the most preferentially oxidized amino acid, but the oxidation reaction was significantly inhibited by its phosphorylation. The study also shows that titanium dioxide photocatalysis provides a fast and easy method to study oxidation reactions of biomolecules, such as peptides.

  20. Glucose-mediated tyrosine nitration in adipocytes: Targets and consequences

    PubMed Central

    Koeck, Thomas; Willard, Belinda; Crabb, John W.; Kinter, Mike; Stuehr, Dennis J.; Aulak, Kulwant S.

    2010-01-01

    Hyperglycemia, a key factor in insulin resistance and diabetic pathology, is associated with cellular oxidative stress that promotes oxidative protein modifications. We report that protein nitration is responsive to changes in glucose concentrations in 3T3-L1 adipocytes. Alterations in the extent of tyrosine nitration as well as the cellular nitroproteome profile correlated tightly with changing glucose concentrations. The target proteins we identified are involved in fatty acid binding, cell signaling, protein folding, energy metabolism, antioxidant capacity, and membrane permeability. The nitration of adipocyte fatty acid binding protein (FABP4) at Tyr19 decreases, similar to phosphorylation, the binding of palmitic acid to the fatty acid-free protein. This potentially alters intracellular fatty acid transport, nuclear translocation of FABP4, and agonism of PPAR gamma. Our results suggest that protein tyrosine nitration may be a factor in obesity, insulin resistance, and the pathogenesis of diabetes. PMID:19135148

  1. Nitric oxide rapidly scavenges tyrosine and tryptophan radicals.

    PubMed Central

    Eiserich, J P; Butler, J; van der Vliet, A; Cross, C E; Halliwell, B

    1995-01-01

    By utilizing a pulse-radiolytic technique, we demonstrate for the first time that the rate constant for the reaction of nitric oxide (.NO) with biologically relevant tyrosine and tryptophan radicals (Tyr. and Trp. respectively) in amino acids, peptides and proteins is of the order of (1-2) x 10(9) M-1.s-1. We also show that .NO effectively interferes with electron-transfer processes between tryptophan and tyrosine residues in proteins subjected to pulse radiolysis. The near diffusion-controlled rates of these reactions, coupled with the increasingly recognized role of protein radicals in enzyme catalysis and oxidative damage, suggest that Tyr. and Trp. are likely and important targets for .NO generated in vivo. PMID:7575405

  2. The role of nitisinone in tyrosine pathway disorders.

    PubMed

    Lock, Edward; Ranganath, Lakshminarayan R; Timmis, Oliver

    2014-11-01

    Nitisinone 2-(2-nitro-4-trifluoromethylbenzoyl)cyclohexane-1,3-dione (NTBC), an effective herbicide, is the licensed treatment for the human condition, hereditary tyrosinaemia type 1 (HT-1). Its mode of action interrupts tyrosine metabolism through inhibition of 4-hydroxyphenylpyruvate dioxygenase (HPPD). Nitisinone is a remarkable safe drug to use with few side effects reported. Therefore, we propose that it should be investigated as a potential treatment for other disorders of tyrosine metabolism. These include alkaptonuria (AKU), a rare disease resulting is severe, early-onset osteoarthritis. We present a case study from the disease, and attempts to use the drug both off-label and in clinical research through the DevelopAKUre consortium.

  3. Targeting Angiogenesis in Colorectal Cancer: Tyrosine Kinase Inhibitors.

    PubMed

    Kircher, Sheetal Mehta; Nimeiri, Halla S; Benson, Al B

    2016-01-01

    Colorectal cancer is commonly diagnosed throughout the world, and treatment options have greatly expanded over the last 2 decades. Targeting angiogenesis has been a major focus of study in a variety of malignancy types. Targeting angiogenesis has been achieved by several mechanisms in colorectal cancer, including use of antiangiogenic small molecule tyrosine kinase inhibitors (TKIs). There have been many attempts and failures to prove efficacy of TKIs in the treatment of colorectal cancer including sorafenib, sunitinib, vatalanib, and tivozanib. Regorafenib was the first TKI to demonstrate efficacy and is an orally active inhibitor of angiogenic (including the vascular endothelial growth factor receptors 1, 2, and 3), stromal, and oncogenic receptor tyrosine kinases. There are ongoing investigations of both regorafenib and ninetanib; however, there remains a critical need to better understand novel combinations with TKIs that could prove more efficacious than available options. PMID:27341596

  4. Reaction of tyrosine oxidation products with proteins of the lens

    PubMed Central

    Pirie, Antoinette

    1968-01-01

    Oxidation of tyrosine in the presence of bovine lens proteins leads to the formation of brown or black melanoproteins. Both tyrosinase and the oxidizing system of ferrous sulphate–ascorbic acid–EDTA are effective. The fluorescence of the lens proteins is both altered and enhanced by the tyrosine-oxidizing systems. Their fluorescence spectra resemble those of urea-insoluble proteins of human cataractous lens and of 1,2-naphthaquinone–proteins of naphthalene cataract. The lens proteins lose their thiol groups and, in acid hydrolysates of treated β-and γ-crystallins, a substance has been detected chromatographically that behaves similarly to a compound formed when 3,4-dihydroxyphenylalanine (dopa) is oxidized by tyrosinase in the presence of cysteine. Analysis and behaviour of this substance from hydrolysates of lens proteins suggest that it is a compound of cysteine and dopa. PMID:4971287

  5. Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding

    PubMed Central

    Ting, Pamela Y.; Johnson, Christian W.; Fang, Cong; Cao, Xiaoqing; Graeber, Thomas G.; Mattos, Carla; Colicelli, John

    2015-01-01

    RAS proteins are signal transduction gatekeepers that mediate cell growth, survival, and differentiation through interactions with multiple effector proteins. The RAS effector RAS- and RAB-interacting protein 1 (RIN1) activates its own downstream effectors, the small GTPase RAB5 and the tyrosine kinase Abelson tyrosine-protein kinase (ABL), to modulate endocytosis and cytoskeleton remodeling. To identify ABL substrates downstream of RAS-to-RIN1 signaling, we examined human HEK293T cells overexpressing components of this pathway. Proteomic analysis revealed several novel phosphotyrosine peptides, including Harvey rat sarcoma oncogene (HRAS)-pTyr137. Here we report that ABL phosphorylates tyrosine 137 of H-, K-, and NRAS. Increased RIN1 levels enhanced HRAS-Tyr137 phosphorylation by nearly 5-fold, suggesting that RAS-stimulated RIN1 can drive ABL-mediated RAS modification in a feedback circuit. Tyr137 is well conserved among RAS orthologs and is part of a transprotein H-bond network. Crystal structures of HRASY137F and HRASY137E revealed conformation changes radiating from the mutated residue. Although consistent with Tyr137 participation in allosteric control of HRAS function, the mutations did not alter intrinsic GTP hydrolysis rates in vitro. HRAS-Tyr137 phosphorylation enhanced HRAS signaling capacity in cells, however, as reflected by a 4-fold increase in the association of phosphorylated HRASG12V with its effector protein RAF proto-oncogene serine/threonine protein kinase 1 (RAF1). These data suggest that RAS phosphorylation at Tyr137 allosterically alters protein conformation and effector binding, providing a mechanism for effector-initiated modulation of RAS signaling.—Ting, P. Y., Johnson, C. W., Fang, C., Cao, X., Graeber, T. G., Mattos, C., Colicelli, J. Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding. PMID:25999467

  6. Comparative Analysis of Mutant Tyrosine Kinase Chemical Rescue†

    PubMed Central

    Muratore, Kathryn E.; Seeliger, Markus A.; Wang, Zhihong; Fomina, Dina; Neiswinger, Johnathan; Havranek, James J.; Baker, David; Kuriyan, John; Cole, Philip A.

    2009-01-01

    Protein tyrosine kinases are critical cell signaling enzymes. These enzymes have a highly conserved Arg residue in their catalytic loop which is present two residues or four residues downstream from an absolutely conserved Asp catalytic base. Prior studies on protein tyrosine kinases Csk and Src revealed the potential for chemical rescue of catalytically-deficient mutant kinases (Arg to Ala mutations) by small diamino compounds, particularly imidazole, however the potency and efficiency of rescue was greater for Src. This current study further examines the structural and kinetic basis of rescue for mutant Src as compared to mutant Abl tyrosine kinase. An X-ray crystal structure of R388A Src revealed the surprising finding that a histidine residue of the N-terminus of a symmetry-related kinase inserts into the active site of the adjacent Src and mimics the hydrogen bonding pattern seen in wild-type protein tyrosine kinases. Abl R367A shows potent and efficient rescue more comparable to Src, even though its catalytic loop is more like that of Csk. Various enzyme redesigns of the active sites indicate that the degree and specificity of rescue is somewhat flexible, but the overall properties of the enzymes and rescue agents play an overarching role. The newly discovered rescue agent 2-aminoimidazole is about as efficient as imidazole in rescuing R/A Src and Abl. Rate vs. pH studies with these imidazole analogs suggest that the protonated imidazolium is the preferred form for chemical rescue, consistent with structural models. The efficient rescue seen with mutant Abl points to the potential of this approach to be used effectively to analyze Abl phosphorylation pathways in cells. PMID:19260709

  7. Characterization of Phospho-(Tyrosine)-Mimetic Calmodulin Mutants

    PubMed Central

    Stateva, Silviya R.; Salas, Valentina; Benaim, Gustavo; Menéndez, Margarita; Solís, Dolores; Villalobo, Antonio

    2015-01-01

    Calmodulin (CaM) phosphorylated at different serine/threonine and tyrosine residues is known to exert differential regulatory effects on a variety of CaM-binding enzymes as compared to non-phosphorylated CaM. In this report we describe the preparation and characterization of a series of phospho-(Y)-mimetic CaM mutants in which either one or the two tyrosine residues present in CaM (Y99 and Y138) were substituted to aspartic acid or glutamic acid. It was expected that the negative charge of the respective carboxyl group of these amino acids mimics the negative charge of phosphate and reproduce the effects that distinct phospho-(Y)-CaM species may have on target proteins. We describe some physicochemical properties of these CaM mutants as compared to wild type CaM, after their expression in Escherichia coli and purification to homogeneity, including: i) changes in their electrophoretic mobility in the absence and presence of Ca2+; ii) ultraviolet (UV) light absorption spectra, far- and near-UV circular dichroism data; iii) thermal stability in the absence and presence of Ca2+; and iv) Tb3+-emitted fluorescence upon tyrosine excitation. We also describe some biochemical properties of these CaM mutants, such as their differential phosphorylation by the tyrosine kinase c-Src, and their action as compared to wild type CaM, on the activity of two CaM-dependent enzymes: cyclic nucleotide phosphodiesterase 1 (PDE1) and endothelial nitric oxide synthase (eNOS) assayed in vitro. PMID:25830911

  8. Characterization of a PRL protein tyrosine phosphatase from Plasmodium falciparum.

    PubMed

    Pendyala, Prakash Rao; Ayong, Lawrence; Eatrides, Jennifer; Schreiber, Melissa; Pham, Connie; Chakrabarti, Ratna; Fidock, David A; Allen, Charles M; Chakrabarti, Debopam

    2008-03-01

    Isoprenylated proteins have important functions in cell growth and differentiation of eukaryotic cells. Inhibitors of protein prenylation in malaria have recently shown strong promise as effective antimalarials. In studying protein prenylation in the malaria protozoan parasite Plasmodium falciparum, we have shown earlier that the incubation of P. falciparum cells with (3)H-prenol precursors resulted in various size classes of labeled proteins. To understand the physiological function of prenylated proteins of malaria parasites, that are targets of prenyltransferase inhibitors, we searched the PlasmoDB database for proteins containing the C-terminus prenylation motif. We have identified, among other potentially prenylated proteins, an orthologue of a PRL (protein of regenerating liver) subgroup protein tyrosine phosphatases, termed PfPRL. Here, we show that PfPRL is expressed in the parasite's intraerythrocytic stages, where it partially associates with endoplasmic reticulum and within a subcompartment of the food vacuole. Additionally, PfPRL targeting parallels that of apical membrane antigen-1 in developing merozoites. Recombinant PfPRL shows phosphatase activity that is preferentially inhibited by a tyrosine phosphatase inhibitor suggesting that PfPRL functions as a tyrosine phosphatase. Recombinant PfPRL can also be farnesylated in vitro. Inhibition of malarial farnesyltransferase activity can be achieved with the heptapetide RKCHFM, which corresponds to the C-terminus of PfPRL. This study provides the first evidence for expression of enzymatically active PRL-related protein tyrosine phosphatases in malarial parasites, and demonstrates the potential of peptides derived from Plasmodium prenylated proteins as malarial farnesyltransferase inhibitors.

  9. Perspective: Dynamics of receptor tyrosine kinase signaling complexes.

    PubMed

    Mayer, Bruce J

    2012-08-14

    Textbook descriptions of signal transduction complexes provide a static snapshot view of highly dynamic events. Despite enormous strides in identifying the key components of signaling complexes and the underlying mechanisms of signal transduction, our understanding of the dynamic behavior of these complexes has lagged behind. Using the example of receptor tyrosine kinases, this perspective takes a fresh look at the dynamics of the system and their potential impact on signal processing. PMID:22584051

  10. Temporal patterns of tyrosine nitration in embryo heart development

    PubMed Central

    Viera, Liliana; Radmilovich, Milka; Vargas, Marcelo R.; Dennys, Cassandra N.; Wilson, Landon; Barnes, Stephen; Franco, Maria Clara; Beckman, Joseph S.; Estévez, Alvaro G.

    2012-01-01

    Tyrosine nitration is a biomarker for the production of peroxynitrite and other reactive nitrogen species. Nitrotyrosine immunoreactivity is present in many pathological conditions including several cardiac diseases. Because the events observed during heart failure may recapitulate some aspects of development, we tested whether nitrotyrosine is present during normal development of the rat embryo heart and its potential relationship in cardiac remodeling through apoptosis. Nitric oxide (NO) production is highly dynamic during development, but whether peroxynitrite and nitrotyrosine are formed during normal embryonic development has received little attention. Rat embryo hearts exhibited strong nitrotyrosine immunoreactivity in endocardial and myocardial cells of the atria and ventricles from E12 to E18. After E18, nitrotyrosine staining faded and disappeared entirely by birth. Tyrosine nitration in the myocardial tissue coincided with elevated protein expression of nitric oxide synthases (eNOS and iNOS). The immunoreactivity for these NOS isoforms remained elevated even after nitrotyrosine had disappeared. Tyrosine nitration did not correlate with cell death or proliferation of cardiac cells. Analysis of tryptic peptides by MALDI-TOF shows that nitration occurs in actin, myosin, and the mitochondrial ATP synthase alpha chain. These results suggest that reactive nitrogen species are not restricted to pathological conditions but may play a role during normal embryonic development. PMID:23195686

  11. Tyrosine phosphorylation of clathrin heavy chain under oxidative stress.

    PubMed

    Ihara, Yoshito; Yasuoka, Chie; Kageyama, Kan; Wada, Yoshinao; Kondo, Takahito

    2002-09-20

    In mouse pancreatic insulin-producing betaTC cells, oxidative stress due to H(2)O(2) causes tyrosine phosphorylation in various proteins. To identify proteins bearing phosphotyrosine under stress, the proteins were affinity purified using an anti-phosphotyrosine antibody-conjugated agarose column. A protein of 180kDa was identified as clathrin heavy chain (CHC) by electrophoresis and mass spectrometry. Immunoprecipitated CHC showed tyrosine phosphorylation upon H(2)O(2) treatment and the phosphorylation was suppressed by the Src kinase inhibitor, PP2. The phosphorylation status of CHC affected the intracellular localization of CHC and the clathrin-dependent endocytosis of transferrin under oxidative stress. In conclusion, CHC is a protein that is phosphorylated at tyrosine by H(2)O(2) and this phosphorylation status is implicated in the intracellular localization and functions of CHC under oxidative stress. The present study demonstrates that oxidative stress affects intracellular vesicular trafficking via the alteration of clathrin-dependent vesicular trafficking. PMID:12237126

  12. Eph-mediated tyrosine phosphorylation of citron kinase controls abscission.

    PubMed

    Jungas, Thomas; Perchey, Renaud T; Fawal, Mohamad; Callot, Caroline; Froment, Carine; Burlet-Schiltz, Odile; Besson, Arnaud; Davy, Alice

    2016-08-29

    Cytokinesis is the last step of cell division, culminating in the physical separation of daughter cells at the end of mitosis. Cytokinesis is a tightly regulated process that until recently was mostly viewed as a cell-autonomous event. Here, we investigated the role of Ephrin/Eph signaling, a well-known local cell-to-cell communication pathway, in cell division. We show that activation of Eph signaling in vitro leads to multinucleation and polyploidy, and we demonstrate that this is caused by alteration of the ultimate step of cytokinesis, abscission. Control of abscission requires Eph kinase activity, and Src and citron kinase (CitK) are downstream effectors in the Eph-induced signal transduction cascade. CitK is phosphorylated on tyrosines in neural progenitors in vivo, and Src kinase directly phosphorylates CitK. We have identified the specific tyrosine residues of CitK that are phosphorylated and show that tyrosine phosphorylation of CitK impairs cytokinesis. Finally, we show that, similar to CitK, Ephrin/Eph signaling controls neuronal ploidy in the developing neocortex. Our study indicates that CitK integrates intracellular and extracellular signals provided by the local environment to coordinate completion of cytokinesis. PMID:27551053

  13. Protein tyrosine phosphorylation during meiotic divisions of starfish oocytes

    SciTech Connect

    Peaucellier, G.; Andersen, A.C.; Kinsey, W.H. )

    1990-04-01

    We have used an antibody specific for phosphotyrosine to investigate protein phosphorylation on tyrosine during hormone-induced maturation of starfish oocytes. Analysis of immunoprecipitates from cortices of in vivo labeled Marthasterias glacialis oocytes revealed the presence of labeled phosphotyrosine-containing proteins only after hormone addition. Six major phosphoproteins of 195, 155, 100, 85, 45, and 35 kDa were detected. Total activity in immunoprecipitates increased until first polar body emission and was greatly reduced upon completion of meiosis but some proteins exhibited different kinetics. The labeling of the 155-kDa protein reached a maximum at germinal vesicle breakdown, while the 35-kDa appeared later and disappeared after polar body emission. Similar results were obtained with Asterias rubens oocytes. In vitro phosphorylation of cortices showed that tyrosine kinase activity is a major protein kinase activity in this fraction, the main endogenous substrate being a 68-kDa protein. The proteins phosphorylated on tyrosine in vitro were almost similar in extracts from oocytes treated or not with the hormone.

  14. Protein Tyrosine Phosphatases in Hypothalamic Insulin and Leptin Signaling.

    PubMed

    Zhang, Zhong-Yin; Dodd, Garron T; Tiganis, Tony

    2015-10-01

    The hypothalamus is critical to the coordination of energy balance and glucose homeostasis. It responds to peripheral factors, such as insulin and leptin, that convey to the brain the degree of adiposity and the metabolic status of the organism. The development of leptin and insulin resistance in hypothalamic neurons appears to have a key role in the exacerbation of diet-induced obesity. In rodents, this has been attributed partly to the increased expression of the tyrosine phosphatases Protein Tyrosine Phosphatase 1B (PTP1B) and T cell protein tyrosine phosphatase (TCPTP), which attenuate leptin and insulin signaling. Deficiencies in PTP1B and TCPTP in the brain, or specific neurons, promote insulin and leptin signaling and prevent diet-induced obesity, type 2 diabetes mellitus (T2DM), and fatty liver disease. Although targeting phosphatases and hypothalamic circuits remains challenging, recent advances indicate that such hurdles might be overcome. Here, we focus on the roles of PTP1B and TCPTP in insulin and leptin signaling and explore their potential as therapeutic targets.

  15. cap alpha. -Methyl-p-tyrosine shifts circadian temperature rhythms

    SciTech Connect

    Cahill, A.L.; Ehret, C.F.

    1982-09-01

    ..cap alpha..-Methyl-p-tyrosine shifts the acrophase (time of highest temperature) of the circadian temperature rhythm of the rat to earlier or later times of day depending on the phase of the cicadian cycle at which the drug is administered. When ..cap alpha..-methyl-p-tyrosine methyl ester HCl is injected intraperitoneally at a dose of 100 mg/kg late in the projected 8-h light phase, the acrophase of the intraperitoneal temperature rhythm is delayed by up to 3 h.However, when the same dose of drug is given 9-10 h into the projected 16-h dark phase of the daily cycle, the acrophase of the temperature rhythm occurs about 2 h earlier than expected. The times of ..cap alpha..-methyl-p-tyrosine administration leading to maximal phase delays or advances are correlated with the times of minimal and maximal turnover of norepinephrine in the hypothalamus. These results suggest that changing rates of norepinephrine turnover in the hypothalamus may regulate the circadian temperature rhythm in rats. The results also emphasize the fact that the effects of drugs may vary as a function of the time of administration. This fact must be taken into account in pharmacologic testing.

  16. Tyrosine kinase blockers: new hope for successful cancer therapy.

    PubMed

    Pytel, Dariusz; Sliwinski, Tomasz; Poplawski, Tomasz; Ferriola, Deborah; Majsterek, Ireneusz

    2009-01-01

    Tyrosine kinases (TKs) are attractive targets for cancer therapy, as quite often their abnormal signaling has been linked with tumor development and growth. Constitutive activated TKs stimulate multiple signaling pathways responsible for DNA repair, apoptosis, and cell proliferation. During the last few years, thorough analysis of the mechanism underlying tyrosine kinase's activity led to novel cancer therapy using TKs blockers. These drugs are remarkably effective in the treatment of various human tumors including head and neck, gastric, prostate and breast cancer and leukemias. The most successful example of kinase blockers is Imatinib (Imatinib mesylate, Gleevec, STI571), the inhibitor of Bcr/Abl oncoprotein, which has become a first-line therapy for chronic myelogenous leukemia. The introduction of STI571 for the treatment of leukemia in clinical oncology has had a dramatic impact on how this disease is currently managed. Others kinase inhibitors used recently in cancer therapy include Dasatinib (BMS-354825) specific for ABL non-receptor cytoplasmic kinase, Gefitinib (Iressa), Erlotinib (OSI-774, Tarceva) and Sunitinib (SU 11248, Sutent) specific for VEGF receptor kinase, AMN107 (Nilotinib) and INNO-406 (NS-187) specific for c-KIT kinase. The following TK blockers for treatment of various human tumors are in clinical development: Lapatinib (Lapatinib ditosylate, Tykerb, GW-572016), Canertinib (CI-1033), Zactima (ZD6474), Vatalanib (PTK787/ZK 222584), Sorafenib (Bay 43-9006, Nexavar), and Leflunomide (SU101, Arava). Herein, we discuss the chemistry, biological activity and clinical potential of new drugs with tyrosine kinase blockers for cancer treatment.

  17. Regulation of receptor protein-tyrosine phosphatase dimerization.

    PubMed

    van der Wijk, Thea; Blanchetot, Christophe; den Hertog, Jeroen

    2005-01-01

    Receptor protein-tyrosine phosphatases (RPTPs) are single membrane spanning proteins belonging to the family of PTPs that, together with the antagonistically acting protein-tyrosine kinases (PTKs), regulate the protein phosphotyrosine levels in cells. Protein-tyrosine phosphorylation is an important post-translational modification that has a major role in cell signaling by affecting protein-protein interactions and enzymatic activities. Increasing evidence indicates that RPTPs, like RPTKs, are regulated by dimerization. For RPTPalpha, we have shown that rotational coupling of the constitutive dimers in the cell membrane determines enzyme activity. Furthermore, oxidative stress, identified as an important second messenger during the past decade, is a regulator of rotational coupling of RPTPalpha dimers. In this review, we discuss the biochemical and cell biological techniques that we use to study the regulation of RPTPs by dimerization. These techniques include (co-) immunoprecipitation, RPTP activity assays, chemical and genetic cross-linking, detection of cell surface proteins by biotinylation, and analysis of RPTPalpha dimers, using conformation-sensitive antibody binding.

  18. Tyrosine, phenylalanine, and tryptophan in gastroesophageal malignancy: a systematic review.

    PubMed

    Wiggins, Tom; Kumar, Sacheen; Markar, Sheraz R; Antonowicz, Stefan; Hanna, George B

    2015-01-01

    Gastroesophageal cancer has a rapidly increasing incidence worldwide and reliable biomarkers are urgently required to facilitate earlier diagnosis and improve survival. The aromatic amino acids tyrosine, phenylalanine, and tryptophan represent potential biomarkers and their relation to gastroesophageal cancer will be evaluated in this review. An electronic literature search was performed to identify all published research relating to the measurement of tyrosine, phenylalanine, or tryptophan in the biofluids or tissues of patients with gastroesophageal cancer. Sixteen studies were included in this systematic review. Six studies investigated serum concentrations, which all found decreased concentrations of these aromatic amino acids, except one study that found increased phenylalanine. Five studies reported increased concentrations within gastric content of these patients and two reported increased urinary concentrations. Tissue concentrations of these aromatic amino acids were increased in three studies. Tyrosine, phenylalanine, and tryptophan represent potential biomarkers of gastroesophageal cancer, and further research is necessary to definitively establish the mechanism responsible for altered concentrations of these compounds in patients with gastroesophageal cancer.

  19. The Croonian Lecture 1997. The phosphorylation of proteins on tyrosine: its role in cell growth and disease.

    PubMed Central

    Hunter, T

    1998-01-01

    The reversible phosphorylation of tyrosines in proteins plays a key role in regulating many different processes in eukaryotic organisms, such as growth control, cell cycle control, differentiation cell shape and movement, gene transcription, synaptic transmission, and insulin action. Phosphorylation of proteins is brought about by enzymes called protein-tyrosine kinases that add phosphate to specific tyrosines in target proteins; phosphate is removed from phosphorylated tyrosines by enzymes called protein-tyrosine phosphatases. Phosphorylated tyrosines are recognized by specialized binding domains on other proteins, and such interactions are used to initiate intracellular signaling pathways. Currently, more than 95 protein-tyrosine kinases and more than 55 protein-tyrosine phosphatase genes are known in Homo sapiens. Aberrant tyrosine phosphorylation is a hallmark of many types of cancer and other human diseases. Drugs are being developed that antagonize the responsible protein-tyrosine kinases and phosphatases in order to combat these diseases. PMID:9602534

  20. Tyrosine Phosphorylation and Dephosphorylation in Burkholderia cenocepacia Affect Biofilm Formation, Growth under Nutritional Deprivation, and Pathogenicity

    PubMed Central

    Andrade, Angel; Tavares-Carreón, Faviola; Khodai-Kalaki, Maryam

    2015-01-01

    Burkholderia cenocepacia, a member of the B. cepacia complex (Bcc), is an opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis. Tyrosine phosphorylation has emerged as an important posttranslational modification modulating the physiology and pathogenicity of Bcc bacteria. Here, we investigated the predicted bacterial tyrosine kinases BCAM1331 and BceF and the low-molecular-weight protein tyrosine phosphatases BCAM0208, BceD, and BCAL2200 of B. cenocepacia K56-2. We show that BCAM1331, BceF, BCAM0208, and BceD contribute to biofilm formation, while BCAL2200 is required for growth under nutrient-limited conditions. Multiple deletions of either tyrosine kinase or low-molecular-weight protein tyrosine phosphatase genes resulted in the attenuation of B. cenocepacia intramacrophage survival and reduced pathogenicity in the Galleria mellonella larval infection model. Experimental evidence indicates that BCAM1331 displays reduced tyrosine autophosphorylation activity compared to that of BceF. With the artificial substrate p-nitrophenyl phosphate, the phosphatase activities of the three low-molecular-weight protein tyrosine phosphatases demonstrated similar kinetic parameters. However, only BCAM0208 and BceD could dephosphorylate BceF. Further, BCAL2200 became tyrosine phosphorylated in vivo and catalyzed its autodephosphorylation. Together, our data suggest that despite having similar biochemical activities, low-molecular-weight protein tyrosine phosphatases and tyrosine kinases have both overlapping and specific roles in the physiology of B. cenocepacia. PMID:26590274

  1. Effects of hemorrhagic hypotension on tyrosine concentrations in rat spinal cord and plasma

    NASA Technical Reports Server (NTRS)

    Conlay, L. A.; Maher, T. J.; Roberts, C. H.; Wurtman, R. J.

    1988-01-01

    Tyrosine is the precursor for catecholamine neurotransmitters. When catecholamine-containing neurons are physiologically active (as sympathoadrenal cells are in hypotension), tyrosine administration increases catecholamine synthesis and release. Since hypotension can alter plasma amino acid composition, the effects of an acute hypotensive insult on tyrosine concentrations in plasma and spinal cord were examined. Rats were cannulated and bled until the systolic blood pressure was 50 mmHg, or were kept normotensive for 1 h. Tyrosine and other large neutral amino acids (LNAA) known to compete with tyrosine for brain uptake were assayed in plasma and spinal cord. The rate at which intra-arterial (H-3)tyrosine disappeared from the plasma was also estimated in hemorrhaged and control rats. In plasma of hemorrhaged animals, both the tyrosine concentration and the tyrosine/LNAA ratio was elevated; moreover, the disappearance of (H-3)tyrosine was slowed. Tyrosine concentrations also increased in spinal cords of hemorrhaged-hypotensive rats when compared to normotensive controls. Changes in plasma amino acid patterns may thus influence spinal cord concentrations of amino acid precursors for neurotransmitters during the stress of hemorrhagic shock.

  2. Tyrosine phosphatases as key regulators of StAR induction and cholesterol transport: SHP2 as a potential tyrosine phosphatase involved in steroid synthesis.

    PubMed

    Cooke, Mariana; Mele, Pablo; Maloberti, Paula; Duarte, Alejandra; Poderoso, Cecilia; Orlando, Ulises; Paz, Cristina; Cornejo Maciel, Fabiana; Podestá, Ernesto J

    2011-04-10

    The phospho-dephosphorylation of intermediate proteins is a key event in the regulation of steroid biosynthesis. In this regard, it is well accepted that steroidogenic hormones act through the activation of serine/threonine (Ser/Thr) protein kinases. Although many cellular processes can be regulated by a crosstalk between different kinases and phosphatases, the relationship of Ser/Thr phosphorylation and tyrosine (Tyr)-dephosphorylation is a recently explored field in the regulation of steroid synthesis. Indeed in steroidogenic cells, one of the targets of hormone-induced Ser/Thr phosphorylation is a protein tyrosine phosphatase. Whereas protein tyrosine phosphatases were initially regarded as household enzymes with constitutive activity, dephosphorylating all the substrates they encountered, evidence is now accumulating that protein tyrosine phosphatases are tightly regulated by various mechanisms. Here, we will describe the role of protein tyrosine phosphatases in the regulation of steroid biosynthesis, relating them to steroidogenic acute regulatory protein, arachidonic acid metabolism and mitochondrial rearrangement.

  3. PSTPIP: A Tyrosine Phosphorylated Cleavage Furrow–associated Protein that Is a Substrate for a PEST Tyrosine Phosphatase

    PubMed Central

    Spencer, Susan; Dowbenko, Donald; Cheng, Jill; Li, Wenlu; Brush, Jennifer; Utzig, Suzan; Simanis, Viesturs; Lasky, Laurence A.

    1997-01-01

    We have investigated proteins which interact with the PEST-type protein tyrosine phosphatase, PTP hematopoietic stem cell fraction (HSCF), using the yeast two-hybrid system. This resulted in the identification of proline, serine, threonine phosphatase interacting protein (PSTPIP), a novel member of the actin- associated protein family that is homologous to Schizosaccharomyces pombe CDC15p, a phosphorylated protein involved with the assembly of the actin ring in the cytokinetic cleavage furrow. The binding of PTP HSCF to PSTPIP was induced by a novel interaction between the putative coiled-coil region of PSTPIP and the COOH-terminal, proline-rich region of the phosphatase. PSTPIP is tyrosine phosphorylated both endogenously and in v-Src transfected COS cells, and cotransfection of dominant-negative PTP HSCF results in hyperphosphorylation of PSTPIP. This dominant-negative effect is dependent upon the inclusion of the COOH-terminal, proline-rich PSTPIP-binding region of the phosphatase. Confocal microscopy analysis of endogenous PSTPIP revealed colocalization with the cortical actin cytoskeleton, lamellipodia, and actin-rich cytokinetic cleavage furrow. Overexpression of PSTPIP in 3T3 cells resulted in the formation of extended filopodia, consistent with a role for this protein in actin reorganization. Finally, overexpression of mammalian PSTPIP in exponentially growing S. pombe results in a dominant-negative inhibition of cytokinesis. PSTPIP is therefore a novel actin-associated protein, potentially involved with cytokinesis, whose tyrosine phosphorylation is regulated by PTP HSCF. PMID:9265651

  4. Constitutive tyrosine phosphorylation of the T-cell receptor (TCR) zeta subunit: regulation of TCR-associated protein tyrosine kinase activity by TCR zeta.

    PubMed Central

    van Oers, N S; Tao, W; Watts, J D; Johnson, P; Aebersold, R; Teh, H S

    1993-01-01

    The T-cell receptor (TCR) zeta subunit is an important component of the TCR complex, involved in signal transduction events following TCR engagement. In this study, we showed that the TCR zeta chain is constitutively tyrosine phosphorylated to similar extents in thymocytes and lymph node T cells. Approximately 35% of the tyrosine-phosphorylated TCR zeta (phospho zeta) precipitated from total cell lysates appeared to be surface associated. Furthermore, constitutive phosphorylation of TCR zeta in T cells occurred independently of antigen stimulation and did not require CD4 or CD8 coreceptor expression. In lymph node T cells that constitutively express tyrosine-phosphorylated TCR zeta, there was a direct correlation between surface TCR-associated protein tyrosine kinase (PTK) activity and expression of phospho zeta. TCR stimulation of these cells resulted in an increase in PTK activity that coprecipitated with the surface TCR complex and a corresponding increase in the levels of phospho zeta. TCR ligations also contributed to the detection of several additional phosphoproteins that coprecipitated with surface TCR complexes, including a 72-kDa tyrosine-phosphorylated protein. The presence of TCR-associated PTK activity also correlated with the binding of a 72-kDa protein, which became tyrosine phosphorylated in vitro kinase assays, to tyrosine phosphorylated TCR zeta. The cytoplasmic region of the TCR zeta chain was synthesized, tyrosine phosphorylated, and conjugated to Sepharose beads. Only tyrosine-phosphorylated, not nonphosphorylated, TCR zeta beads were capable of immunoprecipitating the 72-kDa protein from total cell lysates. This 72-kDa protein is likely the murine equivalent of human PTK ZAP-70, which has been shown to associate specifically with phospho zeta. These results suggest that TCR-associated PTK activity is regulated, at least in part, by the tyrosine phosphorylation status of TCR zeta. Images PMID:7689151

  5. Tyrosine fluorescence probing of the surfactant-induced conformational changes of albumin.

    PubMed

    Zhdanova, Nadezda G; Shirshin, Evgeny A; Maksimov, Eugene G; Panchishin, Ivan M; Saletsky, Alexander M; Fadeev, Victor V

    2015-05-01

    Tyrosine fluorescence in native proteins is known to be effectively quenched, whereas its emission increases upon proteins' unfolding. This suggests that tyrosine fluorescence could be exploited for probing structural rearrangements of proteins in addition to the extensively used tryptophan emission. We studied the possibility of using tyrosine fluorescence as an indicator of surfactant-induced conformational changes in albumins. It was shown that fluorescence of tyrosine residues, which are uniformly distributed all over the protein molecules, allows the detection of subtle structural rearrangements of proteins upon surfactant binding, which do not influence the properties of a single tryptophan residue buried in the inner hydrophobic region of human serum albumin. Tyrosine fluorescence properties, including its fluorescence lifetime, revealed the multistage character of surfactant binding to albumin, consistent with the data provided by other methods. The obtained results demonstrate the possibility of probing conformational changes in proteins using tyrosine photophysical parameters as indicators.

  6. Centrosomal targeting of tyrosine kinase activity does not enhance oncogenicity in chronic myeloproliferative disorders.

    PubMed

    Bochtler, T; Kirsch, M; Maier, B; Bachmann, J; Klingmüller, U; Anderhub, S; Ho, A D; Krämer, A

    2012-04-01

    Constitutive tyrosine kinase activation by reciprocal chromosomal translocation is a common pathogenetic mechanism in chronic myeloproliferative disorders. Since centrosomal proteins have been recurrently identified as translocation partners of tyrosine kinases FGFR1, JAK2, PDGFRα and PDGFRβ in these diseases, a role for the centrosome in oncogenic transformation has been hypothesized. In this study, we addressed the functional role of centrosomally targeted tyrosine kinase activity. First, centrosomal localization was not routinely found for all chimeric fusion proteins tested. Second, targeting of tyrosine kinases to the centrosome by creating artificial chimeric fusion kinases with the centrosomal targeting domain of AKAP450 failed to enhance the oncogenic transforming potential in both Ba/F3 and U2OS cells, although phospho-tyrosine-mediated signal transduction pathways were initiated at the centrosome. We conclude that the centrosomal localization of constitutively activated tyrosine kinases does not contribute to disease pathogenesis in chronic myeloproliferative disorders. PMID:22015771

  7. Virulence genotypes and drug resistance of Helicobacter pylori from Vladivostok, Russia: another feature in the Far East.

    PubMed

    Reva, Ivan; Takano, Tomomi; Higuchi, Wataru; Iwao, Yasuhisa; Taneike, Ikue; Nakagawa, Saori; Ike, Masami; Pererva, Oleg; Tarankov, Alexey; Agapov, Mikhail; Rizhkov, Evgeniy; Singur, Olga; Reva, Galina; Potapov, Vladimir; Yamamoto, Tatsuo

    2012-03-01

    Helicobacter pylori in Vladivostok, Far Eastern Russia, was investigated during 2004 to 2009. The genotype cagA(+) vacA(+) (s1/m1 or m2) accounted for 74.7%, with cagA(-) vacA(+) (s2/m2) at 11.2%. The CagA EPIYA type was mainly Western ABC, with minor types (ABCCC and novel AAABC) or non-Western/non-East Asia type (AB). Regarding drug resistance, metronidazole resistance was the highest, with a marked decrease in 6 years (from 71.4% to 30.8%); in contrast, levofloxacin and clarithromycin resistance increased. The data indicate that in Vladivostok, H. pylori was mainly the Western (not East Asian) type and dynamic changes in drug resistance occurred during 6 years.

  8. Intracellular Growth Is Dependent on Tyrosine Catabolism in the Dimorphic Fungal Pathogen Penicillium marneffei

    PubMed Central

    Boyce, Kylie J.; McLauchlan, Alisha; Schreider, Lena; Andrianopoulos, Alex

    2015-01-01

    During infection, pathogens must utilise the available nutrient sources in order to grow while simultaneously evading or tolerating the host’s defence systems. Amino acids are an important nutritional source for pathogenic fungi and can be assimilated from host proteins to provide both carbon and nitrogen. The hpdA gene of the dimorphic fungus Penicillium marneffei, which encodes an enzyme which catalyses the second step of tyrosine catabolism, was identified as up-regulated in pathogenic yeast cells. As well as enabling the fungus to acquire carbon and nitrogen, tyrosine is also a precursor in the formation of two types of protective melanin; DOPA melanin and pyomelanin. Chemical inhibition of HpdA in P. marneffei inhibits ex vivo yeast cell production suggesting that tyrosine is a key nutrient source during infectious growth. The genes required for tyrosine catabolism, including hpdA, are located in a gene cluster and the expression of these genes is induced in the presence of tyrosine. A gene (hmgR) encoding a Zn(II)2-Cys6 binuclear cluster transcription factor is present within the cluster and is required for tyrosine induced expression and repression in the presence of a preferred nitrogen source. AreA, the GATA-type transcription factor which regulates the global response to limiting nitrogen conditions negatively regulates expression of cluster genes in the absence of tyrosine and is required for nitrogen metabolite repression. Deletion of the tyrosine catabolic genes in the cluster affects growth on tyrosine as either a nitrogen or carbon source and affects pyomelanin, but not DOPA melanin, production. In contrast to other genes of the tyrosine catabolic cluster, deletion of hpdA results in no growth within macrophages. This suggests that the ability to catabolise tyrosine is not required for macrophage infection and that HpdA has an additional novel role to that of tyrosine catabolism and pyomelanin production during growth in host cells. PMID:25812137

  9. Effects of systemic L-tyrosine on dopamine release from rat corpus striatum and nucleus accumbens

    NASA Technical Reports Server (NTRS)

    During, Matthew J.; Acworth, Ian N.; Wurtman, Richard J.

    1988-01-01

    Intracerebral dialysis was used to monitor extracellular fluid from rat striatum and nucleus accumbens following the intraperitoneal administration of tyrosine. Dopamine concentrations in dialysates from both the striatum and the nucleus accumbens increased significantly in response to the tyrosine. The magnitude of the tyrosine effect was greater in the nucleus accumbens than in the striatum. Hence, mesolimbic dopaminergic neurons may be especially responsive to precursor availability.

  10. Chemical inhibition of bacterial protein tyrosine phosphatase suppresses capsule production.

    PubMed

    Standish, Alistair J; Salim, Angela A; Zhang, Hua; Capon, Robert J; Morona, Renato

    2012-01-01

    Capsule polysaccharide is a major virulence factor for a wide range of bacterial pathogens, including Streptococcus pneumoniae. The biosynthesis of Wzy-dependent capsules in both gram-negative and -positive bacteria is regulated by a system involving a protein tyrosine phosphatase (PTP) and a protein tyrosine kinase. However, how the system functions is still controversial. In Streptococcus pneumoniae, a major human pathogen, the system is present in all but 2 of the 93 serotypes found to date. In order to study this regulation further, we performed a screen to find inhibitors of the phosphatase, CpsB. This led to the observation that a recently discovered marine sponge metabolite, fascioquinol E, inhibited CpsB phosphatase activity both in vitro and in vivo at concentrations that did not affect the growth of the bacteria. This inhibition resulted in decreased capsule synthesis in D39 and Type 1 S. pneumoniae. Furthermore, concentrations of Fascioquinol E that inhibited capsule also lead to increased attachment of pneumococci to a macrophage cell line, suggesting that this compound would inhibit the virulence of the pathogen. Interestingly, this compound also inhibited the phosphatase activity of the structurally unrelated gram-negative PTP, Wzb, which belongs to separate family of protein tyrosine phosphatases. Furthermore, incubation with Klebsiella pneumoniae, which contains a homologous phosphatase, resulted in decreased capsule synthesis. Taken together, these data provide evidence that PTPs are critical for Wzy-dependent capsule production across a spectrum of bacteria, and as such represents a valuable new molecular target for the development of anti-virulence antibacterials.

  11. Tyrosine Aminotransferase: Biochemical and Structural Properties and Molecular Dynamics Simulations

    SciTech Connect

    P Mehere; Q Han; J Lemkul; C Vavricka; H Robinson; D Bevan; J Li

    2011-12-31

    Tyrosine aminotransferase (TAT) catalyzes the transamination of tyrosine and other aromatic amino acids. The enzyme is thought to play a role in tyrosinemia type II, hepatitis and hepatic carcinoma recovery. The objective of this study is to investigate its biochemical and structural characteristics and substrate specificity in order to provide insight regarding its involvement in these diseases. Mouse TAT (mTAT) was cloned from a mouse cDNA library, and its recombinant protein was produced using Escherichia coli cells and purified using various chromatographic techniques. The recombinant mTAT is able to catalyze the transamination of tyrosine using {alpha}-ketoglutaric acid as an amino group acceptor at neutral pH. The enzyme also can use glutamate and phenylalanine as amino group donors and p-hydroxy-phenylpyruvate, phenylpyruvate and alpha-ketocaproic acid as amino group acceptors. Through macromolecular crystallography we have determined the mTAT crystal structure at 2.9 {angstrom} resolution. The crystal structure revealed the interaction between the pyridoxal-5'-phosphate cofactor and the enzyme, as well as the formation of a disulphide bond. The detection of disulphide bond provides some rational explanation regarding previously observed TAT inactivation under oxidative conditions and reactivation of the inactive TAT in the presence of a reducing agent. Molecular dynamics simulations using the crystal structures of Trypanosoma cruzi TAT and human TAT provided further insight regarding the substrate-enzyme interactions and substrate specificity. The biochemical and structural properties of TAT and the binding of its cofactor and the substrate may help in elucidation of the mechanism of TAT inhibition and activation.

  12. Tyrosine aminotransferase: biochemical and structural properties and molecular dynamics simulations

    SciTech Connect

    Mehere, P.; Robinson, H.; Han, Q.; Lemkul, J. A.; Vavricka, C. J.; Bevan, D. R.; Li, J.

    2010-11-01

    Tyrosine aminotransferase (TAT) catalyzes the transamination of tyrosine and other aromatic amino acids. The enzyme is thought to play a role in tyrosinemia type II, hepatitis and hepatic carcinoma recovery. The objective of this study is to investigate its biochemical and structural characteristics and substrate specificity in order to provide insight regarding its involvement in these diseases. Mouse TAT (mTAT) was cloned from a mouse cDNA library, and its recombinant protein was produced using Escherichia coli cells and purified using various chromatographic techniques. The recombinant mTAT is able to catalyze the transamination of tyrosine using {alpha}-ketoglutaric acid as an amino group acceptor at neutral pH. The enzyme also can use glutamate and phenylalanine as amino group donors and p-hydroxy-phenylpyruvate, phenylpyruvate and alpha-ketocaproic acid as amino group acceptors. Through macromolecular crystallography we have determined the mTAT crystal structure at 2.9 {angstrom} resolution. The crystal structure revealed the interaction between the pyridoxal-5'-phosphate cofactor and the enzyme, as well as the formation of a disulphide bond. The detection of disulphide bond provides some rational explanation regarding previously observed TAT inactivation under oxidative conditions and reactivation of the inactive TAT in the presence of a reducing agent. Molecular dynamics simulations using the crystal structures of Trypanosoma cruzi TAT and human TAT provided further insight regarding the substrate-enzyme interactions and substrate specificity. The biochemical and structural properties of TAT and the binding of its cofactor and the substrate may help in elucidation of the mechanism of TAT inhibition and activation.

  13. Tyrosine aminotransferase: biochemical and structural properties and molecular dynamics simulations.

    PubMed

    Mehere, Prajwalini; Han, Qian; Lemkul, Justin A; Vavricka, Christopher J; Robinson, Howard; Bevan, David R; Li, Jianyong

    2010-11-01

    Tyrosine aminotransferase (TAT) catalyzes the transamination of tyrosine and other aromatic amino acids. The enzyme is thought to play a role in tyrosinemia type II, hepatitis and hepatic carcinoma recovery. The objective of this study is to investigate its biochemical and structural characteristics and substrate specificity in order to provide insight regarding its involvement in these diseases. Mouse TAT (mTAT) was cloned from a mouse cDNA library, and its recombinant protein was produced using Escherichia coli cells and purified using various chromatographic techniques. The recombinant mTAT is able to catalyze the transamination of tyrosine using α-ketoglutaric acid as an amino group acceptor at neutral pH. The enzyme also can use glutamate and phenylalanine as amino group donors and p-hydroxy-phenylpyruvate, phenylpyruvate and alpha-ketocaproic acid as amino group acceptors. Through macromolecular crystallography we have determined the mTAT crystal structure at 2.9 Å resolution. The crystal structure revealed the interaction between the pyridoxal-5'-phosphate cofactor and the enzyme, as well as the formation of a disulphide bond. The detection of disulphide bond provides some rational explanation regarding previously observed TAT inactivation under oxidative conditions and reactivation of the inactive TAT in the presence of a reducing agent. Molecular dynamics simulations using the crystal structures of Trypanosoma cruzi TAT and human TAT provided further insight regarding the substrate-enzyme interactions and substrate specificity. The biochemical and structural properties of TAT and the binding of its cofactor and the substrate may help in elucidation of the mechanism of TAT inhibition and activation.

