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Sample records for caga epiya tyrosine

  1. Application of PCR amplicon sequencing using a single primer pair in PCR amplification to assess variations in Helicobacter pylori CagA EPIYA tyrosine phosphorylation motifs

    PubMed Central

    2010-01-01

    Background The presence of various EPIYA tyrosine phosphorylation motifs in the CagA protein of Helicobacter pylori has been suggested to contribute to pathogenesis in adults. In this study, a unique PCR assay and sequencing strategy was developed to establish the number and variation of cagA EPIYA motifs. Findings MDA-DNA derived from gastric biopsy specimens from eleven subjects with gastritis was used with M13- and T7-sequence-tagged primers for amplification of the cagA EPIYA motif region. Automated capillary electrophoresis using a high resolution kit and amplicon sequencing confirmed variations in the cagA EPIYA motif region. In nine cases, sequencing revealed the presence of AB, ABC, or ABCC (Western type) cagA EPIYA motif, respectively. In two cases, double cagA EPIYA motifs were detected (ABC/ABCC or ABC/AB), indicating the presence of two H. pylori strains in the same biopsy. Conclusion Automated capillary electrophoresis and Amplicon sequencing using a single, M13- and T7-sequence-tagged primer pair in PCR amplification enabled a rapid molecular typing of cagA EPIYA motifs. Moreover, the techniques described allowed for a rapid detection of mixed H. pylori strains present in the same biopsy specimen. PMID:20181142

  2. Simple Method for Determination of the Number of Helicobacter pylori CagA Variable-Region EPIYA Tyrosine Phosphorylation Motifs by PCR

    PubMed Central

    Argent, Richard H.; Zhang, Youli; Atherton, John C.

    2005-01-01

    Helicobacter pylori strains possessing the cag pathogenicity island are associated with the development of gastric cancer. The CagA protein is translocated into epithelial cells and becomes phosphorylated on tyrosine residues within EPIYA motifs, which may be repeated within the variable region of the protein. Strains possessing CagA with greater numbers of these repeats have been more closely associated with gastric carcinogenesis. Phosphorylated CagA leads to epithelial cell elongation, which is dependent on the number of variable-region EPIYA motifs. Thus, determination of the degree of CagA phosphorylation and the number of EPIYA motifs appears to be more important than detection of cagA alone. Determination of the number of EPIYA motifs by nucleotide sequencing, however, is a laborious and expensive process. We describe here a novel and rapid PCR method for determination of the pattern of repeats containing the EPIYA motif. This will aid in the identification of those strains that may be more likely to cause disease. PMID:15695681

  3. Prevalence of cagA EPIYA motifs in Helicobacter pylori among dyspeptic patients in northeast Thailand.

    PubMed

    Chomvarin, Chariya; Phusri, Karnchanawadee; Sawadpanich, Kookwan; Mairiang, Pisaln; Namwat, Wises; Wongkham, Chaisiri; Hahnvajanawong, Chariya

    2012-01-01

    The aims of this study were to determine the prevalence of cagA type in Helicobacter pylori isolated from dyspeptic patients in northeastern Thailand and to determine whether the pattern of cagA EPIYA motifs were associated with clinical outcomes. One hundred and forty-seven H. pylori-infected dyspeptic patients were enrolled, of whom 68 had non-ulcer dyspepsia (NUD), 57 peptic ulcer disease (PUD), 18 gastric cancer (GCA), and 4 other gastroduodenal diseases. PCR and DNA sequence analysis were used to determine the cagA genotype and the pattern of EPIYA motifs. cagA-positive H. pylori were identified in 138 (94%) of H. pylori-infected dyspeptic patients of whom 75 (54%) were of the Western-type, 44 (32%) the East Asian type and 19 (14%) of the other types. The Western type is significantly found in PUD patients (p = 0.0175). The majority of cagA EPIYA was EPIYA-ABC (43%) and EPIYA-ABD (28%). There is no significant correlation between the increase in number of EPIYA-C motifs and clinical outcomes. Thus, the most frequent cagA type found among northeastern Thai dyspeptic patients was the Western cagA type, which is significantly associated with PUD indicating a possible predictive parameter for clinical outcome. PMID:23082560

  4. Strategy To Characterize the Number and Type of Repeating EPIYA Phosphorylation Motifs in the Carboxyl Terminus of CagA Protein in Helicobacter pylori Clinical Isolates▿ †

    PubMed Central

    Panayotopoulou, Effrosini G.; Sgouras, Dionyssios N.; Papadakos, Konstantinos; Kalliaropoulos, Antonios; Papatheodoridis, George; Mentis, Andreas F.; Archimandritis, Athanasios J.

    2007-01-01

    Cytotoxin-associated gene A (CagA) diversity with regard to EPIYA-A, -B, -C, or -D phosphorylation motifs may play an important role in Helicobacter pylori pathogenesis, and therefore determination of these motifs in H. pylori clinical isolates can become a useful prognostic tool. We propose a strategy for the accurate determination of CagA EPIYA motifs in clinical strains, based upon one-step PCR amplification using primers that flank the EPIYA coding region. We thus analyzed 135 H. pylori isolates derived from 75 adults and 60 children Greek patients. A total of 34 cases were found to be EPIYA PCR negative and were consequently verified as cagA negative by cagA-specific PCR, empty-site cagA PCR, and Western blotting. Sequencing of the remaining 101 PCR-positive amplicons confirmed that an accurate prediction of the number of EPIYA motifs on the basis of size distribution of the PCR products was feasible in all cases. Furthermore, our assay could identify closely related H. pylori subclones within the same patient, harboring different numbers of EPIYA repeats. The prevalence of CagA proteins with three EPIYA motifs (ABC) or four EPIYA motifs (ABCC) was the same within the adult and children groups. However, CagA species with more than four EPIYA motifs were observed exclusively within adults (8.6%), suggesting that CagA-positive strains may acquire additional EPIYA-C motifs throughout adulthood. Our strategy requires no initial cagA screening of the clinical isolates and can accurately predict the number of EPIYA repeats in single or multiple closely related subclones bearing different numbers of EPIYA motifs in their CagA, which may coexist within the same patient. PMID:17151214

  5. 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

  6. 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

  7. Five-year monitoring of considerable changes in tyrosine phosphorylation motifs of the Helicobacter pylori cagA gene in Iran.

    PubMed

    Kargar, Mohammad; Ghorbani-Dalini, Sadegh; Doosti, Abbas; Najafi, Akram

    2014-08-01

    CagA is a major virulence factor of Helicobacter pylori involved in host cell modulation. The C-terminal part of CagA containing the EPIYA motifs is highly variable and is important for the biological activity of the protein. The aim of this study was consideration of the changes in cagA tyrosine phosphorylation motifs (TPMs) of H. pylori. A set of 302 H. pylori DNA samples from the Iranian population from 2006 to 2011 was selected for the proposed study. The cagA gene and its TPMs were assessed by using polymerase chain reaction (PCR) and specific primers. The prevalence of the cagA gene in our study ranged from 91.43% to 97.06% (with an average of 95.03%). Out of the cagA-positive samples, the prevalence of TPMs A and B increased from 12.5% and 23.44% to 71.2% and 63.63%, respectively. Also, the prevalence of samples infected with Western and East Asian types of H. pylori ranged from 64.06% to 5.73% for the Western type and 17.19% to 51.59% for the East Asian type. Overall, our results showed a high prevalence of the cagA gene. Also, it seems that cagA TPMs of H. pylori is undergoing a change from the Western type to the East Asian type in Iran. PMID:24771298

  8. Analysis of the 3' variable region of the cagA gene of Helicobacter pylori isolated in Koreans.

    PubMed

    Choi, Kee Don; Kim, Nayoung; Lee, Dong Ho; Kim, Jung Mogg; Kim, Joo Sung; Jung, Hyun Chae; Song, In Sung

    2007-04-01

    CagA protein of Helicobacter pylori is injected into epithelial cells, and it undergoes tyrosine phosphorylation, resulting in inducing cytoskeletal rearrangements. A few studies have suggested that the number of CagA tyrosine phosphorylation motifs (EPIYA) and subtypes of CagA were associated with gastric cancer. This study was performed to characterize the 3' variable regions of the cagA gene of H. pylori and to investigate whether or not there is any relationship between the diversities of cagA and the disease outcome in Korea. Seventy-nine patients (chronic gastritis, 15; duodenal ulcer, 27; benign gastric ulcer, 18; gastric cancer, 19) were enrolled. Biopsy specimens were taken from the antrum for H. pylori culture, and genomic DNA was extracted. PCR and DNA sequence analysis was carried out for the 3' variable region of the cagA gene. Seventy-eight strains (98.8%) contained three EPIYA motifs and one strain (1.2%) isolated from a patient with duodenal ulcer contained four EPIYA motifs. Seventy-six strains (96.2%) were the East Asian type. In conclusion, there was no significant difference between the number of EPIYA motifs or CagA subtypes and various gastroduodenal diseases in Korea. PMID:17342405

  9. Tyrosine Phosphorylation of CagA from Chinese Helicobacter pylori Isolates in AGS Gastric Epithelial Cells

    PubMed Central

    Zhang, Youli; Argent, Richard H.; Letley, Darren P.; Thomas, Rachael J.; Atherton, John C.

    2005-01-01

    Helicobacter pylori strains possessing the cag pathogenicity island (PaI) are associated with the development of gastroduodenal diseases, including gastric cancer. cag PaI products induce the secretion of interleukin-8 (IL-8) from epithelial cells and facilitate the translocation of CagA into the cell cytosol. In East Asia, where the incidence of gastric cancer is high, most strains possess the cag PaI. To date, however, no cag PaI phenotypic data have been provided for strains isolated in mainland China. Here we used 31 Chinese strains to determine the genotypic and phenotypic status of the cag PaI. All strains possessed cagA and cagE, and we observed a variation in the length of cagA variable regions. Nucleotide sequencing of the cagA variable region revealed that CagA was of two types, a short “Western” form with two tyrosine phosphorylation sites and a longer “East Asian” form with three tyrosine phosphorylation sites. Coculture of strains with AGS epithelial cells showed that strains could induce IL-8 secretion from the cells and that CagA with three phosphorylation sites became more phosphorylated than that with two and could induce significantly (P < 0.001) more cells to elongate. We hypothesize that the preponderance of the more active East Asian form of cagA may underlie the high rate of gastric cancer in China. PMID:15695680

  10. Helicobacter pylori CagA protein polymorphisms and their lack of association with pathogenesis

    PubMed Central

    Acosta, Nicole; Quiroga, Andrés; Delgado, Pilar; Bravo, María Mercedes; Jaramillo, Carlos

    2010-01-01

    AIM: To investigate Helicobacter pylori (H. pylori) CagA diversity and to evaluate the association between protein polymorphisms and the occurrence of gastric pathologies. METHODS: One hundred and twenty-two clinical isolates of H. pylori cultured from gastric biopsies obtained from Colombian patients with dyspepsia were included as study material. DNA extracted from isolates was used to determine cagA status, amplifying the C-terminal cagA gene region by polymerase chain reaction. One hundred and six strains with a single amplicon were sequenced and results were used to characterize the 3’ variable region of the cagA gene. To establish the number and type of tyrosine phosphorylation motifs Glutamine acid-Proline-Isoleucine-Tyrosine-Alanine (EPIYA) bioinformatic analysis using Amino Acid Sequence Analyzer-Amino Acid Sequence Analyzer software was conducted. Analysis of the association between the number of EPIYA motifs and the gastric pathology was performed using χ2 test and analysis of the presence of EPIYA-C motifs in relation to the pathology was made by logistic regression odds ratios. Comparisons among EPIYA types found and those reported in GenBank were performed using a proportion test in Statistix Analytical Software version 8.0. RESULTS: After amplification of the 3’ of the cagA gene, 106 from 122 isolates presented a single amplicon and 16 showed multiple amplicons. As expected, diversity in the size of the cagA unique fragments among isolates was observed. The 106 strains that presented a single amplicon after 3’ cagA amplification came from patients with gastritis (19 patients), atrophic gastritis (21), intestinal metaplasia (26), duodenal ulcer (22) and gastric cancer. DNA sequence analysis showed that the differences in size of 3’ cagA unique fragments was attributable to the number of EPIYA motifs: 1.9% had two EPIYA motifs, 62.3% had three, 33.0% had four and 2.8% had five motifs. The majority of tested clinical strains (62.3%) were found

  11. C-terminal Src kinase-mediated EPIYA phosphorylation of Pragmin creates a feed-forward C-terminal Src kinase activation loop that promotes cell motility.

    PubMed

    Senda, Yoshie; Murata-Kamiya, Naoko; Hatakeyama, Masanori

    2016-07-01

    Pragmin is one of the few mammalian proteins containing the Glu-Pro-Ile-Tyr-Ala (EPIYA) tyrosine-phosphorylation motif that was originally discovered in the Helicobacter pylori CagA oncoprotein. Following delivery into gastric epithelial cells by type IV secretion and subsequent tyrosine phosphorylation at the EPIYA motifs, CagA serves as an oncogenic scaffold/adaptor that promiscuously interacts with SH2 domain-containing mammalian proteins such as the Src homology 2 (SH2) domain-containing protein tyrosine phosphatase-2 (SHP2) and the C-terminal Src kinase (Csk), a negative regulator of Src family kinases. Like CagA, Pragmin also forms a physical complex with Csk. In the present study, we found that Pragmin directly binds to Csk by the tyrosine-phosphorylated EPIYA motif. The complex formation potentiates kinase activity of Csk, which in turn phosphorylates Pragmin on tyrosine-238 (Y238), Y343, and Y391. As Y391 of Pragmin comprises the EPIYA motif, Pragmin-Csk interaction creates a feed-forward regulatory loop of Csk activation. Together with the finding that Pragmin and Csk are colocalized to focal adhesions, these observations indicate that the Pragmin-Csk interaction, triggered by Pragmin EPIYA phosphorylation, robustly stimulates the kinase activity of Csk at focal adhesions, which direct cell-matrix adhesion that regulates cell morphology and cell motility. As a consequence, expression of Pragmin and/or Csk in epithelial cells induces an elongated cell shape with elevated cell scattering in a manner that is mutually dependent on Pragmin and Csk. Deregulation of the Pragmin-Csk axis may therefore induce aberrant cell migration that contributes to tumor invasion and metastasis. PMID:27116701

  12. Conformational Analysis of Isolated Domains of Helicobacter pylori CagA

    PubMed Central

    Alonso, Hernan; Saijo-Hamano, Yumiko; Kwok, Terry; Roujeinikova, Anna

    2013-01-01

    The CagA protein of Helicobacter pylori is associated with increased virulence and gastric cancer risk. CagA is translocated into the host cell by a H. pylori type IV secretion system via mechanisms that are poorly understood. Translocated CagA interacts with numerous host factors, altering a variety of host signalling pathways. The recently determined crystal structure of C-terminally-truncated CagA indicated the presence of two domains: the smaller, flexible N-terminal domain and the larger, middle domain. In this study, we have investigated the conformation, oligomeric state and stability of the N-terminal, middle and glutamate-proline-isoleucine-tyrosine-alanine (EPIYA)-repeats domains. All three domains are monomeric, suggesting that the multimerisation of CagA observed in infected cells is likely to be mediated not by CagA itself but by its interacting partners. The middle and the C-terminal domains, but not the N-terminal domain, are capable of refolding spontaneously upon heat denaturation, lending support to the hypothesis that unfolded CagA is threaded C-terminus first through the type IV secretion channel with its N-terminal domain, which likely requires interactions with other domains to refold, being threaded last. Our findings also revealed that the C-terminal EPIYA-repeats domain of CagA exists in an intrinsically disordered premolten globule state with regions in PPII conformation - a feature that is shared by many scaffold proteins that bind multiple protein components of signalling pathways. Taken together, these results provide a deeper understanding of the physicochemical properties of CagA that underpin its complex cellular and oncogenic functions. PMID:24223932

  13. Natural variant of the Helicobacter pylori CagA oncoprotein that lost the ability to interact with PAR1.

    PubMed

    Hashi, Kana; Murata-Kamiya, Naoko; Varon, Christine; Mégraud, Francis; Dominguez-Bello, Maria Gloria; Hatakeyama, Masanori

    2014-03-01

    Helicobacter pylori strains carrying the cagA gene are associated with severe disease outcomes, most notably gastric cancer. CagA protein is delivered into gastric epithelial cells by a type IV secretion system. The translocated CagA undergoes tyrosine phosphorylation at the C-terminal EPIYA motifs by host cell kinases. Tyrosine-phosphorylated CagA acquires the ability to interact with and activate SHP2, thereby activating mitogenic signaling and inducing cell morphological transformation (hummingbird phenotype). CagA also interacts with PAR1b via the CM sequence, resulting in induction of junctional and polarity defects. Furthermore, CagA-PAR1b interaction stabilizes the CagA-SHP2 complex. Because transgenic mice systemically expressing CagA develop gastrointestinal and hematological malignancies, CagA is recognized as a bacterium-derived oncoprotein. Interestingly, the C-terminal region of CagA displays a large diversity among H. pylori strains, which influences the ability of CagA to bind to SHP2 and PAR1b. In the present study, we investigated the biological activity of v225d CagA, an Amerindian CagA of H. pylori isolated from a Venezuelan Piaroa Amerindian subject, because the variant CagA does not possess a canonical CM sequence. We found that v225d CagA interacts with SHP2 but not PAR1b. Furthermore, SHP2-binding activity of v225d CagA was much lower than that of CagA of H. pylori isolated from Western countries (Western CagA). v225d CagA also displayed a reduced ability to induce the hummingbird phenotype than that of Western CagA. Given that perturbation of PAR1b and SHP2 by CagA underlies the oncogenic potential of CagA, the v225d strain is considered to be less oncogenic than other well-studied cagA-positive H. pylori strains. PMID:24354359

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

    PubMed

    Krisch, Linda M; Posselt, Gernot; Hammerl, Peter; Wessler, Silja

    2016-09-01

    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

  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. CagA C-terminal variations in Helicobacter pylori strains from Colombian patients with gastric precancerous lesions

    PubMed Central

    Sicinschi, L. A.; Correa, P.; Peek, R. M.; Camargo, M. C.; Piazuelo, M. B.; Romero-Gallo, J.; Hobbs, S. S.; Krishna, U.; Delgado, A.; Mera, R.; Bravo, L. E.; Schneider, B. G.

    2010-01-01

    The C-terminus of the Helicobacter pylori CagA protein is polymorphic, bearing different EPIYA sequences (EPIYA-A, B, C or D), and one or more CagA multimerization (CM) motifs. The number of EPIYA-C motifs is associated with precancerous lesions and gastric cancer (GC). The relationship between EPIYA, CM motifs and gastric lesions was examined in H. pylori-infected Colombian patients from areas of high and low risk for GC. Genomic DNA was extracted from H. pylori strains cultured from gastric biopsies from 80 adults with dyspeptic symptoms. Sixty-seven (83.8%) of 80 strains were cagA positive. The 3′ region of cagA was sequenced, and EPIYA and CM motifs were identified. CagA proteins contained one (64.2%), two (34.3%) or three EPIYA-C motifs (1.5%), all with Western type CagA-specific sequences. Strains with one EPIYA-C motif were associated with less severe gastric lesions (non-atrophic and multifocal atrophic gastritis), whereas strains with multiple EPIYA-C motifs were associated with more severe lesions (intestinal metaplasia and dysplasia) (p <0.001). In 54 strains, the CM motifs were identical to those common in Western strains. Thirteen strains from the low-risk area contained two different CM motifs: one of Western type located within the EPIYA-C segment and another following the EPIYA-C segment and resembling the CM motif found in East Asian strains. These strains induced significantly shorter projections in AGS cells and an attenuated reduction in levels of CagA upon immunodepletion of SHP-2 than strains possessing Western/Western motifs. This novel finding may partially explain the difference in GC incidence in these populations. PMID:19456839

  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. Variant of Helicobacter pylori CagA proteins induce different magnitude of morphological changes in gastric epithelial cells.

    PubMed

    Alfizah, Hanafiah; Ramelah, Mohamed

    2012-06-01

    Infection with Helicobacter pylori cagA-positive strains is associated with gastroduodenal diseases. The CagA protein is injected into gastric epithelial cells and supposedly induces morphological changes termed the 'hummingbird phenotype', which is associated with scattering and increased cell motility. The molecular mechanisms leading to the CagA-dependent morphological changes are only partially known. The present study was carried out to investigate the effect of CagA variants on the magnitude of gastric epithelial cell morphological changes. Recombinant 3' terminal domains of cagA were cloned and expressed in a gastric epithelial cell line and the hummingbird phenotype was quantified by microscopy. The 3' region of the cagA gene of Malaysian H. pylori isolates showed six sub-genotypes that differed in the structural organization of the EPIYA repeat sequences. The percentage of hummingbird cells induced by CagA increased with duration of transfection. The hummingbird phenotype was observed to be more pronounced when CagA with 4 EPIYA motifs rather than 3 or 2 EPIYA motifs was produced. The activity of different CagA variants in the induction of the hummingbird phenotype in gastric epithelial cells depends at least in part on EPIYA motif variability. The difference in CagA genotypes might influence the potential of individual CagAs to cause morphological changes in host cells. Depending on the relative exposure of cells to CagA genotypes, this may contribute to the various disease outcomes caused by H. pylori infection in different individuals. PMID:22870595

  20. Study of the Cytoxin-Associated Gene A (CagA Gene) in Helicobacter pylori Using Gastric Biopsies of Iraqi Patients

    PubMed Central

    Kalaf, Elham A.; Al-Khafaji, Zahra M.; Yassen, Nahi Y.; AL-Abbudi, Fadel A.; Sadwen, Saad N.

    2013-01-01

    Background and Aims: The Helicobacter pylori CagA gene is a major virulence factor that plays an important role in gastric pathologies. The size variation of CagA gene, which is dependent on the 3’ repeat region, contains one or more Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs and CagA multimerization (CM) motifs. Four segments flanking the EPIYA motifs, EPIYA −A, −B, −C, or −D, were reported to play a crucial role in the pathogenesis of H. pylori infection. The aim was to determine the roles of EPIYA segments and CM motifs in gastroduodenal pathogenesis in an Iraqi population. Patients and Methods: Gastric biopsies were collected from 210 patients with gastritis, duodenal ulcer (DU), gastric ulcer (GU), and gastric cancer (GC). The EPIYA motif genotyping was determined by polymerase chain reaction and sequencing. The differences in age, gender, and CagA EPIYA motifs of H. pylori between GC, DU, GU and gastritis patients were analyzed using a χ2 -test. Results A total of 22 (45.8%) strains had three copies of EPIYA (ABC type), 2 (4.16%) had four copies (ABCC type), 6 (12.7%) had five copies (ABCCC type), 13 (27.08%) had two copies (AB type), 3 (6.25%) had the BC, and 2 (4.17%) had AC motif. The alignment of the deduced protein sequences confirmed that there were no East Asian type EPIYA-D sequences in Iraqi strains. A significant association was found between increase in number of EPIYA-C motifs and GU (P ≤ 0.01) compared with gastritis. Conclusions: The structure of the 3’ region of the CagA gene in Iraqi strains was Western type with a variable number of EPIYA-C and CM motifs. A significant association was found between increase in number of EPIYA-C motifs and GU compared with gastritis indicating predictive association with the severity of the disease. The GenBank accession numbers for the partial CagA nucleotide sequences determined in this study are JX164093-JX164112. PMID:23481132

  1. Analysis of 3′-end variable region of the cagA gene in Helicobacter pylori isolated from Iranian population

    PubMed Central

    Shokrzadeh, Leila; Baghaei, Kaveh; Yamaoka, Yoshio; Dabiri, Hossein; Jafari, Fereshteh; Sahebekhtiari, Navid; Tahami, Ali; Sugimoto, Mitsushige; Zojaji, Homayon; Zali, Mohammad Reza

    2009-01-01

    Background and Aims The 3′ region of the cagA gene, the most well-known virulence factor of Helicobacter pylori, contains Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs. Four segments flanking the EPIYA motifs, EPIYA-A, -B, -C, or -D, were reported to play important roles in H. pylori-related gastroduodenal pathogenesis. The aim was to determine the roles of EPIYA segments in gastroduodenal pathogenesis in an Iranian population. Methods A total of 92 cagA-positive Iranian strains isolated from dyspepsia patients with non-ulcer dyspepsia (n = 77), peptic ulcer (n = 11) and gastric cancer (n = 4) were studied. The EPIYA motif genotyping was determined by polymerase chain reaction and sequencing. Results A total of 86 (93.5%) strains had three copies of EPIYA (ABC type), three (3.3%) had four copies (ABCC type) and three (3.3%) had two copies (AB type). The alignment of the deduced protein sequences confirmed that there were no East Asian type EPIYA-D sequences (EPIYATIDFDEANQAG) in Iranian strains. When the prevalence of strains with multiple EPIYA-C segments in Iran was compared with previously published data, it was much lower than that in Colombia and Italy, but was higher than that of Iraq, and the patterns were parallel to the incidence of gastric cancer in these countries. Conclusion The structure of the 3′ region of the cagA gene in Iranian strains was Western type. Although we could not find differences between EPIYA types and clinical outcomes, low prevalence of strains with multiple EPIYA-C segments might be reasons for low incidence of gastric cancer in Iran. PMID:19793167

  2. [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

  3. A Comprehensive Sequence and Disease Correlation Analyses for the C-Terminal Region of CagA Protein of Helicobacter pylori

    PubMed Central

    Xia, Youlin; Yamaoka, Yoshio; Zhu, Qi; Matha, Ivan; Gao, Xiaolian

    2009-01-01

    Chronic Helicobacter pylori infection is known to be associated with the development of peptic ulcer, gastric cancer and gastric lymphoma. Currently, the bacterial factors of H. pylori are reported to be important in the development of gastroduodenal diseases. CagA protein, encoded by the cagA, is the best studied virulence factor of H. pylori. The pathogenic CagA protein contains a highly polymorphic Glu-Pro-Ile-Tyr-Ala (EPIYA) repeat region in the C-terminal. This repeat region is reported to be involved in the pathogenesis of gastroduodenal diseases. The segments containing EPIYA motifs have been designated as segments A, B, C, and D; however the classification and disease relation are still unclear. This study used 560 unique CagA sequences containing 1,796 EPIYA motifs collected from public resources, including 274 Western and 286 East Asian strains with clinical data obtained from 433 entries. Fifteen types of EPIYA or EPIYA-like sequences are defined. In addition to four previously reported major segment types, several minor segment types (e.g., segment B′, B′′) and more than 30 sequence types (e.g., ABC, ABD) were defined using our classification method. We confirm that the sequences from Western and East Asian strains contain segment C and D, respectively. We also confirm that strains with two EPIYA segment C have a greater chance of developing gastric cancer than those with one segment C. Our results shed light on the relationships between the types of CagAs, the country of origin of each sequence type, and the frequency of gastric disease. PMID:19893742

  4. Association between cagA and vacA genotypes and pathogenesis in a Helicobacter pylori infected population from South-eastern Sweden

    PubMed Central

    2012-01-01

    Background Chronic gastritis, peptic ulcer disease, and gastric cancer have been shown to be related to infection with Helicobacter pylori (H. pylori). Two major virulence factors of H. pylori, CagA and VacA, have been associated with these sequelae of the infection. In this study, total DNA was isolated from gastric biopsy specimens to assess the cagA and vacA genotypes. Results Variations in H. pylori cagA EPIYA motifs and the mosaic structure of vacA s/m/i/d regions were analysed in 155 H. pylori-positive gastric biopsies from 71 individuals using PCR and sequencing. Analysis of a possible association between cagA and vacA genotypes and gastroduodenal pathogenesis was made by logistic regression analysis. We found that H. pylori strains with variation in the number of cagA EPIYA motif variants present in the same biopsy correlated with peptic ulcer, while occurrence of two or more EPIYA-C motifs was associated with atrophy in the gastric mucosa. No statistically significant relation between vacA genotypes and gastroduodenal pathogenesis was observed. Conclusions The results of this study indicate that cagA genotypes may be important determinants in the development of gastroduodenal sequelae of H. pylori infection. In contrast to other studies, vacA genotypes were not related to disease progression or outcome. In order to fully understand the relations between cagA, vacA and gastroduodenal pathogenesis, the mechanisms by which CagA and VacA act and interact need to be further investigated. PMID:22747681

  5. [The role of CagA in H. pylori infection].

    PubMed

    Tanaka, Hiroshi; Yoshida, Masaru; Azuma, Takeshi

    2009-12-01

    Helicobacter pylori (H. pylori) chronically colonizes human gastric epithelium and induces various diseases. But the mechanism of carcinogenesis in H. pylori infection remains to be assessed. We described that after attachment of H. pylori to gastric epithelial cells, CagA is injected directly from the bacteria into the cells and undergoes tyrosine phosphorylation. Tyrosine phosphorylated CagA can bind to SHP-2. Deregulation of SHP -2 by CagA may induce abnormal proliferation and movement of gastric epithelial cells. There are two patterns of CagA motifs between East Asian strains and Western strains. East Asian-type CagA confers stronger SHP-2 binding and transforming activities than Western-type CagA. We assessed the association between CagA diversity and clinical outcome in Asian countries, where mortalities from gastric cancer is different. As results, H. pylori infection with East Asian-type CagA was associated with gastric atrophy and cancer. Therefore, persistent active inflammation induced by the East Asian CagA-positive strain may play a role in the pathogenesis of disease. PMID:19999107

  6. 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.

  7. 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

  8. 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

  9. Attenuated CagA oncoprotein in Helicobacter pylori from Amerindians in Peruvian Amazon.

    PubMed

    Suzuki, Masato; Kiga, Kotaro; Kersulyte, Dangeruta; Cok, Jaime; Hooper, Catherine C; Mimuro, Hitomi; Sanada, Takahito; Suzuki, Shiho; Oyama, Masaaki; Kozuka-Hata, Hiroko; Kamiya, Shigeru; Zou, Quan-Ming; Gilman, Robert H; Berg, Douglas E; Sasakawa, Chihiro

    2011-08-26

    Population genetic analyses of bacterial genes whose products interact with host tissues can give new understanding of infection and disease processes. Here we show that strains of the genetically diverse gastric pathogen Helicobacter pylori from Amerindians from the remote Peruvian Amazon contain novel alleles of cagA, a major virulence gene, and reveal distinctive properties of their encoded CagA proteins. CagA is injected into the gastric epithelium where it hijacks pleiotropic signaling pathways, helps Hp exploit its special gastric mucosal niche, and affects the risk that infection will result in overt gastroduodenal diseases including gastric cancer. The Amerindian CagA proteins contain unusual but functional tyrosine phosphorylation motifs and attenuated CRPIA motifs, which affect gastric epithelial proliferation, inflammation, and bacterial pathogenesis. Amerindian CagA proteins induced less production of IL-8 and cancer-associated Mucin 2 than did those of prototype Western or East Asian strains and behaved as dominant negative inhibitors of action of prototype CagA during mixed infection of Mongolian gerbils. We suggest that Amerindian cagA is of relatively low virulence, that this may have been selected in ancestral strains during infection of the people who migrated from Asia into the Americas many thousands of years ago, and that such attenuated CagA proteins could be useful therapeutically. PMID:21757722

  10. J-Western Forms of Helicobacter pylori cagA Constitute a Distinct Phylogenetic Group with a Widespread Geographic Distribution

    PubMed Central

    Duncan, Stacy S.; Valk, Pieter L.; Shaffer, Carrie L.

    2012-01-01

    Chronic infection with Helicobacter pylori strains expressing the bacterial oncoprotein CagA confers an increased risk of gastric cancer. While much is known about the ancestry and molecular evolution of Western, East Asian, and Amerindian cagA sequences, relatively little is understood about a fourth group, known as “J-Western,” which has been detected mainly in strains from Okinawa, Japan. We show here that J-Western cagA sequences have a more widespread global distribution than previously recognized, occur in strains with multiple different ancestral origins (based on multilocus sequence typing [MLST] analysis), and did not arise recently. As shown by comparisons of Western and J-Western forms of CagA, there are 45 fixed or nearly fixed amino acid differences, and J-Western forms contain a unique 4-amino-acid insertion. The mean nucleotide diversity of synonymous sites (πs) is slightly lower in the J-Western group than in the Western and East Asian groups (0.066, 0.086, and 0.083, respectively), which suggests that the three groups have comparable, but not equivalent, effective population sizes. The reduced πs of the J-Western group is attributable to ancestral recombination events within the 5′ region of cagA. Population genetic analyses suggest that within the cagA region encoding EPIYA motifs, the East Asian group underwent a marked reduction in effective population size compared to the Western and J-Western groups, in association with positive selection. Finally, we show that J-Western cagA sequences are found mainly in strains producing m2 forms of the secreted VacA toxin and propose that these functionally interacting proteins coevolved to optimize the gastric colonization capacity of H. pylori. PMID:22247512

  11. Diverse characteristics of the CagA gene of Helicobacter pylori strains collected from patients from southern vietnam with gastric cancer and peptic ulcer.

    PubMed

    Truong, Bui Xuan; Mai, Vo Thi Chi; Tanaka, Hiroshi; Ly, Le Thanh; Thong, Tran Minh; Hai, Hoang Hoa; Van Long, Dao; Furumatsu, Keisuke; Yoshida, Masaru; Kutsumi, Hiromu; Azuma, Takeshi

    2009-12-01

    The pathogenesis of gastroduodenal diseases is related to the diversity of Helicobacter pylori strains. CagA-positive strains are more likely to cause gastric cancer than CagA-negative strains. Based on EPIYA (Glu-Pro-Ile-Tyr-Ala) motifs at the carboxyl terminus corresponding to phosphorylation sites, H. pylori CagA is divided into East Asian CagA and Western CagA. The former type prevails in East Asia and is more closely associated with gastric cancer. The present study used full sequences of the cagA gene and CagA protein of 22 H. pylori strains in gastric cancer and peptic ulcer patients from Southern Vietnam to make a comparison of genetic homology among Vietnamese strains and between them and other strains in East Asia. A phylogenetic tree was constructed based on full amino acid sequences of 22 Vietnamese strains in accordance with 54 references from around the world. The cagA gene was found in all Vietnamese H. pylori strains. Twenty-one of 22 (95.5%) strains belonged to the East Asian type and had similar characteristics of amino acid sequence at the carboxyl terminus to other strains from the East Asian region. From evidence of East Asian CagA and epidemiologic cancerous lesions in Vietnam, H. pylori-infected Vietnamese can be classified into a high-risk group for gastric cancer, but further studies on the interaction among environmental and virulence factors should be done. Finally, phylogenetic data support that there is a Japanese subtype in the Western CagA type. PMID:19846630

  12. Diverse Characteristics of the cagA Gene of Helicobacter pylori Strains Collected from Patients from Southern Vietnam with Gastric Cancer and Peptic Ulcer▿

    PubMed Central

    Truong, Bui Xuan; Mai, Vo Thi Chi; Tanaka, Hiroshi; Ly, Le Thanh; Thong, Tran Minh; Hai, Hoang Hoa; Van Long, Dao; Furumatsu, Keisuke; Yoshida, Masaru; Kutsumi, Hiromu; Azuma, Takeshi

    2009-01-01

    The pathogenesis of gastroduodenal diseases is related to the diversity of Helicobacter pylori strains. CagA-positive strains are more likely to cause gastric cancer than CagA-negative strains. Based on EPIYA (Glu-Pro-Ile-Tyr-Ala) motifs at the carboxyl terminus corresponding to phosphorylation sites, H. pylori CagA is divided into East Asian CagA and Western CagA. The former type prevails in East Asia and is more closely associated with gastric cancer. The present study used full sequences of the cagA gene and CagA protein of 22 H. pylori strains in gastric cancer and peptic ulcer patients from Southern Vietnam to make a comparison of genetic homology among Vietnamese strains and between them and other strains in East Asia. A phylogenetic tree was constructed based on full amino acid sequences of 22 Vietnamese strains in accordance with 54 references from around the world. The cagA gene was found in all Vietnamese H. pylori strains. Twenty-one of 22 (95.5%) strains belonged to the East Asian type and had similar characteristics of amino acid sequence at the carboxyl terminus to other strains from the East Asian region. From evidence of East Asian CagA and epidemiologic cancerous lesions in Vietnam, H. pylori-infected Vietnamese can be classified into a high-risk group for gastric cancer, but further studies on the interaction among environmental and virulence factors should be done. Finally, phylogenetic data support that there is a Japanese subtype in the Western CagA type. PMID:19846630

  13. Identification of Helicobacter pylori and the cagA genotype in gastric biopsies using highly sensitive real-time PCR as a new diagnostic tool.

    PubMed

    Yamazaki, Shiho; Kato, Shunji; Matsukura, Norio; Ohtani, Masahiro; Ito, Yoshiyuki; Suto, Hiroyuki; Yamazaki, Yukinao; Yamakawa, Akiyo; Tokudome, Shinkan; Higashi, Hideaki; Hatakeyama, Masanori; Azuma, Takeshi

    2005-06-01

    The CagA protein is one of the virulence factors of Helicobacter pylori, and two major subtypes of CagA have been observed, the Western and East Asian type. CagA is injected from the bacteria into gastric epithelial cells, undergoes tyrosine phosphorylation, and binds to Src homology 2 domain-containing protein-tyrosine phosphatase SHP-2. The East Asian type CagA binds to SHP-2 more strongly than the Western type CagA. Here, we tried to distinguish the CagA type by highly sensitive real-time PCR with the objective of establishing a system to detect H. pylori and CagA subtypes from gastric biopsies. We designed primers and probe sets for Western or East Asian-cagA at Western-specific or East Asian-specific sequence regions, respectively, and H. pylori 16S rRNA. We could detect the H. pylori 16S rRNA gene, Western and East Asian-cagA gene from DNA of gastric biopsies. The sensitivity and specificity for H. pylori infection was 100% in this system. In Thai patients, 87.8% (36/41) were cagA-positive; 26.8% (11/41) were Western-cagA positive and 53.7% (22/41) were East Asian-cagA positive, while 7.3% (3/41) reacted with both types of cagA. These results suggest that this real-time PCR system provides a highly sensitive assessment of CagA type as a new diagnostic tool for the pathogenicity of H. pylori infection. PMID:15907447

  14. 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

  15. Gastrokine 1 inhibits the carcinogenic potentials of Helicobacter pylori CagA

    PubMed Central

    Yoon, Jung Hwan; Seo, Ho Suk; Choi, Sung Sook; Chae, Hyun Suk; Choi, Won Seok; Kim, Olga; Ashktorab, Hassan; Smoot, Duane T.; Nam, Suk Woo; Lee, Jung Young; Park, Won Sang

    2014-01-01

    Helicobacter pylori CagA directly injected by the bacterium into epithelial cells via a type IV secretion system, leads to cellular changes such as morphology, apoptosis, proliferation and cell motility, and stimulates gastric carcinogenesis. We investigated the effects of cytotoxin-associated gene A (CagA) and gastrokine 1 (GKN1) on cell proliferation, apoptosis, reactive oxygen species (ROS) production, epithelial–mesenchymal transition (EMT) and cell migration in CagA- or GKN1-transfected gastric epithelial cells and mucosal tissues from humans and mice infected with H.pylori. On the molecular level, H.pylori CagA induced increased cell proliferation, ROS production, antiapoptotic activity, cell migration and invasion. Moreover, CagA induced activation of NF-κB and PI3K/Akt signaling pathways and EMT-related proteins. In addition, H.pylori CagA reduced GKN1 gene copy number and expression in gastric cells and mucosal tissues of humans and mice. However, GKN1 overexpression successfully suppressed the carcinogenic effects of CagA through binding to CagA. These results suggest that GKN1 might be a target to inhibit the effects from H.pylori CagA. PMID:25239641

  16. 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.

  17. Effect of growth phase and acid shock on Helicobacter pylori cagA expression.

    PubMed Central

    Karita, M; Tummuru, M K; Wirth, H P; Blaser, M J

    1996-01-01

    Helicobacter pylori strains possessing cagA are associated with peptic ulceration. To understand the regulation of expression of cagA, picB, associated with interleukin-8 induction, and ureA, encoding the small urease subunit, we created gene fusions of cagA, ureA, and picB of strain 3401, using a promoterless reporter (xylE). Expression of XylE after growth in broth culture revealed that basal levels of expression of cagA and urea in H. pylori were substantially greater than for picB. For cagA expression in stationary-phase cells, brief exposure to acid pH caused a significant increase in xylE expression compared with neutral pH. In contrast, expression of xylE in urea or picB decreased after parallel exposure to acid pH (pH 7 > 6 > 5 > 4), regardless of the growth phase. Expression of the CagA protein varied with growth phase and pH exposure in parallel with the observed transcriptional variation. The concentration of CagA in a cell membrane-enriched fraction after growth at pH 6 was significantly higher than after growth at pH 5 or 7. We conclude that the promoterless reporter xylE is useful for studying the regulation of gene expression in H. pylori and that regulation of CagA production occurs mainly at the transcriptional level. PMID:8890198

  18. Serum Helicobacter pylori CagA antibody titer was a useful marker for advanced inflammation in the stomach in Japan

    PubMed Central

    Shiota, Seiji; Murakami, Kazunari; Okimoto, Tadayoshi; Kodama, Masaaki; Yamaoka, Yoshio

    2013-01-01

    Background and aim Subjects infected with H. pylori containing cagA do not always induce serum CagA antibody. Our previous meta-analysis showed that serum CagA seropositivity was associated with gastric cancer even in East Asian countries. However, it remains unclear why serum CagA positive status is associated with gastric cancer. In this study, we aimed to examine the relationship between anti CagA antibody titer and the levels of pepsinogen, and histological score. Methods Eighty-eight H. pylori positive Japanese patients with gastritis were included. Serum CagA antibody titer, pepsinogen (PG) I and PG II were evaluated by enzyme-linked immunosorbent assay. Histological scores were evaluated according to Update Sydney System. CagA expression was examined by immunoblot. Results Seroprevalence of CagA antibody was found in 75.0%. Interestingly, serum CagA antibody titer was significantly correlated with PG I and PG II levels (P = 0.003 and 0.004, respectively). Serum CagA antibody titer was also significantly correlated with mucosal inflammation in the corpus (P = 0.04). On the other hand, bacterial density was not related with CagA antibody titer. CagA expression level of the strains was irrespective of the status of PG and serum CagA antibody. Conclusions Subjects with higher serum CagA antibody titer can be considered as high risk population for the development of gastric cancer from the point of strong gastric inflammatory response even in Japan. Host recognition rather than bacterial colonization might be associated with the difference of serum CagA antibody titer. PMID:24033876

  19. Functional association between the Helicobacter pylori virulence factors VacA and CagA.

    PubMed

    Argent, Richard H; Thomas, Rachael J; Letley, Darren P; Rittig, Michael G; Hardie, Kim R; Atherton, John C

    2008-02-01

    The Helicobacter pylori virulence factors CagA and VacA are implicated in the development of gastroduodenal diseases. Most strains possessing CagA also possess the more virulent vacuolating form of VacA. This study assessed the significance of possession of both virulence factors in terms of their effect on gastric epithelial cells, using a set of minimally passaged, isogenic VacA, CagA and CagE mutants in H. pylori strains 60190 and 84-183. The cagA and cagE mutants were found to significantly increase VacA-induced vacuolation of epithelial cells, and the vacA mutants significantly increased CagA-induced cellular elongations, compared with wild-type strains, indicating that CagA reduces vacuolation and VacA reduces hummingbird formation. Although epithelial cells incubated with the wild-type H. pylori strains may display both vacuolation and hummingbird formation, it was found that (i) hummingbird length was significantly reduced in vacuolated cells compared with those without vacuolation; (ii) the number of vacuoles was significantly reduced in vacuolated cells with hummingbird formation compared with those without hummingbirds; and (iii) cells displaying extensive vacuolation did not subsequently form hummingbirds and vice versa. VacA did not affect the phosphorylation of CagA. These data show that VacA and CagA downregulate each other's effects on epithelial cells, potentially allowing H. pylori interaction with cells whilst avoiding excessive cellular damage. PMID:18201978

  20. Exosomes as nanocarriers for systemic delivery of the Helicobacter pylori virulence factor CagA

    PubMed Central

    Shimoda, Asako; Ueda, Koji; Nishiumi, Shin; Murata-Kamiya, Naoko; Mukai, Sada-atsu; Sawada, Shin-ichi; Azuma, Takeshi; Hatakeyama, Masanori; Akiyoshi, Kazunari

    2016-01-01

    CagA, encoded by cytotoxin-associated gene A (cagA), is a major virulence factor of Helicobacter pylori, a gastric pathogen involved in the development of upper gastrointestinal diseases. Infection with cagA-positive H. pylori may also be associated with diseases outside the stomach, although the mechanisms through which H. pylori infection promotes extragastric diseases remain unknown. Here, we report that CagA is present in serum-derived extracellular vesicles, known as exosomes, in patients infected with cagA-positive H. pylori (n = 4). We also found that gastric epithelial cells inducibly expressing CagA secrete exosomes containing CagA. Addition of purified CagA-containing exosomes to gastric epithelial cells induced an elongated cell shape, indicating that the exosomes deliver functional CagA into cells. These findings indicated that exosomes secreted from CagA-expressing gastric epithelial cells may enter into circulation, delivering CagA to distant organs and tissues. Thus, CagA-containing exosomes may be involved in the development of extragastric disorders associated with cagA-positive H. pylori infection. PMID:26739388

  1. 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

  2. Sequence analysis of East Asian cagA of Helicobacter pylori isolated from asymptomatic healthy Japanese and Thai individuals.