  14. Involvement of the N-terminal unique domain of Chk tyrosine kinase in Chk-induced tyrosine phosphorylation in the nucleus

    SciTech Connect

    Nakayama, Yuji; Kawana, Akiko; Igarashi, Asae; Yamaguchi, Naoto . E-mail: nyama@p.chiba-u.ac.jp

    2006-07-15

    Chk tyrosine kinase phosphorylates Src-family kinases and suppresses their kinase activity. We recently showed that Chk localizes to the nucleus as well as the cytoplasm and inhibits cell proliferation. In this study, we explored the role of the N-terminal unique domain of Chk in nuclear localization and Chk-induced tyrosine phosphorylation in the nucleus. In situ binding experiments showed that the N-terminal domain of Chk was associated with the nucleus and the nuclear matrix. The presence of the N-terminal domain of Chk led to a fourfold increase in cell population exhibiting Chk-induced tyrosine phosphorylation in the nucleus. Expression of Chk but not kinase-deficient Chk induced tyrosine phosphorylation of a variety of proteins ranging from 23 kDa to {approx}200 kDa, especially in Triton X-100-insoluble fraction that included chromatin and the nuclear matrix. Intriguingly, in situ subnuclear fractionations revealed that Chk induced tyrosine phosphorylation of proteins that were associated with the nuclear matrix. These results suggest that various unidentified substrates of Chk, besides Src-family kinases, may be present in the nucleus. Thus, our findings indicate that the importance of the N-terminal domain to Chk-induced tyrosine phosphorylation in the nucleus, implicating that these nuclear tyrosine-phosphorylated proteins may contribute to inhibition of cell proliferation.

  15. Analysis of receptor tyrosine kinase internalization using flow cytometry.

    PubMed

    Li, Ning; Hill, Kristen S; Elferink, Lisa A

    2008-01-01

    The internalization of activated receptor tyrosine kinases (RTKs) by endocytosis and their subsequent down regulation in lysosomes plays a critical role in regulating the duration and intensity of downstream signaling events. Uncoupling of the RTK cMet from ligand-induced degradation was recently shown to correlate with sustained receptor signaling and increased cell tumorigenicity, suggesting that the corruption of these endocytic mechanisms could contribute to increased cMet signaling in metastatic cancers. To understand how cMet signaling for normal cell growth is controlled by endocytosis and how these mechanisms are dysregulated in metastatic cancers, we developed flow cytometry-based assays to examine cMet internalization.

  16. Gα13 Stimulates the Tyrosine Phosphorylation of Ric-8A

    PubMed Central

    Yan, Mingda; Ha, Ji Hee

    2015-01-01

    The G12 family of heterotrimeric G proteins is defined by their α-subunits, Gα12 and Gα13. These α-subunits regulate cellular homeostasis, cell migration, and oncogenesis in a context-specific manner primarily through their interactions with distinct proteins partners that include diverse effector molecules and scaffold proteins. With a focus on identifying any other novel regulatory protein(s) that can directly interact with Gα13, we subjected Gα13 to tandem affinity purification-coupled mass spectrometric analysis. Our results from such analysis indicate that Gα13 potently interacts with mammalian Ric-8A. Our mass spectrometric analysis data also indicates that Ric-8A, which was tandem affinity purified along with Gα13, is phosphorylated at Ser-436, Thr-441, Thr-443 and Tyr-435. Using a serial deletion approach, we have defined that the C-terminus of Gα13 containing the guanine-ring interaction site is essential and sufficient for its interaction with Ric-8A. Evaluation of Gα13-specific signaling pathways in SKOV3 or HeyA8 ovarian cancer cell lines indicate that Ric-8A potentiates Gα13-mediated activation of RhoA, Cdc42, and the downstream p38MAPK. We also establish that the tyrosine phosphorylation of Ric-8A, thus far unidentified, is potently stimulated by Gα13. Our results also indicate that the stimulation of tyrosine-phosphorylation of Ric-8A by Gα13 is partially sensitive to inhibitors of Src-family of kinases, namely PP2 and SI. Furthermore, we demonstrate that Gα13 promotes the translocation of Ric-8A to plasma membrane and this translocation is attenuated by the Src-inhibitors, SI1 and PP2. Thus, our results demonstrate for the first time that Gα13 stimulates the tyrosine phosphorylation of Ric-8A and Gα13-mediated tyrosine-phosphorylation plays a critical role in the translocation of Ric-8A to plasma membrane. PMID:27096001

  17. Second-generation inhibitors of Bruton tyrosine kinase.

    PubMed

    Wu, Jingjing; Liu, Christina; Tsui, Stella T; Liu, Delong

    2016-01-01

    Bruton tyrosine kinase (BTK) is a critical effector molecule for B cell development and plays a major role in lymphoma genesis. Ibrutinib is the first-generation BTK inhibitor. Ibrutinib has off-target effects on EGFR, ITK, and Tec family kinases, which explains the untoward effects of ibrutinib. Resistance to ibrutinib was also reported. The C481S mutation in the BTK kinase domain was reported to be a major mechanism of resistance to ibrutinib. This review summarizes the clinical development of novel BTK inhibitors, ACP-196 (acalabrutinib), ONO/GS-4059, and BGB-3111. PMID:27590878

  18. Eliminating tyrosine sequence variants in CHO cell lines producing recombinant monoclonal antibodies.

    PubMed

    Feeney, Lauren; Carvalhal, Veronica; Yu, X Christopher; Chan, Betty; Michels, David A; Wang, Yajun Jennifer; Shen, Amy; Ressl, Jan; Dusel, Brendon; Laird, Michael W

    2013-04-01

    Amino acid sequence variants are defined as unintended amino acid sequence changes that contribute to product variation with potential impact to product safety, immunogenicity, and efficacy. Therefore, it is important to understand the propensity for sequence variant (SV) formation during the production of recombinant proteins for therapeutic use. During the development of clinical therapeutic products, several monoclonal antibodies (mAbs) produced from Chinese Hamster Ovary (CHO) cells exhibited SVs at low levels (≤3%) in multiple locations throughout the mAbs. In these examples, the cell culture process depleted tyrosine, and the tyrosine residues in the recombinant mAbs were replaced with phenylalanine or histidine. In this work, it is demonstrated that tyrosine supplementation eliminated the tyrosine SVs, while early tyrosine starvation significantly increased the SV level in all mAbs tested. Additionally, it was determined that phenylalanine is the amino acid preferentially misincorporated in the absence of tyrosine over histidine, with no other amino acid misincorporated in the absence of tyrosine, phenylalanine, and histidine. The data support that the tyrosine SVs are due to mistranslation and not DNA mutation, most likely due to tRNA(Tyr) mischarging due to the structural similarities between tyrosine and phenylalanine.

  19. Induction of protein tyrosine phosphorylation in macrophages incubated with tumor cells.

    PubMed

    Sodhi, A; Shrivastava, A; Kumar, R

    1995-03-01

    The cellular and molecular interaction between monocyte/macrophage and tumor cells leading to macrophage activation is not clearly understood. Since protein tyrosine phosphorylation appears to be a major intracellular signalling event, we checked whether the tumor cells alter tyrosine phosphorylation of proteins in macrophages. We found that both L929 and Yac-1 tumor cells induced increased tyrosine phosphorylation of several polypeptides in peritoneal as well as P388D-1 and IC-21 macrophages. Macrophages co-cultured with tumor cells also showed increased fluorescence with anti-phosphotyrosine-FITC antibody. These observations suggest that increased tyrosine phosphorylation plays a role in tumor cell-induced activation of macrophages. PMID:7539664

  20. Diurnal variations in response of rat liver tyrosine aminotransferase activity to food intake.

    PubMed

    Kato, H; Saito, M

    1980-01-01

    Effects of fasting and refeeding on the hepatic tyrosine aminotransferase activity were examined in rats that had been fed during the night. The tyrosine aminotransferase activity showed clear diurnal variations, with a maximal activity after the feeding time. The tyrosine aminotransferase rhythm persisted even under starvation, though the amplitude decreased remarkably. When the starved rats were refed at night, the tyrosine aminotransferase activity increased rapidly to a high level, but it increased slowly to a rather lower level when they were refed in daytime.

  1. Spectral analysis of interaction between carotenoid and tyrosine in ethanol-water solution

    NASA Astrophysics Data System (ADS)

    Lu, Liping; Liu, Guiling; Ni, Xiaowu; Luo, Xiaosen

    2015-03-01

    In this study we have applied UV/vis absorption spectroscopy, steady state fluorescence, Raman spectra to investigate the effects of tyrosine on the aggregates of lutein and β-carotene. Absorption spectra analysis revealed that hydroxyl and amino groups of tyrosine can affect the aggregate of lutein to a certain extent. In Raman spectra the effect of tyrosine on the length of conjugation was observed in the case of lutein molecule. In addition tyrosine also had a great effect on the excited electronic state of carotenoids, and internal energy transferring among aggregates.

  2. Cross talk of tyrosine kinases with the DNA damage signaling pathways

    PubMed Central

    Mahajan, Kiran; Mahajan, Nupam P.

    2015-01-01

    Tyrosine kinases respond to extracellular and intracellular cues by activating specific cellular signaling cascades to regulate cell cycle, growth, proliferation, differentiation and survival. Likewise, DNA damage response proteins (DDR) activated by DNA lesions or chromatin alterations recruit the DNA repair and cell cycle checkpoint machinery to restore genome integrity and cellular homeostasis. Several new examples have been uncovered in recent studies which reveal novel epigenetic and non-epigenetic mechanisms by which tyrosine kinases interact with DDR proteins to dictate cell fate, i.e. survival or apoptosis, following DNA damage. These studies reveal the ability of tyrosine kinases to directly regulate the activity of DNA repair and cell cycle check point proteins by tyrosine phosphorylation. In addition, tyrosine kinases epigenetically regulate DNA damage signaling pathways by modifying the core histones as well as chromatin modifiers at critical tyrosine residues. Thus, deregulated tyrosine kinase driven epigenomic alterations have profound implications in cancer, aging and genetic disorders. Consequently, targeting oncogenic tyrosine kinase induced epigenetic alterations has gained significant traction in overcoming cancer cell resistance to various therapies. This review discusses mechanisms by which tyrosine kinases interact with DDR pathways to regulate processes critical for maintaining genome integrity as well as clinical strategies for targeted cancer therapies. PMID:26546517

  3. Low dose alpha-methyl-para-tyrosine (AMPT) in the treatment of dystonia and dyskinesia.

    PubMed

    Ankenman, Ralph; Salvatore, Michael F

    2007-01-01

    AMPT (alpha-methyl-para-tyrosine) is an inhibitor of tyrosine hydroxylase, the rate-limiting enzyme in dopamine biosynthesis. In clinical settings, AMPT is approved to treat pheochromocytoma. Dystonias and dyskinesias seem to have their origin in inconsistent regulation of dopamine function in dopamine pathways. This paper presents case histories of the clinical efficacy of AMPT for treating certain individuals with neuroleptic-induced dystonia or dyskinesia. The authors propose that a special utility of AMPT in tardive disorders may be related to a downregulation of tyrosine hydroxylase activity that may be increased by neuroleptic-induced effects on tyrosine hydroxylase phosphorylation.

  4. Interferon signaling is dependent on specific tyrosines located within the intracellular domain of IFNAR2c. Expression of IFNAR2c tyrosine mutants in U5A cells.

    PubMed

    Wagner, T Charis; Velichko, Sharlene; Vogel, David; Rani, M R Sandhya; Leung, Stewart; Ransohoff, Richard M; Stark, George R; Perez, H Daniel; Croze, Ed

    2002-01-11

    Type I interferons (IFNs) are cytokines that play a central role in mediating antiviral, antiproliferative, and immunomodulatory activities in virtually all cells. These activities are entirely dependent on the interaction of IFNs with their particular cell surface receptor. In this report, we identify two specific tyrosine residues located within the cytoplasmic domain of IFNAR2c that are obligatory for IFN-dependent signaling. Various IFNAR2c tyrosine mutants were expressed in a human lung fibroscarcoma cell line lacking IFNAR2c (U5A). Stable clones expressing these mutants were analyzed for their ability to induce STAT1 and STAT2 activation, ISGF3 transcriptional complex formation, gene expression, and cell growth regulation in response to stimulation with type I IFNs. The replacement of all seven cytoplasmic tyrosine residues of IFNAR2c with phenylalanine resulted in a receptor unable to respond to IFN stimulation. Substitution of single tyrosines at amino acid residue 269, 316, 318, 337, or 512 with phenylalanine had no effect on IFN-dependent signaling, suggesting that no single tyrosine is essential for IFN receptor-mediated signaling. In addition, IFNAR2c retaining five proximal tyrosines residues (269, 306, 316, 318, and 337) or either two distal tyrosine residues (411 or 512) continued to be responsive to IFN stimulation. Surprisingly, the presence of only a single tyrosine at either position 337 or 512 was sufficient to restore a complete IFN response. These results indicate that IFN-dependent signaling proceeds through the redundant usage of two tyrosine residues in the cytoplasmic domain of IFNAR2c.

  5. Tyrosine-rich Conopeptides Affect Voltage-gated K+ Channels*

    PubMed Central

    Imperial, Julita S.; Chen, Ping; Sporning, Annett; Terlau, Heinrich; Daly, Norelle L.; Craik, David J.; Alewood, Paul F.; Olivera, Baldomero M.

    2008-01-01

    Two venom peptides, CPY-Pl1 (EU000528) and CPY-Fe1 (EU000529), characterized from the vermivorous marine snails Conus planorbis and Conus ferrugineus, define a new class of conopeptides, the conopeptide Y (CPY) family. The peptides have no disulfide cross-links and are 30 amino acids long; the high content of tyrosine is unprecedented for any native gene product. The CPY peptides were chemically synthesized and shown to be biologically active upon injection into both mice and Caenorhabditis elegans; activity on mammalian Kv1 channel isoforms was demonstrated using an oocyte heterologous expression system, and selectivity for Kv1.6 was found. NMR spectroscopy revealed that the peptides were unstructured in aqueous solution; however, a helical region including residues 12–18 for one peptide, CPY-Pl1, formed in trifluoroethanol buffer. Clones obtained from cDNA of both species encoded prepropeptide precursors that shared a unique signal sequence, indicating that these peptides are encoded by a novel gene family. This is the first report of tyrosine-rich bioactive peptides in Conus venom. PMID:18505731

  6. Receptor tyrosine kinases and schistosome reproduction: new targets for chemotherapy

    PubMed Central

    Morel, Marion; Vanderstraete, Mathieu; Hahnel, Steffen; Grevelding, Christoph G.; Dissous, Colette

    2014-01-01

    Schistosome parasites still represent a serious public health concern and a major economic problem in developing countries. Pathology of schistosomiasis is mainly due to massive egg production by these parasites and to inflammatory responses raised against the eggs which are trapped in host tissues. Tyrosine kinases (TKs) are key molecules that control cell differentiation and proliferation and they already represent important targets in cancer therapy. During recent years, it has been shown that receptor tyrosine kinases (RTK) signaling was active in reproductive organs and that it could regulate sexual maturation of schistosomes and egg production. This opens interesting perspectives for the control of transmission and pathogenesis of schistosomiasis based on new therapies targeting schistosome RTKs. This review relates the numerous data showing the major roles of kinase signaling in schistosome reproduction. It describes the conserved and particular features of schistosome RTKs, their implication in gametogenesis and reproduction processes and summarizes recent works indicating that RTKs and their signaling partners are interesting chemotherapeutical targets in new programs of control. PMID:25101117

  7. Nitration of Hsp90 on Tyrosine 33 Regulates Mitochondrial Metabolism*

    PubMed Central

    Franco, Maria C.; Ricart, Karina C.; Gonzalez, Analía S.; Dennys, Cassandra N.; Nelson, Pascal A.; Janes, Michael S.; Mehl, Ryan A.; Landar, Aimee; Estévez, Alvaro G.

    2015-01-01

    Peroxynitrite production and tyrosine nitration are present in several pathological conditions, including neurodegeneration, stroke, aging, and cancer. Nitration of the pro-survival chaperone heat shock protein 90 (Hsp90) in position 33 and 56 induces motor neuron death through a toxic gain-of-function. Here we show that nitrated Hsp90 regulates mitochondrial metabolism independently of the induction of cell death. In PC12 cells, a small fraction of nitrated Hsp90 was located on the mitochondrial outer membrane and down-regulated mitochondrial membrane potential, oxygen consumption, and ATP production. Neither endogenous Hsp90 present in the homogenate nor unmodified and fully active recombinant Hsp90 was able to compete with the nitrated protein for the binding to mitochondria. Moreover, endogenous or recombinant Hsp90 did not prevent the decrease in mitochondrial activity but supported nitrated Hsp90 mitochondrial gain-of-function. Nitrotyrosine in position 33, but not in any of the other four tyrosine residues prone to nitration in Hsp90, was sufficient to down-regulate mitochondrial activity. Thus, in addition to induction of cell death, nitrated Hsp90 can also regulate mitochondrial metabolism, suggesting that depending on the cell type, distinct Hsp90 nitration states regulate different aspects of cellular metabolism. This regulation of mitochondrial homeostasis by nitrated Hsp90 could be of particular relevance in cancer cells. PMID:26085096

  8. Tyrosine motifs are required for prestin basolateral membrane targeting

    PubMed Central

    Zhang, Yifan; Moeini-Naghani, Iman; Bai, JunPing; Santos-Sacchi, Joseph; Navaratnam, Dhasakumar S.

    2015-01-01

    ABSTRACT Prestin is targeted to the lateral wall of outer hair cells (OHCs) where its electromotility is critical for cochlear amplification. Using MDCK cells as a model system for polarized epithelial sorting, we demonstrate that prestin uses tyrosine residues, in a YXXΦ motif, to target the basolateral surface. Both Y520 and Y667 are important for basolateral targeting of prestin. Mutation of these residues to glutamine or alanine resulted in retention within the Golgi and delayed egress from the Golgi in Y667Q. Basolateral targeting is restored upon mutation to phenylalanine suggesting the importance of a phenol ring in the tyrosine side chain. We also demonstrate that prestin targeting to the basolateral surface is dependent on AP1B (μ1B), and that prestin uses transferrin containing early endosomes in its passage from the Golgi to the basolateral plasma membrane. The presence of AP1B (μ1B) in OHCs, and parallels between prestin targeting to the basolateral surface of OHCs and polarized epithelial cells suggest that outer hair cells resemble polarized epithelia rather than neurons in this important phenotypic measure. PMID:25596279

  9. Signaling Network Map of Endothelial TEK Tyrosine Kinase.

    PubMed

    Khan, Aafaque Ahmad; Sandhya, Varot K; Singh, Priyata; Parthasarathy, Deepak; Kumar, Awinav; Advani, Jayshree; Gattu, Rudrappa; Ranjit, Dhanya V; Vaidyanathan, Rama; Mathur, Premendu Prakash; Prasad, T S Keshava; Mac Gabhann, F; Pandey, Akhilesh; Raju, Rajesh; Gowda, Harsha

    2014-01-01

    TEK tyrosine kinase is primarily expressed on endothelial cells and is most commonly referred to as TIE2. TIE2 is a receptor tyrosine kinase modulated by its ligands, angiopoietins, to regulate the development and remodeling of vascular system. It is also one of the critical pathways associated with tumor angiogenesis and familial venous malformations. Apart from the vascular system, TIE2 signaling is also associated with postnatal hematopoiesis. Despite the involvement of TIE2-angiopoietin system in several diseases, the downstream molecular events of TIE2-angiopoietin signaling are not reported in any pathway repository. Therefore, carrying out a detailed review of published literature, we have documented molecular signaling events mediated by TIE2 in response to angiopoietins and developed a network map of TIE2 signaling. The pathway information is freely available to the scientific community through NetPath, a manually curated resource of signaling pathways. We hope that this pathway resource will provide an in-depth view of TIE2-angiopoietin signaling and will lead to identification of potential therapeutic targets for TIE2-angiopoietin associated disorders.

  10. Inhibition of lymphoid tyrosine phosphatase by benzofuran salicylic acids.

    PubMed

    Vang, Torkel; Xie, Yuli; Liu, Wallace H; Vidović, Dusica; Liu, Yidong; Wu, Shuangding; Smith, Deborah H; Rinderspacher, Alison; Chung, Caty; Gong, Gangli; Mustelin, Tomas; Landry, Donald W; Rickert, Robert C; Schürer, Stephan C; Deng, Shi-Xian; Tautz, Lutz

    2011-01-27

    The lymphoid tyrosine phosphatase (Lyp, PTPN22) is a critical negative regulator of T cell antigen receptor (TCR) signaling. A single-nucleotide polymorphism (SNP) in the ptpn22 gene correlates with the incidence of various autoimmune diseases, including type 1 diabetes, rheumatoid arthritis, and systemic lupus erythematosus. Since the disease-associated allele is a more potent inhibitor of TCR signaling, specific Lyp inhibitors may become valuable in treating autoimmunity. Using a structure-based approach, we synthesized a library of 34 compounds that inhibited Lyp with IC(50) values between 0.27 and 6.2 μM. A reporter assay was employed to screen for compounds that enhanced TCR signaling in cells, and several inhibitors displayed a dose-dependent, activating effect. Subsequent probing for Lyp's direct physiological targets by immunoblot analysis confirmed the ability of the compounds to inhibit Lyp in T cells. Selectivity profiling against closely related tyrosine phosphatases and in silico docking studies with the crystal structure of Lyp yielded valuable information for the design of Lyp-specific compounds. PMID:21190368

  11. Role of Receptor Tyrosine Kinase Signaling in Renal Fibrosis

    PubMed Central

    Liu, Feng; Zhuang, Shougang

    2016-01-01

    Renal fibrosis can be induced in different renal diseases, but ultimately progresses to end stage renal disease. Although the pathophysiologic process of renal fibrosis have not been fully elucidated, it is characterized by glomerulosclerosis and/or tubular interstitial fibrosis, and is believed to be caused by the proliferation of renal inherent cells, including glomerular epithelial cells, mesangial cells, and endothelial cells, along with defective kidney repair, renal interstitial fibroblasts activation, and extracellular matrix deposition. Receptor tyrosine kinases (RTKs) regulate a variety of cell physiological processes, including metabolism, growth, differentiation, and survival. Many studies from in vitro and animal models have provided evidence that RTKs play important roles in the pathogenic process of renal fibrosis. It is also showed that tyrosine kinases inhibitors (TKIs) have anti-fibrotic effects in basic research and clinical trials. In this review, we summarize the evidence for involvement of specific RTKs in renal fibrosis process and the employment of TKIs as a therapeutic approach for renal fibrosis. PMID:27331812

  12. Tyrosine kinase inhibitors: New class of antimalarials on the horizon?

    PubMed

    Pathak, Vrushali; Colah, Roshan; Ghosh, Kanjaksha

    2015-08-01

    Development of the antimalarial drug resistant strains has currently become a major public health challenge. There is an urgent need to develop new antimalarial drugs. Tyrosine kinase inhibitors (TKIs) are receiving increasing attention as anticancer therapy. It has revolutionarised the management of CML to say the least. TKIs are also increasingly being implicated in complicated but vital life cycle of malaria parasite. Hence we tested two commonly used but different classes of TKIs (imatinib and sorafenib) in-vitro for their antimalarial activity and possible synergistic activity with existing antimalarial drug. Antimalarial activity was tested with the help of modified WHO microtest technique in-vitro for five different Plasmodium falciparum laboratory strains (3D7, Dd2, 7G8, MRC2, PKL9). Imatinib and sorafenib showed a promising antimalarial activity with all the strains. These compounds caused dose dependent inhibition of parasite maturation. The isobologram analysis of the interactions of these TKIs with standard antimalarial drug, artesunate revealed distinct patterns of synergism, additivity and antagonism at different ratios. Imatinib showed worthwhile synergism with artesunate indicating imatinib and other tyrosine kinase inhibitors may have significant antimalarial activity and can be used in combination therapy. PMID:26142327

  13. Hydration of protonated aromatic amino acids: phenylalanine, tryptophan, and tyrosine.

    PubMed

    Gao, Bing; Wyttenbach, Thomas; Bowers, Michael T

    2009-04-01

    The first steps of hydration of the protonated aromatic amino acids phenylalanine, tryptophan, and tyrosine were studied experimentally employing a mass spectrometer equipped with a drift cell to examine the sequential addition of individual water molecules in equilibrium experiments and theoretically by a combination of molecular mechanics and electronic structure calculations (B3LYP/6-311++G**) on the three amino acid systems including up to five water molecules. It is found that both the ammonium and carboxyl groups offer good water binding sites with binding energies of the order of 13 kcal/mol for the first water molecule. Subsequent water molecules bind less strongly, in the range of 7-11 kcal/mol for the second through fifth water molecules. The ammonium group is able to host up to three water molecules and the carboxyl group one water molecule before additional water molecules bind either to the amino acid side chain as in tyrosine or to already-bound water in a second solvation shell around the ammonium group. Reasons for the surprisingly high water affinity of the neutral carboxyl group, comparable to that of the charge-carrying ammonium group, are found to be high intrinsic hydrophilicity, favorable charge-dipole alignment, and--for the case of multiply hydrated species--favorable dipole-dipole interaction among water molecules and the lack of alternative fully exposed hydration sites.

  14. Inhibition of the Hematopoietic Protein Tyrosine Phosphatase by Phenoxyacetic Acids.

    PubMed

    Bobkova, Ekaterina V; Liu, Wallace H; Colayco, Sharon; Rascon, Justin; Vasile, Stefan; Gasior, Carlton; Critton, David A; Chan, Xochella; Dahl, Russell; Su, Ying; Sergienko, Eduard; Chung, Thomas D Y; Mustelin, Tomas; Page, Rebecca; Tautz, Lutz

    2011-02-01

    Protein tyrosine phosphatases (PTPs) have only recently become the focus of attention in the search for novel drug targets despite the fact that they play vital roles in numerous cellular processes and are implicated in many human diseases. The hematopoietic protein tyrosine phosphatase (HePTP) is often found dysregulated in preleukemic myelodysplastic syndrome (MDS), as well as in acute myelogenous leukemia (AML). Physiological substrates of HePTP include the mitogen-activated protein kinases (MAPKs) ERK1/2 and p38. Specific modulators of HePTP catalytic activity will be useful for elucidating mechanisms of MAPK regulation in hematopietic cells, and may also provide treatments for hematopoietic malignancies such as AML. Here we report the discovery of phenoxyacetic acids as inhibitors of HePTP. Structure-activity relationship (SAR) analysis and in silico docking studies reveal the molecular basis of HePTP inhibition by these compounds. We also show that these compounds are able to penetrate cell membranes and inhibit HePTP in human T lymphocytes.

  15. Robust versatile tyrosine kinase assay for HTS in drug discovery

    NASA Astrophysics Data System (ADS)

    Deshpande, Sudhir S.; Mineyev, I.; Owicki, John C.

    1999-04-01

    A fluorescence polarization assay was developed as an alternative to the radiolabeled SPA assays currently used to monitor the activity of tyrosine kinases in drug discovery. The assay can be used with enzymes having substrate specificity similar to that of the insulin receptor, the EGF receptor and the Src kinase receptor enzymes. The assay is easy to configure in 96, 384 and 1536-well microplates in assay volumes ranging from (mu) L with minimal efforts. The reconstituted reagents are stable for up to 24 hr at ambient temperatures, thereby minimizing the need for replenishing the stock solutions during the course of a high-throughput screen. Because of the stability and equilibrium kinetics, the assay allows the user the luxury of scheduling the reading of plates any time up to 24 hr after the completion of the assay without substantial deterioration in the assay signal. The antibody and the tracer solutions can also be premixed and added as a preformed complex in a single step. The performance of the assay with the insulin receptor kinase is described. In addition, given the diversity of the substrates used in measuring the activity of different tyrosine kinases, LJL's on-going efforts to provide different antibodies of wide ranging specificity and sensitivity are described.

  16. Phospho-tyrosine dependent protein–protein interaction network

    PubMed Central

    Grossmann, Arndt; Benlasfer, Nouhad; Birth, Petra; Hegele, Anna; Wachsmuth, Franziska; Apelt, Luise; Stelzl, Ulrich

    2015-01-01

    Post-translational protein modifications, such as tyrosine phosphorylation, regulate protein–protein interactions (PPIs) critical for signal processing and cellular phenotypes. We extended an established yeast two-hybrid system employing human protein kinases for the analyses of phospho-tyrosine (pY)-dependent PPIs in a direct experimental, large-scale approach. We identified 292 mostly novel pY-dependent PPIs which showed high specificity with respect to kinases and interacting proteins and validated a large fraction in co-immunoprecipitation experiments from mammalian cells. About one-sixth of the interactions are mediated by known linear sequence binding motifs while the majority of pY-PPIs are mediated by other linear epitopes or governed by alternative recognition modes. Network analysis revealed that pY-mediated recognition events are tied to a highly connected protein module dedicated to signaling and cell growth pathways related to cancer. Using binding assays, protein complementation and phenotypic readouts to characterize the pY-dependent interactions of TSPAN2 (tetraspanin 2) and GRB2 or PIK3R3 (p55γ), we exemplarily provide evidence that the two pY-dependent PPIs dictate cellular cancer phenotypes. PMID:25814554

  17. Acute administration of l-tyrosine alters energetic metabolism of hippocampus and striatum of infant rats.

    PubMed

    Ramos, Andrea C; Ferreira, Gabriela K; Carvalho-Silva, Milena; Furlanetto, Camila B; Gonçalves, Cinara L; Ferreira, Gustavo C; Schuck, Patrícia F; Streck, Emilio L

    2013-08-01

    Tyrosinemia type II is an inborn error of metabolism caused by mutations in the gene that encodes tyrosine aminotransferase, which leads to increased blood tyrosine levels. Considering that tyrosine levels are highly elevated in fluids of patients with tyrosinemia type II, and that previous studies demonstrated significant alterations in brain energy metabolism of young rats caused by l-tyrosine, the present study aimed to evaluate the effect of acute administration of l-tyrosine on the activities of citrate synthase, malate dehydrogenase, succinate dehydrogenase, and mitochondrial respiratory chain complexes I, II, II-III, and IV in posterior cortex, hippocampus, and striatum of infant rats. Wistar rats (10 days old) were killed 1h after a single intraperitoneal injection of tyrosine (500 mg/kg) or saline. The activities of energy metabolism enzymes were evaluated in brain of rats. Our results demonstrated that acute administration of l-tyrosine inhibited the activity of citrate synthase activity in striatum and increased the activities of malate dehydrogenase and succinate dehydrogenase in hippocampus. On the other hand, these enzymes were not affected in posterior cortex. The activities of complex I and complex II were inhibited by acute administration of l-tyrosine in striatum. On the other hand, the acute administration of l-tyrosine increased the activity of activity of complex II-III in hippocampus. Complex IV was not affected by acute administration of l-tyrosine in infant rats. Our results indicate an alteration in the energy metabolism in hippocampus and striatum of infant rats after acute administration of l-tyrosine. If the same effects occur in the brain of the patients, it is possible that energy metabolism impairment may be contribute to possible damage in memory and cognitive processes in patients with tyrosinemia type II. PMID:23602810

  18. Tyrosine inhibits creatine kinase activity in cerebral cortex of young rats.

    PubMed

    de Andrade, Rodrigo Binkowski; Gemelli, Tanise; Rojas, Denise Bertin; Funchal, Cláudia; Dutra-Filho, Carlos Severo; Wannmacher, Clovis Milton Duval

    2011-09-01

    Tyrosine accumulates in inborn errors of tyrosine catabolism, especially in tyrosinemia type II, where tyrosine levels are highly elevated in tissues and physiological fluids of affected patients. Tyrosinemia type II is a disorder of autosomal recessive inheritance characterized by neurological symptoms similar to those observed in patients with creatine deficiency syndromes. Considering that the mechanisms of brain damage in these disorders are poorly known, in the present study our main objective was to investigate the in vivo and in vitro effects of different concentrations and preincubation times of tyrosine on cytosolic and mitochondrial creatine kinase activities of the cerebral cortex from 14-day-old Wistar rats. The cytosolic CK was reduced by 15% at 1 mM and 32% at 2 mM tyrosine. Similarly, the mitochondrial CK was inhibited by 15% at 1 mM and 22% at 2 mM tyrosine. We observed that the inhibition caused by tyrosine was concentration-dependent and was prevented by reduced glutathione. Results also indicated that mitochondrial, but not cytosolic creatine kinase activity was inhibited by tyrosine in a time-dependent way. Finally, a single injection of L-Tyrosine methyl ester administered i.p. decreased cytosolic (31%) and mitochondrial (18%) creatine kinase activities of brain cortex from rats. Considering that creatine kinase is an enzyme dependent of thiol residues for its function and tyrosine induces oxidative stress, the results suggest that the inhibition caused by tyrosine might occur by oxidation of essential sulfhydryl groups of the enzyme. In case this also occurs in patients with tyrosinemia, it is possible that creatine kinase inhibition may contribute to the neurological dysfunction characteristic of tyrosinemia.

  19. Acute administration of l-tyrosine alters energetic metabolism of hippocampus and striatum of infant rats.

    PubMed

    Ramos, Andrea C; Ferreira, Gabriela K; Carvalho-Silva, Milena; Furlanetto, Camila B; Gonçalves, Cinara L; Ferreira, Gustavo C; Schuck, Patrícia F; Streck, Emilio L

    2013-08-01

    Tyrosinemia type II is an inborn error of metabolism caused by mutations in the gene that encodes tyrosine aminotransferase, which leads to increased blood tyrosine levels. Considering that tyrosine levels are highly elevated in fluids of patients with tyrosinemia type II, and that previous studies demonstrated significant alterations in brain energy metabolism of young rats caused by l-tyrosine, the present study aimed to evaluate the effect of acute administration of l-tyrosine on the activities of citrate synthase, malate dehydrogenase, succinate dehydrogenase, and mitochondrial respiratory chain complexes I, II, II-III, and IV in posterior cortex, hippocampus, and striatum of infant rats. Wistar rats (10 days old) were killed 1h after a single intraperitoneal injection of tyrosine (500 mg/kg) or saline. The activities of energy metabolism enzymes were evaluated in brain of rats. Our results demonstrated that acute administration of l-tyrosine inhibited the activity of citrate synthase activity in striatum and increased the activities of malate dehydrogenase and succinate dehydrogenase in hippocampus. On the other hand, these enzymes were not affected in posterior cortex. The activities of complex I and complex II were inhibited by acute administration of l-tyrosine in striatum. On the other hand, the acute administration of l-tyrosine increased the activity of activity of complex II-III in hippocampus. Complex IV was not affected by acute administration of l-tyrosine in infant rats. Our results indicate an alteration in the energy metabolism in hippocampus and striatum of infant rats after acute administration of l-tyrosine. If the same effects occur in the brain of the patients, it is possible that energy metabolism impairment may be contribute to possible damage in memory and cognitive processes in patients with tyrosinemia type II.

  20. Tyrosine O-prenyltransferase SirD catalyzes S-, C-, and N-prenylations on tyrosine and tryptophan derivatives.

    PubMed

    Rudolf, Jeffrey D; Poulter, C Dale

    2013-12-20

    The tyrosine O-prenyltransferase SirD in Leptosphaeria maculans catalyzes normal prenylation of the hydroxyl group in tyrosine as the first committed step in the biosynthesis of the phytotoxin sirodesmin PL. SirD also catalyzes normal N-prenylation of 4-aminophenylalanine and normal C-prenylation at C7 of tryptophan. In this study, we found that 4-mercaptophenylalanine and several derivatives of tryptophan are also substrates for prenylation by dimethylallyl diphosphate. Incubation of SirD with 4-mercaptophenylalanine gave normal S-prenylated mercaptophenylalanine. We found that incubation of the enzyme with tryptophan gave reverse prenylation at N1 in addition to the previously reported normal prenylation at C7. 4-Methyltryptophan also gave normal prenylation at C7 and reverse prenylation at N1, whereas 4-methoxytryptophan gave normal and reverse prenylation at C7, and 7-methyltryptophan gave normal prenylation at C6 and reverse prenylation at N1. The ability of SirD to prenylate at three different sites on the indole nucleus, with normal and reverse prenylation at one of the sites, is similar to behavior seen for dimethylallyltryptophan synthase. The multiple products produced by SirD suggests it and dimethylallyltryptophan synthase use a dissociative electrophilic mechanism for alkylation of amino acid substrates.

  1. Unlocking Doors without Keys: Activation of Src by Truncated C-terminal Intracellular Receptor Tyrosine Kinases Lacking Tyrosine Kinase Activity

    PubMed Central

    Mezquita, Belén; Mezquita, Pau; Pau, Montserrat; Mezquita, Jovita; Mezquita, Cristóbal

    2014-01-01

    One of the best examples of the renaissance of Src as an open door to cancer has been the demonstration that just five min of Src activation is sufficient for transformation and also for induction and maintenance of cancer stem cells [1]. Many tyrosine kinase receptors, through the binding of their ligands, become the keys that unlock the structure of Src and activate its oncogenic transduction pathways. Furthermore, intracellular isoforms of these receptors, devoid of any tyrosine kinase activity, still retain the ability to unlock Src. This has been shown with a truncated isoform of KIT (tr-KIT) and a truncated isoform of VEGFR-1 (i21-VEGFR-1), which are intracellular and require no ligand binding, but are nonetheless able to activate Src and induce cell migration and invasion of cancer cells. Expression of the i21-VEGFR-1 is upregulated by the Notch signaling pathway and repressed by miR-200c and retinoic acid in breast cancer cells. Both Notch inhibitors and retinoic acid have been proposed as potential therapies for invasive breast cancer. PMID:24709904

  2. Tyrosination of α-tubulin controls the initiation of processive dynein-dynactin motility.

    PubMed

    McKenney, Richard J; Huynh, Walter; Vale, Ronald D; Sirajuddin, Minhajuddin

    2016-06-01

    Post-translational modifications (PTMs) of α/β-tubulin are believed to regulate interactions with microtubule-binding proteins. A well-characterized PTM involves in the removal and re-ligation of the C-terminal tyrosine on α-tubulin, but the purpose of this tyrosination-detyrosination cycle remains elusive. Here, we examined the processive motility of mammalian dynein complexed with dynactin and BicD2 (DDB) on tyrosinated versus detyrosinated microtubules. Motility was decreased ~fourfold on detyrosinated microtubules, constituting the largest effect of a tubulin PTM on motor function observed to date. This preference is mediated by dynactin's microtubule-binding p150 subunit rather than dynein itself. Interestingly, on a bipartite microtubule consisting of tyrosinated and detyrosinated segments, DDB molecules that initiated movement on tyrosinated tubulin continued moving into the segment composed of detyrosinated tubulin. This result indicates that the α-tubulin tyrosine facilitates initial motor-tubulin encounters, but is not needed for subsequent motility. Our results reveal a strong effect of the C-terminal α-tubulin tyrosine on dynein-dynactin motility and suggest that the tubulin tyrosination cycle could modulate the initiation of dynein-driven motility in cells.

  3. Post-translational Tyrosine Nitration of Eosinophil Granule Toxins Mediated by Eosinophil Peroxidase*

    PubMed Central

    Ulrich, Martina; Petre, Alina; Youhnovski, Nikolay; Prömm, Franziska; Schirle, Markus; Schumm, Michael; Pero, Ralph S.; Doyle, Alfred; Checkel, James; Kita, Hirohito; Thiyagarajan, Nethaji; Acharya, K. Ravi; Schmid-Grendelmeier, Peter; Simon, Hans-Uwe; Schwarz, Heinz; Tsutsui, Masato; Shimokawa, Hiroaki; Bellon, Gabriel; Lee, James J.; Przybylski, Michael; Döring, Gerd

    2008-01-01

    Nitration of tyrosine residues has been observed during various acute and chronic inflammatory diseases. However, the mechanism of tyrosine nitration and the nature of the proteins that become tyrosine nitrated during inflammation remain unclear. Here we show that eosinophils but not other cell types including neutrophils contain nitrotyrosine-positive proteins in specific granules. Furthermore, we demonstrate that the human eosinophil toxins, eosinophil peroxidase (EPO), major basic protein, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), and the respective murine toxins, are post-translationally modified by nitration at tyrosine residues during cell maturation. High resolution affinity-mass spectrometry identified specific single nitration sites at Tyr349 in EPO and Tyr33 in both ECP and EDN. ECP and EDN crystal structures revealed and EPO structure modeling suggested that the nitrated tyrosine residues in the toxins are surface exposed. Studies in EPO-/-, gp91phox-/-, and NOS-/- mice revealed that tyrosine nitration of these toxins is mediated by EPO in the presence of hydrogen peroxide and minute amounts of NOx. Tyrosine nitration of eosinophil granule toxins occurs during maturation of eosinophils, independent of inflammation. These results provide evidence that post-translational tyrosine nitration is unique to eosinophils. PMID:18694936

  4. Novel Anthraquinone-based Derivatives as Potent Inhibitors for Receptor Tyrosine Kinases

    PubMed Central

    Stasevych, M.; Zvarych, V.; Lunin, V.; Halenova, T.; Savchuk, O.; Dudchak, O.; Vovk, M.; Novikov, V.

    2015-01-01

    The influence of new derivatives of 9,10-anthraquinone with benzoylthiourea, thiazole, triazole and amino acid fragments on the activity of membrane-associated tyrosine kinases was investigated. Inhibitors of protein tyrosine kinase activity of the membrane fraction, as promising agents to search for new potential anticancer agents among the studied compounds, were discovered. PMID:26798182

  5. Kinetic Characterization of O-Phospho-L-Tyrosine Phosphohydrolase Activity of Two Fungal Phytases.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fungal phytases belonging to 'Histidine Acid Phosphatase' or HAP class of phosphomonoesterase that catalyzes the hydrolysis of phytic acid could also hydrolyze O-phospho-tyrosine. Two phytases from Aspergillus niger and Aspergillus awamori with pH optima 2.5 were tested for phospho-tyrosine hydrola...

  6. Tyrosine impairs enzymes of energy metabolism in cerebral cortex of rats.

    PubMed

    de Andrade, Rodrigo Binkowski; Gemelli, Tanise; Rojas, Denise Bertin; Funchal, Cláudia; Dutra-Filho, Carlos Severo; Wannmacher, Clovis Milton Duval

    2012-05-01

    Tyrosine levels are abnormally elevated in tissues and physiological fluids of patients with inborn errors of tyrosine catabolism, especially in tyrosinemia type II, which is caused by deficiency of tyrosine aminotransferase and provokes eyes, skin, and central nervous system disturbances. Considering that the mechanisms of brain damage in these disorders are poorly known, in this study, we investigated the in vivo and in vitro effects of tyrosine on some parameters of energy metabolism in cerebral cortex of 14-day-old Wistar rats. We observed that 2 mM tyrosine inhibited in vitro the pyruvate kinase (PK) activity and that this inhibition was prevented by 1 mM reduced glutathione with 30, 60, and 90 min of preincubation. Moreover, administration of tyrosine methyl ester (TME) (0.5 mg/g of body weight) decreased the activity of PK and this reduction was prevented by pre-treatment with creatine (Cr). On the other hand, tyrosine did not alter adenylate kinase (AK) activity in vitro, but administration of TME enhanced AK activity not prevented by Cr pre-treatment. Finally, TME administration decreased the activity of CK from cytosolic and mitochondrial fractions and this diminution was prevented by Cr pre-treatment. The results suggest that tyrosine alters essential sulfhydryl groups necessary for CK and PK functions, possibly through oxidative stress. In case this also occurs in the patients, it is possible that energy metabolism alterations may contribute, along with other mechanisms, to the neurological dysfunction of hypertyrosinemias.

  7. Tyrosine Phosphorylation of SGEF Regulates RhoG Activity and Cell Migration

    PubMed Central

    Okuyama, Yusuke; Umeda, Kentaro; Negishi, Manabu; Katoh, Hironori

    2016-01-01

    SGEF and Ephexin4 are members of the Ephexin subfamily of RhoGEFs that specifically activate the small GTPase RhoG. It is reported that Ephexin1 and Ephexin5, two well-characterized Ephexin subfamily RhoGEFs, are tyrosine-phosphorylated by Src, and that their phosphorylation affect their activities and functions. In this study, we show that SGEF, but not Ephexin4, is tyrosine-phosphorylated by Src. Tyrosine phosphorylation of SGEF suppresses its interaction with RhoG, the elevation of RhoG activity, and SGEF-mediated promotion of cell migration. We identified tyrosine 530 (Y530), which is located within the Dbl homology domain, as a major phosphorylation site of SGEF by Src, and Y530F mutation blocked the inhibitory effect of Src on SGEF. Taken together, these results suggest that the activity of SGEF is negatively regulated by tyrosine phosphorylation of the DH domain. PMID:27437949

  8. The Antidepressant Effect of L-Tyrosine-Loaded Nanoparticles: Behavioral Aspects

    PubMed Central

    Alabsi, Abdelrahman; Khoudary, Adel Charbel; Abdelwahed, Wassim

    2016-01-01

    Background Depression has been linked to disruption in the cerebral levels of specific neurotransmitters. L-tyrosine is a precursor of more than one of the neurotransmitters affected by depression. Even though setbacks of monoamines precursors include high doses and low efficiency, many studies have suggested using L-tyrosine as antidepressant. Purpose The purpose of this study was to explore the possible antidepressant effect of L-tyrosine loaded in a nanoparticle-designed formula, using behavioral tests in acute and chronic mild stress (CMS) models of depression in rats. Methods Animals from both models received L-tyrosine-loaded nanoparticles (5 or 10 mg/kg), L-tyrosine solution (10 mg/kg), fluoxetine (10 mg/kg) or placebo daily for 21 days. Rats from the acute stress model of depression were subjected to open field and forced swim tests (FSTs). For the CMS model, sucrose preference test was carried out. Additionally, 3 profiles of the nanoparticles formula were tested in vitro. High dissolution rate and entrapment efficiency were obtained from the in vitro tests. Moreover, L-tyrosine-loaded nanoparticles 10 mg/kg and fluoxetine 10 mg/kg significantly decreased the immobility time in the FST, concomitant with restoration of the basal levels of locomotor activity, distance travelled and rearing counts. Also, an increase of the sucrose consumption was recorded in the sucrose preference test after treatment with L-tyrosine-loaded nanoparticles 10 mg/kg and fluoxetine 10 mg/kg. Results The positive results after treatment with L-tyrosine-loaded nanoparticles, through behavioral tests, are probably attributed to restorating the basal levels of the cerebral noradrenaline. Conclusion The effects of L-tyrosine administration on the cerebral levels of tyrosine hydroxylase and corticotropin-releasing factor should be further investigated.

  9. Tyrosine monitoring in children with early and continuously treated phenylketonuria: results of an international practice survey.

    PubMed

    Sharman, Rachael; Sullivan, Karen A; Young, Ross McD; McGill, James J

    2010-12-01

    Investigations into the biochemical markers associated with executive function (EF) impairment in children with early and continuously treated phenylketonuria (ECT-PKU) remain largely phenylalanine-only focused, despite experimental data showing that a high phenylalanine:tyrosine (phe:tyr) ratio is more strongly associated with EF deficit than phe alone. A high phe:tyr ratio is hypothesized to lead to a reduction in dopamine synthesis within the brain, which in turn results in the development of EF impairment. This paper provides a snapshot of current practice in the monitoring and/or treatment of tyrosine levels in children with PKU, across 12 countries from Australasia, North America and Europe. Tyrosine monitoring in this population has increased over the last 5 years, with over 80% of clinics surveyed reporting routine monitoring of tyrosine levels in infancy alongside phe levels. Twenty-five percent of clinics surveyed reported actively treating/managing tyrosine levels (with supplemental tyrosine above that contained in PKU formulas) to ensure tyrosine levels remain within normal ranges. Anecdotally, supplemental tyrosine has been reported to ameliorate symptoms of both attention deficit hyperactivity disorder and depression in this population. EF assessment of children with ECT-PKU was likewise highly variable, with 50% of clinics surveyed reporting routine assessments of intellectual function. However when function was assessed, test instruments chosen tended towards global measures of IQ prior to school entry, rather than specific assessment of EF development. Further investigation of the role of tyrosine and its relationship with phe and EF development is needed to establish whether routine tyrosine monitoring and increased supplementation is recommended. PMID:20882350

  10. The Antidepressant Effect of L-Tyrosine-Loaded Nanoparticles: Behavioral Aspects

    PubMed Central

    Alabsi, Abdelrahman; Khoudary, Adel Charbel; Abdelwahed, Wassim

    2016-01-01

    Background Depression has been linked to disruption in the cerebral levels of specific neurotransmitters. L-tyrosine is a precursor of more than one of the neurotransmitters affected by depression. Even though setbacks of monoamines precursors include high doses and low efficiency, many studies have suggested using L-tyrosine as antidepressant. Purpose The purpose of this study was to explore the possible antidepressant effect of L-tyrosine loaded in a nanoparticle-designed formula, using behavioral tests in acute and chronic mild stress (CMS) models of depression in rats. Methods Animals from both models received L-tyrosine-loaded nanoparticles (5 or 10 mg/kg), L-tyrosine solution (10 mg/kg), fluoxetine (10 mg/kg) or placebo daily for 21 days. Rats from the acute stress model of depression were subjected to open field and forced swim tests (FSTs). For the CMS model, sucrose preference test was carried out. Additionally, 3 profiles of the nanoparticles formula were tested in vitro. High dissolution rate and entrapment efficiency were obtained from the in vitro tests. Moreover, L-tyrosine-loaded nanoparticles 10 mg/kg and fluoxetine 10 mg/kg significantly decreased the immobility time in the FST, concomitant with restoration of the basal levels of locomotor activity, distance travelled and rearing counts. Also, an increase of the sucrose consumption was recorded in the sucrose preference test after treatment with L-tyrosine-loaded nanoparticles 10 mg/kg and fluoxetine 10 mg/kg. Results The positive results after treatment with L-tyrosine-loaded nanoparticles, through behavioral tests, are probably attributed to restorating the basal levels of the cerebral noradrenaline. Conclusion The effects of L-tyrosine administration on the cerebral levels of tyrosine hydroxylase and corticotropin-releasing factor should be further investigated. PMID:27647959

  11. Methods to monitor classical protein-tyrosine phosphatase oxidation

    PubMed Central

    Karisch, Robert; Neel, Benjamin G.

    2012-01-01

    SUMMARY Reactive oxygen species (ROS), particularly H2O2, act as intracellular second messengers in many signaling pathways. Protein-tyrosine phosphatases (PTPs) are now believed to be important targets of ROS. PTPs contain a conserved catalytic cysteine with an unusually low pKa. This property allows PTPs to execute nucleophilic attack on substrate phosphotyrosyl residues, but also renders them highly susceptible to oxidation. Reversible oxidation, which inactivates PTPs, is emerging as an important cellular regulatory mechanism and might contribute to human diseases, including cancer. Given their potential toxicity, it seems likely that ROS generation is highly controlled within cells to restrict oxidation to those PTPs that must be inactivated for signaling to proceed. Thus, identifying ROS-inactivated PTPs could be tantamount to finding the PTP(s) that critically regulate a specific signaling pathway. This article provides an overview of the methods currently available to identify and quantify PTP oxidation and outlines future challenges in redox signaling. PMID:22577968

  12. Current Management of Chronic Myeloid Leukemia with Tyrosine Kinase Inhibitors

    PubMed Central

    Haznedaroğlu, İbrahim C.

    2013-01-01

    The clinical outcomes and survival of tyrosine kinase inhibitor (TKI)-treated patients with chronic myeloid leukemia (CML) have been significantly improved. The aim of this editorial is to outline critical steps of TKI administration practices during the long-term clinical course of CML based on data obtained from randomized clinical trials and international recommendations. The efficacy of TKI treatment, TKI side effects, off-target complications, and long-term morbidities due to both the disease and the drug are common arguments in the management of CML. Complete hematological response, early complete cytogenetic response, faster major molecular response, and deeper, more durable molecular responses (MR4, MR4.5, MR5) are the ultimate goals for TKI-receiving patients with CML. Conflict of interest:None declared. PMID:24385803

  13. Tyrosine Kinase Receptor Landscape in Lung Cancer: Therapeutical Implications.

    PubMed

    Quintanal-Villalonga, A; Paz-Ares, Luis; Ferrer, Irene; Molina-Pinelo, S

    2016-01-01

    Lung cancer is a heterogeneous disease responsible for the most cases of cancer-related deaths. The majority of patients are clinically diagnosed at advanced stages, with a poor survival rate. For this reason, the identification of oncodrivers and novel biomarkers is decisive for the future clinical management of this pathology. The rise of high throughput technologies popularly referred to as "omics" has accelerated the discovery of new biomarkers and drivers for this pathology. Within them, tyrosine kinase receptors (TKRs) have proven to be of importance as diagnostic, prognostic, and predictive tools and, due to their molecular nature, as therapeutic targets. Along this review, the role of TKRs in the different lung cancer histologies, research on improvement of anti-TKR therapy, and the current approaches to manage anti-TKR resistance will be discussed. PMID:27528792

  14. Tyrosine Kinase Inhibitors Regulate OPG through Inhibition of PDGFRβ

    PubMed Central

    Tay, Mei Lin; Lin, Jian-Ming; Bava, Usha; Callon, Karen; Cornish, Jillian; Naot, Dorit; Grey, Andrew

    2016-01-01

    Nilotinib and imatinib are tyrosine kinase inhibitors (TKIs) used in the treatment of chronic myeloid leukemia (CML) and gastrointestinal stromal tumors (GIST). In vitro, imatinib and nilotinib inhibit osteoclastogenesis, and in patients they reduce levels of bone resorption. One of the mechanisms that might underlie these effects is an increase in the production of osteoprotegerin (OPG). In the current work we report that platelet-derived growth factor receptor beta (PDGFRβ) signaling regulates OPG production in vitro. In addition, we have shown that TKIs have effects on RANKL signaling through inhibition of the PDGFRβ and other target receptors. These findings have implications for our understanding of the mechanisms by which TKIs affect osteoclastogenesis, and the role of PDGFRβ signaling in regulating osteoclastogenesis. Further studies are indicated to confirm the clinical effects of PDGFRβ-inhibitors and to elaborate the intracellular pathways that underpin these effects. PMID:27737004

  15. Novel interaction partners of the TPR/MET tyrosine kinase.

    PubMed

    Schaaf, Christian P; Benzing, Jörg; Schmitt, Thomas; Erz, Dorothee H R; Tewes, Magdalena; Bartram, Claus R; Janssen, Johannes W G

    2005-02-01

    A large variety of biological processes is mediated by stimulation of the receptor tyrosine kinase MET. Screening a mouse embryo cDNA library, we were able to identify several novel, putative intracellular TPR/MET-substrates: SNAPIN, DCOHM, VAV-1, Sorting nexin 2, Death associated protein kinase 3, SMC-1, Centromeric protein C, and hTID-1. Interactions as identified by yeast two-hybrid analysis were validated in vitro and in vivo by mammalian two-hybrid studies, a far-western assay and coimmunoprecipitation. Participation in apoptosis-regulating mechanisms through interaction with DAPK-3 and cell cycle control via binding to nuclear proteins such as CENPC and SMC-1 are possible new aspects of intracellular MET signaling.

  16. Effects of Membrane Trafficking on Signaling by Receptor Tyrosine Kinases

    PubMed Central

    Miaczynska, Marta

    2013-01-01

    The intracellular trafficking machinery contributes to the spatial and temporal control of signaling by receptor tyrosine kinases (RTKs). The primary role in this process is played by endocytic trafficking, which regulates the localization of RTKs and their downstream effectors, as well as the duration and the extent of their activity. The key regulatory points along the endocytic pathway are internalization of RTKs from the plasma membrane, their sorting to degradation or recycling, and their residence in various endosomal compartments. Here I will review factors and mechanisms that modulate RTK signaling by (1) affecting receptor internalization, (2) regulating the balance between degradation and recycling of RTK, and (3) compartmentalization of signals in endosomes and other organelles. Cumulatively, these mechanisms illustrate a multilayered control of RTK signaling exerted by the trafficking machinery. PMID:24186066

  17. MET Receptor Tyrosine Kinase as an Autism Genetic Risk Factor

    PubMed Central

    Peng, Yun; Huentelman, Matthew; Smith, Christopher; Qiu, Shenfeng

    2014-01-01

    In this chapter, we will briefly discuss recent literature on the role of MET receptor tyrosine kinase (RTK) in brain development and how perturbation of MET signaling may alter normal neurodevelopmental outcomes. Recent human genetic studies have established MET as a risk factor for autism, and the molecular and cellular underpinnings of this genetic risk are only beginning to emerge from obscurity. Unlike many autism risk genes that encode synaptic proteins, the spatial and temporal expression pattern of MET RTK indicates this signaling system is ideally situated to regulate neuronal growth, functional maturation, and establishment of functional brain circuits, particularly in those brain structures involved in higher levels of cognition, social skills, and executive functions. PMID:24290385

  18. Receptor Tyrosine Kinases: Molecular Switches Regulating CNS Axon Regeneration

    PubMed Central

    Vigneswara, Vasanthy; Kundi, Sarina; Ahmed, Zubair

    2012-01-01

    The poor or lack of injured adult central nervous system (CNS) axon regeneration results in devastating consequences and poor functional recovery. The interplay between the intrinsic and extrinsic factors contributes to robust inhibition of axon regeneration of injured CNS neurons. The insufficient or lack of trophic support for injured neurons is considered as one of the major obstacles contributing to their failure to survive and regrow their axons after injury. In the CNS, many of the signalling pathways associated with neuronal survival and axon regeneration are regulated by several classes of receptor tyrosine kinases (RTK) that respond to a variety of ligands. This paper highlights and summarises the most relevant recent findings pertinent to different classes of the RTK family of molecules, with a particular focus on elucidating their role in CNS axon regeneration. PMID:22848811

  19. Mechanisms of resistance to EGFR tyrosine kinase inhibitors.

    PubMed

    Huang, Lihua; Fu, Liwu

    2015-09-01

    Since the discovery that non-small cell lung cancer (NSCLC) is driven by epidermal growth factor receptor (EGFR) mutations, the EGFR tyrosine kinase inhibitors (EGFR-TKIs, e.g., gefitinib and elrotinib) have been effectively used for clinical treatment. However, patients eventually develop drug resistance. Resistance to EGFR-TKIs is inevitable due to various mechanisms, such as the secondary mutation (T790M), activation of alternative pathways (c-Met, HGF, AXL), aberrance of the downstream pathways (K-RAS mutations, loss of PTEN), impairment of the EGFR-TKIs-mediated apoptosis pathway (BCL2-like 11/BIM deletion polymorphism), histologic transformation, ATP binding cassette (ABC) transporter effusion, etc. Here we review and summarize the known resistant mechanisms to EGFR-TKIs and provide potential targets for development of new therapeutic strategies.

  20. Tie2 and Eph Receptor Tyrosine Kinase Activation and Signaling

    PubMed Central

    Barton, William A.; Dalton, Annamarie C.; Seegar, Tom C.M.; Himanen, Juha P.

    2014-01-01

    The Eph and Tie cell surface receptors mediate a variety of signaling events during development and in the adult organism. As other receptor tyrosine kinases, they are activated on binding of extracellular ligands and their catalytic activity is tightly regulated on multiple levels. The Eph and Tie receptors display some unique characteristics, including the requirement of ligand-induced receptor clustering for efficient signaling. Interestingly, both Ephs and Ties can mediate different, even opposite, biological effects depending on the specific ligand eliciting the response and on the cellular context. Here we discuss the structural features of these receptors, their interactions with various ligands, as well as functional implications for downstream signaling initiation. The Eph/ephrin structures are already well reviewed and we only provide a brief overview on the initial binding events. We go into more detail discussing the Tie-angiopoietin structures and recognition. PMID:24478383

  1. Recent developments in receptor tyrosine kinases targeted anticancer therapy

    PubMed Central

    Raval, Samir H.; Singh, Ratn D.; Joshi, Dilip V.; Patel, Hitesh B.; Mody, Shailesh K.

    2016-01-01

    Novel concepts and understanding of receptors lead to discoveries and optimization of many small molecules and antibodies as anti-cancerous drugs. Receptor tyrosine kinases (RTKs) are such a promising class of receptors under the investigation in past three decades. RTKs are one of the essential mediators of cell signaling mechanism for various cellular processes. Transformations such as overexpression, dysregulation, or mutations of RTKs may result into malignancy, and thus are an important target for anticancer therapy. Numerous subfamilies of RTKs, such as epidermal growth factor receptor, vascular endothelial growth factor receptor, fibroblast growth factor receptors, insulin-like growth factor receptor, and hepatocyte growth factor receptor, have been being investigated in recent years as target for anticancer therapy. The present review focuses several small molecules drugs as well as monoclonal antibodies targeting aforesaid subfamilies either approved or under investigation to treat the various cancers. PMID:27051190

  2. Protein-Tyrosine Phosphatase 1B Substrates and Metabolic Regulation

    PubMed Central

    Bakke, Jesse; Haj, Fawaz G.

    2014-01-01

    Metabolic homeostasis requires integration of complex signaling networks which, when deregulated, contribute to metabolic syndrome and related disorders. Protein-tyrosine phosphatase 1B (PTP1B) has emerged as a key regulator of signaling networks that are implicated in metabolic diseases such as obesity and type 2 diabetes. In this review, we examine mechanisms that regulate PTP1B-substrate interaction, enzymatic activity and experimental approaches to identify PTP1B substrates. We then highlight findings that implicate PTP1B in metabolic regulation. In particular, insulin and leptin signaling are discussed as well as recently identified PTP1B substrates that are involved in endoplasmic reticulum stress response, cell-cell communication, energy balance and vesicle trafficking. In summary, PTP1B exhibits exquisite substrate specificity and is an outstanding pharmaceutical target for obesity and type 2 diabetes. PMID:25263014

  3. DIRS-1 and the other tyrosine recombinase retrotransposons.

    PubMed

    Poulter, R T M; Goodwin, T J D

    2005-01-01

    DIRS-1 is a retroelement from the slime mold Dictyostelium discoideum. Until recently only two related retrotransposons had been described: PAT from the nematode Panagrellus redivivus and Prt1 from the zygomycete fungus Phycomyces blakesleeanus. Analyses of the reverse transcriptase sequences encoded by these three elements suggested that they were closely related to each other and more distantly related to the Ty3/gypsy Long Terminal Repeat (LTR) retroelements. They have several unusual structural features that distinguish them from typical LTR elements. For instance, they each encode a tyrosine recombinase (YR), but not a DDE-type integrase or an aspartic protease. Although the DIRS-1-related elements are bordered by terminal repeats these differ from typical LTRs in a number of ways. In DIRS-1, for example, the terminal repeats are inverted (complementary), non-identical in sequence, and the outer edges of the terminal sequences are repeated (adjacent to each other) in the internal region. PAT has so-called "split" direct repeats in which the unrelated terminal sequences appear as direct repeats adjacent to each other in the internal region. The only repetition displayed by Prt1 is the presence of short inverted terminal repeats, but the sequenced copy of this element is believed to be a truncated version of an element with a structure resembling DIRS-1. The unusual structure of the terminal repeats of the DIRS1-like elements appears to be related to their replication via free circular intermediates. Site-specific recombination is believed to integrate the circle without creating duplications of the target sites. In recognition of these important distinctions it is proposed that the retrotransposons that encode tyrosine recombinases be called the tyrosine recombinase (or YR) retrotransposons. Recently a large number of additional YR retrotransposons have been described, including elements from fungi (zygomycetes and basidiomycetes), plants (green algae) and a

  4. Precision Therapy for Lung Cancer: Tyrosine Kinase Inhibitors and Beyond.

    PubMed

    Rajan, Arun; Schrump, David S

    2015-01-01

    For patients with advanced cancers there has been a concerted effort to transition from a generic treatment paradigm to one based on tumor-specific biologic, and patient-specific clinical characteristics. This approach, known as precision therapy has been made possible owing to widespread availability and a reduction in the cost of cutting-edge technologies that are used to study the genomic, proteomic, and metabolic attributes of individual tumors. This review traces the evolution of precision therapy for lung cancer from the identification of molecular subsets of the disease to the development and approval of tyrosine kinase, as well as immune checkpoint inhibitors for lung cancer therapy. Challenges of the precision therapy era including the emergence of acquired resistance, identification of untargetable mutations, and the effect on clinical trial design are discussed. We conclude by highlighting newer applications for the concept of precision therapy. PMID:26074108

  5. Tyrosine Kinase Receptor Landscape in Lung Cancer: Therapeutical Implications

    PubMed Central

    Quintanal-Villalonga, A.; Paz-Ares, Luis

    2016-01-01

    Lung cancer is a heterogeneous disease responsible for the most cases of cancer-related deaths. The majority of patients are clinically diagnosed at advanced stages, with a poor survival rate. For this reason, the identification of oncodrivers and novel biomarkers is decisive for the future clinical management of this pathology. The rise of high throughput technologies popularly referred to as “omics” has accelerated the discovery of new biomarkers and drivers for this pathology. Within them, tyrosine kinase receptors (TKRs) have proven to be of importance as diagnostic, prognostic, and predictive tools and, due to their molecular nature, as therapeutic targets. Along this review, the role of TKRs in the different lung cancer histologies, research on improvement of anti-TKR therapy, and the current approaches to manage anti-TKR resistance will be discussed. PMID:27528792

  6. Examination of tyrosine/adenine stacking interactions in protein complexes.

    PubMed

    Copeland, Kari L; Pellock, Samuel J; Cox, James R; Cafiero, Mauricio L; Tschumper, Gregory S

    2013-11-14

    The π-stacking interactions between tyrosine amino acid side chains and adenine-bearing ligands are examined. Crystalline protein structures from the protein data bank (PDB) exhibiting face-to-face tyrosine/adenine arrangements were used to construct 20 unique 4-methylphenol/N9-methyladenine (p-cresol/9MeA) model systems. Full geometry optimization of the 20 crystal structures with the M06-2X density functional theory method identified 11 unique low-energy conformations. CCSD(T) complete basis set (CBS) limit interaction energies were estimated for all of the structures to determine the magnitude of the interaction between the two ring systems. CCSD(T) computations with double-ζ basis sets (e.g., 6-31G*(0.25) and aug-cc-pVDZ) indicate that the MP2 method overbinds by as much as 3.07 kcal mol(-1) for the crystal structures and 3.90 kcal mol(-1) for the optimized structures. In the 20 crystal structures, the estimated CCSD(T) CBS limit interaction energy ranges from -4.00 to -6.83 kcal mol(-1), with an average interaction energy of -5.47 kcal mol(-1), values remarkably similar to the corresponding data for phenylalanine/adenine stacking interactions. Geometry optimization significantly increases the interaction energies of the p-cresol/9MeA model systems. The average estimated CCSD(T) CBS limit interaction energy of the 11 optimized structures is 3.23 kcal mol(-1) larger than that for the 20 crystal structures.

  7. Deregulated tyrosine-phenylalanine metabolism in pulmonary tuberculosis patients.

    PubMed

    Das, Mrinal Kumar; Bishwal, Subasa Chandra; Das, Aleena; Dabral, Deepti; Badireddy, Vinod Kumar; Pandit, Bhaswati; Varghese, George M; Nanda, Ranjan Kumar

    2015-04-01

    Metabolic profiling of biofluids from tuberculosis (TB) patients would help us in understanding the disease pathophysiology and may also be useful for the development of novel diagnostics and host-directed therapy. In this pilot study we have compared the urine metabolic profiles of two groups of subjects having similar TB symptoms and categorized as active TB (ATB, n = 21) and non-TB (NTB, n = 21) based on GeneXpert test results. Silylation, gas chromatography mass spectrometry, and standard chemometric methods were employed to identify the important molecules and deregulated metabolic pathways. Eleven active TB patients were followed up on longitudinally for comparative urine metabolic profiling with healthy controls (n = 11). A set of 42 features qualified to have a variable importance parameter score of > 1.5 of a partial least-squares discriminate analysis model and fold change of > 1.5 at p value < 0.05 between ATB and NTB. Using these variables, a receiver operating characteristics curve was plotted and the area under the curve was calculated to be 0.85 (95% CI: 0.72-0.96). Several of these variables that represent norepinephrine, gentisic acid, 4-hydroxybenzoic acid, hydroquinone, and 4-hydroxyhippuric acid are part of the tyrosine-phenylalanine metabolic pathway. In the longitudinal study we observed a treatment-dependent trend in the urine metabolome of follow-up samples, and subjects declared as clinically cured showed similar metabolic profile as those of asymptomatic healthy subjects. The deregulated tyrosine-phenylalanine axis reveals a potential target for diagnostics and intervention in TB. PMID:25693719

  8. Deregulated tyrosine-phenylalanine metabolism in pulmonary tuberculosis patients.

    PubMed

    Das, Mrinal Kumar; Bishwal, Subasa Chandra; Das, Aleena; Dabral, Deepti; Badireddy, Vinod Kumar; Pandit, Bhaswati; Varghese, George M; Nanda, Ranjan Kumar

    2015-04-01

    Metabolic profiling of biofluids from tuberculosis (TB) patients would help us in understanding the disease pathophysiology and may also be useful for the development of novel diagnostics and host-directed therapy. In this pilot study we have compared the urine metabolic profiles of two groups of subjects having similar TB symptoms and categorized as active TB (ATB, n = 21) and non-TB (NTB, n = 21) based on GeneXpert test results. Silylation, gas chromatography mass spectrometry, and standard chemometric methods were employed to identify the important molecules and deregulated metabolic pathways. Eleven active TB patients were followed up on longitudinally for comparative urine metabolic profiling with healthy controls (n = 11). A set of 42 features qualified to have a variable importance parameter score of > 1.5 of a partial least-squares discriminate analysis model and fold change of > 1.5 at p value < 0.05 between ATB and NTB. Using these variables, a receiver operating characteristics curve was plotted and the area under the curve was calculated to be 0.85 (95% CI: 0.72-0.96). Several of these variables that represent norepinephrine, gentisic acid, 4-hydroxybenzoic acid, hydroquinone, and 4-hydroxyhippuric acid are part of the tyrosine-phenylalanine metabolic pathway. In the longitudinal study we observed a treatment-dependent trend in the urine metabolome of follow-up samples, and subjects declared as clinically cured showed similar metabolic profile as those of asymptomatic healthy subjects. The deregulated tyrosine-phenylalanine axis reveals a potential target for diagnostics and intervention in TB.

  9. Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite.

    PubMed

    Reiter, C D; Teng, R J; Beckman, J S

    2000-10-20

    Tyrosine nitration is a widely used marker of peroxynitrite (ONOO(-)) produced from the reaction of nitric oxide with superoxide. Pfeiffer and Mayer (Pfeiffer, S., and Mayer, B. (1998) J. Biol. Chem. 273, 27280-27285) reported that superoxide produced from hypoxanthine plus xanthine oxidase in combination with nitric oxide produced from spermine NONOate did not nitrate tyrosine at neutral pH. They suggested that nitric oxide and superoxide at neutral pH form a less reactive intermediate distinct from preformed alkaline peroxynitrite that does not nitrate tyrosine. Using a stopped-flow spectrophotometer to rapidly mix potassium superoxide with nitric oxide at pH 7.4, we report that an intermediate spectrally and kinetically identical to preformed alkaline cis-peroxynitrite was formed in 100% yield. Furthermore, this intermediate nitrated tyrosine in the same yield and at the same rate as preformed peroxynitrite. Equivalent concentrations of nitric oxide under aerobic conditions in the absence of superoxide did not produce detectable concentrations of nitrotyrosine. Carbon dioxide increased the efficiency of nitration by nitric oxide plus superoxide to the same extent as peroxynitrite. In experiments using xanthine oxidase as a source of superoxide, tyrosine nitration was substantially inhibited by urate formed from hypoxanthine oxidation, which was sufficient to account for the lack of tyrosine nitration previously reported. We conclude that peroxynitrite formed from the reaction of nitric oxide with superoxide at physiological pH remains an important species responsible for tyrosine nitration in vivo. PMID:10906340

  10. ROLE OF TYROSINE-SULFATED PROTEINS IN RETINAL STRUCTURE AND FUNCTION

    PubMed Central

    Kanan, Y.; Al-Ubaidi, M.R.

    2014-01-01

    The extracellular matrix (ECM) plays a significant role in cellular and retinal health. The study of retinal tyrosine-sulfated proteins is an important first step toward understanding the role of ECM in retinal health and diseases. These secreted proteins are members of the retinal ECM. Tyrosine sulfation was shown to be necessary for the development of proper retinal structure and function. The importance of tyrosine sulfation is further demonstrated by the evolutionary presence of tyrosylprotein sulfotransferases, enzymes that catalyze proteins’ tyrosine sulfation, and the compensatory abilities of these enzymes. Research has identified four tyrosine-sulfated retinal proteins: fibulin 2, vitronectin, complement factor H (CFH), and opticin. Vitronectin and CFH regulate the activation of the complement system and are involved in the etiology of some cases of age-related macular degeneration. Analysis of the role of tyrosine sulfation in fibulin function showed that sulfation influences the protein's ability to regulate growth and migration. Although opticin was recently shown to exhibit anti-angiogenic properties, it is not yet determined what role sulfation plays in that function. Future studies focusing on identifying all of the tyrosine-sulfated retinal proteins would be instrumental in determining the impact of sulfation on retinal protein function in retinal homeostasis and diseases. PMID:25819460

  11. Conformational and vibrational analyses of meta-tyrosine: An experimental and theoretical study.

    PubMed

    Yao, Guohua; Zhang, Jingjing; Huang, Qing

    2015-01-01

    M-tyrosine is one kind of positional isomer of tyrosine which is widely applied in agrichemical, medicinal chemistry, and food science. However, the structural and vibrational features of m-tyrosine have not been reported or systematically investigated. In this work, potential energy surface (PES) calculations were used for searching and determining the stable zwitterionic conformers of m-tyrosine, and the Raman spectra of m-tyrosine and deuterated m-tyrosine were measured and interpreted based on theoretical computation. For the spectral simulation, several DFT-based quantum chemistry (QC) methods were employed, and the M06-2X functional with SMD solvent model was found to be best in reproducing the Raman spectra and geometrical property. As such, this study has not only presented a detailed study of m-tyrosine's vibrational property which is lack in the literature, but also may shed some light on the optimal choice of QC methods for calculation of conformations and vibrational properties of zwitterionic amino acids.

  12. Novel post-translational incorporation of tyrosine in PMA-activated polymorphonuclear leukocytes (PMN)

    SciTech Connect

    Nath, J.; Oliver, C.; Ohno, Y.; Gallin, J.I.

    1986-03-05

    During studies undertaken to determine whether stimulation of tubulin tyrosinolation occurs in PMA-activated PMN, a distinctly different and novel post-translational incorporation of tyrosine into multiple PMN proteins was observed. The reaction also occurred in organelle-depleted neutrophil cytoplasts and was highly exaggerated in organelle-enriched karyogranuloplasts. The incorporation was specific for tyrosine, did not require extracellular Ca/sup 2 +/ and was inhibited in the presence of a variety of reducing agents, intracellular scavengers of oxygen radicals and inhibitors of peroxidase-mediated reactions. The PMA-induced incorporation of tyrosine was completely absent in PMN from patients with chronic granulomatous disease, but occurred normally in PMN of a patient with myeloperoxidase deficiency. Moreover, the incorporation of tyrosine was blocked by N-acetyl-L-tyrosine but not by phenylalanine suggesting a requirement for the phenolic group. A two-fold increase in stable protein carbonyl derivatives was demonstrated suggesting an increased oxidative modification of the proteins. SDS urea PAGE and reversed phase HPLC did not reveal any detectable changes in the extent of protein cross-linking. The PMN tyrosine pool was approximately 900 ..mu..M and yet only 1 ..mu..M tyrosine was added in these experiments. The functional significance of this reaction is not yet clear.

  13. Partial purification and characterization of an enzyme from pea nuclei with protein tyrosine phosphatase activity.

    PubMed

    Guo, Y L; Roux, S J

    1995-01-01

    A pea (Pisum sativum L.) nuclear enzyme with protein tyrosine phosphatase activity has been partially purified and characterized. The enzyme has a molecular mass of 90 kD as judged by molecular sieve column chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Like animal protein tyrosine phosphatases it can be inhibited by low concentrations of molybdate and vanadate. It is also inhibited by heparin and spermine but not by either the acid phosphatase inhibitors citrate and tartrate or the protein serine/threonine phosphatase inhibitor okadaic acid. The enzyme does not require Ca2+, Mg2+, or Mn2+ for its activity but is stimulated by ethylenediaminetetraacetate and by ethyleneglycolbis(beta-aminoethyl ether)-N,N'-tetraacetic acid. It dephosphorylates phosphotyrosine residues on the four different 32P-tyrosine-labeled peptides tested but not the phosphoserine/threonine residues on casein and histone. Like some animal protein tyrosine phosphatases, it has a variable pH optimum depending on the substrate used: the optimum is 5.5 when the substrate is [32P]tyrosine-labeled lysozyme, but it is 7.0 when the substrate is [32P]tyrosine-labeled poly(glutamic acid, tyrosine). It has a Km of 4 microM when the lysozyme protein is used as a substrate.

  14. Quantitation of tyrosine hydroxylase, protein levels: Spot immunolabeling with an affinity-purified antibody

    SciTech Connect

    Haycock, J.W. )

    1989-09-01

    Tyrosine hydroxylase was purified from bovine adrenal chromaffin cells and rat pheochromocytoma using a rapid (less than 2 days) procedure performed at room temperature. Rabbits were immunized with purified enzyme that was denatured with sodium dodecylsulfate, and antibodies to tyrosine hydroxylase were affinity-purified from immune sera. A Western blot procedure using the affinity-purified antibodies and {sup 125}I-protein A demonstrated a selective labeling of a single Mr approximately 62,000 band in samples from a number of different tissues. The relative lack of background {sup 125}I-protein A binding permitted the development of a quantitative spot immunolabeling procedure for tyrosine hydroxylase protein. The sensitivity of the assay is 1-2 ng of enzyme. Essentially identical standard curves were obtained with tyrosine hydroxylase purified from rat pheochromocytoma, rat corpus striatum, and bovine adrenal medulla. An extract of PC 12 cells (clonal rat pheochromocytoma cells) was calibrated against purified rat pheochromocytoma tyrosine hydroxylase and used as an external standard against which levels of tyrosine hydroxylase in PC12 cells and other tissue were quantified. With this procedure, qualitative assessment of tyrosine hydroxylase protein levels can be obtained in a few hours and quantitative assessment can be obtained in less than a day.

  15. Production of tyrosine through phenylalanine hydroxylation bypasses the intrinsic feedback inhibition in Escherichia coli.

    PubMed

    Huang, Jin; Lin, Yuheng; Yuan, Qipeng; Yan, Yajun

    2015-04-01

    Tyrosine is a proteinogenic aromatic amino acid that is often used as a supplement of food and animal feed, as well as a (bio-)synthetic precursor to various pharmaceutically or industrially important molecules. Extensive metabolic engineering efforts have been made towards the efficient and cost-effective microbial production of tyrosine. Conventional strategies usually focus on eliminating intrinsic feedback inhibition and redirecting carbon flux into the shikimate pathway. In this study, we found that continuous conversion of phenylalanine into tyrosine by the action of tetrahydromonapterin (MH4)-utilizing phenylalanine 4-hydroxylase (P4H) can bypass the feedback inhibition in Escherichia coli, leading to tyrosine accumulation in the cultures. First, expression of the P4H from Xanthomonas campestris in combination with an MH4 recycling system in wild-type E. coli allowed the strain to accumulate tyrosine at 262 mg/L. On this basis, enhanced expression of the key enzymes associated with the shikimate pathway and the MH4 biosynthetic pathway resulted in the elevation of tyrosine production up to 401 mg/L in shake flasks. This work demonstrated a novel approach to tyrosine production and verified the possibility to alleviate feedback inhibition by creating a phenylalanine sink.

  16. Tyrosine administration decreases glutathione and stimulates lipid and protein oxidation in rat cerebral cortex.

    PubMed

    Sgaravatti, Angela M; Magnusson, Alessandra S; de Oliveira, Amanda S; Rosa, Andréa P; Mescka, Caroline Paula; Zanin, Fernanda R; Pederzolli, Carolina D; Wyse, Angela T S; Wannmacher, Clóvis M D; Wajner, Moacir; Dutra-Filho, Carlos Severo

    2009-09-01

    Tyrosine levels are abnormally elevated in tissues and physiological fluids of patients with inborn errors of tyrosine catabolism especially in tyrosinemia type II which is caused by deficiency of tyrosine aminotransferase (TAT) and provokes eyes, skin and central nervous system disturbances. We have recently reported that tyrosine promoted oxidative stress in vitro but the exact mechanisms of brain damage in these disorder are poorly known. In the present study, we investigated the in vivo effect of L-tyrosine (500 mg/Kg) on oxidative stress indices in cerebral cortex homogenates of 14-day-old Wistar rats. A single injection of L-tyrosine decreased glutathione (GSH) and thiol-disulfide redox state (SH/SS ratio) while thiobarbituric acid-reactive substances, protein carbonyl content and glucose-6-phosphate dehydrogenase activity were enhanced. In contrast, the treatment did not affect ascorbic acid content, and the activities of superoxide dismutase, catalase and glutathione peroxidase. These results indicate that acute administration of L-tyrosine may impair antioxidant defenses and stimulate oxidative damage to lipids and proteins in cerebral cortex of young rats in vivo. This suggests that oxidative stress may represent a pathophysiological mechanism in hypetyrosinemic patients.

  17. Influence of the thyroid hormone status on tyrosine hydroxylase in central and peripheral catecholaminergic structures.

    PubMed

    Claustre, J; Balende, C; Pujol, J F

    1996-03-01

    We investigated the effect of hyper- and hypothyroidism on tyrosine hydroxylase protein concentration in the locus coeruleus (divided into anterior and posterior parts), the substantia nigra and the adrenals of adult rats. Rats were made hypothyroid with propylthiouracile (PTU, 0.02% in drinking water for 21 days) or hyperthyroid by thyroxine injection (100 or 250 micrograms/kg/day), for 3 or 17 days. PTU treatment resulted in statistically significant decrease of tyrosine hydroxylase in the anterior locus coeruleus (-13%) and the adrenals (-14%). After thyroxine treatment, in the anterior locus coeruleus, tyrosine hydroxylase was significantly higher (2 way ANOVA) after the 3 day treatment than after the 17 day treatment: tyrosine hydroxylase showed a trend to increase the 3 day treatment (+20% with the 250 micrograms/kg dose) and to decrease after the 17 day treatment (-15% with the 250 micrograms/kg dose). In the adrenals, tyrosine hydroxylase was increased by the 3 day treatment (+42% after the 250 micrograms/kg dose), but this increase was not observed after 17 days of treatment. Tyrosine hydroxylase was not altered in the posterior locus coeruleus and the substantia nigra, whatever the treatment. Together, our results support the hypothesis that in the anterior locus coeruleus and in the adrenals tyrosine hydroxylase level is positively modulated by thyroid hormones. After long-term treatment (17 days) this effect is not observed. PMID:8813245

  18. Identification of nitrated tyrosine residues of protein kinase G-Iα by mass spectrometry.

    PubMed

    Lu, Jingshan; Yao, Ikuko; Shimojo, Masahito; Katano, Tayo; Uchida, Hitoshi; Setou, Mitsutoshi; Ito, Seiji

    2014-02-01

    The nitration of tyrosine to 3-nitrotyrosine is an oxidative modification of tyrosine by nitric oxide and is associated with many diseases, and targeting of protein kinase G (PKG)-I represents a potential therapeutic strategy for pulmonary hypertension and chronic pain. The direct assignment of tyrosine residues of PKG-I has remained to be made due to the low sensitivity of the current proteomic approach. In order to assign modified tyrosine residues of PKG-I, we nitrated purified PKG-Iα expressed in insect Sf9 cells by use of peroxynitrite in vitro and analyzed the trypsin-digested fragments by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Among the 21 tyrosine residues of PKG-Iα, 16 tyrosine residues were assigned in 13 fragments; and six tyrosine residues were nitrated, those at Y71, Y141, Y212, Y336, Y345, and Y567, in the peroxynitrite-treated sample. Single mutation of tyrosine residues at Y71, Y212, and Y336 to phenylalanine significantly reduced the nitration of PKG-Iα; and four mutations at Y71, Y141, Y212, and Y336 (Y4F mutant) reduced it additively. PKG-Iα activity was inhibited by peroxynitrite in a concentration-dependent manner from 30 μM to 1 mM, and this inhibition was attenuated in the Y4F mutant. These results demonstrated that PKG-Iα was nitrated at multiple tyrosine residues and that its activity was reduced by nitration of these residues.

  19. Peroxidase induced oligo-tyrosine cross-links during polymerization of α-lactalbumin.

    PubMed

    Dhayal, Surender Kumar; Sforza, Stefano; Wierenga, Peter A; Gruppen, Harry

    2015-12-01

    Horseradish peroxidase (HRP) induced cross-linking of proteins has been reported to proceed through formation of di-tyrosine cross-links. In the case of low molar mass phenolic substrates, the enzymatic oxidation is reported to lead to polymerization of the phenols. The aim of this work was to investigate if during oxidative cross-linking of proteins oligo-tyrosine cross-links are formed in addition to dityrosine. To this end, α-lactalbumin (α-LA) was cross-linked using horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂). The reaction products were acid hydrolysed, after which the cross-linked amino acids were investigated by LC-MS and MALDI-MS. To test the effect of the size of the substrate, the cross-linking reaction was also performed with L-tyrosine, N-acetyl L-tyrosinamide and angiotensin. These products were analyzed by LC-MS directly, as well as after acid hydrolysis. In the acid hydrolysates of all samples oligo-tyrosine (Yn, n=3-8) was found in addition to di-tyrosine (Y2). Two stages of cross-linking of α-LA were identified: a) 1-2 cross-links were formed per monomer until the monomers were converted into oligomers, and b) subsequent cross-linking of oligomers formed in the first stage to form nanoparticles containing 3-4 cross-links per monomer. The transition from first stage to the second stage coincided with the point where di-tyrosine started to decrease and more oligo-tyrosines were formed. In conclusion, extensive polymerization of α-LA using HRP via oligo-tyrosine cross-links is possible, as is the case for low molar mass tyrosine containing substrates. PMID:26282909

  20. Interleukin-2 induces tyrosine phosphorylation of the vav proto-oncogene product in human T cells: lack of requirement for the tyrosine kinase lck.

    PubMed Central

    Evans, G A; Howard, O M; Erwin, R; Farrar, W L

    1993-01-01

    The haematopoietic protein, p95vav, has been shown to be a tyrosine kinase substrate and to have tyrosine kinase-modulated guanine-nucleotide-releasing-factor activity. This implies a function in the control of ras or ras-like proteins. Because ras activation has been shown to be a downstream event following stimulation of the interleukin-2 (IL-2) receptor, we investigated the possibility that vav was involved in IL-2 signal transduction pathways, using human T cells as a model. We found rapid tyrosine phosphorylation of vav in response to IL-2 within 1 min, with maximum increase of phosphorylation of 5-fold occurring by 5 min after treatment in normal human T cells. IL-2 stimulation of the human T-cell line YT and a subclone of the YT cell line (YTlck-) that does not express message for the src-family kinase p56lck also results in a rapid rate of tyrosine phosphorylation of vav of more than 5-fold by 5 min. These results suggest that vav may play an important role in IL-2-stimulated signal transduction and that there is not a strict requirement for the tyrosine kinase p56lck. Images Figure 1 Figure 3 Figure 4 PMID:7690544

  1. DMBA induces tyrosine phosphorylation of PLC-[gamma]1 and activates the tyrosine kinases lck and fyn in the HPB-ALL human T-cell line

    SciTech Connect

    Archuleta, M.M.; Schieven, G.L.; Ledbetter, J.A.; Burchiel, S.W. . Coll. of Pharmacy)

    1993-01-01

    Previous studies in this laboratory have demonstrated that DMBA alters biochemical events associated with lymphocyte activation including formation of the second messenger IP[sub 3] and the release of intracellular Ca[sup 2+]. The purpose of the present studies was to evaluate the mechanisms by which DMBA induces IP[sub 3] formation and Ca[sup 2+] release by examining phosphorylation of membrane associated proteins and activation of protein tyrosine kinases lck and fyn. These studies demonstrated that exposure of HPB-ALL cells to 10[mu]M DMBA resulted in a time- and dose-dependent increase in tyrosine phosphorylation of PLC-[gamma]1 that correlated with our earlier findings of IP[sub 3] formation and Ca[sup 2+] release. These results indicate that the effects of DMBA on the PI-PLC signaling pathway are in part, the result of DMBA-induced tyrosine phosphorylation of the PLC-[gamma]1 enzyme. The mechanism of DMBA- induced tyrosine phosphorylation of PLC-[gamma]1 may be due to activation of fyn or lck kinase activity, since it was found that DMBA increased the activity of these PTKs by more than 2-fold. Therefore, these studies demonstrate that DMBA may disrupt T cell activation by stimulating PTK activation with concomitant tyrosine phosphorylation of PLC-[gamma]1, release of IP[sub 3], and mobilization of intracellular Ca[sup 2+].

  2. DMBA induces tyrosine phosphorylation of PLC-{gamma}1 and activates the tyrosine kinases lck and fyn in the HPB-ALL human T-cell line

    SciTech Connect

    Archuleta, M.M.; Schieven, G.L.; Ledbetter, J.A.; Burchiel, S.W.

    1993-02-01

    Previous studies in this laboratory have demonstrated that DMBA alters biochemical events associated with lymphocyte activation including formation of the second messenger IP{sub 3} and the release of intracellular Ca{sup 2+}. The purpose of the present studies was to evaluate the mechanisms by which DMBA induces IP{sub 3} formation and Ca{sup 2+} release by examining phosphorylation of membrane associated proteins and activation of protein tyrosine kinases lck and fyn. These studies demonstrated that exposure of HPB-ALL cells to 10{mu}M DMBA resulted in a time- and dose-dependent increase in tyrosine phosphorylation of PLC-{gamma}1 that correlated with our earlier findings of IP{sub 3} formation and Ca{sup 2+} release. These results indicate that the effects of DMBA on the PI-PLC signaling pathway are in part, the result of DMBA-induced tyrosine phosphorylation of the PLC-{gamma}1 enzyme. The mechanism of DMBA- induced tyrosine phosphorylation of PLC-{gamma}1 may be due to activation of fyn or lck kinase activity, since it was found that DMBA increased the activity of these PTKs by more than 2-fold. Therefore, these studies demonstrate that DMBA may disrupt T cell activation by stimulating PTK activation with concomitant tyrosine phosphorylation of PLC-{gamma}1, release of IP{sub 3}, and mobilization of intracellular Ca{sup 2+}.

  3. Phosphonate derivatives of tetraazamacrocycles as new inhibitors of protein tyrosine phosphatases.

    PubMed

    Kobzar, Oleksandr L; Shevchuk, Michael V; Lyashenko, Alesya N; Tanchuk, Vsevolod Yu; Romanenko, Vadim D; Kobelev, Sergei M; Averin, Alexei D; Beletskaya, Irina P; Vovk, Andriy I; Kukhar, Valery P

    2015-07-21

    α,α-Difluoro-β-ketophosphonated derivatives of tetraazamacrocycles were synthesized and found to be potential inhibitors of protein tyrosine phosphatases. N-Substituted conjugates of cyclam and cyclen with bioisosteric phosphonate groups displayed good activities toward T-cell protein tyrosine phosphatase with IC50 values in the micromolar to nanomolar range and showed selectivity over PTP1B, CD45, SHP2, and PTPβ. Kinetic studies indicated that the inhibitors can occupy the region of the active site of TC-PTP. This study demonstrates a new approach which employs tetraazamacrocycles as a molecular platform for designing inhibitors of protein tyrosine phosphatases. PMID:26058329

  4. Degradation of Tyrosine in Anaerobically Stored Piggery Wastes and in Pig Feces

    PubMed Central

    Spoelstra, Sierk F.

    1978-01-01

    Radioactively labeled compounds that might be intermediates in the anaerobic degradation of tyrosine were added to pig feces and to stored piggery wastes. Changes in the compounds were followed by using thin-layer and gas chromatography. In feces, p-cresol and 3-phenylpropionic acid were the end products of tyrosine metabolism; in anaerobically stored mixed wastes, phenol, p-cresol, and minor quantities of phenylpropionic acid were formed. Schemes were proposed for the degradation of tyrosine in pig feces and in mixed wastes. PMID:16345325

  5. Ultraviolet and laser Raman investigation of the buried tyrosines in fd phage.

    PubMed

    Dunker, A K; Williams, R W; Peticolas, W L

    1979-07-25

    The tyrosines of the filamentous phage fd have been found to be inaccessible to solvent by pH titration while monitoring the ultraviolet spectrum or the laser Raman spectrum. The uv spectra suggest that the tyrosines do not become deprotonated unless the phage becomes disrupted. One possible explanation of the Raman spectra is that the tyrosine OH groups are the recipients of hydrogen-bonded protons arising from fairly acidic donors, yet these acidic donors do not become titrated over the pH 7 to 12 range.

  6. The use of the tyrosine phosphatase antagonist orthovanadate in the study of a cell proliferation inhibitor

    NASA Technical Reports Server (NTRS)

    Enebo, D. J.; Hanek, G.; Fattaey, H. K.; Johnson, T. C.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    Incubation of murine fibroblasts with orthovanadate, a global tyrosine phosphatase inhibitor, was shown to confer a "pseudo-transformed" phenotype with regard to cell morphology and growth characteristics. This alteration was manifested by both an increasing refractile appearance of the cells, consistent with many transformed cell lines, as well as an increase in maximum cell density was attained. Despite the abrogation of cellular tyrosine phosphatase activity, orthovanadate-treated cells remained sensitive to the biological activity of a naturally occurring sialoglycopeptide (SGP) cell surface proliferation inhibitor. The results indicated that tyrosine phosphatase activity, inhibited by orthovanadate, was not involved in the signal transduction pathway of the SGP.

  7. Palmitylation of Src family tyrosine kinases regulates functional interaction with a B cell substrate.

    PubMed

    Saouaf, S J; Wolven, A; Resh, M D; Bolen, J B

    1997-05-19

    Palmitylation of Src family tyrosine kinases has been shown to play a role in directing their membrane localization. Here we demonstrate that palmitylation can also regulate recognition and tyrosine phosphorylation of the B cell Src kinase substrate Ig alpha. Blk and Src, which are not palmitylated, phosphorylate co-expressed Ig alpha in Cos cells, whereas palmitylated Src kinases do not. Addition of a palmitylation site to Blk abrogates its phosphorylation of the substrate, while mutation of Fyn's palmitylation sites results in recognition and phosphorylation of Ig alpha. These results indicate that palmitylation, a reversible protein modification, aids in regulating recognition of physiologic substrates by Src family tyrosine kinases. PMID:9177269

  8. Regulation of the EphA2 kinase by the low molecular weight tyrosine phosphatase induces transformation.

    PubMed

    Kikawa, Keith D; Vidale, Derika R; Van Etten, Robert L; Kinch, Michael S

    2002-10-18

    Intracellular signaling by protein tyrosine phosphorylation is generally understood to govern many aspects of cellular behavior. The biological consequences of this signaling pathway are important because the levels of protein tyrosine phosphorylation are frequently elevated in cancer cells. In the classic paradigm, tyrosine kinases promote tumor cell growth, survival, and invasiveness, whereas tyrosine phosphatases negatively regulate these same behaviors. Here, we identify one particular tyrosine phosphatase, low molecular weight tyrosine phosphatase (LMW-PTP), which is frequently overexpressed in transformed cells. We also show that overexpression of LMW-PTP is sufficient to confer transformation upon non-transformed epithelial cells. Notably, we show that the EphA2 receptor tyrosine kinase is a prominent substrate for LMW-PTP and that the oncogenic activities of LMW-PTP result from altered EphA2 expression and function. These results suggest a role for LMW-PTP in transformation progression and link its oncogenic potential to EphA2.

  9. A Tyrosine Aminomutase from Rice (Oryza sativa) Isomerizes (S)-α- to (R)-β-Tyrosine with Unique High Enantioselectivity and Retention of Configuration.

    PubMed

    Walter, Tyler; King, Zayna; Walker, Kevin D

    2016-01-12

    A recently discovered 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO)-dependent tyrosine aminomutase (OsTAM) from rice [Yan, J., et al. (2015) Plant Cell 27, 1265] converts (S)-α-tyrosine to a mixture of (R)- and (S)-β-tyrosines, with high (94%) enantiomeric excess, which does not change with pH, like it does for two bacterial TAMs. The K(M) of 490 μM and the k(cat) of 0.005 s(-1) are similar for other TAM enzymes. OsTAM is unique and also catalyzes (R)-β- from (S)-α-phenylalanine. OsTAM principally retains the configuration at the reactive C(α) and C(β) centers during catalysis much like the phenylalanine aminomutase on the Taxol biosynthetic pathway in Taxus plants.

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

    PubMed

    Marx, Kenneth A; Zhou, Tiean; Long, Dang

    2005-01-01

    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

  11. Fyn is a downstream target of the pleiotrophin/receptor protein tyrosine phosphatase beta/zeta-signaling pathway: regulation of tyrosine phosphorylation of Fyn by pleiotrophin.