    PubMed

    Hirai, Itaru; Yoshinaga, Aya; Kimoto, Ai; Sasaki, Tadahiro; Yamamoto, Yoshimasa

    2011-03-01

    CagA, especially East Asian type, is one of the most important virulence factors of Helicobacter pylori, which is believed to contribute to the gastric cancer development. There is extreme sequence heterogeneity on 3' region of cagA gene, demonstrated by the sequence analysis of cagA of H. pylori strains isolated from gastric disease patients. However, whether such heterogeneity of the cagA gene sequence is related to the pathogenicity of H. pylori in the gastric cancer development is not certain. Therefore, in this study, the 3' region of cagA sequences isolated from asymptomatic healthy individuals in Japan and Thailand, which show high and low gastric cancer prevalence, respectively, were analyzed and compared with those from patients with gastric cancer. The CagA sequences analysis in 21 and 12 H. pylori DNA samples obtained from Japanese and Thai individuals, respectively, by the molecular phylogenetic method showed that the sequences were more conserved in the Thai individuals (concordance rates among Thai sequences, 93.9-100%) than in the Japanese individuals (concordance rates among Japanese sequences, 82.8-100%) as shown by unrooted neighbor-joining (N-J) consensus trees constructed with the sequence between Asn869 and Ala967 in CagA. CagA sequences in gastric cancer patients were obtained from published data; analysis of these sequences revealed that CagA sequences from almost all Thai individuals were concentrated in one branch. In contrast, CagA sequences from Japanese individuals were uniformly distributed throughout the N-J consensus tree. These results suggest that the difference in the CagA sequences between asymptomatic healthy Japanese and Thai individuals may be linked to the incidence of gastric cancer in Japan and Thailand. PMID:21046394

  3. Evaluation of the Anti-East Asian CagA-Specific Antibody for CagA Phenotyping▿

    PubMed Central

    Nguyen, Lam Tung; Uchida, Tomohisa; Kuroda, Akiko; Tsukamoto, Yoshiyuki; Trinh, Tuan Dung; Ta, Long; Mai, Hong Bang; Ho, Dang Quy Dung; Hoang, Hoa Hai; Vilaichone, Ratha-Korn; Mahachai, Varocha; Matsuhisa, Takeshi; Kudo, Yoko; Okimoto, Tadayoshi; Kodama, Masaaki; Murakami, Kazunari; Fujioka, Toshio; Yamaoka, Yoshio; Moriyama, Masatsugu

    2009-01-01

    The determination of the cagA genotype is generally based on sequencing the variable 3′ region of the cagA gene. In a previous study, we successfully generated an anti-East Asian CagA-specific antibody (anti-EAS Ab) immunoreactive only with the East Asian CagA and not with the Western CagA. In a small number of Japanese patients, anti-EAS Ab appeared to be a useful tool for phenotyping CagA immunohistochemically. The present study was conducted to validate the anti-EAS Ab immunohistochemistry method in a larger number of patients from Vietnam and Thailand. A total of 385 Vietnamese and Thais were recruited. Helicobacter pylori status was determined by a combination of three methods, including culture, histology, and immunohistochemistry with anti-H. pylori antibody. The sensitivity, specificity, and accuracy of the anti-EAS Ab immunohistochemistry method for the diagnosis of CagA phenotype were calculated based on the results of the cagA sequencing as the gold standard. The sensitivity, specificity, and accuracy of our immunohistochemistry method were 96.7%, 97.9%, and 97.1%, respectively. Moreover, anti-EAS Ab was not cross-reactive with noninfected gastric mucosa. In conclusion, immunohistochemistry with anti-EAS Ab appears to be a good method for determination of CagA phenotype. PMID:19776193

  4. A novel diagnostic monoclonal antibody specific for Helicobacter pylori CagA of East Asian type.

    PubMed

    Yasuda, Aiko; Uchida, Tomohisa; Nguyen, Lam Tung; Kawazato, Hiroaki; Tanigawa, Masato; Murakami, Kazunari; Kishida, Tetsuko; Fujioka, Toshio; Moriyama, Masatsugu

    2009-12-01

    Molecular biological and epidemiological studies have suggested that Helicobacter pylori producing East Asian CagA protein variant is more virulent than that producing Western CagA. In the present study, we developed and validated an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody specifically recognizing East Asian CagA-positive H. pylori. A total of 32 H. pylori strains were tested and the data were subjected to receiver-operator characteristic (ROC) curve analysis. The accuracy of the test, determined by calculating the area under the curve, was 0.96, which indicated a high level of accuracy. At the ROC optimized cutoff, the sensitivity and specificity of our ELISA method were 88.0% and 100%, respectively. The validated ELISA showed good performance in terms of sensitivity and specificity. These results suggest that this test is suitable for the diagnostic detection of East Asian CagA carrying strains. We also analyzed the localization of the CagA protein in H. pylori-infected gastric mucosa with fluorescence immunohistochemistry, and found that CagA protein expression was up-regulated by adhesion to epithelial cells. PMID:20078554

  5. Geographic differences and the role of cagA gene in gastroduodenal diseases associated with Helicobacter pylori infection.

    PubMed

    Valmaseda Pérez, T; Gisbert, J P; Pajares García, J M

    2001-07-01

    Helicobacter pylori (H. pylori) is the major causal agent of gastritis, peptic ulcer and gastric cancer. Several bacterium genes seem to be involved in the pathogenicity mechanism. One of them, the cagA gene, has been extensively studied and characterized. In this article we have carried out a study of characteristics and genetic variability of cagA gene in different geographic areas of the world. At the same time, we have summarized several studies that evaluate possible relation of cagA with gastroduodenal diseases associated by H. pylori infection. In our study we found that the presence of the cagA gene has been confirmed in more than 60% H. pylori strains distributed throughout the world. The prevalence of cagA genotype is of 65.4% in gastritis patients, 84.2% in patients with peptic ulcer and 86.5% in those with gastric cancer. It shows a high genetic variability of cagA associated with gastroduodenal diseases that could serve as a virulence marker in H. pylori infected subjects. However, the high prevalence of H. pylori cagA positive strains in some geographic areas does not confirm the strong association between cagA and virulence of strains as described in other countries. Nowadays, cagA gene is considered as a marker for the presence of cag pathogenicity island (cag-PAI) in H. pylori genoma. This region contains several genes that has been involved with the production of cytokines that results in an increased inflammation of host gastric mucosa, but its function is unknown. Probably, others bacterium factors, such as susceptibility host and environmental cofactors could influence in the risk of developing different gastroduodenal diseases associated with H. pylori infection. PMID:11685943

  6. 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

  7. Helicobacter pylori cagA 12-bp insertion can be a marker for duodenal ulcer in Okinawa, Japan

    PubMed Central

    Matsuo, Yuichi; Shiota, Seiji; Matsunari, Osamu; Suzuki, Rumiko; Watada, Masahide; Binh, Tran Thanh; Kinjo, Nagisa; Kinjo, Fukunori; Yamaoka, Yoshio

    2013-01-01

    Backgrounds Helicobacter pylori cagA can be classified into mainly two types (East-Asian-type and Western-type cagA) according to the repeat regions located in the 3′ region. Recent studies showed that the Western-type cagA in strains from Okinawa, Japan formed a different cluster (J-Western-type cagA subtype). We also reported that J-Western-type cagA possess a 12-bp insertion located in the 5′ region of cagA sequence. Methods The prevalence of 12-bp insertion in cagA in Okinawa and the United States (U.S.) was examined by DNA sequencing. We then designed the primer pair which can detect the 12-bp insertion only by polymerase chain reaction (PCR). The prevalence of strains with 12-bp insertion was examined in 336 strains isolated from Okinawa by PCR. Results In case of Western-type cagA/vacA s1m2 strains, the prevalence of 12-bp insertion was significantly higher in strains isolated from Okinawa than that from the U.S. (P = 0.002). Phylogenetic tree showed that strains with 12-bp insertion formed two individual clusters within J-Western-type cagA subtype; one is from Okinawa and another is from the U.S. Our designed primer set showed high sensitivity (100%) and specificity (90.8%) in Okinawa. The 12-bp insertion was found in 23.7%, 14.3%, 4.2%, and 4.0% of strains with duodenal ulcer (DU), gastritis, gastric cancer (GC), and gastric ulcer (GU), respectively (P < 0.001 for DU vs. GU) in Okinawa. Conclusions Although the mechanisms are unknown, the presence of 12-bp insertion was associated with the presence of DU and might have a suppressive action on GU and GC. PMID:23190390

  8. 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.

  9. 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

  10. Characterization of Helicobacter pylori cagA and vacA Genotypes among Alaskans and Their Correlation with Clinical Disease▿

    PubMed Central

    Miernyk, Karen; Morris, Julie; Bruden, Dana; McMahon, Brian; Hurlburt, Debby; Sacco, Frank; Parkinson, Alan; Hennessy, Thomas; Bruce, Michael

    2011-01-01

    Helicobacter pylori infection is common in Alaska. The development of severe H. pylori disease is partially determined by the virulence of the infecting strain. Here we present vacA and cagA genotype data for H. pylori strains isolated from Alaskans and their correlation with clinical disease. We enrolled patients scheduled for esophagogastroduodenoscopy and positive for H. pylori infection. Gastric biopsy specimens from the stomach antrum and fundus were cultured. We performed PCR analysis of the H. pylori vacA gene and for the presence of the cagA gene and cagA empty site. We genotyped 515 H. pylori samples from 220 Native and 66 non-Native Alaskans. We detected the cagA gene in 242/286 (85%) persons; of 222 strains that could be subtyped, 95% (212) were non-Asian cagA and 3% (6) were East Asian cagA. After removing mixed infections (n = 17), 83% of H. pylori strains had either the vacA s1m1 (120/269) or s2m2 (103/269) genotype. Sixty-six percent (68/103) of H. pylori strains with the vacA s2m2 genotype also contained the cagA gene. Infection with an H. pylori strain having the cagA gene or vacA s1m1 genotype (compared with s1m2 and s2m2) was associated with a decreased risk of esophagitis (P = 0.003 and 0.0003, respectively). Infection with an H. pylori strain having the vacA s1m1 genotype (compared with s1m2 and s2m2) was associated with an increased risk of peptic ulcer disease (PUD) (P = 0.003). The majority of H. pylori strains in this study carried the non-Asian cagA gene and either the vacA s1m1 or s2m2 genotype. A majority of H. pylori strains with the vacA s2m2 genotype also contained the cagA gene. There was an association of H. pylori genotype with esophagitis and PUD. PMID:21752979

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

    PubMed Central

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

    2015-01-01

    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. PMID:26457016

  12. Cag Type IV Secretion System: CagI Independent Bacterial Surface Localization of CagA

    PubMed Central

    Kumar, Navin; Shariq, Mohd; Kumari, Rajesh; Tyagi, Rakesh K.; Mukhopadhyay, Gauranga

    2013-01-01

    Helicobacter pylori Cag type IV secretion system (Cag-T4SS) is a multi-component transporter of oncoprotein CagA across the bacterial membranes into the host epithelial cells. To understand the role of unique Cag-T4SS component CagI in CagA translocation, we have characterized it by biochemical and microscopic approaches. We observed that CagI is a predominantly membrane attached periplasmic protein partially exposed to the bacterial surface especially on the pili. The association of the protein with membrane appeared to be loose as it could be easily recovered in soluble fraction. We documented that the stability of the protein is dependent on several key components of the secretion system and it has multiple interacting partners including a non-cag-PAI protein HP1489. Translocation of CagA across the bacterial membranes to cell surface is CagI-independent process. The observations made herein are expected to assist in providing an insight into the substrate translocation by the Cag-T4SS system and Helicobacter pylori pathogenesis. PMID:24040297

  13. Helicobacter pylori CagA Suppresses Apoptosis through Activation of AKT in a Nontransformed Epithelial Cell Model of Glandular Acini Formation

    PubMed Central

    Vallejo-Flores, Gabriela; Torres, Javier; Sandoval-Montes, Claudia; Arévalo-Romero, Haruki; Meza, Isaura; Camorlinga-Ponce, Margarita; Torres-Morales, Julián; Chávez-Rueda, Adriana Karina; Legorreta-Haquet, María Victoria; Fuentes-Pananá, Ezequiel M.

    2015-01-01

    H. pylori infection is the most important environmental risk to develop gastric cancer, mainly through its virulence factor CagA. In vitro models of CagA function have demonstrated a phosphoprotein activity targeting multiple cellular signaling pathways, while cagA transgenic mice develop carcinomas of the gastrointestinal tract, supporting oncogenic functions. However, it is still not completely clear how CagA alters cellular processes associated with carcinogenic events. In this study, we evaluated the capacity of H. pylori CagA positive and negative strains to alter nontransformed MCF-10A glandular acini formation. We found that CagA positive strains inhibited lumen formation arguing for an evasion of apoptosis activity of central acini cells. In agreement, CagA positive strains induced a cell survival activity that correlated with phosphorylation of AKT and of proapoptotic proteins BIM and BAD. Anoikis is a specific type of apoptosis characterized by AKT and BIM activation and it is the mechanism responsible for lumen formation of MCF-10A acini in vitro and mammary glands in vivo. Anoikis resistance is also a common mechanism of invading tumor cells. Our data support that CagA positive strains signaling function targets the AKT and BIM signaling pathway and this could contribute to its oncogenic activity through anoikis evasion. PMID:26557697

  14. 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

  15. Influence of H. pylori CagA Coupled with Alcohol Consumption on Cytokine Profiles in Men

    PubMed Central

    Qu, Baoge; Han, Xinghai; Ren, Guangying; Jia, Yiguo; Liu, Yuanxun; Su, Jiliang; Wang, Zhongdong; Wang, Yafei; Wang, Hui; Pan, Jindun; Liu, Li-li; Hu, Wen-Juan

    2016-01-01

    Abstract The aim of this study was to evaluate the effect of Helicobacter pylori (H pylori) cytotoxin-associated gene A (CagA) coupled with chronic alcohol ingestion on cytokine profiles. A total of 215 male subjects were divided into the following 4 groups: 130 alcohol H pylori CagA-negative consumers (CagA−) (group A), 50 alcohol H pylori CagA-positive consumers (CagA+) (group B), 24 nonalcohol H pylori CagA-negative consumers (group C), and 11 nonalcohol H pylori CagA-positive consumers (group D). The serum CagA, C-reactive protein (CRP), interleukin (IL)-6, IL-10, E-selectin, adiponectin (ADP), and tumor necrosis factor-α (TNF-α) levels were measured through enzyme-linked immunosorbent assays (ELISAs). After adjusting for age and mean alcohol drinking history, a multivariable linear regression analysis revealed that the mean daily alcohol consumption, IL-6, TNF-α, and ADP levels were significantly increased with increases in the serum CagA concentrations (P = 0.008, P = 0.000, P = 0.000, and P = 0.006, respectively). The serum IL-6 and IL-10 levels of group A were significantly lower than those of group B (all P = 0.000). Furthermore, the serum IL-6 and IL-10 levels of groups A and C were significantly lower than those of group D (all P = 0.000), and the serum IL-6 and IL-10 levels of group C were significantly lower than those of group B (all P = 0.000). The serum ADP and E-selectin levels of groups B and D were significantly higher than those of group A (P = 0.000). The serum ADP levels of group B were significantly higher than those of group C (P = 0.000), and the serum ADP and E-selectin levels of group C were significantly lower than those of group D (P = 0.000 and P = 0.005, respectively). Finally, the serum TNF-α levels of groups B, C, and D were significantly higher than those of group A (all P = 0.000), and the serum TNF-α levels of group C were significantly higher than those of group D (P = 0

  16. Helicobacter pylori Infection in Thailand: A Nationwide Study of the CagA Phenotype

    PubMed Central

    Uchida, Tomohisa; Miftahussurur, Muhammad; Pittayanon, Rapat; Vilaichone, Ratha-korn; Wisedopas, Naruemon; Ratanachu-ek, Thawee; Kishida, Tetsuko; Moriyama, Masatsugu; Yamaoka, Yoshio; Mahachai, Varocha

    2015-01-01

    Background The risk to develop gastric cancer in Thailand is relatively low among Asian countries. In addition, the age-standardized incidence rate (ASR) of gastric cancer in Thailand varies with geographical distribution; the ASR in the North region is 3.5 times higher than that in the South region. We hypothesized that the prevalence of H. pylori infection and diversity of CagA phenotype contributes to the variety of gastric cancer risk in various regions of Thailand. Methods We conducted a nationwide survey within Thailand. We determined H. pylori infection prevalence by detecting H. pylori, using histochemical and immunohistochemical methods. The anti-CagA antibody and anti-East-Asian type CagA antibody (α-EAS Ab), which showed high accuracy in several East Asian countries, were used to determine CagA phenotype. Results Among 1,546 patients from four regions, including 17 provinces, the overall prevalence of H. pylori infection was 45.9% (710/1,546). Mirroring the prevalence of H. pylori infection, histological scores were the lowest in the South region. Of the 710 H. pylori-positive patients, 93.2% (662) were immunoreactive with the anti-CagA antibody. CagA-negative strain prevalence in the South region was significantly higher than that in other regions (17.9%; 5/28; p < 0.05). Overall, only 77 patients (11.6%) were immunoreactive with the α-EAS Ab. There were no differences in the α-EAS Ab immunoreactive rate across geographical regions. Conclusions This is the first study using immunohistochemistry to confirm H. pylori infections across different regions in Thailand. The prevalence of East-Asian type CagA H. pylori in Thailand was low. The low incidence of gastric cancer in Thailand may be attributed to the low prevalence of precancerous lesions. The low incidence of gastric cancer in the South region might be associated with the lower prevalence of H. pylori infection, precancerous lesions, and CagA-positive H. pylori strains, compared with that in the

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

    PubMed

    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

  18. 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

  19. 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

  20. 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

  1. 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

  2. Role of the Helicobacter pylori virulence factors vacuolating cytotoxin, CagA, and urease in a mouse model of disease.

    PubMed Central

    Ghiara, P; Marchetti, M; Blaser, M J; Tummuru, M K; Cover, T L; Segal, E D; Tompkins, L S; Rappuoli, R

    1995-01-01

    The pathogenic role of Helicobacter pylori virulence factors has been studied with a mouse model of gastric disease. BALB/c mice were treated orally with different amounts of sonic extracts of cytotoxic H. pylori strains (NCTC 11637, 60190, 84-183, and 87A300 [CagA+/Tox+]). The pathological effects on histological sections of gastric mucosae were assessed and were compared with the effects of treatments with extracts from noncytotoxic strains (G21 and G50 [CagA-/Tox-]) and from strains that express either CagA alone (D931 [CagA+/Tox-]) or the cytotoxin alone (G104 [CagA-/Tox+]). The treatment with extracts from cytotoxic strains induced various epithelial lesions (vacuolation, erosions, and ulcerations), recruitment of inflammatory cells in the lamina propria, and a marked reduction of the mucin layer. Extracts of noncytotoxic strains induced mucin depletion but no other significant pathology. Crude extracts of strain D931, expressing CagA alone, caused only mild infiltration of inflammatory cells, whereas extracts of strain G104, expressing cytotoxin alone, induced extensive epithelial damage but little inflammatory reaction. Loss of the mucin layer was not associated with a cytotoxic phenotype, since this loss was observed in mice treated with crude extracts of all strains. The pathogenic roles of CagA, cytotoxin, and urease were further assessed by using extracts of mutant strains of H. pylori defective in the expression of each of these virulence factors. The results obtained suggest that (i) urease activity does not play a significant role in inducing the observed gastric damage, (ii) cytotoxin has an important role in the induction of gastric epithelial cell lesions but not in eliciting inflammation, and (iii) other components present in strains which carry the cagA gene, but distinct from CagA itself, are involved in eliciting the inflammatory response. PMID:7558333

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

    PubMed

    Zhang, Bao-Gui; Hu, Lei; Zang, Ming De; Wang, He-Xiao; Zhao, Wei; Li, Jian-Fang; Su, Li-Ping; Shao, Zhifeng; Zhao, Xiaodong; Zhu, Zheng-Gang; Yan, Min; Liu, Bingya

    2016-03-01

    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

  4. 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

  5. CagA promotes proliferation and secretion of extracellular matrix by inhibiting signaling pathway of apoptosis in rat glomerular mesangial cells.

    PubMed

    Wang, Li; Tan, Rui-Zhi; Chen, Yue; Wang, Hong-Lian; Liu, Yu-Hang; Wen, Dan; Fan, Jun-Ming

    2016-04-01

    Cytotoxin-associated antigen A (CagA), a major virulence factor of Helicobacter pylori (Hp), is associated with the pathogenesis of peptic ulcer and gastric cancer. Recent researches demonstrated that Hp exists in palatine tonsil in all studied IgA nephropathy (IgAN) patients, most of which were CagA-positive, suggesting that CagA may be a causative pathogenic factor of IgAN. However, the underlying molecular mechanisms and signaling pathway are still largely unclear. In the present study, CCK8 assay, enzyme-linked immunosorbent assay, and immunohistochemistry were performed to investigate the effect of CagA on cell proliferation and extracellular matrix secretion in rat glomerular mesangial cells. RT-PCR and western blotting were used to reveal the potential signaling pathway. Rat glomerular mesangial cells were treated with recombinant CagA protein for 72 h, in a dose- and time-dependent manner. We found that CagA promoted cell proliferation and extracellular matrix secretion by inhibiting signaling pathway of apoptosis. Taken together, these findings suggested that CagA induced cellular injury in glomerular mesangium by proliferation and secretion of extracellular matrix, and may play an important role in pathogenesis of IgAN. PMID:26837331

  6. Role of vacuolating cytotoxin VacA and cytotoxin-associated antigen CagA of Helicobacter pylori in the progression of gastric cancer.

    PubMed

    Ki, Mi-Ran; Hwang, Meeyul; Kim, Ah-Young; Lee, Eun-Mi; Lee, Eun-Joo; Lee, Myeong-Mi; Sung, Soo-Eun; Kim, Sang-Hyeob; Lee, Hye Seung; Jeong, Kyu-Shik

    2014-11-01

    Helicobacter (H.) pylori strains that express the cagA and s1a vacA genes are associated with an increased risk for gastric cancer. Here, we examined the association between the products of these virulence genes with the development of gastric cancer by immunohistochemical staining of gastric biopsy specimens taken from 208 routine gastroscopies and 43 gastric cancer patients. The correlation was analyzed by multivariate logistic regression. CagA and VacA expressions in gastric mucosa were significantly associated with chronic gastritis (CG) and intestinal metaplasia (IM), respectively, accompanying CG independent of age. The association of CagA expression with IM accompanying CG was increased in patients over 50-year old (p < 0.01) and that of VacA with CG was significant in patients younger than 50 year (p < 0.05). VacA and CagA were associated with mild IM incidence (p = 0.025 and p = 0.076, respectively) but not advanced IM. In the 43 gastric cancer patients, positivity for VacA was significantly higher in cases of CG and IM than carcinoma (p = 0.042), while that for CagA was slightly higher for individuals with carcinoma than those with CG and IM. These results indicate that CagA and VacA are critical factors for inducing CG and the subsequent progression of IM from CG with an increasing age. PMID:25038872

  7. 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

  8. 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

  9. Transgenic expression of the Helicobacter pylori virulence factor CagA promotes apoptosis or tumorigenesis through JNK activation in Drosophila.

    PubMed

    Wandler, Anica M; Guillemin, Karen

    2012-01-01

    Gastric cancer development is strongly correlated with infection by Helicobacter pylori possessing the effector protein CagA. Using a transgenic Drosophila melanogaster model, we show that CagA expression in the simple model epithelium of the larval wing imaginal disc causes dramatic tissue perturbations and apoptosis when CagA-expressing and non-expressing cells are juxtaposed. This cell death phenotype occurs through activation of JNK signaling and is enhanced by loss of the neoplastic tumor suppressors in CagA-expressing cells or loss of the TNF homolog Eiger in wild type neighboring cells. We further explored the effects of CagA-mediated JNK pathway activation on an epithelium in the context of oncogenic Ras activation, using a Drosophila model of metastasis. In this model, CagA expression in epithelial cells enhances the growth and invasion of tumors in a JNK-dependent manner. These data suggest a potential role for CagA-mediated JNK pathway activation in promoting gastric cancer progression. PMID:23093933

  10. Serum Helicobacter pylori CagA antibody as a biomarker for gastric cancer in east-Asian countries

    PubMed Central

    Shiota, Seiji; Matsunari, Osamu; Watada, Masahide; Yamaoka, Yoshio

    2011-01-01

    Aims In east-Asian countries, while almost all Helicobacter pylori strains possess the cytotokine-associated gene A (CagA) gene, serum CagA antibody is not detected in some infected subjects. We aimed to clarify the association between anti-CagA antibody and gastric cancer in east-Asian countries. Materials & methods We performed a meta-analysis of case–control studies with age- and sex-matched controls, which provided raw data in east-Asian countries. Results Ten studies with a total of 4325 patients were identified in the search. Some reports from Japan, Korea and China showed a positive association between the presence of anti-CagA antibody and gastric cancer; however, the results differed in their various backgrounds. The disparate findings appeared to result from the use of different methods or from variations in the antigens used to detect the anti-CagA antibody. CagA seropositivity was associated with an increased risk of developing gastric cancer. Conclusion Anti-CagA antibody can be used as a biomarker for gastric cancer even in east-Asian countries. PMID:21155667

  11. Genotyping of the Helicobacter pylori cagA Gene Isolated From Gastric Biopsies in Shiraz, Southern Iran: A PCR-RFLP and Sequence Analysis Approach

    PubMed Central

    Moaddeb, Afsaneh; Fattahi, Mohammad Reza; Firouzi, Roya; Derakhshandeh, Abdollah; Farshad, Shohreh

    2016-01-01

    Background Cytotoxin-associated gene A (cagA) is an important virulence factor in the pathogenesis of Helicobacter pylori. Objectives The aim of this study was to genotype the H. pylori cagA gene isolated from antral biopsies of patients with stomach symptoms, using a PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. Patients and Methods A total of 161 gastric biopsies were collected from patients with stomach symptoms. After isolation of H. pylori from the biopsy culture, the cagA gene was assessed using PCR. The PCR products were then digested by the HinfI restriction endonuclease enzyme. A sample of each genotype was also subjected to direct sequencing for further analysis. Results From 161 antral biopsies, 61 (37.9%) were positive for H. pylori in culture. Overall, 24 cagA-positives were detected in the isolates. RFLP indicated three different genotypes (I, II, and III) of cagA with a frequency of 62.5%, 25%, and 12.5% among the isolates, respectively. Genotypes I and II of cagA were predominant in patients who had gastritis. However, genotype III was found in three patients with duodenitis and duodenal ulcers. Alignment of the nucleotide sequences of the three isolated genotypes, with H. pylori 26695 as a reference strain, revealed 12 inserted nucleotides in genotype III. When the sequence of genotype III was aligned with 15 additional H. pylori strains available in GenBank, the same inserted nucleotides were detected in six of them. Conclusions Using the PCR-RFLP method, three distinctive H. pylori cagA genotypes were detected in antral biopsies. Genotype I, which was predominant among the isolates, was significantly associated with gastritis. However, the data showed that cagA genotype III may play a role in duodenitis and duodenal ulcers in patients infected with H. pylori. PMID:27335631

  12. Non-invasive Genotyping of Helicobacter pylori cagA, vacA, and hopQ from Asymptomatic Children

    PubMed Central

    Sicinschi, Liviu A.; Correa, Pelayo; Bravo, Luis E.; Peek, Richard M.; Wilson, Keith T.; Loh, John T.; Yepez, Maria C.; Gold, Benjamin D.; Thompson, Dexter T.; Cover, Timothy L.; Schneider, Barbara G.

    2011-01-01

    Background H. pylori infection is usually acquired in childhood, but little is known about its natural history in asymptomatic children, primarily due to the paucity of non-invasive diagnostic methods. H. pylori strains harboring cagA and specific alleles of hopQ and vacA are associated with increased risk for gastric cancer. Many studies of H. pylori virulence markers in children have the bias that symptomatic subjects are selected for endoscopy, and these children may harbor the most virulent strains. Our aim: to genotype cagA, hopQ and vacA alleles in stool DNA samples of healthy Colombian children residing in an area with high incidence of gastric cancer, in order to avoid selection bias resulting from endoscopy. Methods H. pylori status of 86 asymptomatic children was assessed by 13C-Urea Breath Test (UBT) and PCR. H. pylori 16S rRNA, cagA, hopQ and vacA genes were amplified from stool DNA samples and sequenced. Results UBT was positive in 69 (80.2%) of 86 children; in stool DNA analysis, 78.3% were positive by 16S rRNA PCR. cagA, vacA and hopQ were detected in 66.1%, 84.6%, and 72.3% of stool DNA samples from 16S rRNA positive children. Of the children's DNA samples which revealed vacA and hopQ alleles, 91.7% showed vacA s1 and 73.7% showed type I hopQ. Type I hopQ alleles were associated with cagA-positivity and vacA s1 genotypes (P<0.0001). Conclusions Using stool DNA samples, virulence markers of H. pylori were successfully genotyped in a high percentage of the asymptomatic infected children, revealing a high prevalence of genotypes associated with virulence. Type I hopQ alleles were associated with the presence of cagA and the vacA s1 genotype. PMID:22404439

  13. Prevalence and correlation with clinical diseases of Helicobacter pylori cagA and vacA genotype among gastric patients from Northeast China.

    PubMed

    Aziz, Faisal; Chen, Xin; Yang, Xuesong; Yan, Qiu

    2014-01-01

    Helicobacter pylori vacA and cagA genes have significant genetic heterogenicity, resulting in different clinical outcomes. Northeast part of China has reported high prevalence of H. pylori infections and gastric cancer. Hence, we investigated the H. pylori cagA and vacA genotypes with clinical outcomes in Northeast China. Gastric tissue samples (n = 169), chronic gastritis (GIs), gastric ulcer (GU), and gastric cancer (GC) were analysed for 16S rRNA ureA, cagA, and cagA genotypes by PCR. A total of 141 (84%) cases were found positive for H. pylori by 16S rRNA and ureA. GC showed high H. pylori infection (93%) compared with GIs (72%) and GU (84%). The vacAs1am1 was highly found in GC (40%) and GU (36%), vacAs1am2 in GIs (33%), vacAs1bm1 (14%) and vacAs1bm2 (8%) in GU cases, and s2m1 in normal cases (33%), while vacAs1cm1 showed low frequency in GIs (2%) and GU (3%) and GC showed negative result. The East-Asian cagA strain was highly observed in GC (43%), as compared to GIs (41%) and GU (20%). The East-Asian cagA/vacAs1am1 was significantly higher in GC (23%) than in GU (22%) and GIs (145) patients. The East-Asian type cagA with vacAs1a and vacAm1 is the most predominant genotype in H. pylori strains of Northeast China. PMID:24949419

  14. 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

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

    PubMed

    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

  16. Detection of serum antibodies to CagA and VacA and of serum neutralizing activity for vacuolating cytotoxin in patients with Helicobacter pylori-induced gastritis.

    PubMed Central

    Donati, M; Moreno, S; Storni, E; Tucci, A; Poli, L; Mazzoni, C; Varoli, O; Sambri, V; Farencena, A; Cevenini, R

    1997-01-01

    Thirty patients with dyspepsia, with histological diagnosis of gastritis, and with endoscopic diagnosis of peptic ulcer disease (PUD) (n = 13) or nonulcer dyspepsia (NUD) (n = 17) were admitted to the study. Helicobacter pylori vacuolating cytotoxin-producing strains (Tox+) were isolated from 14 (46.7%) patients, whereas non-cytotoxin-producing (Tox-) H. pylori strains were isolated from the remaining patients. Of 30 patients studied, 20 (66.7%) had serum cytotoxin neutralizing activity in vitro. Fourteen patients with Tox+ H. pylori strains showed serum cytotoxin neutralizing activity and serum immunoglobulin G (IgG) and IgA antibodies reactive with both 87-kDa H. pylori vacuolating cytotoxin (VacA) and 128-kDa cytotoxin-associated gene product (CagA) by immunoblotting using native enriched preparations of VacA and CagA proteins from H. pylori culture supernatants as the antigens. A 94-kDa antigen cross-reacting with the 87-kDa VacA protein could be demonstrated in culture supernatant with immune sera from humans and animals. All patients (n = 10) lacking serum neutralizing activity were also negative for IgG or IgA against VacA antigen, whereas 6 of the 10 patients showed IgG serum antibody responses against CagA antigen. The prevalence of antibodies to VacA and CagA antigens was significantly (P < 0.001) higher in patients with gastritis (20 and 26 patients for VacA and CagA, respectively, of 30 patients) than in H. pylori culture-negative controls (0 of 27 for both VacA and CagA) and in randomly selected blood donors (17 and 21 for VacA and CagA, respectively, of 120 subjects). All patients with PUD had antibodies to CagA, whereas 13 of 17 (76.5%) patients with NUD had anti-CagA antibodies. Serum IgG antibodies to VacA were present in 9 (69.2%) patients with PUD of 13 patients and in 11 (64.7%) patients with NUD of 17 patients. Anti-CagA antibodies seemed to correlate better with PUD than anti-VacA antibodies. PMID:9220168

  17. Heterogeneity in the Helicobacter pylori vacA and cagA genes: association with gastroduodenal disease in South Africa?

    PubMed Central

    Kidd, M; Lastovica, A; Atherton, J; Louw, J

    1999-01-01

    BACKGROUND—Helicobacter pylori infection is universally associated with gastritis, but only sometimes with clinically significant disease. Candidate virulence markers seem to be useful in identifying the pathogenic infections in some populations.
AIMS—To investigate the association between putative virulence markers and disease in an African population.
METHODS—Fifty nine H pylori strains isolated from dyspeptic patients (11 with peptic ulceration, eight with gastric adenocarcinoma, and 28 with no pathology other than gastritis) were studied for differences in the genes vacA and cagA.
RESULTS—Forty seven (80%) of 59 strains had the vacA signal sequence genotype s1 (one s1a, 46 s1b) and 12 (20%) had subtype s2. vacA mid-region analysis revealed that 40 (68%) strains were vacA m1 and 19 (32%) were m2. All 14 strains from patients with peptic ulceration were vacA s1, in contrast to 23 (66%) of 35 strains from patients with gastritis alone (p<0.01). vacA s2 was found exclusively in patients with gastritis alone (p<0.01). All strains isolated from patients with gastric adenocarcinoma were s1b/m1 (p<0.005 versus gastritis alone). cagA was detectable in 56 (95%) of 59 isolates. Strains from patients with peptic ulceration (12/13 versus 19/30 with gastritis alone, p=0.05) had the shortest fragment length in the 3' region of cagA, while 4/10 strains from patients with gastric cancer had the longest fragment length in this region (p<0.02 versus gastritis alone).
CONCLUSION—In this study, the vacA s1 genotype, and fragment length of the 3' region of cagA identified isolates associated with significant clinical disease. The vacA s1bm1 genotype seems to be strongly associated with gastric cancer.


Keywords: adenocarcinoma; cagA; Helicobacter pylori; peptic ulceration; South Africa; vacuolating cytotoxin PMID:10486355

  18. 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.

  19. 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.

  20. A Tale of Two Toxins: Helicobacter Pylori CagA and VacA Modulate Host Pathways that Impact Disease

    PubMed Central

    Jones, Kathleen R.; Whitmire, Jeannette M.; Merrell, D. Scott

    2010-01-01

    Helicobacter pylori is a pathogenic bacterium that colonizes more than 50% of the world's population, which leads to a tremendous medical burden. H. pylori infection is associated with such varied diseases as gastritis, peptic ulcers, and two forms of gastric cancer: gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. This association represents a novel paradigm for cancer development; H. pylori is currently the only bacterium to be recognized as a carcinogen. Therefore, a significant amount of research has been conducted to identify the bacterial factors and the deregulated host cell pathways that are responsible for the progression to more severe disease states. Two of the virulence factors that have been implicated in this process are cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA), which are cytotoxins that are injected and secreted by H. pylori, respectively. Both of these virulence factors are polymorphic and affect a multitude of host cellular pathways. These combined facts could easily contribute to differences in disease severity across the population as various CagA and VacA alleles differentially target some pathways. Herein we highlight the diverse types of cellular pathways and processes targeted by these important toxins. PMID:21687723

  1. 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

  2. CagA status & genetic characterization of metronidazole resistant strains of H. pylori from: A region at high risk of gastric cancer

    PubMed Central

    Yue, Jin-Yong; Yue, Jing; Wang, Ming-Yi; Song, Wen-chong; Gao, Xiao-Zhong

    2014-01-01

    Objective: The aim of study was to determine relationship between cagA and genetic characterization of metronidazole (MTZ) resistant H. pylori strains from a region at high risk of gastric cancer. Methods: 172 H. pylori strains were isolated from the patients with dyspeptic symptoms, and antimicrobial susceptibility testing for MTZ was assessed by E-test. rdxA and frxA genes were amplified using PCR among the MTZ resistant isolates. The status of the plasmid and classes 1~3 integrons were investigated in all isolates. Results: MTZ was detected in 88 isolates (51.16%). Variations in the rdxA gene leading to alterations of amino acids in RdxA proteins were identified in all MTZ resistant strains. FrxA contained missense alterations in 55 MTZ resistant isolates, while the premature truncation of FrxA was caused by frameshift mutations in 9 MTZ resistant strains. Plasmid was found in one MTZ sensitive strain (0.58%), and none of Class 1~3 integrases gene was detected in the studied isolates. The conservative cagA fragment was obtained from all clinical isolates of H. pylori. The sequence of cagA 3' variable region in 164 strains were obtained, including East Asian-type (122, 74.39%) and Western-type (42, 25.61%). Prevalence of Western-type cagA 3' variable region was significantly higher in MTZ resistant (33.73%, 28/83) than those of MTZ-sensitive strains (17.28%, 14/81) (p=0.02). Conclusion: A high prevalence of MTZ resistance was found in the region, and bacterial chromosome mutations in the rdxA and frxA gene still contribute to the high-level MTZ resistance. H. pylori strains characterized with West-type cagA 3’ variable region tend to acquire MTZ resistance in the region. PMID:25097521

  3. 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

    2015-07-01

    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. PMID:26230117

  4. 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

  5. Structural basis of tubulin tyrosination by tubulin tyrosine ligase

    PubMed Central

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

    2013-01-01

    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

  6. 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

  7. XAFS of human tyrosine hydroxylase

    NASA Astrophysics Data System (ADS)

    Meyer, W.; Haavik, J.; Winkler, H.; Trautwein, A. X.; Nolting, H.-F.

    1995-02-01

    Tyrosine hydroxylase (TH) catalyses the rate-limiting step (hydroxylation of tyrosine to form dihydroxyphenylalanine) in the biosynthetic pathway leading to the catecholamines dopamine, noradrenaline and adrenaline. The human enzyme (hTH) is present in four isoforms, generated by splicing of pre-mRNA. The purified apoenzyme (metal free) binds stoichiometric amounts of iron. The incorporation of Fe(II) results in a rapid and up to 40-fold increase of activity [1]. Besides the coordination of the metal centers in native enzyme we studied the purported inhibition of TH by its immediate products. So we analysed Fe-hTH isoform 1 native as well as oxidized with dopamine and Co-hTH isoform 2.

  8. 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

  9. 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

  10. Distinct Diversity of vacA, cagA, and cagE Genes of Helicobacter pylori Associated with Peptic Ulcer in Japan

    PubMed Central

    Yamazaki, Shiho; Yamakawa, Akiyo; Okuda, Tomoyuki; Ohtani, Masahiro; Suto, Hiroyuki; Ito, Yoshiyuki; Yamazaki, Yukinao; Keida, Yoshihide; Higashi, Hideaki; Hatakeyama, Masanori; Azuma, Takeshi

    2005-01-01

    Colonization of the stomach mucosa by Helicobacter pylori is a major cause of acute and chronic gastric pathologies in humans. Several H. pylori virulence genes that may play a role in its pathogenicity have been identified. The most important determinants are vacA and cagA in the cag pathogenicity island (cagPAI) genes. In the present study, to consider the association of molecular genetics between vacA and the cagPAI regarding clinical outcome, we selected H. pylori strains with various genotypes of vacA in Japan and sequenced full-length vacA, cagA, and cagE genes. Sequencing of vacA and cagA genes revealed variable size, whereas the cagE gene was well conserved among strains. Each of the phylogenetic trees based on the deduced amino acid sequences of VacA, CagA, and CagE indicated that all three proteins were divided into two major groups, a Western group and an East Asian group, and the distributions of isolates exhibited similar patterns among the three proteins. The strains with s2 and s1a/m1a vacA genotypes and the Western-type 3′ region cagA genotype were classified into the Western group, and the strains with the s1c/m1b vacA genotype and the East Asian-type 3′ cagA genotype were included in the East Asian group. In addition, the prevalence of infection with the Western group strain was significantly higher in patients with peptic ulcer (90.0%, 9/10) than in patients with chronic gastritis (22.7%, 5/22) (χ2 = 12.64, P = 0.00057). These data suggest that the molecular genetics of vacA and cagPAI are associated and that the Western group with vacA and cagPAI genes is associated with peptic ulcer disease. PMID:16081930

  11. Distinct diversity of vacA, cagA, and cagE genes of Helicobacter pylori associated with peptic ulcer in Japan.

    PubMed

    Yamazaki, Shiho; Yamakawa, Akiyo; Okuda, Tomoyuki; Ohtani, Masahiro; Suto, Hiroyuki; Ito, Yoshiyuki; Yamazaki, Yukinao; Keida, Yoshihide; Higashi, Hideaki; Hatakeyama, Masanori; Azuma, Takeshi

    2005-08-01

    Colonization of the stomach mucosa by Helicobacter pylori is a major cause of acute and chronic gastric pathologies in humans. Several H. pylori virulence genes that may play a role in its pathogenicity have been identified. The most important determinants are vacA and cagA in the cag pathogenicity island (cagPAI) genes. In the present study, to consider the association of molecular genetics between vacA and the cagPAI regarding clinical outcome, we selected H. pylori strains with various genotypes of vacA in Japan and sequenced full-length vacA, cagA, and cagE genes. Sequencing of vacA and cagA genes revealed variable size, whereas the cagE gene was well conserved among strains. Each of the phylogenetic trees based on the deduced amino acid sequences of VacA, CagA, and CagE indicated that all three proteins were divided into two major groups, a Western group and an East Asian group, and the distributions of isolates exhibited similar patterns among the three proteins. The strains with s2 and s1a/m1a vacA genotypes and the Western-type 3' region cagA genotype were classified into the Western group, and the strains with the s1c/m1b vacA genotype and the East Asian-type 3' cagA genotype were included in the East Asian group. In addition, the prevalence of infection with the Western group strain was significantly higher in patients with peptic ulcer (90.0%, 9/10) than in patients with chronic gastritis (22.7%, 5/22) (chi2 = 12.64, P = 0.00057). These data suggest that the molecular genetics of vacA and cagPAI are associated and that the Western group with vacA and cagPAI genes is associated with peptic ulcer disease. PMID:16081930

  12. 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

  13. Zur Biosynthese von Phenylalanin und Tyrosin

    NASA Astrophysics Data System (ADS)

    Lingens, F.; Keller, E.