    PubMed

    Pariser, Harold; Ezquerra, Laura; Herradon, Gonzalo; Perez-Pinera, Pablo; Deuel, Thomas F

    2005-07-01

    Pleiotrophin (PTN the protein, Ptn the gene) signals downstream targets through inactivation of its receptor, the transmembrane receptor protein tyrosine phosphatase (RPTP)beta/zeta, disrupting the balanced activity of RPTPbeta/zeta and the activity of a constitutively active tyrosine kinase. As a consequence of the inactivation of RPTPbeta/zeta, PTN stimulates a sharp increase in the levels of tyrosine phosphorylation of the substrates of RPTPbeta/zeta in PTN-stimulated cells. We now report that the Src family member Fyn interacts with the intracellular domain of RPTPbeta/zeta in a yeast two-hybrid system. We further demonstrate that Fyn is a substrate of RPTPbeta/zeta, and that tyrosine phosphorylation of Fyn is sharply increased in PTN-stimulated cells. In previous studies, we demonstrated that beta-catenin and beta-adducin are targets of the PTN/RPTPbeta/zeta-signaling pathway and defined the mechanisms through which tyrosine phosphorylation of beta-catenin and beta-adducin disrupts cytoskeletal protein complexes. We conclude that Fyn is a downstream target of the PTN/RPTPbeta/zeta-signaling pathway and suggest that PTN coordinately regulates tyrosine phosphorylation of beta-catenin, beta-adducin, and Fyn through the PTN/RPTPbeta/zeta-signaling pathway and that together Fyn, beta-adducin, and beta-catenin may be effectors of the previously described PTN-stimulated disruption of cytoskeletal stability, increased cell plasticity, and loss of cell-cell adhesion that are characteristic of PTN-stimulated cells and a feature of many human malignant cells in which mutations have established constitutive expression of the Ptn gene.

  12. Functional specificity of cytoplasmic and transmembrane tyrosine kinases: identification of 130- and 75-kilodalton substrates of c-fps/fes tyrosine kinase in macrophages.

    PubMed Central

    Areces, L B; Dello Sbarba, P; Jücker, M; Stanley, E R; Feldman, R A

    1994-01-01

    c-fps/fes encodes a 92-kDa protein-tyrosine kinase (NCP92) that is expressed at the highest levels in macrophages. To determine if c-fps/fes can mediate the action of the colony-stimulating factor 1 (CSF-1) receptor (CSF-1R) and to identify potential targets of c-fps/fes in macrophages, we have overexpressed c-fps/fes in a CSF-1-dependent macrophage cell line. A 30- to 50-fold overexpression of c-fps/fes partially released these cells from their factor dependence by a nonautocrine mechanism, and this correlated with the tyrosine phosphorylation of two proteins of 130 and 75 kDa (P130 and P75). c-fps/fes did not cause tyrosine phosphorylation or activation of CSF-1 dependent targets, including CSF-1R, Shc, and phosphatidylinositol 3-kinase, and conversely, CSF-1 did not induce tyrosine phosphorylation of P130 and P75. P75 appears to be a novel phosphotyrosyl protein, whereas P130 cross-reacts with a known substrate of v-src. P130 and P75 may be direct substrates of c-fps/fes: P130 was tightly associated with NCP92, and the src homology 2 domain of NCP92 specifically bound phosphorylated P130 and P75 but not the CSF-1-induced phosphotyrosyl proteins, consistent with the possibility that P130 and P75 are physiological targets of c-fps/fes. We conclude that although c-fps/fes can functionally substitute for CSF-1R to a certain extent, these tyrosine kinases act largely independently of each other and that P130 and P75 are novel targets whose mechanisms of action may be unrelated to the signalling pathways utilized by receptor tyrosine kinases. Images PMID:8007965

  13. Immunoreceptor tyrosine-based inhibitory motif (ITIM)-mediated inhibitory signaling is regulated by sequential phosphorylation mediated by distinct nonreceptor tyrosine kinases: a case study involving PECAM-1.

    PubMed

    Tourdot, Benjamin E; Brenner, Michelle K; Keough, Kathleen C; Holyst, Trudy; Newman, Peter J; Newman, Debra K

    2013-04-16

    The activation state of many blood and vascular cells is tightly controlled by a delicate balance between receptors that contain immunoreceptor tyrosine-based activation motifs (ITAMs) and those that contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Precisely how the timing of cellular activation by ITAM-coupled receptors is regulated by ITIM-containing receptors is, however, poorly understood. Using platelet endothelial cell adhesion molecule 1 (PECAM-1) as a prototypical ITIM-bearing receptor, we demonstrate that initiation of inhibitory signaling occurs via a novel, sequential process in which Src family kinases phosphorylate the C-terminal ITIM, thereby enabling phosphorylation of the N-terminal ITIM of PECAM-1 by other Src homology 2 domain-containing nonreceptor tyrosine kinases (NRTKs). NRTKs capable of mediating the second phosphorylation event include C-terminal Src kinase (Csk) and Bruton's tyrosine kinase (Btk). Btk and Csk function downstream of phosphatidylinositol 3-kinase (PI3K) activation during ITAM-dependent platelet activation. In ITAM-activated platelets that were treated with a PI3K inhibitor, PECAM-1 was phosphorylated but did not bind the tandem SH2 domain-containing tyrosine phosphatase SHP-2, indicating that it was not phosphorylated on its N-terminal ITIM. Csk bound to and phosphorylated PECAM-1 more efficiently than did Btk and required its SH2 domain to perform these functions. Additionally, the phosphorylation of the N-terminal ITIM of Siglec-9 by Csk is enhanced by the prior phosphorylation of its C-terminal ITIM, providing evidence that the ITIMs of other dual ITIM-containing receptors are also sequentially phosphorylated. On the basis of these findings, we propose that sequential ITIM phosphorylation provides a general mechanism for precise temporal control over the recruitment and activation of tandem SH2 domain-containing tyrosine phosphatases that dampen ITAM-dependent signals. PMID:23418871

  14. Unplanned discontinuation of tyrosine kinase inhibitors in chronic myeloid leukemia

    PubMed Central

    TSUTSUMI, YUTAKA; ITO, SHINICHI; OHIGASHI, HIROYUKI; SHIRATORI, SOUICHI; TESHIMA, TAKANORI

    2016-01-01

    This study was conducted to investigate the outcomes of patients with chronic myeloid leukemia (CML) who discontinued tyrosine kinase inhibitor (TKI) treatment. A single-center retrospective analysis was performed, including 46 chronic-phase (CP) CML patients who achieved complete molecular response (CMR) with TKIs. TKI treatment was discontinued in 13 patients based on their requests. The BCR-ABL transcript levels were monitored in the peripheral blood by quantitative polymerase chain reaction analysis following treatment discontinuation. Of the 13 patients who discontinued TKI treatment, 7 remained in CMR, with a median follow-up of 26 months (range, 10–60 months). The remaining 6 patients lost CMR following TKI discontinuation; 2 of these patients achieved a second CMR following re-administration of TKIs, 2 patients spontaneously achieved CMR and 2 remained in complete hematological response (CHR) without TKI treatment with a median follow-up of 29.5 months (range, 10–52 months). In conclusion, the survival of patients who lost CMR following TKI discontinuation may not be affected, even without re-administration of TKIs. Vigilant observation is recommended for such patients. The limitations of this study included the small patient sample, retrospective design and patient heterogeneity. Therefore, the results must be interpreted with caution. PMID:26870364

  15. Tyrosine aminotransferase from Leishmania infantum: A new drug target candidate.

    PubMed

    Moreno, Miguel Angel; Alonso, Ana; Alcolea, Pedro Jose; Abramov, Ariel; de Lacoba, Mario García; Abendroth, Jan; Zhang, Sunny; Edwards, Thomas; Lorimer, Don; Myler, Peter John; Larraga, Vicente

    2014-12-01

    Leishmania infantum is the etiological agent of zoonotic visceral leishmaniasis in the Mediterranean basin. The disease is fatal without treatment, which has been based on antimonial pentavalents for more than 60 years. Due to resistances, relapses and toxicity to current treatment, the development of new drugs is required. The structure of the L. infantum tyrosine aminotransferase (LiTAT) has been recently solved showing important differences with the mammalian orthologue. The characterization of LiTAT is reported herein. This enzyme is cytoplasmic and is over-expressed in the more infective stages and nitric oxide resistant parasites. Unlike the mammalian TAT, LiTAT is able to use ketomethiobutyrate as co-substrate. The pharmacophore model of LiTAT with this specific co-substrate is described herein. This may allow the identification of new inhibitors present in the databases. All the data obtained support that LiTAT is a good target candidate for the development of new anti-leishmanial drugs. PMID:25516846

  16. Recent inventions on receptor tyrosine kinase RET modulation.

    PubMed

    Jurvansuu, Jaana M; Goldman, Adrian

    2008-01-01

    Rearranged during transfection, RET, is a receptor tyrosine kinase expressed in neural crest derived cell lineages. RET is activated by dimerisation facilitated by its binding to the heterodimeric complex formed by Glial cell-derived neurotrophic factor (GDNF) -family ligand (GFL) and GNDF-family receptor (GFR). Both GDNFs and their co-receptors are a small protein family of four members. RET kinase mediated signaling can lead to survival, cell growth, differentiation, and migration. Pharmaceutically RET is of interest due to its involvement in several disease conditions. Oncogenic RET activation by mutations or rearragements predisposes to cancers like multiple endocrine neoplasia type 2 (A and B) and medullary thyroid carcinoma. Loss-of-function mutations in RET are a strong susceptibility factor for Hirschsprung disease, which is characterized by lack of ganglion cells in gastrointestinal tract. All the GFLs promote neuronal survival and GDNF is one of the most potent neurotrophic factors for dopaminergic neurons. Therefore, the neuroprotective capacity of RET activation to override the apoptotic program in neurodegenerative diseases, like in dying midbrain dopaminergic neurons in Parkinson's disease, is of great interest. This article reviews the recent international patents on modulation of RET kinase activity by small-molecule and peptide-based agonists and antagonists.

  17. A novel putative tyrosine kinase receptor with oncogenic potential.

    PubMed

    Janssen, J W; Schulz, A S; Steenvoorden, A C; Schmidberger, M; Strehl, S; Ambros, P F; Bartram, C R

    1991-11-01

    We have detected transforming activity by a tumorigenicity assay using NIH3T3 cells transfected with DNA from a chronic myeloproliferative disorder patient. Here, we report the cDNA cloning of the corresponding oncogene, designated UFO, in allusion to the as yet unidentified function of its protein. Nucleotide sequence analysis of a 3116bp cDNA clone revealed a 2682-bp-long open reading frame capable of directing the synthesis of a 894 amino acid polypeptide. The predicted UFO protein exhibits characteristic features of a transmembrane receptor with associated tyrosine kinase activity. The UFO proto-oncogene maps to human chromosome 19q13.1 and is transcribed into two 5.0 kb and 3.2 kb mRNAs in human bone marrow and human tumor cell lines. The UFO locus is evolutionarily conserved between vertebrate species. A 4.0 kb mRNA of the murine UFO homolog is expressed in a variety of different mouse tissues. We thus have identified a novel element of the complex signaling network involved in the control of cell proliferation and differentiation.

  18. Role of Striatal-Enriched Tyrosine Phosphatase in Neuronal Function.

    PubMed

    Kamceva, Marija; Benedict, Jessie; Nairn, Angus C; Lombroso, Paul J

    2016-01-01

    Striatal-enriched protein tyrosine phosphatase (STEP) is a CNS-enriched protein implicated in multiple neurologic and neuropsychiatric disorders. STEP regulates key signaling proteins required for synaptic strengthening as well as NMDA and AMPA receptor trafficking. Both high and low levels of STEP disrupt synaptic function and contribute to learning and behavioral deficits. High levels of STEP are present in human postmortem samples and animal models of Alzheimer's disease, Parkinson's disease, and schizophrenia and in animal models of fragile X syndrome. Low levels of STEP activity are present in additional disorders that include ischemia, Huntington's chorea, alcohol abuse, and stress disorders. Thus the current model of STEP is that optimal levels are required for optimal synaptic function. Here we focus on the role of STEP in Alzheimer's disease and the mechanisms by which STEP activity is increased in this illness. Both genetic lowering of STEP levels and pharmacological inhibition of STEP activity in mouse models of Alzheimer's disease reverse the biochemical and cognitive abnormalities that are present. These findings suggest that STEP is an important point for modulation of proteins required for synaptic plasticity. PMID:27190655

  19. Regulation of therapeutic resistance in cancers by receptor tyrosine kinases

    PubMed Central

    Chen, Mei-Kuang; Hung, Mien-Chie

    2016-01-01

    In response to DNA damage lesions due to cellular stress, DNA damage response (DDR) pathways are activated to promote cell survival and genetic stability or unrepaired lesion-induced cell death. Current cancer treatments predominantly utilize DNA damaging agents, such as irradiation and chemotherapy drugs, to inhibit cancer cell proliferation and induce cell death through the activation of DDR. However, a portion of cancer patients is reported to develop therapeutic resistance to these DDR-inducing agents. One significant resistance mechanism in cancer cells is oncogenic kinase overexpression, which promotes cell survival by enhancing DNA damage repair pathways and evading cell cycle arrest. Among the oncogenic kinases, overexpression of receptor tyrosine kinases (RTKs) is reported in many of solid tumors, and numerous clinical trials targeting RTKs are currently in progress. As the emerging trend in cancer treatment combines DNA damaging agents and RTK inhibitors, it is important to understand the substrates of RTKs relative to the DDR pathways. In addition, alteration of RTK expression and their phosphorylated substrates can serve as biomarkers to stratify patients for combination therapies. In this review, we summarize the deleterious effects of RTKs on the DDR pathways and the emerging biomarkers for personalized therapy. PMID:27186434

  20. Redundant Regulation of Cdk1 Tyrosine Dephosphorylation in Saccharomyces cerevisiae.

    PubMed

    Kennedy, Erin K; Dysart, Michael; Lianga, Noel; Williams, Elizabeth C; Pilon, Sophie; Doré, Carole; Deneault, Jean-Sebastien; Rudner, Adam D

    2016-03-01

    Cdk1 activity drives both mitotic entry and the metaphase-to-anaphase transition in all eukaryotes. The kinase Wee1 and the phosphatase Cdc25 regulate the mitotic activity of Cdk1 by the reversible phosphorylation of a conserved tyrosine residue. Mutation of cdc25 in Schizosaccharomyces pombe blocks Cdk1 dephosphorylation and causes cell cycle arrest. In contrast, deletion of MIH1, the cdc25 homolog in Saccharomyces cerevisiae, is viable. Although Cdk1-Y19 phosphorylation is elevated during mitosis in mih1∆ cells, Cdk1 is dephosphorylated as cells progress into G1, suggesting that additional phosphatases regulate Cdk1 dephosphorylation. Here we show that the phosphatase Ptp1 also regulates Cdk1 dephosphorylation in vivo and can directly dephosphorylate Cdk1 in vitro. Using a novel in vivo phosphatase assay, we also show that PP2A bound to Rts1, the budding yeast B56-regulatory subunit, regulates dephosphorylation of Cdk1 independently of a function regulating Swe1, Mih1, or Ptp1, suggesting that PP2A(Rts1) either directly dephosphorylates Cdk1-Y19 or regulates an unidentified phosphatase. PMID:26715668

  1. Regulation of Endothelial Adherens Junctions by Tyrosine Phosphorylation

    PubMed Central

    Adam, Alejandro Pablo

    2015-01-01

    Endothelial cells form a semipermeable, regulated barrier that limits the passage of fluid, small molecules, and leukocytes between the bloodstream and the surrounding tissues. The adherens junction, a major mechanism of intercellular adhesion, is comprised of transmembrane cadherins forming homotypic interactions between adjacent cells and associated cytoplasmic catenins linking the cadherins to the cytoskeleton. Inflammatory conditions promote the disassembly of the adherens junction and a loss of intercellular adhesion, creating openings or gaps in the endothelium through which small molecules diffuse and leukocytes transmigrate. Tyrosine kinase signaling has emerged as a central regulator of the inflammatory response, partly through direct phosphorylation and dephosphorylation of the adherens junction components. This review discusses the findings that support and those that argue against a direct effect of cadherin and catenin phosphorylation in the disassembly of the adherens junction. Recent findings indicate a complex interaction between kinases, phosphatases, and the adherens junction components that allow a fine regulation of the endothelial permeability to small molecules, leukocyte migration, and barrier resealing. PMID:26556953

  2. Desferrioxamine Inhibits Protein Tyrosine Nitration: Mechanisms and Implications

    PubMed Central

    Adgent, Margaret A.; Squadrito, Giuseppe L.; Ballinger, Carol A.; Krzywanski, David M.; Lancaster, Jack R.; Postlethwait, Edward M.

    2012-01-01

    Tissues are exposed to exogenous and endogenous nitrogen dioxide (•NO2), which is the terminal agent in protein tyrosine nitration. Besides iron chelation, the hydroxamic acid (HA) desferrioxamine (DFO) shows multiple functionalities including nitration inhibition. To investigate mechanisms whereby DFO affects 3-nitrotyrosine (3-NT) formation, we utilized gas phase •NO2 exposures, to limit introduction of other reactive species, and a lung surface model wherein red cell membranes (RCM) were immobilized under a defined aqueous film. When RCM were exposed to •NO2 covered by +/− DFO: (i) DFO inhibited 3-NT formation more effectively than other HA and non-HA chelators; (ii) 3-NT inhibition occurred at very low [DFO] for prolonged times; and (iii) 3-NT formation was iron independent but inhibition required DFO present. DFO poorly reacted with •NO2 compared to ascorbate, assessed via •NO2 reactive absorption and aqueous phase oxidation rates, yet limited 3-NT formation at far lower concentrations. DFO also inhibited nitration under aqueous bulk phase conditions, and inhibited 3-NT generated by active myeloperoxidase “bound” to RCM. Per the above and kinetic analyses suggesting preferential DFO versus •NO2 reaction within membranes, we conclude that DFO inhibits 3-NT formation predominantly by facile repair of the tyrosyl radical intermediate, which prevents •NO2 addition, and thus nitration, and potentially influences biochemical functionalities. PMID:22705369

  3. Expression of Tyrosine Hydroxylase is Negatively Regulated Via Prion Protein.

    PubMed

    da Luz, Marcio Henrique Mello; Glezer, Isaias; Xavier, Andre Machado; da Silva, Marcelo Alberti Paiva; Pino, Jessica Monteiro Volejnik; Zamith, Thiago Panaro; Vieira, Taynara Fernanda; Antonio, Bruno Brito; Antunes, Hanna Karen Moreira; Martins, Vilma Regina; Lee, Kil Sun

    2016-07-01

    Cellular prion protein (PrP(C)) is a glycoprotein of the plasma membrane that plays pleiotropic functions by interacting with multiple signaling complexes at the cell surface. Recently, a number of studies have reported the involvement of PrP(C) in dopamine metabolism and signaling, including its interactions with tyrosine hydroxylase (TH) and dopamine receptors. However, the outcomes reported by independent studies are still debatable. Therefore in this study, we investigated the effects of PrP(C) on the TH expression during the differentiation of N2a cells with dibutyryl-cAMP, a well-known cAMP analog that activates TH transcription. Upon differentiation, TH was induced with concomitant reduction of PrP(C) at protein level, but not at mRNA level. shRNA-mediated PrP(C) reduction increased the basal level of TH at both mRNA and protein levels without dibutyryl-cAMP treatment. This phenotype was reversed by re-expression of PrP(C). PrP(C) knockdown also potentiated the effect of dibutyryl-cAMP on TH expression. Our findings suggest that PrP(C) has suppressive effects on TH expression. As a consequence, altered PrP(C) functions may affect the regulation of dopamine metabolism and related neurological disorders.

  4. Studying N-linked glycosylation of receptor tyrosine kinases.

    PubMed

    Itkonen, Harri M; Mills, Ian G

    2015-01-01

    Metabolic alterations have been identified as a frequent event in cancer. This is often associated with increased flux through glycolysis, and also a secondary pathway to glycolysis, hexosamine biosynthetic pathway (HBP). HBP provides substrate for N-linked glycosylation, which occurs in the endoplasmic reticulum and the Golgi apparatus. N-linked glycosylation supports protein folding and correct sorting of proteins to plasma membrane and secretion. This process generates complex glycoforms, which can be recognized by other proteins and glycosylation of receptor tyrosine kinases (RTK) can also regulate their plasma-membrane retention time. Of special interest for experimental biologists, plants produce proteins, termed lectins, which bind with high specificity to glyco-conjugates. For the purposes of molecular biology, plant lectins can be conjugated to different moieties, such as agarose beads, which enable precipitation of specifically glycosylated proteins. In this chapter, we describe in detail how to perform pull-down experiments with commercially available lectins to identify changes in the glycosylation of RTKs. PMID:25319893

  5. Pancreatitis with vascular endothelial growth factor receptor tyrosine kinase inhibitors.

    PubMed

    Ghatalia, Pooja; Morgan, Charity J; Choueiri, Toni K; Rocha, Pedro; Naik, Gurudatta; Sonpavde, Guru

    2015-04-01

    A trial-level meta-analysis was conducted to determine the relative risk (RR) of pancreatitis associated with multi-targeted vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors (TKI). Eligible studies included randomized phase 2 and 3 trials comparing arms with and without an FDA-approved VEGFR TKI (sunitinib, sorafenib, pazopanib, axitinib, vandetanib, cabozantinib, ponatinib, regorafenib). Statistical analyses calculated the RR and 95% confidence intervals (CI). A total of 10,578 patients from 16 phase III trials and 6 phase II trials were selected. The RR for all grade and high-grade pancreatitis for the TKI vs. no TKI- arms was 1.95 (p=0.042, 95% CI: 1.02 to 3.70) and 1.89 (p=0.069, 95% CI: 0.95 to 373), respectively. No differential impact of malignancy type or specific TKI agent was seen on RR of all grade of high grade pancreatitis. Better patient selection and monitoring may mitigate the risk of severe pancreatitis.

  6. Role of Receptor Tyrosine Kinases and Their Ligands in Glioblastoma

    PubMed Central

    Carrasco-García, Estefanía; Saceda, Miguel; Martínez-Lacaci, Isabel

    2014-01-01

    Glioblastoma multiforme is the most frequent, aggressive and fatal type of brain tumor. Glioblastomas are characterized by their infiltrating nature, high proliferation rate and resistance to chemotherapy and radiation. Recently, oncologic therapy experienced a rapid evolution towards “targeted therapy,” which is the employment of drugs directed against particular targets that play essential roles in proliferation, survival and invasiveness of cancer cells. A number of molecules involved in signal transduction pathways are used as molecular targets for the treatment of various tumors. In fact, inhibitors of these molecules have already entered the clinic or are undergoing clinical trials. Cellular receptors are clear examples of such targets and in the case of glioblastoma multiforme, some of these receptors and their ligands have become relevant. In this review, the importance of glioblastoma multiforme in signaling pathways initiated by extracellular tyrosine kinase receptors such as EGFR, PDGFR and IGF-1R will be discussed. We will describe their ligands, family members, structure, activation mechanism, downstream molecules, as well as the interaction among these pathways. Lastly, we will provide an up-to-date review of the current targeted therapies in cancer, in particular glioblastoma that employ inhibitors of these pathways and their benefits. PMID:24709958

  7. Regulation of Btk by Src family tyrosine kinases.

    PubMed Central

    Afar, D E; Park, H; Howell, B W; Rawlings, D J; Cooper, J; Witte, O N

    1996-01-01

    Loss of function of Bruton's tyrosine kinase (Btk) results in X-linked immunodeficiencies characterized by a broad spectrum of signaling defects, including those dependent on Src family kinase-linked cell surface receptors. A gain-of-function mutant, Btk*, induces the growth of fibroblasts in soft agar and relieves the interleukin-5 dependence of a pre-B-cell line. To genetically define Btk signaling pathways, we used a strategy to either activate or inactivate Src family kinases in fibroblasts that express Btk*. The transformation potential of Btk* was dramatically increased by coexpression with a partly activated c-Src mutant (E-378 --> G). This synergy was further potentiated by deletion of the Btk Src homology 3 domain. Downregulation of Src family kinases by the C-terminal Src kinase (Csk) suppressed Btk* activation and biological potency. In contrast, kinase-inactive Csk (K-222 --> R), which functioned as a dominant negative molecule, synergized with Btk* in biological transformation. Activation of Btk* correlated with increased phosphotyrosine on transphosphorylation and autophosphorylation sites. These findings suggest that the Src and Btk kinase families form specific signaling units in tissues in which both are expressed. PMID:8668162

  8. Comparative Analysis of Protein Tyrosine Phosphatases Regulating Microglial Activation

    PubMed Central

    Song, Gyun Jee; Kim, Jaehong; Kim, Jong-Heon; Song, Seungeun; Park, Hana; Zhang, Zhong-Yin

    2016-01-01

    Protein tyrosine phosphatases (PTPs) are key regulatory factors in inflammatory signaling pathways. Although PTPs have been extensively studied, little is known about their role in neuroinflammation. In the present study, we examined the expression of 6 different PTPs (PTP1B, TC-PTP, SHP2, MEG2, LYP, and RPTPβ) and their role in glial activation and neuroinflammation. All PTPs were expressed in brain and glia. The expression of PTP1B, SHP2, and LYP was enhanced in the inflamed brain. The expression of PTP1B, TC-PTP, and LYP was increased after treating microglia cells with lipopolysaccharide (LPS). To examine the role of PTPs in microglial activation and neuroinflammation, we used specific pharmacological inhibitors of PTPs. Inhibition of PTP1B, TC-PTP, SHP2, LYP, and RPTPβ suppressed nitric oxide production in LPS-treated microglial cells in a dose-dependent manner. Furthermore, intracerebroventricular injection of PTP1B, TC-PTP, SHP2, and RPTPβ inhibitors downregulated microglial activation in an LPS-induced neuroinflammation model. Our results indicate that multiple PTPs are involved in regulating microglial activation and neuroinflammation, with different expression patterns and specific functions. Thus, PTP inhibitors can be exploited for therapeutic modulation of microglial activation in neuroinflammatory diseases. PMID:27790059

  9. Tyrosine aminotransferase from Leishmania infantum: A new drug target candidate

    PubMed Central

    Moreno, Miguel Angel; Alonso, Ana; Alcolea, Pedro Jose; Abramov, Ariel; de Lacoba, Mario García; Abendroth, Jan; Zhang, Sunny; Edwards, Thomas; Lorimer, Don; Myler, Peter John; Larraga, Vicente

    2014-01-01

    Leishmania infantum is the etiological agent of zoonotic visceral leishmaniasis in the Mediterranean basin. The disease is fatal without treatment, which has been based on antimonial pentavalents for more than 60 years. Due to resistances, relapses and toxicity to current treatment, the development of new drugs is required. The structure of the L. infantum tyrosine aminotransferase (LiTAT) has been recently solved showing important differences with the mammalian orthologue. The characterization of LiTAT is reported herein. This enzyme is cytoplasmic and is over-expressed in the more infective stages and nitric oxide resistant parasites. Unlike the mammalian TAT, LiTAT is able to use ketomethiobutyrate as co-substrate. The pharmacophore model of LiTAT with this specific co-substrate is described herein. This may allow the identification of new inhibitors present in the databases. All the data obtained support that LiTAT is a good target candidate for the development of new anti-leishmanial drugs. PMID:25516846

  10. Mechanisms of Activation of Receptor Tyrosine Kinases: Monomers or Dimers

    PubMed Central

    Maruyama, Ichiro N.

    2014-01-01

    Receptor tyrosine kinases (RTKs) play essential roles in cellular processes, including metabolism, cell-cycle control, survival, proliferation, motility and differentiation. RTKs are all synthesized as single-pass transmembrane proteins and bind polypeptide ligands, mainly growth factors. It has long been thought that all RTKs, except for the insulin receptor (IR) family, are activated by ligand-induced dimerization of the receptors. An increasing number of diverse studies, however, indicate that RTKs, previously thought to exist as monomers, are present as pre-formed, yet inactive, dimers prior to ligand binding. The non-covalently associated dimeric structures are reminiscent of those of the IR family, which has a disulfide-linked dimeric structure. Furthermore, recent progress in structural studies has provided insight into the underpinnings of conformational changes during the activation of RTKs. In this review, I discuss two mutually exclusive models for the mechanisms of activation of the epidermal growth factor receptor, the neurotrophin receptor and IR families, based on these new insights. PMID:24758840

  11. The 2010 patent landscape for spleen tyrosine kinase inhibitors.

    PubMed

    Moretto, Alessandro F; Dehnhardt, Christoph; Kaila, Neelu; Papaioannou, Nikolaos; Thorarensen, Atli

    2012-05-01

    Discovery of small molecular inhibitors for treatment of rheumatoid arthritis is a major ongoing effort within the pharmaceutical industry. Spleen tyrosine kinase (SYK) is one of leading small molecular targets with regard to clinical development primarlly due to efforts by Rigel and Portola. In this review, we provide a comprehensive overview of the SYK patent landscape. The patent literature we evaluated relates to any organization that has filed applications that imply that SYK is the intended target. The interest in SYK was initiated in the early 2000's with many organizations, including several large pharmaceutical companies, and has been active for years. In general, the structural theme of most of the compounds in these applications is a traditional ATP competitive inhibitor with each organization having a different hinge binding element. In general, the attachment to the hinge is an aryl amine that is decorated with a solubilizing group. The other substituents are broadly variable across the numerous companies indicating that SYK has significant flexibility in its interactions in that portion of the kinase. This overview of the SYK patent literature and the learnings of the inhibitors' substitution patterns would be an important reference for anyone working in this area.

  12. Have adjuvant tyrosine kinase inhibitors lost their shine?

    PubMed

    Sabari, Joshua K; Chaft, Jamie E

    2016-08-01

    Despite broad advances in molecularly targeted therapies, lung cancer remains the leading cause of cancer related mortality in the United States. Epidermal growth factor receptor (EGFR) mutations occur in approximately 17% of advanced non-small cell lung cancer (NSCLC) in the US population. The remarkable efficacy of small-molecule EGFR tyrosine kinase inhibitors (TKIs) in this unique subset of patients has revolutionized the therapeutic approach to lung cancer. The success of these agents in the metastatic setting leads to the logical question of what role these drugs may have in the adjuvant setting for patients with earlier stage disease. RADIANT, an international randomized, double-blind, placebo controlled phase III study in patients with completely resected stage IB to IIIA NSLC whose tumors expressed EGFR by IHC and EGFR amplification by FISH, attempted to answer the question of whether erlotinib would improve disease free survival and overall survival in the adjuvant setting. While RADIANT does not conclude for or against adjuvant use of EGFR-TKIs, all data points towards benefit in a selected population. As clinicians, we must continue to enroll to potentially practice changing therapeutic neoadjuvant and adjuvant chemotherapy studies internationally. PMID:27568486

  13. The evolution of tyrosine-recombinase elements in Nematoda.

    PubMed

    Szitenberg, Amir; Koutsovoulos, Georgios; Blaxter, Mark L; Lunt, David H

    2014-01-01

    Transposable elements can be categorised into DNA and RNA elements based on their mechanism of transposition. Tyrosine recombinase elements (YREs) are relatively rare and poorly understood, despite sharing characteristics with both DNA and RNA elements. Previously, the Nematoda have been reported to have a substantially different diversity of YREs compared to other animal phyla: the Dirs1-like YRE retrotransposon was encountered in most animal phyla but not in Nematoda, and a unique Pat1-like YRE retrotransposon has only been recorded from Nematoda. We explored the diversity of YREs in Nematoda by sampling broadly across the phylum and including 34 genomes representing the three classes within Nematoda. We developed a method to isolate and classify YREs based on both feature organization and phylogenetic relationships in an open and reproducible workflow. We also ensured that our phylogenetic approach to YRE classification identified truncated and degenerate elements, informatively increasing the number of elements sampled. We identified Dirs1-like elements (thought to be absent from Nematoda) in the nematode classes Enoplia and Dorylaimia indicating that nematode model species do not adequately represent the diversity of transposable elements in the phylum. Nematode Pat1-like elements were found to be a derived form of another Pat1-like element that is present more widely in animals. Several sequence features used widely for the classification of YREs were found to be homoplasious, highlighting the need for a phylogenetically-based classification scheme. Nematode model species do not represent the diversity of transposable elements in the phylum.

  14. Atherosclerosis: A Link Between Lipid Intake and Protein Tyrosine Nitration

    PubMed Central

    Upmacis, Rita K.

    2009-01-01

    Atherosclerosis, a disease characterized by plaque formation in the arterial wall that can lead to heart attack and stroke, is a principal cause of death in the world. Since the 1990’s, protein nitrotyrosine formation has been known to occur in the atherosclerotic plaque. This potentially damaging reaction occurs as a result of tyrosine modification by reactive nitrogen species, such as nitrogen dioxide radical, which forms upon peroxynitrite decomposition or nitrite oxidation by hydrogen peroxide-activated peroxidase enzymes. The presence of protein-bound nitrotyrosine can be considered an indicator of a loss in the natural balance of oxidants and antioxidants, and as such, there is an emerging view that protein-bound nitrotyrosine may be a risk factor for cardiovascular disease. This review brings together evidence that the accumulation of protein nitrotyrosine during atherogenesis is more widespread than initially thought (as its presence can be detected not only in the lesion but also in the blood stream and other organs) and is closely linked to lipid intake. PMID:20157638

  15. Druggability analysis and classification of protein tyrosine phosphatase active sites

    PubMed Central

    Ghattas, Mohammad A; Raslan, Noor; Sadeq, Asil; Al Sorkhy, Mohammad; Atatreh, Noor

    2016-01-01

    Protein tyrosine phosphatases (PTP) play important roles in the pathogenesis of many diseases. The fact that no PTP inhibitors have reached the market so far has raised many questions about their druggability. In this study, the active sites of 17 PTPs were characterized and assessed for its ability to bind drug-like molecules. Consequently, PTPs were classified according to their druggability scores into four main categories. Only four members showed intermediate to very druggable pocket; interestingly, the rest of them exhibited poor druggability. Particularly focusing on PTP1B, we also demonstrated the influence of several factors on the druggability of PTP active site. For instance, the open conformation showed better druggability than the closed conformation, while the tight-bound water molecules appeared to have minimal effect on the PTP1B druggability. Finally, the allosteric site of PTP1B was found to exhibit superior druggability compared to the catalytic pocket. This analysis can prove useful in the discovery of new PTP inhibitors by assisting researchers in predicting hit rates from high throughput or virtual screening and saving unnecessary cost, time, and efforts via prioritizing PTP targets according to their predicted druggability. PMID:27757011

  16. Have adjuvant tyrosine kinase inhibitors lost their shine?

    PubMed Central

    Sabari, Joshua K.

    2016-01-01

    Despite broad advances in molecularly targeted therapies, lung cancer remains the leading cause of cancer related mortality in the United States. Epidermal growth factor receptor (EGFR) mutations occur in approximately 17% of advanced non-small cell lung cancer (NSCLC) in the US population. The remarkable efficacy of small-molecule EGFR tyrosine kinase inhibitors (TKIs) in this unique subset of patients has revolutionized the therapeutic approach to lung cancer. The success of these agents in the metastatic setting leads to the logical question of what role these drugs may have in the adjuvant setting for patients with earlier stage disease. RADIANT, an international randomized, double-blind, placebo controlled phase III study in patients with completely resected stage IB to IIIA NSLC whose tumors expressed EGFR by IHC and EGFR amplification by FISH, attempted to answer the question of whether erlotinib would improve disease free survival and overall survival in the adjuvant setting. While RADIANT does not conclude for or against adjuvant use of EGFR-TKIs, all data points towards benefit in a selected population. As clinicians, we must continue to enroll to potentially practice changing therapeutic neoadjuvant and adjuvant chemotherapy studies internationally. PMID:27568486

  17. Terreic Acid, a Quinone Epoxide Inhibitor of Bruton's Tyrosine Kinase

    NASA Astrophysics Data System (ADS)

    Kawakami, Yuko; Hartman, Stephen E.; Kinoshita, Eiji; Suzuki, Hidefumi; Kitaura, Jiro; Yao, Libo; Inagaki, Naoki; Franco, Alessandra; Hata, Daisuke; Maeda-Yamamoto, Mari; Fukamachi, Hiromi; Nagai, Hiroichi; Kawakami, Toshiaki

    1999-03-01

    Bruton's tyrosine kinase (Btk) plays pivotal roles in mast cell activation as well as in B cell development. Btk mutations lead to severe impairments in proinflammatory cytokine production induced by cross-linking of high-affinity IgE receptor on mast cells. By using an in vitro assay to measure the activity that blocks the interaction between protein kinase C and the pleckstrin homology domain of Btk, terreic acid (TA) was identified and characterized in this study. This quinone epoxide specifically inhibited the enzymatic activity of Btk in mast cells and cell-free assays. TA faithfully recapitulated the phenotypic defects of btk mutant mast cells in high-affinity IgE receptor-stimulated wild-type mast cells without affecting the enzymatic activities and expressions of many other signaling molecules, including those of protein kinase C. Therefore, this study confirmed the important roles of Btk in mast cell functions and showed the usefulness of TA in probing into the functions of Btk in mast cells and other immune cell systems. Another insight obtained from this study is that the screening method used to identify TA is a useful approach to finding more efficacious Btk inhibitors.

  18. Signal processing by protein tyrosine phosphorylation in plants

    PubMed Central

    2011-01-01

    Protein phosphorylation is a reversible post-translational modification controlling many biological processes. Most phosphorylation occurs on serine and threonine, and to a less extend on tyrosine (Tyr). In animals, Tyr phosphorylation is crucial for the regulation of many responses such as growth or differentiation. Only recently with the development of mass spectrometry, it has been reported that Tyr phosphorylation is as important in plants as in animals. The genes encoding protein Tyr kinases and protein Tyr phosphatases have been identified in the Arabidopsis thaliana genome. Putative substrates of these enzymes, and thus Tyr-phosphorylated proteins have been reported by proteomic studies based on accurate mass spectrometry analysis of the phosphopeptides and phosphoproteins. Biochemical approaches, pharmacology and genetic manipulations have indicated that responses to stress and developmental processes involve changes in protein Tyr phosphorylation. The aim of this review is to present an update on Tyr phosphorylation in plants in order to better assess the role of this post-translational modification in plant physiology. PMID:21628997

  19. Role of Striatal-Enriched Tyrosine Phosphatase in Neuronal Function

    PubMed Central

    Lombroso, Paul J.

    2016-01-01

    Striatal-enriched protein tyrosine phosphatase (STEP) is a CNS-enriched protein implicated in multiple neurologic and neuropsychiatric disorders. STEP regulates key signaling proteins required for synaptic strengthening as well as NMDA and AMPA receptor trafficking. Both high and low levels of STEP disrupt synaptic function and contribute to learning and behavioral deficits. High levels of STEP are present in human postmortem samples and animal models of Alzheimer's disease, Parkinson's disease, and schizophrenia and in animal models of fragile X syndrome. Low levels of STEP activity are present in additional disorders that include ischemia, Huntington's chorea, alcohol abuse, and stress disorders. Thus the current model of STEP is that optimal levels are required for optimal synaptic function. Here we focus on the role of STEP in Alzheimer's disease and the mechanisms by which STEP activity is increased in this illness. Both genetic lowering of STEP levels and pharmacological inhibition of STEP activity in mouse models of Alzheimer's disease reverse the biochemical and cognitive abnormalities that are present. These findings suggest that STEP is an important point for modulation of proteins required for synaptic plasticity. PMID:27190655

  20. Redundant Regulation of Cdk1 Tyrosine Dephosphorylation in Saccharomyces cerevisiae.

    PubMed

    Kennedy, Erin K; Dysart, Michael; Lianga, Noel; Williams, Elizabeth C; Pilon, Sophie; Doré, Carole; Deneault, Jean-Sebastien; Rudner, Adam D

    2016-03-01

    Cdk1 activity drives both mitotic entry and the metaphase-to-anaphase transition in all eukaryotes. The kinase Wee1 and the phosphatase Cdc25 regulate the mitotic activity of Cdk1 by the reversible phosphorylation of a conserved tyrosine residue. Mutation of cdc25 in Schizosaccharomyces pombe blocks Cdk1 dephosphorylation and causes cell cycle arrest. In contrast, deletion of MIH1, the cdc25 homolog in Saccharomyces cerevisiae, is viable. Although Cdk1-Y19 phosphorylation is elevated during mitosis in mih1∆ cells, Cdk1 is dephosphorylated as cells progress into G1, suggesting that additional phosphatases regulate Cdk1 dephosphorylation. Here we show that the phosphatase Ptp1 also regulates Cdk1 dephosphorylation in vivo and can directly dephosphorylate Cdk1 in vitro. Using a novel in vivo phosphatase assay, we also show that PP2A bound to Rts1, the budding yeast B56-regulatory subunit, regulates dephosphorylation of Cdk1 independently of a function regulating Swe1, Mih1, or Ptp1, suggesting that PP2A(Rts1) either directly dephosphorylates Cdk1-Y19 or regulates an unidentified phosphatase.

  1. Differential pre- and postsynaptic modulation of chemical transmission in the squid giant synapse by tyrosine phosphorylation.

    PubMed

    Llinás, R; Moreno, H; Sugimori, M; Mohammadi, M; Schlessinger, J

    1997-03-01

    To assess the role of tyrosine phosphorylation/dephosphorylation balance in synaptic transmission, a set of studies was implemented at the squid giant synapse. Presynaptic induction of tyrosine phosphorylation, following administration of the tyrosine phosphatase inhibitor pervanadate, produced a sizable increase in presynaptic calcium current and a concomitant and paradoxical decrement of the postsynaptic potential amplitude. Presynaptic microinjection of an active protein tyrosine kinase dramatically increased calcium currents and incremented postsynaptic potential amplitude. By contrast, the same procedure at the postsynaptic terminal reduced the size of the postsynaptic potential. This differential effect may be prodromic to long-term plasticity, as postsynaptic sensitivity is momentarily deemphasized, whereas presynaptic second messenger cascades triggered by increased calcium currents are accentuated.

  2. DNA sequence, structure, and tyrosine kinase activity of the Drosophila melanogaster abelson proto-oncogene homolog

    SciTech Connect

    Henkemeyer, M.J.; Bennett, R.L.; Gertler, F.B.; Hoffmann, F.M.

    1988-02-01

    The authors report their molecular characterization of the Drosophila melanogaster Abelson gene (abl), a gene in which recessive loss-of-function mutations result in lethality at the pupal stage of development. This essential gene consists of 10 exons extending over 26 kilobase pairs of genomic DNA. The DNA sequence encodes a protein of 1,520 amino acids with strong sequence similarity to the human c-abl proto-oncogene beginning in the type 1b 5' exon and extending through the region essential for tyrosine kinase activity. When the tyrosine kinase homologous region was expressed in Escherichia coli, phosphorylation of proteins on tyrosine residues was observed with an antiphosphotyrosine antibody. These results show that the abl gene is highly conserved through evolution and encodes a functional tyrosine protein kinase required for Drosophila development.

  3. Conformational Structure of Tyrosine, Tyrosyl-Glycine, and Tyrosyl-Glycyl-Glycine by Double Resonance Spectroscopy

    NASA Technical Reports Server (NTRS)

    Abo-Riziq, Ali; Grace, Louis; Crews, Bridgit; Callahan, Michael P,; van Mourik, Tanja; de Vries, Mattanjah S,

    2011-01-01

    We investigated the variation in conformation for the amino acid tyrosine (Y), alone and in the small peptides tyrosine-glycine (YC) and tyrosine-glycine-glycine (YGG), in the gas phase by using UV-UV and IR-UV double resonance spectroscopy and density functional theory calculations. For tyrosine we found seven different conformations, for YG we found four different conformations, and for YGG we found three different conformations. As the peptides get larger, we observe fewer stable conformers, despite the increasing complexity and number of degrees of freedom. We find structural trends similar to those in phenylalanine-glycine glycine (FGG) and tryptophan-glycine-glycine (WGG)j however) the effect of dispersive forces in FGG for stabilizing a folded structure is replaced by that of hydrogen bonding in YGG.

  4. Protein-tyrosine phosphatase activity of CD45 is activated by sequential phosphorylation by two kinases.

    PubMed Central

    Stover, D R; Walsh, K A

    1994-01-01

    We describe a potential regulatory mechanism for the transmembrane protein-tyrosine phosphatase CD45. Phosphorylation on both tyrosine and serine residues in vitro results in an activation of CD45 specifically toward one artificial substrate but not another. The activation of these kinases appears to be order dependent, as it is enhanced when phosphorylation of tyrosine precedes that of serine but phosphorylation in the reverse order yields no activation. Any of four protein-tyrosine kinases tested, in combination with the protein-serine/threonine kinase, casein kinase II, was capable of mediating this activation in vitro. The time course of phosphorylation of CD45 in response to T-cell activation is consistent with the possibility that this regulatory mechanism is utilized in vivo. Images PMID:7518565

  5. Tyrosine kinase inhibitor STI571 enhances thyroid cancer cell motile response to Hepatocyte Growth Factor.

    PubMed

    Frasca, F; Vigneri, P; Vella, V; Vigneri, R; Wang, J Y

    2001-06-28

    The Hepatocyte Growth Factor (HGF) and its receptor Met are physiological regulators of cell migration. HGF and Met have also been implicated in tumor progression and metastasis. We show here that the tyrosine kinase inhibitor STI571 has a stimulatory effect on HGF-induced migration and branching morphogenesis in thyroid cancer but not in primary or immortalized thyroid epithelial cells. These stimulatory effects of STI571 are observed at a concentration that is clinically relevant. The STI571-enhanced motile response can be correlated with an increase in the Met receptor tyrosine phosphorylation as well as ERK and Akt activation by HGF. Interestingly, one of the targets of STI571, namely the c-Abl tyrosine kinase, is activated by HGF and is recruited at the migrating edge of thyroid cancer cells. These data suggests that c-Abl and/or STI571-inhibited tyrosine kinases can negatively regulate the Met receptor to restrain the motile response in thyroid cancer cells.

  6. Determination of Phenylalanine and Tyrosine by High Performance Liquid Chromatography-Tandem Mass Spectrometry.

    PubMed

    Peat, Judy; Garg, Uttam

    2016-01-01

    Hyperphenylalaninemia/phenylketonuria (PKU) is one of the most common inborn errors of amino acid metabolism affecting about 1:15,000 infants in the United States. PKU is an autosomal recessive disorder that if untreated results in mental retardation. The most common cause of PKU is deficiency of the enzyme phenylalanine hydroxylase that converts phenylalanine to tyrosine. Tyrosine deficiency results in impaired synthesis of catecholamines and thyroxine. Less commonly, it can result from defects in the synthesis or regeneration of tetrahydrobiopterin (BH4), an essential cofactor for the enzyme phenylalanine hydroxylase. Increased phenylalanine and decreased tyrosine in blood are used in the diagnosis and follow-up of patients with PKU. LC/MS/MS method is described for the quantification of phenylalanine and tyrosine.

  7. Administration of supplemental L-tyrosine with phenelzine: a clinical literature review

    PubMed Central

    Hinz, Marty; Stein, Alvin; Cole, Ted; Ryan, Patricia

    2014-01-01

    The subject of this literature review is the alleged relationship between L-tyrosine, phenelzine, and hypertensive crisis. Phenelzine (Nardil®) prescribing information notes: “The potentiation of sympathomimetic substances and related compounds by MAO inhibitors may result in hypertensive crises (see WARNINGS). Therefore, patients being treated with NARDIL should not take […] L-tyrosine […]”. Interest in the scientific foundation of this claim was generated during routine patient care. A comprehensive literature search of Google Scholar and PubMed revealed no reported cases of hypertensive crisis associated with concomitant administration of L-tyrosine and phenelzine. Review of current US Food and Drug Administration nutritional guidelines relating to ongoing phenelzine studies reveals no mention and requires no consideration of L-tyrosine ingestion in combination with phenelzine. This paper is intended to provide an objective review of the science to then allow the reader to formulate the final opinion. PMID:25092999

  8. Pervanadate activation of intracellular kinases leads to tyrosine phosphorylation and shedding of syndecan-1.

    PubMed Central

    Reiland, J; Ott, V L; Lebakken, C S; Yeaman, C; McCarthy, J; Rapraeger, A C

    1996-01-01

    Syndecan-1 is a transmembrane haparan sulphate proteoglycan that binds extracellular matrices and growth factors, making it a candidate to act between these regulatory molecules and intracellular signalling pathways. It has a highly conserved transmembrane/cytoplasmic domain that contains four conserved tyrosines. One of these is in a consensus sequence for tyrosine kinase phosphorylation. As an initial step to investigating whether or not phosphorylation of these tyrosines is part of a signal-transduction pathway, we have monitored the tyrosine phosphorylation of syndecan-1 by cytoplasmic tyrosine kinases in intact cells. Tyrosine phosphorylation of syndecan-1 is observed when NMuMG cells are treated with sodium orthovanadate or pervanadate, which have been shown to activate intracellular tyrosine kinases. Initial studies with sodium orthovanadate demonstrate a slow accumulation of phosphotyrosine on syndecan-1 over the course of several hours. Pervanadate, a more effective inhibitor of phosphatases, allows detection of phosphotyrosine on syndecan-1 within 5 min, with peak phosphorylation seen by 15 min. Concurrently, in a second process activated by pervanadate, syndecan-1 ectodomain is cleaved and released into the culture medium. Two phosphorylated fragments of syndecan-1 of apparent sizes 6 and 8 kDa remain with the cell after shedding of the ectodomain. The 8 kDa size class appears to be a highly phosphorylated form of the 6 kDa product, as it disappears if samples are dephosphorylated. These fragments contain the C-terminus of syndecan-1 and also retain at least a portion of the transmembrane domain, suggesting that they are produced by a cell surface cleavage event. Thus pervanadate treatment of cells results in two effects of syndecan-1: (i) phosphorylation of one or more of its tyrosines via the action of a cytoplasmic kinase(s) and (ii) cleavage and release of the ectodomain into the medium, producing a C-terminal fragment containing the transmembrane

  9. Spectroscopic studies on the interaction of cysteine capped CuS nanoparticles with tyrosine

    SciTech Connect

    Prasanth, S.; Raj, D. Rithesh; Kumar, T. V. Vineesh; Sudarsanakumar, C.

    2015-06-24

    Biocompatible cysteine coated CuS nanoparticles were synthesized by a simple aqueous solution method. Hexagonal phase of the samples were confirmed from X-ray diffraction and particle size found to be 9 nm. The possible interaction between the bioactive cysteine capped CuS nanoparticles and tyrosine were investigated using spectroscopic techniques such as UV-Visible absorption and fluorescence spectroscopy. It is observed that the luminescence intensity of tyrosine molecule enhanced by the addition CuS nanoparticles.

  10. Spectroscopic studies on the interaction of cysteine capped CuS nanoparticles with tyrosine

    NASA Astrophysics Data System (ADS)

    Prasanth, S.; Raj, D. Rithesh; Kumar, T. V. Vineesh; Sudarsanakumar, C.

    2015-06-01

    Biocompatible cysteine coated CuS nanoparticles were synthesized by a simple aqueous solution method. Hexagonal phase of the samples were confirmed from X-ray diffraction and particle size found to be 9 nm. The possible interaction between the bioactive cysteine capped CuS nanoparticles and tyrosine were investigated using spectroscopic techniques such as UV-Visible absorption and fluorescence spectroscopy. It is observed that the luminescence intensity of tyrosine molecule enhanced by the addition CuS nanoparticles.

  11. De novo design of VEGFR-2 tyrosine kinase inhibitors based on a linked-fragment approach.

    PubMed

    Liu, Yi-Zhou; Wang, Xiao-Li; Wang, Xin-Ying; Yu, Ri-Lei; Liu, Dong-Qing; Kang, Cong-Min

    2016-09-01

    Vascular endothelial growth factor receptor-2 (VEGFR-2) tyrosine kinase inhibitors have been demonstrated to possess substantial antitumor activity. VEGFR-2 tyrosine kinase inhibitors are crucial for development of antitumor drugs. Based on the crystal structure of VEGFR-2 tyrosine kinase, a linked-fragment strategy was employed to design novel VEGFR-2 tyrosine kinase inhibitors, and 1000 compounds were generated in this process. Absorption, distribution, metabolism, excretion and toxicity (ADMET) were used to screen the 1000 compounds, and 59 compounds were acceptable. Scaffold hopping was then used for further screening, and only four compounds were obtained in this way. Then, the binding energy of the four molecules to VEGFR-2 tyrosine kinase was calculated using molecular docking, and their values were found to be lower than that of Sorafenib. Finally, molecular dynamics simulations were performed on the complex of the compound with the lowest binding energy with VEGFR-2 tyrosine kinase, and the binding model was analyzed. At the end, four chemical entities with novel structures were obtained, and were suggested for experimental testing in future studies. PMID:27558799

  12. Regulation of the cystic fibrosis transmembrane conductance regulator anion channel by tyrosine phosphorylation

    PubMed Central

    Billet, Arnaud; Jia, Yanlin; Jensen, Tim; Riordan, John R.; Hanrahan, John W.

    2015-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) channel is activated by PKA phosphorylation of a regulatory domain that interacts dynamically with multiple CFTR domains and with other proteins. The large number of consensus sequences for phosphorylation by PKA has naturally focused most attention on regulation by this kinase. We report here that human CFTR is also phosphorylated by the tyrosine kinases p60c-Src (proto-oncogene tyrosine-protein kinase) and the proline-rich tyrosine kinase 2 (Pyk2), and they can also cause robust activation of quiescent CFTR channels. In excised patch-clamp experiments, CFTR activity during exposure to Src or Pyk2 reached ∼80% of that stimulated by PKA. Exposure to PKA after Src or Pyk2 caused a further increase to the level induced by PKA alone, implying a common limiting step. Channels became spontaneously active when v-Src or the catalytic domain of Pyk2 was coexpressed with CFTR and were further stimulated by the tyrosine phosphatase inhibitor dephostatin. Exogenous Src also activated 15SA-CFTR, a variant that lacks 15 potential PKA sites and has little response to PKA. PKA-independent activation by tyrosine phosphorylation has implications for the mechanism of regulation by the R domain and for the physiologic functions of CFTR.—Billet, A., Jia, Y., Jensen, T., Riordan, J. R., Hanrahan, J. W. Regulation of the cystic fibrosis transmembrane conductance regulator anion channel by tyrosine phosphorylation. PMID:26062600

  13. Lack of behavioural effects after acute tyrosine depletion in healthy volunteers.

    PubMed

    Lythe, K E; Anderson, I M; Deakin, J F W; Elliott, R; Strickland, P L

    2005-01-01

    Acute dietary tyrosine depletion has previously been shown to reduce dopamine neurotransmission in both animals and humans. In this study, we investigated the effects of brain dopamine depletion, through acute tyrosine and phenylalanine depletion, on plasma prolactin, mood and neuropsychological function in 12 normal subjects. In a randomized, double-blind, cross-over design, subjects received two amino-acid drinks separated by a week, a nutritionally balanced mixture (Bal) and on the other occasion a tyrosine and phenylalanine deficient mixture (TP-). The plasma ratio of tyrosine and phenylalanine to the other large neutral amino acids decreased significantly on the TP- occasion (-78.7%, p < 0.0001) and there was an increase in plasma prolactin concentration relative to the balanced drink in the seven subjects for whom results were available for both occasions (p < 0.02). Acute tyrosine depletion did not alter mood as measured by visual analogue scale ratings, and measures of memory, attention and behavioural inhibition were also unaffected. Our results are consistent with acute dietary tyrosine depletion causing a reduction in brain dopamine neurotransmission but raise questions about how robust or consistent the effects are on psychological function. PMID:15671123

  14. Verbascoside promotes the regeneration of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra

    PubMed Central

    Liang, Jian-qing; Wang, Li; He, Jian-cheng; Hua, Xian-dong

    2016-01-01

    Tyrosine hydroxylase is a key enzyme in dopamine biosynthesis. Change in tyrosine hydroxylase expression in the nigrostriatal system is closely related to the occurrence and development of Parkinson's disease. Verbascoside, an extract from Radix Rehmanniae Praeparata has been shown to be clinically effective in treating Parkinson's disease. However, the underlying mechanisms remain unclear. It is hypothesized that the effects of verbascoside on Parkinson's disease are related to tyrosine hydroxylase expression change in the nigrostriatal system. Rat models of Parkinson's disease were established and verbascoside (60 mg/kg) was administered intraperitoneally once a day. After 6 weeks of verbascoside treatment, rat rotational behavior was alleviated; tyrosine hydroxylase mRNA and protein expression and the number of tyrosine hydroxylase-immunoreactive neurons in the rat right substantia nigra were significantly higher than the Parkinson's model group. These findings suggest that the mechanism by which verbascoside treats Parkinson's disease is related to the regeneration of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra. PMID:26981096

  15. Tetrodotoxin-insensitive Na+ channel activator palytoxin inhibits tyrosine uptake into cultured bovine adrenal chromaffin cells

    SciTech Connect

    Morita, K.; Teraoka, K.; Azuma, M.; Oka, M.; Hamano, S. )

    1991-07-01

    The effects of the tetrodotoxin-insensitive Na+ channel activator palytoxin on both the secretion of endogenous catecholamines and the formation of 14C-catecholamines from (14C)tyrosine were examined using cultured bovine adrenal chromaffin cells. Palytoxin was shown to cause the stimulation of catecholamine secretion in a concentration-dependent manner. However, this toxin caused the reduction rather than the stimulation of 14C-catecholamine formation at the same concentrations. Palytoxin failed to cause any alteration in the activity of tyrosine hydroxylase prepared from bovine adrenal medulla. Furthermore, the uptake of (14C)tyrosine into the cells was shown to be inhibited by this toxin under the conditions in which the suppression of 14C-catecholamine formation was observed, and this inhibitory action on tyrosine uptake was closely correlated with that on catecholamine formation. The inhibitory action of palytoxin on tyrosine uptake into the cells was observed to be noncompetitive, and this effect was not altered by the removal of Na+ from the incubation mixture. These results suggest that palytoxin may be able to inhibit the uptake of (14C)tyrosine into the cells, resulting in the suppression of 14C-catecholamine formation, probably through its direct action on the plasma membranes of bovine adrenal chromaffin cells.

  16. Tyrosine phosphorylation of band 3 protein in Ca2+/A23187-treated human erythrocytes.

    PubMed Central

    Minetti, G; Piccinini, G; Balduini, C; Seppi, C; Brovelli, A

    1996-01-01

    Human erythrocytes were induced to release membrane vesicles by treatment with Ca2+ and ionophore A23187. In addition to the biochemical changes already known to accompany loading of human erythrocytes with Ca2+, the present study reveals that tyrosine phosphorylation of the anion exchanger band 3 protein also occurs. The relationship between tyrosine phosphorylation of band 3 and membrane vesiculation was analysed using quinine (a non-specific inhibitor of the Ca(2+)-activated K+ channel, and the only known inhibitor of Ca(2+)-induced vesiculation) and charybdotoxin, a specific inhibitor of the apamin-insensitive K(+)-channel. Both inhibitors suppressed tyrosine phosphorylation of band 3. In the presence of quinine, membrane vesiculation was also suppressed. In contrast, at the concentration of charybdotoxin required to suppress tyrosine phosphorylation of band 3, membrane vesiculation was only mildly inhibited (16-23% inhibition), suggesting that tyrosine phosphorylation of band 3 is not necessary for membrane vesiculation. Phosphorylation of band 3 was in fact observed when erythrocytes were induced to shrink in a Ca(2+)-independent manner, e.g. by treatment with the K+ ionophore valinomycin or with hypertonic solutions. These observations suggest that band 3 tyrosine phosphorylation occurs when cell volume regulation is required. PMID:8973551

  17. Effect of tyrosine administration on duodenal ulcer induced by cysteamine in the rat

    SciTech Connect

    Oishi, T.; Szabo, S.

    1987-03-01

    Duodenal ulcers were produced by administering cysteamine to rats. Pretreatment with the catecholamine precursor, L-tyrosine (40 mg/100 g i.p. for 5 days), decreased the intensity of duodenal ulcers induced by cysteamine. Equimolar doses of tyrosine methyl ester (51.2 mg/100 g i.p. or s.c.) were equally effective in reducing ulcer intensity. Other amino acids (i.e., alanine, aspartic acid, glutamic acid, glycine, leucine, lysine, tryptophan and valine) did not prevent experimental duodenal ulcers. Coadministration of other large neutral amino acids (e.g., leucine and valine) that compete with tyrosine for uptake into the brain did not inhibit the effect of tyrosine on duodenal ulcers induced by cysteamine. Gastric, duodenal and brain dopamine concentrations were increased 1 hr after the injection of tyrosine methyl ester (25.6 mg/100 g s.c.). These results suggest that the effect of tyrosine on duodenal ulcer induced by cysteamine may be mediated by changes in gastrointestinal dopamine metabolism.

  18. An experimental and computational investigation into the gas-phase acidities of tyrosine and phenylalanine: three structures for deprotonated tyrosine.

    PubMed

    Bokatzian, Samantha S; Stover, Michele L; Plummer, Chelsea E; Dixon, David A; Cassady, Carolyn J

    2014-11-01

    Using mass spectrometry and correlated molecular orbital theory, three deprotonated structures were revealed for the amino acid tyrosine. The structures were distinguished experimentally by ion/molecule reactions involving proton transfer and trimethylsilyl azide. Gas-phase acidities from proton transfer reactions and from G3(MP2) calculations generally agree well. The lowest energy structure, which was only observed experimentally using electrospray ionization from aprotic solvents, is deprotonated at the carboxylic acid group and is predicted to be highly folded. A second unfolded carboxylate structure is several kcal/mol higher in energy and primarily forms from protic solvents. Protic solvents also yield a structure deprotonated at the phenolic side chain, which experiments find to be intermediate in energy to the two carboxylate forms. G3(MP2) calculations indicate that the three structures differ in energy by only 2.5 kcal/mol, yet they are readily distinguished experimentally. Structural abundance ratios are dependent upon experimental conditions, including the solvent and accumulation time of ions in a hexapole. Under some conditions, carboxylate ions may convert to phenolate ions. For phenylalanine, which lacks a phenolic group, only one deprotonated structure was observed experimentally when electrosprayed from protic solvent. This agrees with G3(MP2) calculations that find the folded and unfolded carboxylate forms to differ by 0.3 kcal/mol.

  19. Interaction of vanadate with phenol and tyrosine: implications for the effects of vanadate on systems regulated by tyrosine phosphorylation

    SciTech Connect

    Tracey, A.S.; Gresser, M.J.

    1986-02-01

    The interaction of vanadate with phenol and N-acetyltyrosine ethyl ester in aqueous solution has been studied by using /sup 51/V nuclear magnetic resonance spectroscopy. On the basis of these studies, it has been concluded that vanadate rapidly esterifies the hydroxyl group of the aromatic ring to yield a phenyl vanadate. For phenol, the equilibrium constant for this reaction in terms of the convention that the activity of liquid water is 1.0 is K/sub 1/ = (phenyl vanadate)/(phenol)(vanadate) = 0.97 +/- 0.02. This value is well over 4 orders of magnitude larger than estimates from the literature for the corresponding equilibrium constant for the esterification of phenol by phosphate. The equilibrium constant for esterification of the phenol moiety of N-acetyltyrosine ethyl ester is similar to that for esterification of phenol. The relevance of these observations to processes that are regulated by reversible phosphorylation/dephosphorylation of tyrosine residues is discussed, in particular the insulin-like effect of vanadate.

  20. An Experimental and Computational Investigation into the Gas-Phase Acidities of Tyrosine and Phenylalanine: Three Structures for Deprotonated Tyrosine

    SciTech Connect

    Bokatzian, Samantha S.; Stover, Michele L.; Plummer, Chelsea E.; Dixon, David A.; Cassady, Carolyn J.

    2014-11-06

    Using mass spectrometry and correlated molecular orbital theory, three deprotonated structures were revealed for the amino acid tyrosine. The structures were distinguished experimentally by ion/molecule reactions involving proton transfer and trimethylsilyl azide. Gas-phase acidities from proton transfer reactions and from G3(MP2) calculations generally agree well. The lowest energy structure, which was only observed experimentally using electrospray ionization from aprotic solvents, is deprotonated at the carboxylic acid group and is predicted to be highly folded. A second unfolded carboxylate structure is several kcal/mol higher in energy and primarily forms from protic solvents. Protic solvents also yield a structure deprotonated at the phenolic side chain, which experiments find to be intermediate in energy to the two carboxylate forms. G3(MP2) calculations indicate that the three structures differ in energy by only 2.5 kcal/mol, yet they are readily distinguished experimentally. Structural abundance ratios are dependent upon experimental conditions, including the solvent and accumulation time of ions in a hexapole. Under some conditions, carboxylate ions may convert to phenolate ions. For phenylalanine, which lacks a phenolic group, only one deprotonated structure was observed experimentally when electrosprayed from protic solvent. This agrees with G3(MP2) calculations that find the folded and unfolded carboxylate forms to differ by 0.3 kcal/mol.

  1. An experimental and computational investigation into the gas-phase acidities of tyrosine and phenylalanine: three structures for deprotonated tyrosine.

    PubMed

    Bokatzian, Samantha S; Stover, Michele L; Plummer, Chelsea E; Dixon, David A; Cassady, Carolyn J

    2014-11-01

    Using mass spectrometry and correlated molecular orbital theory, three deprotonated structures were revealed for the amino acid tyrosine. The structures were distinguished experimentally by ion/molecule reactions involving proton transfer and trimethylsilyl azide. Gas-phase acidities from proton transfer reactions and from G3(MP2) calculations generally agree well. The lowest energy structure, which was only observed experimentally using electrospray ionization from aprotic solvents, is deprotonated at the carboxylic acid group and is predicted to be highly folded. A second unfolded carboxylate structure is several kcal/mol higher in energy and primarily forms from protic solvents. Protic solvents also yield a structure deprotonated at the phenolic side chain, which experiments find to be intermediate in energy to the two carboxylate forms. G3(MP2) calculations indicate that the three structures differ in energy by only 2.5 kcal/mol, yet they are readily distinguished experimentally. Structural abundance ratios are dependent upon experimental conditions, including the solvent and accumulation time of ions in a hexapole. Under some conditions, carboxylate ions may convert to phenolate ions. For phenylalanine, which lacks a phenolic group, only one deprotonated structure was observed experimentally when electrosprayed from protic solvent. This agrees with G3(MP2) calculations that find the folded and unfolded carboxylate forms to differ by 0.3 kcal/mol. PMID:25299802

  2. Pervanadate-induced adhesion of CD4+ T cell to fibronectin is associated with tyrosine phosphorylation of paxillin.

    PubMed

    Miron, S; Kachalsky, S G; Hershkoviz, R; Lider, O

    1997-09-01

    The initial stages of T cell activation involve tyrosine protein kinase-mediated intracellular signaling events. Integrin-mediated adhesion of CD4+ T lymphocytes to extracellular matrix glycoproteins, such as fibronectin, is an activation-dependent process. The involvement of tyrosine protein kinases in the adhesion of CD4+ T cells to fibronectin was examined using pervanadate, a protein-tyrosine phosphatase inhibitor. Pervanadate induced the adhesion of human CD4+ T cells to immobilized fibronectin in a beta1 integrin-mediated fashion, and adhesion was associated with an increase of protein tyrosine phosphorylation. Tyrosine protein kinase inhibitors abrogated both T cell adhesion and intracellular protein tyrosine phosphorylation. Participation of cytoskeletal proteins in the pervanadate-induced T cell adhesion was indicated because cytoskeleton disruption by cytochalasin B inhibited cell adhesion to fibronectin. We demonstrate that the cytoskeletal protein paxillin underwent time-dependent tyrosine phosphorylation simultaneously with pervanadate-induced T cell adhesion to fibronectin. Tyrosine phosphorylation of paxillin was related to cell adhesion, since pretreatment of T cells with cytochalasin B abrogated both adhesion and phosphorylation. This study demonstrates a correlation between activation of protein tyrosine kinases, tyrosine phosphorylation of paxillin, and integrin-mediated T cell adhesion to extracellular matrix glycoproteins. PMID:9307082

  3. Effective L-Tyrosine Hydroxylation by Native and Immobilized Tyrosinase

    PubMed Central

    Lewańczuk, Marcin; Koźlecki, Tomasz; Liesiene, Jolanta; Bryjak, Jolanta

    2016-01-01

    Hydroxylation of L-tyrosine to 3,4-dihydroxyphenylalanine (L-DOPA) by immobilized tyrosinase in the presence of ascorbic acid (AH2), which reduces DOPA-quinone to L-DOPA, is characterized by low reaction yields that are mainly caused by the suicide inactivation of tyrosinase by L-DOPA and AH2. The main aim of this work was to compare processes with native and immobilized tyrosinase to identify the conditions that limit suicide inactivation and produce substrate conversions to L-DOPA of above 50% using HPLC analysis. It was shown that immobilized tyrosinase does not suffer from partitioning and diffusion effects, allowing a direct comparison of the reactions performed with both forms of the enzyme. In typical processes, additional aeration was applied and boron ions to produce the L-DOPA and AH2 complex and hydroxylamine to close the cycle of enzyme active center transformations. It was shown that the commonly used pH 9 buffer increased enzyme stability, with concomitant reduced reactivity of 76%, and that under these conditions, the maximal substrate conversion was approximately 25 (native) to 30% (immobilized enzyme). To increase reaction yield, the pH of the reaction mixture was reduced to 8 and 7, producing L-DOPA yields of approximately 95% (native enzyme) and 70% (immobilized). A three-fold increase in the bound enzyme load achieved 95% conversion in two successive runs, but in the third one, tyrosinase lost its activity due to strong suicide inactivation caused by L-DOPA processing. In this case, the cost of the immobilized enzyme preparation is not overcome by its reuse over time, and native tyrosinase may be more economically feasible for a single use in L-DOPA production. The practical importance of the obtained results is that highly efficient hydroxylation of monophenols by tyrosinase can be obtained by selecting the proper reaction pH and is a compromise between complexation and enzyme reactivity. PMID:27711193

  4. Tyrosine aminotransferase contributes to benzylisoquinoline alkaloid biosynthesis in opium poppy.

    PubMed

    Lee, Eun-Jeong; Facchini, Peter J

    2011-11-01

    Tyrosine aminotransferase (TyrAT) catalyzes the transamination of L-Tyr and α-ketoglutarate, yielding 4-hydroxyphenylpyruvic acid and L-glutamate. The decarboxylation product of 4-hydroxyphenylpyruvic acid, 4-hydroxyphenylacetaldehyde, is a precursor to a large and diverse group of natural products known collectively as benzylisoquinoline alkaloids (BIAs). We have isolated and characterized a TyrAT cDNA from opium poppy (Papaver somniferum), which remains the only commercial source for several pharmaceutical BIAs, including codeine, morphine, and noscapine. TyrAT belongs to group I pyridoxal 5'-phosphate (PLP)-dependent enzymes wherein Schiff base formation occurs between PLP and a specific Lys residue. The amino acid sequence of TyrAT showed considerable homology to other putative plant TyrATs, although few of these have been functionally characterized. Purified, recombinant TyrAT displayed a molecular mass of approximately 46 kD and a substrate preference for L-Tyr and α-ketoglutarate, with apparent K(m) values of 1.82 and 0.35 mm, respectively. No specific requirement for PLP was detected in vitro. Liquid chromatography-tandem mass spectrometry confirmed the conversion of L-Tyr to 4-hydroxyphenylpyruvate. TyrAT gene transcripts were most abundant in roots and stems of mature opium poppy plants. Virus-induced gene silencing was used to evaluate the contribution of TyrAT to BIA metabolism in opium poppy. TyrAT transcript levels were reduced by at least 80% in silenced plants compared with controls and showed a moderate reduction in total alkaloid content. The modest correlation between transcript levels and BIA accumulation in opium poppy supports a role for TyrAT in the generation of alkaloid precursors, but it also suggests the occurrence of other sources for 4-hydroxyphenylacetaldehyde.

  5. From tyrosine to melanin: Signaling pathways and factors regulating melanogenesis.

    PubMed

    Rzepka, Zuzanna; Buszman, Ewa; Beberok, Artur; Wrześniok, Dorota

    2016-01-01

    Melanins are natural pigments of skin, hair and eyes and can be classified into two main types: brown to black eumelanin and yellow to reddish-brown pheomelanin. Biosynthesis of melanins takes place in melanosomes, which are specialized cytoplasmic organelles of melanocytes - dendritic cells located in the basal layer of the epidermis, uveal tract of the eye, hair follicles, as well as in the inner ear, central nervous system and heart. Melanogenesis is a multistep process and begins with the conversion of amino acid L-tyrosine to DOPAquinone. The addition of cysteine or glutathione to DOPAquinone leads to the intermediates formation, followed by subsequent transformations and polymerization to the final product, pheomelanin. In the absence of thiol compounds DOPAquinone undergoes an intramolecular cyclization and oxidation to form DOPAchrome, which is then converted to 5,6-dihydroksyindole (DHI) or 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Eumelanin is formed by polymerization of DHI and DHICA and their quinones. Regulation of melanogenesis is achieved by physical and biochemical factors. The article presents the intracellular signaling pathways: cAMP/PKA/CREB/MITF cascade, MAP kinases cascade, PLC/DAG/PKCβ cascade and NO/cGMP/PKG cascade, which are involved in the regulation of expression and activity of the melanogenesis-related proteins by ultraviolet radiation and endogenous agents (cytokines, hormones). Activity of the key melanogenic enzyme, tyrosinase, is also affected by pH and temperature. Many pharmacologically active substances are able to inhibit or stimulate melanin biosynthesis, as evidenced by in vitro studies on cultured pigment cells. PMID:27356601

  6. Cardiotoxicity Associated with the Tyrosine Kinase Inhibitor Sunitinib

    PubMed Central

    Chu, Tammy F.; Rupnick, Maria A.; Kerkela, Risto; Dallabrida, Susan M.; Zurakowski, David; Nguyen, Lisa; Woulfe, Kathleen; Pravda, Elke; Cassiola, Flavia; Desai, Jayesh; George, Suzanne; Morgan, Jeffrey A.; Harris, David; Ismail, Nesreen S.; Chen, Jey-Hsin; Schoen, Frederick J.

    2008-01-01

    Background Tyrosine kinase inhibitors (TKIs) have advanced cancer treatment. Sunitinib, a recently-approved, multi-targeted TKI, prolongs survival for patients with metastatic renal cell carcinoma (RCC) and gastrointestinal stromal tumor (GIST), but concerns about cardiac safety have arisen with this agent. Methods To determine the cardiovascular risk associated with sunitinib, we reviewed all cardiovascular events in patients with imatinib-resistant, metastatic GIST at the Dana-Farber Cancer Institute enrolled in a Phase I/II protocol evaluating the efficacy of the drug (n=75). Sunitinib’s effects on left ventricular ejection fraction (LVEF) and blood pressure (BP) were also examined. Studies in isolated cardiomyocytes and mice investigated potential mechanisms of sunitinib-associated cardiac effects. Findings Eleven percent (8/75) of subjects suffered a cardiovascular event with congestive heart failure (CHF) occurring in 8% (6/75) of the population. Twenty-eight percent (10/36) of patients treated at the FDA-approved dose had LVEF declines of ≥ 10 EF%, and nineteen percent (7/36) experienced LVEF declines of ≥ 15 EF%. Sunitinib induced significant increases in mean systolic and diastolic BP in patients, and 47% (35/75) of individuals developed hypertension (HTN) (>150/100 mmHg). CHF and LV dysfunction generally responded to withholding drug and instituting medical management. In mice and cultured cardiomyocytes, sunitinib caused mitochondrial injury and cardiomyocyte apoptosis. Interpretation Sunitinib treatment can lead to HTN, LVEF decline, and/or CHF. Experimental studies suggest that this is due, at least in part, to direct cardiomyocyte toxicity which may be exacerbated by HTN. Patients treated with sunitinib should receive close monitoring and prompt treatment for HTN and/or LVEF decline. PMID:18083403

  7. Laser Desorption Supersonic Jet Spectroscopy of Hydrated Tyrosine

    NASA Astrophysics Data System (ADS)

    Oba, Hikari; Shimozono, Yoko; Ishiuchi, Shun-Ichi; Fujii, Masaaki; Carcabal, Pierre

    2013-06-01

    The structure of tyrosine (tyr) consists of amino-acid chain and phenol, and it has roughly two possible binding sites for water, amino-acid site and phenolic OH site. Investigating how water molecule binds to tyr will give fundamental information for hydrations of peptide and protein. Resonance enhanced multi photon ionization (REMPI) spectrum of tyr-water 1:1 cluster has already been reported by de Vries and co-workers, however, no analysis on the hydrated structures has been reported. In the REMPI spectrum, two clusters of bands are observed; one appears at ˜35600 cm^{-1} energy region which is the almost same with 0-0 transitions of tyr monomer, and another is observed at ˜300 cm^{-1} lower than the former. Based on the electronic transition energy of phenylalanine and the hydrated clusters, the former is expected to be derived from a structure that water binds to amino acid site. On the other hand, it is plausibly predicted that the latter originates from a structure that water binds to phenolic OH group, because the electronic transition of mono hydrated phenol is ˜300 cm^{-1} red-shifted from the monomer. We applied IR dip spectroscopy which can measure conformer selective IR spectra to the tyr-(H_{2}O)_{1} clusters by using laser desorption supersonic jet technique to confirm the assignments. Especially in the phenolic OH bound isomer, it was found that the intra molecular hydrogen bond within amino-acid chain, which is far from the water molecule and cannot interact directly with each other, is strengthened by the hydration. A. Abio-Riziq et al., J. Phys. Chem. A, 115, 6077 (2011). Y. Shimozono, et al., Phys. Chem. Chem. Phys., (2013) DOI: 10.1039/c3cp43573c. T. Ebata et al., Phys. Chem. Chem. Phys., 8, 4783 (2006). T. Watanabe et al., J. Chem. Phys., 105, 408 (1996).

  8. Reduced striatal tyrosine hydroxylase in incidental Lewy body disease

    PubMed Central

    Adler, Charles H.; Sue, Lucia I.; Peirce, Jeffrey B.; Bachalakuri, Jyothi; Dalsing-Hernandez, Jessica E.; Lue, Lih Fen; Caviness, John N.; Connor, Donald J.; Sabbagh, Marwan N.; Walker, Douglas G.

    2009-01-01

    Incidental Lewy body disease (ILBD) is the term used when Lewy bodies are found in the nervous system of subjects without clinically documented parkinsonism or dementia. The prevalence of ILBD in the elderly population has been estimated at between 3.8 and 30%, depending on subject age and anatomical site of sampling. It has been speculated that ILBD represents the preclinical stage of Parkinson’s disease (PD) and/or dementia with Lewy bodies (DLB). Studies of ILBD could potentially identify early diagnostic signs of these disorders. At present, however, it is impossible to know whether ILBD is a precursor to PD or DLB or is just a benign finding of normal aging. We hypothesized that, if ILBD represents an early stage of PD or DLB, it should be associated with depletion of striatal dopaminergic markers. Eleven subjects with ILBD and 27 control subjects were studied. The ILBD subjects ranged in age from 74 to 96 years (mean 86.5) while the control subjects’ age ranged from 75 to 102 years (mean 86.7). Controls and subjects did not differ in terms of age, postmortem interval, gender distribution, medical history conditions, brain weight, neuritic plaque density or Braak neurofibrillary stage. Quantitative ELISA measurement of striatal tyrosine hydroxylase (TH), the principal enzyme for dopamine synthesis, showed a 49.8% (P = 0.01) reduction in ILBD cases, as compared with control cases. The finding suggests that ILBD is not a benign condition but is likely a precursor to PD and/or DLB. PMID:17985144

  9. Pharmacophore modeling for protein tyrosine phosphatase 1B inhibitors.

    PubMed

    Bharatham, Kavitha; Bharatham, Nagakumar; Lee, Keun Woo

    2007-05-01

    A three dimensional chemical feature based pharmacophore model was developed for the inhibitors of protein tyrosine phosphatase 1B (PTP1B) using the CATALYST software, which would provide useful knowledge for performing virtual screening to identify new inhibitors targeted toward type II diabetes and obesity. A dataset of 27 inhibitors, with diverse structural properties, and activities ranging from 0.026 to 600 microM, was selected as a training set. Hypol, the most reliable quantitative four featured pharmacophore hypothesis, was generated from a training set composed of compounds with two H-bond acceptors, one hydrophobic aromatic and one ring aromatic features. It has a correlation coefficient, RMSD and cost difference (null cost-total cost) of 0.946, 0.840 and 65.731, respectively. The best hypothesis (Hypol) was validated using four different methods. Firstly, a cross validation was performed by randomizing the data using the Cat-Scramble technique. The results confirmed that the pharmacophore models generated from the training set were valid. Secondly, a test set of 281 molecules was scored, with a correlation of 0.882 obtained between the experimental and predicted activities. Hypol performed well in correctly discriminating the active and inactive molecules. Thirdly, the model was investigated by mapping on two PTP1B inhibitors identified by different pharmaceutical companies. The Hypol model correctly predicted these compounds as being highly active. Finally, docking simulations were performed on few compounds to substantiate the role of the pharmacophore features at the binding site of the protein by analyzing their binding conformations. These multiple validation approaches provided confidence in the utility of this pharmacophore model as a 3D query for virtual screening to retrieve new chemical entities showing potential as potent PTP1B inhibitors.

  10. The tyrosine kinase inhibitor, nilotinib potentiates a prothrombotic state.

    PubMed

    Alhawiti, Naif; Burbury, Kate L; Kwa, Faith A; O'Malley, Cindy J; Shuttleworth, Peter; Alzard, Mohamad; Hamadi, Abdullah; Grigg, Andrew P; Jackson, Denise E

    2016-09-01

    Tyrosine kinase inhibitors (TKI) such as imatinib, nilotinib and dasatinib are now established as highly effective frontline therapies for chronic myeloid leukaemia (CML). Disease control is achieved in the majority of patients and survival is excellent such that recent focus has been on toxicities of these agents. Cumulative data have reported an excess of serious vascular complications, including arterial thrombosis and peripheral arterial occlusive disease, in patients receiving nilotinib in comparison with other TKIs, with resultant interest in delineating the pathophysiology and implications for rationale cardiovascular risk modification. To address this issue, we studied the effects of imatinib, nilotinib and dasatinib on platelet function and thrombus formation in human and mouse models using in vitro, ex vivo and in vivo approaches. In vitro studies demonstrated that dasatinib and imatinib but not nilotinib inhibited ADP, CRP, and collagen-induced platelet aggregation and moreover, that nilotinib potentiated PAR-1-mediated alpha granule release. Pretreatment of wild-type C57BL/6 mice with nilotinib but not imatinib or dasatinib, significantly increased thrombus growth and stability, on type I collagen under ex vivo arterial flow conditions and increased thrombus growth and stability following FeCl3-induced vascular injury of mesenteric arterioles and carotid artery injury in vivo. Whole blood from nilotinib-treated CML patients, demonstrated increased platelet adhesion ex vivo under flow, increased plasma soluble P- and E-selectin, sICAM-1, sVCAM-1, TNF-alpha, IL-6 levels and endogenous thrombin potential (ETP) levels in vivo, despite being on daily low-dose aspirin. These results demonstrate that nilotinib can potentiate platelet and endothelial activation and platelet thrombus formation ex vivo and in vivo.

  11. Tubulin-tyrosine Ligase (TTL)-mediated Increase in Tyrosinated α-Tubulin in Injured Axons Is Required for Retrograde Injury Signaling and Axon Regeneration.

    PubMed

    Song, Wenjun; Cho, Yongcheol; Watt, Dana; Cavalli, Valeria

    2015-06-01

    Injured peripheral neurons successfully activate a pro-regenerative program to enable axon regeneration and functional recovery. The microtubule-dependent retrograde transport of injury signals from the lesion site in the axon back to the cell soma stimulates the increased growth capacity of injured neurons. However, the mechanisms initiating this retrograde transport remain poorly understood. Here we show that tubulin-tyrosine ligase (TTL) is required to increase the levels of tyrosinated α-tubulin at the axon injury site and plays an important role in injury signaling. Preventing the injury-induced increase in tyrosinated α-tubulin by knocking down TTL impairs retrograde organelle transport and delays activation of the pro-regenerative transcription factor c-Jun. In the absence of TTL, axon regeneration is reduced severely. We propose a model in which TTL increases the levels of tyrosinated α-tubulin locally at the injury site to facilitate the retrograde transport of injury signals that are required to activate a pro-regenerative program.

  12. Identification of protein tyrosine phosphatases and dual-specificity phosphatases in mammalian spermatozoa and their role in sperm motility and protein tyrosine phosphorylation.

    PubMed

    González-Fernández, L; Ortega-Ferrusola, C; Macias-Garcia, B; Salido, G M; Peña, F J; Tapia, J A

    2009-06-01

    Protein tyrosine kinases have important roles in spermatozoa; however, little is known about the presence and regulation in these cells of their counterparts in signaling, namely, protein tyrosine phosphatases (PTPs) and dual-specificity phosphatases (DSPs). The objectives of the present study were to identify PTPs and DSPs in boar, stallion, and dog spermatozoa; to characterize their subcellular distribution; and to investigate the roles of tyrosine phosphatases in maintenance of protein tyrosine phosphorylation level and in sperm motility. Using Western blotting with specific antibodies in boar and stallion sperm lysates, we unequivocally identified two PTPs (PTPRB and PTPN11) and two DSPs (DUSP3 and DUSP4). In dog sperm lysates, only PTPN11, DUSP3, and DUSP4 were detected. In all these species, we did not detect the specific signal with anti-PTPRC (CD45), CDKN3, DUSP1, DUSP2, DUSP6, DUSP9, PTPN1, PTPN3, PTPN6, PTPN7, PTPN13, PTPRA, PTPRG, PTPRJ, PTPRK, or PTPRZ antibodies. Positive matches were further investigated by indirect immunofluorescence and confocal microscopy. Results showed that PTPRB was associated with the plasma membrane in the head and tail of boar and stallion spermatozoa. In agreement with Western blotting results, PTPRB antibodies did not show immunoreactivity in dog sperm analyzed by immunofluorescence. In the three species, DUSP4 was mainly found in the tail of spermatozoa, with little or no immunoreactivity in the head. PTPN11 was mainly located in the postacrosomal region in the head, whereas DUSP3 immunoreactivity was extended within the acrosome. PTPN11 and DUSP3 showed immunoreactivity in the tail that was restricted to the midpiece. Finally, we incubated boar, stallion, and dog spermatozoa with pervanadate and sodium orthovanadate, two PTP inhibitors, and analyzed overall protein tyrosine phosphorylation and assessed sperm motility. Sodium orthovanadate and pervanadate showed concentration-dependent inhibition of sperm motility that was

  13. Hyaluronan and the hyaluronan receptor RHAMM promote focal adhesion turnover and transient tyrosine kinase activity

    PubMed Central

    1994-01-01

    The molecular mechanisms whereby hyaluronan (HA) stimulates cell motility was investigated in a C-H-ras transformed 10T 1/2 fibroblast cell line (C3). A significant (p < 0.001) stimulation of C3 cell motility with HA (10 ng/ml) was accompanied by an increase in protein tyrosine phosphorylation as detected by anti-phosphotyrosine antibodies using immunoblot analysis and immunofluorescence staining of cells. Tyrosine phosphorylation of several proteins was found to be both rapid and transient with phosphorylation occurring within 1 min of HA addition and dissipating below control levels 10-15 min later. These responses were also elicited by an antibody generated against a peptide sequence within the HA receptor RHAMM. Treatment of cells with tyrosine kinase inhibitors (genistein, 10 micrograms/ml or herbimycin A, 0.5 micrograms/ml) or microinjection of anti-phosphotyrosine antibodies inhibited the transient protein tyrosine phosphorylation in response to HA as well as prevented HA stimulation of cell motility. To determine a link between HA-stimulated tyrosine phosphorylation and the resulting cell locomotion, cytoskeletal reorganization was examined in C3 cells plated on fibronectin and treated with HA or anti-RHAMM antibody. These agents caused a rapid assembly and disassembly of focal adhesions as revealed by immunofluorescent localization of vinculin. The time course with which HA and antibody induced focal adhesion turnover exactly paralleled the induction of transient protein tyrosine phosphorylation. In addition, phosphotyrosine staining colocalized with vinculin within structures in the lamellapodia of these cells. Notably, the focal adhesion kinase, pp125FAK, was rapidly phosphorylated and dephosphorylated after HA stimulation. These results suggest that HA stimulates locomotion via a rapid and transient protein tyrosine kinase signaling event mediated by RHAMM. They also provide a possible molecular basis for focal adhesion turnover, a process that is

  14. L-tyrosine induces DNA damage in brain and blood of rats.

    PubMed

    De Prá, Samira D T; Ferreira, Gabriela K; Carvalho-Silva, Milena; Vieira, Júlia S; Scaini, Giselli; Leffa, Daniela D; Fagundes, Gabriela E; Bristot, Bruno N; Borges, Gabriela D; Ferreira, Gustavo C; Schuck, Patrícia F; Andrade, Vanessa M; Streck, Emilio L

    2014-01-01

    Mutations in the tyrosine aminotransferase gene have been identified to cause tyrosinemia type II which is inherited in an autosomal recessive manner. Studies have demonstrated that an excessive production of ROS can lead to reactions with macromolecules, such as DNA, lipids, and proteins. Considering that the L-tyrosine may promote oxidative stress, the main objective of this study was to investigate the in vivo effects of L-tyrosine on DNA damage determined by the alkaline comet assay, in brain and blood of rats. In our acute protocol, Wistar rats (30 days old) were killed 1 h after a single intraperitoneal L-tyrosine injection (500 mg/kg) or saline. For chronic administration, the animals received two subcutaneous injections of L-tyrosine (500 mg/kg, 12-h intervals) or saline administered for 24 days starting at postnatal day (PD) 7 (last injection at PD 31), 12 h after the last injection, the animals were killed by decapitation. We observed that acute administration of L-tyrosine increased DNA damage frequency and damage index in cerebral cortex and blood when compared to control group. Moreover, we observed that chronic administration of L-tyrosine increased DNA damage frequency and damage index in hippocampus, striatum, cerebral cortex and blood when compared to control group. In conclusion, the present work demonstrated that DNA damage can be encountered in brain from animal models of hypertyrosinemia, DNA alterations may represent a further means to explain neurological dysfunction in this inherited metabolic disorder and to reinforce the role of oxidative stress in the pathophysiology of tyrosinemia type II. PMID:24297753

  15. An Extensive Survey of Tyrosine Phosphorylation Revealing New Sites in Human Mammary Epithelial Cells

    SciTech Connect

    Heibeck, Tyler H.; Ding, Shi-Jian; Opresko, Lee K.; Zhao, Rui; Schepmoes, Athena A.; Yang, Feng; Tolmachev, Aleksey V.; Monroe, Matthew E.; Camp, David G.; Smith, Richard D.; Wiley, H. S.; Qian, Weijun

    2009-08-01

    Protein tyrosine phosphorylation is a central regulatory mechanism in cell signaling. To extensively characterize the site-specific tyrosine phosphorylation in human cells, we present here a global survey of tyrosine phosphorylation sites in a normal-derived human mammary epithelial cell (HMEC) line by applying anti-phosphotyrosine (pTyr) peptide immunoaffinity purification (IP) coupled with high sensitivity LC-MS/MS. A total of 481 tyrosine phosphorylation sites (covered by 716 unique peptides) from 285 proteins were confidently identified in HMEC following the analysis of both the basal condition and an acute stimulated condition with epidermal growth factor (EGF). The estimated false discovery rate is 1.0% as measured by comparison against a scrambled database search. Comparison of these data to the literature showed significant agreement in site matches. Additionally 281 sites were not previously observed in HMEC culture were found. Twenty-nine of these sites have not been reported in any human cell or tissue system. The global profiling also allowed us to examine the phosphorylation stoichiometry differences based on spectral count information. Comparison of the data to a previous global proteome profiling study illustrates that most of the highly phoshorylated proteins are of relatively low-abundance. Large differences in phosphorylation stoichiometry for sites within the same protein were also observed for many of the identified proteins, suggesting potentially more important functional roles for those highly phosphorylated pTyr sites within a given protein. By mapping to major signaling networks such as EGF receptor and insulin growth factor-1 receptor signaling pathways, many known proteins involved in these pathways were revealed to be tyrosine phosphorylated, which should allow us to select interesting targeted involved in a given pathway for more directed studies. This extensive HMEC tyrosine phosphorylation dataset represents an important database

  16. Reactive oxygen species induce reversible PECAM-1 tyrosine phosphorylation and SHP-2 binding.

    PubMed

    Maas, Matthias; Wang, Ronggang; Paddock, Cathy; Kotamraju, Srigiridhar; Kalyanaraman, Balaraman; Newman, Peter J; Newman, Debra K

    2003-12-01

    Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) functions to control the activation and survival of the cells on which it is expressed. Many of the regulatory functions of PECAM-1 are dependent on its tyrosine phosphorylation and subsequent recruitment of the Src homology (SH2) domain containing protein tyrosine phosphatase SHP-2. The recent demonstration that PECAM-1 tyrosine phosphorylation occurs in cells exposed to the reactive oxygen species hydrogen peroxide (H2O2) suggested that this form of oxidative stress may also support PECAM-1/SHP-2 complex formation. In the present study, we show that PECAM-1 tyrosine phosphorylation in response to exposure of cells to H2O2 is reversible, involves a shift in the balance between kinase and phosphatase activities, and supports binding of SHP-2 and recruitment of this phosphatase to cell-cell borders. We speculate, however, that the unique ability of H2O2 to reversibly oxidize the reactive site cysteine residues of protein tyrosine phosphatases may result in transient inactivation of the SHP-2 that is bound to PECAM-1 under these conditions. Finally, we provide evidence that PECAM-1 tyrosine phosphorylation and SHP-2 binding in endothelial cells requires exposure to an "oxidative burst" of H2O2, but that exposure of these cells to sufficiently high concentrations of H2O2 for a sufficiently long period of time abrogates binding of SHP-2 to tyrosine-phosphorylated PECAM-1. These findings support a role for PECAM-1 as a sensor of oxidative stress, perhaps most importantly during the process of inflammation. PMID:12893640

  17. L-tyrosine induces DNA damage in brain and blood of rats.

    PubMed

    De Prá, Samira D T; Ferreira, Gabriela K; Carvalho-Silva, Milena; Vieira, Júlia S; Scaini, Giselli; Leffa, Daniela D; Fagundes, Gabriela E; Bristot, Bruno N; Borges, Gabriela D; Ferreira, Gustavo C; Schuck, Patrícia F; Andrade, Vanessa M; Streck, Emilio L

    2014-01-01

    Mutations in the tyrosine aminotransferase gene have been identified to cause tyrosinemia type II which is inherited in an autosomal recessive manner. Studies have demonstrated that an excessive production of ROS can lead to reactions with macromolecules, such as DNA, lipids, and proteins. Considering that the L-tyrosine may promote oxidative stress, the main objective of this study was to investigate the in vivo effects of L-tyrosine on DNA damage determined by the alkaline comet assay, in brain and blood of rats. In our acute protocol, Wistar rats (30 days old) were killed 1 h after a single intraperitoneal L-tyrosine injection (500 mg/kg) or saline. For chronic administration, the animals received two subcutaneous injections of L-tyrosine (500 mg/kg, 12-h intervals) or saline administered for 24 days starting at postnatal day (PD) 7 (last injection at PD 31), 12 h after the last injection, the animals were killed by decapitation. We observed that acute administration of L-tyrosine increased DNA damage frequency and damage index in cerebral cortex and blood when compared to control group. Moreover, we observed that chronic administration of L-tyrosine increased DNA damage frequency and damage index in hippocampus, striatum, cerebral cortex and blood when compared to control group. In conclusion, the present work demonstrated that DNA damage can be encountered in brain from animal models of hypertyrosinemia, DNA alterations may represent a further means to explain neurological dysfunction in this inherited metabolic disorder and to reinforce the role of oxidative stress in the pathophysiology of tyrosinemia type II.