    1983-03-01

    With the discovery of arogenic acid two new pathways for the biosynthesis of phenylalanine and tyrosine have been revealed. The occurrence of two, three, or four pathways for the biosynthesis of phenylalanine and tyrosine in microorganisms and plants may be a useful tool for taxonomic classifications. Investigations on enterobacteriaceae, pseudomonads, flavobacteria, streptomycetes, archaebacteria, and on Sphaerotilus, Trichococcus and Leptothrix species from bulking sludge are described. The possible role of arogenate in the evolution of the pathways for tyrosine and phenylalanine biosynthesis is discussed.

  14. Evaluation of Clarithromycin Resistance and cagA and vacA Genotyping of Helicobacter pylori Strains from the West of Ireland Using Line Probe Assays

    PubMed Central

    Ryan, Kieran A.; van Doorn, Leen-Jan; Moran, Anthony P.; Glennon, Maura; Smith, Terry; Maher, Majella

    2001-01-01

    The prevalence of clarithromycin resistance-associated mutations, the cytotoxin-associated gene (cagA), and the various vacuolating cytotoxin (vacA) genotypes was determined in 50 gastric biopsy specimens from Helicobacter pylori-infected patients, using line probe assays. The clarithromycin resistance-associated mutation A2143G was detected in H. pylori strains from 26% of the specimens, which suggested that the high rate of H. pylori treatment failure in Ireland may be partly attributable to the presence of these mutations. All strains examined carried the vacA s1 genotype, and 76% were cagA positive. Of these 50 specimens, 13 (26%) carried H. pylori strains with vacA midregion genotype m1, 29 (58%) carried strains that were m2, 1 (2%) was infected by a strain that was positive for both m1 and m2, and 7 (14%) carried strains that could not be typed. PMID:11326028

  15. 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

  16. Disruption of cagA, the apoprotein gene of chromoprotein antibiotic C-1027, eliminates holo-antibiotic production, but not the cytotoxic chromophore.

    PubMed

    Cui, Zhihui; Wang, Lifei; Wang, Songmei; Li, Guangwei; Hong, Bin

    2009-11-01

    C-1027 is a chromoprotein of the nine-membered enediyne antitumour antibiotic family, comprising apoprotein to stabilize and transport the enediyne chromophore. The disruption of apoprotein gene cagA within the C-1027 biosynthetic gene cluster abolished C-1027 holo-antibiotic production detected by an antibacterial assay, as well as the expression of the apoprotein and C-1027 chromophore extracted following protein precipitation of the culture supernatant. Complementation of the cagA-disrupted mutant AKO with the intact cagA gene restored C-1027 production, suggesting that cagA is indispensable for holo-antibiotic production. Overexpression of cagA in the wild-type strain resulted in a significant increase in C-1027 production as expected. Surprisingly, electrospray ionization (ESI)-MS and ESI-MS/MS analyses suggested that the AKO mutant still produced the C-1027 enediyne chromophore [m/z=844 (M+H)(+)] and its aromatized product [m/z=846 (M+H)(+)]. Consistent with this, the results from gene expression analysis using real-time reverse transcriptase-PCR showed that transcripts of the positive regulator sgcR3 and the structural genes sgcA1, sgcC4, sgcD6 and sgcE were readily detected in the AKO mutant as well as in the wild-type and the complementation strain. These results provided, for the first time, evidence suggesting that the apoprotein of C-1027 is not essential in the self-resistance mechanism for the enediyne chromophore. PMID:19845765

  17. 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

  18. 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.

  19. 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

  20. 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

  1. Simultaneous Identification of Tyrosine Phosphorylation and Sulfation Sites Utilizing Tyrosine-Specific Bromination

    NASA Astrophysics Data System (ADS)

    Kim, Jong-Seo; Song, Si-Uk; Kim, Hie-Joon

    2011-11-01

    Tyrosine phosphorylation and sulfation play many key roles in the cell. Isobaric phosphotyrosine and sulfotyrosine residues in peptides were determined by mass spectrometry using phosphatase or sulfatase to remove the phosphate or the sulfate group. Unique Br signature was introduced to the resulting tyrosine residues by incubation with 32% HBr at -20 °C for 20 min. MS/MS analysis of the brominated peptide enabled unambiguous determination of the phosphotyrosine and the sulfotyrosine sites. When phosphotyrosine and sulfotyrosine as well as free tyrosine were present in the same peptide, they could be determined simultaneously using either phosphatase or sulfatase following acetylation of the free tyrosine.

  2. 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

  3. 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.

  4. Distribution of Helicobacter pylori cagA, cagE, oipA and vacA in different major ethnic groups in Tehran, Iran

    PubMed Central

    Dabiri, Hossein; Maleknejad, Parviz; Yamaoka, Yoshio; Feizabadi, Mohammad M; Jafari, Fereshteh; Rezadehbashi, Maryam; Nakhjavani, Farrokh A; Mirsalehian, Akbar; Zali, Mohammad R

    2011-01-01

    Background and Aim There are geographical variations in Helicobacter pylori virulence genes; cagA, cagE, vacA and oipA. The present study compared the distribution of these genotypes in major ethnic groups residing in Tehran, Iran and their association with clinical outcomes. Methods A total of 124 H. pylori-positive patients living in Tehran were enrolled in this study. The ethnic distribution was 74 Persians, 33 Turks and 17 other ethnics including Kurds, Lurs, Afghanis and Arabs. The presence of the cagA, cagE and oipA genes and vacA alleles (signal [s] and middle [m] region) were determined by polymerase chain reaction (PCR) from H. pylori DNA. Results The cagA-positive status was predominant in all three ethnic groups (e.g. 65% in Persians and 73% in Turks). In contrast, the cagE-positive status was less than half in Persians (47%) and Turks (30%), whereas it was 77% in other ethnicities (P = 0.008). The predominant vacA genotypes were s1 and m1 in all three ethnic groups (e.g. 68% in Persians and 70% in Turks were s1). There was no significant association between cagA and cagE status or vacA genotypes and clinical outcomes. The oipA-positive strains were more common in non-ulcer dyspepsia (NUD) (63%) than in peptic ulcer patients (15%) (P = 0.001) in Persians, but the association was not observed in other ethnic groups. Conclusion There are some differences in the H. pylori genotypes among the ethnic groups in Iran. However, none of these markers seemed to be clinically helpful in predicting the clinical presentation of a H. pylori infection in Iran. PMID:19702906

  5. 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

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

    PubMed Central

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

    2013-01-01

    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

  7. 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

  8. Histone tyrosine phosphorylation comes of age

    PubMed Central

    Singh, Rakesh Kumar

    2011-01-01

    Histones were discovered over a century ago and have since been found to be the most extensively post-translationally modified proteins, although tyrosine phosphorylation of histones had remained elusive until recently. The year 2009 proved to be a landmark year for histone tyrosine (Y) phosphorylation as five research groups independently discovered this modification. Three groups describe phosphorylation of Y142 in the variant histone H2A.X, where it may be involved in the cellular decision making process to either undergo DNA repair or apoptosis in response to DNA damage. Further, one group suggests that phosphorylation of histone H3 on Y99 is crucial for its regulated proteolysis in yeast, while another found that Y41 phosphorylation modulates chromatin architecture and oncogenesis in mammalian cells. These pioneering studies provide the initial conceptual framework for further analyses of the diverse roles of tyrosine phosphorylation on different histones, with far reaching implications for human health and disease. PMID:20935492

  9. Monoclonal antibodies to individual tyrosine-phosphorylated protein substrates of oncogene-encoded tyrosine kinases

    SciTech Connect

    Kanner, S.B.; Reynolds, A.B.; Vines, R.R.; Parsons, J.T. )

    1990-05-01

    Cellular transformation by oncogenic retroviruses encoding protein tyrosine kinases coincides with the tyrosine-specific phosphorylation of multiple protein substrates. Previous studies have shown that tyrosine phosphorylation of a protein of 120 kDa, p120, correlated with src transformation in chicken embryo fibroblasts. Additionally, the authors previously identified two phosphotyrosine-containing cellular proteins, p130 and p110, that formed stable complexes with activated variants of pp60{sup src}, the src-encoded tyrosine kinase. To study transformation-relevant tyrosine kinase substrates, they have generated monoclonal antibodies to individual tyrosine phosphoproteins, including p130, p120, p110, and five additional phosphoproteins (p210, p125, p118, p85, and p185/p64). These antibodies detected several of the same tyrosine phosphoproteins in chicken embryo fibroblasts transformed by avian retroviruses Y73 and CT10, encoding the yes and crk oncogenes, respectively. Protein substrates in mouse, rat, hamster, and human cells overexpressing activated variants of chicken pp60{sup src} were also detected by several of the monoclonal antibodies.

  10. 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

  11. 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...

  12. 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

  13. 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.

  14. 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

  15. Conservation and Early Expression of Zebrafish Tyrosine Kinases Support the Utility of Zebrafish as a Model for Tyrosine Kinase Biology

    PubMed Central

    Challa, Anil Kumar

    2013-01-01

    Abstract 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

  16. Tyrosine and carboxyl protonation changes in the bacteriorhodopsin photocycle. 2. Tyrosine-26 and -64

    SciTech Connect

    Roepe, P.; Scherrer, P.; Ahl, P.L.; Gupta, S.K.D.; Bogomolni, R.A.; Herzfeld, J.; Rothschild, K.J.

    1987-10-20

    Low-temperature Fourier transform infrared (FTIR) and UV difference spectroscopies combined with selective tyrosine nitration and tyrosine isotopic labeling have been used to investigate the participation of tyrosines-26 and -64 in the bacteriorhodopsin (bR) photocycle. Nitration of Tyr-26 has no detectable effect on the FTIR or UV difference spectra of the BR/sub 570/ ..-->.. K/sub 630/ or BR/sub 570/ ..-->.. M/sub 412/ transitions. In contrast, nitration of Tyr-64 causes changes in both the FTIR and UV spectra of these transitions. However, this nitration does not alter tyrosine peaks in the FTIR difference spectra which have previously been associated with the protonation of a tyrosinate by K/sub 630/ and the deprotonation of a tyrosine by M/sub 412/. Instead, Tyr-64 nitration appears to affect other tyrosine peaks. These results and changes in UV difference spectra upon Tyr-64 nitration are consistent with the deprotonation of Tyr-64 by M/sub 412/ as concluded previously. Effects on chromophore vibrations caused by Tyr-64 nitration are unaltered upon reducing the nitrotyrosine to aminotyrosine with sodium dithionite. Finally, nitro-Tyr-64 causes a shift in the frequency of a positive peak at 1739 cm/sup -1/ in the BR/sub 570/ ..-->.. M/sub 412/ FTIR difference spectrum which reflects the protonation of a carboxyl-containing residue. The shift does not occur for samples containing amino-Tyr-64. These data suggest that Tyr-64 may interact with this carboxyl group.

  17. 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

  18. 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

  19. Evaluation of the effect of cagPAI genes of Helicobacter pylori on AGS epithelial cell morphology and IL-8 secretion.

    PubMed

    Salih, Barik A; Guner, Ahmet; Karademir, Ahu; Uslu, Merve; Ovali, Mehmet Akif; Yazici, Duygu; Bolek, Bora Kazim; Arikan, Soykan

    2014-01-01

    Helicobacter pylori cagPAI genes play an important role in pathogenesis, however little is known about their functions in isolates from Turkish patients. We aimed to evaluate the intactness and the effect of the cagPAI genes (cagT, cagM, cagE, cagA) and cagA EPIYA motifs on the AGS morphological changes and IL-8 induction. Of 53 patients 38 were found infected with H. pylori. PCR amplification of the cagPAI genes showed 42.1 % intact, 39.5 % partially deleted and 18.4 % with complete deletions. Isolates from gastritis, duodenal and gastric ulcer patients with intact and partially deleted cagPAI genes induced higher IL-8 secretion than those with complete deletions. Isolates from gastritis patients had higher deletion frequencies of the cagT and cagM genes than the other two genes. Infection of AGS cells with isolates that possess intact cagPAI and EPIYA-ABC resulted in the formation of the hummingbird phenotype. The cagA positive isolates induced higher IL-8 secretion than cagA negative isolates. Isolates from DU patients with more than one EPIYA-C motif induced higher concentrations of IL-8 than those with EPIYA-ABC. In conclusion, the intactness of the cagPAI in our isolates from different patients was not conserved. An intact cagPAI was found to play an important role in the pathogenesis of DU but not GU or gastritis. The cagA gene, but not other cagPAI genes, was associated with the induction of IL-8 and the morphological changes of the AGS cells. An increase in the number of EPIYA-C motifs had noticeable effect on the formation of the hummingbird phenotype. PMID:24170115

  20. 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.

  1. 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

  2. Potential sites of CFTR activation by tyrosine kinases.

    PubMed

    Billet, Arnaud; Jia, Yanlin; Jensen, Timothy J; Hou, Yue-Xian; Chang, Xiu-Bao; Riordan, John R; Hanrahan, John W

    2016-05-01

    The CFTR chloride channel is tightly regulated by phosphorylation at multiple serine residues. Recently it has been proposed that its activity is also regulated by tyrosine kinases, however the tyrosine phosphorylation sites remain to be identified. In this study we examined 2 candidate tyrosine residues near the boundary between the first nucleotide binding domain and the R domain, a region which is important for channel function but devoid of PKA consensus sequences. Mutating tyrosines at positions 625 and 627 dramatically reduced responses to Src or Pyk2 without altering the activation by PKA, suggesting they may contribute to CFTR regulation. PMID:26645934

  3. 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.

  4. 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.

  5. 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

  6. Microtubule-associated protein 1B interaction with tubulin tyrosine ligase contributes to the control of microtubule tyrosination.

    PubMed

    Utreras, Elías; Jiménez-Mateos, Eva Maria; Contreras-Vallejos, Erick; Tortosa, Elena; Pérez, Mar; Rojas, Sebastián; Saragoni, Lorena; Maccioni, Ricardo B; Avila, Jesús; González-Billault, Christian

    2008-01-01

    Microtubule-associated protein 1B (MAP1B) is the first microtubule-associated protein to be expressed during nervous system development. MAP1B belongs to a large family of proteins that contribute to the stabilization and/or enhancement of microtubule polymerization. These functions are related to the control of the dynamic properties of microtubules. The C-terminal domain of the neuronal alpha-tubulin isotype is characterized by the presence of an acidic polypeptide, with the last amino acid being tyrosine. This tyrosine residue may be enzymatically removed from the protein by an unknown carboxypeptidase activity. Subsequently, the tyrosine residue is again incorporated into this tubulin by another enzyme, tubulin tyrosine ligase, to yield tyrosinated tubulin. Because neurons lacking MAP1B have a reduced proportion of tyrosinated microtubules, we analyzed the possible interaction between MAP1B and tubulin tyrosine ligase. Our results show that these proteins indeed interact and that the interaction is not affected by MAP1B phosphorylation. Additionally, neurons lacking MAP1B, when exposed to drugs that reversibly depolymerize microtubules, do not fully recover tyrosinated microtubules upon drug removal. These results suggest that MAP1B regulates tyrosination of alpha-tubulin in neuronal microtubules. This regulation may be important for general processes involved in nervous system development such as axonal guidance and neuronal migration. PMID:18075266

  7. Inhibition of biofilm formation by D-tyrosine: Effect of bacterial type and D-tyrosine concentration.

    PubMed

    Yu, Cong; Li, Xuening; Zhang, Nan; Wen, Donghui; Liu, Charles; Li, Qilin

    2016-04-01

    D-Tyrosine inhibits formation and triggers disassembly of bacterial biofilm and has been proposed for biofouling control applications. This study probes the impact of D-tyrosine in different biofilm formation stages in both G+ and G- bacteria, and reveals a non-monotonic correlation between D-tyrosine concentration and biofilm inhibition effect. In the attachment stage, cell adhesion was studied in a flow chamber, where D-tyrosine caused significant reduction in cell attachment. Biofilms formed by Pseudomonas aeruginosa and Bacillus subtilis were characterized by confocal laser scanning microscopy as well as quantitative analysis of cellular biomass and extracellular polymeric substances. D-Tyrosine exhibited strong inhibitive effects on both biofilms with an effective concentration as low as 5 nM; the biofilms responded to D-tyrosine concentration change in a non-monotonic, bi-modal pattern. In addition, D-tyrosine showed notable and different impact on EPS production by G+ and G- bacteria. Extracellular protein was decreased in P. aeruginosa biofilms, but increased in those of B. subtilis. Exopolysaccharides production by P. aeruginosa was increased at low concentrations and reduced at high concentrations while no impact was found in B. subtilis. These results suggest that distinct mechanisms are at play at different D-tyrosine concentrations and they may be species specific. Dosage of D-tyrosine must be carefully controlled for biofouling control applications. PMID:26854605

  8. Tyrosine for the treatment of depression.

    PubMed

    Gelenberg, A J; Gibson, C J

    1984-01-01

    The two most widely held biochemical models of depression--the catecholamine (CA) and indoleamine (IA) hypotheses--explain depression as a result of deficient transmission of the CA norepinephrine (NE) or the IA serotonin (5-hydroxytryptamine, 5-HT) respectively. Until recently, all drugs used to treat depression appeared to enhance neurotransmission in one or both of these systems, which was used to explain their antidepressant actions (Gelenberg and Klerman, 1978). In fact, it was this action of antidepressants that gave rise to the models of depression. Another way to increase brain levels of NE and 5-HT, and potentially to increase presynaptic activity, would be the systemic administration of the precursors of the neurotransmitters, an approach something like organic gardening in the brain. For this purpose, the 5-HT precursors tryptophan and 5-hydroxtryptophan (5-HTP), and the NE precursors tyrosine and dihydroxyphenylalanine (DOPA), have been administered to depressed patients. This paper reviews some of the theoretical background and clinical experience with the precursor strategy, focusing primarily on work with L-tyrosine. All four precursors as possible antidepressants have been recently reviewed (Gelenberg, 1982). PMID:6443584

  9. 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

  10. Bruton tyrosine kinase (Btk) suppresses osteoblastic differentiation.

    PubMed

    Kaneshiro, Shoichi; Ebina, Kosuke; Shi, Kenrin; Yoshida, Kiyoshi; Otsuki, Dai; Yoshikawa, Hideki; Higuchi, Chikahisa

    2015-09-01

    The Tec family of nonreceptor tyrosine kinases has been shown to play a key role in inflammation and bone destruction. Bruton tyrosine kinase (Btk) has been the most widely studied because of its critical role in B cells. Furthermore, recent evidence has demonstrated that blocking Btk signaling is effective in ameliorating lymphoma progression and experimental arthritis. The role of Btk in osteoblastic differentiation has not been well elucidated. In this study, we demonstrated the role of Btk in osteoblastic differentiation and investigated the effects of a Btk inhibitor on osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells, primary calvarial osteoblasts, and bone marrow stromal ST2 cells. Btk expression was detected in all three cell lines. Btk inhibition stimulated mRNA expression of osteoblastic markers (alkaline phosphatase, osteocalcin, and osterix) and promoted mineralization of the extracellular matrix. In addition, Btk knockdown caused increased mRNA expression of osteoblastic markers. Furthermore, Btk inhibition suppressed the phosphorylation of mitogen-activated protein kinase (MAPK), nuclear factor kappa B (NFκB), and protein kinase Cα (PKCα). Our results indicate that Btk may regulate osteoblastic differentiation through the MAPK, NFκB, and PKCα signaling pathways. PMID:25230818

  11. Receptor Tyrosine Kinases with Intracellular Pseudokinase Domains

    PubMed Central

    Mendrola, Jeannine M.; Shi, Fumin; Park, Jin H.; Lemmon, Mark A.

    2013-01-01

    As with other groups of protein kinases, approximately 10% of the receptor tyrosine kinases (RTKs) in the human proteome contain intracellular pseudokinases that lack one or more conserved catalytically important residues. These include ErbB3, a member of the epidermal growth factor receptor (EGFR) family, and a series of unconventional Wnt receptors. We recently showed that, despite its reputation as a pseudokinase, the ErbB3 tyrosine kinase domain (TKD) does retain significant – albeit weak – kinase activity. This led us to suggest that a subgroup of RTKs may be able to signal even with very inefficient kinases. Recent work suggests that this is not the case, however. Other pseudokinase RTKs have not revealed significant kinase activity, and mutations that impair ErbB3’s weak kinase activity have not so far been found to exhibit signaling defects. These findings therefore point to models in which the TKDs of pseudokinase RTKs participate in receptor signaling by allosterically regulating associated kinases (such as ErbB3 regulation of ErbB2) and/or function as regulated ‘scaffolds’ for other intermolecular interactions central to signal propagation. Further structural and functional studies – particularly of the pseudokinase RTKs involved in Wnt signaling – are required to shed new light on these intriguing signaling mechanisms. PMID:23863174

  12. 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

  13. 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...

  14. 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

  15. Tyrosine kinase inhibitors and the thyroid.

    PubMed

    Sherman, Steven I

    2009-12-01

    Protein tyrosine kinase inhibitors (TKIs) have emerged as significant targets for novel cancer therapies. For patients with differentiated or medullary carcinomas unresponsive to conventional treatments, multiple novel therapies primarily targeting angiogenesis have entered clinical trials. Partial response rates up to 30% have been reported in single-agent studies, but prolonged disease stabilisation is more commonly seen. The most successful agents target the vascular endothelial growth factor receptors. Sorafenib and sunitinib have had promising preliminary results reported and are being used selectively for patients who do not qualify for clinical trials. Treatment for patients with metastatic or advanced thyroid carcinoma now emphasises clinical trial opportunities for novel agents with considerable promise. Adverse effects on thyroid function and thyroid hormone metabolism have also been seen with several TKIs, necessitating prospective thyroid function testing for all patients starting therapy. PMID:19942148

  16. The Ror receptor tyrosine kinase family.

    PubMed

    Forrester, W C

    2002-01-01

    Receptor tyrosine kinases (RTKs) participate in numerous developmental decisions. Ror RTKs are a family of orphan receptors that are related to muscle specific kinase (MuSK) and Trk neurotrophin receptors. MuSK assembles acetylcholine receptors at the neuromuscular junction, and Trk receptors function in the developing nervous system (reviewed in [3-5]). Rors have been identified in nematodes, insects and mammals. Recent studies have begun to shed light on Ror function during development. In most species, Rors are expressed in many tissue types during development. Analyses of mutants that are defective in the single nematode Ror demonstrate a role in cell migration and in orienting cell polarity. Mice lacking one of the two Ror gene products display defects in bone and heart formation. Similarly, two different human bone development disorders, dominant brachydactyly B and recessive Robinow syndrome, result from mutations in one of the human Ror genes. PMID:11846036

  17. 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

  18. 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

  19. Food for creativity: tyrosine promotes deep thinking.

    PubMed

    Colzato, Lorenza S; de Haan, Annelies M; Hommel, Bernhard

    2015-09-01

    Anecdotal evidence suggests that creative people sometimes use food to overcome mental blocks and lack of inspiration, but empirical support for this possibility is still lacking. In this study, we investigated whether creativity in convergent- and divergent-thinking tasks is promoted by the food supplement L-Tyrosine (TYR)-a biochemical precursor of dopamine, which is assumed to drive cognitive control and creativity. We found no evidence for an impact of TYR on divergent thinking ("brainstorming") but it did promote convergent ("deep") thinking. As convergent thinking arguably requires more cognitive top-down control, this finding suggests that TYR can facilitate control-hungry creative operations. Hence, the food we eat may affect the way we think. PMID:25257259

  20. The tyrosine kinase FER is responsible for the capacitation-associated increase in tyrosine phosphorylation in murine sperm.

    PubMed

    Alvau, Antonio; Battistone, Maria Agustina; Gervasi, Maria Gracia; Navarrete, Felipe A; Xu, Xinran; Sánchez-Cárdenas, Claudia; De la Vega-Beltran, Jose Luis; Da Ros, Vanina G; Greer, Peter A; Darszon, Alberto; Krapf, Diego; Salicioni, Ana Maria; Cuasnicu, Patricia S; Visconti, Pablo E

    2016-07-01

    Sperm capacitation is required for fertilization. At the molecular level, this process is associated with fast activation of protein kinase A. Downstream of this event, capacitating conditions lead to an increase in tyrosine phosphorylation. The identity of the tyrosine kinase(s) mediating this process has not been conclusively demonstrated. Recent experiments using stallion and human sperm have suggested a role for PYK2 based on the use of small molecule inhibitors directed against this kinase. However, crucially, loss-of-function experiments have not been reported. Here, we used both pharmacological inhibitors and genetically modified mice models to investigate the identity of the tyrosine kinase(s) mediating the increase in tyrosine phosphorylation in mouse sperm. Similar to stallion and human, PF431396 blocks the capacitation-associated increase in tyrosine phosphorylation. Yet, sperm from Pyk2(-/-) mice displayed a normal increase in tyrosine phosphorylation, implying that PYK2 is not responsible for this phosphorylation process. Here, we show that PF431396 can also inhibit FER, a tyrosine kinase known to be present in sperm. Sperm from mice targeted with a kinase-inactivating mutation in Fer failed to undergo capacitation-associated increases in tyrosine phosphorylation. Although these mice are fertile, their sperm displayed a reduced ability to fertilize metaphase II-arrested eggs in vitro. PMID:27226326

  1. 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

  2. pH regulation of an egg cortex tyrosine kinase.

    PubMed

    Jiang, W P; Veno, P A; Wood, R W; Peaucellier, G; Kinsey, W H

    1991-07-01

    Fertilization of the echinoderm egg is known to result in the phosphorylation, on tyrosine, of a high-molecular-weight cortical protein (HMWCP) localized in the egg cortex. Studies using various parthenogenic agents indicate that this phosphorylation event occurs in response to the alkaline shift in cytoplasmic pHi which normally occurs 1 to 2 min after fertilization. In the present study, the purified egg cell surface complex was used as in vitro system to determine whether a small alkaline shift in pH, such as occurs upon fertilization, could stimulate the activity of the egg cortex-associated tyrosine kinase toward endogenous protein substrates. The results demonstrated that the cell surface complex is highly enriched in a tyrosine kinase activity which accounts for the majority of the protein kinase activity in this preparation. The activity of this tyrosine kinase toward the HMWCP and other cortical proteins was highly dependent on pH over the range pH 6.8 to 7.3. This indicates that the fertilization-associated change in cytoplasmic pH would be sufficient to trigger increased tyrosine phosphorylation of the high-molecular-weight cortical protein in vivo. The regulation of tyrosine phosphorylation by small changes in pH represents a novel control mechanism in which a tyrosine protein kinase may act as a pH-sensitive transducer. PMID:2060713

  3. Tyrosine depletion lowers in vivo DOPA synthesis in ventral hippocampus.

    PubMed

    Bongiovanni, Rodolfo; Kyser, Abby N; Jaskiw, George E

    2012-12-01

    In vivo dopamine synthesis in the medial prefrontal cortex of the rat is sensitive to the availability of tyrosine. Whether other limbic cortical dopamine terminal regions are similarly tyrosine-dependent is not known. In this study we examined the effects of tyrosine depletion on dopamine synthesis and catecholamine levels in the ventral hippocampus. A tyrosine- and phenylalanine-free neutral amino acid mixture was used to lower brain tyrosine levels in rats undergoing in vivo microdialysis. In one group, NSD-1015 was included in perfusate to permit measurement of DOPA levels. In a second group, NSD-1015 was not included in perfusate so that catecholamine levels could be assayed. Tyrosine depletion significantly lowered DOPA levels in the NSD-1015 treated group and lowered DOPAC but not dopamine or noradrenaline levels in the group not exposed to NSD-1015. We conclude that while catecholamine synthesis in the ventral hippocampus declines when tyrosine availability is lowered, under basal conditions, compensatory mechanisms are able to maintain stable extracellular catecholamine levels. PMID:23022716

  4. 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

  5. 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.

  6. Tyrosine hydroxylase deficiency with severe clinical course.

    PubMed

    Zafeiriou, D I; Willemsen, M A; Verbeek, M M; Vargiami, E; Ververi, A; Wevers, R

    2009-05-01

    Tyrosine hydroxylase (TH) deficiency is a rare autosomal recessive disorder mapped to chromosome 11p15.5. Its clinical expression varies with presentations as dopa-responsive dystonia (recessive Segawa's disease), dopa-responsive infantile parkinsonism, dopa-responsive spastic paraplegia, progressive infantile encephalopathy or dopa-non-responsive dystonia. We describe a 7-year-old boy with progressive infantile encephalopathy and non-responsiveness to dopamine. The patient demonstrated generalized hypotonia, pyramidal tract dysfunction and temperature instability after the second month of life. Dystonia, tremor and oculogyric crises complicated the clinical picture during the following months. Neurotransmitter analysis in CSF disclosed almost undetectable levels of HVA and MHPG, whereas serum prolactin was profoundly increased. Subsequent molecular analysis revealed homozygosity for a missense mutation (c.707T>C) in the TH gene. l-Dopa therapy in both high and low doses resulted in massive hyperkinesias, while substitution with selegiline exerted only a mild beneficial effect. Today, at the age of 7 years, the patient demonstrates severe developmental retardation with marked trunkal hypotonia, hypokinesia and occasionally dystonic and/or hyperkinetic crises. He is the third Greek patient with TH deficiency to be reported. Since all three patients carry the same pathogenetic mutation, a founder effect is suspected. PMID:19282209

  7. 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

  8. Structure of tyrosine aminotransferase from Leishmania infantum

    PubMed Central

    Moreno, M. A.; Abramov, A.; Abendroth, J.; Alonso, A.; Zhang, S.; Alcolea, P. J.; Edwards, T.; Lorimer, D.; Myler, P. J.; Larraga, V.

    2014-01-01

    The trypanosomatid parasite Leishmania infantum is the causative agent of visceral leishmaniasis (VL), which is usually fatal unless treated. VL has an incidence of 0.5 million cases every year and is an important opportunistic co-infection in HIV/AIDS. Tyrosine aminotransferase (TAT) has an important role in the metabolism of trypanosomatids, catalyzing the first step in the degradation pathway of aromatic amino acids, which are ultimately converted into their corresponding l-2-oxoacids. Unlike the enzyme in Trypanosoma cruzi and mammals, L. infantum TAT (LiTAT) is not able to transaminate ketoglutarate. Here, the structure of LiTAT at 2.35 Å resolution is reported, and it is confirmed that the presence of two Leishmania-specific residues (Gln55 and Asn58) explains, at least in part, this specific reactivity. The difference in substrate specificity between leishmanial and mammalian TAT and the importance of this enzyme in parasite metabolism suggest that it may be a useful target in the development of new drugs against leishmaniasis. PMID:24817714

  9. [Side effect management of tyrosine kinase inhibitors in urology : Hypertension].

    PubMed

    Sikic, D; Meidenbauer, N; Lieb, V; Keck, B

    2016-07-01

    Tyrosine kinase inhibitors like sunitinib, sorafenib, pazopanib or axintinib are regarded the standard of care in the systemic therapy of metastatic renal cell carcinoma. However, the many side effects associated with this therapy pose challenges for the treating physician and the patient. This review offers an overview of the classification and the treatment of hypertension, which is one of the major side effects induced by all tyrosine kinase inhibitors, in order to improve treatment efficacy and patient compliance. PMID:27146871

  10. 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.

  11. 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.

  12. 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

  13. Are Striatal Tyrosine Hydroxylase Interneurons Dopaminergic?

    PubMed Central

    Xenias, Harry S.; Ibáñez-Sandoval, Osvaldo; Koós, Tibor

    2015-01-01

    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. 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

  15. 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

  16. Heterologous production of caffeic acid from tyrosine in Escherichia coli.

    PubMed

    Rodrigues, J L; Araújo, R G; Prather, K L J; Kluskens, L D; Rodrigues, L R

    2015-04-01

    Caffeic acid is a plant secondary metabolite and its biological synthesis has attracted increased attention due to its beneficial effects on human health. In this study, Escherichia coli was engineered for the production of caffeic acid using tyrosine as the initial precursor of the pathway. The pathway design included tyrosine ammonia lyase (TAL) from Rhodotorula glutinis to convert tyrosine to p-coumaric acid and 4-coumarate 3-hydroxylase (C3H) from Saccharothrix espanaensis or cytochrome P450 CYP199A2 from Rhodopseudomonas palustris to convert p-coumaric acid to caffeic acid. The genes were codon-optimized and different combinations of plasmids were used to improve the titer of caffeic acid. TAL was able to efficiently convert 3mM of tyrosine to p-coumaric acid with the highest production obtained being 2.62mM (472mg/L). CYP199A2 exhibited higher catalytic activity towards p-coumaric acid than C3H. The highest caffeic acid production obtained using TAL and CYP199A2 and TAL and C3H was 1.56mM (280mg/L) and 1mM (180mg/L), respectively. This is the first study that shows caffeic acid production using CYP199A2 and tyrosine as the initial precursor. This study suggests the possibility of further producing more complex plant secondary metabolites like flavonoids and curcuminoids. PMID:25765308

  17. 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

  18. A chimeric tyrosine/tryptophan hydroxylase. The tyrosine hydroxylase regulatory domain serves to stabilize enzyme activity.

    PubMed

    Mockus, S M; Kumer, S C; Vrana, K E

    1997-08-01

    The neurotransmitter biosynthetic enzymes, tyrosine hydroxylase (TH), and tryptophan hydroxylase (TPH) are each composed of an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. A chimeric hydroxylase was generated by coupling the regulatory domain of TH (TH-R) to the catalytic domain of TPH (TPH-C) and expressing the recombinant enzyme in bacteria. The chimeric junction was created at proline 165 in TH and proline 106 in TPH because this residue is within a conserved five amino-acid span (ValProTrpPhePro) that defines the beginning of the highly homologous catalytic domains of TH and TPH. Radioenzymatic activity assays demonstrated that the TH-R/TPH-C chimera hydroxylates tryptophan, but not tyrosine. Therefore, the regulatory domain does not confer substrate specificity. Although the TH-R/TPH-C enzyme did serve as a substrate for protein kinase (PKA), activation was not observed following phosphorylation. Phosphorylation studies in combination with kinetic data provided evidence that TH-R does not exert a dominant influence on TPH-C. Stability assays revealed that, whereas TH exhibited a t1/2 of 84 min at 37 degrees C, TPH was much less stable (t1/2 = 28.3 min). The stability profile of TH-R/TPH-C, however, was superimposable on that of TH. Removal of the regulatory domain (a deletion of 165 amino acids from the N-terminus) of TH rendered the catalytic domain highly unstable, as demonstrated by a t1/2 of 14 min. The authors conclude that the regulatory domain of TH functions as a stabilizer of enzyme activity. As a corollary, the well-characterized instability of TPH may be attributed to the inability of its regulatory domain to stabilize the catalytic domain. PMID:9356925

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

    PubMed

    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 2(nd) and 3(rd) 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- + orthotyrosine)/ 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

  20. Enrichment and detection of tyrosine-nitrated proteins.

    PubMed

    Dekker, Frank; Abello, Nicolas; Wisastra, Rosalina; Bischoff, Rainer

    2012-08-01

    Nitrotyrosine is a post-translationally modified amino acid with distinctly different properties than tyrosine or any other of the genetically encoded amino acids. Detecting proteins containing nitrotyrosine is the first step towards a better understanding of the role of nitrotyrosine in health and disease. Moreover, quantifying the extent of nitrotyrosine and determining its location in a protein forms the basis for a better understanding of the effect of tyrosine nitration on biological function. Described in this unit is a method to detect tyrosine-nitrated proteins in tissue sections and on western blots after creating a fluorescent complex between aminotyrosine, salicylaldehyde, and Al(3+). In addition, an approach is detailed for labeling aminotyrosine with biotin to enrich peptides from complex samples. Both methods require reduction of nitrotyrosine to aminotyrosine, which can be achieved with sodium dithionite or hemin plus dithiothreitol. PMID:22851496

  1. Cysteine mutations cause defective tyrosine phosphorylation in MEGF10 myopathy

    PubMed Central

    Mitsuhashi, Satomi; Mitsuhashi, Hiroaki; Alexander, Matthew S; Sugimoto, Hiroyuki; Kang, Peter B

    2013-01-01

    Recessive mutations in MEGF10 are known to cause a congenital myopathy in humans. Two mutations in the extracellular EGF-like domains of MEGF10, C326R and C774R, were associated with decreased tyrosine phosphorylation of MEGF10 in vitro. Y1030 was identified to be the major tyrosine phosphorylation site in MEGF10 and is phosphorylated at least in part by c-Src. Overexpression of wild-type MEGF10 enhanced C2C12 myoblast proliferation, while overexpression of Y1030F mutated MEGF10 did not. We conclude that MEGF10-mediated signaling via tyrosine phosphorylation helps to regulate myoblast proliferation. Defects in this signaling pathway may contribute to the disease mechanism of MEGF10 myopathy. PMID:23954233

  2. Tyrosine Kinase Inhibitors and Diabetes: A Novel Treatment Paradigm?

    PubMed

    Fountas, Athanasios; Diamantopoulos, Leonidas-Nikolaos; Tsatsoulis, Agathocles

    2015-11-01

    Deregulation of protein tyrosine kinase (PTK) activity is implicated in various proliferative conditions. Multi-target tyrosine kinase inhibitors (TKIs) are increasingly used for the treatment of different malignancies. Recently, several clinical cases of the reversal of both type 1 and 2 diabetes mellitus (T1DM, T2DM) during TKI administration have been reported. Experimental in vivo and in vitro studies have elucidated some of the mechanisms behind this effect. For example, inhibition of Abelson tyrosine kinase (c-Abl) results in β cell survival and enhanced insulin secretion, while platelet-derived growth factor receptor (PDGFR) and epidermal growth factor receptor (EGFR) inhibition leads to improvement in insulin sensitivity. In addition, inhibition of vascular endothelial growth factor receptor 2 (VEGFR2) reduces the degree of islet cell inflammation (insulitis). Therefore, targeting several PTKs may provide a novel approach for correcting the pathophysiologic disturbances of diabetes. PMID:26492832

  3. Characterization of the PEST family protein tyrosine phosphatase BDP1.

    PubMed

    Kim, Y W; Wang, H; Sures, I; Lammers, R; Martell, K J; Ullrich, A

    1996-11-21

    Using a polymerase chain reaction (PCR) amplification strategy, we identified a novel protein tyrosine phosphatase (PTPase) designated Brain Derived Phosphatase (BDP1). The full length sequence encoded an open reading frame of 459 amino acids with no transmembrane domain and had a calculated molecular weight of 50 kDa. The predicted amino acid sequence contained a PEST motif and accordingly, BDP1 shared the greatest homology with members of the PTP-PEST family. When transiently expressed in 293 cells BDP1 hydrolyzed p-Nitrophenylphosphate, confirming it as a functional protein tyrosine phosphatase. Northern blot analysis indicated that BDP1 was expressed not only in brain, but also in colon and several different tumor-derived cell lines. Furthermore, BDP1 was found to differentially dephosphorylate autophosphorylated tyrosine kinases which are known to be overexpressed in tumor tissues. PMID:8950995

  4. 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

  5. Modular engineering of L-tyrosine production in Escherichia coli.

    PubMed

    Juminaga, Darmawi; Baidoo, Edward E K; Redding-Johanson, Alyssa M; Batth, Tanveer S; Burd, Helcio; Mukhopadhyay, Aindrila; Petzold, Christopher J; Keasling, Jay D

    2012-01-01

    Efficient biosynthesis of L-tyrosine from glucose is necessary to make biological production economically viable. To this end, we designed and constructed a modular biosynthetic pathway for L-tyrosine production in E. coli MG1655 by encoding the enzymes for converting erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) to L-tyrosine on two plasmids. Rational engineering to improve L-tyrosine production and to identify pathway bottlenecks was directed by targeted proteomics and metabolite profiling. The bottlenecks in the pathway were relieved by modifications in plasmid copy numbers, promoter strength, gene codon usage, and the placement of genes in operons. One major bottleneck was due to the bifunctional activities of quinate/shikimate dehydrogenase (YdiB), which caused accumulation of the intermediates dehydroquinate (DHQ) and dehydroshikimate (DHS) and the side product quinate; this bottleneck was relieved by replacing YdiB with its paralog AroE, resulting in the production of over 700 mg/liter of shikimate. Another bottleneck in shikimate production, due to low expression of the dehydroquinate synthase (AroB), was alleviated by optimizing the first 15 codons of the gene. Shikimate conversion to L-tyrosine was improved by replacing the shikimate kinase AroK with its isozyme, AroL, which effectively consumed all intermediates formed in the first half of the pathway. Guided by the protein and metabolite measurements, the best producer, consisting of two medium-copy-number, dual-operon plasmids, was optimized to produce >2 g/liter L-tyrosine at 80% of the theoretical yield. This work demonstrates the utility of targeted proteomics and metabolite profiling in pathway construction and optimization, which should be applicable to other metabolic pathways. PMID:22020510

  6. Phosphorylated tyrosine in the flagellum filament protein of Pseudomonas aeruginosa

    SciTech Connect

    Kelly-Wintenberg, K.; Anderson, T.; Montie, T.C. )

    1990-09-01

    Purified flagella from two strains of {sup 32}P-labeled Pseudomonas aeruginosa were shown to be phosphorylated. This was confirmed by autoradiography of flagellin protein in polyacrylamide gels. Thin-layer electrophoresis and autoradiography of flagellin partial hydrolysates indicated that phosphotyrosine was the major phosphorylated amino acid. High-pressure liquid chromatographic analysis confirmed the presence of phosphotyrosine in flagellum filament protein. Preliminary data indicated that less than one tyrosine per subunit was phosphorylated. No evidence was found for phosphorylation of serine or threonine. A function related to tyrosine phosphorylation has not been determined.