  18. The human p50csk tyrosine kinase phosphorylates p56lck at Tyr-505 and down regulates its catalytic activity.

    PubMed Central

    Bergman, M; Mustelin, T; Oetken, C; Partanen, J; Flint, N A; Amrein, K E; Autero, M; Burn, P; Alitalo, K

    1992-01-01

    Protein tyrosine kinases participate in the transduction and modulation of signals that regulate proliferation and differentiation of cells. Excessive or deregulated protein tyrosine kinase activity can cause malignant transformation. The catalytic activity of the T cell protein tyrosine kinase p56lck is normally suppressed by phosphorylation of a carboxyl-terminal tyrosine, Tyr-505, by another cellular protein tyrosine kinase. Here we characterize a human cytosolic 50 kDa protein tyrosine kinase, p50csk, which specifically phosphorylates Tyr-505 of p56lck and a synthetic peptide containing this site. Phosphorylation of Tyr-505 suppressed the catalytic activity of p56lck. We suggest that p50csk negatively regulates p56lck, and perhaps other cellular src family kinases. Images PMID:1639064

  19. Phosphorylation of Tip60 Tyrosine 327 by Abl Kinase Inhibits HAT Activity through Association with FE65

    PubMed Central

    Shin, Sung Hwa; Kang, Sang Sun

    2013-01-01

    The transfer of acetyl groups from acetyl coenzyme A to the ε amino group of internal lysine residues is catalyzed by Tip60, which is in the MYST family of nuclear histone acetyltransferases (HATs). The tyrosine phosphorylation of Tip60 seems to be a unique modification. We present evidence that Tip60 is modified on tyrosine 327 by Abl kinase. We show that this causes functional changes in HAT activity and the subcellular localization of TIP60, which forms a complex with Abl kinase. The Tip60 mutation Y327F abolished tyrosine phosphorylation, reduced the inhibition of Tip60 HAT activity, and caused G0-G1 arrest and association with FE65. Thus, our findings for the first time suggested a novel regulation mechanism of Tip60. Regulation was through phosphorylation of tyrosine 327 by Abl tyrosine kinase and depended on environmental conditions, suggesting that the tyrosine residue of Tip60 is important for the activation process. PMID:24044023

  20. Electropolymerized tyrosine-based thin films: selective cell binding via peptide recognition to novel electropolymerized biomimetic tyrosine RGDY films.

    PubMed

    Marx, Kenneth A; Zhou, Tiean; McIntosh, Donna; Braunhut, Susan J

    2009-01-01

    We have created thin films by cyclic voltammetry (CV) electropolymerizations of the following phenolic functional group-based monomers: phenol; tyrosineamide; the tetrapeptide RGDY-containing the integrin membrane adhesion protein recognition tripeptide RGD; RDGY, a nonsense control tetrapeptide; and 1:3 mixtures of tyrosineamide with the two tetrapeptide monomers. The film formation process and description of the film properties were obtained by repetitive CV cycling using the oscillating quartz frequency shift, Deltaf, and motional resistance shift, DeltaR, parameters obtained with the electrochemical quartz crystal microbalance technique. Only the poly(phenol) film exhibited close chain packing-based self-limiting behavior, where all film synthesis ceased after approximately 7 CV cycles. All other films continued to form by electropolymerization with successive CV cycles out to the maximum cycle number (30 cycles) we measured. All of the films exhibited little energy dissipation behavior. Using the quartz crystal microbalance, we next compared the time course of cell attachment with the washed films and demonstrated that cells bound best to films in the following order: RGDY sense peptide:tyrosineamide films>RDGY nonsense peptide:tyrosineamide films=tyrosineamide films>phenol films. Cell enumeration after washing and trypsinization revealed firm protein-based cell attachment to the underlying extracellular matrix for the RGDY-containing films. These sense peptide films bound and retained two- to fivefold as many cells as the other films, with cells exhibiting a normal morphology. These results suggest the operation of specific cell attachment to the electropolymerized films via the RGD binding site for cellular integrin membrane proteins. The electropolymerization method we studied here forms a cassette system for creating electropolymerized films tailored to specific attachment of different cell types by varying the electropolymerized Y(tyrosine

  1. Morin inhibits STAT3 tyrosine 705 phosphorylation in tumor cells through activation of protein tyrosine phosphatase SHP1.

    PubMed

    Gupta, Subash C; Phromnoi, Kanokkarn; Aggarwal, Bharat B

    2013-04-01

    The major goal of cancer drug discovery is to find an agent that is safe and affordable, yet effective against cancer. Here we show that morin (3,5,7,2',4'-pentahydroxyflavone) has potential against cancer cells through suppression of the signal transducer and activator of transcription 3 (STAT3) pathway, which is closely linked to the transformation, survival, proliferation, and metastasis of cancer. We found that morin completely suppressed inducible and constitutively activated STAT3 and blocked the nuclear translocation of STAT3 and its DNA binding in multiple myeloma and head and neck squamous carcinoma cells. Morin inhibited activated Src, JAK-1, and JAK-2, all of which are linked to STAT3 activation, while up-regulating a protein inhibitor of activated STAT3, PIAS3. Pervanadate reversed the effects of morin on STAT3 phosphorylation, indicating the role of a protein tyrosine phosphatase. Furthermore, morin induced SHP1 expression at both the mRNA and protein levels, and silencing of SHP1 abrogated the effect of morin on STAT3 phosphorylation, indicating that morin mediates its effects on STAT3 through SHP1. Suppression of STAT3 correlated with the down-regulation of various gene products linked to tumor survival, proliferation, and angiogenesis and led to sensitization of tumor cells to thalidomide and bortezomib. Comparing the activities of morin with those of four structurally related flavonols demonstrated the importance of hydroxyl groups in the B ring in inhibiting STAT3 activation. These findings suggest that morin suppresses the STAT3 pathway, leading to the down-regulation of STAT3-dependent gene expression and chemosensitization of tumor cells.

  2. Induction of multiple sclerosis and response to tyrosine kinase inhibitors.

    PubMed

    Moawad, Emad Y

    2014-10-01

    The goal of this work is to determine the role of the autoimmune cells in multiple sclerosis (MS) induction and the immunomodulatory mechanism of therapy with tyrosine kinase inhibitors (TKIs) in MS attenuation. Samples (5 × 10(5) cells per well) of C6 and primary rat astrocytes were stimulated with 10 ng/mL of platelet-derived growth factor (PDGFbb) as a positive control forming a mouse model of MS. PDGFbb was added to the astrocytes in the absence or presence of 0.1 and 1 μM of imatinib. Proliferation of C6 and primary rat astrocytes samples were assessed for samples staging by the addition of 1 μCi of (3)H-thymidine per well. Samples of RAW 264.7 cells were stimulated for 48 h with 10 ng/mL of PDGFbb in the absence or presence of 0.1 and 1 μM of sorafenib. Tumour necrotic factor (TNF) levels in culture supernatants from RAW 264.7 cells were measured by ELISA. The histologic grade (HG) and the level of TNF of the mouse model of MS was 1/5 and 5 times respectively of those in the control one to clarify that MS induction is due to a major decrease in HG inversely proportional to the accompanied increase in TNF level perpetuating local inflammation and demyelination in MS lesion. The addition of 0.1 and 1 μM doses of imatinib increased HG of the mouse model of MS by 6 and 11 times respectively while 0.1 and 1 μM doses of sorafenib decreased TNF level to be 1/2 and 1/5 of that in the mouse model of MS respectively restoring normal rate of TNF level of normal lesion to show that HGand TNF level would be strongly inversely correlated (r = -0.99) in attenuating MS effectively by TKIs therapy but not in an inverse proportion as in MS induction. PMID:25298631

  3. Novel bone-targeted Src tyrosine kinase inhibitor drug discovery.

    PubMed

    Shakespeare, William C; Metcalf, Chester A; Wang, Yihan; Sundaramoorthi, Raji; Keenan, Terence; Weigele, Manfred; Bohacek, Regine S; Dalgarno, David C; Sawyer, Tomi K

    2003-09-01

    Bone-targeted Src tyrosine kinase (STK) inhibitors have recently been developed for the treatment of osteoporosis and cancer-related bone diseases. The concept of bone targeting derives from bisphosphonates, and from the evolution of such molecules in terms of therapeutic efficacy for the treatment of bone disorders. Interestingly, some of the earliest bisphosphonates were recognized for their ability to inhibit calcium carbonate precipitation (scaling) by virtue of their affinity to chelate calcium. This chelating property was subsequently exploited in the development of bisphosphonate analogs as inhibitors of the bone-resorbing cells known as osteoclasts, giving rise to breakthrough medicines, such as Fosamax (for the treatment of osteoporosis) and Zometa (for the treatment of osteoporosis and bone metastases). Relative to these milestone achievements, there is a tremendous opportunity to explore beyond the limited chemical space (functional group diversity) of such bisphosphonates to design novel bone-targeting moieties, which may be used to develop other classes of promising small-molecule drugs affecting different biological pathways. Here, we review studies focused on bone-targeted inhibitors of STK, a key enzyme in osteoclast-dependent bone resorption. Two strategies are described relative to bone-targeted STK inhibitor drug discovery: (i) the development of novel Src homology (SH)-2 inhibitors incorporating non-hydrolyzable phosphotyrosine mimics and exhibiting molecular recognition and bone-targeting properties, leading to the in vivo-effective lead compound AP-22408; and (ii) the development of novel ATP-based Src kinase inhibitors incorporating bone-targeting moieties, leading to the in vivo-effective lead compound AP-23236. In summary, AP-22408 and AP-23236, which differ mechanistically by virtue of blocking Src-dependent non-catalytic or catalytic activities in osteoclasts, exemplify ARIAD Pharmaceuticals' structure-based design of novel bone

  4. Cellular interactions with biodegradable polyurethanes formulated from L-tyrosine.

    PubMed

    Shah, Parth N; Yun, Yang H

    2013-05-01

    L-Tyrosine polyurethanes (LTUs) have been synthesized by structural modification of the poly (amino acid) backbone to circumvent the problems associated with the processing of poly (amino acids) arising from their high crystallinity, insolubility in common organic solvents, and high glass-transition and melting temperatures. Additionally, problems such as unpredictable swelling characteristics, change in conformation, and uncontrolled enzymatic degradation have severely restricted the use of poly (amino acids). In contrast, LTUs are designed to retain their superior physico-chemical properties, while incorporating biodegradability through enzymatic, hydrolytic, and oxidative pathways. The aim of this study is to evaluate initially the biocompatibility of LTUs and their degradation products. Studies involving primary dermal human fibroblasts cultured in contact with LTU films or degradation products suggest a lack of toxicity (cell viabilities >93% with p < 0.05 compared to the control for all studies). The diversity of LTU polymer chemistry and the ability of LTUs to phase separate seem to present a heterogeneous surface with variable wettability. This phenomenon influences the adhesion and proliferation of human fibroblasts on polymeric surfaces, wherein fibroblast adhesion on polycaprolactone diol (PCL) based LTUs is characterized by higher cell counts (81,250 ± 18,390 for PCL-C-DTH (desaminotyrosine-tyrosyl hexyl, DTH), 58,360 ± 7370 for PCL-L-DTH, 38,480 ± 12,680 for PEG-C-DTH (polyethylene glycol, PEG), and 46,430 ± 16,000 for PEG-L-DTH at 120 h with p < 0.001 for comparison between PCL-C-DTH and all other LTUs), more rapid cellular proliferation (doubling time of 37-49 h for PCL-based LTUs compared to 68-90 h for PEG-based LTUs), and a uniform cell distribution compared to PEG-based LTUs. However, immunofluorescence assay for F-actin suggests that the cells are well attached. Thus, the lack of cytotoxicity and the ability to control cellular adhesion

  5. Pathway optimization by re-design of untranslated regions for L-tyrosine production in Escherichia coli.

    PubMed

    Kim, Seong Cheol; Min, Byung Eun; Hwang, Hyun Gyu; Seo, Sang Woo; Jung, Gyoo Yeol

    2015-09-08

    L-tyrosine is a commercially important compound in the food, pharmaceutical, chemical, and cosmetic industries. Although several attempts have been made to improve L-tyrosine production, translation-level expression control and carbon flux rebalancing around phosphoenolpyruvate (PEP) node still remain to be achieved for optimizing the pathway. Here, we demonstrate pathway optimization by altering gene expression levels for L-tyrosine production in Escherichia coli. To optimize the L-tyrosine biosynthetic pathway, a synthetic constitutive promoter and a synthetic 5'-untranslated region (5'-UTR) were introduced for each gene of interest to allow for control at both transcription and translation levels. Carbon flux rebalancing was achieved by controlling the expression level of PEP synthetase using UTR Designer. The L-tyrosine productivity of the engineered E. coli strain was increased through pathway optimization resulting in 3.0 g/L of L-tyrosine titer, 0.0354 g L-tyrosine/h/g DCW of productivity, and 0.102 g L-tyrosine/g glucose yield. Thus, this work demonstrates that pathway optimization by 5'-UTR redesign is an effective strategy for the development of efficient L-tyrosine-producing bacteria.

  6. Pathway optimization by re-design of untranslated regions for L-tyrosine production in Escherichia coli

    PubMed Central

    Cheol Kim, Seong; Eun Min, Byung; Gyu Hwang, Hyun; Woo Seo, Sang; Yeol Jung, Gyoo

    2015-01-01

    L-tyrosine is a commercially important compound in the food, pharmaceutical, chemical, and cosmetic industries. Although several attempts have been made to improve L-tyrosine production, translation-level expression control and carbon flux rebalancing around phosphoenolpyruvate (PEP) node still remain to be achieved for optimizing the pathway. Here, we demonstrate pathway optimization by altering gene expression levels for L-tyrosine production in Escherichia coli. To optimize the L-tyrosine biosynthetic pathway, a synthetic constitutive promoter and a synthetic 5′-untranslated region (5′-UTR) were introduced for each gene of interest to allow for control at both transcription and translation levels. Carbon flux rebalancing was achieved by controlling the expression level of PEP synthetase using UTR Designer. The L-tyrosine productivity of the engineered E. coli strain was increased through pathway optimization resulting in 3.0 g/L of L-tyrosine titer, 0.0354 g L-tyrosine/h/g DCW of productivity, and 0.102 g L-tyrosine/g glucose yield. Thus, this work demonstrates that pathway optimization by 5′-UTR redesign is an effective strategy for the development of efficient L-tyrosine-producing bacteria. PMID:26346938

  7. Laboratory simulation of ultraviolet irradiation from the Sun on amino acids. III. irradiation of glycine-tyrosine

    NASA Astrophysics Data System (ADS)

    Scappini, F.; Capobianco, M. L.; Casadei, F.; Zamboni, R.

    2009-04-01

    The effects of near ultraviolet (UV) radiation on water solutions of tyrosine and glycine-tyrosine are investigated using a broadband xenon lamp in the region 200-800 nm. These experiments form a contribution in the laboratory simulation of the solar irradiation on the building blocks of life with regard to the origin of life. Results are presented showing the photodecomposition of tyrosine and glycine-tyrosine, at different concentrations, against UV doses. The analysis of the irradiated solutions is carried out by spectroscopic and analytical techniques. The findings of our laboratory simulations are used to constrain the early stages of the life emerging process.

  8. Novel Regulation of Parkin Function Through c-Abl-Mediated Tyrosine Phosphorylation: Implications for Parkinson's Disease

    PubMed Central

    Imam, Syed Z.; Zhou, Qing; Yamamoto, Ayako; Valente, Anthony J.; Ali, Syed F.; Bains, Mona; Roberts, James L.; Kahle, Philipp J.; Clark, Robert A.; Li, Senlin

    2011-01-01

    Mutations in parkin, an E3 ubiquitin ligase, are most common cause of autosomal-recessive Parkinson's disease (PD). Here, we show that the stress-signaling non-receptor tyrosine-kinase c-Abl links parkin to sporadic forms of PD via tyrosine phosphorylation. Under oxidative and dopaminergic stress, c-Abl was activated in cultured neuronal cells and in striatum of adult C57 mice. Activated c-Abl was found in the striatum of PD patients. Concomitantly, parkin was tyrosine-phosphorylated, causing loss ofit's ubiquitin ligase and cytoprotective activities, and the accumulation of parkin substrates, AIMP2 (p38/JTV-1) and FBP-1. STI-571, a selective c-Abl inhibitor, prevented tyrosine phosphorylation of parkin and restored its E3 ligase activity and cytoprotective function both in vitro and in vivo. Our results suggest that tyrosine phosphorylation of parkin by c-Abl is a major post-translational modification that leads to loss of parkin function and disease progression in sporadic PD. Moreover, inhibition of c-Abl offers new therapeutic opportunities for blocking PD progression. PMID:21209200

  9. Selective Sensing of Tyrosine Phosphorylation in Peptides Using Terbium(III) Complexes

    PubMed Central

    Sumaoka, Jun; Akiba, Hiroki; Komiyama, Makoto

    2016-01-01

    Phosphorylation of tyrosine residues in proteins, as well as their dephosphorylation, is closely related to various diseases. However, this phosphorylation is usually accompanied by more abundant phosphorylation of serine and threonine residues in the proteins and covers only 0.05% of the total phosphorylation. Accordingly, highly selective detection of phosphorylated tyrosine in proteins is an urgent subject. In this review, recent developments in this field are described. Monomeric and binuclear TbIII complexes, which emit notable luminescence only in the presence of phosphotyrosine (pTyr), have been developed. There, the benzene ring of pTyr functions as an antenna and transfers its photoexcitation energy to the TbIII ion as the emission center. Even in the coexistence of phosphoserine (pSer) and phosphothreonine (pThr), pTyr can be efficintly detected with high selectivity. Simply by adding these TbIII complexes to the solutions, phosphorylation of tyrosine in peptides by protein tyrosine kinases and dephosphorylation by protein tyrosine phosphatases can be successfully visualized in a real-time fashion. Furthermore, the activities of various inhibitors on these enzymes are quantitatively evaluated, indicating a strong potential of the method for efficient screening of eminent inhibitors from a number of candidates. PMID:27375742

  10. New tyrosinase inhibitory decapeptide: Molecular insights into the role of tyrosine residues.

    PubMed

    Ochiai, Akihito; Tanaka, Seiya; Imai, Yuta; Yoshida, Hisashi; Kanaoka, Takumi; Tanaka, Takaaki; Taniguchi, Masayuki

    2016-06-01

    Tyrosinase, a rate-limiting enzyme in melanin biosynthesis, catalyzes the hydroxylation of l-tyrosine to 3,4-dihydroxy-l-phenylalanine (l-dopa) (monophenolase reaction) and the subsequent oxidation of l-dopa to l-dopaquinone (diphenolase reaction). Thus, tyrosinase inhibitors have been proposed as skin-lightening agents; however, many of the existing inhibitors cannot be widely used in the cosmetic industry due to their high cytotoxicity and instability. On the other hand, some tyrosinase inhibitory peptides have been reported as safe. In this study, we found that the peptide TH10, which has a similar sequence to the characterized inhibitory peptide P4, strongly inhibits the monophenolase reaction with a half-maximal inhibitory concentration of 102 μM. Seven of the ten amino acid residues in TH10 were identical to P4; however, TH10 possesses one N-terminal tyrosine, whereas P4 contains three tyrosine residues located at its N-terminus, center, and C-terminus. Subsequent analysis using sequence-shuffled variants indicated that the tyrosine residues located at the N-terminus and center of P4 have little to no contribution to its inhibitory activity. Furthermore, docking simulation analysis of these peptides with mushroom tyrosinase demonstrated that the active tyrosine residue was positioned close to copper ions, suggesting that TH10 and P4 bind to tyrosinase as a substrate analogue. PMID:26589783

  11. Chemical modification of cysteine and tyrosine residues in formyltetrahydrofolate synthetase from Clostridium thermoaceticum

    SciTech Connect

    Elliott, J.I.; Ljungdahl, L.G.

    1982-04-01

    The chemical modification of cysteine and tyrosine residues in formyltetrahydrofolate synthetase from Clostridium thermoaceticum has been examined relative to enzymatic activity and reactivity of these groups in the native protein. 4,4'-Dipyridyl disulfide, dansylaziridine, and fluorescein mercuric acetate all reacted with just one of six sulfhydryls per enzyme subunit, resulting in activities of 100, 95 and 70%, respectively. The K/sub m/ values for MgATP, formate, and tetrahydrofolate were unaltered in the modified enzymes. ATP did produce a 2.5-fold reduction in the rate of reaction between the enzyme and 4,4'-dipyridyl disulfide. Tetranitromethane reacted most rapidly with a single sulfhydryl group per subunit to produce a 20 to 30% loss in activity. Subsequent additions of tetranitromethane modified 2.2 tyrosines per subunit which was proportional to the loss of the remaining enzymatic activity. Folic acid, a competitive inhibitor, protected against modification of the tyrosines and the associated activity losses; however, the oxidation of the single sulfhydryl group and the initial 20 to 30% activity loss were unaffected. In the presence of folic acid, higher concentrations of tetranitromethane produced a loss of the remaining activity proportional to the modification of 1.2 tyrosines per subunit. It is proposed that at least 1 tyrosine critical for enzymatic activity is located at or near the folic acid/tetrahydrofolate binding site.

  12. Src drives the Warburg effect and therapy resistance by inactivating pyruvate dehydrogenase through tyrosine-289 phosphorylation

    PubMed Central

    Shenoy, Anitha K.; Lim, Sangbin; Zhang, Ying; Charles, Steve; Tarrash, Miriam; Fu, Xueqi; Kamarajugadda, Sushama; Trevino, Jose G.; Tan, Ming; Lu, Jianrong

    2016-01-01

    The Warburg effect, which reflects cancer cells' preference for aerobic glycolysis over glucose oxidation, contributes to tumor growth, progression and therapy resistance. The restraint on pyruvate flux into mitochondrial oxidative metabolism in cancer cells is in part attributed to the inhibition of pyruvate dehydrogenase (PDH) complex. Src is a prominent oncogenic non-receptor tyrosine kinase that promotes cancer cell proliferation, invasion, metastasis and resistance to conventional and targeted therapies. However, the potential role of Src in tumor metabolism remained unclear. Here we report that activation of Src attenuated PDH activity and generation of reactive oxygen species (ROS). Conversely, Src inhibitors activated PDH and increased cellular ROS levels. Src inactivated PDH through direct phosphorylation of tyrosine-289 of PDH E1α subunit (PDHA1). Indeed, Src was the main kinase responsible for PDHA1 tyrosine phosphorylation in cancer cells. Expression of a tyrosine-289 non-phosphorable PDHA1 mutant in Src-hyperactivated cancer cells restored PDH activity, increased mitochondrial respiration and oxidative stress, decreased experimental metastasis, and sensitized cancer cells to pro-oxidant treatment. The results suggest that Src contributes to the Warburg phenotype by inactivating PDH through tyrosine phosphorylation, and the metabolic effect of Src is essential for Src-driven malignancy and therapy resistance. Combination therapies consisting of both Src inhibitors and pro-oxidants may improve anticancer efficacy. PMID:26848621

  13. Activation of spleen tyrosine kinase (Syk) at fertilization in Rhinella arenarum eggs.

    PubMed

    Mouguelar, Valeria S; Coux, Gabriela

    2014-01-01

    Recently, we have provided evidence for the involvement of a cytosolic tyrosine-phosphorylatable 70 kDa oocyte protein in Rhinella arenarum (Anura: Bufonidae) fertilization. The aim of the present work was to characterize its phosphorylation, determine the identity of this protein and establish its biological role during the fertilization process. Tyrosine phosphorylation of the 70 kDa protein was not observed in eggs activated with the calcium ionophore A23187. Pretreatment of oocytes with the tyrosine kinase inhibitor genistein effectively blocked the fertilization-dependent phosphorylation of the 70 kDa protein. In order to identify this protein, we examined the presence in amphibian oocytes of non-receptor 70 kDa tyrosine kinase members of the Syk/Zap70 and Tec families by RT-PCR using degenerate primers. We found that R. arenarum oocytes contain the transcripts coding for Syk and Tec kinases. Western blot analysis confirmed the presence of Syk protein in unfertilized oocytes and eggs. Studies using phospho-Syk specific antibodies showed that fertilization rapidly (less than 10 minutes) induces phosphorylation on Syk tyrosine residues (352 and 525/526) that are necessary for the activation of the enzyme. Finally, specific inhibition of Syk with the R406 compound provoked a diminished fertilization score, thereby confirming a functional role of the Syk protein during R. arenarum fertilization. To our knowledge this is the first time that Syk is described as a player in the signaling cascade activated after fertilization.

  14. Two Dictyostelium tyrosine kinase–like kinases function in parallel, stress-induced STAT activation pathways

    PubMed Central

    Araki, Tsuyoshi; Vu, Linh Hai; Sasaki, Norimitsu; Kawata, Takefumi; Eichinger, Ludwig; Williams, Jeffrey G.

    2014-01-01

    When Dictyostelium cells are hyperosmotically stressed, STATc is activated by tyrosine phosphorylation. Unusually, activation is regulated by serine phosphorylation and consequent inhibition of a tyrosine phosphatase: PTP3. The identity of the cognate tyrosine kinase is unknown, and we show that two tyrosine kinase–like (TKL) enzymes, Pyk2 and Pyk3, share this function; thus, for stress-induced STATc activation, single null mutants are only marginally impaired, but the double mutant is nonactivatable. When cells are stressed, Pyk2 and Pyk3 undergo increased autocatalytic tyrosine phosphorylation. The site(s) that are generated bind the SH2 domain of STATc, and then STATc becomes the target of further kinase action. The signaling pathways that activate Pyk2 and Pyk3 are only partially overlapping, and there may be a structural basis for this difference because Pyk3 contains both a TKL domain and a pseudokinase domain. The latter functions, like the JH2 domain of metazoan JAKs, as a negative regulator of the kinase domain. The fact that two differently regulated kinases catalyze the same phosphorylation event may facilitate specific targeting because under stress, Pyk3 and Pyk2 accumulate in different parts of the cell; Pyk3 moves from the cytosol to the cortex, whereas Pyk2 accumulates in cytosolic granules that colocalize with PTP3. PMID:25143406

  15. Biochemical evaluation of a parsley tyrosine decarboxylase results in a novel 4-hydroxyphenylacetaldehyde synthase enzyme.

    PubMed

    Torrens-Spence, Michael P; Gillaspy, Glenda; Zhao, Bingyu; Harich, Kim; White, Robert H; Li, Jianyong

    2012-02-10

    Plant aromatic amino acid decarboxylases (AAADs) are effectively indistinguishable from plant aromatic acetaldehyde syntheses (AASs) through primary sequence comparison. Spectroscopic analyses of several characterized AASs and AAADs were performed to look for absorbance spectral identifiers. Although this limited survey proved inconclusive, the resulting work enabled the reevaluation of several characterized plant AAS and AAAD enzymes. Upon completion, a previously reported parsley AAAD protein was demonstrated to have AAS activity. Substrate specificity tests demonstrate that this novel AAS enzyme has a unique substrate specificity towards tyrosine (km 0.46mM) and dopa (km 1.40mM). Metabolite analysis established the abundance of tyrosine and absence of dopa in parsley extracts. Such analysis indicates that tyrosine is likely to be the sole physiological substrate. The resulting information suggests that this gene is responsible for the in vivo production of 4-hydroxyphenylacetaldehyde (4-HPAA). This is the first reported case of an AAS enzyme utilizing tyrosine as a primary substrate and the first report of a single enzyme capable of producing 4-HPAA from tyrosine.

  16. A Multifeatures Fusion and Discrete Firefly Optimization Method for Prediction of Protein Tyrosine Sulfation Residues

    PubMed Central

    Liu, Chunhua; Zhou, Peng; Li, Yanling

    2016-01-01

    Tyrosine sulfation is one of the ubiquitous protein posttranslational modifications, where some sulfate groups are added to the tyrosine residues. It plays significant roles in various physiological processes in eukaryotic cells. To explore the molecular mechanism of tyrosine sulfation, one of the prerequisites is to correctly identify possible protein tyrosine sulfation residues. In this paper, a novel method was presented to predict protein tyrosine sulfation residues from primary sequences. By means of informative feature construction and elaborate feature selection and parameter optimization scheme, the proposed predictor achieved promising results and outperformed many other state-of-the-art predictors. Using the optimal features subset, the proposed method achieved mean MCC of 94.41% on the benchmark dataset, and a MCC of 90.09% on the independent dataset. The experimental performance indicated that our new proposed method could be effective in identifying the important protein posttranslational modifications and the feature selection scheme would be powerful in protein functional residues prediction research fields. PMID:27034949

  17. Effects of Protonation State on a Tyrosine-Histidine Bioinspired Redox Mediator

    SciTech Connect

    Moore, Gary F.; Hambourger, Michael; Kodis, Gerdenis; Michl, Weston; Gust, Devens; Moore, Thomas A.; Moore, Ana L.

    2010-11-18

    The conversion of tyrosine to the corresponding tyrosyl radical in photosytem II (PSII) is an example of proton-coupled electron transfer. Although the tyrosine moiety (TyrZ) is known to function as a redox mediator between the photo-oxidized primary donor (P680 •+) and the Mn-containing oxygen-evolving complex, the protonation states involved in the course of the reaction remain an active area of investigation. Herein, we report on the optical, structural, and electrochemical properties of tyrosine-histidine constructs, which model the function of their naturally occurring counterparts in PSII. Electrochemical studies show that the phenoxyl/phenol couple of the model is chemically reversible and thermodynamically capable of water oxidation. Studies under acidic and basic conditions provide clear evidence that an ionizable proton controls the electrochemical potential of the tyrosine-histidine mimic and that an exogenous base or acid can be used to generate a low-potential or high-potential mediator, respectively. The phenoxyl/phenoxide couple associated with the low-potential mediator is thermodynamically incapable of water oxidation, whereas the relay associated with the high-potential mediator is thermodynamically incapable of reducing an attached photoexcited porphyrin. These studies provide insight regarding the mechanistic role of the tyrosine-histidine complex in water oxidation and strategies for making use of hydrogen bonds to affect the coupling between proton and electron transfer in artificial photosynthetic systems.

  18. Tumor-promoting phorbol ester stimulates tyrosine phosphorylation in U-937 monocytes.

    PubMed Central

    Grunberger, G; Zick, Y; Taylor, S I; Gorden, P

    1984-01-01

    Solubilized lectin-purified extracts from human monocyte-like cells (U-937) and freshly isolated human mononuclear cells preincubated in the presence of phorbol 12-myristate 13-acetate (PMA) stimulated phosphorylation of synthetic tyrosine-containing polymers and of casein. Tyrosine phosphorylation was confirmed by phospho amino acid analysis. PMA stimulated phosphorylation of exogenous substrates in a time- and concentration-dependent manner. This phosphorylation reaction did not require addition of phospholipid, diolein, or calcium. Biologically inactive phorbol compounds did not stimulate phosphorylation in this system. In addition, PMA enhanced phosphorylation of a Mr approximately equal to 140,000 protein as well as several other endogenous proteins in the U-937 extracts. PMA treatment stimulated predominantly phosphorylation on tyrosine residues of the Mr 140,000 protein. Tyrosine phosphorylation, typical of growth-promoting peptides such as insulin or epidermal growth factor, is believed to play a role in regulating normal and disordered cellular growth and proliferation. The demonstration of PMA-stimulated tyrosine phosphorylation might provide a clue to the mechanism of cellular differentiation and proliferation induced by the tumor promoter. Images PMID:6201862

  19. [Spatial structure and mechanism of tyrosine phenol-lyase and tryptophan indole-lyase].

    PubMed

    Demidkina, T V; Anston, A A; Faleev, N G; Phillips, R S; Zakomyrdina, L N

    2009-01-01

    The bacterial tyrosine phenol-lyase (EC 4.1.99.2) and tryptoptophan indole-lyase (EC 4.1.99.1) belong to pyridoxal-5'-phosphate dependent beta-eliminating lyases, catalysing the reversible decomposition of L-tyrosine and L-tryptophan to pyruvate, ammonia, and phenol or indole correspondingly. Data on the three dimentional structures of the holoenzymes of tyrosine phenol-lyase and tryptophan indole-lyase and several enzyme-inhibitor complexes, modeling distinct reaction stages of the beta-elimination of L-tyrosine are described in the paper and structural bases of monovalent cations influence of activity of the enzymes are discussed. The spectral and catalytic properties of the mutant enzymes were studied. The data thus obtained have allowed us to elucidate the catalytic functions of a number of amino acid residues and conclude that the acid-base properties of the catalytic groups of the enzymes under the optimal for the catalysis conditions in hydrophobic active sites of tyrosine phenol-lyase and tryptoptophan indol-lyase are different from those in water solutions. Study of the mechanisms of labilization of Calpha-proton of the bound amino acids and activation of the leaving groups of the substrates during the catalytic process has demonstrated that in certain cases concerted reaction pathways are realized instead of stepwise ones. PMID:19425498

  20. Bombesin, vasopressin, and endothelin rapidly stimulate tyrosine phosphorylation in intact Swiss 3T3 cells

    SciTech Connect

    Zachary, I.; Gil, J.; Lehmann, W.; Sinnett-Smith, J.; Rozengurt, E. )

    1991-06-01

    The mitogenic neuropeptides bombesin and vasopressin markedly increased tyrosine and serine phosphorylation of multiple substrates in quiescent Swiss 3T3 fibroblasts, including two major bands of M{sub r} 90,000 and 115,000. Tyrosine phosphorylation of these proteins was increased as judged by immunoprecipitation of {sup 32}P{sub i}-labeled cells and immunoblotting of unlabeled cells with monoclonal antiphosphotyrosine antibodies, elution with phenyl phosphate, and phospho amino acid analysis. Phosphotyrosyl proteins generated by bombesin and vasopressin did not correspond either by apparent molecular weight or by immunological and biochemical criteria to several known tyrosine kinase substrates, including phospholipase C{sub {gamma}}, the microtubule-associated protein 2 kinase, GTPase-activating protein, or phosphatidylinositol kinase. The effect was rapid (within seconds), concentration dependent, and inhibited by specific receptor antagonists for both bombesin and vasopressin. The endothelin-related peptide, vasoactive intestinal contractor, also elicited a rapid and concentration-dependent tyrosine/serine phosphorylation of a similar set of substrates. These results demonstrate that neuropeptides, acting through receptors linked to GTP-binding proteins, stimulate tyrosine phosphorylation of a common set of substrates in quiescent Swiss 3T3 cells and suggest the existence of an additional signal transduction pathway in neuropeptide-induced mitogenesis.

  1. Serine/threonine/tyrosine phosphorylation regulates DNA binding of bacterial transcriptional regulators.

    PubMed

    Kalantari, Aida; Derouiche, Abderahmane; Shi, Lei; Mijakovic, Ivan

    2015-09-01

    Reversible phosphorylation of bacterial transcriptional regulators (TRs) belonging to the family of two-component systems (TCSs) is a well-established mechanism for regulating gene expression. Recent evidence points to the fact that reversible phosphorylation of bacterial TRs on other types of residue, i.e. serine, threonine, tyrosine and cysteine, is also quite common. The phosphorylation of the ester type (phospho-serine/threonine/tyrosine) is more stable than the aspartate phosphorylation of TCSs. The kinases which catalyse these phosphorylation events (Hanks-type serine/threonine protein kinases and bacterial protein tyrosine kinases) are also much more promiscuous than the TCS kinases, i.e. each of them can phosphorylate several substrate proteins. As a consequence, the dynamics and topology of the signal transduction networks depending on these kinases differ significantly from the TCSs. Here, we present an overview of different classes of bacterial TR phosphorylated and regulated by serine/threonine and tyrosine kinases. Particular attention is given to examples when serine/threonine and tyrosine kinases interact with TCSs, phosphorylating either the histidine kinases or the response regulators. We argue that these promiscuous kinases connect several signal transduction pathways and serve the role of signal integration. PMID:26220449

  2. Mechanism of Oxygen Reduction in Cytochrome c Oxidase and the Role of the Active Site Tyrosine.

    PubMed

    Blomberg, Margareta R A

    2016-01-26

    Cytochrome c oxidase, the terminal enzyme in the respiratory chain, reduces molecular oxygen to water and stores the released energy through electrogenic chemistry and proton pumping across the membrane. Apart from the heme-copper binuclear center, there is a conserved tyrosine residue in the active site (BNC). The tyrosine delivers both an electron and a proton during the O-O bond cleavage step, forming a tyrosyl radical. The catalytic cycle then occurs in four reduction steps, each taking up one proton for the chemistry (water formation) and one proton to be pumped. It is here suggested that in three of the reduction steps the chemical proton enters the center of the BNC, leaving the tyrosine unprotonated with radical character. The reproprotonation of the tyrosine occurs first in the final reduction step before binding the next oxygen molecule. It is also suggested that this reduction mechanism and the presence of the tyrosine are essential for the proton pumping. Density functional theory calculations on large cluster models of the active site show that only the intermediates with the proton in the center of the BNC and with an unprotonated tyrosyl radical have a high electron affinity of similar size as the electron donor, which is essential for the ability to take up two protons per electron and thus for the proton pumping. This type of reduction mechanism is also the only one that gives a free energy profile in accordance with experimental observations for the amount of proton pumping in the working enzyme.

  3. A Novel Isoform of the B Cell Tyrosine Kinase BTK Protects Breast Cancer Cells from Apoptosis

    PubMed Central

    Eifert, Cheryl; Wang, Xianhui; Kokabee, Leila; Kourtidis, Antonis; Jain, Ritu; Gerdes, Michael J.; Conklin, Douglas S.

    2016-01-01

    Tyrosine kinases orchestrate key cellular signaling pathways and their dysregulation is often associated with cellular transformation. Several recent cases in which inhibitors of tyrosine kinases have been successfully used as anticancer agents have underscored the importance of this class of proteins in the development of targeted cancer therapies. We have carried out a large-scale loss-of-function analysis of the human tyrosine kinases using RNA interference to identify novel survival factors for breast cancer cells. In addition to kinases with known roles in breast and other cancers, we identified several kinases that were previously unknown to be required for breast cancer cell survival. The most surprising of these was the cytosolic, nonreceptor tyrosine kinase, Bruton’s tyrosine kinase (BTK), which has been extensively studied in B cell development. Down regulation of this protein with RNAi or inhibition with pharmacological inhibitors causes apoptosis; overexpression inhibits apoptosis induced by Doxorubicin in breast cancer cells. Our results surprisingly show that BTK is expressed in several breast cancer cell lines and tumors. The predominant form of BTK found in tumor cells is transcribed from an alternative promoter and results in a protein with an amino-terminal extension. This alternate form of BTK is expressed at significantly higher levels in tumorigenic breast cells than in normal breast cells. Since this protein is a survival factor for these cells, it represents both a potential marker and novel therapeutic target for breast cancer. PMID:23913792

  4. Lincomycin Biosynthesis Involves a Tyrosine Hydroxylating Heme Protein of an Unusual Enzyme Family

    PubMed Central

    Novotna, Jitka; Olsovska, Jana; Novak, Petr; Mojzes, Peter; Chaloupkova, Radka; Kamenik, Zdenek; Spizek, Jaroslav; Kutejova, Eva; Mareckova, Marketa; Tichy, Pavel; Damborsky, Jiri; Janata, Jiri

    2013-01-01

    The gene lmbB2 of the lincomycin biosynthetic gene cluster of Streptomyces lincolnensis ATCC 25466 was shown to code for an unusual tyrosine hydroxylating enzyme involved in the biosynthetic pathway of this clinically important antibiotic. LmbB2 was expressed in Escherichia coli, purified near to homogeneity and shown to convert tyrosine to 3,4-dihydroxyphenylalanine (DOPA). In contrast to the well-known tyrosine hydroxylases (EC 1.14.16.2) and tyrosinases (EC 1.14.18.1), LmbB2 was identified as a heme protein. Mass spectrometry and Soret band-excited Raman spectroscopy of LmbB2 showed that LmbB2 contains heme b as prosthetic group. The CO-reduced differential absorption spectra of LmbB2 showed that the coordination of Fe was different from that of cytochrome P450 enzymes. LmbB2 exhibits sequence similarity to Orf13 of the anthramycin biosynthetic gene cluster, which has recently been classified as a heme peroxidase. Tyrosine hydroxylating activity of LmbB2 yielding DOPA in the presence of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) was also observed. Reaction mechanism of this unique heme peroxidases family is discussed. Also, tyrosine hydroxylation was confirmed as the first step of the amino acid branch of the lincomycin biosynthesis. PMID:24324587

  5. No change in spontaneous behavior of rats after acute oral doses of aspartame, phenylalanine, and tyrosine.

    PubMed

    Mullenix, P J; Tassinari, M S; Schunior, A; Kernan, W J

    1991-04-01

    Spontaneous behavior subsequent to acute oral administration of high doses of aspartame, phenylalanine, or tyrosine was analyzed using a computer pattern recognition system. Sprague-Dawley male rats (250-300 g) were dosed orally with aspartame (500 or 1000 mg/kg), phenylalanine (281 or 562 mg/kg), or tyrosine (309 or 618 mg/kg), and their behavior was analyzed 1 hr after dosing. The computer pattern recognition system recorded and classified 13 different behavioral acts performed by the animals during the first 15-min exploration of a novel environment. Three measures that provide independent information concerning motor output from the central nervous system were taken: the number of behavioral initiations, total time, and time structure. These results were compared with the effects induced by d-amphetamine. Plasma concentrations of phenylalanine and tyrosine were determined from blood samples taken immediately after behavioral examination. Data analysis revealed that these doses of aspartame, phenylalanine, and tyrosine did not induce any significant changes in spontaneous behavior. Unlike low doses of amphetamine and despite high plasma concentrations of phenylalanine and tyrosine, no behavioral alteration was detected by the computer pattern recognition system. Absence of behavioral changes in this study using an objective analysis of behavior raises questions concerning the relationship between amino acid precursor loading and purported anecdotal changes in behavior following aspartame administration.

  6. The Crystal Structure of Aquifex aeolicus Prephenate Dehydrogenase Reveals the Mode of Tyrosine Inhibition

    SciTech Connect

    Sun, Warren; Shahinas, Dea; Bonvin, Julie; Hou, Wenjuan; Kimber, Matthew S.; Turnbull, Joanne; Christendat, Dinesh

    2009-08-14

    TyrA proteins belong to a family of dehydrogenases that are dedicated to l-tyrosine biosynthesis. The three TyrA subclasses are distinguished by their substrate specificities, namely the prephenate dehydrogenases, the arogenate dehydrogenases, and the cyclohexadienyl dehydrogenases, which utilize prephenate, l-arogenate, or both substrates, respectively. The molecular mechanism responsible for TyrA substrate selectivity and regulation is unknown. To further our understanding of TyrA-catalyzed reactions, we have determined the crystal structures of Aquifex aeolicus prephenate dehydrogenase bound with NAD(+) plus either 4-hydroxyphenylpyuvate, 4-hydroxyphenylpropionate, or l-tyrosine and have used these structures as guides to target active site residues for site-directed mutagenesis. From a combination of mutational and structural analyses, we have demonstrated that His-147 and Arg-250 are key catalytic and binding groups, respectively, and Ser-126 participates in both catalysis and substrate binding through the ligand 4-hydroxyl group. The crystal structure revealed that tyrosine, a known inhibitor, binds directly to the active site of the enzyme and not to an allosteric site. The most interesting finding though, is that mutating His-217 relieved the inhibitory effect of tyrosine on A. aeolicus prephenate dehydrogenase. The identification of a tyrosine-insensitive mutant provides a novel avenue for designing an unregulated enzyme for application in metabolic engineering.

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

    PubMed Central

    1994-01-01

    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

  8. Asymmetric Receptor Contact is Required for Tyrosine Autophosphorylation of Fibroblast Growth Factor Receptor in Living Cells

    SciTech Connect

    Bae, J.; Boggon, T; Tomé, F; Mandiyan, V; Lax, I; Schlessinge, J

    2010-01-01

    Tyrosine autophosphorylation of receptor tyrosine kinases plays a critical role in regulation of kinase activity and in recruitment and activation of intracellular signaling pathways. Autophosphorylation is mediated by a sequential and precisely ordered intermolecular (trans) reaction. In this report we present structural and biochemical experiments demonstrating that formation of an asymmetric dimer between activated FGFR1 kinase domains is required for transphosphorylation of FGFR1 in FGF-stimulated cells. Transphosphorylation is mediated by specific asymmetric contacts between the N-lobe of one kinase molecule, which serves as an active enzyme, and specific docking sites on the C-lobe of a second kinase molecule, which serves a substrate. Pathological loss-of-function mutations or oncogenic activating mutations in this interface may hinder or facilitate asymmetric dimer formation and transphosphorylation, respectively. The experiments presented in this report provide the molecular basis underlying the control of transphosphorylation of FGF receptors and other receptor tyrosine kinases.

  9. Keratinocyte-derived laminin-332 protein promotes melanin synthesis via regulation of tyrosine uptake.

    PubMed

    Chung, Heesung; Jung, Hyejung; Lee, Jung-Hyun; Oh, Hye Yun; Kim, Ok Bin; Han, Inn-Oc; Oh, Eok-Soo

    2014-08-01

    Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake.

  10. TIE2-mediated tyrosine phosphorylation of H4 regulates DNA damage response by recruiting ABL1

    PubMed Central

    Hossain, Mohammad B.; Shifat, Rehnuma; Johnson, David G.; Bedford, Mark T.; Gabrusiewicz, Konrad R.; Cortes-Santiago, Nahir; Luo, Xuemei; Lu, Zhimin; Ezhilarasan, Ravesanker; Sulman, Erik P.; Jiang, Hong; Li, Shawn S. C.; Lang, Frederick F.; Tyler, Jessica; Hung, Mien-Chie; Fueyo, Juan; Gomez-Manzano, Candelaria

    2016-01-01

    DNA repair pathways enable cancer cells to survive DNA damage induced after genotoxic therapies. Tyrosine kinase receptors (TKRs) have been reported as regulators of the DNA repair machinery. TIE2 is a TKR overexpressed in human gliomas at levels that correlate with the degree of increasing malignancy. Following ionizing radiation, TIE2 translocates to the nucleus, conferring cells with an enhanced nonhomologous end-joining mechanism of DNA repair that results in a radioresistant phenotype. Nuclear TIE2 binds to key components of DNA repair and phosphorylates H4 at tyrosine 51, which, in turn, is recognized by the proto-oncogene ABL1, indicating a role for nuclear TIE2 as a sensor for genotoxic stress by action as a histone modifier. H4Y51 constitutes the first tyrosine phosphorylation of core histones recognized by ABL1, defining this histone modification as a direct signal to couple genotoxic stress with the DNA repair machinery.

  11. Horseradish-peroxidase-catalyzed polymerization of amphiphilic tyrosine derivatives in micelles

    NASA Astrophysics Data System (ADS)

    Sarma, Rupmoni; Alva, Shridhara; Marx, Kenneth A.; Akkara, Joseph A.; Kaplan, David L.; Tripathy, Sukant K.

    1998-04-01

    There has been much interest in enzyme catalyzed organic synthesis because it allows the design and synthesis of new materials via chemically mild reaction schemes. This study reports on the horseradish peroxidase catalyzed polymerization of the amphiphilic, C10 alkyl monomer derivative of d and l isomers of tyrosine in micellar solutions. The methodology has been developed to improve the solubility and hence processability of these phenolic polymers. The technique involves the formation of emulsions or micelles of the amphiphilic tyrosines in aqueous medium through manipulation of the solution pH and subsequent enzymatic polymerization. The solution pH, concentrations of the tyrosine derivatives, hydrogen peroxide and the enzyme have been optimized for maximum conversion. The physico- chemical properties of the resulting polymers have been studied by various spectroscopic techniques. Limited stereo- specificity of the reaction has been demonstrated by kinetic methods. Thin films of these polymeric materials have been fabricated using the Langmuir-Blodgett film technique.

  12. Inhibition of an Erythrocyte Tyrosine Kinase with Imatinib Prevents Plasmodium falciparum Egress and Terminates Parasitemia

    PubMed Central

    Kesely, Kristina R.; Pantaleo, Antonella; Turrini, Francesco M.; Olupot-Olupot, Peter

    2016-01-01

    With half of the world’s population at risk for malaria infection and with drug resistance on the rise, the search for mutation-resistant therapies has intensified. We report here a therapy for Plasmodium falciparum malaria that acts by inhibiting the phosphorylation of erythrocyte membrane band 3 by an erythrocyte tyrosine kinase. Because tyrosine phosphorylation of band 3 causes a destabilization of the erythrocyte membrane required for parasite egress, inhibition of the erythrocyte tyrosine kinase leads to parasite entrapment and termination of the infection. Moreover, because one of the kinase inhibitors to demonstrate antimalarial activity is imatinib, i.e. an FDA-approved drug authorized for use in children, translation of the therapy into the clinic will be facilitated. At a time when drug resistant strains of P. falciparum are emerging, a strategy that targets a host enzyme that cannot be mutated by the parasite should constitute a therapeutic mechanism that will retard evolution of resistance. PMID:27768734

  13. DNA-Catalyzed Introduction of Azide at Tyrosine for Peptide Modification

    PubMed Central

    Wang, Puzhou; Silverman, Scott K.