  7. Biosynthesis of brominated tyrosine metabolites by Aplysina fistularis.

    PubMed

    Carney, J R; Rinehart, K L

    1995-07-01

    The biosynthesis of brominated tyrosine metabolites by the marine sponge Aplysina fistularis was investigated. [U-14C]-L-Tyrosine, [U-14C]-L-3-bromotyrosine, and [U-14C]-L-3,5-dibromotyrosine were incorporated into both dibromoverongiaquinol [1] and aeroplysinin-1 [2], and [methyl-14C]methionine was specifically incorporated into the O-methyl group group of 2. [Methyl-14C]-L-O-methyltyrosine, [methyl-14C]-L-3,5-dibromo-O-methyltyrosine, and several putative nitrile precursors were not incorporated into 1 or 2. PMID:7561906

  8. Growth and characterisation of ?-tyrosine-doped TGS crystals

    NASA Astrophysics Data System (ADS)

    Meera, K.; Aravazhi, S.; Santhana Raghavan, P.; Ramasamy, P.

    2000-04-01

    Single crystals of L-tyrosine-doped triglycine sulphate (LTTGS) were grown by low-temperature solution method. Morphological changes were observed on the grown crystals. Crystalline quality and cell parameter values were determined using rocking curve and powder X-ray diffraction analysis. The presence of L-tyrosine in LTTGS was estimated qualitatively by FTIR analysis. Microhardness studies carried out using Leitz Weitzler hardness tester at room temperature showed a decrease due to doping. Domain studies on b-cut plates were observed using scanning electron microscopy.

  9. Cell-free expression of functional receptor tyrosine kinases

    PubMed Central

    He, Wei; Scharadin, Tiffany M.; Saldana, Matthew; Gellner, Candice; Hoang-Phou, Steven; Takanishi, Christina; Hura, Gregory L.; Tainer, John A; Carraway III, Kermit L.; Henderson, Paul T.; Coleman, Matthew A.

    2015-01-01

    Receptor tyrosine kinases (RTKs) play critical roles in physiological and pathological processes, and are important anticancer drug targets. In vitro mechanistic and drug discovery studies of full-length RTKs require protein that is both fully functional and free from contaminating proteins. Here we describe a rapid cell-free and detergent-free co-translation method for producing full-length and functional ERBB2 and EGFR receptor tyrosine kinases supported by water-soluble apolipoprotein A-I based nanolipoprotein particles. PMID:26274523

  10. ZDHHC3 Tyrosine Phosphorylation Regulates Neural Cell Adhesion Molecule Palmitoylation.

    PubMed

    Lievens, Patricia Marie-Jeanne; Kuznetsova, Tatiana; Kochlamazashvili, Gaga; Cesca, Fabrizia; Gorinski, Natalya; Galil, Dalia Abdel; Cherkas, Volodimir; Ronkina, Natalia; Lafera, Juri; Gaestel, Matthias; Ponimaskin, Evgeni; Dityatev, Alexander

    2016-09-01

    The neural cell adhesion molecule (NCAM) mediates cell-cell and cell-matrix adhesion. It is broadly expressed in the nervous system and regulates neurite outgrowth, synaptogenesis, and synaptic plasticity. Previous in vitro studies revealed that palmitoylation of NCAM is required for fibroblast growth factor 2 (FGF2)-stimulated neurite outgrowth and identified the zinc finger DHHC (Asp-His-His-Cys)-containing proteins ZDHHC3 and ZDHHC7 as specific NCAM-palmitoylating enzymes. Here, we verified that FGF2 controlled NCAM palmitoylation in vivo and investigated molecular mechanisms regulating NCAM palmitoylation by ZDHHC3. Experiments with overexpression and pharmacological inhibition of FGF receptor (FGFR) and Src revealed that these kinases control tyrosine phosphorylation of ZDHHC3 and that ZDHHC3 is phosphorylated by endogenously expressed FGFR and Src proteins. By site-directed mutagenesis, we found that Tyr18 is an FGFR1-specific ZDHHC3 phosphorylation site, while Tyr295 and Tyr297 are specifically phosphorylated by Src kinase in cell-based and cell-free assays. Abrogation of tyrosine phosphorylation increased ZDHHC3 autopalmitoylation, enhanced interaction with NCAM, and upregulated NCAM palmitoylation. Expression of ZDHHC3 with tyrosine mutated in cultured hippocampal neurons promoted neurite outgrowth. Our findings for the first time highlight that FGFR- and Src-mediated tyrosine phosphorylation of ZDHHC3 modulates ZDHHC3 enzymatic activity and plays a role in neuronal morphogenesis. PMID:27247265

  11. Purification of a specific reversible tyrosine-O-phosphate phosphatase.

    PubMed Central

    Fukami, Y; Lipmann, F

    1982-01-01

    A phosphatase specific for tyrosine-O-phosphate (Tyr-P) was separated from several nonspecific phosphatases present in the third instar larvae of Drosophila melanogaster. The enzyme hydrolyzed L-Tyr-P, with an apparent Km of 0.14 mM, but not D-Tyr-P after being freed from hydrolytic activity toward p-nitrophenyl phosphate, the common phosphatase substrate. Such purified preparations also catalyzed a reversible phosphate transfer reaction from unlabeled Tyr-P to [3H]tyrosine. The transfer activity was L4-14% of the hydrolytic activity, depending on the initial concentration of tyrosine (0.25-4.0 mM). The two activities coincided throughout purification. However, they differed in pH optimum, that of hydrolysis being 6.5-7 and that of phosphate transfer being 7.7.5. The two activities were also differentially inhibited by 1-p-bromotetramisole oxalate in the presence of EDTA and by Mn2+. Addition of Mg2+ did not affect either hydrolysis or phosphate transfer, but 5 mM Zn2+ was 65% inhibitory to both. Sodium fluoride strongly inhibited both reactions, and this inhibition was reversed by EDTA, while EDTA itself had no effect. Pi had no effect and no detectable incorporation of 32Pi into Tyr-P was observed, indicating that the phosphate transfer reaction is not a simple reversal of hydrolysis. No ATP-linked phosphorylation of tyrosine was found. PMID:6181504

  12. The mitochondrial ATPase. Evidence for a single essential tyrosine residue.

    PubMed

    Ferguson, S J; Lloyd, W J; Lyons, M H; Radda, G K

    1975-05-01

    1. Evidence is presented which indicates that inactivation of the mitochondrial ATPase from bovine heart by the reagent 4-chloro-7-nitrobenzofurazan results from modification of one tyrosine residue per enzyme molecule. Activity can be restored by a variety of sulphydryl reagents. 2. In sodium dodecyl sulphate, the nitrogenzofurazan group on tyrosine is transfered to newly exposed sulphydryl groups on the enzyme. 3. The rate of transfer of the nitrobenzofurazan moiety from theenzyme to sulphydryl compounds is compared with that for transfer from the model compound N-acetyl-tyrosine-0(7-nitrobenzo-furazan) ethyl ester, the synthesis and properties of which are also described. 4. The ligands ATP and ADP exert a protective effect on the rate of reaction between the mitochondrial ATPase and 4-chloro-7-nitrobenzofurazan. The variation in rate of this reaction with change in pH has also been examined and a pKa of 9.5 estimated for the tyrosine residue. 5. The modification does not prevent substrate binding as judged by changes in the fluorescence of aurovertin, an antibiotic with specific affinity for mitochondiral ATPases. 6. When the ATPase activity of submitochondrial particles is inhibited by 4-chloro-7-nitrobenzo-furazan, there is a parallel decrease in the extent of the energy-linked fluorescence enhancement of 1-anilino-naphthalene-8-sulphonate induced by ATP hydrolysis. Both ATPase activity and the fluorescence enhancement are restored by sluphydryl reagents. PMID:238839

  13. Inhibition of Bcr serine kinase by tyrosine phosphorylation.

    PubMed Central

    Liu, J; Wu, Y; Ma, G Z; Lu, D; Haataja, L; Heisterkamp, N; Groffen, J; Arlinghaus, R B

    1996-01-01

    The first exon of the BCR gene encodes a new serine/threonine protein kinase. Abnormal fusion of the BCR and ABL genes, resulting from the formation of the Philadelphia chromosome (Ph), is the hallmark of Ph-positive leukemia. We have previously demonstrated that the Bcr protein is tyrosine phosphorylated within first-exon sequences by the Bcr-Abl oncoprotein. Here we report that in addition to tyrose 177 (Y-177), Y-360 and Y283 are phosphorylated in Bcr-Abl proteins in vitro. Moreover, Bcr tyrosine 360 is phosphorylated in vivo within both Bcr-Abl and Bcr. Bcr mutant Y177F had a greatly reduced ability to transphosphorylate casein and histone H1, whereas Bcr mutants Y177F and Y283F had wild-type activities. In contrast, the Y360F mutation had little effect on Bcr's autophosphorylation activity. Tyrosine-phosphorylated Bcr, phosphorylated in vitro by Bcr-Abl, was greatly inhibited in its serine/threonine kinase activity, impairing both auto- and transkinase activities of Bcr. Similarly, the isolation of Bcr from cells expressing Bcr-Abl under conditions that preserve phosphotyrosine residues also reduced Bcr's kinase activity. These results indicate that tyrosine 360 of Bcr is critical for the transphosphorylation activity of Bcr and that in Ph-positive leukemia, Bcr serine/threonine kinase activity is seriously impaired. PMID:8622703

  14. 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

  15. 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....

  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. 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....

  18. 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....

  19. 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....

  20. 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.

  1. 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.

  2. Redundant and selective roles for erythropoietin receptor tyrosines in erythropoiesis in vivo.

    PubMed

    Longmore, G D; You, Y; Molden, J; Liu, K D; Mikami, A; Lai, S Y; Pharr, P; Goldsmith, M A

    1998-02-01

    Cytokine receptors have been shown in cell culture systems to use phosphotyrosine residues as docking sites for certain signal transduction intermediates. Studies using various cellular backgrounds have yielded conflicting information about the importance of such residues. The present studies were undertaken to determine whether or not tyrosine residues within the erythropoietin receptor (EPOR) are essential for biologic activity during hematopoiesis in vivo. A variant of the EPOR was constructed that contains both a substitution (R129C) causing constitutive receptor activation as well as replacement of all eight cytoplasmic tyrosines by phenylalanines (cEPORYF). A comparison between animals exposed to recombinant retroviruses expressing cEPOR and cEPORYF showed that efficient red blood cell (RBC) development in vivo is dependent on the pressence of tyrosine residues in the cytoplasmic domain of the EPOR. In addition, an inefficient EPOR tyrosine independent pathway supporting RBC development was detected. Tyrosine add-back mutants showed that multiple individual tyrosines have the capacity to restore full erythropoietic potential to the EPOR as determined in whole animals. The analysis of primary erythroid progenitors transduced with the various cEPOR tyrosine mutants and tyrosine add-backs showed that only tyrosine 343 (Y1) and tyrosine 479 (Y8) were capable of supporting immature burst-forming unit-erythroid progenitor development. Thus, this receptor is characterized by striking functional redundancy of tyrosines in a biologically relevant context. However, selective tyrosine residues may be uniquely important for early signals supporting erythroid development. PMID:9446647

  3. 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

  4. Inverse correlation between tyrosine phosphorylation and collagenase production in chondrocytes.

    PubMed Central

    Cruz, T F; Mills, G; Pritzker, K P; Kandel, R A

    1990-01-01

    Collagenase production by chondrocytes appears to play a major role in the development of osteoarthritis. Although the mechanisms regulating collagenase production by chondrocytes are not known, incubation of bovine chondrocytes in serum markedly decreases collagenase production. Since serum has been demonstrated to increase levels of phosphotyrosine (P-Tyr) in several cell types, we determined the effect of altering intracellular levels of P-Tyr on collagenase production. Both orthovanadate, a potent inhibitor of tyrosine phosphatases, and serum caused a marked increase in tyrosine phosphorylation. The increase in P-Tyr was associated with a decrease in the production of collagenase, suggesting that two processes may be linked. Orthovanadate caused an increase in P-Tyr in the absence of serum, suggesting that P-Tyr levels in resting chondrocytes are regulated through activity of both tyrosine kinases and phosphatases. Orthovanadate and serum induced a synergistic increase in P-Tyr levels, suggesting that serum functions through increasing kinase activity rather than decreasing phosphatase activity. In the absence of serum, concentrations of orthovanadate which maximally inhibited collagenase production primarily increased phosphorylation of a 36 kDa protein, suggesting that the phosphorylation of this protein may play a major role in regulating collagenase production. Orthovanadate had limited effects on chondrocyte proteoglycan synthesis, morphology or viability in the presence or absence of serum, suggesting that the decrease in collagenase production was not due to non-specific inhibition of protein synthesis or cellular toxicity. Inhibition of tyrosine phosphatases by orthovanadate or activation of tyrosine kinases by addition of serum correlated with the inhibition of collagenase production. Images Fig. 1. Fig. 2. PMID:1697163

  5. 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

  6. 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

  7. Co-expression of protein tyrosine kinases EGFR-2 and PDGFRβ with protein tyrosine phosphatase 1B in Pichia pastoris.

    PubMed

    Tu, Pham Ngoc; Wang, Yamin; Cai, Menghao; Zhou, Xiangshan; Zhang, Yuanxing

    2014-02-28

    The regulation of protein tyrosine phosphorylation is mediated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) and is essential for cellular homeostasis. Coexpression of PTKs with PTPs in Pichia pastoris was used to facilitate the expression of active PTKs by neutralizing their apparent toxicity to cells. In this study, the gene encoding phosphatase PTP1B with or without a blue fluorescent protein or peroxisomal targeting signal 1 was cloned into the expression vector pAG32 to produce four vectors. These vectors were subsequently transformed into P. pastoris GS115. The tyrosine kinases EGFR-2 and PDGFRβ were expressed from vector pPIC3.5K and were fused with a His-tag and green fluorescent protein at the N-terminus. The two plasmids were transformed into P. pastoris with or without PTP1B, resulting in 10 strains. The EGFR-2 and PDGFRβ fusion proteins were purified by Ni(2+) affinity chromatography. In the recombinant P. pastoris, the PTKs co-expressed with PTP1B exhibited higher kinase catalytic activity than did those expressing the PTKs alone. The highest activities were achieved by targeting the PTKs and PTP1B into peroxisomes. Therefore, the EGFR-2 and PDGFRβ fusion proteins expressed in P. pastoris may be attractive drug screening targets for anticancer therapeutics. PMID:24248091

  8. Tyrosine Administration Decreases Vulnerability to Ventricular Fibrillation in the Normal Canine Heart

    NASA Astrophysics Data System (ADS)

    Scott, Neal A.; Desilva, Regis A.; Lown, Bernard; Wurtman, Richard J.

    1981-02-01

    Intravenous infusion of tyrosine (1, 2, or 4 milligrams per kilogram) for 20 to 30 minutes caused dose-dependent increases in the ventricular fibrillation threshold in normal dogs. Administration of valine, a neutral amino acid that competes with tyrosine for uptake at the blood-brain barrier, in a dose equimolar to the most effective dose of tyrosine, slightly decreased the ventricular fibrillation threshold when given alone and significantly blocked elevation of the ventricular fibrillation threshold after tyrosine infusion. Hence, tyrosine, presumably acting in the central nervous system, can protect against certain ventricular arrhythmias.

  9. 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

  10. 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

  11. Stabilization and purification of tyrosine aminotransferase from rat liver.

    PubMed

    Hargrove, J L

    1990-01-01

    Purification of unmodified tyrosine aminotransferase from rat liver requires that the activity of cathepsin T be minimized, and that losses of enzyme due to dilution or oxidation by prevented. The enzyme was stabilized by pyridoxal 5'-phosphate, dithiothreitol, and potassium phosphate, but was destabilized by L-tyrosine or L-glutamate. A rapid, efficient method for purification of this enzyme included the following steps: twenty-fold induction with a high-casein diet plus dexamethasone phosphate administered in the drinking water; a heat step (65 degrees C) followed by precipitation from 0.20 M sucrose at pH 5.0; and small-scale chromatography on DEAE-cellulose, hydroxyapatite and CM-Sephadex C50 at pH 6.0. These steps yielded more than 10 mg of native enzyme from 35 rats, with a recovery of 68% of the initial activity. PMID:1973296

  12. Bone sialoprotein II synthesized by cultured osteoblasts contains tyrosine sulfate

    SciTech Connect

    Ecarot-Charrier, B.; Bouchard, F.; Delloye, C. )

    1989-11-25

    Isolated mouse osteoblasts that retain their osteogenic activity in culture were incubated with (35S) sulfate. Two radiolabeled proteins, in addition to proteoglycans, were extracted from the calcified matrix of osteoblast cultures. All the sulfate label in both proteins was in the form of tyrosine sulfate as assessed by amino acid analysis and thin layer chromatography following alkaline hydrolysis. The elution behavior on DEAE-Sephacel of the major sulfated protein and the apparent Mr on sodium dodecyl sulfate gels were characteristic of bone sialoprotein II extracted from rat. This protein was shown to cross-react with an antiserum raised against bovine bone sialoprotein II, indicating that bone sialoprotein II synthesized by cultured mouse osteoblasts is a tyrosine-sulfated protein. The minor sulfated protein was tentatively identified as bone sialoprotein I or osteopontin based on its elution properties on DEAE-Sephacel and anomalous behavior on sodium dodecyl sulfate gels similar to those reported for rat bone sialoprotein I.

  13. 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

  14. 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. PMID:25266991

  15. 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. PMID:27268440

  16. 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

  17. 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

  18. 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

  19. Novel Bruton’s tyrosine kinase inhibitors currently in development

    PubMed Central

    D’Cruz, Osmond J; Uckun, Fatih M

    2013-01-01

    Bruton’s tyrosine kinase (Btk) is intimately involved in multiple signal-transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. Btk is overexpressed and constitutively active in several B-lineage lymphoid malignancies. Btk has emerged as a new antiapoptotic molecular target for treatment of B-lineage leukemias and lymphomas. Preclinical and early clinical results indicate that Btk inhibitors may be useful in the treatment of leukemias and lymphomas. PMID:23493945

  20. Transformation of erythroid progenitors by viral and cellular tyrosine kinases.

    PubMed

    Beug, H; Schroeder, C; Wessely, O; Deiner, E; Meyer, S; Ischenko, I D; Hayman, M J

    1995-08-01

    Recently, two different normal avian erythroid progenitors were described. They differ in the receptor tyrosine kinases they express and in their ability to undergo self-renewal in culture. A common progenitor, termed stem cell factor (SCF) progenitor, expresses the receptor for avian SCF c-Kit, and undergoes short-term self-renewal when grown in the presence of avian SCF. A second progenitor, referred to as SCF/transforming growth factor-alpha progenitor, coexpresses c-Kit and the avian epidermal growth factor receptor homologue c-ErbB. These progenitors undergo sustained self-renewal when grown in the presence of transforming growth factor-alpha plus estradiol. The phenotype of the normal SCF/transforming growth factor-alpha progenitors closely corresponded to that of erythroid cells transformed by the tyrosine kinase oncogenes v-erbB or v-sea. This suggested that these cells, but not the SCF progenitors, would be the target cells for erythroblast transformation by these oncogenes. However, we demonstrate that both progenitor cells can be transformed by the v-erbB and v-sea oncogenes and also by the ligand-activated proto-oncogene product c-ErbB. We conclude that the target cell specificity of certain tyrosine kinase oncoproteins for erythroid cells is a reflection of their ability to provide signals for self-renewal that normally emanate from the endogenous c-ErbB protein. PMID:8547228

  1. 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.

  2. MERTK receptor tyrosine kinase is a therapeutic target in melanoma

    PubMed Central

    Schlegel, Jennifer; Sambade, Maria J.; Sather, Susan; Moschos, Stergios J.; Tan, Aik-Choon; Winges, Amanda; DeRyckere, Deborah; Carson, Craig C.; Trembath, Dimitri G.; Tentler, John J.; Eckhardt, S. Gail; Kuan, Pei-Fen; Hamilton, Ronald L.; Duncan, Lyn M.; Miller, C. Ryan; Nikolaishvili-Feinberg, Nana; Midkiff, Bentley R.; Liu, Jing; Zhang, Weihe; Yang, Chao; Wang, Xiaodong; Frye, Stephen V.; Earp, H. Shelton; Shields, Janiel M.; Graham, Douglas K.

    2013-01-01

    Metastatic melanoma is one of the most aggressive forms of cutaneous cancers. Although recent therapeutic advances have prolonged patient survival, the prognosis remains dismal. C-MER proto-oncogene tyrosine kinase (MERTK) is a receptor tyrosine kinase with oncogenic properties that is often overexpressed or activated in various malignancies. Using both protein immunohistochemistry and microarray analyses, we demonstrate that MERTK expression correlates with disease progression. MERTK expression was highest in metastatic melanomas, followed by primary melanomas, while the lowest expression was observed in nevi. Additionally, over half of melanoma cell lines overexpressed MERTK compared with normal human melanocytes; however, overexpression did not correlate with mutations in BRAF or RAS. Stimulation of melanoma cells with the MERTK ligand GAS6 resulted in the activation of several downstream signaling pathways including MAPK/ERK, PI3K/AKT, and JAK/STAT. MERTK inhibition via shRNA reduced MERTK-mediated downstream signaling, reduced colony formation by up to 59%, and diminished tumor volume by 60% in a human melanoma murine xenograft model. Treatment of melanoma cells with UNC1062, a novel MERTK-selective small-molecule tyrosine kinase inhibitor, reduced activation of MERTK-mediated downstream signaling, induced apoptosis in culture, reduced colony formation in soft agar, and inhibited invasion of melanoma cells. This work establishes MERTK as a therapeutic target in melanoma and provides a rationale for the continued development of MERTK-targeted therapies. PMID:23585477

  3. Direct Binding of GTP Cyclohydrolase and Tyrosine Hydroxylase

    PubMed Central

    Bowling, Kevin M.; Huang, Zhinong; Xu, Dong; Ferdousy, Faiza; Funderburk, Christopher D.; Karnik, Nirmala; Neckameyer, Wendi; O'Donnell, Janis M.

    2008-01-01

    The signaling functions of dopamine require a finely tuned regulatory network for rapid induction and suppression of output. A key target of regulation is the enzyme tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, which is activated by phosphorylation and modulated by the availability of its cofactor, tetrahydrobiopterin. The first enzyme in the cofactor synthesis pathway, GTP cyclohydrolase I, is activated by phosphorylation and inhibited by tetrahydrobiopterin. We previously reported that deficits in GTP cyclohydrolase activity in Drosophila heterozygous for mutant alleles of the gene encoding this enzyme led to tightly corresponding diminution of in vivo tyrosine hydroxylase activity that could not be rescued by exogenous cofactor. We also found that the two enzymes could be coimmunoprecipitated from tissue extracts and proposed functional interactions between the enzymes that extended beyond provision of cofactor by one pathway for another. Here, we confirm the physical association of these enzymes, identifying interacting regions in both, and we demonstrate that their association can be regulated by phosphorylation. The functional consequences of the interaction include an increase in GTP cyclohydrolase activity, with concomitant protection from end-product feedback inhibition. In vivo, this effect would in turn provide sufficient cofactor when demand for catecholamine synthesis is greatest. The activity of tyrosine hydroxylase is also increased by this interaction, in excess of the stimulation resulting from phosphorylation alone. Vmax is elevated, with no change in Km. These results demonstrate that these enzymes engage in mutual positive regulation. PMID:18801743

  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. PMID:12237126

  5. 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.

  6. Involvement of the Tyrosine Kinase Fer in Cell Adhesion

    PubMed Central

    Rosato, Roberto; Veltmaat, Jacqueline M.; Groffen, John; Heisterkamp, Nora

    1998-01-01

    The Fer protein belongs to the fes/fps family of nontransmembrane receptor tyrosine kinases. Lack of success in attempts to establish a permanent cell line overexpressing it at significant levels suggested a strong negative selection against too much Fer protein and pointed to a critical cellular function for Fer. Using a tetracycline-regulatable expression system, overexpression of Fer in embryonic fibroblasts was shown to evoke a massive rounding up, and the subsequent detachment of the cells from the substratum, which eventually led to cell death. Induction of Fer expression coincided with increased complex formation between Fer and the cadherin/src-associated substrate p120cas and elevated tyrosine phosphorylation of p120cas. β-Catenin also exhibited clearly increased phosphotyrosine levels, and Fer and β-catenin were found to be in complex. Significantly, although the levels of α-catenin, β-catenin, and E-cadherin were unaffected by Fer overexpression, decreased amounts of α-catenin and β-catenin were coimmunoprecipitated with E-cadherin, demonstrating a dissolution of adherens junction complexes. A concomitant decrease in levels of phosphotyrosine in the focal adhesion-associated protein p130 was also observed. Together, these results provide a mechanism for explaining the phenotype of cells overexpressing Fer and indicate that the Fer tyrosine kinase has a function in the regulation of cell-cell adhesion. PMID:9742093

  7. 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

  8. eps15, a novel tyrosine kinase substrate, exhibits transforming activity.

    PubMed Central

    Fazioli, F; Minichiello, L; Matoskova, B; Wong, W T; Di Fiore, P P

    1993-01-01

    An expression cloning method which allows direct isolation of cDNAs encoding substrates for tyrosine kinases was applied to the study of the epidermal growth factor (EGF) receptor (EGFR) signaling pathway. A previously undescribed cDNA was isolated and designated eps15. The structural features of the predicted eps15 gene product allow its subdivision into three domains. Domain I contains signatures of a regulatory domain, including a candidate tyrosine phosphorylation site and EF-hand-type calcium-binding domains. Domain II presents the characteristic heptad repeats of coiled-coil rod-like proteins, and domain III displays a repeated aspartic acid-proline-phenylalanine motif similar to a consensus sequence of several methylases. Antibodies specific for the eps15 gene product recognize two proteins: a major species of 142 kDa and a minor component of 155 kDa, both of which are phosphorylated on tyrosine following EGFR activation by EGF in vivo. EGFR is also able to directly phosphorylate the eps15 product in vitro. In addition, phosphorylation of the eps15 gene product in vivo is relatively receptor specific, since the erbB-2 kinase phosphorylates it very inefficiently. Finally, overexpression of eps15 is sufficient to transform NIH 3T3 cells, thus suggesting that the eps15 gene product is involved in the regulation of mitogenic signals. Images PMID:7689153

  9. Protein Tyrosine Phosphatase φ Regulates Paxillin Tyrosine Phosphorylation and Mediates Colony-Stimulating Factor 1-Induced Morphological Changes in Macrophages

    PubMed Central

    Pixley, Fiona J.; Lee, Pierre S. W.; Condeelis, John S.; Stanley, E. Richard

    2001-01-01

    Removal of colony-stimulating factor 1 (CSF-1) causes macrophages to round up and to increase their expression of protein tyrosine phosphatase φ (PTPφ). This is accompanied by the disruption of focal complexes and the formation of ruffles. Here we have overexpressed wild-type (WT) PTPφ and a phosphatase-inactive (C325S) mutant in a macrophage cell line in the presence and absence of CSF-1. In the presence of CSF-1, WT PTPφ induces cell rounding and ruffle formation, while C325S PTPφ has no effect. In contrast, in CSF-1-starved cells, C325S PTPφ behaves in a dominant negative fashion, preventing rounding and ruffling. Furthermore, C325S PTPφ increases adhesion in cycling cells, while WT PTPφ enhances motility. In WT PTPφ-overexpressing cells, the focal contact protein paxillin is selectively depleted from focal complexes and specifically dephosphorylated on tyrosine. In contrast, paxillin is hyperphosphorylated in C325S PTPφ-expressing cells. Moreover, a complex containing PTPφ, paxillin, and a paxillin-associated tyrosine kinase, Pyk2, can be immunoprecipitated from macrophage lysates, and the catalytic domain of PTPφ selectively binds paxillin and Pyk2 in vitro. Although PTPφ and Pyk2 do not colocalize with paxillin in focal complexes, all three proteins are colocalized in dorsal ruffles. The results suggest that paxillin is dephosphorylated by PTPφ in dorsal ruffles, using Pyk2 as a bridging molecule, resulting in a reduced pool of tyrosine-phosphorylated paxillin available for incorporation into focal complexes, thereby mediating CSF-1 regulation of macrophage morphology, adhesion, and motility. PMID:11238916

  10. 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

  11. 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.

  12. Para-Tyrosine Supplementation Improves Insulin- and Liraglutide- Induced Vasorelaxation in Cholesterol-Fed Rats.

    PubMed

    Sélley, Eszter; Kun, Szilárd; Kürthy, Mária; Kovács, Tibor; Wittmann, István; Molnár, Gergo A

    2015-01-01

    Former data of our workgroup indicated that the accumulation of oxidized amino acids (meta- and ortho-tyrosine) due to oxidative stress may play an important role in the impaired insulininduced vasoactive properties of different arterial segments. There are evidences, that incorporation of these amino acids into cellular proteins leads to certain hormonal resistances, which might be restored by supplementation with the physiologic isoform, para-tyrosine. Rats in the control group were kept on a regular diet, rats in the cholesterol-fed group received high-fat diet, while the third group of rats received high-fat diet with para-tyrosine supplementation for 16 weeks. Plasma cholesterol level was significantly higher in the cholesterol-fed group, while the level of cholesterol in the cholesterol+para-tyrosine group did not differ significantly from that of the controls. Plasma level of insulin after glucose stimulation was decreased in the cholesterol-fed group, while that in the para-tyrosine supplemented group did not differ significantly from the controls. Vascular para-, meta- and ortho-tyrosine content was measured with HPLC. Elevated vascular meta-tyrosine/para-tyrosine ratio of cholesterol fed rats could be avoided by para-tyrosine supplementation. Vascular response of the thoracic aorta to insulin and liraglutide was assessed by a DMT multi-myograph. Cholesterol feeding resulted in vascular insulin-and liraglutide resistance, which was restored by para-tyrosine supplementation. Incorporation of the oxidative stress induced pathological tyrosine isoforms leads to vascular-hormone-resistances. We show that the physiological amino acid para-tyrosine is capable of restoring hypercholesterolemia-induced increased meta-tyrosine content of the vascular wall, thus attenuating functional vascular damage. PMID:26202368

  13. 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

  14. 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.

  15. Genomic organization of Bruton`s tyrosine kinase

    SciTech Connect

    Rohrer, J.; Conley, M.E.

    1994-09-01

    Bruton`s tyrosine kinase (Btk), is a nonreceptor tyrosine kinase that has been identified as the defective gene in X-linked agammaglobulinemia (XLA). XLA patients have profound hypogammaglobulinemia and markedly reduced numbers of B cells while their T cell and phagocyte numbers remain normal. To determine the genomic organization of Btk, intron/exon borders were identified by sequencing cosmid DNA using cDNA primers. Nineteen exons spanning 37 kb of genomic DNA were identified. All the intron/exon splice junctions followed the GT/AG rule. The translational ATG start codon was in exon 2 which was 6 kb downstream of exon 1. Exon 19, 519 bp in length and 3.8 kb distal to exon 18, was the largest exon and included the 450 bp of the 3{prime} untranslated region. Exons 6 through 18 formed the largest cluster of exons with no intron being longer than 1550 bp. There was no apparent correlation between the exon boundaries of Btk and the functional domains of the protein or the exon boundaries of src, the nonreceptor protein tyrosine kinase prototype. The region 500 bp upstream of the presumed transcriptional start site was sequenced and found to have a G+C content of 52%. No TATA-type promoter elements in the -20 bp to -30 bp region were identified. However, at position -48 bp, a TGTGAA motif was found that bears some similarity to the TATA box. This sequence was preceded by a perfect inverted CCAAT box at position -90 bp. Three retinoic acid binding sites were also identified at positions -50 bp, -83 bp and -197 bp. Defining the genomic structure of Btk will permit us to identify regulatory elements in this gene and to identify mutations in genomic DNA of patients with XLA.

  16. Bosutinib: a novel second-generation tyrosine kinase inhibitor.

    PubMed

    Isfort, Susanne; Keller-v Amsberg, Gunhild; Schafhausen, Philippe; Koschmieder, Steffen; Brümmendorf, Tim H

    2014-01-01

    Bosutinib (SKI-606) is a 4-anilino-3-quinoline carbonitrile, which acts as a dual inhibitor of Src and ABL kinases. In addition, the BCR-ABL fusion gene product, a constitutively activated tyrosine kinase which is crucial for the development of chronic myeloid leukemia (CML), is highly sensitive to bosutinib. Interestingly, distinctly lower concentrations of bosutinib are required to ablate BCR-ABL phosphorylation when compared to the first-generation tyrosine kinase inhibitor imatinib (IM). Bosutinib is a potent inhibitor of CML cell proliferation in vitro and has demonstrated promising activity in CML patients resistant or intolerant to IM as well as in newly diagnosed patients with chronic phase CML (CML-CP). Remarkably, bosutinib has been found to be capable of overcoming the majority of IM-resistant BCR-ABL mutations. Bosutinib has the potency to induce deep and fast responses in second- and third-/fourth-line treatment, and as a consequence, the drug has recently been licensed for patients previously treated with one or more tyrosine kinase inhibitor(s) and for whom imatinib, nilotinib, and dasatinib are not considered appropriate treatment options. Due to its potency and differing toxicity profile, it promises to be a good therapeutic option for a defined cohort of patients. The most common side effects are gastrointestinal with most of the patients suffering from nausea, vomiting, or diarrhea. For the most part, these gastrointestinal symptoms occur early after treatment initiation, are manageable, and often self-limiting. Continuous monitoring of liver enzymes upon treatment initiation is necessary during bosutinib treatment. In addition to CML treatment, bosutinib has shown some efficacy in selected patients suffering from advanced-stage solid tumors. In conclusion, bosutinib is a promising novel small molecule inhibitor approved now for targeted therapy of CML and in clinical development for other malignancies. PMID:24756786

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. Receptor tyrosine kinases: mechanisms of activation and signaling

    PubMed Central

    Hubbard, Stevan R.; Miller, W. Todd

    2008-01-01

    Receptor tyrosine kinases (RTKs) are essential components of signal transduction pathways that mediate cell-to-cell communication. These single-pass transmembrane receptors, which bind polypeptide ligands — mainly growth factors — play key roles in processes such as cellular growth, differentiation, metabolism and motility. Recent progress has been achieved towards an understanding of the precise (and varied) mechanisms by which RTKs are activated by ligand binding and by which signals are propagated from the activated receptors to downstream targets in the cell. PMID:17306972

  2. Monitoring Conformational Changes in the Receptor Tyrosine Kinase EGFR.

    PubMed

    Becker, Christian; Öcal, Sinan; Nguyen, Hoang D; Phan, Trang; Keul, Marina; Simard, Jeffrey R; Rauh, Daniel

    2016-06-01

    The receptor tyrosine kinase EGFR is regulated by complex conformational changes, and this conformational control is disturbed in certain types of cancer. Many ligands are known to bind EGFR in its active conformation, thereby preventing ATP from binding. Only a few ligands are known to stabilize EGFR in its inactive conformation, thus providing novel strategies for perturbing EGFR activity. We report a direct binding assay that enables the identification of novel ligands that bind to and stabilize the inactive conformation of EGFR. PMID:26991964

  3. Tyrosine kinase receptors as molecular targets In pheochromocytomas and paragangliomas

    PubMed Central

    Cassol, Clarissa A.; Winer, Daniel; Liu, Wei; Guo, Miao; Ezzat, Shereen; Asa, Sylvia L.

    2016-01-01

    Pheochromocytomas and paragangliomas are neuroendocrine tumors shown to be responsive to multi-targeted tyrosine kinase inhibitor treatment. Despite growing knowledge regarding their genetic basis, the ability to predict behavior in these tumors remains challenging. There is also limited knowledge of their tyrosine kinase receptor expression and whether the clinical response observed to the tyrosine kinase inhibitor Sunitinib relates only to its anti-angiogenic properties or also due to a direct effect on tumor cells. To answer these questions, an in vitro model of sunitinib treatment of a pheochromocytoma cell line was created. Sunitinib targets (VEGFRs, PDGFRs, C-KIT), FGFRs and cell cycle regulatory proteins were investigated in human tissue microarrays. SDHB immunohistochemistry was used as a surrogate marker for the presence of succinate dehydrogenase mutations. The FGFR4 G388R SNP was also investigated. Sunitinib treatment in vitro decreases cell proliferation mainly by targeting cell cycle, DNA metabolism, and cell organization genes. FGFR1, -2 and -4, VEGFR2, PDGFRα and p16 were overexpressed in primary human pheochromocytomas and paragangliomas. Discordant results were observed for VEGFR1, p27 and p21 (overexpressed in paragangliomas but underexpressed in pheochromoctyomas); PDGFRβ, Rb and Cyclin D1 (overexpressed in paragangliomas only) and FGFR3 (overexpressed in pheochromocytomas and underexpressed in paragangliomas). Low expression of C-KIT, p53, Aurora Kinase A and B was observed. Nuclear FGFR2 expression was associated with increased risk of metastasis (odds ratio [OR]=7.61; p=0.008), as was membranous PDGFRα (OR= 13.71, p=0.015), membranous VEGFR1 (OR=8.01; p=0.037), nuclear MIB1 (OR=1.26, p=0.008) and cytoplasmic p27 (OR=1.037, p=0.030). FGFR3, VEGFR2 and C-KIT levels were associated with decreased risk of metastasis. We provide new insights into the mechanistic actions of sunitinib in pheochromoctyomas and paragangliomas and support current

  4. Phenylalanine and tyrosine levels in newborn screening blood samples.

    PubMed Central

    Morris, A F; Holton, J B; Burman, D; Colley, J R

    1983-01-01

    A previously described difference in newborn blood phenylalanine concentrations between those living in urban and non-urban areas in the south west of England has been confirmed and shown to be independent of the type of feed. Several factors including the place of abode, type of feed, birthweight, and the accuracy of the test have been found to affect the measured, phenylalanine concentration in the newborn screening blood spot, and the importance of these results to screening practice is considered. Blood tyrosine also varied with the above factors, but severe, neonatal tyrosinaemia was shown to be a rare problem. PMID:6847230

  5. Organization and evolution of the rat tyrosine hydroxylase gene

    SciTech Connect

    Brown, E.R.; Coker, G.T. III; O'Malley, K.L.

    1987-08-11

    This report describes the organization of the rat tyrosine hydroxylase (TH) gene and compares its structure with the human phenylalanine hydroxylase gene. Both genes are single copy and contain 13 exons separated by 12 introns. Remarkably, the positions of 10 out 12 intron/exon boundaries are identical for the two genes. These results support the idea that these hydroxylases genes are members of a gene family which has a common evolutionary origin. The authors predict that this ancestral gene would have encoded exons similar to those of TH prior to evolutionary drift to other members of this gene family.

  6. 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

  7. 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

  8. 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.

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

    PubMed

    Mahajan, Kiran; Mahajan, Nupam P

    2015-12-15

    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

  10. Distinct tyrosine autophosphorylation sites negatively and positively modulate neu-mediated transformation.

    PubMed Central

    Dankort, D L; Wang, Z; Blackmore, V; Moran, M F; Muller, W J

    1997-01-01

    A number of cytoplasmic signaling molecules are thought to mediate mitogenic signaling from the activated Neu receptor tyrosine kinase through binding specific phosphotyrosine residues located within the intracellular portion of Neu/c-ErbB-2. An activated neu oncogene containing tyrosine-to-phenylalanine substitutions at each of the known autophosphorylation sites was generated and assessed for its specific transforming potential in Rat1 and NIH 3T3 fibroblasts. Mutation of these sites resulted in a dramatic impairment of the transforming potential of neu. To assess the role of these tyrosine phosphorylation sites in cellular transformation, the transforming potential of a series of mutants in which individual tyrosine residues were restored to this transformation-debilitated neu mutant was evaluated. Reversion of any one of four mutated sites to tyrosine residues restored wild-type transforming activity. While each of these transforming mutants displayed Ras-dependent signaling, the transforming activity of two of these mutants was correlated with their ability to bind either the GRB2 or SHC adapter molecules that couple receptor tyrosine kinases to the Ras signaling pathway. By contrast, restoration of a tyrosine residue located at position 1028 completely suppressed the basal transforming activity of this mutated neu molecule or other transforming neu molecules which possessed single tyrosine residues. These data argue that the transforming potential of activated neu is mediated both by positive and negative regulatory tyrosine phosphorylation sites. PMID:9271418

  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. Activity-based kinase profiling of approved tyrosine kinase inhibitors.

    PubMed

    Kitagawa, Daisuke; Yokota, Koichi; Gouda, Masaki; Narumi, Yugo; Ohmoto, Hiroshi; Nishiwaki, Eiji; Akita, Kensaku; Kirii, Yasuyuki

    2013-02-01

    The specificities of nine approved tyrosine kinase inhibitors (imatinib, dasatinib, nilotinib, gefitinib, erlotinib, lapatinib, sorafenib, sunitinib, and pazopanib) were determined by activity-based kinase profiling using a large panel of human recombinant active kinases. This panel consisted of 79 tyrosine kinases, 199 serine/threonine kinases, three lipid kinases, and 29 disease-relevant mutant kinases. Many potential targets of each inhibitor were identified by kinase profiling at the K(m) for ATP. In addition, profiling at a physiological ATP concentration (1 mm) was carried out, and the IC(50) values of the inhibitors against each kinase were compared with the estimated plasma-free concentration (calculated from published pharmacokinetic parameters of plasma C(trough) and C(max) values). This analysis revealed that the approved kinase inhibitors were well optimized for their target kinases. This profiling also implicates activity at particular off-target kinases in drug side effects. Thus, large-scale kinase profiling at both K(m) and physiological ATP concentrations could be useful in characterizing the targets and off-targets of kinase inhibitors. PMID:23279183

  13. Tyrosine-selective protein alkylation using pi-allylpalladium complexes.

    PubMed

    Tilley, S David; Francis, Matthew B

    2006-02-01

    A new protein modification reaction has been developed based on a palladium-catalyzed allylic alkylation of tyrosine residues. This technique employs electrophilic pi-allyl intermediates derived from allylic acetate and carbamate precursors and can be used to modify proteins in aqueous solution at room temperature. To facilitate the detection of modified proteins using SDS-PAGE analysis, a fluorescent allyl acetate was synthesized and coupled to chymotrypsinogen A and bacteriophage MS2. The tyrosine selectivity of the reaction was confirmed through trypsin digest analysis. The utility of the reaction was demonstrated by using taurine-derived carbamates as water solubilizing groups that are cleaved upon protein functionalization. This solubility switching technique was used to install hydrophobic farnesyl and C(17) chains on chymotrypsinogen A in water using little or no cosolvent. Following this, the C(17) alkylated proteins were found to associate with lipid vesicles. In addition to providing a new protein modification strategy targeting an under-utilized amino acid side chain, this method provides convenient access to synthetic lipoproteins. PMID:16433516

  14. The Solution Structure of the Regulatory Domain of Tyrosine Hydroxylase

    PubMed Central

    Zhang, Shengnan; Huang, Tao; Ilangovan, Udayar; Hinck, Andrew P.; Fitzpatrick, Paul F.