    2016-01-01

    We show that DNA enzymes (deoxyribozymes) can introduce azide functional groups at tyrosine residues in peptide substrates. Using in vitro selection, we identified deoxyribozymes that transfer the 2′-azido-2′-deoxyadenosine 5′-monophosphoryl group (2′-Az-dAMP) from the analogous 5′-triphosphate (2′-Az-dATP) onto the tyrosine hydroxyl group of a peptide, which is either tethered to a DNA anchor or free. Some of the new deoxyribozymes are general with regard to the amino acid residues surrounding the tyrosine, while other DNA enzymes are sequence-selective. We use one of the new deoxyribozymes to modify free peptide substrates by attaching PEG moieties and fluorescent labels. PMID:27391404

  14. Inactivation of tyrosine hydroxylase activity by ascorbate in vitro and in rat PC12 cells.

    PubMed

    Wilgus, H; Roskoski, R

    1988-10-01

    Tyrosine hydroxylase activity is reversibly modulated by the actions of a number of protein kinases and phosphoprotein phosphatases. A previous report from this laboratory showed that low-molecular-weight substances present in striatal extracts lead to an irreversible loss of tyrosine hydroxylase activity under cyclic AMP-dependent phosphorylation conditions. We report here that ascorbate is one agent that inactivates striatal tyrosine hydroxylase activity with an EC50 of 5.9 microM under phosphorylating conditions. Much higher concentrations (100 mM) fail to inactivate the enzyme under nonphosphorylating conditions. Isoascorbate (EC50, 11 microM) and dehydroascorbate (EC50, 970 microM) also inactivated tyrosine hydroxylase under phosphorylating but not under nonphosphorylating conditions. In contrast, ascorbate sulfate was inactive under phosphorylating conditions at concentrations up to 100 mM. Since the reduced compounds generate several reactive species in the presence of oxygen, the possible protecting effects of catalase, peroxidase, and superoxide dismutase were examined. None of these three enzymes, however, afforded any protection against inactivation. We also examined the effects of ascorbate and its congeners on the activity of tyrosine hydroxylase purified to near homogeneity from a rat pheochromocytoma. This purified enzyme was also inactivated by the same agents that inactivated the impure corpus striatal enzyme. Under conditions in which ascorbate almost completely abolished enzyme activity, we found no indication for significant proteolysis of the purified enzyme as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We also found that pretreatment of PC12 cells in culture for 4 h with 1 mM ascorbate, dehydroascorbate, or isoascorbate (but not ascorbate sulfate) also decreased tyrosine hydroxylase activity 25-50%.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2901463

  15. Infarction-induced cytokines cause local depletion of tyrosine hydroxylase in cardiac sympathetic nerves

    PubMed Central

    Parrish, Diana C.; Alston, Eric N.; Rohrer, Hermann; Nkadi, Paul; Woodward, William R.; Schütz, Günther; Habecker, Beth A.

    2010-01-01

    Myocardial infarction causes heterogeneity of noradrenergic transmission that contributes to the development of ventricular arrhythmias and sudden cardiac death. Ischemia-induced alterations in sympathetic transmission include regional variations in cardiac norepinephrine (NE) and in tyrosine hydroxylase, the rate-limiting enzyme in NE synthesis. Inflammatory cytokines that act through gp130 are elevated in the heart after myocardial infarction. These cytokines decrease expression of tyrosine hydroxylase in sympathetic neurons, and indirect evidence suggests they contribute to the local depletion of tyrosine hydroxylase in the damaged left ventricle. However, gp130 cytokines are also important for the survival of cardiac myocytes following damage to the heart. To examine the effect of cytokines on tyrosine hydroxylase and NE content in cardiac nerves we used gp130DBH-Cre/lox mice, which have a deletion of the gp130 receptor in neurons expressing dopamine beta hydroxylase. The absence of neuronal gp130 prevented the loss of tyrosine hydroxylase in cardiac sympathetic nerves innervating the left ventricle one week after ischemia-reperfusion. Surprisingly, restoring tyrosine hydroxylase in the damaged ventricle did not return neuronal NE content to normal levels. NE uptake into cardiac nerves was significantly lower in gp130 KO mice, contributing to the lack of neuronal NE stores. There were no significant differences in left ventricular peak systolic pressure, dP/dtMAX, or dP/dtMIN between the two genotypes after myocardial infarction, but ganglionic blockade revealed differences in autonomic tone between the genotypes. Stimulating the heart with dobutamine or releasing endogenous NE with tyramine generated similar responses in both genotypes. Thus, the removal of gp130 from sympathetic neurons prevents the post-infarct depletion of TH in the left ventricle, but does not alter NE content or cardiac function. PMID:19880537

  16. Inhibition of lactoperoxidase-catalyzed 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and tyrosine oxidation by tyrosine-containing random amino acid copolymers.

    PubMed

    Clausen, Morten R; Skibsted, Leif H; Stagsted, Jan

    2008-09-24

    Oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) by lactoperoxidase was found to be inhibited by tyrosine-containing random amino acid copolymers but not by tyrosine. Both electrostatic effects and polymer size were found to be important by comparison of negatively and positively charged copolymers of varying lengths, with poly(Glu, Tyr)4:1 ([E 4Y 1] approximately 40) as the strongest competitive inhibitor (EC 50 approximately 20 nM). This polymer did not form dityrosine in the presence of lactoperoxidase (LPO) and peroxide. Furthermore, incubation with tert-butyl hydroperoxide, as opposed to hydrogen peroxide, resulted in a peculiar long lag phase of the reaction between the redox intermediate compound II and [E 4Y 1] approximately 40, indicating a very tight association between enzyme and inhibitor. We propose that interactions between multiple positively charged areas on the surface of LPO and the polymer are required for optimal inhibition.

  17. Tyrosine kinase inhibition: A therapeutic target for the management of chronic-phase chronic myeloid leukemia

    PubMed Central

    Jabbour, Elias J; Cortes, Jorge E; Kantarjian, Hagop M

    2014-01-01

    Chronic myeloid leukemia (CML) is a hematologic neoplasm with a progressive, ultimately terminal, disease course. In most cases, CML arises owing to the aberrant formation of a chimeric gene for a constitutively active tyrosine kinase. Inhibition of the signaling activity of this kinase has proved to be a highly successful treatment target transforming the prognosis of patients with CML. New tyrosine kinase inhibitors (TKIs) continue to improve the management of CML, offering alternative options for those resistant to or intolerant of standard TKIs. Here we review the pathobiology of CML and explore emerging strategies to optimize the management of chronic-phase CML, particularly first-line treatment. PMID:24236822

  18. Versatile and Efficient Site-Specific Protein Functionalization by Tubulin Tyrosine Ligase.

    PubMed

    Schumacher, Dominik; Helma, Jonas; Mann, Florian A; Pichler, Garwin; Natale, Francesco; Krause, Eberhard; Cardoso, M Cristina; Hackenberger, Christian P R; Leonhardt, Heinrich

    2015-11-01

    A novel chemoenzymatic approach for simple and fast site-specific protein labeling is reported. Recombinant tubulin tyrosine ligase (TTL) was repurposed to attach various unnatural tyrosine derivatives as small bioorthogonal handles to proteins containing a short tubulin-derived recognition sequence (Tub-tag). This novel strategy enables a broad range of high-yielding and fast chemoselective C-terminal protein modifications on isolated proteins or in cell lysates for applications in biochemistry, cell biology, and beyond, as demonstrated by the site-specific labeling of nanobodies, GFP, and ubiquitin. PMID:26404067

  19. A Molecular Brake in the Kinase Hinge Region Regulates the Activity of Receptor Tyrosine Kinases

    SciTech Connect

    Chen,H.; Ma, J.; Li, W.; Eliseenkova, A.; Xu, C.; Neubert, T.; Miller, W.; Mohammadi, M.

    2007-01-01

    Activating mutations in the tyrosine kinase domain of receptor tyrosine kinases (RTKs) cause cancer and skeletal disorders. Comparison of the crystal structures of unphosphorylated and phosphorylated wild-type FGFR2 kinase domains with those of seven unphosphorylated pathogenic mutants reveals an autoinhibitory 'molecular brake' mediated by a triad of residues in the kinase hinge region of all FGFRs. Structural analysis shows that many other RTKs, including PDGFRs, VEGFRs, KIT, CSF1R, FLT3, TEK, and TIE, are also subject to regulation by this brake. Pathogenic mutations activate FGFRs and other RTKs by disengaging the brake either directly or indirectly.

  20. Structure and regulation of the c-Fes protein-tyrosine kinase.

    PubMed

    Hellwig, Sabine; Smithgall, Thomas E

    2011-06-01

    The c-Fes protein-tyrosine kinase is the normal cellular ortholog of several avian and feline retroviral oncoproteins. Unlike its transforming viral counterparts, c-Fes tyrosine kinase activity is tightly regulated in vivo through a mechanism involving coiled-coil oligomerization domains and other unique structural features found in its long N-terminal region. This review is focused on the regulatory features and structural biology of c-Fes, which has been implicated in normal cellular growth regulation, the innate immune response, and tumorigenesis.

  1. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors from the natural origin: a recent perspective.

    PubMed

    Patel, Harun M; Rane, Rajesh; Thapliyal, Neeta; Palkar, Mahesh; Shaikh, Mahamadhanif; Karpoormath, Rajshekhar

    2015-01-01

    Overexpression of epidermal growth factor receptor (EGFR) is seen in a number of human tumors like prostate, colon, breast and ovarian. Their expression is correlated with vascularity and often difficult to diagnose. Though a number of active inhibitors and anticancer drugs against EGFR-tyrosine kinase are known, increase in resistance together with many side effects designate the need for new and improved treatments. Natural products and their analoges have significant contribution in the cancer drug discovery and development process. Therefore in the current review we mainly discuss design, synthesis and structural activity relationship of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors from the natural origin.

  2. Determination of Tyrosine and Tryptophan Metabolites in Body Ruids Using Electrochemical Detection

    NASA Astrophysics Data System (ADS)

    Davis, Gregory C.; Koch, David D.; Kissinger, Peter T.; Bruntlett, Craig S.; Shoup, Ronald E.

    The amino acids tyrosine and tryptophan are precursors for a number of important physiological compounds. The catecholamines, which are metabolites of tyrosine, serve as neurotransmitters in the central and peripheral nervous systems. Serotonin, a major metabolite of tryptophan, is a potent neurotransmitter and vasoconstrictor. Without doubt, these compounds have been some of the most intensely studied molecules in the last twenty years. One of the benefits that often accrues from basic biochemical research is clinical data of diagnostic and prognostic significance. In this case, however, the results have been disappointing. In only a few instances has the measurement of metabolites of these two amino acids been shown to have real clinical significance.

  3. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors from the natural origin: a recent perspective.

    PubMed

    Patel, Harun M; Rane, Rajesh; Thapliyal, Neeta; Palkar, Mahesh; Shaikh, Mahamadhanif; Karpoormath, Rajshekhar

    2015-01-01

    Overexpression of epidermal growth factor receptor (EGFR) is seen in a number of human tumors like prostate, colon, breast and ovarian. Their expression is correlated with vascularity and often difficult to diagnose. Though a number of active inhibitors and anticancer drugs against EGFR-tyrosine kinase are known, increase in resistance together with many side effects designate the need for new and improved treatments. Natural products and their analoges have significant contribution in the cancer drug discovery and development process. Therefore in the current review we mainly discuss design, synthesis and structural activity relationship of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors from the natural origin. PMID:25763933

  4. Soft X-ray induced decomposition of phenylalanine and tyrosine: Acomparative study

    SciTech Connect

    Zubavichus, Y.; Zharnikov, M.; Shaporenko, A.; Fuchs, O.; Weinhart, L.; Heske, C.; Umbach, E.; Denlinger, J.D.; Grunze, M.

    2003-11-19

    The pristine state and soft X-ray induced decomposition of two aromatic amino acids, viz. phenylalanine and tyrosine, have been studied by means of XPS and NEXAFS. The spectroscopic data on the radiation decomposition have been supplemented by a mass-spectral analysis of desorbed species in the residual gas. Despite very similar chemical structures, the two amino acids show a drastically different behavior towards ionizing radiation: phenylalanine degrades very quickly whereas tyrosine shows a prominent stability against radiation damage. Reasons for this difference are discussed in relation to radical-mediated reactions responsible for the decomposition.

  5. Stimulatory actions of bioflavenoids on tyrosine uptake into cultured bovine adrenal chromaffin cells

    SciTech Connect

    Morita, K.; Hamano, S.; Oka, M.; Teraoka, K. )

    1990-09-28

    The effects of flavenoids on L-({sup 14}C)tyrosine uptake into cultured adrenal chromaffin cells were examined. Flavone markedly stimulated tyrosine uptake into these cells in a manner dependent on its concentration. Apigenin also caused a moderate stimulatory action, but quercetin had no significant effect on the uptake. Flavone also stimulated the uptake of histidine, but did not affect the uptake of serine, lysine, or glutamic acid. These results are considered to propose the possibility that flavonoids may be able to stimulate the precursor uptake into the cells, resulting in an enhancement of the biogenic amine production.

  6. Redundant kinase activation and resistance of EGFR-tyrosine kinase inhibitors

    PubMed Central

    Luo, Min; Fu, Li-Wu

    2014-01-01

    Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic effects against that tumors harboring EGFR activating mutations in the EGFR intracytoplasmic tyrosine kinase domain and resulted in cell apoptosis. Unfortunately, a number of patients ultimately developed resistance by multiple mechanisms. Thus, elucidation of the mechanism of resistance to EGFR-TKIs can provide strategies for blocking or reversing the situation. Recent studies suggested that redundant kinase activation plays pivotal roles in escaping from the effects of EGFR-TKIs. Herein, we aimed to characterize several molecular events involved in the resistance to EGFR-TKIs mediated by redundant kinase activation. PMID:25520855

  7. Chondroid syringoma with tyrosine crystals: case report and review of the literature.

    PubMed

    Constantinescu, Monica B; Chan, Jessica B; Cassarino, David S

    2010-04-01

    Chondroid syringoma (CS) is a relatively rare cutaneous mixed tumor arising from sweat glands. It usually presents in the head and neck area as an asymptomatic, slow-growing, firm, circumscribed, lobulated nodule within the dermis or subcutaneous fat. CSs share morphologic similarities with their salivary gland counterparts, pleomorphic adenomas (benign mixed tumors). Although the presence of tyrosine-rich crystalloids in mixed tumors of the salivary gland is well recognized, to our knowledge, this finding has not been previously described in mixed tumors of the skin. We report a case of tyrosine crystalline structures in a CS and review the pertinent literature.

  8. The tyrosine 343 residue of nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) is important for its interaction with SHP1, a cytoplasmic tyrosine phosphatase with tumor suppressor functions.

    PubMed

    Hegazy, Samar A; Wang, Peng; Anand, Mona; Ingham, Robert J; Gelebart, Pascal; Lai, Raymond

    2010-06-25

    The cytoplasmic tyrosine phosphatase SHP1 has been shown to inhibit the oncogenic fusion protein nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK), and loss of SHP1 contributes to NPM-ALK-mediated tumorigenesis. In this study, we aimed to further understand how SHP1 interacts and regulates NPM-ALK. We employed an in vitro model in which GP293 cells were transfected with various combinations of NPM-ALK (or mutants) and SHP1 (or mutants) expression vectors. We found that SHP1 co-immunoprecipitated with NPM-ALK, but not the enzymatically inactive NPM-ALK(K210R) mutant, or the mutant in which all three functionally important tyrosine residues (namely, Tyr(338), Tyr(342), and Tyr(343)) in the kinase activation loop (KAL) of ALK were mutated. Interestingly, whereas mutation of Tyr(338) or Tyr(342) did not result in any substantial change in the NPM-ALK/SHP1 binding (assessed by co-immunoprecipitation), mutation of Tyr(343) abrogated this interaction. Furthermore, the NPM-ALK/SHP1 binding was readily detectable when each of the remaining 8 tyrosine residues known to be phosphorylated were mutated. Although the expression of SHP1 effectively reduced the level of tyrosine phosphorylation of NPM-ALK, it did not affect that of the NPM-ALK(Y343F) mutant. In soft agar clonogenic assay, SHP1 expression significantly reduced the tumorigenicity of NPM-ALK but not that of NPM-ALK(Y343F). In conclusion, we identified Tyr(343) of NPM-ALK as the crucial site for mediating the NPM-ALK/SHP1 interaction. Our results also support the notion that the tumor suppressor effects of SHP1 on NPM-ALK are dependent on its ability to bind to this oncogenic protein.

  9. Phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) serine-511 by the combined action of tyrosine kinases and CK2: the implication of tyrosine-512 and phenylalanine-508.

    PubMed

    Cesaro, Luca; Marin, Oriano; Venerando, Andrea; Donella-Deana, Arianna; Pinna, Lorenzo A

    2013-12-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) harbors, close to Phe-508, whose deletion is the commonest cause of cystic fibrosis, a conserved potential CK2 phospho-acceptor site (Ser511), which however is not susceptible to phosphorylation by CK2. To shed light on this apparent paradox, a series of systematically substituted peptides encompassing Ser511 were assayed for their ability to be phosphorylated. The main outcomes of our study are the following: (a) Tyr512 plays a prominent role as a negative determinant as its replacement by Ala restores Ser511 phosphorylation by CK2; (b) an even more pronounced phosphorylation of Ser511 is promoted if Tyr512 is replaced by phospho-tyrosine instead of alanine; (c) Tyr512 and, to a lesser extent, Tyr515 are readily phosphorylated by Lyn, a protein tyrosine kinase of the Src family, in a manner which is enhanced by the concomitant Phe508 deletion. Collectively taken, our data, in conjunction with the notion that Tyr515 is phosphorylated in vivo, disclose the possibility that CFTR Ser511 can be phosphorylated by the combined action of tyrosine kinases and CK2 and disclose a new mechanism of hierarchical phosphorylation where the role of the priming kinase is that of removing negative determinant(s).

  10. Characterization of Cbl tyrosine phosphorylation and a Cbl-Syk complex in RBL-2H3 cells

    PubMed Central

    1996-01-01

    Tyrosine phosphorylation of the Cbl protooncogene has been shown to occur after engagement of a number of different receptors on hematopoietic cells. However, the mechanisms by which these receptors induce Cbl tyrosine phosphorylation are poorly understood. Here we demonstrate that engagement of the high affinity IgE receptor (Fc epsilon R1) leads to the tyrosine phosphorylation of Cbl and analyze how this occurs. We show that at least part of Fc epsilon R1-induced Cbl tyrosine phosphorylation is mediated by the Syk tyrosine kinase, and that the Syk-dependent tyrosine phosphorylation of Cbl occurs mainly distal to the Cbl proline-rich region within the COOH-terminal 250 amino acids. Furthermore, we show by coprecipitation that Cbl is present in a complex with Syk before receptor engagement, that the proline-rich region of Cbl and a region of Syk comprised of the two SH2 domains and intradomain linker are required for formation of the complex, and that little or no tyrosine-phosphorylated Cbl is detected in complex with Syk. Overexpression of truncation mutants of Cbl capable of binding Syk has the effect of blocking tyrosine phosphorylation of endogenous Cbl. These results define a potentially important intramolecular interaction in mast cells and suggest a complex function for Cbl in intracellular signaling pathways. PMID:8920860

  11. Biodegradation of the allelopathic chemical m-tyrosine by Bacillus aquimaris SSC5 involves the homogentisate central pathway.

    PubMed

    Khan, Fazlurrahman; Kumari, Munesh; Cameotra, Swaranjit Singh

    2013-01-01

    m-Tyrosine is an amino acid analogue, exuded from the roots of fescue grasses, which acts as a potent allelopathic and a broad spectrum herbicidal chemical. Although the production and toxic effects of m-tyrosine are known, its microbial degradation has not been documented yet. A soil microcosm study showed efficient degradation of m-tyrosine by the inhabitant microorganisms. A bacterial strain designated SSC5, that was able to utilize m-tyrosine as the sole source of carbon, nitrogen, and energy, was isolated from the soil microcosm and was characterized as Bacillus aquimaris. Analytical methods such as HPLC, GC-MS, and (1)H-NMR performed on the resting cell samples identified the formation of 3-hydroxyphenylpyruvate (3-OH-PPA), 3-hydroxyphenylacetate (3-OH-PhAc), and homogentisate (HMG) as major intermediates in the m-tyrosine degradation pathway. Enzymatic assays carried out on cell-free lysates of m-tyrosine-induced cells confirmed transamination reaction as the first step of m-tyrosine degradation. The intermediate 3-OH-PhAc thus obtained was further funneled into the HMG central pathway as revealed by a hydroxylase enzyme assay. Subsequent degradation of HMG occurred by ring cleavage catalyzed by the enzyme homogentisate 1, 2-dioxygenase. This study has significant implications in terms of understanding the environmental fate of m-tyrosine as well as regulation of its phytotoxic effect by soil microorganisms. PMID:24098407

  12. Effect of inhibition of tyrosine phosphatases on voltage-operated calcium channel currents in rabbit isolated ear artery cells

    PubMed Central

    Wijetunge, S; Lymn, J S; Hughes, A D

    1998-01-01

    The effect of increasing cellular tyrosine phosphorylation by inhibiting endogenous tyrosine phosphatases was examined on voltage-operated calcium channel currents in vascular smooth muscle cells.In single ear artery smooth muscle cells of the rabbit, studied by the whole cell voltage clamp technique, intracellular application of the tyrosine phosphatase inhibitors, sodium orthovanadate (100 μM) and peroxyvanadate (100 μM orthovanadate+1 mM H2O2) increased voltage-operated calcium channel currents by 56% and 83%, respectively.Bath application of two other membrane permeant tyrosine phosphatase inhibitors, phenylarsine oxide (100 μM) and dephostatin (50 μM) also increased voltage-operated calcium channel currents by 48% and 52%, respectively.The selective tyrosine kinase inhibitor, tyrphostin-23 (100 μM) reduced calcium channel currents by 41%. Pre-incubation with tyrphostin-23 abolished the effects of peroxyvanadate, phenylarsine oxide and dephostatin on calcium channels.Western blot analysis of rabbit ear artery cell lysates showed increased tyrosine phosphorylation of several endogenous proteins following treatment with peroxyvanadate.These results indicate that a number of structurally dissimilar inhibitors of tyrosine phosphatases increase voltage-operated calcium channel currents in arterial smooth muscle cells presumably due to increased tyrosine phosphorylation. PMID:9641547

  13. The conversion of phenylalanine to tyrosine in man. Direct measurement by continuous intravenous tracer infusions of L-(ring-/sup 2/H5)phenylalanine and L-(1-/sup 13/C) tyrosine in the postabsorptive state

    SciTech Connect

    Clarke, J.T.; Bier, D.M.

    1982-10-01

    Steady state phenylalanine and tyrosine turnover and the rate of conversion of phenylalanine of tyrosine in vivo were determined in 6 healthy postabsorptive adult volunteers. Continuous infusions of tracer amounts of L-(ring-/sup 2/H5)phenylalanine were determined intravenously for 13-14 hr. After 9-10 hr, a priming dose followed by a continuous infusion of L-(1-/sup 13/C)tyrosine was added and maintained, along with the (/sup 2/H5)phenylalanine infusion, for 4 hr. Venous plasma samples were obtained before the initiation of each infusion and every 30 min during the course of the combined (/sup 2/H5)phenylalanine and (/sup 13/C)tyrosine infusion for determination of isotopic enrichments of (/sup 2/H5)phenylalanine, (/sup 13/C)tyrosine, and (/sup 2/H4)tyrosine by gas chromatograph-mass spectrometric analysis of the N-trifluoroacetyl-, methyl ester derivatives of the amino acids. Calculated from the observed enrichments, free phenylalanine and tyrosine turnover rates were 36.1 +/- 5.1 mumole . kg-1 . h-1 and 39.8 +/- 3.5 mumole . kg-1 . h-1, respectively. Phenylalanine was converted to tyrosine at the rate of 5.83 +/- 0.59 mumole . kg-1 . h-1, accounting for approximately 16% of either the phenylalanine or the tyrosine flux. The results indicate that the normal basal steady state phenylalanine hydroxylase activity in vivo in man is lower than that obtained from phenylalanine loading studies. This supports the existence of some type of substance activation of the enzyme as reflected in the previously reported exponential relationship between phenylalanine concentration and phenylalanine hydroxylase activity in vitro. The use of continuous simultaneous infusions of tracer amounts of stable isotope-labeled phenylalanine and tyrosine provides a direct means for studying physiological regulation of phenylalanine hydroxylase activity in vivo.

  14. SOCS3 tyrosine phosphorylation as a potential bio-marker for myeloproliferative neoplasms associated with mutant JAK2 kinases

    PubMed Central

    Elliott, Joanne; Suessmuth, Yvonne; Scott, Linda M.; Nahlik, Krystyna; McMullin, Mary Frances; Constantinescu, Stefan N.; Green, Anthony R.; Johnston, James A.

    2009-01-01

    JAK2 V617F, identified in the majority of patients with myeloproliferative neoplasms, tyrosine phosphorylates SOCS3 and escapes its inhibition. Here, we demonstrate that the JAK2 exon 12 mutants described in a subset of V617F-negative MPN cases, also stabilize tyrosine phosphorylated SOCS3. SOCS3 tyrosine phosphorylation was also observed in peripheral blood mononuclear cells and granulocytes isolated from patients with JAK2 H538QK539L or JAK2 F537-K539delinsL mutations. JAK kinase inhibitors, which effectively inhibited the proliferation of cells expressing V617F or K539L, also caused a dose-dependent reduction in both mutant JAK2 and SOCS3 tyrosine phosphorylation. We propose, therefore, that SOCS3 tyrosine phosphorylation may be a novel bio-marker of myeloproliferative neoplasms resulting from a JAK2 mutation and a potential reporter of effective JAK2 inhibitor therapy currently in clinical development. PMID:19229050

  15. Role of the Yes and Csk tyrosine kinases in the development of a pathological state in the human retina.

    PubMed

    Baranova, Lyudmila; Emelyanova, Valentina; Volotovski, Igor

    2010-07-01

    Amplification and a cloning of fragments of genes of human retina tyrosine kinases, the nucleotide sequences of which feature a high homology to the gene families of the Yes and Csk tyrosine kinases, and a cloning of the complete coding sequence of the cDNA of the Csk tyrosine kinase gene of the human lymphocytes have been carried out. It has been established that this sequence contains 1,624 bp and encodes a protein that, with a 99% homology, corresponds to the human tyrosine kinase. A comparative analysis of the nucleotide sequences of the full-size cDNA of the Csk tyrosine kinase of the lymphocytes of healthy donors and of patients with an eye choroidal melanoma has shown that a risk of development of an eye choroidal melanoma can be estimated by the frequency of occurrence of a mutant allele in the 10th exon.

  16. Protein tyrosine kinase and mitogen-activated protein kinase signalling pathways contribute to differences in heterophil-mediated innate immune responsiveness between two lines of broilers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein tyrosine phosphorylation mediates signal transduction of cellular processes, with protein tyrosine kinases (PTKs) regulating virtually all signaling events. The mitogen-activated protein kinase (MAPK) super-family consists of three conserved pathways that convert receptor activation into ce...

  17. Siglec-5 (CD170) can mediate inhibitory signaling in the absence of immunoreceptor tyrosine-based inhibitory motif phosphorylation.

    PubMed

    Avril, Tony; Freeman, Sylvie D; Attrill, Helen; Clarke, Rosemary G; Crocker, Paul R

    2005-05-20

    Siglec-5 (CD170) is a member of the recently described human CD33-related siglec subgroup of sialic acid binding Ig-like lectins and is expressed on myeloid cells of the hemopoietic system. Similar to other CD33-related siglecs, Siglec-5 contains two tyrosine-based motifs in its cytoplasmic tail implicated in signaling functions. To investigate the role of these motifs in Siglec-5-dependent signaling, we used transfected rat basophil leukemia cells as a model system. Tyrosine phosphorylation of Siglec-5 led to recruitment of the tyrosine phosphatases SHP-1 and SHP-2, as seen in both pull-down assays and microscopy. Siglec-5 could efficiently inhibit FcepsilonRI-mediated calcium fluxing and serotonin release after co-cross-linking. Surprisingly, a double tyrosine to alanine mutant of Siglec-5 could still mediate strong inhibition of serotonin release in the absence of detectable tyrosine phosphorylation, whereas a double tyrosine to phenylalanine mutant lost all inhibitory activity. In comparison, suppression of Siglec-5-dependent adhesion to red blood cells was reversed by either tyrosine to alanine or tyrosine to phenylalanine mutations of the membrane proximal tyrosine-based motif. Using an in vitro phosphatase assay with synthetic and recombinant forms of the cytoplasmic tail, it was shown that a double alanine mutant of Siglec-5 had weak, but significant SHP-1 activating properties similar to those of wild type, non-phosphorylated cytoplasmic tail, whereas a double phenylalanine mutant was inactive. These findings establish that Siglec-5 can be classified as an inhibitory receptor with the potential to mediate SHP-1 and/or SHP-2-dependent signaling in the absence of tyrosine phosphorylation. PMID:15769739

  18. Brain catecholamine depletion and motor impairment in a Th knock-in mouse with type B tyrosine hydroxylase deficiency.

    PubMed

    Korner, Germaine; Noain, Daniela; Ying, Ming; Hole, Magnus; Flydal, Marte I; Scherer, Tanja; Allegri, Gabriella; Rassi, Anahita; Fingerhut, Ralph; Becu-Villalobos, Damasia; Pillai, Samyuktha; Wueest, Stephan; Konrad, Daniel; Lauber-Biason, Anna; Baumann, Christian R; Bindoff, Laurence A; Martinez, Aurora; Thöny, Beat

    2015-10-01

    Tyrosine hydroxylase catalyses the hydroxylation of L-tyrosine to l-DOPA, the rate-limiting step in the synthesis of catecholamines. Mutations in the TH gene encoding tyrosine hydroxylase are associated with the autosomal recessive disorder tyrosine hydroxylase deficiency, which manifests phenotypes varying from infantile parkinsonism and DOPA-responsive dystonia, also termed type A, to complex encephalopathy with perinatal onset, termed type B. We generated homozygous Th knock-in mice with the mutation Th-p.R203H, equivalent to the most recurrent human mutation associated with type B tyrosine hydroxylase deficiency (TH-p.R233H), often unresponsive to l-DOPA treatment. The Th knock-in mice showed normal survival and food intake, but hypotension, hypokinesia, reduced motor coordination, wide-based gate and catalepsy. This phenotype was associated with a gradual loss of central catecholamines and the serious manifestations of motor impairment presented diurnal fluctuation but did not improve with standard l-DOPA treatment. The mutant tyrosine hydroxylase enzyme was unstable and exhibited deficient stabilization by catecholamines, leading to decline of brain tyrosine hydroxylase-immunoreactivity in the Th knock-in mice. In fact the substantia nigra presented an almost normal level of mutant tyrosine hydroxylase protein but distinct absence of the enzyme was observed in the striatum, indicating a mutation-associated mislocalization of tyrosine hydroxylase in the nigrostriatal pathway. This hypomorphic mouse model thus provides understanding on pathomechanisms in type B tyrosine hydroxylase deficiency and a platform for the evaluation of novel therapeutics for movement disorders with loss of dopaminergic input to the striatum.

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

    PubMed

    Kraker, A J; Hartl, B G; Amar, A M; Barvian, M R; Showalter, H D; Moore, C W

    2000-10-01

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

  20. Diagnosis and prognosis of male infertility in mammal: the focusing of tyrosine phosphorylation and phosphotyrosine proteins.

    PubMed

    Kwon, Woo-Sung; Rahman, Md Saidur; Pang, Myung-Geol

    2014-11-01

    Male infertility refers to the inability of a man to achieve a pregnancy in a fertile female. In more than one-third of cases, infertility arises due to the male factor. Therefore, developing strategies for the diagnosis and prognosis of male infertility is critical. Simultaneously, a satisfactory model for the cellular mechanisms that regulate normal sperm function must be established. In this regard, tyrosine phosphorylation is one of the most common mechanisms through which several signal transduction pathways are adjusted in spermatozoa. It regulates the various aspects of sperm function, for example, motility, hyperactivation, capacitation, the acrosome reaction, fertilization, and beyond. Several recent large-scale studies have identified the proteins that are phosphorylated in spermatozoa to acquire fertilization competence. However, most of these studies are basal and have not presented an overall mechanism through which tyrosine phosphorylation regulates male infertility. In this review, we focus of this mechanism, discussing most of the tyrosine-phosphorylated proteins in spermatozoa that have been identified to date. We categorized tyrosine-phosphorylated proteins in spermatozoa that regulate male infertility using MedScan Reader (v5.0) and Pathway Studio (v9.0).

  1. Quantitative profiling of spreading-coupled protein tyrosine phosphorylation in migratory cells

    PubMed Central

    Xie, Yajun; Wang, Jinlong; Zhang, Yuanya; Liu, Xiaofei; Wang, Xiaorong; Liu, Kehui; Huang, Xiahe; Wang, Yingchun

    2016-01-01

    Protein tyrosine phosphorylation is an important mechanism that regulates cytoskeleton reorganization and cell spreading of migratory cells. A number of cytoskeletal proteins are known to be tyrosine phosphorylated (pY) in different cellular processes. However, the profile of pY proteins during different stages of cell spreading has not been available. Using immunoafffinity enrichment of pY proteins coupled with label free quantitative proteomics, we quantitatively identified 447 pY proteins in the migratory ECV-304 cells at the early spreading (adhesion) and the active spreading stages. We found that pY levels of the majority of the quantified proteins were significantly increased in the active spreading stage compared with the early spreading stage, suggesting that active cell spreading is concomitant with extra tyrosine phosphorylation. The major categories of proteins impacted by tyrosine phosphorylation are involved in cytoskeleton and focal adhesion regulation, protein translation and degradation. Our findings, for the first time, dissect the cell spreading-specific pY signals from the adhesion induced pY signals, and provide a valuable resource for the future mechanistic research regarding the regulation of cell spreading. PMID:27554326

  2. Phosphorylation of talin at tyrosine in Rous sarcoma virus-transformed cells.

    PubMed Central

    DeClue, J E; Martin, G S

    1987-01-01

    The cytoskeletal protein talin was found to undergo enhanced phosphorylation at tyrosine residues in chicken embryo fibroblasts following transformation by Rous sarcoma virus. An increase in the tyrosine phosphorylation of talin was also observed within 6 h in cells infected by the temperature-sensitive mutant tsNY68 after a shift from the nonpermissive to the permissive temperature. The overall extent of phosphorylation was 0.07 mol of phosphate per mol of talin and was not appreciably altered by transformation. In uninfected cells talin was shown to be phosphorylated at multiple sites by tryptic peptide mapping. Following transformation most of these sites remained phosphorylated, to the same or to a lesser extent, while novel, phosphotyrosine-containing phosphopeptides appeared. Talin was phosphorylated at tyrosine in cells infected by Rous sarcoma virus mutants which induce altered or partial transformation morphologies; thus the increased phosphorylation of talin at tyrosine occurred irrespective of the morphology induced. Transformation by Y73 also induced elevated levels of phosphotyrosine in talin, whereas transformation by the avian erythroblastosis and Fujinami sarcoma viruses did not. Images PMID:3031468

  3. Engagement of CD81 induces ezrin tyrosine phosphorylation and its cellular redistribution with filamentous actin

    SciTech Connect

    Coffey, Greg P.; Rajapaksa, Ranjani; Liu, Raymond; Sharpe, Orr; Kuo, Chiung-Chi; Wald Krauss, Sharon; Sagi, Yael; Davis, R. Eric; Staudt, Louis M.; Sharman, Jeff P.; Robinson, William H.; Levy, Shoshana

    2009-06-09

    CD81 is a tetraspanin family member involved in diverse cellular interactions in the immune and nervous systems and in cell fusion events. However, the mechanism of action of CD81 and of other tetraspanins has not been defined. We reasoned that identifying signaling molecules downstream of CD81 would provide mechanistic clues. We engaged CD81 on the surface of Blymphocytes and identified the induced tyrosine-phosphorylated proteins by mass spectrometry. This analysis showed that the most prominent tyrosine phosphorylated protein was ezrin, an actin binding protein and a member of the ezrin-radixin-moesin family. We also found that CD81 engagement induces spleen tyrosine kinase (Syk) and that Syk was involved in tyrosine phosphorylation of ezrin. Ezrin colocalized with CD81 and F-actin upon stimulation and this association was disrupted when Syk activation was blocked. Taken together, these studies suggest a model in which CD81 interfaces between the plasma membrane and the cytoskeleton by activating Syk, mobilizing ezrin, and recruiting F-actin to facilitate cytoskeletal reorganization and cell signaling. This may be a mechanism explaining the pleiotropic effects induced in response to stimulating cells by anti-CD81 antibodies or by the hepatitis C virus, which uses this molecule as its key receptor.

  4. A cytosolic activator of DNA replication is tyrosine phosphorylated in its active form.

    PubMed

    Fresa, K L; Autieri, M V; Coffman, F D; Georgoff, I; Cohen, S

    1993-04-01

    Cytosolic extracts from actively dividing lymphoid cells have been shown to induce DNA synthesis in isolated, quiescent nuclei. An initiating factor in such extracts (activator of DNA replication; ADR) is a > 90-kDa aprotinin-binding protein whose activity is inhibitable not only by aprotinin, but also by several other protease inhibitors as well. Although cytosol from non-proliferating lymphocytes is devoid of ADR activity, we have shown that these preparations can be induced to express ADR activity by brief exposure to a membrane-enriched fraction of spontaneously proliferating MOLT-4 cells via a kinase-dependent mechanism. In the present study, we examine the role of tyrosine kinases in this process. Three inhibitors of tyrosine kinases (genistein, kaempferol, and quercetin) can inhibit the in vitro generation of ADR activity. In vitro generation of ADR activity is associated with the de novo phosphorylation of several proteins, many of which are detectable using anti-phosphotyrosine monoclonal antibodies. ADR itself may be tyrosine phosphorylated in active form as immunoprecipitation using such monoclonal antibodies leads to the depletion of its activity. Moreover, immunoprecipitation results in the removal of several de novo tyrosine-phosphorylated proteins, including species at approximately 122, 105, 93, 86, 79, and 65 kDa. A subset of de novo-phosphorylated proteins, migrating at approximately 105, 93, and 70 kDa, also bound to aprotinin, suggesting that at least one of these proteins may represent ADR itself. PMID:7683270

  5. Vector Biology: Tyrosine Degradation Protects Blood Feeders from Death via La Grande Bouffe.

    PubMed

    Kopáček, Petr; Perner, Jan

    2016-08-22

    Blood-feeding arthropods digest vast amounts of host-blood nutrients. A new study suggests that tyrosine degradation is essential for the survival of blood-fed kissing bugs, mosquitoes, and ticks. This finding presents a promising target for the control of these disease vectors. PMID:27554655

  6. Engagement of CD81 induces ezrin tyrosine phosphorylation and its cellular redistribution with filamentous actin

    PubMed Central

    Coffey, Greg P.; Rajapaksa, Ranjani; Liu, Raymond; Sharpe, Orr; Kuo, Chiung-Chi; Krauss, Sharon Wald; Sagi, Yael; Davis, R. Eric; Staudt, Louis M.; Sharman, Jeff P.; Robinson, William H.; Levy, Shoshana

    2009-01-01

    Summary CD81 is a tetraspanin family member involved in diverse cellular interactions in the immune and nervous systems and in cell fusion events. However, the mechanism of action of CD81 and of other tetraspanins has not been defined. We reasoned that identifying signaling molecules downstream of CD81 would provide mechanistic clues. We engaged CD81 on the surface of B-lymphocytes and identified the induced tyrosine-phosphorylated proteins by mass spectrometry. This analysis showed that the most prominent tyrosine phosphorylated protein was ezrin, an actin-binding protein and a member of the ezrin-radixin-moesin family. We also found that CD81 engagement induces spleen tyrosine kinase (Syk) and that Syk was involved in tyrosine phosphorylation of ezrin. After engagement of CD81, it colocalized with ezrin and F-actin, and this association was disrupted when Syk activation was blocked. Taken together, these studies suggest a model in which CD81 interfaces between the plasma membrane and the cytoskeleton by activating Syk, mobilizing ezrin, and recruiting F-actin to facilitate cytoskeletal reorganization and cell signaling. This mechanism might explain the pleiotropic effects induced in response to stimulation of cells by anti-CD81 antibodies or by the hepatitis C virus, which uses this molecule as its key receptor. PMID:19654214

  7. Activating Mutations in PIK3CA Lead to Widespread Modulation of the Tyrosine Phosphoproteome

    PubMed Central

    Blair, Brian G.; Pinto, Sneha M.; Nirujogi, Raja S.; Jelinek, Christine A.; Malhotra, Radhika; Kim, Min-Sik; Park, Ben Ho; Pandey, Akhilesh

    2015-01-01

    The human oncogene PIK3CA is frequently mutated in human cancers. Two hotspot mutations in PIK3CA, E545K and H1047R, have been shown to regulate widespread signaling events downstream of AKT, leading to increased cell proliferation, growth, survival, and motility. We used quantitative mass spectrometry to profile the global phosphotyrosine proteome of isogenic knock-in cell lines containing these activating mutations, where we identified 824 unique phosphopeptides. Although it is well understood that these mutations result in hyperactivation of the serine/threonine kinase AKT, we found a surprisingly widespread modulation of tyrosine phosphorylation levels of proteins in the mutant cells. In the tyrosine kinome alone, 29 tyrosine kinases were altered in their phosphorylation status. Many of the regulated phosphosites that we identified were located in the kinase domain or the canonical activation sites, indicating that these kinases and their downstream signaling pathways were activated. Our study demonstrates that there is frequent and unexpected cross-talk that occurs between tyrosine signaling pathways and serine/threonine signaling pathways activated by the canonical PI3K-AKT axis. PMID:26267517

  8. MECHANISM OF PROTEIN TYROSINE PHOSPHATASE INHIBITION IN HUMAN AIRWAY EPITHELIAL CELLS (HAEC) EXPOSED TO ZN2+

    EPA Science Inventory

    A number of studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to Zn2+ inhibits protein tyrosine phosphatase (PTP) activity and leads to activation of epidermal growth factor receptor (EGFR) signaling in ...

  9. Platelet immunoreceptor tyrosine-based activation motif (ITAM) signaling and vascular integrity.

    PubMed

    Boulaftali, Yacine; Hess, Paul R; Kahn, Mark L; Bergmeier, Wolfgang

    2014-03-28

    Platelets are well-known for their critical role in hemostasis, that is, the prevention of blood loss at sites of mechanical vessel injury. Inappropriate platelet activation and adhesion, however, can lead to thrombotic complications, such as myocardial infarction and stroke. To fulfill its role in hemostasis, the platelet is equipped with various G protein-coupled receptors that mediate the response to soluble agonists such as thrombin, ADP, and thromboxane A2. In addition to G protein-coupled receptors, platelets express 3 glycoproteins that belong to the family of immunoreceptor tyrosine-based activation motif receptors: Fc receptor γ chain, which is noncovalently associated with the glycoprotein VI collagen receptor, C-type lectin 2, the receptor for podoplanin, and Fc receptor γII A, a low-affinity receptor for immune complexes. Although both genetic and chemical approaches have documented a critical role for platelet G protein-coupled receptors in hemostasis, the contribution of immunoreceptor tyrosine-based activation motif receptors to this process is less defined. Studies performed during the past decade, however, have identified new roles for platelet immunoreceptor tyrosine-based activation motif signaling in vascular integrity in utero and at sites of inflammation. The purpose of this review is to summarize recent findings on how platelet immunoreceptor tyrosine-based activation motif signaling controls vascular integrity, both in the presence and absence of mechanical injury. PMID:24677237

  10. Tyrosine-sulfated V2 peptides inhibit HIV-1 infection via coreceptor mimicry.

    PubMed

    Cimbro, Raffaello; Peterson, Francis C; Liu, Qingbo; Guzzo, Christina; Zhang, Peng; Miao, Huiyi; Van Ryk, Donald; Ambroggio, Xavier; Hurt, Darrell E; De Gioia, Luca; Volkman, Brian F; Dolan, Michael A; Lusso, Paolo

    2016-08-01

    Tyrosine sulfation is a post-translational modification that facilitates protein-protein interaction. Two sulfated tyrosines (Tys173 and Tys177) were recently identified within the second variable (V2) loop of the major HIV-1 envelope glycoprotein, gp120, and shown to contribute to stabilizing the intramolecular interaction between V2 and the third variable (V3) loop. Here, we report that tyrosine-sulfated peptides derived from V2 act as structural and functional mimics of the CCR5 N-terminus and potently block HIV-1 infection. Nuclear magnetic and surface plasmon resonance analyses indicate that a tyrosine-sulfated V2 peptide (pV2α-Tys) adopts a CCR5-like helical conformation and directly interacts with gp120 in a CD4-dependent fashion, competing with a CCR5 N-terminal peptide. Sulfated V2 mimics, but not their non-sulfated counterparts, inhibit HIV-1 entry and fusion by preventing coreceptor utilization, with the highly conserved C-terminal sulfotyrosine, Tys177, playing a dominant role. Unlike CCR5 N-terminal peptides, V2 mimics inhibit a broad range of HIV-1 strains irrespective of their coreceptor tropism, highlighting the overall structural conservation of the coreceptor-binding site in gp120. These results document the use of receptor mimicry by a retrovirus to occlude a key neutralization target site and provide leads for the design of therapeutic strategies against HIV-1. PMID:27389109

  11. Platelet immunoreceptor tyrosine-based activation motif (ITAM) signaling and vascular integrity.

    PubMed

    Boulaftali, Yacine; Hess, Paul R; Kahn, Mark L; Bergmeier, Wolfgang

    2014-03-28

    Platelets are well-known for their critical role in hemostasis, that is, the prevention of blood loss at sites of mechanical vessel injury. Inappropriate platelet activation and adhesion, however, can lead to thrombotic complications, such as myocardial infarction and stroke. To fulfill its role in hemostasis, the platelet is equipped with various G protein-coupled receptors that mediate the response to soluble agonists such as thrombin, ADP, and thromboxane A2. In addition to G protein-coupled receptors, platelets express 3 glycoproteins that belong to the family of immunoreceptor tyrosine-based activation motif receptors: Fc receptor γ chain, which is noncovalently associated with the glycoprotein VI collagen receptor, C-type lectin 2, the receptor for podoplanin, and Fc receptor γII A, a low-affinity receptor for immune complexes. Although both genetic and chemical approaches have documented a critical role for platelet G protein-coupled receptors in hemostasis, the contribution of immunoreceptor tyrosine-based activation motif receptors to this process is less defined. Studies performed during the past decade, however, have identified new roles for platelet immunoreceptor tyrosine-based activation motif signaling in vascular integrity in utero and at sites of inflammation. The purpose of this review is to summarize recent findings on how platelet immunoreceptor tyrosine-based activation motif signaling controls vascular integrity, both in the presence and absence of mechanical injury.

  12. Crystal structure of human tyrosylprotein sulfotransferase-2 reveals the mechanism of protein tyrosine sulfation reaction

    PubMed Central

    Teramoto, Takamasa; Fujikawa, Yukari; Kawaguchi, Yoshirou; Kurogi, Katsuhisa; Soejima, Masayuki; Adachi, Rumi; Nakanishi, Yuichi; Mishiro-Sato, Emi; Liu, Ming-Cheh; Sakakibara, Yoichi; Suiko, Masahito; Kimura, Makoto; Kakuta, Yoshimitsu

    2013-01-01

    Post-translational protein modification by tyrosine-sulfation plays an important role in extracellular protein-protein interactions. The protein tyrosine sulfation reaction is catalyzed by the Golgi-enzyme called the tyrosylprotein sulfotransferase (TPST). To date, no crystal structure is available for TPST. Detailed mechanism of protein tyrosine sulfation reaction has thus remained unclear. Here we present the first crystal structure of the human TPST isoform 2 (TPST2) complexed with a substrate peptide (C4P5Y3) derived from complement C4 and 3’-phosphoadenosine-5’-phosphate (PAP) at 1.9Å resolution. Structural and complementary mutational analyses revealed the molecular basis for catalysis being an SN2-like in-line displacement mechanism. TPST2 appeared to recognize the C4 peptide in a deep cleft by using a short parallel β-sheet type interaction, and the bound C4P5Y3 forms an L-shaped structure. Surprisingly, the mode of substrate peptide recognition observed in the TPST2 structure resembles that observed for the receptor type tyrosine kinases. PMID:23481380

  13. Identification of the Enterococcus faecalis Tyrosine Decarboxylase Operon Involved in Tyramine Production

    PubMed Central

    Connil, Nathalie; Le Breton, Yoann; Dousset, Xavier; Auffray, Yanick; Rincé, Alain; Prévost, Hervé

    2002-01-01

    Screening of a library of Enterococcus faecalis insertional mutants allowed isolation of a mutant affected in tyramine production. The growth of this mutant was similar to that of the wild-type E. faecalis JH2-2 strain in Maijala broth, whereas high-performance liquid chromatography analyses showed that tyramine production, which reached 1,000 μg ml−1 for the wild-type strain, was completely abolished. Genetic analysis of the insertion locus revealed a gene encoding a decarboxylase with similarity to eukaryotic tyrosine decarboxylases. Sequence analysis revealed a pyridoxal phosphate binding site, indicating that this enzyme belongs to the family of amino acid decarboxylases using this cofactor. Reverse transcription-PCR analyses demonstrated that the gene (tdc) encoding the putative tyrosine decarboxylase of E. faecalis JH2-2 is cotranscribed with the downstream gene encoding a putative tyrosine-tyramine antiporter and with the upstream tyrosyl-tRNA synthetase gene. This study is the first description of a tyrosine decarboxylase gene in prokaryotes. PMID:12089039

  14. Assays to measure the activation of membrane tyrosine kinase receptors: focus on cellular methods.

    PubMed

    Minor, Lisa K

    2003-09-01

    Many methods have been explored as means to measure the activation and inhibition of tyrosine kinase receptors, in vitro using the isolated kinase domain, and in living cells. Kinase activity has been measured in enzyme assays using a peptide substrate, but with different detection systems. These include the radioactive FlashPlate assay, the fluorescent resonance energy transfer (FRET) assay, the dissociation-enhance lanthanide fluorescence immunoassay (DELFIA) and other formats. These methods have successfully identified inhibitors of receptor activity. Cell-based assays have recently emerged to measure receptor activation and inhibition. When membrane tyrosine kinase receptors become activated, they increase their state of phosphorylation. This phosphorylation may lead to an increase in tyrosine kinase-specific activity. Methods have been developed that take advantage of these properties. These include measuring the ligand-stimulated total tyrosine phosphorylation of the receptor using a DELFIA or an ELISA assay, measuring ligand-stimulated enzyme activation of the receptor by quantifying enzyme activity, and dimerization of the activated receptor using bioluminescence resonance energy transfer (BRET). Although cell-based assays are still in their infancy, these techniques may prove a valuable addition to the receptor screening strategy.