    2014-01-01

    Tyrosine hydroxylase (TyrH) catalyzes the hydroxylation of tyrosine to form 3,4-dihydroxyphenylalanine in the biosynthesis of the catecholamine neurotransmitters. The activity of the enzyme is regulated by phosphorylation of serine residues in a regulatory domain and by binding of catecholamines to the active site. Available structures of TyrH lack the regulatory domain, limiting the understanding of the effect of regulation on structure. We report the use of NMR spectroscopy to analyze the solution structure of the isolated regulatory domain of rat TyrH. The protein is composed of a largely unstructured N-terminal region (residues 1-71) and a well-folded C-terminal portion (residues 72-159). The structure of a truncated version of the regulatory domain containing residues 65-159 has been determined and establishes that it is an ACT domain. The isolated domain is a homodimer in solution, with the structure of each monomer very similar to that of the core of the regulatory domain of phenylalanine hydroxylase. Two TyrH regulatory domain monomers form an ACT domain dimer composed of a sheet of eight strands with four α-helices on one side of the sheet. Backbone dynamic analyses were carried out to characterize the conformational flexibility of TyrH65-159. The results provide molecular details critical for understanding the regulatory mechanism of TyrH. PMID:24361276

  15. Growth inhibition by tyrosine kinase inhibitors in mesothelioma cell lines.

    PubMed

    Nutt, Joyce E; O'Toole, Kieran; Gonzalez, David; Lunec, John

    2009-06-01

    Clinical outcome following chemotherapy for malignant pleural mesothelioma is poor and improvements are needed. This preclinical study investigates the effect of five tyrosine kinase inhibitors (PTK787, ZD6474, ZD1839, SU6668 and SU11248) on the growth of three mesothelioma cell lines (NCI H226, NCI H28 and MSTO 211H), the presence of growth factor receptors and inhibition of their downstream signalling pathways. GI50 values were determined: ZD6474 and SU11248, mainly VEGFR2 inhibitors, gave the lowest GI50 across all cell lines (3.5-6.9 microM) whereas ZD1839 gave a GI50 in this range only in H28 cells. All cell lines were positive for EGFR, but only H226 cells were positive for VEGFR2 by Western blotting. ZD6474 and ZD1839 inhibited EGF-induced phosphorylation of EGFR, AKT and ERK, whereas VEGF-induced phosphorylation of VEGFR2 was completely inhibited with 0.1 microM SU11248. VEGFR2 was detected in tumour samples by immunohistochemistry. VEGFR2 tyrosine kinase inhibitors warrant further investigation in mesothelioma. PMID:19318229

  16. 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

  17. A Crystallographic Snapshot of Tyrosine Trans-phosphorylation in Action

    SciTech Connect

    Chen, H.; Xu, C; Ma, J; Eliseenkova, A; Li, W; Pollock, P; Pitteloud, N; Miller, W; Neubert, T; Mohammadi, M

    2008-01-01

    Tyrosine trans-phosphorylation is a key event in receptor tyrosine kinase signaling, yet, the structural basis for this process has eluded definition. Here, we present the crystal structure of the FGF receptor 2 kinases caught in the act of trans-phosphorylation of Y769, the major C-terminal phosphorylation site. The structure reveals that enzyme- and substrate-acting kinases engage each other through elaborate and specific interactions not only in the immediate vicinity of Y769 and the enzyme active site, but also in regions that are as much of 18 {angstrom} away from D626, the catalytic base in the enzyme active site. These interactions lead to an unprecedented level of specificity and precision during the trans-phosphorylation on Y769. Time-resolved mass spectrometry analysis supports the observed mechanism of trans-phosphorylation. Our data provide a molecular framework for understanding the mechanism of action of Kallmann syndrome mutations and the order of trans-phosphorylation reactions in FGFRs. We propose that the salient mechanistic features of Y769 trans-phosphorylation are applicable to trans-phosphorylation of the equivalent major phosphorylation sites in many other RTKs.

  18. 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

  19. Zebrafish tyrosine hydroxylase 2 gene encodes tryptophan hydroxylase.

    PubMed

    Ren, Guiqi; Li, Song; Zhong, Hanbing; Lin, Shuo

    2013-08-01

    The primary pathological hallmark of Parkinson disease (PD) is the profound loss of dopaminergic neurons in the substantia nigra pars compacta. To facilitate the understanding of the underling mechanism of PD, several zebrafish PD models have been generated to recapitulate the characteristics of dopaminergic (DA) neuron loss. In zebrafish studies, tyrosine hydroxylase 1 (th1) has been frequently used as a molecular marker of DA neurons. However, th1 also labels norepinephrine and epinephrine neurons. Recently, a homologue of th1, named tyrosine hydroxylase 2 (th2), was identified based on the sequence homology and subsequently used as a novel marker of DA neurons. In this study, we present evidence that th2 co-localizes with serotonin in the ventral diencephalon and caudal hypothalamus in zebrafish embryos. In addition, knockdown of th2 reduces the level of serotonin in the corresponding th2-positive neurons. This phenotype can be rescued by both zebrafish th2 and mouse tryptophan hydroxylase 1 (Tph1) mRNA as well as by 5-hydroxytryptophan, the product of tryptophan hydroxylase. Moreover, the purified Th2 protein has tryptophan hydroxylase activity comparable with that of the mouse TPH1 protein in vitro. Based on these in vivo and in vitro results, we conclude that th2 is a gene encoding for tryptophan hydroxylase and should be used as a marker gene of serotonergic neurons. PMID:23754283

  20. Role of Receptor Tyrosine Kinase Signaling in Renal Fibrosis.

    PubMed

    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

  1. 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

  2. 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. PMID:22211953

  3. 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

  4. Electrical stimulation increases phosphorylation of tyrosine hydroxylase in superior cervical ganglion of rat.

    PubMed Central

    Cahill, A L; Perlman, R L

    1984-01-01

    Electrical stimulation of the superior cervical ganglion of the rat increased the phosphorylation of tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) in this tissue. Ganglia were incubated with [32P]Pi for 90 min and were then electrically stimulated via the preganglionic nerve. Tyrosine hydroxylase was isolated from homogenates of the ganglia by immunoprecipitation followed by polyacrylamide gel electrophoresis. 32P-labeled tyrosine hydroxylase was visualized by radioautography, and the incorporation of 32P into the enzyme was quantitated by densitometry of the radioautograms. Stimulation of ganglia at 20 Hz for 5 min increased the incorporation of 32P into tyrosine hydroxylase to a level 5-fold that found in unstimulated control ganglia. The increase in phosphorylation of tyrosine hydroxylase was dependent on the duration and frequency of stimulation. Preganglionic stimulation did not increase the phosphorylation of tyrosine hydroxylase in a medium that contained low Ca2+ and high Mg2+. Increases in phosphorylation were reversible; within 30 min after the cessation of stimulation, the incorporation of 32P into tyrosine hydroxylase decreased to the level found in unstimulated ganglia. The nicotinic antagonist hexamethonium reduced the increase in 32P incorporation into tyrosine hydroxylase by about 50%, while the muscarinic antagonist atropine had no effect. Thus, preganglionic stimulation appeared to increase the phosphorylation of tyrosine hydroxylase in part by a nicotinic mechanism and in part by a noncholinergic mechanism. Antidromic stimulation of ganglia also increased the phosphorylation of tyrosine hydroxylase. Two-dimensional gel electrophoresis revealed that electrical stimulation also increased the incorporation of 32P into at least six other phosphoproteins in the ganglion. Images PMID:6150485

  5. 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

  6. Defining the role of tyrosine and rational tuning of oxidase activity by genetic incorporation of unnatural tyrosine analogs.

    PubMed

    Yu, Yang; Lv, Xiaoxuan; Li, Jiasong; Zhou, Qing; Cui, Chang; Hosseinzadeh, Parisa; Mukherjee, Arnab; Nilges, Mark J; Wang, Jiangyun; Lu, Yi

    2015-04-15

    While a conserved tyrosine (Tyr) is found in oxidases, the roles of phenol ring pKa and reduction potential in O2 reduction have not been defined despite many years of research on numerous oxidases and their models. These issues represent major challenges in our understanding of O2 reduction mechanism in bioenergetics. Through genetic incorporation of unnatural amino acid analogs of Tyr, with progressively decreasing pKa of the phenol ring and increasing reduction potential, in the active site of a functional model of oxidase in myoglobin, a linear dependence of both the O2 reduction activity and the fraction of H2O formation with the pKa of the phenol ring has been established. By using these unnatural amino acids as spectroscopic probe, we have provided conclusive evidence for the location of a Tyr radical generated during reaction with H2O2, by the distinctive hyperfine splitting patterns of the halogenated tyrosines and one of its deuterated derivatives incorporated at the 33 position of the protein. These results demonstrate for the first time that enhancing the proton donation ability of the Tyr enhances the oxidase activity, allowing the Tyr analogs to augment enzymatic activity beyond that of natural Tyr. PMID:25672571

  7. 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...

  8. 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

  9. The Next Wave of EGFR Tyrosine Kinase Inhibitors Enter the Clinic.

    PubMed

    Politi, Katerina; Ayeni, Deborah; Lynch, Thomas

    2015-06-01

    The T790M mutation in EGFR accounts for approximately half of all lung cancer cases with acquired resistance to the current clinical EGFR tyrosine kinase inhibitors. In tyrosine kinase inhibitor-resistant lung tumors, rociletinib and AZD9291 are highly active when T790M is present and modestly active when T790M is absent. PMID:26058074

  10. Design and synthesis of constrained analogs of LCRF-0004 as potent RON tyrosine kinase inhibitors.

    PubMed

    Raeppel, Stéphane L; Therrien, Eric; Raeppel, Franck

    2015-09-01

    New fused bicyclic lactam head groups as rigidified analogs of thieno[3,2-b]pyridine-based kinase inhibitor LCRF-0004 were designed and synthesized. Depending on the functionalities and the size of these bicyclic head groups, potent inhibitors of RON tyrosine kinase with various level of selectivity against c-Met tyrosine kinase were obtained. PMID:26112445

  11. Phosphotyrosine enrichment identifies focal adhesion kinase and other tyrosine kinases for targeting in canine hemangiosarcoma.

    PubMed

    Marley, K; Maier, C S; Helfand, S C

    2012-09-01

    Canine hemangiosarcoma (HSA) is an endothelial cell malignancy driven, in part, by activating mutations in receptor and non-receptor tyrosine kinases. Proteomics, Western blots and a tyrosine kinase inhibitor were used to elucidate activating mechanisms in HSA cell lines. Phosphotyrosine peptides from focal adhesion kinase (FAK) STAT3, Lyn, Fyn and other signal transduction kinases were identified by mass spectrometry. FAK was constitutively activated at tyrosine 397, the autophosphorylation site, and this was reversible with high concentrations of a FAK inhibitor. FAK inhibitor-14 suppressed migration and phosphorylation of FAK tyrosine 397 and tyrosines 576/577 and was cytotoxic to HSA cells suggesting FAK signalling may be an important contributor to canine HSA survival. PMID:22487216

  12. Phosphorylation of tyrosine residues of calmodulin in Rous sarcoma virus-transformed cells.

    PubMed Central

    Fukami, Y; Nakamura, T; Nakayama, A; Kanehisa, T

    1986-01-01

    Calmodulin, a wide-spread eukaryotic Ca2+-binding protein, was phosphorylated at its tyrosine residues in Rous sarcoma virus (RSV)-transformed chicken and rat cells but not in normal chicken embryo fibroblasts. In contrast, serine and threonine phosphorylation of calmodulin was found to occur in both normal and virus-transformed cells. In an in vitro system containing purified src kinase from RSV-transformed cells, tyrosine phosphorylation of calmodulin by the src kinase was inhibited by Ca2+. Furthermore, the tyrosine-phosphorylated calmodulin showed slower mobility than that of nonphosphorylated calmodulin in NaDodSO4/polyacrylamide gel electrophoresis when Ca2+ was present. These results suggest that the structure of calmodulin Ca2+ complex may be altered by tyrosine phosphorylation. It is thus inferred that Ca2+ may regulate the level of tyrosine phosphorylation of calmodulin in RSV-transformed cells, and phosphorylation in turn may attenuate the function of this protein in vivo. Images PMID:2424020

  13. 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

  14. Muscarinic agonists and phorbol esters increase tyrosine phosphorylation of a 40-kilodalton protein in hippocampal slices

    SciTech Connect

    Stratton, K.R.; Worley, P.F.; Huganir, R.L.; Baraban, J.M. )

    1989-04-01

    The authors have used the hippocampal slice preparation to investigate the regulation of protein tyrosine phosphorylation in brain. After pharmacological treatment of intact slices, proteins were separated by electrophoresis, and levels of protein tyrosine phosphorylation were assessed by immunoblotting with specific anti-phosphotyrosine antibodies. Phorbol esters, activators of the serine- and threonine-phosphorylating enzyme protein kinase C, selectively increase tyrosine phosphorylation of a soluble protein with an apparent molecular mass of approximately 40 kilodaltons. Muscarinic agonists such as carbachol and oxotremorine M that strongly activate the inositol phospholipid system also increase tyrosine phosphorylation of this protein. Neurotransmitter activation of the inositol phospholipid system and protein kinase C appears to trigger a cascade leading to increased tyrosine phosphorylation.

  15. 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

  16. WEE1 Tyrosine Kinase, A Novel Epigenetic Modifier

    PubMed Central

    Mahajan, Kiran; Mahajan, Nupam P.

    2013-01-01

    The cell cycle requires cells to duplicate their chromatin, DNA, and histones, while retaining a subset of epigenetic marks, in a highly coordinated manner. The WEE1 kinase was identified as an important regulator during S phase, preventing entry into mitosis until DNA replication has been completed. Interestingly, WEE1 has also emerged as a key player in regulating histone synthesis. It phosphorylates histone H2B at tyrosine 37 in the nucleosomes found upstream of the histone gene cluster, which suppresses histone transcription in late S phase. These observations highlight a dual role for WEE1 as both a mitotic gatekeeper and a surveyor of chromatin synthesis, providing a direct link between epigenetics and cell cycle progression. Importantly, this link has implications for the design of novel epigenetic inhibitors targeting cancers that display elevated expression of this kinase. PMID:23537585

  17. 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

  18. 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

  19. Biological Screening of a Novel Nickel (II) Tyrosine Complex

    PubMed Central

    Islam, Md. Rafiqul; Islam, S. M. Rafiqul; Noman, Abu Shadat Mohammod; Khanam, Jahan Ara; Ali, Shaikh Mohammad Mohsin; Alam, Shahidul

    2007-01-01

    A newly synthesized Nickel (II) tyrosine complex was screened as potential antimicrobial agent against a number of medically important bacteria (Bacillus subtilis, Streptococcus β-haemolytica, Escherichia coli, Shigella dysenterae) and fungi (Aspergillus fumigatus, Candida albicans, Aspergillus niger, Aspergillus flavus, Penicillium sp.) strains. were used for antifungal activity. The antimicrobial activity was evaluated using the Agar Disc method. Moreover, the minimum inhibitory concentration of the complexes was determined against the same pathogenic bacteria and the values were found between 4~64 µg ml-1. Brine shrimp bioassay was carried out for cytotoxicity measurements of the complexes. The LC50 values were calculated after probit transformation of the resulting mortality data and found to be 6 µg ml-1. PMID:24015064

  20. 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

  1. 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

  2. Getting Syk: Spleen Tyrosine Kinase as a Therapeutic Target

    PubMed Central

    Geahlen, Robert L.

    2014-01-01

    Syk is a cytoplasmic protein-tyrosine kinase well known for its ability to couple immune cell receptors to intracellular signaling pathways that regulate cellular responses to extracellular antigens and antigen-immunoglobulin complexes of particular importance to the initiation of inflammatory responses. Thus, Syk is an attractive target for therapeutic kinase inhibitors designed to ameliorate symptoms and consequences of acute and chronic inflammation. Its more recently recognized role as a promoter of cell survival in numerous cancer cell types ranging from leukemia to retinoblastoma has attracted considerable interest as a target for a new generation of anticancer drugs. This review discusses the biological processes in which Syk participates that have made this kinase such a compelling drug target. PMID:24975478

  3. 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. PMID:19066037

  4. Receptor tyrosine kinase inhibitors: Are they real tumor killers?

    PubMed

    Gaumann, Andreas K A; Kiefer, Friedemann; Alfer, Joachim; Lang, Sven A; Geissler, Edward K; Breier, Georg

    2016-02-01

    Inhibiting tumor growth by targeting the tumor vasculature was first proposed by Judah Folkman almost 40 years ago. Since then, different approaches and numerous drugs and agents have been developed to achieve this goal, either with the aim of inhibiting tumor neoangiogenesis or normalizing the tumor vasculature. Among the most promising therapeutic targets are receptor tyrosine kinases (RTKs), some of which are predominantly expressed on tumor endothelial cells, although they are sometimes also present on tumor cells. The majority of RTK inhibitors investigated over the past two decades competes with ATP at the active site of the kinase and therefore block the phosphorylation of intracellular targets. Some of these drugs have been approved for therapy, whereas others are still in clinical trials. Here, we discuss the scientific basis, current status, problems and future prospects of RTK inhibition in anti-tumor therapy. PMID:25716346

  5. Mechanisms of resistance to EGFR tyrosine kinase inhibitors

    PubMed Central

    Huang, Lihua; Fu, Liwu

    2015-01-01

    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. PMID:26579470

  6. 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

  7. 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

  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. PMID:25693719

  9. Characterization of the protein tyrosine phosphatase PRL from Entamoeba histolytica.

    PubMed

    Ramírez-Tapia, Ana Lilia; Baylón-Pacheco, Lidia; Espíritu-Gordillo, Patricia; Rosales-Encina, José Luis

    2015-12-01

    Protein tyrosine phosphatase of regenerating liver (PRL) is a group of phosphatases that has not been broadly studied in protozoan parasites. In humans, PRLs are involved in metastatic cancer, the promotion of cell migration and invasion. PTPs have been increasingly recognized as important effectors of host-pathogen interactions. We characterized the only putative protein tyrosine phosphatase PRL (PTP EhPRL) in the eukaryotic human intestinal parasite Entamoeba histolytica. Here, we reported that the EhPRL protein possessed the classical HCX5R catalytic motif of PTPs and the CAAX box characteristic of the PRL family and exhibited 31-32% homology with the three human PRL isoforms. In amebae, the protein was expressed at low but detectable levels. The recombinant protein (rEhPRL) had enzymatic activity with the 3-o-methyl fluorescein phosphate (OMFP) substrate; this enzymatic activity was inhibited by the PTP inhibitor o-vanadate. Using immunofluorescence we showed that native EhPRL was localized to the cytoplasm and plasma membrane. When the trophozoites interacted with collagen, EhPRL relocalized over time to vesicle-like structures. Interaction with fibronectin increased the presence of the enzyme in the cytoplasm. Using RT-PCR, we demonstrated that EhPRL mRNA expression was upregulated when the trophozoites interacted with collagen but not with fibronectin. Trophozoites recovered from amoebic liver abscesses showed higher EhPRL mRNA expression levels than normal trophozoites. These results strongly suggest that EhPRL may play an important role in the biology and adaptive response of the parasite to the host environment during amoebic liver abscess development, thereby participating in the pathogenic mechanism. PMID:26431820

  10. Kyotorphin (tyrosine-arginine) synthetase in rat brain synaptosomes.

    PubMed

    Ueda, H; Yoshihara, Y; Fukushima, N; Shiomi, H; Nakamura, A; Takagi, H

    1987-06-15

    Kyotorphin (Tyr-Arg) is a unique neuropeptide which produces analgesia by releasing Met-enkephalin from slices of the brain and spinal cord. Recent studies revealed that kyotorphin possesses the properties of neurotransmitter/neuroregulator. In the present study, we identified a kyotorphin synthetase in the soluble fraction of rat brain synaptosomes (synaptosol) and characterized it. The enzyme partially purified with Sephacryl S-300 showed an absolute requirement for ATP, MgCl2, tyrosine, and arginine. The optimal pH was 7.5-9.0 and the pI was determined to be 6.1-6.2 by isoelectric focusing. The Km was 25.6 microM for tyrosine, 926 microM for arginine, 294 microM for ATP, and 442 microM for MgCl2. The Vmax was 34.0 pmol/mg of protein/h. The apparent molecular size of this "kyotorphin synthetase" further purified by the DE52 column was 240,000-245,000 daltons, estimated using TSKgel G4000SW column chromatography. The enzyme reaction is represented by the following equation: Tyr + Arg + ATP + MgCl2 + kyotorphin synthetase----Tyr-Arg (kyotorphin) + AMP + PPi + MgCl2 + kyotorphin synthetase. The regional distribution and subcellular localization of the synthetase showed a close correlation to that of kyotorphin levels in the rat brain. The amounts of kyotorphin formed from amino acids by the synthetase in the dialyzed synaptosol was 3.0-4.0 times higher than that from precursor proteins by processing enzymes within the 30 min incubation. PMID:3597366

  11. Antigen receptor signaling: integration of protein tyrosine kinase functions.

    PubMed

    Tamir, I; Cambier, J C

    1998-09-17

    Antigen receptors on T and B cells function to transduce signals leading to a variety of biologic responses minimally including antigen receptor editing, apoptotic death, developmental progression, cell activation, proliferation and survival. The response to antigen depends upon antigen affinity and valence, involvement of coreceptors in signaling and differentiative stage of the responding cell. The requirement that these receptors integrate signals that drive an array of responses may explain their evolved structural complexity. Antigen receptors are composed of multiple subunits compartmentalized to provide antigen recognition and signal transduction function. In lieu of on-board enzymatic activity these receptors rely on associated Protein Tyrosine Kinases (PTKs) for their signaling function. By aggregating the receptors, and hence their appended PTKs, antigens induce PTK transphosphorylation, activating them to phosphorylate the receptor within conserved motifs termed Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) found in transducer subunits. The tyrosyl phosphorylated ITAMs then interact with Src Homology 2 (SH2) domains within the PTKs leading to their further activation. As receptor phosphorylation is amplified, other effectors, such as Shc, dock by virtue of SH2 binding, and serve, in-turn, as substrates for these PTKs. This sequence of events not only provides a signal amplification mechanism by combining multiple consecutive steps with positive feedback, but also allows for signal diversification by differential recruitment of effectors that provide access to distinct parallel downstream signaling pathways. The subject of antigen receptor signaling has been recently reviewed in depth (DeFranco, 1997; Kurosaki, 1997). Here we discuss the biochemical basis of antigen receptor signal transduction, using the B cell receptor (BCR) as a paradigm, with specific emphasis on the involved PTKs. We review several specific mechanisms by which responses

  12. Bruton tyrosine kinase inhibition in chronic lymphocytic leukemia.

    PubMed

    Maddocks, Kami; Jones, Jeffrey A

    2016-04-01

    Chronic lymphocytic leukemia (CLL) is the most common adult leukemia and remains incurable outside of the setting of allogeneic stem cell transplant. While the standard therapy for both initial and relapsed CLL has traditionally included monoclonal antibody therapy in combination with chemotherapy, there are patients with high-risk disease features including unmutated IgVH, del(11q22) and del(17p13) that are associated with poor overall responses to these therapies with short time to relapse and shortened overall survival. Additionally, many of these therapies have a high rate of infectious toxicity in a population already at increased risk. Targeting the B-cell receptor (BCR) signaling pathway has emerged as a promising therapeutic advance in a variety of B-cell malignancies, including CLL. Bruton agammaglobulinemia tyrosine kinase (Btk) is a tyrosine kinase in the BCR pathway critical to the survival of both normal and malignant B cells and inhibition of this kinase has shown to block the progression of CLL. Ibrutinib, a first in class oral inhibitor of Btk, has shown promise as a very effective agent in the treatment of CLL-in both relapsed and upfront therapy, alone and in combination with other therapies, and in patients of all-risk disease-which has led to its approval in relapsed CLL and as frontline therapy in patients with the high-risk del(17p13) disease. Several studies are ongoing to evaluate the efficacy and safety of ibrutinib in combination with chemotherapy as frontline treatment for CLL and investigation into newer-generation Btk inhibitors is also underway. PMID:27040703

  13. 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

  14. 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.

  15. 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.

  16. Grass roots chemistry: meta-Tyrosine, an herbicidal nonprotein amino acid

    PubMed Central

    Bertin, Cécile; Weston, Leslie A.; Huang, Tengfang; Jander, Georg; Owens, Thomas; Meinwald, Jerrold; Schroeder, Frank C.

    2007-01-01

    Fine fescue grasses displace neighboring plants by depositing large quantities of an aqueous phytotoxic root exudate in the soil rhizosphere. Via activity-guided fractionation, we have isolated and identified the nonprotein amino acid m-tyrosine as the major active component. m-Tyrosine is significantly more phytotoxic than its structural isomers o- and p-tyrosine. We show that m-tyrosine exposure results in growth inhibition for a wide range of plant species and propose that the release of this nonprotein amino acid interferes with root development of competing plants. Acid hydrolysis of total root protein from Arabidopsis thaliana showed incorporation of m-tyrosine, suggesting this as a possible mechanism of phytotoxicity. m-Tyrosine inhibition of A. thaliana root growth is counteracted by exogenous addition of protein amino acids, with phenylalanine having the most significant effect. The discovery of m-tyrosine, as well as a further understanding of its mode(s) of action, could lead to the development of biorational approaches to weed control. PMID:17940026

  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. Regulatory Interactions between a Bacterial Tyrosine Kinase and Its Cognate Phosphatase*

    PubMed Central

    Temel, Deniz B.; Dutta, Kaushik; Alphonse, Sébastien; Nourikyan, Julien; Grangeasse, Christophe; Ghose, Ranajeet

    2013-01-01

    The cyclic process of autophosphorylation of the C-terminal tyrosine cluster (YC) of a bacterial tyrosine kinase and its subsequent dephosphorylation following interactions with a counteracting tyrosine phosphatase regulates diverse physiological processes, including the biosynthesis and export of polysaccharides responsible for the formation of biofilms or virulence-determining capsules. We provide here the first detailed insight into this hitherto uncharacterized regulatory interaction at residue-specific resolution using Escherichia coli Wzc, a canonical bacterial tyrosine kinase, and its opposing tyrosine phosphatase, Wzb. The phosphatase Wzb utilizes a surface distal to the catalytic elements of the kinase, Wzc, to dock onto its catalytic domain (WzcCD). WzcCD binds in a largely YC-independent fashion near the Wzb catalytic site, inducing allosteric changes therein. YC dephosphorylation is proximity-mediated and reliant on the elevated concentration of phosphorylated YC near the Wzb active site resulting from WzcCD docking. Wzb principally recognizes the phosphate of its phosphotyrosine substrate and further stabilizes the tyrosine moiety through ring stacking interactions with a conserved active site tyrosine. PMID:23543749

  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. The role of non-receptor protein tyrosine kinases in the excitotoxicity induced by the overactivation of NMDA receptors.

    PubMed

    Sun, Yongjun; Chen, You; Zhan, Liying; Zhang, Linan; Hu, Jie; Gao, Zibin

    2016-04-01

    Protein tyrosine phosphorylation is one of the primary modes of regulation of N-methyl-d-aspartate (NMDA) receptors. The non-receptor tyrosine kinases are one of the two types of protein tyrosine kinases that are involved in this process. The overactivation of NMDA receptors is a primary reason for neuron death following cerebral ischemia. Many studies have illustrated the important role of non-receptor tyrosine kinases in ischemia insults. This review introduces the roles of Src, Fyn, focal adhesion kinase, and proline-rich tyrosine kinase 2 in the excitotoxicity induced by the overactivation of NMDA receptors following cerebral ischemia. PMID:26540220

  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. 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.

  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. 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

  6. Pyridazinone derivatives displaying highly potent and selective inhibitory activities against c-Met tyrosine kinase.

    PubMed

    Liu, Yang; Jin, Shiyu; Peng, Xia; Lu, Dong; Zeng, Limin; Sun, Yiming; Ai, Jing; Geng, Meiyu; Hu, Youhong

    2016-01-27

    Over activation of c-Met tyrosine kinase is known to promote tumorigenesis and metastasis, as well as to cause therapeutic resistance. Herein we describe the design, synthesis and biological activities of novel, ATP-competitive, c-Met tyrosine kinase inhibitors that are members of the 6-aryl-2-(3-(heteroarylamino)benzyl)pyridazinone family. A structure-activity relationship (SAR) study of these substances led to identification of pyridazinone 19 as a highly selective and potent c-Met tyrosine inhibitor, which displays favorable pharmacokinetic properties in mice and significant antitumor activity against a c-Met driven EBC-1 tumor xenograft. PMID:26698536

  7. Evaluation of toxicity on epithelial and tumor cells of biaryl dipeptide tyrosines.

    PubMed

    Vasconcelos, Stanley N S; Drewes, Carine C; de Vinci Kanda Kupa, Leonard; Farsky, Sandra H P; Stefani, Hélio A

    2016-05-23

    We report a method to obtain biaryl dipeptide tyrosine via Suzuki-Miyaura and alkynyl dipeptide tyrosine by Sonogashira cross-coupling reactions. Analysis of the biological action of biaryl dipeptide tyrosine 4d compound showed its ability to impair the metabolism and proliferation of SK-Mel-28 human melanoma lineage cells, independently of mitochondrial membrane depolarization, apoptosis and necrosis. Moreover, 4d compound did not cause toxicity to human umbilical vein endothelial cells (HUVEC), suggesting its toxic specificity to cancer cells. PMID:26974369

  8. 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. PMID:26709535

  9. 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

  10. Myoclonus-dystonia syndrome due to tyrosine hydroxylase deficiency

    PubMed Central

    Mencacci, Niccolo E.; Cordivari, Carla; Batla, Amit; Wood, Nick W.; Houlden, Henry; Hardy, John; Bhatia, Kailash P.

    2012-01-01

    Objective: To present a new family with tyrosine hydroxylase deficiency (THD) that presented with a new phenotype of predominant, levodopa-responsive myoclonus with dystonia due to compound heterozygosity of one previously reported mutation in the promoter region and a novel nonsynonymous mutation in the other allele, thus expanding the clinical and genetic spectrum of this disorder. Methods: We performed detailed clinical examination of the family and electrophysiology to characterize the myoclonus. We performed analysis of the TH gene and in silico prediction of the possible effect of nonsynonymous substitutions on protein structure. Results: Electrophysiology suggested that the myoclonus was of subcortical origin. Genetic analysis of the TH gene revealed compound heterozygosity of a point mutation in the promoter region (c.1-71 C>T) and a novel nonsynonymous substitution in exon 12 (c.1282G>A, p.Gly428Arg). The latter is a novel variant, predicted to have a deleterious effect on the TH protein function and is the first pathogenic TH mutation in patients of African ancestry. Conclusion: We presented a THD family with predominant myoclonus-dystonia and a new genotype. It is important to consider THD in the differential diagnosis of myoclonus-dystonia, because early treatment with levodopa is crucial for these patients. PMID:22815559

  11. Tyrosine Phosphorylation in Toll-Like Receptor Signaling

    PubMed Central

    Chattopadhyay, Saurabh; Sen, Ganes C.

    2014-01-01

    There is a wealth of knowledge about how different Ser/Thr protein kinases participate in Toll-like receptor (TLR) signaling. In many cases, we know the identities of the Ser/Thr residues of various components of the TLR-signaling pathways that are phosphorylated, the functional consequences of the phosphorylation and the responsible protein kinases. In contrast, the analysis of Tyr-phosphorylation of TLRs and their signaling proteins is currently incomplete, because several existing analyses are not systematic or they do not rely on robust experimental data. Nevertheless, it is clear that many TLRs require, for signaling, ligand-dependent phosphorylation of specific Tyr residues in their cytoplasmic domains; the list includes TLR2, TLR3, TLR4, TLR5, TLR8 and TLR9. In this article, we discuss the current status of knowledge on the effect of Tyr-phosphorylation of TLRs and their signaling proteins on their biochemical and biological functions, the possible identities of the relevant protein tyrosine kinases (PTKs) and the nature of regulations of PTK-mediated activation of TLR signaling pathways. PMID:25022196

  12. 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

  13. Sulfation of tyrosine residues in coagulation factor V

    SciTech Connect

    Hortin, G.L. )

    1990-09-01

    Sulfation of human coagulation factor V was investigated by biosynthetically labeling the products of HepG2 cells with ({sup 35}S)sulfate. There was abundant incorporation of the sulfate label into a product identified as factor V by immunoprecipitation, lability to proteases, affinity for the lectin jacalin, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two or more sites in factor V incorporated sulfate as indicated by labeling of different peptide chains of factor Va. The 150-Kd activation fragment of factor Va incorporated the greatest amounts of sulfate. This fragment of factor Va was bound selectively by jacalin-agarose, reflecting its content of O-linked oligosaccharides. Analysis of an alkaline hydrolysate of sulfate-labeled factor Va by anion-exchange chromatography showed that the sulfate occurred partly in tyrosine sulfate residues and partly in alkaline-labile linkages. Sulfate groups are potentially important structural and functional elements in factor V, and labeling with (35S)sulfate provides a useful approach for examining the biosynthesis and processing of this protein. The hypothesis is advanced that sites of sulfation in factor V and several other plasma proteins contribute to the affinity and specificity of thrombin for these molecules, just as it does for the interaction of thrombin with the potent inhibitor hirudin from leeches.

  14. 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

  15. 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

  16. 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

  17. Mutations in Bruton's tyrosine kinase impair IgA responses.

    PubMed

    Mitsuiki, Noriko; Yang, Xi; Bartol, Sophinus J W; Grosserichter-Wagener, Christina; Kosaka, Yoshiyuki; Takada, Hidetoshi; Imai, Kohsuke; Kanegane, Hirokazu; Mizutani, Shuki; van der Burg, Mirjam; van Zelm, Menno C; Ohara, Osamu; Morio, Tomohiro

    2015-03-01

    X-linked agammaglobulinemia (XLA) is a primary immunodeficiency caused by mutations in Bruton's tyrosine kinase (BTK), and is characterized by markedly decreased numbers of blood B cells and an absence of all immunoglobulin isotypes. We performed whole exome sequencing in a male pediatric patient with dysgammaglobulinemia with IgA deficiency. Genetic analysis revealed a BTK missense mutation (Thr316Ala). To investigate whether a BTK mutation underlay this antibody deficiency with marked decrease of IgA in this patient, we performed functional analyses of B cells and phagocytes, and molecular analyses of somatic hypermutation and class switch recombination. The BTK missense mutation resulted in B cells with reduced BTK and high IgM expression. Equal proportions of CD19(low) and CD19(normal) fractions were observed, and both included naïve and memory B cells. Calcium influx and phospholipase Cγ2 phosphorylation upon IgM stimulation were marginally impaired in CD19(low), but not in CD19(+) B cells. Similar to XLA patients, IgA transcripts showed low SHM levels, whereas IgG transcripts were hardly affected. Our analyses suggest that the BTK mutation likely underlies the disease in this case, and that hypomorphic BTK mutations can result in normal circulating B cell numbers, but specifically impair IgA responses. PMID:25589397

  18. Bruton's tyrosine kinase inhibitors for the treatment of rheumatoid arthritis.

    PubMed

    Whang, Jennifer A; Chang, Betty Y

    2014-08-01

    The function and role of Bruton's tyrosine kinase (BTK) in human B cell development was demonstrated by its association with X-linked agammaglobulinemia (XLA) manifested by a substantial reduction in immunoglobulins and B cells. BTK has a crucial role in pre-B cell receptor (BCR) and BCR signaling during normal B cell development and activation. Aberrant BCR signaling is associated with autoimmune diseases, such as rheumatoid arthritis (RA). In addition, BTK is also expressed in myeloid cell populations, including monocytes, macrophages, neutrophils and mast cells. These innate cells infiltrate the synovial cavity and produce inflammatory cytokines, aggravating arthritic symptoms. In myeloid cell populations, BTK functions downstream of the Fcγ receptors (FcγR) and Fcɛ receptors (FcɛR). In the absence of BTK, FcR-mediated functions, such as cytokine production, are impaired. In addition, Xid mice, which have a mutation in BTK, have decreased susceptibility to developing collagen-induced arthritis (CIA). Given that BTK is involved in multiple signaling pathways downstream of the BCR and FcR, it is an attractive therapeutic target for RA. PMID:24721226

  19. 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

  20. Function of redox-active tyrosine in photosystem II.

    PubMed

    Ishikita, Hiroshi; Knapp, Ernst-Walter

    2006-06-01

    Water oxidation at photosystem II Mn-cluster is mediated by the redox-active tyrosine Y(Z). We calculated the redox potential (E(m)) of Y(Z) and its symmetrical counterpart Y(D), by solving the linearized Poisson-Boltzmann equation. The calculated E(m)(Y( )/Y(-)) were +926 mV/+694 mV for Y(Z)/Y(D) with the Mn-cluster in S2 state. Together with the asymmetric position of the Mn-cluster relative to Y(Z/D), differences in H-bond network between Y(Z) (Y(Z)/D1-His(190)/D1-Asn(298)) and Y(D) (Y(D)/D2-His(189)/D2-Arg(294)/CP47-Glu(364)) are crucial for E(m)(Y(Z/D)). When D1-His(190) is protonated, corresponding to a thermally activated state, the calculated E(m)(Y(Z)) was +1216 mV, which is as high as the E(m) for P(D1/D2). We observed deprotonation at CP43-Arg(357) upon S-state transition, which may suggest its involvement in the proton exit pathway. E(m)(Y(D)) was affected by formation of P(D2)(+) (but not P(D1)(+)) and sensitive to the protonation state of D2-Arg(180). This points to an electrostatic link between Y(D) and P(D2). PMID:16513785

  1. 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

  2. The Evolution of Tyrosine-Recombinase Elements in Nematoda

    PubMed Central

    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. PMID:25197791

  3. Estrus cycle effect on muscle tyrosine kinase activity in bitches.

    PubMed

    Gomes Pöppl, Álan; Costa Valle, Sandra; Hilário Díaz González, Félix; de Castro Beck, Carlos Afonso; Kucharski, Luiz Carlos; Silveira Martins Da Silva, Roselis

    2012-03-01

    Estrus cycle is a well recognized cause of insulin resistance in bitches. The insulin receptor (IR) as well as the insulin-like growth factor-I receptor belong to the same subfamily of tyrosine kinase (TK) receptors. The objective of this study was to evaluate basal TK activity in muscle tissue of bitches during the estrus cycle. Twenty-four bitches were used in the study (7 in anestrus, 7 in estrus, and 10 in diestrus). Muscle samples, taken after spaying surgery to determine TK activity, were immediately frozen in liquid nitrogen and then stored at -80°C until the membranes were prepared by sequential centrifugation after being homogenized. TK activity was determined by Poly (Glu 4:Tyr 1) phosphorylation and expressed in cpm/μg of protein. TK activity was significantly lower (P < 0.001) in the animals in estrus (104.5 ± 11.9 cpm/μg of protein) and diestrus (94.5 ± 16.9 cpm/μg of protein) when compared with bitches in anestrus (183.2 ± 39.2 cpm/μg of protein). These results demonstrate, for the first time, lower basal TK activity in the muscle tissue of female dogs during estrus and diestrus, which may represent lower insulin signaling capacity, opening a new field of investigation into the molecular mechanisms of insulin resistance in dogs. PMID:22139063

  4. 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

  5. New functional aspects of the atypical protein tyrosine phosphatase VHZ

    PubMed Central

    Kuznetsov, Vyacheslav I.; Hengge, Alvan C.