  15. Response to pentagastrin after acute phenylalanine and tyrosine depletion in healthy men: a pilot study.

    PubMed Central

    Coupland, N; Zedkova, L; Sanghera, G; Leyton, M; Le Mellédo, J M

    2001-01-01

    OBJECTIVE: To assess the effects of the acute depletion of the catecholamine precursors phenylalanine and tyrosine on mood and pentagastrin-induced anxiety. DESIGN: Randomized, double-blind controlled multiple crossover study. SETTING: University department of psychiatry. PARTICIPANTS: 6 healthy male volunteers. INTERVENTIONS: 3 treatments were compared: pretreatment with a nutritionally balanced amino acid mixture, followed 5 hours later by a bolus injection of normal saline placebo; pretreatment with a balanced amino acid mixture, followed by a bolus injection of pentagastrin (0.6 microgram/kg); and pretreatment with an amino acid mixture without the catecholamine precursors phenylalanine or tyrosine, followed by pentagastrin (0.6 microgram/kg). OUTCOME MEASURES: Scores on the panic symptom scale, a visual analogue scale for anxiety, the Borg scale of respiratory exertion and the Profile of Mood States Elation-Depression Scale. RESULTS: Pentagastrin produced the expected increases in anxiety symptoms, but there was no significant or discernible influence of acute phenylalanine and tyrosine depletion on anxiety or mood. CONCLUSIONS: These pilot data do not support further study using the same design in healthy men. Under these study conditions, phenylalanine and tyrosine depletion may have larger effects on dopamine than noradrenaline. Alternative protocols to assess the role of catecholamines in mood and anxiety are proposed. PMID:11394194

  16. Role of Tyrosine Kinase Inhibitors in Indolent and Other Mature B-Cell Neoplasms

    PubMed Central

    Kutsch, Nadine; Marks, Reinhard; Ratei, Richard; Held, Thomas K; Schmidt-Hieber, Martin

    2015-01-01

    Targeting tyrosine kinases represents a highly specific treatment approach for different malignancies. This also includes non-Hodgkin lymphoma since it is well known that these enzymes are frequently involved in the lymphomagenesis. Hereby, tyrosine kinases might either be dysregulated intrinsically or be activated within signal transduction pathways leading to tumor survival and growth. Among others, Bruton’s tyrosine kinase (Btk) is of particular interest as a potential therapeutic target. Btk is stimulated by B-cell receptor signaling and activates different transcription factors such as nuclear factor κB. The Btk inhibitor ibrutinib has been approved for the treatment of chronic lymphocytic leukemia and mantle-cell lymphoma recently. Numerous clinical trials evaluating this agent in different combinations (eg, with rituximab or classical chemotherapeutic agents) as a treatment option for aggressive and indolent lymphoma are under way. Here, we summarize the role of tyrosine kinase inhibitors in the treatment of indolent and other non-Hodgkin lymphomas (eg, mantle-cell lymphoma). PMID:26327780

  17. Ecology drives the distribution of specialized tyrosine metabolism modules in fungi.

    PubMed

    Greene, George H; McGary, Kriston L; Rokas, Antonis; Slot, Jason C

    2014-01-01

    Gene clusters encoding accessory or environmentally specialized metabolic pathways likely play a significant role in the evolution of fungal genomes. Two such gene clusters encoding enzymes associated with the tyrosine metabolism pathway (KEGG #00350) have been identified in the filamentous fungus Aspergillus fumigatus. The l-tyrosine degradation (TD) gene cluster encodes a functional module that facilitates breakdown of the phenolic amino acid, l-tyrosine through a homogentisate intermediate, but is also involved in the production of pyomelanin, a fungal pathogenicity factor. The gentisate catabolism (GC) gene cluster encodes a functional module likely involved in phenolic compound degradation, which may enable metabolism of biphenolic stilbenes in multiple lineages. Our investigation of the evolution of the TD and GC gene clusters in 214 fungal genomes revealed spotty distributions partially shaped by gene cluster loss and horizontal gene transfer (HGT). Specifically, a TD gene cluster shows evidence of HGT between the extremophilic, melanized fungi Exophiala dermatitidis and Baudoinia compniacensis, and a GC gene cluster shows evidence of HGT between Sordariomycete and Dothideomycete grass pathogens. These results suggest that the distribution of specialized tyrosine metabolism modules is influenced by both the ecology and phylogeny of fungal species. PMID:24391152

  18. IN VITRO CARDIOTOXICITY OF AIR POLLUTION PARTICLES: ROLE OF BIOAVAILABLE CONSTITUENTS, OXIDATIVE STRESS AND TYROSINE PHOSPHORYLATION

    EPA Science Inventory

    IN VITRO CARDIOTOXICITY OF AIR POLLUTION PARTICLES: ROLE OF BIOAVAILABLE CONSTITUENTS, OXIDATIVE STRESS AND TYROSINE PHOSPHORYLATION.

    T. L. Knuckles1 R. Jaskot2, J. Richards2, and K.Dreher2.
    1Department of Molecular and Biomedical Sciences, College of Veterinary Medicin...

  19. Seleninate in Place of Phosphate: Irreversible Inhibition of Protein Tyrosine Phosphatases

    SciTech Connect

    Abdo, Mohannad; Liu, Sijiu; Zhou, Bo; Walls, Chad D.; Wu, Li; Knapp, Spencer; Zhang, Zhong-Yin

    2009-02-16

    A homotyrosine based seleninic acid irreversibly inhibits protein tyrosine phosphatases by forming a covalent selenosulfide linkage with the active site cysteine sulfhydryl specifically. The details of the event are revealed by model synthetic studies and by kinetic, mass spectrometric, and crystallographic characterization.

  20. Tyrosine requirement during the rapid catch-up growth phase of recovery from severe childhood undernutrition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The requirement for aromatic amino acids, during the rapid catch-up in weight phase of recovery from severe childhood under nutrition (SCU) is not clearly established. As a first step, the present study aimed to estimate the tyrosine requirement of children with SCU during the catch-up growth phase ...

  1. Standard enthalpies of formation for glycyl-tyrosine and products of its dissociation in aqueous solutions

    NASA Astrophysics Data System (ADS)

    Kochergina, L. A.; Badelin, V. G.; Krutova, O. N.; Volkov, A. V.; Damrina, K. V.

    2015-07-01

    The enthalpies of solution of crystalline glycyl-tyrosine in water and potassium hydroxide aqueous solutions are determined at 298.15 K by means of direct calorimetry. Standard enthalpies of formation for dipeptide and its products of dissociation in an aqueous solution are calculated.

  2. Regulation of the Src family tyrosine kinase Blk through E6AP-mediated ubiquitination

    PubMed Central

    Oda, Hideaki; Kumar, Sushant; Howley, Peter M.

    1999-01-01

    The Src family of nonreceptor tyrosine kinases are important regulators of a variety of cellular processes, including cytoskeletal organization, cell–cell contact, and cell–matrix adhesion. Activation of Src family kinases also can induce DNA synthesis and cellular proliferation; therefore, tight regulation of their kinase activities is important for the cell to maintain proliferative control. Posttranslational phosphorylation and dephosphorylation are recognized as the principle modifications by which the activities of the Src family of tyrosine kinases are regulated. We have discovered that this family of kinases also is regulated by ubiquitin-mediated proteolysis. Studies aimed at the identification of cellular targets for E6AP, an E3 ubiquitin protein ligase involved in ubquitin-mediated degradation, led us to the identification of members of the Src family kinases as potential substrates for E6AP. We have found that E6AP can bind to several of the Src family tyrosine kinases. Here we show that activated Blk is preferentially degraded by the ubiquitin–proteasome pathway and that its ubiquitination is mediated by E6AP. Identification of members of the Src tyrosine kinase family as substrates of the E6AP ubiquitin-protein ligase implicates a role for the ubiquitin pathway in regulating the activities of individual members of this important family of signaling molecules. PMID:10449731

  3. Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases.

    PubMed

    Chen, Ying-Nan P; LaMarche, Matthew J; Chan, Ho Man; Fekkes, Peter; Garcia-Fortanet, Jorge; Acker, Michael G; Antonakos, Brandon; Chen, Christine Hiu-Tung; Chen, Zhouliang; Cooke, Vesselina G; Dobson, Jason R; Deng, Zhan; Fei, Feng; Firestone, Brant; Fodor, Michelle; Fridrich, Cary; Gao, Hui; Grunenfelder, Denise; Hao, Huai-Xiang; Jacob, Jaison; Ho, Samuel; Hsiao, Kathy; Kang, Zhao B; Karki, Rajesh; Kato, Mitsunori; Larrow, Jay; La Bonte, Laura R; Lenoir, Francois; Liu, Gang; Liu, Shumei; Majumdar, Dyuti; Meyer, Matthew J; Palermo, Mark; Perez, Lawrence; Pu, Minying; Price, Edmund; Quinn, Christopher; Shakya, Subarna; Shultz, Michael D; Slisz, Joanna; Venkatesan, Kavitha; Wang, Ping; Warmuth, Markus; Williams, Sarah; Yang, Guizhi; Yuan, Jing; Zhang, Ji-Hu; Zhu, Ping; Ramsey, Timothy; Keen, Nicholas J; Sellers, William R; Stams, Travis; Fortin, Pascal D

    2016-07-01

    The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS–ERK signalling pathway. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy. Here we report the discovery of a highly potent (IC50 = 0.071 μM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS–ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers. PMID:27362227

  4. Ecology Drives the Distribution of Specialized Tyrosine Metabolism Modules in Fungi

    PubMed Central

    Greene, George H.; McGary, Kriston L.; Rokas, Antonis; Slot, Jason C.

    2014-01-01

    Gene clusters encoding accessory or environmentally specialized metabolic pathways likely play a significant role in the evolution of fungal genomes. Two such gene clusters encoding enzymes associated with the tyrosine metabolism pathway (KEGG #00350) have been identified in the filamentous fungus Aspergillus fumigatus. The l-tyrosine degradation (TD) gene cluster encodes a functional module that facilitates breakdown of the phenolic amino acid, l-tyrosine through a homogentisate intermediate, but is also involved in the production of pyomelanin, a fungal pathogenicity factor. The gentisate catabolism (GC) gene cluster encodes a functional module likely involved in phenolic compound degradation, which may enable metabolism of biphenolic stilbenes in multiple lineages. Our investigation of the evolution of the TD and GC gene clusters in 214 fungal genomes revealed spotty distributions partially shaped by gene cluster loss and horizontal gene transfer (HGT). Specifically, a TD gene cluster shows evidence of HGT between the extremophilic, melanized fungi Exophiala dermatitidis and Baudoinia compniacensis, and a GC gene cluster shows evidence of HGT between Sordariomycete and Dothideomycete grass pathogens. These results suggest that the distribution of specialized tyrosine metabolism modules is influenced by both the ecology and phylogeny of fungal species. PMID:24391152

  5. Deciphering the Role of Tyrosine Sulfation in Xanthomonas oryzae pv. oryzae Using Shotgun Proteomic Analysis

    PubMed Central

    Park, Hye-Jee; Park, Chang-Jin; Bae, Nahee; Han, Sang-Wook

    2016-01-01

    A bacterial tyrosine sulfotransferase, RaxST, is required for activation of rice XA21-mediated immunity, and it catalyzes sulfation of tyrosine residues of Omp1X and RaxX in Xanthomonas oryzae pv. oryzae, a causal agent of bacterial blight in rice. Although RaxST is biochemically well-characterized, biological functions of tyrosine sulfation have not been fully elucidated. We compared protein expression patterns between the wildtype and a raxST knockout mutant using shotgun proteomic analysis. Forty nine proteins displayed a more than 1.5-fold difference in their expression between the wildtype and the mutant strains. Clusters of orthologous groups analysis revealed that proteins involved in cell motility were most abundant, and phenotypic observation also showed that the twitching motility of the mutant was dramatically changed. These results indicate that tyrosine sulfation by RaxST is essential for Xoo movement, and they provide new insights into the biological roles of RaxST in cellular processes. PMID:27298602

  6. Tyrosine phosphorylation of measles virus nucleocapsid protein in persistently infected neuroblastoma cells.

    PubMed Central

    Segev, Y; Ofir, R; Salzberg, S; Heller, A; Weinstein, Y; Isakov, N; Udem, S; Wolfson, M; Rager-Zisman, B

    1995-01-01

    Subacute sclerosing panencephalitis is a slowly progressing fatal human disease of the central nervous system which is a delayed sequel of measles virus (MV) infection. A typical pathological feature of this disease is the presence of viral ribonucleocapsid structures in the form of inclusion bodies and the absence of infectious virus or budding viral particles. The mechanisms governing the establishment and maintenance of a persistent MV infection in brain cells are still largely unknown. To understand the mechanisms underlying MV persistence in neuronal cells, a tissue culture model was studied. Clone NS20Y/MS of the murine neuroblastoma C1300 persistently infected with the wild-type Edmonston strain of MV secretes relatively high levels of alpha/beta interferon (IFN). As shown previously, treatment of the persistently infected cultures with anti-IFN serum converted the persistent state into a productive infection indicated by the appearance of multinucleated giant cells. In this study, we have investigated whether alpha/beta IFN produced by NS20Y/MS cells activates cellular protein tyrosine kinases which will induce tyrosine phosphorylating activity specific to virus-infected cells. We present data to show augmented protein tyrosine kinase activity in the persistently infected cells. We demonstrate that the MV N protein is phosphorylated on tyrosine in addition to serine and threonine in the persistent state but not in NS20Y cells acutely infected with MV. PMID:7884896

  7. Tyrosine phosphorylation of measles virus nucleocapsid protein in persistently infected neuroblastoma cells.

    PubMed

    Segev, Y; Ofir, R; Salzberg, S; Heller, A; Weinstein, Y; Isakov, N; Udem, S; Wolfson, M; Rager-Zisman, B

    1995-04-01

    Subacute sclerosing panencephalitis is a slowly progressing fatal human disease of the central nervous system which is a delayed sequel of measles virus (MV) infection. A typical pathological feature of this disease is the presence of viral ribonucleocapsid structures in the form of inclusion bodies and the absence of infectious virus or budding viral particles. The mechanisms governing the establishment and maintenance of a persistent MV infection in brain cells are still largely unknown. To understand the mechanisms underlying MV persistence in neuronal cells, a tissue culture model was studied. Clone NS20Y/MS of the murine neuroblastoma C1300 persistently infected with the wild-type Edmonston strain of MV secretes relatively high levels of alpha/beta interferon (IFN). As shown previously, treatment of the persistently infected cultures with anti-IFN serum converted the persistent state into a productive infection indicated by the appearance of multinucleated giant cells. In this study, we have investigated whether alpha/beta IFN produced by NS20Y/MS cells activates cellular protein tyrosine kinases which will induce tyrosine phosphorylating activity specific to virus-infected cells. We present data to show augmented protein tyrosine kinase activity in the persistently infected cells. We demonstrate that the MV N protein is phosphorylated on tyrosine in addition to serine and threonine in the persistent state but not in NS20Y cells acutely infected with MV.

  8. Quantitative profiling of spreading-coupled protein tyrosine phosphorylation in migratory cells.

    PubMed

    Xie, Yajun; Wang, Jinlong; Zhang, Yuanya; Liu, Xiaofei; Wang, Xiaorong; Liu, Kehui; Huang, Xiahe; Wang, Yingchun

    2016-01-01

    Protein tyrosine phosphorylation is an important mechanism that regulates cytoskeleton reorganization and cell spreading of migratory cells. A number of cytoskeletal proteins are known to be tyrosine phosphorylated (pY) in different cellular processes. However, the profile of pY proteins during different stages of cell spreading has not been available. Using immunoafffinity enrichment of pY proteins coupled with label free quantitative proteomics, we quantitatively identified 447 pY proteins in the migratory ECV-304 cells at the early spreading (adhesion) and the active spreading stages. We found that pY levels of the majority of the quantified proteins were significantly increased in the active spreading stage compared with the early spreading stage, suggesting that active cell spreading is concomitant with extra tyrosine phosphorylation. The major categories of proteins impacted by tyrosine phosphorylation are involved in cytoskeleton and focal adhesion regulation, protein translation and degradation. Our findings, for the first time, dissect the cell spreading-specific pY signals from the adhesion induced pY signals, and provide a valuable resource for the future mechanistic research regarding the regulation of cell spreading. PMID:27554326

  9. Two Closely Spaced Tyrosines Regulate NFAT Signaling in B Cells via Syk Association with Vav▿

    PubMed Central

    Chen, Chih-Hong; Martin, Victoria A.; Gorenstein, Nina M.; Geahlen, Robert L.; Post, Carol Beth

    2011-01-01

    Activated Syk, an essential tyrosine kinase in B cell signaling, interacts with Vav guanine nucleotide exchange factors and regulates Vav activity through tyrosine phosphorylation. The Vav SH2 domain binds Syk linker B by an unusual recognition of two closely spaced Syk tyrosines: Y342 and Y346. The binding affinity is highest when both Y342 and Y346 are phosphorylated. An investigation in B cells of the dependence of Vav phosphorylation and NFAT activation on phosphorylation of Y342 and Y346 finds that cellular response levels match the relative binding affinities of the Vav1 SH2 domain for singly and doubly phosphorylated linker B peptides. This key result suggests that the uncommon recognition determinant of these two closely spaced tyrosines is a limiting factor in signaling. Interestingly, differences in affinities for binding singly and doubly phosphorylated peptides are reflected in the on rate, not the off rate. Such a control mechanism would be highly effective for regulating binding among competing Syk binding partners. The nuclear magnetic resonance (NMR) structure of Vav1 SH2 in complex with a doubly phosphorylated linker B peptide reveals diverse conformations associated with the unusual SH2 recognition of two phosphotyrosines. NMR relaxation indicates compensatory changes in loop fluctuations upon binding, with implications for nonphosphotyrosine interactions of Vav1 SH2. PMID:21606197

  10. Study of Interaction Between Tryptophan, Tyrosine, and Phenylalanine Separately with Silver Nanoparticles by Fluorescence Quenching Method

    NASA Astrophysics Data System (ADS)

    Roy, S.; Das, T. K.

    2015-09-01

    Using the spectroscopic method, the individual interaction of the three biochemically important amino acids, which are constituents of protein, namely, tryptophan, tyrosine, and phenylalanine with biologically synthesized silver nanoparticles has been investigated. The obtained UV-Vis spectra show the formation of ground-state complexes between tryptophan, tyrosine, and phenylalanine with silver nanoparticles. Silver nanoparticles possess the ability to quench the intrinsic fluorescence of the aforesaid amino acids by a dynamic quenching process. The binding constant, number of binding sites, and corresponding thermodynamic parameters (Δ H, Δ S, and Δ G) based on the interaction system were calculated for 293, 303, and 313 K. In the case of tryptophan and phenylalanine, with increase in temperature, the binding constant K was found to decrease; conversely, it was found to increase with increase in temperature in the case of tyrosine. The thermodynamic results revealed that the binding process was spontaneous; hydrogen bonding and van der Waals interaction were the predominant forces responsible for the complex stabilization in the case of tryptophan and phenylalanine, respectively, whereas in the case of tyrosine, hydrophobic interaction was the sole force conferring stability. Moreover, the Förster non-radiation energy transfer theory has been applied to calculate the average binding distance among the above amino acids and silver nanoparticles. The results show a binding distance of <7 nm, which ensures that energy transfer does occur between the said amino acids and silver nanoparticles.

  11. Perturbations of tyrosine metabolism promote the indolepyruvate pathway via tryptophan in host and microbiome.

    PubMed

    Gertsman, Ilya; Gangoiti, Jon A; Nyhan, William L; Barshop, Bruce A

    2015-03-01

    The drug nitisinone (NTBC) is used to treat tyrosinemia type I, and more recently has been also used for the treatment of another disorder of tyrosine metabolism, alkaptonuria. While studying the dose effects of NTBC treatment on alkaptonuria, untargeted metabolomics revealed perturbations in a completely separate pathway, that of tryptophan metabolism. Significant elevations in several indolic compounds associated with the indolepyruvate pathway of tryptophan metabolism were present in NTBC-treated patient sera and correlated with elevations of an intermediate of tyrosine metabolism. Indolic compounds of this pathway have long been associated with commensal bacterial and plant metabolism. These exogenous sources of indoles have been more recently implicated in affecting mammalian cell function and disease. We studied the correlation of these indolic compounds in other disorders of tyrosine metabolism including tyrosinemia types I and II as well as transient tyrosinemia, and demonstrated that 4-hydroxyphenylpyruvate (4-HPP) was directly responsible for the promotion of this pathway. We then investigated the regulation of the indolepyruvate pathway and the role of 4-HPP further in both mammalian cells and intestinal microbial cultures. We demonstrated that several of the indolic products, including indolepyruvate and indolelactate, were in fact generated by human cell metabolism, while the downstream indole metabolite, indolecarboxaldehyde, was produced exclusively by microbial cultures of human gut flora. This study describes a symbiotic perturbation in host and microbiome tryptophan metabolism in response to elevations related to defects of tyrosine metabolism and concomitant drug treatment.

  12. Protein-tyrosine-phosphatase SHPTP2 is a required positive effector for insulin downstream signaling.

    PubMed Central

    Yamauchi, K; Milarski, K L; Saltiel, A R; Pessin, J E

    1995-01-01

    SHPTP2 is a ubiquitously expressed tyrosine-specific protein phosphatase that contains two amino-terminal Src homology 2 (SH2) domains responsible for its association with tyrosine-phosphorylated proteins. In this study, expression of dominant interfering mutants of SHPTP2 was found to inhibit insulin stimulation of c-fos reporter gene expression and activation of the 42-kDa (Erk2) and 44-kDa (Erk1) mitogen-activated protein kinases. Cotransfection of dominant interfering SHPTP2 mutants with v-Ras or Grb2 indicated that SHPTP2 regulated insulin signaling either upstream of or in parallel to Ras function. Furthermore, phosphotyrosine blotting and immunoprecipitation identified the 125-kDa focal adhesion kinase (pp125FAK) as a substrate for insulin-dependent tyrosine dephosphorylation. These data demonstrate that SHPTP2 functions as a positive regulator of insulin action and that insulin signaling results in the dephosphorylation of tyrosine-phosphorylated pp125FAK. Images Fig. 2 Fig. 4 Fig. 5 PMID:7531337

  13. Tyrosine Phosphorylation Modulates the Vascular Responses of Mesenteric Arteries from Human Colorectal Tumors

    PubMed Central

    Ferrero, Eduardo; Mauricio, María Dolores; Granado, Miriam; García-Villar, Oscar; Aldasoro, Martín; Vila, José María; Hidalgo, Manuel; Ferrero, Jorge Luis; Fernández, Nuria; García-Villalón, Ángel Luis

    2013-01-01

    The aim of this study was to analyze whether tyrosine phosphorylation in tumoral arteries may modulate their vascular response. To do this, mesenteric arteries supplying blood flow to colorectal tumors or to normal intestine were obtained during surgery and prepared for isometric tension recording in an organ bath. Increasing tyrosine phosphorylation with the phosphatase inhibitor, sodium orthovanadate produced arterial contraction which was lower in tumoral than in control arteries, whereas it reduced the contraction to noradrenaline in tumoral but not in control arteries and reduced the relaxation to bradykinin in control but not in tumoral arteries. Protein expression of VEGF-A and of the VEGF receptor FLT1 was similar in control and tumoral arteries, but expression of the VEGF receptor KDR was increased in tumoral compared with control arteries. This suggests that tyrosine phosphorylation may produce inhibition of the contraction in tumoral mesenteric arteries, which may increase blood flow to the tumor when tyrosine phosphorylation is increased by stimulation of VEGF receptors. PMID:24324963

  14. CDPKs are dual-specificity protein kinases and tyrosine autophosphorylation attenuates kinase activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Calcium-dependent protein kinases (CDPKs or CPKs) are classified as serine/threonine protein kinases but we made the surprising observation that soybean CDPK' and several Arabidopsis isoforms (AtCPK4 and AtCPK34) could also autophosphorylate on tyrosine residues. In studies with His6-GmCDPK', we ide...

  15. Ultrafast Spectroscopic and AB Initio Computational Investigations on Solvation Dynamics of Neutral and Deprotonated Tyrosine.

    NASA Astrophysics Data System (ADS)

    Fujiwara, Takashige; Zgierski, Marek Z.

    2015-06-01

    We have studied one of the aromatic amino acids, tyrosine, regarding its photophysical properties in various solvent conditions by using a femtosecond fluorescence up-conversion technique and high-level TDDFT and CC2 computations. In this talk, profound details not only on ultrafast solvation dynamics on a neutral tyrosine in various solvents, but also on the excited-state dynamics for a single- (or doubly-) deprotonated tyrosine under various pH solutions will be presented. In high basicity, a tyrosine shows different absorption/emission spectra, and a total spectrum consists of a combination of these individual spectra that depend on the pH of the solution. The time scale of acid--base equilibrium is essential in solvation dynamics; whereas the protonation is simply controlled by diffusion, the de-protonation is considered to be slow process such that acid--base equilibrium may not be reached in the short-lived excited state after photo-excitation. Experimental and computational approaches taken and insights obtained in this concerted work will be described.

  16. Effects of modification of the tyrosine residues of bacteriorhodopsin with tetranitromethane.

    PubMed Central

    Campos-Cavieres, M; Moore, T A; Perham, R N

    1979-01-01

    Treatment of the purple membrane of Halobacterium halobium with tetranitromethane led to modification of tyrosine residues. Modification of more than 3-4 tyrosine residues per bacteriorhodopsin monomer caused a decrease in the light-induced proton-pumping ability of purple membrane in synthetic lipid vesicles, loss of the sharp X-ray-diffraction patterns characteristic of the crystal lattice, loss of the absorbance maximum at 560 nm, and change in the buoyant density of the membrane. No modification of lipid was detected. These changes were interpreted as a gradual denaturation of the protein component such that when 8-9 tyrosine residues are modified, no proton pumping is observed. Modification of less than 3-4 tyrosine residues with tetranitromethane caused an increse in light-induced proton pumping. It was possible to generate partly modified purple membrane which had completely lost the property of diffracting X-rays into the sharp pattern observed with native purple membrane, but which still retained the ability to pump protons in a vectorial manner. Retention of crystal lattice is not essential for proton pumping. Images Fig. 3. PMID:475758

  17. Tyrosine kinase inhibitors target cancer stem cells in renal cell cancer.

    PubMed

    Czarnecka, Anna M; Solarek, Wojciech; Kornakiewicz, Anna; Szczylik, Cezary

    2016-03-01

    This study was designed to analyze the impact of multi-targeted tyrosine kinase inhibitors on the cancer stem cell subpopulation in renal cell cancer. The second objective was to evaluate the effect of tumor growth inhibition related to a tumor niche factor - oxygen deprivation - as hypoxia develops along with the anti-angiogenic activity of tyrosine kinase inhibitors in renal tumors. Cells were treated with tyrosine kinase inhibitors, sunitinib, sorafenib and axitinib, in 2D and 3D culture conditions. Cell proliferation along with drug toxicity were evaluated. It was shown that the proliferation rate of cancer stem cells was decreased by the tyrosine kinase inhibitors. The efficacy of the growth inhibition was limited by hypoxic conditions and 3D intratumoral cell-cell interactions. We conclude that understanding the complex molecular interaction feedback loops between differentiated cancer cells, cancer stem cells and the tumor microenvironment in 3D culture should aid the identification of novel treatment targets and to evalute the efficacy of renal cancer therapies. Cell-cell interaction may represent a critical microenvironmental factor regulating cancer stem cell self-renewal potential, enhancing the stem cell phenotype and limiting drug toxicity. At the same time the role of hypoxia in renal cancer stem cell biology is also significant.

  18. Fluorescence lifetimes of tyrosine residues in cytochrome c'' as local probes to study protein unfolding.

    PubMed

    Noronha, Melinda; Santos, Raquel; Paci, Emanuele; Santos, Helena; Maçanita, António L

    2009-04-01

    Time-resolved fluorescence spectroscopy was used to show that multiple tyrosine residues of a protein can serve as localized probes of structural changes during thermal unfolding. Cytochrome c'' from Methylophilus methylotrophus, which has four tyrosine residues, was chosen as a model protein. The procedure involved, first, the assignment of the experimental decay times to the tyrosine residues, followed by the interpretation of the changes in the decay times and pre-exponential coefficients with temperature. We found that the fluorescence decays of cytochrome c'' are double-exponential from 23 to 80 degrees C, with decay times much shorter than those of the parent compound N-acetyl-tyrosinamide; this quenching was ascribed to dipole-dipole energy transfer from the tyrosine residues to the heme. The tyrosine-heme distances (R) and theoretical decay times, tau(comp), were estimated for each tyrosine residue. The analysis of the simulated decay generated with tau(comp), showed that a double-exponential fit is sufficient to describe the four decay times with two pre-exponential coefficients close to values observed from the experimental decay. Therefore, the decay times at 23 degrees C could be assigned to the individual tyrosine residues as tau(1) to Tyr-10 and Tyr-23 (at 20.3 A) and tau(2) to Tyr-12 and Tyr-115 (at 12-14 A). On the basis of this assignment and MD simulations, the temperature dependence of the decay times and pre-exponential coefficients suggest that upon unfolding, Tyr-12 is displaced from the heme, with loss of the structure of alpha-helix I. Moreover, Tyr-115 remains close to the heme and the structure in this region of the protein is not altered significantly. Altogether the data support the view that the protein core, comprising the heme and the four alpha-helices II to V, is clearly more stable than the remaining region that includes alpha-helix I and the loop between residues 19-27.

  19. Endothelial Bmx tyrosine kinase activity is essential for myocardial hypertrophy and remodeling.

    PubMed

    Holopainen, Tanja; Räsänen, Markus; Anisimov, Andrey; Tuomainen, Tomi; Zheng, Wei; Tvorogov, Denis; Hulmi, Juha J; Andersson, Leif C; Cenni, Bruno; Tavi, Pasi; Mervaala, Eero; Kivelä, Riikka; Alitalo, Kari

    2015-10-20

    Cardiac hypertrophy accompanies many forms of heart disease, including ischemic disease, hypertension, heart failure, and valvular disease, and it is a strong predictor of increased cardiovascular morbidity and mortality. Deletion of bone marrow kinase in chromosome X (Bmx), an arterial nonreceptor tyrosine kinase, has been shown to inhibit cardiac hypertrophy in mice. This finding raised the possibility of therapeutic use of Bmx tyrosine kinase inhibitors, which we have addressed here by analyzing cardiac hypertrophy in gene-targeted mice deficient in Bmx tyrosine kinase activity. We found that angiotensin II (Ang II)-induced cardiac hypertrophy is significantly reduced in mice deficient in Bmx and in mice with inactivated Bmx tyrosine kinase compared with WT mice. Genome-wide transcriptomic profiling showed that Bmx inactivation suppresses myocardial expression of genes related to Ang II-induced inflammatory and extracellular matrix responses whereas expression of RNAs encoding mitochondrial proteins after Ang II administration was maintained in Bmx-inactivated hearts. Very little or no Bmx mRNA was expressed in human cardiomyocytes whereas human cardiac endothelial cells expressed abundant amounts. Ang II stimulation of endothelial cells increased Bmx phosphorylation, and Bmx gene silencing inhibited downstream STAT3 signaling, which has been implicated in cardiac hypertrophy. Furthermore, activation of the mechanistic target of rapamycin complex 1 pathway by Ang II treatment was decreased in the Bmx-deficient hearts. Our results demonstrate that inhibition of the cross-talk between endothelial cells and cardiomyocytes by Bmx inactivation suppresses Ang II-induced signals for cardiac hypertrophy. These results suggest that the endothelial Bmx tyrosine kinase could provide a target to attenuate the development of cardiac hypertrophy. PMID:26430242

  20. Endothelial Bmx tyrosine kinase activity is essential for myocardial hypertrophy and remodeling

    PubMed Central

    Holopainen, Tanja; Räsänen, Markus; Anisimov, Andrey; Tuomainen, Tomi; Zheng, Wei; Tvorogov, Denis; Hulmi, Juha J.; Andersson, Leif C.; Cenni, Bruno; Tavi, Pasi; Mervaala, Eero; Kivelä, Riikka; Alitalo, Kari

    2015-01-01

    Cardiac hypertrophy accompanies many forms of heart disease, including ischemic disease, hypertension, heart failure, and valvular disease, and it is a strong predictor of increased cardiovascular morbidity and mortality. Deletion of bone marrow kinase in chromosome X (Bmx), an arterial nonreceptor tyrosine kinase, has been shown to inhibit cardiac hypertrophy in mice. This finding raised the possibility of therapeutic use of Bmx tyrosine kinase inhibitors, which we have addressed here by analyzing cardiac hypertrophy in gene-targeted mice deficient in Bmx tyrosine kinase activity. We found that angiotensin II (Ang II)-induced cardiac hypertrophy is significantly reduced in mice deficient in Bmx and in mice with inactivated Bmx tyrosine kinase compared with WT mice. Genome-wide transcriptomic profiling showed that Bmx inactivation suppresses myocardial expression of genes related to Ang II-induced inflammatory and extracellular matrix responses whereas expression of RNAs encoding mitochondrial proteins after Ang II administration was maintained in Bmx-inactivated hearts. Very little or no Bmx mRNA was expressed in human cardiomyocytes whereas human cardiac endothelial cells expressed abundant amounts. Ang II stimulation of endothelial cells increased Bmx phosphorylation, and Bmx gene silencing inhibited downstream STAT3 signaling, which has been implicated in cardiac hypertrophy. Furthermore, activation of the mechanistic target of rapamycin complex 1 pathway by Ang II treatment was decreased in the Bmx-deficient hearts. Our results demonstrate that inhibition of the cross-talk between endothelial cells and cardiomyocytes by Bmx inactivation suppresses Ang II-induced signals for cardiac hypertrophy. These results suggest that the endothelial Bmx tyrosine kinase could provide a target to attenuate the development of cardiac hypertrophy. PMID:26430242

  1. Alterations in connexin 43 during diabetic cardiomyopathy: competition of tyrosine nitration versus phosphorylation

    PubMed Central

    COOK, Angela C.; SCHANBACHER, Brandon L.; BAUER, John Anthony

    2014-01-01

    Objective Cardiac conduction abnormalities are observed early in the progression of Type I diabetes, but the mechanism(s) involved are undefined. Connexin 43, a critical component of ventricular gap junctions, depends on tyrosine phosphorylation status to modulate channel conductance - alterations in connexin 43 content, distributions, and/or phosphorylation status may be involved in cardiac rhythm disturbances. We tested the hypothesis that cardiac content/distribution of connexin 43 are altered in a rat model of Type I diabetic cardiomyopathy, investigating a mechanistic role for tyrosine. Methods We conducted electrocardiographic analyses during the progression of diabetic cardiomyopathy in rats dosed with streptozotocin (65mg/kg), at 3, 7, and 35 days post-induction of diabetes. Following functional analyses, we conducted immunohistochemical and immunoprecipitation studies to assess alterations in connexin 43. Results We observed significant evidence of ventricular conduction abnormalities (QRS complex, Q-T interval) as early as 7 days post-streptozotocin, persisting throughout the study. Connexin 43 levels were increased 7d post- streptozotocin and remained elevated throughout the study. Connexin 40 content was unchanged relative to controls throughout the study. Changes in Connexin 43 distribution were also observed; connexin 43 staining was dispersed from myocyte short axis junctions. Connexin 43 tyrosine phosphorylation declined during the progression of diabetes, with concurrent increases in tyrosine nitration. Conclusions These data suggest that alterations in connexin 43 content and distribution occur during experimental diabetes and likely contribute to alterations in cardiac function, and that oxidative modification of tyrosine-mediated signaling may play a mechanistic role. PMID:24796789

  2. K depletion increases protein tyrosine kinase-mediated phosphorylation of ROMK

    PubMed Central

    Lin, Dao-Hong; Sterling, Hyacinth; Lerea, Kenneth M.; Welling, Paul; Jin, Lianhong; Giebisch, Gerhard; Wang, Wen-Hui

    2010-01-01

    We purified Histagged ROMK1 and carried out in vitro phosphorylation assays with 32P-radiolabeled ATP to determine whether ROMK1 protein is a substrate for PTK. Addition of active c-Src and [32P]ATP to the purified ROMK1 protein resulted in the phosphorylation of the ROMK1 protein. However, c-Src did not phosphorylate R1Y337A in which tyrosine residue 337 was mutated to alanine. Furthermore, phosphopeptide mapping identified two phosphopeptides from the trypsin-digested ROMK1 protein. In contrast, no phosphorylated peptide has been found in the trypsin-digested R1Y337A protein. This suggested that two phosphorylated peptides might contain the same tyrosine residue. Also, addition of c-Src and [32P]ATP phosphorylated the synthesized peptide corresponding to amino acid sequence 333–362 of the COOH terminus of ROMK1. We then examined the effect of dietary K intake on the tyrosine-phosphorylated ROMK level. Although the ROMK channels pulled down by immunoprecipitation with ROMK antibody were the same from rats on a K-deficient diet or on a high-K diet, more ROMK channels were phosphorylated by PTK in rats on a K-deficient diet than those on a high-K diet. We conclude that ROMK1 can be phosphorylated by PTK and that tyrosine residue 337 is the key site for the phosphorylation. Also, the tyrosine phosphorylation of ROMK is modulated by dietary K intake. This strongly suggests that PTK is an important member of the aldosterone-independent signal transduction pathway for regulating renal K secretion. PMID:12217858

  3. Review of L-tyrosine confirming its safe human use as an adjuvant.

    PubMed

    Baldrick, Paul; Richardson, Derek; Wheeler, Alan W

    2002-01-01

    Although there is a long history of exposure to allergy vaccines containing L-tyrosine, there has been no central publication reviewing its adjuvant properties in animal and human studies together with an assessment of its safe use. This paper summarizes a range of investigational data (unpublished) available to the authors as well as published literature reports. An array of in vitro and in vivo studies showed that L-tyrosine has ideal adjuvant properties, comprising a high adsorptive power for proteins, enhancement of IgG antibody induction with no stimulatory effect on IgE antibody level and action as a short-term depot adjuvant, delaying the bioavailability of allergenic materials rather than directly influencing immunocompetent cells. A series of preclinical safety investigations comprised single-dose parenteral studies in the mouse and rat, repeat-dose parenteral toxicity studies over 28 days in the rat and dog (up to 25 mg kg(-1) day(-1)) plus genotoxicity and local tolerance studies. No signs of toxicity or genotoxicity were seen; repeat-dose toxicity studies showed expected white cell and spleen weight immunostimulatory effects; local-dose site reactions were also seen and were confirmed in local tolerance studies. Findings from a range of clinical studies using allergy vaccines containing L-tyrosine reflected the lack of toxicity seen in animal work and showed evidence of enhanced immunostimulatory activity. Local injection site reactions (a common response to any form of clinical vaccination) in these studies were likely to be due to the presence of L-tyrosine per se. The lack of findings of toxicological concern found during this review supports the hypothesis that L-tyrosine is a safe adjuvant for human use.

  4. ACK1/TNK2 tyrosine kinase: molecular signaling and evolving role in cancers.

    PubMed

    Mahajan, K; Mahajan, N P

    2015-08-01

    Deregulated tyrosine kinase signaling alters cellular homeostasis to drive cancer progression. The emergence of a non-receptor tyrosine kinase (non-RTK), ACK1 (also known as activated Cdc42-associated kinase 1 or TNK2) as an oncogenic kinase, has uncovered novel mechanisms by which tyrosine kinase signaling promotes cancer progression. Although early studies focused on ACK1 as a cytosolic effector of activated transmembrane RTKs, wherein it shuttles between the cytosol and the nucleus to rapidly transduce extracellular signals from the RTKs to the intracellular effectors, recent data unfold a new aspect of its functionality as an epigenetic regulator. ACK1 interacts with the estrogen receptor (ER)/histone demethylase KDM3A (JHDM2a) complex, which modifies KDM3A by tyrosine phosphorylation to regulate the transcriptional outcome at HOXA1 locus to promote the growth of tamoxifen-resistant breast cancer. It is also well established that ACK1 regulates the activity of androgen receptor (AR) by tyrosine phosphorylation to fuel the growth of hormone-refractory prostate cancers. Further, recent explosion in genomic sequencing has revealed recurrent ACK1 gene amplification and somatic mutations in a variety of human malignancies, providing a molecular basis for its role in neoplastic transformation. In this review, we will discuss the various facets of ACK1 signaling, including its newly uncovered epigenetic regulator function, which enables cells to bypass the blockade to major survival pathways to promote resistance to standard cancer treatments. Not surprisingly, cancer cells appear to acquire an 'addiction' to ACK1-mediated survival, particularly under stress conditions, such as growth factor deprivation or genotoxic insults or hormone deprivation. With the accelerated development of potent and selective ACK1 inhibitors, targeted treatment for cancers harboring aberrant ACK1 activity may soon become a clinical reality.

  5. Tyrosine residues as redox cofactors in human hemoglobin: implications for engineering nontoxic blood substitutes.

    PubMed

    Reeder, Brandon J; Grey, Marie; Silaghi-Dumitrescu, Radu-Lucian; Svistunenko, Dimitri A; Bülow, Leif; Cooper, Chris E; Wilson, Michael T

    2008-11-01

    Respiratory proteins such as myoglobin and hemoglobin can, under oxidative conditions, form ferryl heme iron and protein-based free radicals. Ferryl myoglobin can safely be returned to the ferric oxidation state by electron donation from exogenous reductants via a mechanism that involves two distinct pathways. In addition to direct transfer between the electron donor and ferryl heme edge, there is a second pathway that involves "through-protein" electron transfer via a tyrosine residue (tyrosine 103, sperm whale myoglobin). Here we show that the heterogeneous subunits of human hemoglobin, the alpha and beta chains, display significantly different kinetics for ferryl reduction by exogenous reductants. By using selected hemoglobin mutants, we show that the alpha chain possesses two electron transfer pathways, similar to myoglobin. Furthermore, tyrosine 42 is shown to be a critical component of the high affinity, through-protein electron transfer pathway. We also show that the beta chain of hemoglobin, lacking the homologous tyrosine, does not possess this through-protein electron transfer pathway. However, such a pathway can be engineered into the protein by mutation of a specific phenylalanine residue to a tyrosine. High affinity through-protein electron transfer pathways, whether native or engineered, enhance the kinetics of ferryl removal by reductants, particularly at low reductant concentrations. Ferryl iron has been suggested to be a major cause of the oxidative toxicity of hemoglobin-based blood substitutes. Engineering hemoglobin with enhanced rates of ferryl removal, as we show here, is therefore likely to result in molecules better suited for in vivo oxygen delivery.

  6. Activation of protein tyrosine kinase p72syk by Fc epsilon RI aggregation in rat basophilic leukemia cells. p72syk is a minor component but the major protein tyrosine kinase of pp72.

    PubMed

    Minoguchi, K; Benhamou, M; Swaim, W D; Kawakami, Y; Kawakami, T; Siraganian, R P

    1994-06-17

    Aggregation of the high affinity IgE receptors (Fc epsilon RI) on rat basophilic leukemia RBL-2H3 cells results in protein tyrosine phosphorylations. Previously we reported that there is prominent tyrosine phosphorylation of approximately 72-kDa proteins (pp72) and that the tyrosine kinase p72syk is one component of pp72. Here we studied further the relationship of p72syk to pp72. The aggregation of Fc epsilon RI induced the activation of p72syk which was parallel to its tyrosine phosphorylation. By in vitro kinase assay of immune complexes purified with anti-phosphotyrosine antibodies, p72syk was the major pp72 tyrosine kinase. However, by immunoblotting with anti-phosphotyrosine antibodies, p72syk was a minor component of pp72. The heterogeneous nature of pp72 was indicated by different studies. Under optimum conditions of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, pp72 consisted of a heterogeneous group of 69-, 71-, and 72-kDa tyrosine-phosphorylated proteins. There were differences in the tyrosine phosphorylation of these proteins in cells activated in the absence of extracellular calcium or when stimulation was with the calcium ionophore A23187 or with phorbol myristate acetate. One of the proteins migrating at 69 kDa was p72syk. By two-dimensional gel electrophoresis pp72 was found to consist of multiple tyrosine-phosphorylated protens including 71-80-kDa proteins that associate with p53/56lyn. A 75-kDa tyrosine-phosphorylated protein, different from pp72, was identified as p75HS1 (SPY75). These results demonstrate the heterogeneous nature of the pp72 and that p72syk is activated after Fc epsilon RI aggregation. PMID:7515887

  7. Reconstitution of interactions between tyrosine kinases and the high affinity IgE receptor which are controlled by receptor clustering.

    PubMed Central

    Scharenberg, A M; Lin, S; Cuenod, B; Yamamura, H; Kinet, J P

    1995-01-01

    High affinity IgE receptor (Fc epsilon RI) signaling after contact with antigen occurs in response to receptor clustering. This paper describes methodology, based on vaccinia virus driven protein expression, for probing signaling pathways and its application to Fc epsilon RI interactions with the lyn and syk tyrosine kinases. Reconstitution of the complete tetrameric Fc epsilon RI receptor, lyn and syk in a non-hematopoietic 'null' cell line is sufficient to reconstruct clustering-controlled receptor tyrosine phosphorylation and activation of syk, without apparent requirement for hematopoietic specific phosphatases. The src family kinase lyn phosphorylates Fc epsilon RI in response to receptor clustering, resulting in syk binding to the phosphorylated Fc epsilon RI. Lyn also participates in the tyrosine phosphorylation and activation of syk in a manner which is dependent on phosphorylated Fc epsilon RI. Using overexpression of active and dominant negative syk proteins in a mast cell line which naturally expresses Fc epsilon RI, we corroborate syk's role downstream of receptor phosphorylation, and demonstrate that syk SH2 domains protect receptor ITAMs from ongoing dephosphorylation. Based on these results, we propose that receptor clustering controls lyn-mediated Fc epsilon RI tyrosine phosphorylation by shifting a balance between phosphorylation and dephosphorylation towards accumulation of tyrosine phosphorylated Fc epsilon RI. Fc epsilon RI tyrosine phosphorylation functions to bring syk into a microenvironment where it becomes tyrosine phosphorylated and activated, thereby allowing clustering to indirectly control syk activity. Images PMID:7628439

  8. High-throughput analysis of genome-wide receptor tyrosine kinase expression in human cancers identifies potential novel drug targets.

    PubMed

    Müller-Tidow, Carsten; Schwäble, Joachim; Steffen, Björn; Tidow, Nicola; Brandt, Burkhardt; Becker, Kerstin; Schulze-Bahr, Eric; Halfter, Har