    2013-01-01

    LDP3 (VHZ) is the smallest classical protein tyrosine phosphatase (PTP) known to date, and was originally misclassified as an atypical dual specificity phosphatase (DSP). Kinetic isotope effects with steady state and pre-steady state kinetics of VHZ and mutants with para-nitrophenol phosphate (pNPP) have revealed several unusual properties. VHZ is significantly more active than previously reported, but remains one of the least active PTPs. Highly unusual for a PTP, VHZ possesses two acidic residues (E134 and D65) in the active site. D65 occupies the position corresponding to the typical general acid in the PTP family. However, VHZ primarily utilizes E134 as the general acid, with D65 taking over this role when E134 is mutated. This unusual behavior is facilitated by two coexisting, but unequally populated, substrate binding modes. Unlike most classical PTPs, VHZ exhibits phosphotransferase activity. Despite the presence of the Q-loop that normally prevents alcoholysis of the phosphoenzyme intermediate in other classical PTPs, VHZ readily phosphorylates ethylene glycol. Although mutations to Q-loop residues affect this phosphotransferase activity, mutations on the IPD-loop that contains the general acid exert more control over this process. A single P68V substitution on this loop completely abolishes phosphotransferase activity. The ability of native VHZ to catalyze transphosphorylation may lead to an imbalance of intracellular phosphorylation, which could explain the correlation of its overexpression with several types of cancer. PMID:24073992

  6. 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

  7. Intersubunit binding domains within tyrosine hydroxylase and tryptophan hydroxylase.

    PubMed

    Yohrling, G J; Jiang, G C; Mockus, S M; Vrana, K E

    2000-08-01

    Tryptophan hydroxylase (TPH), the rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin (5-HT) belongs to the aromatic amino acid hydroxylase superfamily, which includes phenylalanine hydroxylase (PAH) and tyrosine hydroxylase (TH). The crystal structures for both PAH and TH have been reported, but a crystallographic model of TPH remains elusive. For this reason, we have utilized the information presented in the TH crystal structure in combination with primary sequence alignments to design point mutations in potential structural domains of the TPH protein. Mutation of a TH salt bridge (K170E) was sufficient to alter enzyme macromolecular assembly. We found that the disruption of the cognate intersubunit dimerization salt bridge (K111-E223) in TPH, however, did not affect the macromolecular assembly of TPH. Enzyme peaks representing only tetramers were observed with size exclusion chromatography. By contrast, a single-point mutation within the tetramerization domain of TPH (L435A) was sufficient to disrupt the normal homotetrameric assembly of TPH. These studies indicate that, although the proposed salt bridge dimerization interface of TH is conserved in TPH, this hypothetical TPH intersubunit binding domain, K111-E223, is not required for the proper macromolecular assembly of the protein. However, leucine 435 within the tetramerization domain is necessary for the proper macromolecular assembly of TPH. PMID:10900078

  8. Tyrosine hydroxylase and tryptophan hydroxylase do not form heterotetramers.

    PubMed

    Mockus, S M; Yohrling, G J; Vrana, K E

    1998-02-01

    Tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) both contain a C-terminal tetramerization domain composed of a leucine heptad repeat embedded within a 4,3-hydrophobic repeat. Previous mutagenesis experiments and X-ray crystallographic studies have demonstrated that these repeats are required for tetramer assembly of the hydroxylase enzymes via coiled-coil interactions. The specificity of these particular C-terminal intersubunit binding motifs was investigated by determining if TH and TPH can form heterotetramers when coexpressed in bacteria. Bacterial cells were contransformed with TH and TPH expression plasmids under kanamycin and ampicillin selection, respectively. Immunoprecipitation of induced bacterial supernatants with a TPH monoclonal antibody demonstrated that, unlike the human TH isoforms, TH and TPH do not form heterotetramers. The data suggest that specificity of oligomerization of the aromatic amino acid hydroxylases may be partially determined by polar amino acids interspersed within the coiled-coil. This finding should be influential in the development of eukaryotic expression systems and ultimately in gene therapy approaches. PMID:9589369

  9. Protein tyrosine phosphatase 1B inhibitors isolated from Artemisia roxburghiana.

    PubMed

    Shah, Muhammad Raza; Ishtiaq; Hizbullah, Syed Muhammad; Habtemariam, Solomon; Zarrelli, Armando; Muhammad, Akhtar; Collina, Simona; Khan, Inamulllah

    2016-08-01

    Artemisia roxburghiana is used in traditional medicine for treating various diseases including diabetes. The present study was designed to evaluate the antidiabetic potential of active constituents by using protein tyrosine phosphatase 1B (PTP1B) as a validated target for management of diabetes. Various compounds were isolated as active principles from the crude methanolic extract of aerial parts of A. roxburghiana. All compounds were screened for PTP1B inhibitory activity. Molecular docking simulations were performed to investigate the mechanism behind PTP1B inhibition of the isolated compound and positive control, ursolic acid. Betulinic acid, betulin and taraxeryl acetate were the active PTP1B principles with IC50 values 3.49 ± 0.02, 4.17 ± 0.03 and 87.52 ± 0.03 µM, respectively. Molecular docking studies showed significant molecular interactions of the triterpene inhibitors with Gly220, Cys215, Gly218 and Asp48 inside the active site of PTP1B. The antidiabetic activity of A. roxburghiana could be attributed due to PTP1B inhibition by its triterpene constituents, betulin, betulinic acid and taraxeryl acetate. Computational insights of this study revealed that the C-3 and C-17 positions of the compounds needs extensive optimization for the development of new lead compounds. PMID:26118418

  10. Cheminfomatic-based Drug Discovery of Human Tyrosine Kinase Inhibitors.

    PubMed

    Reid, Terry-Elinor; Fortunak, Joseph M; Wutoh, Anthony; Simon Wang, Xiang

    2016-01-01

    Receptor Tyrosine Kinases (RTKs) are essential components for regulating cell-cell signaling and communication events in cell growth, proliferation, differentiation, survival and metabolism. Deregulation of RTKs and their associated signaling pathways can lead to a wide variety of human diseases such as immunodeficiency, diabetes, arterosclerosis, psoriasis and cancer. Thus RTKs have become one of the most important drug targets families in recent decade. Pharmaceutical companies have dedicated their research efforts towards the discovery of small-molecule inhibitors of RTKs, many of which had been approved by the U.S. Food and Drug Administration (US FDA) or are currently in clinical trials. The great successes in the development of small-molecule inhibitors of RTKs are largely attributed to the use of modern cheminformatic approaches to identifying lead scaffolds. Those include the quantitative structure-activity relationship (QSAR) modeling, as well as the structure-, and ligand-based pharmacophore modeling techniques in this case. Herein we inspected the literature thoroughly in an effort to conduct a comparative analysis of major findings regarding the essential structure-activity relationships (SARs)/pharmacophore features of known active RTK inhibitors, most of which were collected from cheminformatic modeling approaches. PMID:26369823

  11. Bruton's tyrosine kinase inhibitors in chronic lymphocytic leukemia and lymphoma.

    PubMed

    Varma, Gaurav; Johnson, Tyler P; Advani, Ranjana H

    2016-07-01

    The development of Bruton's tyrosine kinase (BTK) inhibitors and their introduction into clinical practice represent a major advance in the treatment of chronic lymphocytic leukemia (CLL) and other B-cell lymphomas. Although ibrutinib is the only BTK inhibitor that has been approved by the US Food and Drug Administration, several others are under investigation. Ibrutinib is currently approved for use in relapsed/refractory CLL, CLL with 17p deletion (del[17p]), relapsed or refractory mantle cell lymphoma, and Waldenström macroglobulinemia. Although it is clear that ibrutinib has altered treatment paradigms and outcomes in these diseases, several questions remain regarding (1) its role in frontline vs salvage therapy; (2) its use as a single agent vs in combination with biologic agents, other small molecules, or traditional chemoimmunotherapy; (3) the optimal duration of treatment; and (4) the treatment of patients who cannot tolerate or have disease resistant to ibrutinib. Because sparse clinical data are available on other BTK inhibitors, it is unclear at present whether their clinical efficacy and toxicity will differ from those of ibrutinib. PMID:27379948

  12. 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

  13. 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

  14. Protein tyrosine phosphatases expression during development of mouse superior colliculus.

    PubMed

    Reinhard, Jacqueline; Horvat-Bröcker, Andrea; Illes, Sebastian; Zaremba, Angelika; Knyazev, Piotr; Ullrich, Axel; Faissner, Andreas

    2009-12-01

    Protein tyrosine phosphatases (PTPs) are key regulators of different processes during development of the central nervous system. However, expression patterns and potential roles of PTPs in the developing superior colliculus remain poorly investigated. In this study, a degenerate primer-based reverse transcription-polymerase chain reaction (RT-PCR) approach was used to isolate seven different intracellular PTPs and nine different receptor-type PTPs (RPTPs) from embryonic E15 mouse superior colliculus. Subsequently, the expression patterns of 11 PTPs (TC-PTP, PTP1C, PTP1D, PTP-MEG2, PTP-PEST, RPTPJ, RPTPε, RPTPRR, RPTPσ, RPTPκ and RPTPγ) were further analyzed in detail in superior colliculus from embryonic E13 to postnatal P20 stages by quantitative real-time RT-PCR, Western blotting and immunohistochemistry. Each of the 11 PTPs exhibits distinct spatiotemporal regulation of mRNAs and proteins in the developing superior colliculus suggesting their versatile roles in genesis of neuronal and glial cells and retinocollicular topographic mapping. At E13, additional double-immunohistochemical analysis revealed the expression of PTPs in collicular nestin-positive neural progenitor cells and RC-2-immunoreactive radial glia cells, indicating the potential functional importance of PTPs in neurogenesis and gliogenesis. PMID:19727691

  15. Emerging Role of Tyrosine Kinases as Drugable Targets in Cancer

    PubMed Central

    Na, Il-Kang; le Coutre, Philipp

    2015-01-01

    Tyrosine kinases (TKs) play a significant role in cancerogenesis and cancer cell function. Initial developments in this field go back to the early 80s, but the success story really started with the selective BCR-ABL inhibitor, imatinib. Owing to the cancer-driving role of BCR-ABL in chronic myeloid leukemia (CML), excellent response rates lead to fast FDA approval in both the first and second treatments of CML patients. Since then, numerous TKs were identified. TK inhibitors have been developed accordingly, and technology to test for ideal drug–target interactions has profoundly improved. By now, medical oncologists and hematologists struggle to have a pool of potential TK inhibitors, where the most efficient one could be picked out to treat a specific cancer patient, which might also help overcome the occurring resistance mechanisms against TK inhibitors. Whether disease eradication can be achieved via single or sequential TK inhibitor treatment(s) needs to be tested in the present and in the future. PMID:26401097

  16. 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

  17. Spleen tyrosine kinase suppresses osteoblastic differentiation through MAPK and PKCα.

    PubMed

    Yoshida, Kiyoshi; Higuchi, Chikahisa; Nakura, Akio; Yoshikawa, Hideki

    2011-08-12

    Spleen tyrosine kinase (Syk) is a non-receptor protein kinase present in abundance in a wide range of hematopoietic cells. Syk reportedly plays a crucial role in immune signaling in B cells and cells bearing Fcγ-activation receptors. The role of syk in osteoblastic differentiation has not been well elucidated. We report herein the role of syk in osteoblastic differentiation. We investigated the effects of two syk inhibitors on osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells and bone marrow stromal ST2 cells. Expression of syk was detected in these two cell lines. Two syk inhibitors stimulated mRNA expression of osteoblastic markers (ALP, Runx2, Osterix). Mineralization of extracellular matrix was also promoted by treatment with syk inhibitors. Knockdown of Syk caused increased mRNA expression of osteoblastic markers. In addition, syk inhibitor and knockdown of Syk suppressed phosphorylation of mitogen-activated protein kinase (MAPK) and protein kinase Cα (PKCα). Our results indicate that syk might regulate osteoblastic differentiation through MAPK and PKCα. PMID:21782794

  18. Proteolytic cleavage, trafficking, and functions of nuclear receptor tyrosine kinases.

    PubMed

    Chen, Mei-Kuang; Hung, Mien-Chie

    2015-10-01

    Intracellular localization has been reported for over three-quarters of receptor tyrosine kinase (RTK) families in response to environmental stimuli. Internalized RTK may bind to non-canonical substrates and affect various cellular processes. Many of the intracellular RTKs exist as fragmented forms that are generated by γ-secretase cleavage of the full-length receptor, shedding, alternative splicing, or alternative translation initiation. Soluble RTK fragments are stabilized and intracellularly transported into subcellular compartments, such as the nucleus, by binding to chaperone or transcription factors, while membrane-bound RTKs (full-length or truncated) are transported from the plasma membrane to the ER through the well-established Rab- or clathrin adaptor protein-coated vesicle retrograde trafficking pathways. Subsequent nuclear transport of membrane-bound RTK may occur via two pathways, INFS or INTERNET, with the former characterized by release of receptors from the ER into the cytosol and the latter characterized by release of membrane-bound receptor from the ER into the nucleoplasm through the inner nuclear membrane. Although most non-canonical intracellular RTK signaling is related to transcriptional regulation, there may be other functions that have yet to be discovered. In this review, we summarize the proteolytic processing, intracellular trafficking and nuclear functions of RTKs, and discuss how they promote cancer progression, and their clinical implications. PMID:26096795

  19. Redox and zinc signalling pathways converging on protein tyrosine phosphatases.

    PubMed

    Bellomo, Elisa; Hogstrand, Christer; Maret, Wolfgang

    2014-10-01

    Zinc ions, though redox-inert, have either pro-antioxidant or pro-oxidant functions at critical junctures in redox metabolism and redox signalling. They are released from cells and in cells, e.g. from metallothionein, a protein that transduces redox signals into zinc signals (1). The released zinc ions inhibit enzymes such as protein tyrosine phosphatases (PTPs), key regulatory enzymes of cellular phosphorylation signalling. The Ki(Zn) value for inhibition of receptor PTPB is 21pM (2). The binding is about as tight as the binding of zinc to zinc metalloenzymes and suggests tonic zinc inhibition. PTP1-B (PTPN1), an enzyme regulating the insulin and leptin receptors and involved in cancer and diabetes pathobiochemistry, has a Ki(Zn) value of about 5nM (3). Zinc ions bind to the enzyme in the closed conformation when additional metal-binding ligands are brought into the vicinity of the active site. In contrast, redox reactions target cysteines in the active sites of PTPs in the open conformation. This work provides a molecular basis how hydrogen peroxide and free zinc ions generated by growth factor signalling stimulate phosphorylation signalling differentially. (Supported by the Biotechnology and Biological Sciences Research Council UK, grant BB/K001442/1.). PMID:26461422

  20. 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

  1. The Role of Tyrosine Kinase Receptors in Peritoneal Fibrosis.

    PubMed

    Wang, Li; Zhuang, Shougang

    2015-01-01

    Peritoneal dialysis (PD) is a modality for treatment of patients with end-stage renal disease (ESRD) that depends on the structural and functional integrity of the peritoneal membrane. However, long-term PD can lead to morphological and functional changes in the peritoneum; in particular, peritoneal fibrosis has become one of the most common complications that ultimately results in ultrafiltration failure (UFF) and discontinuation of PD. Several factors and mechanisms such as inflammation and overproduction of transforming growth factor-β1 have been implicated in the development of peritoneal fibrosis, but there is no effective therapy to prevent or delay this process. Recent studies have shown that activation of multiple receptor tyrosine kinases (RTKs) is associated with the development and progression of tissue fibrosis in various organs, and there are also reports indicating the involvement of some RTKs in peritoneal fibrosis. This review will describe the role and mechanisms of RTKs in peritoneal fibrosis and discuss the possibility of using them as therapeutic targets for prevention and treatment of this complication. PMID:26450477

  2. 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

  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. PMID:26975317

  4. The EGFR Family: Not So Prototypical Receptor Tyrosine Kinases

    PubMed Central

    Lemmon, Mark A.; Schlessinger, Joseph; Ferguson, Kathryn M.

    2014-01-01

    The epidermal growth factor receptor (EGFR) was among the first receptor tyrosine kinases (RTKs) for which ligand binding was studied and for which the importance of ligand-induced dimerization was established. As a result, EGFR and its relatives have frequently been termed “prototypical” RTKs. Many years of mechanistic studies, however, have revealed that—far from being prototypical—the EGFR family is quite unique. As we discuss in this review, the EGFR family uses a distinctive “receptor-mediated” dimerization mechanism, with ligand binding inducing a dramatic conformational change that exposes a dimerization arm. Intracellular kinase domain regulation in this family is also unique, being driven by allosteric changes induced by asymmetric dimer formation rather than the more typical activation-loop phosphorylation. EGFR family members also distinguish themselves from other RTKs in having an intracellular juxtamembrane (JM) domain that activates (rather than autoinhibits) the receptor and a very large carboxy-terminal tail that contains autophosphorylation sites and serves an autoregulatory function. We discuss recent advances in mechanistic aspects of all of these components of EGFR family members, attempting to integrate them into a view of how RTKs in this important class are regulated at the cell surface. PMID:24691965

  5. Synaptic Clustering of PSD-95 Is Regulated by c-Abl through Tyrosine Phosphorylation

    PubMed Central

    de Arce, Karen Perez; Varela-Nallar, Lorena; Farias, Olivia; Cifuentes, Alejandra; Bull, Paulina; Couch, Brian A.; Koleske, Anthony J.; Inestrosa, Nibaldo C.; Alvarez, Alejandra R.

    2010-01-01

    The c-Abl tyrosine kinase is present in mouse brain synapses, but its precise synaptic function is unknown. We found that c-Abl levels in the rat hippocampus increase postnatally, with expression peaking at the first postnatal week. In 14 d in vitro hippocampal neuron cultures, c-Abl localizes primarily to the postsynaptic compartment, in which it colocalizes with the postsynaptic scaffold protein postsynaptic density protein-95 (PSD-95) in apposition to presynaptic markers. c-Abl associates with PSD-95, and chemical or genetic inhibition of c-Abl kinase activity reduces PSD-95 tyrosine phosphorylation, leading to reduced PSD-95 clustering and reduced synapses in treated neurons. c-Abl can phosphorylate PSD-95 on tyrosine 533, and mutation of this residue reduces the ability of PSD-95 to cluster at postsynaptic sites. Our results indicate that c-Abl regulates synapse formation by mediating tyrosine phosphorylation and clustering of PSD-95. PMID:20220006

  6. Related proteins are phosphorylated at tyrosine in response to mitogenic stimuli and at meiosis.

    PubMed Central

    Cooper, J A

    1989-01-01

    Forty-two-kilodalton proteins that contain phosphotyrosine in metaphase-arrested Xenopus laevis eggs are closely related to p42, a protein that is phosphorylated at tyrosine when somatic cells are exposed to mitogenic stimuli. Images PMID:2779558

  7. PARGYLINE AND Y-BUTYROLACTONE ENHANCE TYROSINE HYDROXYLASE IMMUNOSTAINING OF NIGROSTRIATAL AXONS

    EPA Science Inventory

    Administration of pargyline or q-butyrolactone enhanced immunostaining of tyrosine hydroxylase immunoreactive axons within the striatum. he qualitative characteristics of this enhancement were compound dependent and the enhancement was not associated with an increase in the amoun...

  8. Synthesis of Hydroxylated Bicyclic Amino Acids from l-Tyrosine: Octahydro-1H-indole Carboxylates

    PubMed Central

    Pierce, Joshua G.; Kasi, Dhanalakshmi; Fushimi, Makoto; Cuzzupe, Anthony; Wipf, Peter

    2009-01-01

    A stereoselective approach to polyhydroxylated l-Choi derivatives has been developed. The oxidative cyclization of l-tyrosine was optimized to avoid partial racemization and to allow a more efficient scale-up. PMID:18767800

  9. 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.

  10. Kinetics of steroid induction and deinduction of tyrosine aminotransferase synthesis in cultured hepatoma cells.

    PubMed Central

    Steinberg, R A; Levinson, B B; Tomkins, G M

    1975-01-01

    The specific rate of synthesis of tyrosine aminotransferase (EC 2.6.1.5; L-tyrosine:2-oxoglutarate aminotransferase) is used as a measure of the level of functional, cytoplasmic, tyrosine aminotransferase-specific mRNA in cultured rat hepatoma cells. An analysis of the kinetics of change in this rate after the addition or withdrawal of glucocorticosteroids sets an upper limit on the half-life of the enzyme-specific mRNA of 1-1.5 hr, whether or not steroid is present. The inactivation rate of the enzyme mRNA is independent of the growth condition of the cells, occuring equally rapidly in the presence or absence of serum or insulin, both of which induce tyrosine aminotransferase in these cells. The implications of these results for the mechanism of steroid induction are discussed. PMID:237268

  11. Tyrosine phosphorylation of mitochondrial pyruvate dehydrogenase kinase 1 is important for cancer metabolism

    PubMed Central

    Hitosugi, Taro; Fan, Jun; Chung, Tae-Wook; Lythgoe, Katherine; Wang, Xu; Xie, Jianxin; Ge, Qingyuan; Gu, Ting-Lei; Polakiewicz, Roberto D.; Roesel, Johannes L.; Chen, Zhuo (Georgia); Boggon, Titus J.; Lonial, Sagar; Fu, Haian; Khuri, Fadlo R.; Kang, Sumin; Chen, Jing

    2011-01-01

    SUMMARY Many tumor cells rely on aerobic glycolysis instead of oxidative phosphorylation for their continued proliferation and survival. Myc and HIF-1 are believed to promote such a metabolic switch by, in part, upregulating gene expression of pyruvate dehydrogenase (PDH) kinase 1 (PDHK1), which phosphorylates and inactivates mitochondrial PDH and consequently pyruvate dehydrogenase complex (PDC). Here we report that tyrosine phosphorylation enhances PDHK1 kinase activity by promoting ATP and PDC binding. Functional PDC can form in mitochondria outside of matrix in some cancer cells and PDHK1 is commonly tyrosine phosphorylated in human cancers by diverse oncogenic tyrosine kinases localized to different mitochondrial compartments. Expression of phosphorylation-deficient, catalytic hypomorph PDHK1 mutants in cancer cells leads to decreased cell proliferation under hypoxia and increased oxidative phosphorylation with enhanced mitochondrial utilization of pyruvate, and reduced tumor growth in xenograft nude mice. Together, tyrosine phosphorylation activates PDHK1 to promote the Warburg effect and tumor growth. PMID:22195962

  12. Cadmium inhibits mouse sperm motility through inducing tyrosine phosphorylation in a specific subset of proteins.

    PubMed

    Wang, Lirui; Li, Yuhua; Fu, Jieli; Zhen, Linqing; Zhao, Na; Yang, Qiangzhen; Li, Sisi; Li, Xinhong

    2016-08-01

    Cadmium (Cd) has been reported to impair male fertility, primarily by disrupting sperm motility, but the underlying molecular mechanism remains unclear. Here we investigated the effects of Cd on sperm motility, tyrosine phosphorylation, AMP-activated protein kinase (AMPK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, and ATP levels in vitro. Our results demonstrated that Cd inhibited sperm motility, GAPDH activity, AMPK activity and ATP production, and induced tyrosine phosphorylation of 55-57KDa proteins. Importantly, all the parameters affected by Cd were restored to normal levels when incubated with 10μM Cd in the presence of 30μM ethylene diamine tetraacetic acid (EDTA). Interestingly, changes of tyrosine phosphorylation levels of 55-57KDa proteins are completely contrary to that of other parameters. These results suggest that Cd-induced tyrosine phosphorylation of 55-57KDa proteins might act as an engine to block intracellular energy metabolism and thus decrease sperm motility. PMID:27233480

  13. 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

  14. 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.

  15. Mycobacterium avium subsp. paratuberculosis PtpA Is an Endogenous Tyrosine Phosphatase Secreted during Infection▿

    PubMed Central

    Bach, Horacio; Sun, Jim; Hmama, Zakaria; Av-Gay, Yossef

    2006-01-01

    Adaptive gene expression in prokaryotes is mediated by protein kinases and phosphatases. These regulatory proteins mediate phosphorylation of histidine or aspartate in two-component systems and serine/threonine or tyrosine in eukaryotic and eukaryote-like protein kinase systems. The genome sequence of Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease, does not possess a defined tyrosine kinase. Nevertheless, it encodes for protein tyrosine phosphatases. Here, we report that Map1985, is a functional low-molecular tyrosine phosphatase that is secreted intracellularly upon macrophage infection. This finding suggests that Map1985 might contribute to the pathogenesis of Mycobacterium avium subsp. paratuberculosis by dephosphorylating essential macrophage signaling and/or adaptor molecules. PMID:16982836

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. DIRECT MODULATION OF THE PROTEIN KINASE A CATALYTIC SUBUNIT α BY GROWTH FACTOR RECEPTOR TYROSINE KINASES

    PubMed Central

    Caldwell, George B.; Howe, Alan K.; Nickl, Christian K.; Dostmann, Wolfgang R.; Ballif, Bryan A.; Deming, Paula B.

    2011-01-01

    The cyclic-AMP-dependent protein kinase A (PKA) regulates processes such as cell proliferation and migration following activation of growth factor receptor tyrosine kinases (RTKs), yet the signaling mechanisms that link PKA with growth factor receptors remain largely undefined. Here we report that RTKs can directly modulate the function of the catalytic subunit of PKA (PKA-C) through post-translational modification. In vitro kinase assays revealed that both the epidermal growth factor and platelet derived growth factor receptors (EGFR and PDGFR, respectively) tyrosine phosphorylate PKA-C. Mass spectrometry identified tyrosine 330 (Y330) as a receptor-mediated phosphorylation site and mutation of Y330 to phenylalanine (Y330F) all but abolished the RTK-mediated phosphorylation of PKA-C in vitro. Y330 resides within a conserved region at the C-terminal tail of PKA-C that allosterically regulates enzymatic activity. Therefore, the effect of phosphorylation at Y330 on the activity of PKA-C was investigated. The Km for a peptide substrate was markedly decreased when PKA-C subunits were tyrosine phosphorylated by the receptors as compared to un-phosphorylated controls. Importantly, tyrosine-phosphorylated PKA-C subunits were detected in cells stimulated with EGF, PDGF and FGF2 and in fibroblasts undergoing PDGF-mediated chemotaxis. These results demonstrate a direct, functional interaction between RTKs and PKA-C and identify tyrosine phosphorylation as a novel mechansim for regulating PKA activity. PMID:21866565

  1. 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

  2. Intracellular Ca2+ stores modulate SOCCs and NMDA receptors via tyrosine kinases in rat hippocampal neurons.

    PubMed

    Koss, David J; Riedel, Gernot; Platt, Bettina

    2009-07-01

    The regulation of intracellular Ca(2+) signalling by phosphorylation processes remains poorly defined, particularly with regards to tyrosine phosphorylation. Evidence from non-excitable cells implicates tyrosine phosphorylation in the activation of so-called store-operated Ca(2+) channels (SOCCs), but their involvement in neuronal Ca(2+) signalling is still elusive. In the present study, we determined the role of protein tyrosine kinases (PTKs) and tyrosine phosphatases (PTPs) in the coupling between intracellular Ca(2+) stores and SOCCs in neonatal rat hippocampal neurons by Fura-2 Ca(2+) imaging. An early Ca(2+) response from intracellular stores was triggered with thapsigargin, and followed by a secondary plasma membrane Ca(2+) response. This phase was blocked by the non-specific Ca(2+) channel blocker NiCl and the SOCC blocker, 2-aminoethoxydiphenyl borate (2-APB). Interestingly, two structurally distinct PTK inhibitors, genistein and AG126, also inhibited this secondary response. Application of the PTP inhibitor sodium orthovanadate (OV) also activated a sustained and tyrosine kinase dependent Ca(2+) response, blocked by NiCl and 2-APB. In addition, OV resulted in a Ca(2+) store dependent enhancement of NMDA responses, corresponding to, and occluding the signalling pathway for group I metabotropic glutamate receptors (mGluRs). This study provides first evidence for tyrosine based phospho-regulation of SOCCs and NMDA signalling in neurons. PMID:19423160

  3. 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

  4. Pyomelanin production in Penicillium chrysogenum is stimulated by L-tyrosine.

    PubMed

    Vasanthakumar, Archana; DeAraujo, Alice; Mazurek, Joy; Schilling, Michael; Mitchell, Ralph

    2015-06-01

    From a tomb in Upper Egypt we isolated a strain of Penicillium chrysogenum that was capable of producing brown pigment in vitro when grown in a minimal salts medium containing tyrosine. We present evidence that this pigment is a pyomelanin, a compound that is known to assist in the survival of some micro-organisms in adverse environments. We tested type strains of Pe. chrysogenum, which were also able to produce this pigment under similar conditions. Inhibitors of the DHN and DOPA melanin pathways were unable to inhibit the formation of the pigment. Fourier transform IR analysis indicated that this brown pigment is similar to pyomelanin. Pyrolysis-GC/MS revealed the presence of phenolic compounds. Using LC/MS, homogentisic acid, the monomeric precursor of pyomelanin, was detected in supernatants of Pe. chrysogenum cultures growing in tyrosine medium but not in cultures lacking tyrosine. Partial regions of the genes encoding two enzymes in the homogentisic acid pathway of tyrosine degradation were amplified. Data from reverse-transcription PCR demonstrated that hmgA transcription was increased in cultures grown in tyrosine medium, suggesting that tyrosine induced the transcription. PMID:25568259

  5. Bacterial single-stranded DNA-binding proteins are phosphorylated on tyrosine

    PubMed Central

    Mijakovic, Ivan; Petranovic, Dina; Macek, Boris; Cepo, Tina; Mann, Matthias; Davies, Julian; Jensen, Peter R.; Vujaklija, Dusica

    2006-01-01

    Single-stranded DNA-binding proteins (SSBs) are required for repair, recombination and replication in all organisms. Eukaryotic SSBs are regulated by phosphorylation on serine and threonine residues. To our knowledge, phosphorylation of SSBs in bacteria has not been reported. A systematic search for phosphotyrosine-containing proteins in Streptomyces griseus by immunoaffinity chromatography identified bacterial SSBs as a novel target of bacterial tyrosine kinases. Since genes encoding protein-tyrosine kinases (PTKs) have not been recognized in streptomycetes, and SSBs from Streptomyces coelicolor (ScSSB) and Bacillus subtilis (BsSSB) share 38.7% identity, we used a B.subtilis protein-tyrosine kinase YwqD to phosphorylate two cognate SSBs (BsSSB and YwpH) in vitro. We demonstrate that in vivo phosphorylation of B.subtilis SSB occurs on tyrosine residue 82, and this reaction is affected antagonistically by kinase YwqD and phosphatase YwqE. Phosphorylation of B.subtilis SSB increased binding almost 200-fold to single-stranded DNA in vitro. Tyrosine phosphorylation of B.subtilis, S.coelicolor and Escherichia coli SSBs occured while they were expressed in E.coli, indicating that tyrosine phosphorylation of SSBs is a conserved process of post-translational modification in taxonomically distant bacteria. PMID:16549871

  6. Formation of 3-nitrotyrosine by riboflavin photosensitized oxidation of tyrosine in the presence of nitrite.

    PubMed

    Fontana, Mario; Blarzino, Carla; Pecci, Laura

    2012-05-01

    The results of the present investigation show the susceptibility of tyrosine to undergo visible light-induced photomodification to 3-nitrotyrosine in the presence of nitrite and riboflavin, as sensitizer. By changing H2O by D2O, it could be established that singlet oxygen has a minor role in the reaction. The finding that nitration of tyrosine still occurred to a large extent under anaerobic conditions indicates that the process proceeds mainly through a type I mechanism, which involves the direct interaction of the excited state of riboflavin with tyrosine and nitrite to give tyrosyl radical and nitrogen dioxide radical, respectively. The tyrosyl radicals can either dimerize to yield 3,3'-dityrosine or combine with nitrogen dioxide radical to form 3-nitrotyrosine. The formation of 3-nitrotyrosine was found to increase with the concentration of nitrite added and was accompanied by a decrease in the recovery of 3,3'-dityrosine, suggesting that tyrosine nitration competes with dimerization reaction. The riboflavin photosensitizing reaction in the presence of nitrite was also able to induce nitration of tyrosine residues in proteins as revealed by the spectral changes at 430 nm, a characteristic absorbance of 3-nitrotyrosine, and by immunoreactivity using 3-nitrotyrosine antibodies. Since riboflavin and nitrite are both present endogenously in living organism, it is suggested that this pathway of tyrosine nitration may potentially occur in tissues and organs exposed to sunlight such as skin and eye. PMID:21479936

  7. Tyrosine phosphorylation of WIP releases bound WASP and impairs podosome assembly in macrophages

    PubMed Central

    Vijayakumar, Vineetha; Monypenny, James; Chen, Xing Judy; Machesky, Laura M.; Lilla, Sergio; Thrasher, Adrian J.; Antón, Inés M.; Calle, Yolanda; Jones, Gareth E.

    2015-01-01

    ABSTRACT Podosomes are integrin-containing adhesion structures commonly found in migrating leukocytes of the monocytic lineage. The actin cytoskeletal organisation of podosomes is based on a WASP- and Arp2/3-mediated mechanism. WASP also associates with a second protein, WIP (also known as WIPF1), and they co-localise in podosome cores. Here, we report for the first time that WIP can be phosphorylated on tyrosine residues and that tyrosine phosphorylation of WIP is a trigger for release of WASP from the WIP–WASP complex. Using a knockdown approach together with expression of WIP phosphomimics, we show that in the absence of WIP–WASP binding, cellular WASP is rapidly degraded, leading to disruption of podosomes and a failure of cells to degrade an underlying matrix. In the absence of tyrosine phosphorylation, the WIP–WASP complex remains intact and podosome lifetimes are extended. A screen of candidate kinases and inhibitor-based assays identified Bruton's tyrosine kinase (Btk) as a regulator of WIP tyrosine phosphorylation. We conclude that tyrosine phosphorylation of WIP is a crucial regulator of WASP stability and function as an actin-nucleation-promoting factor. PMID:25413351

  8. 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

  9. 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.

  10. 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

  11. Agonistic TAM-163 antibody targeting tyrosine kinase receptor-B

    PubMed Central

    Vugmeyster, Yulia; Rohde, Cynthia; Perreault, Mylene; Gimeno, Ruth E.; Singh, Pratap

    2013-01-01

    TAM-163, an agonist monoclonal antibody targeting tyrosine receptor kinase-B (TrkB), is currently being investigated as a potential body weight modulatory agent in humans. To support the selection of the dose range for the first-in-human (FIH) trial of TAM-163, we conducted a mechanistic analysis of the pharmacokinetic (PK) and pharmacodynamic (PD) data (e.g., body weight gain) obtained in lean cynomolgus and obese rhesus monkeys following single doses ranging from 0.3 to 60 mg/kg. A target-mediated drug disposition (TMDD) model was used to describe the observed nonlinear PK and Emax approach was used to describe the observed dose-dependent PD effect. The TMDD model development was supported by the experimental determination of the binding affinity constant (9.4 nM) and internalization rate of the drug-target complex (2.08 h−1). These mechanistic analyses enabled linking of exposure, target (TrkB) coverage, and pharmacological activity (e.g., PD) in monkeys, and indicated that ≥ 38% target coverage (time-average) was required to achieve significant body weight gain in monkeys. Based on the scaling of the TMDD model from monkeys to humans and assuming similar relationship between the target coverage and pharmacological activity between monkey and humans, subcutaneous (SC) doses of 1 and 15 mg/kg in humans were projected to be the minimally and the fully pharmacologically active doses, respectively. Based on the minimal anticipated biological effect level (MABEL) approach for starting dose selection, the dose of 0.05 mg/kg (3 mg for a 60 kg human) SC was recommended as the starting dose for FIH trials, because at this dose level < 10% target coverage was projected at Cmax (and all other time points). This study illustrates a rational mechanistic approach for the selection of FIH dose range for a therapeutic protein with a complex model of action. PMID:23529133

  12. 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

  13. 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. PMID:17615669

  14. The sustained phase of tyrosine hydroxylase activation in vivo.

    PubMed

    Ong, Lin Kooi; Sominsky, Luba; Dickson, Phillip W; Hodgson, Deborah M; Dunkley, Peter R

    2012-09-01

    Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthetic pathway for catecholamine synthesis. Stress triggers an increase in TH activity, resulting in increased release of catecholamines from both neurons and the adrenal medulla. In response to stress three phases of TH activation have been identified (acute, sustained and chronic) and each phase has a unique mechanism. The acute and chronic phases have been studied in vivo in a number of animal models, but to date the sustained phase has only been characterised in vitro. We aimed to investigate the effects of dual exposure to lipopolysaccharide (LPS) in neonatal rats on TH protein, TH phosphorylation at serine residues 19, 31 and 40 and TH activity in the adrenal gland over the sustained phase. Wistar rats were administered LPS (0.05 mg/kg, intraperitoneal injection) or an equivolume of non-pyrogenic saline on days 3 and 5 postpartum. Adrenal glands were collected at 4, 24 and 48 h after the drug exposure on day 5. Neonatal LPS treatment resulted in increases in TH phosphorylation of Ser40 at 4 and 24 h, TH phosphorylation of Ser31 at 24 h, TH activity at 4 and 24 h and TH protein at 48 h. We therefore have provided evidence for the first time that TH phosphorylation at Ser31 and Ser40 occurs for up to 24 h in vivo and leads to TH activation independent of TH protein synthesis, suggesting that the sustained phase of TH activation occurs in vivo. PMID:22684282

  15. Cellular Biochemistry Methods for Investigating Protein Tyrosine Phosphatases

    PubMed Central

    Stanford, Stephanie M.; Ahmed, Vanessa

    2014-01-01

    Abstract Significance: The protein tyrosine phosphatases (PTPs) are a family of proteins that play critical roles in cellular signaling and influence many aspects of human health and disease. Although a wealth of information has been collected about PTPs since their discovery, many questions regarding their regulation and function still remain. Critical Issues: Of particular importance are the elucidation of the biological substrates of individual PTPs and understanding of the chemical and biological basis for temporal and spatial resolution of PTP activity within a cell. Recent Advances: Drawing from recent advances in both biology and chemistry, innovative approaches have been developed to study the intracellular biochemistry and physiology of PTPs. We provide a summary of PTP-tailored techniques and approaches, emphasizing methodologies to study PTP activity within a cellular context. We first provide a discussion of methods for identifying PTP substrates, including substrate-trapping mutants and synthetic peptide libraries for substrate selectivity profiling. We next provide an overview of approaches for monitoring intracellular PTP activity, including a discussion of mechanistic-based probes, gel-based assays, substrates that can be used intracellularly, and assays tied to cell growth. Finally, we review approaches used for monitoring PTP oxidation, a key regulatory pathway for these enzymes, discussing the biotin switch method and variants of this approach, along with affinity trapping techniques and probes designed to detect PTP oxidation. Future Directions: Further development of approaches to investigate the intracellular PTP activity and functions will provide specific insight into their mechanisms of action and control of diverse signaling pathways. Antioxid. Redox Signal. 20, 2160–2178. PMID:24294920

  16. In Vivo Gene Delivery with L-Tyrosine Polyphosphate Nanoparticles

    PubMed Central

    Ditto, Andrew J.; Reho, John J.; Shah, Kush N.; Smolen, Justin A.; Holda, James H.; Ramirez, Rolando J.; Yun, Yang H.

    2013-01-01

    The concept of gene therapy is promising; however, the perceived risks and side effects associated with this technology have severely dampened the researchers’ enthusiasm.1–3 Thus, the development of a non-viral gene vector without immunological effects and with high transfection efficiency is necessary. Currently, most non-viral vectors have failed to achieve the in vivo transfection efficiencies of viral vectors due their toxicity,4 rapid clearance,5, 6 and/or inappropriate release rates.7 Although our previous studies have successfully demonstrated the controlled-release of plasmid DNA (pDNA) polyplexes encapsulated into nanoparticles formulated with L-tyrosine polyphosphate (LTP-pDNA nanoparticles),8, 9 the in vivo transfection capabilities and immunogenicity of this delivery system has yet to be examined. Thus, we evaluate LTP-pDNA nanoparticles in an in vivo setting via injection into rodent uterine tissue. Our results demonstrate through X-gal staining and immunohistochemistry of uterine tissue that transfection has successfully occurred after a nine-day incubation. In contrast, the results for the control nanoparticles show similar results to shams. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) from the injected tissues confirms the transfection in vivo. To examine the immunogenicity, the LTP nanoparticles have been evaluated in a mouse model. No significant differences in the activation of the innate immune system are observed. These data provide the first report for the potential use of controlled-release nanoparticles formulated from an amino acid based polymer as an in vivo non-viral vector for gene therapy. PMID:23510151

  17. 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

  18. 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).

  19. State-by-state investigation of destructive interference in resonance Raman spectra of neutral tyrosine and the tyrosinate anion with the simplified sum-over-states approach.

    PubMed

    Cabalo, Jerry B; Saikin, Semion K; Emmons, Erik D; Rappoport, Dmitrij; Aspuru-Guzik, Alán

    2014-10-16

    UV resonance Raman scattering is uniquely sensitive to the molecular electronic structure as well as intermolecular interactions. To better understand the relationship between electronic structure and resonance Raman cross section, we carried out combined experimental and theoretical studies of neutral tyrosine and the tyrosinate anion. We studied the Raman cross sections of four vibrational modes as a function of excitation wavelength, and we analyzed them in terms of the contributions of the individual electronic states as well as of the Albrecht A and B terms. Our model, which is based on time-dependent density functional theory (TDDFT), reproduced the experimental resonance Raman spectra and Raman excitation profiles for both studied molecules with good agreement. We found that for the studied modes, the contributions of Albrecht's B terms in the Raman cross sections were important across the frequency range spanning the L(a,b) and B(a,b) electronic excitations in tyrosine and the tyrosinate anion. Furthermore, we demonstrated that interference with high-energy states had a significant impact and could not be neglected even when in resonance with a lower-energy state. The symmetry of the vibrational modes served as an indicator of the dominance of the A or B mechanisms. Excitation profiles calculated with a damping constant estimated from line widths of the electronic absorption bands had the best consistency with experimental results. PMID:25233377

  20. 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

  1. 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

  2. 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

  3. Clustering of Neuronal K+-Cl− Cotransporters in Lipid Rafts by Tyrosine Phosphorylation*

    PubMed Central

    Watanabe, Miho; Wake, Hiroaki; Moorhouse, Andrew J.; Nabekura, Junichi

    2009-01-01

    The neuronal K+-Cl− cotransporter (KCC2) is a membrane transport protein that extrudes Cl− from neurons and helps maintain low intracellular [Cl−] and hyperpolarizing GABAergic synaptic potentials. Depolarizing γ-aminobutyric acid (GABA) responses in neonatal neurons and following various forms of neuronal injury are associated with reduced levels of KCC2 expression. Despite the importance for plasticity of inhibitory transmission, less is known about cellular mechanisms involved in more dynamic changes in KCC2 function. In this study, we investigated the role of tyrosine phosphorylation in KCC2 localization and function in hippocampal neurons and in cultured GT1-7 cells. Mutation to the putative tyrosine phosphorylation site within the long intracellular carboxyl terminus of KCC2(Y1087D) or application of the tyrosine kinase inhibitor genistein shifted the GABA reversal potential (EGABA) to more depolarized values, indicating reduced KCC2 function. This was associated with a change in the expression pattern of KCC2 from a punctate distribution to a more uniform distribution, suggesting that functional tyrosine-phosphorylated KCC2 forms clusters in restricted membrane domains. Sodium vanadate, a tyrosine phosphatase inhibitor, increased the proportion of KCC2 associated with lipid rafts membrane domains. Loss of tyrosine phosphorylation also reduced oligomerization of KCC2. A loss of the punctuate distribution and oligomerization of KCC2 and a more depolarized EGABA were seen when the 28-amino-acid carboxyl terminus of KCC2 was deleted. These results indicate that direct tyrosine phosphorylation of KCC2 results in membrane clusters and functional transport activity, suggesting a mechanism by which intracellular Cl− concentrations and GABA responses can be rapidly modulated. PMID:19679663

  4. Urine homogentisic acid and tyrosine: simultaneous analysis by liquid chromatography tandem mass spectrometry.

    PubMed

    Hughes, A T; Milan, A M; Christensen, P; Ross, G; Davison, A S; Gallagher, J A; Dutton, J J; Ranganath, L R

    2014-07-15

    Alkaptonuria (AKU) is a rare debilitating autosomal recessive disorder of tyrosine metabolism. Deficiency of homogentisate 1,2-dioxygenase results in increased homogentisic acid (HGA) which although excreted in gram quantities in the urine, is deposited as an ochronotic pigment in connective tissues, especially cartilage. Ochronosis leads to a severe, early-onset form of osteoarthritis, increased renal and prostatic stone formation and hardening of heart vessels. Treatment with the orphan drug, Nitisinone, an inhibitor of the enzyme 4-hydroxyphenylpyruvate dioxygenase has been shown to reduce urinary excretion of HGA, resulting in accumulation of the upstream pre-cursor, tyrosine. Using reverse phase LC-MS/MS, a method has been developed to simultaneously quantify urinary HGA and tyrosine. Using matrix-matched calibration standards, two product ion transitions were identified for each compound and their appropriate isotopically labelled internal standards. Validation was performed across the AKU and post-treatment concentrations expected. Intrabatch accuracy for acidified urine was 96-109% for tyrosine and 94-107% for HGA; interbatch accuracy (n=20 across ten assays) was 95-110% for tyrosine and 91-109% for HGA. Precision, both intra- and interbatch was <10% for tyrosine and <5% for HGA. Matrix effects observed with acidified urine (12% decrease, CV 5.6%) were normalised by the internal standard. Tyrosine and HGA were proved stable under various storage conditions and no carryover, was observed. Overall the method developed and validated shows good precision, accuracy and linearity appropriate for the monitoring of patients with AKU, pre and post-nitisinone therapy. PMID:24952314

  5. 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

  6. The nature of heme/iron-induced protein tyrosine nitration

    PubMed Central

    Bian, Ka; Gao, Zhonghong; Weisbrodt, Norman; Murad, Ferid

    2003-01-01

    Recently, substantial evidence has emerged that revealed a very close association between the formation of nitrotyrosine and the presence of activated granulocytes containing peroxidases, such as myeloperoxidase. Peroxidases share heme-containing homology and can use H2O2 to oxidize substrates. Heme is a complex of iron with protoporphyrin IX, and the iron-containing structure of heme has been shown to be an oxidant in several model systems where the prooxidant effects of free iron, heme, and hemoproteins may be attributed to the formation of hypervalent states of the heme iron. In the current study, we have tested the hypothesis that free heme and iron play a crucial role in NO2-Tyr formation. The data from our study indicate that: (i) heme/iron catalyzes nitration of tyrosine residues by using hydrogen peroxide and nitrite, a reaction that revealed the mechanism underlying the protein nitration by peroxidase, H2O2, and NO\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{_{2}^{-}}}\\end{equation*}\\end{document}; (ii) H2O2 plays a key role in the protein oxidation that forms the basis for the protein nitration, whereas nitrite is an essential element that facilitates nitration by the heme(Fe), H2O2, and the NO\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{_{2}^{-}}}\\end{equation*}\\end{document} system; (iii) the formation of a Fe(IV) hypervalent compound may be essential for heme(Fe)-catalyzed nitration, whereas O\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage

  7. Contractile effects of angiotensin peptides in rat aorta are differentially dependent on tyrosine kinase activity.

    PubMed

    Petrescu, G; Costuleanu, M; Slatineanu, S M; Costuleanu, N; Foia, L; Costuleanu, A

    2001-09-01

    It has been suggested that tyrosine kinase activity participates in the regulation of signal transduction associated with angiotensin II (Ang II)-induced pharmaco-mechanical coupling in rat aortic smooth muscle. We further tested the effects of genistein, a tyrosine-kinase inhibitor, and its inactive analogue, daidzein, on angiotensin I (Ang I), angiotensin III (Ang III) and angiotensin IV (Ang IV) contractions, as compared with those on Ang II. Genistein partially inhibited Ang II- and Ang I-induced contractions. The genistein-induced inhibition was more evident on Ang III and especially important on Ang IV contractile effects. Thus, Ang IV- and Ang III-induced contractions seem to be more dependent on tyrosine kinase activity than those evoked by Ang II or Ang I. Daidzein did not significantly affect the contractile effects of any of angiotensin peptides tested. These results clearly suggest that the inhibition of the action of angiotensin peptides actions by genistein is mediated by inhibition of endogenous tyrosine kinase activity. Furthermore, our data show that the type and/or intensity of tyrosine kinase activity is differentially associated with the contractile effects of different angiotensin peptides in rat aorta. Nifedipine, a blocker of membrane L-type Ca2+ channels, strongly inhibited Ang IV-induced contractions. At the same time, it significantly inhibited Ang III contractile effects as compared with Ang II and Ang I contractions. Meanwhile, we observed a close relationship between calcium influx and tyrosine kinase phosphorylation activity under the stimulatory effects of angiotensin peptides. Furthermore, genistein did not significantly influence the phasic contractions induced by angiotensin peptides in Ca2+-free Krebs-Henseleit solution. Thus, it appears that Ca2+ influx, rather than the release of Ca2+ from IP3-sensitive stores, may play a major role in the contractile effects of angiotensin peptides in rat aorta via tyrosine kinase activation

  8. A bacterial tyrosine aminomutase proceeds through retention or inversion of stereochemistry to catalyze its isomerization reaction.

    PubMed

    Wanninayake, Udayanga; Walker, Kevin D

    2013-07-31

    β-Amino acids are biologically active compounds of interest in medicinal chemistry. A class I lyase-like family of aminomutases isomerizes (S)-α-arylalanines to the corresponding β-amino acids by exchange of the NH2/H pair. This family uses a 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) group within the active site to initiate the reaction. The absolute stereochemistry of the product is known for an MIO-dependent tyrosine aminomutase from Chondromyces crocatus (CcTAM) that isomerizes (S)-α-tyrosine to (R)-β-tyrosine. To evaluate the cryptic stereochemistry of the CcTAM mechanism, (2S,3S)-[2,3-(2)H2]- and (2S,3R)-[3-(2)H]-α-tyrosine were stereoselectively synthesized from unlabeled (or [(2)H]-labeled) (4'-hydroxyphenyl)acrylic acids by reduction with D2 (or H2) gas and a chiral Rh-Prophos catalyst. GC/EIMS analysis of the [(2)H]-β-tyrosine biosynthesized by CcTAM revealed that the α-amino group was transferred to Cβ of the phenylpropanoid skeleton with retention of configuration. These labeled substrates also showed that the pro-(3S) proton exchanges with protons from the bulk media during its migration to Cα during catalysis. (1)H- and (2)H NMR analyses of the [(2)H]-β-tyrosine derived from (2S)-[3,3-(2)H2]-α-tyrosine by CcTAM catalysis showed that the migratory proton attached to Cα of the product also with retention of configuration. CcTAM is stereoselective for (R)-β-tyrosine (85%) yet also forms the (S)-β-tyrosine enantiomer (15%) through inversion of configuration at both migration termini, as described herein. The proportion of the (S)-β-isomer made by CcTAM during steady state interestingly increased with solvent pH, and this effect on the proposed reaction mechanism is also discussed. PMID:23800193

  9. 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

  10. Pathway optimization by re-design of untranslated regions for L-tyrosine production in Escherichia coli.

    PubMed

    Kim, Seong Cheol; Min, Byung Eun; Gyu Hwang, Hyun; Seo, Sang Woo; Jung, Gyoo Yeol

    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

  11. PSD-93 MEDIATES TYROSINE-PHOSPHORYLATION OF THE N-METHYL-D-ASPARTATE RECEPTORS

    PubMed Central

    Sato, Yuko; Tao, Yuan-Xiang; Su, Qingning; Johns, Roger A

    2009-01-01

    Src family protein kinases (SFKs)-mediated tyrosine-phosphorylation regulates N-methyl-D-aspartate (NMDA) receptor synaptic function. Some members of the membrane-associated guanylate kinase (MAGUK) family of proteins bind to both SFKs and NMDA receptors, but it is unclear whether the MAGUK family of proteins is required for SFKs-mediated tyrosine-phosphorylation of the NMDA receptors. Here, we showed by co-immunoprecipitation that PSD-93, a member of the MAGUK family of proteins, interacts with the NMDA receptor subunits NR2A and NR2B as well as with Fyn, a member of the SFKs, in mouse cerebral cortex. Using a biochemical fractionation approach to isolate subcellular compartments revealed that the expression of Fyn, but not of other members of the SFKs (Lyn, Src, and Yes), was significantly decreased in synaptosomal membrane fractions derived from the cerebral cortex of PSD-93 knockout mice. Interestingly, we found that PSD-93 disruption causes reduction of tyrosine-phosphorylated NR2A and NR2B in the same fraction. Moreover, PSD-93 deletion markedly blocked the SFKs-mediated increase in tyrosine-phosphorylated NR2A and NR2B through the protein kinase C pathway after induction with 4β-PMA in cultured cortical neurons. Our findings indicate that PSD-93 appears to mediate tyrosine-phosphorylation of the NMDA receptors and synaptic localization of Fyn. PMID:18423999

  12. Identification of novel inhibitors of BCR-ABL tyrosine kinase via virtual screening.

    PubMed

    Peng, Hui; Huang, Niu; Qi, Jing; Xie, Ping; Xu, Chen; Wang, Jianxiang; Yang, Chunzheng

    2003-11-01

    Inhibition of BCR-ABL tyrosine kinase activity has shown to be essential for the treatment of chronic myelogenous leukemia (CML). However, drug resistance has quickly arisen in recent clinical trials for STI571 (Gleevec), which is the first approved drug of CML by inhibiting ABL tyrosine kinase. It is desirable to develop new types of ABL tyrosine kinase inhibitors that may overcome this drug resistance problem. Here we present the discovery of novel inhibitors targeted at the catalytic domain of ABL tyrosine kinase by using three-dimensional database searching techniques. From a database containing 200,000 commercially available compounds, the top 1000 compounds with the best DOCK energy score were selected and subjected to structural diversity and drug likeness analysis, 15 compounds were submitted for biological assay. Eight out of the 15 showed inhibitory activity against K562 cells with IC(50) value ranging from 10 to 200 microM. Two promising compounds showed inhibition in further ABL tyrosine phosphorylation assay. It is anticipated that those two compounds can serve as lead compounds for further drug design and optimization. PMID:14552760

  13. The adjuvant effect of stearyl tyrosine on a recombinant subunit hepatitis B surface antigen.

    PubMed

    Nixon-George, A; Moran, T; Dionne, G; Penney, C L; Lafleur, D; Bona, C A

    1990-06-15

    We describe the enhancement of the antibody response against hepatitis B surface Ag by octadecyl L-tyrosine, a synthetic adjuvant designed to exert its adjuvant effect in a manner similar to that of alum because it binds soluble Ag and releases it slowly from the site of injection. Our data demonstrate that octadecyl L-tyrosine showed a significant enhancement of the antihepatitis B surface Ag response compared to that of alum in the secondary response. The most striking difference between octadecyl L-tyrosine and alum in the antihepatitis B surface Ag antibody response was the absence of IgE-specific antibodies subsequent to immunization of the Ag in octadecyl L-tyrosine. Both the optical isomers of the octadecyl esters of tyrosine were adjuvant active, however, the racemic mixture showed a significantly lowe adjuvant activity. This adjuvant has great potential to be used in humans because it is devoid of side effects as assessed by the lack of acute and chronic toxicity in mice and rats, pyrogenicity in rabbits, formation of granuloma in cats, and adjuvant arthritis in rats. PMID:2351829

  14. Activation of signal transduction in platelets by the tyrosine phosphatase inhibitor pervanadate (vanadyl hydroperoxide).

    PubMed Central

    Pumiglia, K M; Lau, L F; Huang, C K; Burroughs, S; Feinstein, M B

    1992-01-01

    The protein tyrosine phosphatase (PTPase) inhibitor pervanadate (vanadyl hydroperoxide) stimulated protein tyrosine phosphorylation 29-fold more than did thrombin in intact and saponin-permeabilized platelets. Increased tyrosine phosphorylation preceded, or was coincident with, a fall in PtdIns(4,5)P2 levels, production of PtdIns(3,4)P2 and phosphatidic acid, mobilization of intracellular Ca2+, stimulation of protein kinase C-dependent protein phosphorylation, secretion of dense and alpha-granules, increased actin polymerization, shape change and aggregation which required fibrinogen and was mediated by increased surface expression of GPIIb-IIIa. The tyrosine kinase inhibitor RG 50864 totally prevented induction of tyrosine phosphorylation by pervanadate, as well as all other responses measured; in contrast, the inactive structural analogue, tyrphostin #1, had no effect. Dense-granule secretion induced by pervanadate required protein kinase C activity; however, aggregation and alpha-granule secretion were independent of protein kinase C. In saponin-permeabilized platelets pervanadate and thrombin stimulated phospholipase C activity by GTP-independent and GTP-dependent mechanisms respectively. We conclude that PTPases are important regulators of signal transduction in platelets. Images Fig. 1. Fig. 2. PMID:1530576

  15. 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

  16. Selective Sensing of Tyrosine Phosphorylation in Peptides Using Terbium(III) Complexes.

    PubMed

    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 Tb(III) 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 Tb(III) 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 Tb(III) 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

  17. 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

  18. 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.

  19. A Multifeatures Fusion and Discrete Firefly Optimization Method for Prediction of Protein Tyrosine Sulfation Residues.

    PubMed

    Guo, Song; 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

  20. Tyrosine 402 phosphorylation of Pyk2 is involved in ionomycin-induced neurotransmitter release.

    PubMed

    Zhang, Zhao; Zhang, Yun; Mou, Zheng; Chu, Shifeng; Chen, Xiaoyu; He, Wenbin; Guo, Xiaofeng; Yuan, Yuhe; Takahashi, Masami; Chen, Naihong

    2014-01-01

    Protein tyrosine kinases, which are highly expressed in the central nervous system, are implicated in many neural processes. However, the relationship between protein tyrosine kinases and neurotransmitter release remains unknown. In this study, we found that ionomycin, a Ca²⁺ ionophore, concurrently induced asynchronous neurotransmitter release and phosphorylation of a non-receptor protein tyrosine kinase, proline-rich tyrosine kinase 2 (Pyk2), in clonal rat pheochromocytoma PC12 cells and cerebellar granule cells, whereas introduction of Pyk2 siRNA dramatically suppressed ionomycin-induced neurotransmitter release. Further study indicated that Tyr-402 (Y402) in Pyk2, instead of other tyrosine sites, underwent rapid phosphorylation after ionomycin induction in 1 min to 2 min. We demonstrated that the mutant of Pyk2 Y402 could abolish ionomycin-induced dopamine (DA) release by transfecting cells with recombinant Pyk2 and its mutants (Y402F, Y579F, Y580F, and Y881F). In addition, Src inhibition could prolong phosphorylation of Pyk2 Y402 and increase DA release. These findings suggested that Pyk2 was involved in ionomycin-induced neurotransmitter release through phosphorylation of Y402. PMID:24718602

  1. 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

  2. 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

  3. 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

  4. Tyrosine phosphorylation modulates store-operated calcium entry in cultured rat epididymal basal cells.

    PubMed

    Zuo, Wu-Lin; Du, Jian-Yang; Huang, Jie-Hong; Li, Sheng; Zhang, Geng; Chen, Si-Liang; Ruan, Ye-Chun; Cheng, Christopher H K; Zhou, Wen-Liang

    2011-04-01

    Store-operated calcium entry (SOCE) is essential for many cellular processes. In this study, we investigated modulation of SOCE by tyrosine phosphorylation in rat epididymal basal cells. The intracellular Ca(2+) ([Ca(2+)]i) measurement showed that SOCE occurred in rat epididymal basal cells by pretreating the cells with thapsigargin (Tg), the inhibitor of sarco-endoplasmic reticulum Ca(2+)-ATPase. To identify the role of Ca(2+) channels in this response, we examined the effects of transient receptor potential canonical channel blockers 2-aminoethoxydiphenyl borate (2-APB), 1-[β-[3-(4-methoxyphenyl)pro-poxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride(SKF96365), Gd(3+), and non-selective cation channel blocker Ni(2+) respectively on SOCE and found that these blockers could inhibit the Ca(2+) influx to different extent. Furthermore, we studied the regulation of SOCE by tyrosine kinase pathway. The inhibitor of tyrosine kinase genistein remarkably suppressed the SOCE response, whereas sodium orthovanadate, the inhibitor of tyrosine phosphatase, greatly enhanced it. The results suggest that tyrosine kinase pathway plays a significant role in the initiation of SOCE and positively modulates SOCE in epididymal basal cells. PMID:20857412

  5. The relationship between low leucocyte ascorbic-acid levels and tyrosine metabolism in the elderly.

    PubMed

    Windsor, A C; Hobbs, C B; Treby, D A; Gupta, C B

    1975-11-01

    Twenty-seven elderly men aged 69-94 years had tyrosine tolerance tests measured before and after receiving ascorbic acid 1 g daily for four days. There was a significant rise in the fasting and half-hourly mean plasma tyrosine levels after ascorbic acid administration in those subjects with an initial leucocyte ascorbic-acid level (LAA) below 15 micrograms/10(8) w.c.c. The peak of the tyrosine tolerance curve was also advanced following administration of ascorbic acid in those subjects with an initial LAA below 15 micrograms/10(8) w.c.c. A further ten elderly men aged 73-89 years had simultaneous measurements of the circadian rhythms of LAA, plasma cortisol and plasma tyrosine levels before and after receiving ascorbic acid 1 g daily for four days. All ten subjects showed a peak LAA level at 5 p.m. when saturated with vitamin C but there was no significant correlation between plasma cortisol changes and plasma tyrosine changes. Possible explanations for the results are discussed. PMID:803133

  6. Identification and analysis of a novel protein-tyrosine kinase from bovine thymus

    SciTech Connect

    Zioncheck, T.F.; Harrison, M.L.; Geahlen, R.L.

    1986-05-01

    A cytosolic protein-tyrosine kinase has been identified and purified to near homogeneity from calf thymus by using the phosphorylation of the tyrosine-containing peptide angiotensin I as an assay. Specific peptide phosphorylating activity was enhanced by carrying out the assay at high ionic strength (2M NaCl). The inclusion of NaCl at this concentration acts to stimulate endogenous protein-tyrosine kinase activity while simultaneously inhibiting other endogenous kinases. The purification procedure involved extraction of the enzyme from calf-thymus and sequential chromatography on columns of DEAE-cellulose, heparin-agarose, casein-sepharose, butylagarose, and Sephadex G-75. Analysis of the most highly purified preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single Coomassie blue-stained band of 41 KDa. This molecular weight was consistent with results obtained from gel filtration, indicating that the enzyme exists as a monomer. The enzyme has also been found to catalyze an autophosphorylation reaction. Incubation of the enzyme with Mn/sup 2 +/ and (..gamma..-/sup 32/P)ATP led to its modification on a tyrosine residue. Phosphopeptide mapping experiments indicated that the 41 KDa kinase was distinct from p56, the major membrane-associated protein-tyrosine kinase in T lymphocytes.

  7. Regulation of the cystic fibrosis transmembrane conductance regulator anion channel by tyrosine phosphorylation.

    PubMed

    Billet, Arnaud; Jia, Yanlin; Jensen, Tim; Riordan, John R; Hanrahan, John W

    2015-09-01

    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. PMID:26062600

  8. 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

  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. 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.

  11. 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.

  12. Dimerization mediated through a leucine zipper activates the oncogenic potential of the met receptor tyrosine kinase.

    PubMed Central

    Rodrigues, G A; Park, M

    1993-01-01

    Oncogenic activation of the met (hepatocyte growth factor/scatter factor) receptor tyrosine kinase involves a genomic rearrangement that generates a hybrid protein containing tpr-encoded sequences at its amino terminus fused directly to the met-encoded receptor kinase domain. Deletion of Tpr sequences abolishes the transforming ability of this protein, implicating this region in oncogenic activation. We demonstrate, by site-directed mutagenesis and coimmunoprecipitation experiments, that a leucine zipper motif within Tpr mediates dimerization of the tpr-met product and is essential for the transforming activity of the met oncogene. By analogy with ligand-stimulated activation of receptor tyrosine kinases, we propose that constitutive dimerization mediated by a leucine zipper motif within Tpr is responsible for oncogenic activation of the Met kinase. The possibility that this mechanism of activation represents a paradigm for a class of receptor tyrosine kinase oncogenes activated by DNA rearrangement is discussed. Images PMID:8413267

  13. The role of tyrosine kinases in systemic lupus erythematosus and their potential as therapeutic targets

    PubMed Central

    Shao, Wen-Hai; Cohen, Philip L

    2014-01-01

    The autoimmune disease systemic lupus erythematosus is characterized by loss of tolerance to nuclear antigens. Breakdown of tolerance is associated with alterations in T-cell and B-cell receptor signal transduction, including increased protein phosphorylation that may underlie pathogenesis and explain the characteristic hyperactivity of T and B cells and other immune cells in active disease. Tyrosine kinases play a central role in signaling processes in cells known to be important in the pathogenesis of autoimmune diseases. Considerable progress has been made in understanding the function of tyrosine kinases in immune cell signaling pathways. In this review, we will summarize the function of tyrosine kinases and their novel inhibitors from studies made in animal lupus models and systemic lupus erythematosus patients. PMID:24678775

  14. Could tyrosine and tryptophan serve multiple roles in biological redox processes?

    PubMed Central

    Winkler, Jay R.; Gray, Harry B.

    2015-01-01

    Single-step electron tunnelling reactions can transport charges over distances of 15–20 Åin proteins. Longer-range transfer requires multi-step tunnelling processes along redox chains, often referred to as hopping. Long-range hopping via oxidized radicals of tryptophan and tyrosine, which has been identified in several natural enzymes, has been demonstrated in artificial constructs of the blue copper protein azurin. Tryptophan and tyrosine serve as hopping way stations in high-potential charge transport processes. It may be no coincidence that these two residues occur with greater-than-average frequency in O2- and H2O2-reactive enzymes. We suggest that appropriately placed tyrosine and/or tryptophan residues prevent damage from high-potential reactive intermediates by reduction followed by transfer of the oxidizing equivalent to less harmful sites or out of the protein altogether. PMID:25666062

  15. 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

  16. 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.

  17. Keratinocyte-derived Laminin-332 Protein Promotes Melanin Synthesis via Regulation of Tyrosine Uptake*

    PubMed Central

    Chung, Heesung; Jung, Hyejung; Lee, Jung-hyun; Oh, Hye Yun; Kim, Ok Bin; Han, Inn-Oc; Oh, Eok-Soo

    2014-01-01

    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. PMID:24951591

  18. Functions of the major tyrosine phosphorylation site of the PDGF receptor beta subunit.

    PubMed Central

    Kazlauskas, A; Durden, D L; Cooper, J A

    1991-01-01

    Two tyrosine phosphorylation sites in the human platelet-derived growth factor receptor (PDGFR) beta subunit have been mapped previously to tyrosine (Y)751, in the kinase insert, and Y857, in the kinase domain. Y857 is the major site of tyrosine phosphorylation in PDGF-stimulated cells. To evaluate the importance of these phosphorylations, we have characterized the wild-type (WT) and mutant human PDGF receptor beta subunits in dog kidney epithelial cells. Replacement of either Y751 or Y857 with phenylalanine (F) reduced PDGF-stimulated DNA synthesis to approximately 50% of the WT level. A mutant receptor with both tyrosines mutated was unable to initiate DNA synthesis, as was a kinase-inactive mutant receptor. Transmodulation of the epidermal growth factor receptor required Y857 but not Y751. We also tested the effects of phosphorylation site mutations on PDGF-stimulated receptor kinase activity. PDGF-induced tyrosine phosphorylation of two cellular proteins, phospholipase C gamma 1 (PLC gamma 1) and the GTPase activating protein of Ras (GAP), was assayed in epithelial cells expressing each of the mutant receptors. Tyrosine phosphorylation of GAP and PLC gamma 1 was reduced markedly by the F857 mutation but not significantly by the F751 mutation. Reduced kinase activity of F857 receptors was also evident in vitro. Immunoprecipitated WT receptors showed a two- to fourfold increase in specific kinase activity if immunoprecipitated from PDGF-stimulated cells. The F751 receptors showed a similar increase in activity, but F857 receptors did not. Our data suggest that phosphorylation of Y857 may be important for stimulation of kinase activity of the receptors and for downstream actions such as epidermal growth factor receptor transmodulation and mitogenesis. Images PMID:1653029

  19. Effect of ethanol on tyrosine hydroxylation in brain regions of long and short sleep mice.

    PubMed

    French, T A; Weiner, N

    1984-01-01

    The effect of ethanol on the in vivo rate of tyrosine hydroxylation in 6 brain regions was examined in two lines of mice selectively bred for a differential sensitivity to ethanol. The mice are designated long-sleep (LS) and short-sleep (SS) and lose their righting reflex for a duration of 100 minutes (LS) and 13 minutes (SS) following an intraperitoneal dose of ethanol of 4.0 g/kg. DOPA accumulation after NSD-1015 administration was measured in the absence and presence of ethanol (4.0 g/kg, IP) in the periods 5-35 minutes and 85-115 minutes after saline or ethanol. There were no differences between the lines in either basal catecholamine levels or basal tyrosine hydroxylation rates (as measured by DOPA accumulation) in any brain region except the cerebellum, where the norepinephrine content in the SS mice is 33% greater and the tyrosine hydroxylation rate is 25% higher than that in the LS mice. In the presence of ethanol, there was a differential effect on the in vivo tyrosine hydroxylation rate. In the cerebellum of both LS and SS mice there was a decreased rate of tyrosine hydroxylation in the early period after ethanol, but the rate in the cerebellum of SS mice returned to the control value at 85-115 min. At that time, the rate in LS mice is still decreased. In the locus ceruleus, hypothalamus and frontal cortex, ethanol has no effect on the rate of tyrosine hydroxylation in either LS or SS mice during the early period, but ethanol decreases the rate during the later period in the LS mice only.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:6443068

  20. ACK1/TNK2 Tyrosine Kinase: Molecular Signaling and Evolving Role in Cancers

    PubMed Central

    Mahajan, Kiran; Mahajan, Nupam P.

    2014-01-01

    Deregulated tyrosine kinase signaling alters cellular homeostasis to drive cancer progression. The emergence of a non-receptor tyrosine kinase, ACK1 as an oncogenic kinase, has uncovered novel mechanisms by which tyrosine kinase signaling promotes cancer progression. While early studies focused on ACK1 (also known as activated Cdc42-associated kinase 1 or TNK2) as a cytosolic effecter of activated transmembrane receptor tyrosine kinases (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, modifies KDM3A by tyrosine phosphorylation to regulate 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. PMID:25347744

  1. Enhancement of cytosolic tyrosine kinase activity by propylthiouracil-induced hyperplasia in the rat thyroid.

    PubMed

    Polychronakos, C; Piscina, R; Fantus, I G

    1989-01-01

    Hyperplasia of the thyroid gland induced by propylthiouracil (PTU) is a well established model of rapid cell proliferation in vivo. Recent evidence indicates that tyrosine kinase activity is associated with growth factor receptors and oncogene protein products and may have an important regulatory action in the control of cell growth. Thus, we examined tyrosine kinase activity in rat thyroid membrane and cytosol preparations at rest and during PTU-induced hyperplasia. Although kinase activity was present in a crude microsomal membrane preparation, no change was observed during thyroid growth. In contrast, tyrosine kinase activity assayed with the artificial substrate poly(Glu,Na:Tyr) 4:1 was present in normal rat thyroid cytosol and increased 2- to 6-fold during the rapid phase of hyperplasia in the first 5-10 days of PTU treatment. It declined to control values by day 15, when the size and DNA content of the thyroid reached a plateau. Preincubation of the cytosolic preparations with several peptides known to bind to and activate growth factor receptor tyrosine kinases failed to enhance the activity, suggesting, along with the cytosolic localization, that the activity was distinct from these receptors. By gel filtration chromatography and polyacrylamide gel electrophoresis, tyrosine kinase activity was associated with a 55 kDa protein. Partial purification over a poly(Glu,Na:Tyr)4:1-Sepharose column, yielded a protein that appeared capable of autophosphorylation. It is suggested that this tyrosine kinase plays a role in mediating the growth-promoting effects of this model of thyroid cell hyperplasia. PMID:2909378

  2. 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

  3. 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.

  4. Thrombin Ca(2+)-dependently stimulates protein tyrosine phosphorylation in BC3H1 muscle cells.

    PubMed Central

    Offermanns, S; Bombien, E; Schultz, G

    1993-01-01

    The proteinase thrombin, known to act via heptahelical G-protein-coupled receptors, is a mitogenic agent for different cell types, including the mouse muscle cell line BC3H1. In this study, the effect of thrombin on tyrosine phosphorylation was examined using anti-phosphotyrosine antibodies. Thrombin was found to induce phosphorylation of 65-70 and 110-120 kDa proteins in BC3H1 cells. The effect of thrombin was concentration-dependent, being half-maximal and maximal at concentrations of 0.03 and 1 unit/ml respectively. The thrombin-induced increase in phosphorylation was rapid (< or = 10 s) and transient, with a peak response after about 1-2 min. The effect of thrombin could be mimicked by the thrombin receptor agonist peptide SFLLRN-NH2. Preincubation of cells with pertussis toxin (PT) had no effect on thrombin-induced tyrosine phosphorylation. Epidermal growth factor, platelet-derived growth factor and insulin stimulated tyrosine phosphorylation of different proteins, among which were 65-70 and 110-120 kDa proteins. The phorbol ester 12-myristate 13-acetate (PMA) as well as the Ca2+ ionophore A23187 both stimulated tyrosine phosphorylation of proteins identical to those phosphorylated by thrombin, suggesting that activation of protein kinase C (PKC) and elevation of the cytosolic Ca2+ concentration alone are sufficient to induce tyrosine phosphorylation. However, calphostin C and other PKC inhibitors, which completely inhibited tyrosine phosphorylation induced by PMA, had no influence on the effect of thrombin, whereas loading of cells with the intracellular Ca2+ chelator bis-(O-aminophenoxy)ethane-NNN'N'-tetra-acetic acid totally blocked thrombin-stimulated tyrosine phosphorylation. Thus tyrosine phosphorylation stimulated by thrombin is an early PT-insensitive cellular response which is either directly mediated by elevation of cytosolic Ca2+ concentration or by a presently unknown mechanism that requires an elevated cytosolic Ca2+ concentration. Images Figure 1

  5. Induction of macrophage procoagulant activity by murine hepatitis virus strain 3: role of tyrosine phosphorylation.

    PubMed Central

    Dackiw, A P; Zakrzewski, K; Nathens, A B; Cheung, P Y; Fingerote, R; Levy, G A; Rotstein, O D

    1995-01-01

    The induction of a unique macrophage procoagulant molecule by murine hepatitis virus strain 3 correlates with the severity of viral hepatitis. The role of tyrosine phosphorylation in the signalling pathway leading to procoagulant expression was studied. Murine hepatitis virus strain 3 initiated a rapid increase in phosphotyrosine accumulation. Tyrosine kinase inhibition precluded this increase and abrogated expression of the virus-induced procoagulant mouse fibrinogen-like protein (musfiblp) gene. These findings suggest that manipulation of this signalling pathway in vivo might represent a novel approach to treating this disease. PMID:7543590

  6. 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.

  7. 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

  8. Using ovality to predict nonmutagenic, orally efficacious pyridazine amides as cell specific spleen tyrosine kinase inhibitors.

    PubMed

    Lucas, Matthew C; Bhagirath, Niala; Chiao, Eric; Goldstein, David M; Hermann, Johannes C; Hsu, Pei-Yuan; Kirchner, Stephan; Kennedy-Smith, Joshua J; Kuglstatter, Andreas; Lukacs, Christine; Menke, John; Niu, Linghao; Padilla, Fernando; Peng, Ying; Polonchuk, Liudmila; Railkar, Aruna; Slade, Michelle; Soth, Michael; Xu, Daigen; Yadava, Preeti; Yee, Calvin; Zhou, Mingyan; Liao, Cheng

    2014-03-27

    Inhibition of spleen tyrosine kinase has attracted much attention as a mechanism for the treatment of cancers and autoimmune diseases such as asthma, rheumatoid arthritis, and systemic lupus erythematous. We report the structure-guided optimization of pyridazine amide spleen tyrosine kinase inhibitors. Early representatives of this scaffold were highly potent and selective but mutagenic in an Ames assay. An approach that led to the successful identification of nonmutagenic examples, as well as further optimization to compounds with reduced cardiovascular liabilities is described. Select pharmacokinetic and in vivo efficacy data are presented. PMID:24520947

  9. Identification of tyrosines 154 and 307 in the extracellular domain and 653 and 766 in the intracellular domain as phosphorylation sites in the heparin-binding fibroblast growth factor receptor tyrosine kinase (flg).

    PubMed Central

    Hou, J.; McKeehan, K.; Kan, M.; Carr, S. A.; Huddleston, M. J.; Crabb, J. W.; McKeehan, W. L.

    1993-01-01

    Four tyrosine residues have been identified as phosphorylation sites in the tyrosine kinase isoform of the heparin-binding fibroblast growth factor receptor flg (FGF-R1). Baculoviral-insect cell-derived recombinant FGF-R1 was phosphorylated and fragmented with trypsin while immobilized on heparin-agarose beads. Phosphotyrosine peptides were purified by chromatography on immobilized anti-phosphotyrosine antibody and analyzed by Edman degradation and electrospray tandem mass spectrometry. Tyrosine residue 653, which is in a homologous spatial position to major autophosphorylation sites in the catalytic domain of the src and insulin receptor kinases, is the major intracellular FGF-R1 phosphorylation site. Residue 766 in the COOH-terminus outside the kinase domain is a secondary site. Tyrosine residues 154 and 307, which are in the extracellular domain of transmembrane receptor isoforms and are in an unusual sequence context for tyrosine phosphorylation, were also phosphorylated. PMID:8443592

  10. 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

  11. Biodegradation of the Allelopathic Chemical m-Tyrosine by Bacillus aquimaris SSC5 Involves the Homogentisate Central Pathway

    PubMed Central

    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 1H-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. 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

  13. 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

  14. A heme peroxidase with a functional role as an L-tyrosine hydroxylase in the biosynthesis of anthramycin.

    PubMed

    Connor, Katherine L; Colabroy, Keri L; Gerratana, Barbara

    2011-10-18

    We report the first characterization and classification of Orf13 (S. refuineus) as a heme-dependent peroxidase catalyzing the ortho-hydroxylation of L-tyrosine to L-DOPA. The putative tyrosine hydroxylase coded by orf13 of the anthramycin biosynthesis gene cluster has been expressed and purified. Heme b has been identified as the required cofactor for catalysis, and maximal L-tyrosine conversion to L-DOPA is observed in the presence of hydrogen peroxide. Preincubation of L-tyrosine with Orf13 prior to the addition of hydrogen peroxide is required for L-DOPA production. However, the enzyme becomes inactivated by hydrogen peroxide during catalysis. Steady-state kinetic analysis of L-tyrosine hydroxylation revealed similar catalytic efficiency for both L-tyrosine and hydrogen peroxide. Spectroscopic data from a reduced-CO(g) UV-vis spectrum of Orf13 and electron paramagnetic resonance of ferric heme Orf13 are consistent with heme peroxidases that have a histidyl-ligated heme iron. Contrary to the classical heme peroxidase oxidation reaction with hydrogen peroxide that produces coupled aromatic products such as o,o'-dityrosine, Orf13 is novel in its ability to catalyze aromatic amino acid hydroxylation with hydrogen peroxide, in the substrate addition order and for its substrate specificity for L-tyrosine. Peroxygenase activity of Orf13 for the ortho-hydroxylation of L-tyrosine to L-DOPA by a molecular oxygen dependent pathway in the presence of dihydroxyfumaric acid is also observed. This reaction behavior is consistent with peroxygenase activity reported with horseradish peroxidase for the hydroxylation of phenol. Overall, the putative function of Orf13 as a tyrosine hydroxylase has been confirmed and establishes the first bacterial class of tyrosine hydroxylases. PMID:21919439

  15. 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

  16. 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.

  17. Tyrosine kinase activity, cytoskeletal organization, and motility in human vascular endothelial cells.

    PubMed Central

    Romer, L H; McLean, N; Turner, C E; Burridge, K

    1994-01-01

    Tyrosine phosphorylation of cytoskeletal proteins occurs during integrin-mediated cell adhesion to extracellular matrix proteins. We have investigated the role of tyrosine phosphorylation in the migration and initial spreading of human umbilical vein endothelial cells (HUVEC). Elevated phosphotyrosine concentrations were noted in the focal adhesions of HUVEC migrating into wounds. Anti-phosphotyrosine Western blots of extracts of wounded HUVEC monolayers demonstrated increased phosphorylation at 120-130 kDa when compared with extracts of intact monolayers. The pp125FAK immunoprecipitated from wounded monolayers exhibited increased kinase activity as compared to pp125FAK from intact monolayers. The time to wound closure in HUVEC monolayers was doubled by tyrphostin AG 213 treatment. The same concentration of AG 213 interfered with HUVEC focal adhesion and stress fiber formation. AG 213 inhibited adhesion-associated tyrosine phosphorylation of pp125FAK in HUVEC. Tyrphostins AG 213 and AG 808 inhibited pp125FAK activity in in vitro kinase assays. pp125FAK immunoprecipitates from HUVEC treated with both of these inhibitors also had kinase activity in vitro that was below levels seen in untreated HUVEC. These findings suggest that tyrosine phosphorylation of cytoskeletal proteins may be important in HUVEC spreading and migration and that pp125FAK may mediate phosphotyrosine formation during these processes. Images PMID:8049526

  18. 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

  19. 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

  20. 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.

  1. Modulation of protein tyrosine nitration and inflammatory mediators by isoprenylhydroquinone glucoside.

    PubMed

    Olmos, Ana; Giner, Rosa-María; Recio, María-Carmen; Ríos, José-Luis; Máñez, Salvador

    2007-03-01

    The nitration of tyrosine caused by peroxynitrite and other reactive nitrogen species is clearly detrimental for some physiological processes; however, its signalling role is still open to controversy. Among the natural phenolics known for their ability to oppose free tyrosine nitration, isoprenylhydroquinone glucoside is investigated due to its unusual structure, which contains a simple hydroxybenzene alkylated by a hemiterpenoid moiety. This hydroquinone was shown to be an effective inhibitor of peroxynitrite-induced protein tyrosine nitration in 3T3 fibroblasts. When tested on bovine seroalbumin nitration, however, the potency was reduced by half and the effect was almost abolished in the presence of bicarbonate. In contrast, addition of this anion had no effect on the nitrite/hydrogen peroxide/hemin system. Isoprenylhydroquinone glucoside was also active in the microM range on intra- and extracellular protein-bound tyrosine nitration by phorbol 12-myristate 13-acetate-stimulated neutrophils. The effects on nitric oxide synthase expression, interleukin-1beta and tumor necrosis factor-alpha production by lipopolysaccharide-stimulated macrophages were quite moderate. Thus, isoprenylhydroquinone glucoside is an inhibitor of protein nitration in situ, but lacks effect on the generation of either nitric oxide or inflammatory cytokines. PMID:17161592

  2. 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...

  3. MALDI mass sequencing and biochemical characterization of Setaria cervi protein tyrosine phosphatase.

    PubMed

    Rai, Reeta; Singh, Neetu; Elesela, Srikanth; Tiwari, Savitri; Rathaur, Sushma

    2013-01-01

    A 30-kDa acid phosphatase with protein tyrosine phosphatase activity was identified in Setaria cervi (ScPTP). The enzyme was purified to homogeneity using three-step column chromatography. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of purified ScPTP yielded a total of eight peptides matching most closely to phosphoprotein phosphatase of Ricinus communis (RcPP). A hydrophilicity plot of RcPP revealed the presence of these peptides in the hydrophilic region, suggesting their antigenic nature. The substrate specificity of ScPTP with ortho-phospho-L-tyrosine and inhibition with sodium orthovanadate and ammonium molybdate affirmed it as a protein tyrosine phosphatase. ScPTP was also found to be tartrate resistant. The Km and Vmax were 6.60 mM and 83.3 μM/ml/min, respectively, with pNPP and 8.0 mM and 111 μM/ml/min, respectively, with ortho-phospho-L-tyrosine as the substrate. The Ki value with sodium orthovanadate was calculated to be 16.10 mM. Active site modification with DEPC, EDAC and pHMB suggested the presence of histidine, cysteine and aspartate at its active site. Thus, on the basis of MALDI-TOF and biochemical studies, it was confirmed that purified acid phosphatase is a PTP. PMID:23052758

  4. 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

  5. The effect of oxygen, antioxidants, and superoxide radical on tyrosine phenoxyl radical dimerization

    SciTech Connect

    Hunter, E.P.; Desrosiers, M.F.; Simic, M.G. )

    1989-01-01

    Dimerization of tyrosine phenoxyl radical yields bityrosine (BT) which can easily be monitored by its characteristic fluorescence at 400 nm. The reactivity of tyrosine phenoxyl radical with O{sub 2} was examined by a variety of techniques. BT fluorescence was measured as a function of O{sub 2} concentration. Over a range of pH values (4-12) there was no effect of oxygen on BT production ((O{sub 2}) less than or equal to 0.72 mM). In addition, oxygen uptake by the phenoxyl radical was measured directly with an oxygen electrode. It was determined by this technique that oxygen does not react with the phenoxyl radical with a rate constant greater than 10(3) M-1 s-1. Tyrosine phenoxyl radical repair by superoxide and physiological antioxidants was examined by BT fluorescence quenching as well as pulse radiolysis. Implications of these results as to the fate of tyrosine phenoxyl radicals produced in biological systems is discussed.

  6. Activation of a protein tyrosine phosphatase and inactivation of Raf-1 by somatostatin.

    PubMed Central

    Reardon, D B; Wood, S L; Brautigan, D L; Bell, G I; Dent, P; Sturgill, T W

    1996-01-01

    Human somatostatin receptor 3 ('hsstr3') was transiently expressed in NIH 3T3 cells stably transformed with Ha-Ras (G12V). Somatostatin activated a protein tyrosine phosphatase and inactivated the constitutively active, membrane-associated form of the Raf-1 serine kinase present in these cells in vivo and in vitro. PMID:8670047

  7. 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

  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. 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...

  10. 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 ...

  11. Determination of L-tyrosine by β-cyclodextrin sensitized fluorescence quenching method

    NASA Astrophysics Data System (ADS)

    Zhu, Xiashi; Xu, Suqin

    2010-10-01

    A novel β-cyclodextrin (β-CD) sensitized fluorescence quenching method for the determination of L-tyrosine ( L-Tyr) with Mo(VI)-phenyl-fluorone (PF) as a fluorescence probe has been developed. The fluorescence intensity of Mo(VI)-PF-β-CD was diminished as the L-tyrosine was added, the fluorescence quenching value Δ F = Fβ-CD-Mo-PF - Fβ-CD-Mo-PF- L-Tyr was enhanced in β-CD and there was a linear relationship between the Δ F and the concentration of L-Tyr. Under the optimal conditions, the linear range of calibration curve for the determination of L-tyrosine was 0.3-20.0 μg mL -1; the detection limit was 0.094 μg mL -1. NaOH (10%, w/v) is the best reagent of hydrolysis in sample preparation. The sensitized mechanism of β-cyclodextrin was discussed. The method has been applied to the determination of L-tyrosine in spirulina and food samples with satisfactory results.

  12. Endostatin-induced tyrosine kinase signaling through the Shb adaptor protein regulates endothelial cell apoptosis.

    PubMed

    Dixelius, J; Larsson, H; Sasaki, T; Holmqvist, K; Lu, L; Engström, A; Timpl, R; Welsh, M; Claesson-Welsh, L

    2000-06-01

    Endostatin, which corresponds to the C-terminal fragment of collagen XVIII, is a potent inhibitor of angiogenesis. Fibroblast growth factor-2 (FGF-2)-induced angiogenesis in the chicken chorioallantoic membrane was inhibited by endostatin, but not by an endostatin mutant R158/270A, lacking heparin-binding ability. Endostatin was internalized by endothelial cells, but not by mouse fibroblasts. Treatment of murine brain endothelial (IBE) cells with endostatin reduced the proportion of cells in S phase, whereas growth-arrested IBE cells in collagen gels treated with endostatin displayed enhanced tubular morphogenesis. IBE cells overexpressing Shb, an adaptor protein implicated in angiostatin-induced apoptosis, displayed elevated apoptosis and decreased tubular morphogenesis in collagen gels in response to endostatin when added together with FGF-2. Induction of apoptosis was dependent on the heparin-binding ability of endostatin and the expression of Shb with a functional Src homology 2 (SH2)-domain. Endostatin treatment for 10 minutes or 24 hours induced tyrosine phosphorylation of Shb and formation of multiprotein complexes. An Shb SH2 domain fusion protein precipitated a 125-kd phosphotyrosyl protein in endostatin-treated cells. The 125-kd component either contained intrinsic tyrosine kinase activity or occurred in complex with a tyrosine kinase. In conclusion, our data show that endostatin induces tyrosine kinase activity and enhanced apoptosis in FGF-treated endothelial cells. PMID:10828022

  13. 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

  14. Angiopoietin-1 Regulates Brain Endothelial Permeability through PTPN-2 Mediated Tyrosine Dephosphorylation of Occludin

    PubMed Central

    Siddiqui, M. Rizwan; Mayanil, Chandra S.; Kim, Kwang Sik; Tomita, Tadanori

    2015-01-01

    Objective Blood brain barrier (BBB) breakdown and increased endothelial permeability is a hallmark of neuro-vascular inflammation. Angiopoietin-1 (Ang-1), a Tie-2 receptor agonist ligand, is known to modulate barrier function of endothelial cells; however the molecular mechanisms related to Ang-1 mediated repair of Tight Junctions (TJs) in brain endothelium still remain elusive. In this study, we investigated a novel role of non-receptor protein tyrosine phosphatase N-2 (PTPN-2) in Ang-1 mediated stabilization of tight junction proteins. Method and Result To study the barrier protective mechanism of Ang-1, we challenged human brain microvascular endothelial cells in-vitro, with a potent inflammatory mediator thrombin. By using confocal microscopy and transwell permeability assay, we show that pretreatment of brain endothelial cells with Ang-1 diminish thrombin mediated disruption of TJs and increase in endothelial permeability. We also found that Ang-1 inhibits thrombin induced tyrosine phosphorylation of Occludin and promote Occludin interaction with Zona Occludens-1 (ZO-1) to stabilize TJs. Interestingly, our study revealed that depletion of PTPN-2 by siRNAs abolishes Ang-1 ability to promote tyrosine dephosphorylation of Occludin, resulting Occludin disassociation from ZO-1 and endothelial hyperpermeability. Summary Collectively, our findings suggest that in brain endothelial cells blocking PTPN-2 mediated tyrosine phosphorylation of Occludin is a novel mechanism to maintain BBB function, and may offer a key therapeutic strategy for neuro-inflammatory disorders associated with BBB disruption. PMID:26090670

  15. 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

  16. Computer-aided identification of EGFR tyrosine kinase inhibitors using ginsenosides from Panax ginseng.

    PubMed

    Sathishkumar, Natarajan; Karpagam, Veerappan; Sathiyamoorthy, Subramaniyam; Woo, Min Jin; Kim, Yeon-Ju; Yang, Deok-Chun

    2013-07-01

    Natural products have served as structural resources in the history of drug discovery for cancer therapy. Among these natural products, Korean Panax ginseng serves as a potential anti-cancer medicinal plant. To determine the anti-cancer activities of Korean P. ginseng active compounds, we performed pharmacophore-based virtual screening and molecular docking studies on EGFR (epidermal growth factor receptor) tyrosine kinase domain. The EGFR family tyrosine kinase receptor is a cell surface receptor that regulates diverse biological processes including cell proliferation, differentiation, survival, and apoptosis. Over expression of EGFR tyrosine kinase domain associated with the development and progression of numerous human cancers. In our study, we developed the best pharmacophore model (Hypo1) using a diverse training set and validated by Fischer's randomization, a test set, and a decoy set. The best validated model was employed in the virtual screening of P. ginseng compound database. Further, chosen molecules were evaluated by applying ADMET screening and molecular docking studies. Finally, 14 compounds were obtained based on binding affinity scores and interactions with protein active site residues. These final lead compounds from P. ginseng can be used in the designing of new EGFR tyrosine kinase inhibitors. PMID:23668355

  17. 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.

  18. 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. PMID:25680927

  19. 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

  20. 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

  1. An immunochemical approach to detect oxidized protein tyrosine phosphatases using a selective C-nucleophile tag.

    PubMed

    Garcia, Francisco J; Carroll, Kate S

    2016-05-24

    Protein tyrosine phosphatases are crucial regulators of signal transduction and function as antagonists towards protein tyrosine kinases to control reversible tyrosine phosphorylation, thereby regulating fundamental physiological processes. Growing evidence has supported the notion that reversible oxidative inactivation of the catalytic cysteine residue in protein tyrosine phosphatases serves as an oxidative post-translational modification that regulates its activity to influence downstream signaling by promoting phosphorylation and induction of the signaling cascade. The oxidation of cysteine to the sulfenic acid is often transient and difficult to detect, thus making it problematic in understanding the role that this oxidative post-translational modification plays in redox-biology and pathogenesis. Several methods to detect cysteine oxidation in biological systems have been developed, though targeted approaches to directly detect oxidized phosphatases are still lacking. Herein we describe the development of a novel immunochemical approach to directly profile oxidized phosphatases. This immunochemical approach consists of an antibody designed to recognize the conserved sequence of the PTP active site (VHCDMDSAG) harboring the catalytic cysteine modified with dimedone (CDMD), a nucleophile that chemoselectively reacts with cysteine sulfenic acids to form a stable thioether adduct. Additionally, we provide biochemical and mass spectrometry workflows to be used in conjugation with this newly developed immunochemical approach to assist in the identification and quantification of basal and oxidized phosphatases. PMID:26757830

  2. Effect of sialylation on EGFR phosphorylation and resistance to tyrosine kinase inhibition.

    PubMed

    Yen, Hsin-Yung; Liu, Ying-Chih; Chen, Nai-Yu; Tsai, Chia-Feng; Wang, Yi-Ting; Chen, Yu-Ju; Hsu, Tsui-Ling; Yang, Pan-Chyr; Wong, Chi-Huey

    2015-06-01

    Epidermal growth factor receptor (EGFR) is a heavily glycosylated transmembrane receptor tyrosine kinase. Upon EGF-binding, EGFR undergoes conformational changes to dimerize, resulting in kinase activation and autophosphorylation and downstream signaling. Tyrosine kinase inhibitors (TKIs) have been used to treat lung cancer by inhibiting EGFR phosphorylation. Previously, we demonstrated that EGFR sialylation suppresses its dimerization and phosphorylation. In this report, we further investigated the effect of sialylation on the phosphorylation profile of EGFR in TKI-sensitive and TKI-resistant cells. Sialylation was induced in cancer progression to inhibit the association of EGFR with EGF and the subsequent autophosphorylation. In the absence of EGF the TKI-resistant EGFR mutant (L858R/T790M) had a higher degree of sialylation and phosphorylation at Y1068, Y1086, and Y1173 than the TKI-sensitive EGFR. In addition, although sialylation in the TKI-resistant mutants suppresses EGFR tyrosine phosphorylation, with the most significant effect on the Y1173 site, the sialylation effect is not strong enough to stop cancer progression by inhibiting the phosphorylation of these three sites. These findings were supported further by the observation that the L858R/T790M EGFR mutant, when treated with sialidase or sialyltransferase inhibitor, showed an increase in tyrosine phosphorylation, and the sensitivity of the corresponding resistant lung cancer cells to gefitinib was reduced by desialylation and was enhanced by sialylation. PMID:25971727

  3. Estradiol receptor: phosphorylation on tyrosine in uterus and interaction with anti-phosphotyrosine antibody.

    PubMed Central

    Migliaccio, A; Rotondi, A; Auricchio, F

    1986-01-01

    Estradiol receptor from rat uteri incubated with [32P] orthophosphate has been purified by diethylstilbestrol--Sepharose followed by heparin--Sepharose chromatography. The purified receptor, analyzed by centrifugation through sucrose gradients after incubation with monoclonal antibodies against purified estradiol receptor, appears to be labeled with 32P. The receptor preparation has been further purified by immunoaffinity chromatography and submitted to SDS--poly-acrylamide gel electrophoresis. A heavily 32P-labeled 68 kd protein and a very lightly 32P-labeled 48 kd protein, probably a proteolytic product of the 68 kd protein, were detected. Phosphoamino acid analysis of the receptor eluted from the immunoaffinity column shows that its 32P-labeling occurs exclusively on tyrosine. This is the first report on phosphorylation on tyrosine of a steroid receptor in tissue. It is consistent with our previous finding that a uterus estradiol receptor-kinase, which confers hormone binding ability to the estradiol receptor, in vitro phosphorylates this receptor exclusively on tyrosine. Calf uterus receptor binds with high specificity and affinity to monoclonal anti-phosphotyrosine antibodies covalently bound to Sepharose (Kd = 0.28 nM). Dephosphorylation of the receptor by nuclei containing the calf uterus nuclear phosphatase abolishes the interaction with antibodies. These results suggest that also in calf uterus, estradiol receptor is phosphorylated on tyrosine. Anti-phosphotyrosine antibodies bound to Sepharose have been used to partially purify the estradiol receptor from calf uterus. Images Fig. 2. Fig. 3. PMID:2431901

  4. 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

  5. Protein tyrosine nitration of mitochondrial carbamoyl phosphate synthetase 1 and its functional consequences.

    PubMed

    Takakusa, Hideo; Mohar, Isaac; Kavanagh, Terrance J; Kelly, Edward J; Kaspera, Rüdiger; Nelson, Sidney D

    2012-03-30

    Mitochondria are the primary locus for the generation of reactive nitrogen species including peroxynitrite and subsequent protein tyrosine nitration. Protein tyrosine nitration may have important functional and biological consequences such as alteration of enzyme catalytic activity. In the present study, mouse liver mitochondria were incubated with peroxynitrite, and the mitochondrial proteins were separated by 1D and 2D gel electrophoresis. Nitrotyrosinylated proteins were detected with an anti-nitrotyrosine antibody. One of the major proteins nitrated by peroxynitrite was carbamoyl phosphate synthetase 1 (CPS1) as identified by LC-MS protein analysis and Western blotting. The band intensity of nitration normalized to CPS1 was increased in a peroxynitrite concentration-dependent manner. In addition, CPS1 activity was decreased by treatment with peroxynitrite in a peroxynitrite concentration- and time-dependent manner. The decreased CPS1 activity was not recovered by treatment with reduced glutathione, suggesting that the decrease of the CPS1 activity is due to tyrosine nitration rather than cysteine oxidation. LC-MS analysis of in-gel digested samples, and a Popitam-based modification search located 5 out of 36 tyrosine residues in CPS1 that were nitrated. Taken together with previous findings regarding CPS1 structure and function, homology modeling of mouse CPS1 suggested that nitration at Y1450 in an α-helix of allosteric domain prevents activation of CPS1 by its activator, N-acetyl-l-glutamate. In conclusion, this study demonstrated the tyrosine nitration of CPS1 by peroxynitrite and its functional consequence. Since CPS1 is responsible for ammonia removal in the urea cycle, nitration of CPS1 with attenuated function might be involved in some diseases and drug-induced toxicities associated with mitochondrial dysfunction. PMID:22402285

  6. A GAPDH Mutant Defective in Src-Dependent Tyrosine Phosphorylation Impedes Rab2-Mediated Events

    PubMed Central

    Tisdale, Ellen J.; Artalejo, Cristina R.

    2013-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has multiple intracellular activities in addition to its role in gluconeogenesis. Indeed, we have reported that GAPDH is required for Rab2-mediated retrograde transport from vesicular tubular clusters (VTCs). These diverse GAPDH activities are the result of post-translational modifications that confer a new function to the enzyme. In that regard, GAPDH is tyrosine phosphorylated by Src. To establish the functional significance of this modification for GAPDH activity in Rab2-dependent events, an amino acid substitution was made at tyrosine 41 (GAPDH Y41F). The inability of Src to phosphorylate purified recombinant GAPDH Y41F was confirmed in an in vitro kinase assay. The mutant was then employed in a quantitative membrane-binding assay that measures Rab2 recruitment of soluble components to VTCs. As we observed with GAPDH wild type, Rab2 promoted GAPDH Y41F binding to membranes in a dose-dependent manner indicating that GAPDH tyrosine phosphorylation is not required for VTC association. However, GAPDH was tyrosine phosphorylated on VTCs. Importantly, GAPDH Y41F blocked VSV-G transport in an assay that reconstitutes endoplasmic reticulum (ER) to Golgi trafficking indicating that phosphorylation of tyrosine 41 is essential for GAPDH activity in the early secretory pathway. The block in transport is due to the decreased binding of atypical protein kinase C iota/lambda (aPKCι/λ) to GAPDH Y41F, which reduces β-COP association with the VTC and subsequent formation of Rab2-mediated retrograde vesicles. Our results suggest that Src plays a pivotal role in regulating the interaction of Rab2 effectors on the VTC. PMID:17488287

  7. Nephrin Tyrosine Phosphorylation Is Required to Stabilize and Restore Podocyte Foot Process Architecture.

    PubMed

    New, Laura A; Martin, Claire E; Scott, Rizaldy P; Platt, Mathew J; Keyvani Chahi, Ava; Stringer, Colin D; Lu, Peihua; Samborska, Bozena; Eremina, Vera; Takano, Tomoko; Simpson, Jeremy A; Quaggin, Susan E; Jones, Nina

    2016-08-01

    Podocytes are specialized epithelial cells of the kidney blood filtration barrier that contribute to permselectivity via a series of interdigitating actin-rich foot processes. Positioned between adjacent projections is a unique cell junction known as the slit diaphragm, which is physically connected to the actin cytoskeleton via the transmembrane protein nephrin. Evidence indicates that tyrosine phosphorylation of the intracellular tail of nephrin initiates signaling events, including recruitment of cytoplasmic adaptor proteins Nck1 and Nck2 that regulate actin cytoskeletal dynamics. Nephrin tyrosine phosphorylation is altered in human and experimental renal diseases characterized by pathologic foot process remodeling, prompting the hypothesis that phosphonephrin signaling directly influences podocyte morphology. To explore this possibility, we generated and analyzed knockin mice with mutations that disrupt nephrin tyrosine phosphorylation and Nck1/2 binding (nephrin(Y3F/Y3F) mice). Homozygous nephrin(Y3F/Y3F) mice developed progressive proteinuria accompanied by structural changes in the filtration barrier, including podocyte foot process effacement, irregular thickening of the glomerular basement membrane, and dilated capillary loops, with a similar but later onset phenotype in heterozygous animals. Furthermore, compared with wild-type mice, nephrin(Y3F/Y3F) mice displayed delayed recovery in podocyte injury models. Profiling of nephrin tyrosine phosphorylation dynamics in wild-type mice subjected to podocyte injury indicated site-specific differences in phosphorylation at baseline, injury, and recovery, which correlated with loss of nephrin-Nck1/2 association during foot process effacement. Our results define an essential requirement for nephrin tyrosine phosphorylation in stabilizing podocyte morphology and suggest a model in which dynamic changes in phosphotyrosine-based signaling confer plasticity to the podocyte actin cytoskeleton. PMID:26802179

  8. 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

  9. 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

  10. Adhesion of fibroblasts to fibronectin stimulates both serine and tyrosine phosphorylation of paxillin.

    PubMed Central

    Bellis, S L; Perrotta, J A; Curtis, M S; Turner, C E

    1997-01-01

    Tyrosine phosphorylation of paxillin by the focal adhesion kinase (FAK) has been implicated as a signal transduction mechanism associated with cell adhesion and cytoskeletal reorganization. The potential role of serine phosphorylation of paxillin in these events has not been well characterized. In this study we have examined the phosphorylation profile of paxillin both in vitro and in vivo. By using glutathione S-transferase-paxillin fusion proteins in precipitation-kinase assays in vitro we observed that a fusion protein spanning amino acid residues 54-313 of paxillin, and containing a FAK-binding site, precipitated substantial serine kinase activity as well as FAK activity from a smooth-muscle lysate. Together these kinases phosphorylated paxillin on tyrosine residue 118, a site that has been identified previously as a target for FAK phosphorylation, and on serine residues 188 and/or 190. The binding site for the serine kinase, the identity of which is currently unknown, was further mapped to residues 168-191 of paxillin. To assess the physiological relevance of these sites phosphorylated in vitro, the profile of paxillin phosphorylation in vivo stimulated by seeding fibroblasts on fibronectin was characterized. As expected, plating cells on fibronectin enhanced the tyrosine phosphorylation of paxillin. However, 96% of the phosphorylation of paxillin occurred on serine residues. Comparison by two-dimensional phosphopeptide analyses indicated that the major sites of tyrosine and serine phosphorylation detected in the assays in vitro co-migrate with phosphopeptides derived from paxillin phosphorylated in vivo in response to plating cells on fibronectin. These findings support a role for both tyrosine and serine kinases in the signal transduction pathway linking integrin activation to paxillin phosphorylation. PMID:9230116

  11. 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

  12. Tyrosine Phosphorylation of Caspase-8 Abrogates Its Apoptotic Activity and Promotes Activation of c-Src

    PubMed Central

    Tsang, Jennifer LY; Jia, Song Hui; Parodo, Jean; Plant, Pamela; Lodyga, Monika; Charbonney, Emmanuel; Szaszi, Katalin; Kapus, Andras; Marshall, John C.

    2016-01-01

    Src family tyrosine kinases (SFKs) phosphorylate caspase-8A at tyrosine (Y) 397 resulting in suppression of apoptosis. In addition, the phosphorylation of caspase-8A at other sites including Y465 has been implicated in the regulation of caspase-8 activity. However, the functional consequences of these modifications on caspase-8 processing/activity have not been elucidated. Moreover, various Src substrates are known to act as potent Src regulators, but no such role has been explored for caspase-8. We asked whether the newly identified caspase-8 phosphorylation sites might regulate caspase-8 activation and conversely, whether caspase-8 phosphorylation might affect Src activity. Here we show that Src phosphorylates caspase-8A at multiple tyrosine sites; of these, we have focused on Y397 within the linker region and Y465 within the p12 subunit of caspase-8A. We show that phosphomimetic mutation of caspase-8A at Y465 prevents its cleavage and the subsequent activation of caspase-3 and suppresses apoptosis. Furthermore, simultaneous phosphomimetic mutation of caspase-8A at Y397 and Y465 promotes the phosphorylation of c-Src at Y416 and increases c-Src activity. Finally, we demonstrate that caspase-8 activity prevents its own tyrosine phosphorylation by Src. Together these data reveal that dual phosphorylation converts caspase-8 from a pro-apoptotic to a pro-survival mediator. Specifically, tyrosine phosphorylation by Src renders caspase-8 uncleavable and thereby inactive, and at the same time converts it to a Src activator. This novel dynamic interplay between Src and caspase-8 likely acts as a potent signal-integrating switch directing the cell towards apoptosis or survival. PMID:27101103

  13. Analysis of virulence factors of Helicobacter pylori isolated from a Vietnamese population

    PubMed Central

    2009-01-01

    Background The incidence of gastric cancer differs among countries in Asia, and it has been suggested that virulence factors associated with Helicobacter pylori are partly responsible. The aim of this study was to investigate several genetic factors regarded as virulence or molecular epidemiologic markers in H. pylori isolates from Vietnamese subjects. Results The cagA, vacA and cag right-end junction genotypes of 103 H. pylori strains from Vietnam (54 from Hanoi and 49 from Ho Chi Minh) were determined by PCR and sequencing. Three types of deletion in the region located upstream of the cagA Glu-Pro-Ile-Tyr-Ala (EPIYA) repeat region were identified: the 39-bp deletion type, the 18-bp deletion type, and the no-deletion type. The majority of strains studied (77%; 80/103) had the 18-bp deletion irrespective of geographical location in the country or clinical outcome. All of the 39-bp and 18-bp deletion-type strains possessed the East Asian type cagA repeat region. The type II cag right-end junction genotype was predominant (84%). The vacA m1 genotype was significantly more common in strains isolated in Hanoi, where the incidence of gastric cancer is higher, than in strains from Ho Chi Minh. Conclusion Pre-EPIYA-region typing of the cagA gene could provide a new genetic marker of H. pylori genomic diversity. Our data support the hypothesis that vacA m1 is closely associated with gastric carcinogenesis. PMID:19698173

  14. Ligand-independent tyrosine kinase signalling in RTH 149 trout hepatoma cells: comparison among heavy metals and pro-oxidants.

    PubMed

    Burlando, Bruno; Magnelli, Valeria; Panfoli, Isabella; Berti, Elena; Viarengo, Aldo

    2003-01-01

    Tyrosine phosphorylation depends on the activity of receptor and non-receptor tyrosine kinases and promote cell growth, differentiation and apoptosis. Different stressors are known to stimulate tyrosine kinase activities and this could explain a wide spectrum of effects that these agents produce on different organisms. We studied the effects of heavy metals and pro-oxidants on tyrosine kinase signalling in trout hepatoma cells (RTH 149) by Western immunoblotting. Use of antiphosphotyrosine showed that Hg(2+) and Cu(2+)in the microM range, and H(2)O(2) in the mM range, induced tyrosine phosphorylation. The effect of Cu(2+)was prevented by pre-incubation with genistein, while those of Hg(2+)and H(2)O(2) were only decreased, probably due to tyrosine kinase stimulation coupled to phosphatase inhibition. Phosphospecific antibodies against the three types of MAPKs showed that ERK is activated by heavy metals only, while p38 and SAPK/JNK are activated by H(2)O(2), Hg(2+), and Cu(2+) plus low H(2)O(2). Cell pre-incubation with p38 inhibitors indicated that ERK activation by H(2)O(2) is prevented by concomitant activation of p38. Phosphospecific STAT antibodies revealed activation by H(2)O(2) only. In conclusion, fish cell exposure to heavy metals and pro-oxidants produce specific tyrosine kinase responses, involving cross talk and redox modulatory effects. PMID:12876385

  15. Influence of the tyrosine environment on the second harmonic generation of iturinic antimicrobial lipopeptides at the air-water interface.

    PubMed

    Nasir, Mehmet Nail; Benichou, Emmanuel; Loison, Claire; Russier-Antoine, Isabelle; Besson, Françoise; Brevet, Pierre-François

    2013-12-01

    The second harmonic generation (SHG) response at the air-water interface from the tyrosine-containing natural iturinic cyclo-lipopeptides mycosubtilin, iturin A and bacillomycin D is reported. It is shown that this response is dominated by the single tyrosine residue present in these molecules owing to the large first hyperpolarizability arising from the non-centrosymmetric aromatic ring structure of this amino acid. The SHG response of these iturinic antibiotics is also compared to the response of surfactin, a cyclo-lipopeptide with a similar l,d-amino acid sequence but lacking a tyrosine residue, and PalmATA, a synthetic linear lipopeptide possessing a single tyrosine residue but lacking the amino acid sequence structuring the cycle of the iturinic antibiotics. From the light polarization analysis of the SHG response, it is shown that the tyrosine local environment is critical in defining the SHG response of these peptides at the air-water interface. Our results demonstrate that tyrosine, similar to tryptophan, can be used as an endogenous molecular probe of peptides and proteins for SHG at the air-water interface, paving the way for SHG studies of other tyrosine-containing bioactive molecules. PMID:24149982

  16. Identification of the flavoprotein of succinate dehydrogenase and aconitase as in vitro mitochondrial substrates of Fgr tyrosine kinase.

    PubMed

    Salvi, Mauro; Morrice, Nick A; Brunati, Anna Maria; Toninello, Antonio

    2007-12-11

    Overlooked until recently, mitochondrial protein phosphorylation is now emerging as a key post-translational mechanism in the regulation of mitochondrial functions. In particular, tyrosine phosphorylation represents a promising field to discover new mechanisms of bioenergetic regulation. Tyrosine kinases belonging to the Src kinase family have been observed in mitochondrial compartments, however their substrates are almost unknown. Here, we provide evidence that the flavoprotein of succinate dehydrogenase and aconitase are "in vitro" substrates of Fgr tyrosine kinase. Fgr phosphorylates flavoprotein of succinate dehydrogenase at Y535 and Y596 and aconitase at Y71, Y544 and Y665. The significance of these findings is discussed. PMID:17997986

  17. Inducible nitric oxide synthase (NOS2) expressed in septic patients is nitrated on selected tyrosine residues: implications for enzymic activity.

    PubMed Central

    Lanone, Sophie; Manivet, Philippe; Callebert, Jacques; Launay, Jean-Marie; Payen, Didier; Aubier, Michel; Boczkowski, Jorge; Mebazaa, Alexandre

    2002-01-01

    Tyrosine nitration is a post-translational protein modification with potentially significant biological implications. In the present study we demonstrate, for the first time, that tyrosine residues of human inducible nitric oxide synthase (NOS2) can be nitrated by peroxynitrite in vitro, leading to a decreased activity. Moreover, we show that NOS2 expressed in a skeletal muscle from septic patients is nitrated on selective tyrosine residues belonging to a canonic sequence. This phenomenon could be an endogenous mechanism of in vivo modulation of NOS2 enzymic activity. PMID:12097137

  18. Identification of targets of c-Src tyrosine kinase by chemical complementation and phosphoproteomics.

    PubMed

    Ferrando, Isabel Martinez; Chaerkady, Raghothama; Zhong, Jun; Molina, Henrik; Jacob, Harrys K C; Herbst-Robinson, Katie; Dancy, Beverley M; Katju, Vikram; Bose, Ron; Zhang, Jin; Pandey, Akhilesh; Cole, Philip A

    2012-08-01

    The cellular proto-oncogene c-Src is a nonreceptor tyrosine kinase involved in cell growth and cytoskeletal regulation. Despite being dysregulated in a variety of human cancers, its precise functions are not fully understood. Identification of the substrates of c-Src remains a major challenge, because there is no simple way to directly stimulate its activity. Here we combine the chemical rescue of mutant c-Src and global quantitative phosphoproteomics to obtain the first high resolution snapshot of the range of tyrosine phosphorylation events that occur in the cell immediately after specific c-Src stimulation. After enrichment by anti-phosphotyrosine antibodies, we identified 29 potential novel c-Src substrate proteins. Tyrosine phosphopeptide mapping allowed the identification of 382 nonredundant tyrosine phosphopeptides on 213 phosphoproteins. Stable isotope labeling of amino acids in cell culture-based quantitation allowed the detection of 97 nonredundant tyrosine phosphopeptides whose level of phosphorylation is increased by c-Src. A large number of previously uncharacterized c-Src putative protein targets and phosphorylation sites are presented here, a majority of which play key roles in signaling and cytoskeletal networks, particularly in cell adhesion. Integrin signaling and focal adhesion kinase signaling pathway are two of the most altered pathways upon c-Src activation through chemical rescue. In this context, our study revealed the temporal connection between c-Src activation and the GTPase Rap1, known to stimulate integrin-dependent adhesion. Chemical rescue of c-Src provided a tool to dissect the spatiotemporal mechanism of activation of the Rap1 guanine exchange factor, C3G, one of the identified potential c-Src substrates that plays a role in focal adhesion signaling. In addition to unveiling the role of c-Src in the cell and, specifically, in the Crk-C3G-Rap1 pathway, these results exemplify a strategy for obtaining a comprehensive understanding of

  19. Effects of L-tyrosine and carbohydrate ingestion on endurance exercise performance.

    PubMed

    Chinevere, Troy D; Sawyer, Robert D; Creer, Andrew R; Conlee, Robert K; Parcell, Allen C

    2002-11-01

    To test the effects of tyrosine ingestion with or without carbohydrate supplementation on endurance performance, nine competitive cyclists cycled at 70% peak oxygen uptake for 90 min under four different feeding conditions followed immediately by a time trial. At 30-min intervals, beginning 60 min before exercise, each subject consumed either 5 ml/kg body wt of water sweetened with aspartame [placebo (Pla)], polydextrose (70 g/l) (CHO), L-tyrosine (25 mg/kg body wt) (Tyr), or polydextrose (70 g/l) and L-tyrosine (25 mg/kg body wt) (CHO+Tyr). The experimental trials were given in random order and were carried out by using a counterbalanced double-blind design. No differences were found between treatments for oxygen uptake, heart rate, or rating of perceived exertion at any time during the 90-min ride. Plasma tyrosine rose significantly from 60 min before exercise to test termination (TT) in Tyr (means +/- SE) (480 +/- 26 micromol) and CHO+Tyr (463 +/- 34 micromol) and was significantly higher in these groups from 30 min before exercise to TT vs. CHO (90 +/- 3 micromol) and Pla (111 +/- 7 micromol) (P < 0.05). Plasma free tryptophan was higher after 90 min of exercise, 15 min into the endurance time trial, and at TT in Tyr (10.1 +/- 0.9, 10.4 +/- 0.8, and 12.0 +/- 0.9 micromol, respectively) and Pla (9.7 +/- 0.5, 10.0 +/- 0.3, and 11.7 +/- 0.5 micromol, respectively) vs. CHO (7.8 +/- 0.5, 8.6 +/- 0.5, and 9.3 +/- 0.6 micromol, respectively) and CHO+Tyr (7.8 +/- 0.5, 8.5 +/- 0.5, 9.4 +/- 0.5 micromol, respectively) (P < 0.05). The plasma tyrosine-to-free tryptophan ratio was significantly higher in Tyr and CHO+Tyr vs. CHO and Pla from 30 min before exercise to TT (P < 0.05). CHO (27.1 +/- 0.9 min) and CHO+Tyr (26.1 +/- 1.1 min) treatments resulted in a reduced time to complete the endurance time trial compared with Pla (34.4 +/- 2.9 min) and Tyr (32.6 +/- 3.0 min) (P < 0.05). These findings demonstrate that tyrosine ingestion did not enhance performance during a

  20. Optimization of tyrosine-derived polycarbonate terpolymers for bone regeneration scaffolds

    NASA Astrophysics Data System (ADS)

    Resurreccion-Magno, Maria Hanshella C.

    Tyrosine-derived polycarbonates (TyrPC) are a versatile class of polymers highly suitable for bone tissue engineering. Among the tyrosine-derived polycarbonates, poly(DTE carbonate) has an FDA masterfile that documents its biocompatibility and non-toxicity and has shown potential utility in orthopedics due to its osteoconductive properties and strength. DTE stands for desaminotyrosyl-tyrosine ethyl ester and is the most commonly used tyrosine-derived monomer. However, in vitro degradation studies showed that poly(DTE carbonate) did not completely resorb even after four years of incubation in phosphate buffered saline. Thus for bone regeneration, which only requires a temporary implant until the bone heals, poly(DTE carbonate) would not be the best choice. The goal of the present research was to optimize a scaffold composition for bone regeneration that is based on desaminotyrosyl-tyrosine alkyl ester (DTR), desaminotyrosyl-tyrosine (DT) and poly(ethylene glycol) (PEG). Five areas of research were presented: (1) synthesis and characterization of a focused library of TyrPC terpolymers; (2) evaluation of the effects of how small changes on the composition affected the mechanism and kinetics of polymer degradation and erosion; (3) fabrication of bioactive three-dimensional porous scaffold constructs for bone regeneration; (4) assessment of osteogenic properties in vitro using pre-osteoblasts; and (5) evaluation of bone regeneration potential, with or without recombinant human bone morphogenetic protein-2 (rhBMP-2), in vivo using a critical sized defect (CSD) rabbit calvaria (cranium) model. Small changes in the composition, such as changing the R group of DTR from ethyl to methyl, varying the mole percentages of DT and PEG, and using a different PEG block length, affected the overall properties of these polymers. Porous scaffolds were prepared by a combination of solvent casting, porogen leaching and phase separation techniques. Calcium phosphate was coated on the

  1. Tyrosine analogues for probing proton-coupled electron transfer processes in peptides and proteins.

    PubMed

    Nara, Susheel J; Valgimigli, Luca; Pedulli, Gian Franco; Pratt, Derek A

    2010-01-20

    A series of amino acids analogous to tyrosine, but differing in the physicochemical properties of the aryl alcohol side chain, have been prepared and characterized. These compounds are expected to be useful in understanding the relationships between structure, thermodynamics, and kinetics in long-range proton-coupled electron transfer processes in peptides and proteins. Systematic changes in the acidity, redox potential, and O-H bond strength of the tyrosine side chain could be induced upon substituting the phenol for pyridinol and pyrimidinol moieties. Further modulation was possible by introducing methyl and t-butyl substitution in the position ortho to the phenolic hydroxyl. The unnatural amino acids were prepared by Pd-catalyzed cross-coupling of the corresponding halogenated aryl alcohol protected as their benzyl ethers with an organozinc reagent derived from N-Boc L-serine carboxymethyl ester. Subsequent debenzylation by catalytic hydrogenation yielded the tyrosine analogues in good yield. Spectrophotometric titrations revealed a decrease in tyrosine pK(a) of ca. 1.5 log units per included nitrogen atom, along with a corresponding increase in the oxidation (peak) potentials of ca. 200 mV, respectively. All told, the six novel amino acids described here have phenol-like side chains with pK(a)'s that span a range of 7.0 to greater than 10, and an oxidation (peak) potential range of greater than 600 mV at and around physiological pH. Radical equilibration EPR experiments were carried out to reveal that the O-H bond strengths increase systematically upon nitrogen incorporation (by ca. 0.5-1.0 kcal/mol), and radical stability and persistence increase systematically upon introduction of alkyl substitution in the ortho positions. The EPR spectra of the aryloxyl radicals derived from tyrosine and each of the analogues could be determined at room temperature, and each featured distinct spectral properties. The uniqueness of their spectra will be helpful in discerning

  2. Rhizobiales-like Phosphatase 2 from Arabidopsis thaliana Is a Novel Phospho-tyrosine-specific Phospho-protein Phosphatase (PPP) Family Protein Phosphatase.

    PubMed

    Uhrig, R Glen; Labandera, Anne-Marie; Muhammad, Jamshed; Samuel, Marcus; Moorhead, Greg B

    2016-03-11

    Cellular signaling through protein tyrosine phosphorylation is well established in mammalian cells. Although lacking the classic tyrosine kinases present in humans, plants have a tyrosine phospho-proteome that rivals human cells. Here we report a novel plant tyrosine phosphatase from Arabidopsis thaliana (AtRLPH2) that, surprisingly, has the sequence hallmarks of a phospho-serine/threonine phosphatase belonging to the PPP family. Rhizobiales/Rhodobacterales/Rhodospirillaceae-like phosphatases (RLPHs) are conserved in plants and several other eukaryotes, but not in animals. We demonstrate that AtRLPH2 is localized to the plant cell cytosol, is resistant to the classic serine/threonine phosphatase inhibitors okadaic acid and microcystin, but is inhibited by the tyrosine phosphatase inhibitor orthovanadate and is particularly sensitive to inhibition by the adenylates, ATP and ADP. AtRLPH2 displays remarkable selectivity toward tyrosine-phosphorylated peptides versus serine/threonine phospho-peptides and readily dephosphorylates a classic tyrosine phosphatase protein substrate, suggesting that in vivo it is a tyrosine phosphatase. To date, only one other tyrosine phosphatase is known in plants; thus AtRLPH2 represents one of the missing pieces in the plant tyrosine phosphatase repertoire and supports the concept of protein tyrosine phosphorylation as a key regulatory event in plants. PMID:26742850

  3. Solution interactions between the uranyl cation [UO2(2+)] and histidine, N-acetyl-histidine, tyrosine, and N-acetyl-tyrosine.

    PubMed

    Xie, Wei; Badawi, Ahmed; Huang, Huan; Van Horn, J David

    2009-01-01

    Complexes of the uranyl cation [UO(2)(2+)] with histidine (His), N-acetyl-histidine (NAH), tyrosine (Tyr), and N-acetyl-tyrosine (NAT) were studied by UV-visible and NMR spectroscopy, and by potentiometric titration. Protonation constants for each ligand are reported, as are cumulative formation constants for uranyl-amino acid complexes. Coupling constant data (J(CH)) for uranyl-histidine complexes indicate that inner-sphere solution interactions between histidine and uranyl cation are solely at the carboxylate site. At 25 degrees C the major uranyl-histidine complex has a cumulative formation constant of logbeta(110)=8.53, and a proposed formula of [UO(2)HisH(2)(OH)(2)](+); the stepwise formation constant, logK(UL), is estimated to be 5.6 ( approximately 8.53-(-6.1)-(-6.1)-15.15). Outer-sphere interactions, H-bonding or electrostatic interactions, are proposed as contributing a significant portion of the stability to the ternary uranyl-hydroxo-amino acid complexes. The temperature dependent protonation constants of histidine and formation constants between uranyl cation and histidine are reported from 10 to 35 degrees C; at 25 degrees C, DeltaG=-43.3 kJ/mol. PMID:18947879

  4. Protein Tyrosine Phosphatase N2 Is a Positive Regulator of Lipopolysaccharide Signaling in Raw264.7 Cell through Derepression of Src Tyrosine Kinase.

    PubMed

    Ha Thi, Huyen Trang; Choi, Seo-Won; Kim, Young-Mi; Kim, Hye-Youn; Hong, Suntaek

    2016-01-01

    T cell protein tyrosine phosphatase N2 (PTPN2) is a phosphotyrosine-specific nonreceptor phosphatase and is ubiquitously expressed in tissues. Although PTPN2 functions as an important regulator in different signaling pathways, it is still unclear what is specific target protein of PTPN2 and how is regulated in lipopolysaccharide (LPS)-induced inflammatory signaling pathway. Here, we found that PTPN2 deficiency downregulated the expression of LPS-mediated pro-inflammtory cytokine genes. Conversely, overexpression of PTPN2 in Raw264.7 cells enhanced the expression and secretion of those cytokines. The activation of MAPK and NF-κB signaling pathways by LPS was reduced in PTPN2-knockdowned cells and ectopic expression of PTPN2 reversed these effects. Furthermore, we found that PTNP2 directly interacted with Src and removed the inhibitory Tyr527 phosphorylation of Src to enhance the activatory phosphorylation of Tyr416 residue. These results suggested that PTPN2 is a positive regulator of LPS-induced inflammatory response by enhancing the activity of Src through targeting the inhibitory phosphor-tyrosine527 of Src. PMID:27611995

  5. Dephosphorylation of Tyrosine 393 in Argonaute 2 by Protein Tyrosine Phosphatase 1B Regulates Gene Silencing in Oncogenic RAS-Induced Senescence

    PubMed Central

    Yang, Ming; Haase, Astrid D.; Huang, Fang-Ke; Coulis, Gérald; Rivera, Keith D.; Dickinson, Bryan C.; Chang, Christopher J.; Pappin, Darryl J.; Neubert, Thomas A.; Hannon, Gregory J.; Boivin, Benoit; Tonks, Nicholas K.

    2014-01-01

    SUMMARY Oncogenic RAS (H-RASV12) induces premature senescence in primary cells by triggering production of reactive oxygen species (ROS), but the molecular role of ROS in senescence remains elusive. We investigated whether inhibition of protein tyrosine phosphatases by ROS contributed to H-RASV12-induced senescence. We identified protein tyrosine phosphatase 1B (PTP1B) as a major target of H-RASV12-induced ROS. Inactivation of PTP1B was necessary and sufficient to induce premature senescence in H-RASV12-expressing IMR90 fibroblasts. We identified phospho-Tyr 393 of argonaute 2 (AGO2) as a direct substrate of PTP1B. Phosphorylation of AGO2 at Tyr 393 inhibited loading with microRNAs (miRNA) and thus miRNA-mediated gene silencing, which counteracted the function of H-RASV12-induced oncogenic miRNAs. Overall, our data illustrate that premature senescence in H-RASV12-transformed primary cells is a consequence of oxidative inactivation of PTP1B and inhibition of miRNA-mediated gene silencing. PMID:25175024

  6. Discovery and enantiocontrol of axially chiral urazoles via organocatalytic tyrosine click reaction

    PubMed Central

    Zhang, Ji-Wei; Xu, Jin-Hui; Cheng, Dao-Juan; Shi, Chuan; Liu, Xin-Yuan; Tan, Bin

    2016-01-01

    Axially chiral compounds play an important role in areas such as asymmetric catalysis. The tyrosine click-like reaction is an efficient approach for synthesis of urazoles with potential applications in pharmaceutical and asymmetric catalysis. Here we discover a class of urazole with axial chirality by restricted rotation around an N–Ar bond. By using bifunctional organocatalyst, we successfully develop an organocatalytic asymmetric tyrosine click-like reaction in high yields with excellent enantioselectivity under mild reaction conditions. The excellent remote enantiocontrol of the strategy originates from the efficient discrimination of the two reactive sites in the triazoledione and transferring the stereochemical information of the catalyst into the axial chirality of urazoles at the remote position far from the reactive site. PMID:26864510

  7. Synthesis of Chondramide A Analogues with Modified β-Tyrosine and Their Biological Evaluation

    PubMed Central

    Zhdanko, Alexander; Schmauder, Anke; Ma, Christopher I.; Sibley, L. David; Sept, D. David; Sasse, Florenz

    2011-01-01

    Starting from cinnamates 9, obtained by Wittig reaction or Heck coupling, the diols 17 were prepared by asymmetric dihydroxylation. This was followed by a regioselective substitution of the 3-OH group with hydrazoic acid under Mitsunobu conditions. Methylation of the 2-OH group and reduction of the azide group led to the β-tyrosine derivatives 8. Condensation with the dipeptide acid 6 furnished the tripeptide part of the chondramides. The derived acids 21 were combined with the hydroxy ester 7 to the esters 22. Cleavage of the tert-butyl groups and intramolecular lactam formation gave rise to the chondramide A analogues 2b–k. Growth inhibition assays showed most of the analogues to be biologically active. Some of them even reach the activity of jasplakinolide. It can be concluded that the 4-position of the aryl ring in the β-tyrosine of chondramide A tolerates structural modifications quite well. PMID:22012705

  8. Tyrosine binding and promiscuity in the arginine repressor from the pathogenic bacterium Corynebacterium pseudotuberculosis.

    PubMed

    Mariutti, Ricardo Barros; Ullah, Anwar; Araujo, Gabriela Campos; Murakami, Mario Tyago; Arni, Raghuvir Krishnaswamy

    2016-07-01

    The arginine repressor (ArgR) regulates arginine biosynthesis in a number of microorganisms and consists of two domains interlinked by a short peptide; the N-terminal domain is involved in DNA binding and the C-terminal domain binds arginine and forms a hexamer made-up of a dimer of trimers. The crystal structure of the C-terminal domain of ArgR from the pathogenic Corynebacterium pseudotuberculosis determined at 1.9 Å resolution contains a tightly bound tyrosine at the arginine-binding site indicating hitherto unobserved promiscuity. Structural analysis of the binding pocket displays clear molecular adaptations to accommodate tyrosine binding suggesting the possible existence of an alternative regulatory process in this pathogenic bacterium. PMID:27233609

  9. Novel morpholin-3-one fused quinazoline derivatives as EGFR tyrosine kinase inhibitors.

    PubMed

    Qin, Xuemei; Lv, Yongjuan; Liu, Peng; Li, Zhipeng; Hu, Liming; Zeng, Chengchu; Yang, Leifu

    2016-03-15

    A series of novel morpholin-3-one-fused quinazoline derivatives were designed, synthesized and evaluated as EGFR tyrosine kinase inhibitors. Nineteen compounds showed significant inhibitory activities against EGFR(wt) kinase (IC50<1 μM). Compound a8 demonstrated the most potent inhibitory activity toward EGFR(wt) (IC50=53.1 nM). Compound a7 and a8 showed excellent inhibitory activities against mutant EGFR(T790M/L858R) and strong antiproliferative activity against H358 and A549 cell lines. Finally, molecular docking studies were performed to predict the possible binding mode of the target compounds. It is believed that this work would be very useful for designing a new series of tyrosine kinase inhibitors targeting EGFR. PMID:26879314

  10. LACK OF PROTEIN-TYROSINE SULFATION DISRUPTS PHOTORECEPTOR OUTER SEGMENT MORPHOGENESIS, RETINAL FUNCTION AND RETINAL ANATOMY

    PubMed Central

    Sherry, David M.; Murray, Anne R.; Kanan, Yogita; Arbogast, Kelsey L.; Hamilton, Robert A.; Fliesler, Steven J.; Burns, Marie E.; Moore, Kevin L.; Al-Ubaidi, Muayyad R.

    2010-01-01

    To investigate the role(s) of protein-tyrosine sulfation in the retina, we examined retinal function and structure in mice lacking tyrosylprotein sulfotransferases (TPST) 1 and 2. Tpst double knockout (DKO; Tpst1−/−/Tpst2−/−) retinas had drastically reduced electroretinographic responses, although their photoreceptors exhibited normal responses in single cell recordings. These retinas appeared normal histologically; however, the rod photoreceptors had ultrastructurally abnormal outer segments, with membrane evulsions into the extracellular space, irregular disc membrane spacing, and expanded intradiscal space. Photoreceptor synaptic terminals were disorganized in Tpst DKO retinas, but established ultrastructurally normal synapses, as did bipolar and amacrine cells; however, the morphology and organization of neuronal processes in the inner retina were abnormal. These results indicate that protein-tyrosine sulfation is essential for proper outer segment morphogenesis and synaptic function, but is not critical for overall retinal structure or synapse formation, and may serve broader functions in neuronal development and maintenance. PMID:21039965

  11. Targeting Bruton's tyrosine kinase signaling as an emerging therapeutic agent of B-cell malignancies

    PubMed Central

    XIA, BING; QU, FULIAN; YUAN, TIAN; ZHANG, YIZHUO

    2015-01-01

    It is becoming increasingly evident that B-cell receptor (BCR) signaling is central to the development and function of B cells. BCR signaling has emerged as a pivotal pathway and a key driver of numerous B-cell lymphomas. Disruption of BCR signaling can be lethal to malignant B cells. Recently, kinase inhibitors that target BCR signaling have induced notable clinical responses. These inhibitors include spleen tyrosine kinase, mammalian target of rapamycin, phosphoinositide 3′-kinase and Bruton's tyrosine kinase (BTK). Ibrutinib, an oral irreversible BTK inhibitor, has emerged as a promising targeted therapy for patients with B-cell malignancies. The present review discusses the current understanding of BTK-mediated BCR signaling in the biology and pathobiology of normal and malignant B cells, and the cellular interaction with the tumor microenvironment. The data on ibrutinib in the preclinical and clinical settings is also discussed, and perspectives for the future use of ibrutinib are outlined. PMID:26788133

  12. [Possibilities for inhibiting tumor-induced angiogenesis: results with multi-target tyrosine kinase inhibitors].

    PubMed

    Török, Szilvia; Döme, Balázs

    2012-03-01

    Functional blood vasculature is essential for tumor progression. The main signalization pathways that play a key role in the survival and growth of tumor vessels originate from the VEGF-, PDGF- and FGF tyrosine kinase receptors. In the past decade, significant results have been published on receptor tyrosine kinase inhibitors (RTKIs). In this paper, the mechanisms of action and the results so far available of experimental and clinical studies on multi-target antiangiogenic TKIs are discussed. On the one hand, notable achievements have been made recently and these drugs are already used in clinical practice in some patient populations. On the other hand, the optimal combination and dosage of these drugs, selection of the apropriate biomarker and better understanding of the conflicting role of PDGFR and FGFR signaling in angiogenesis remain future challenges. PMID:22403757

  13. How tyrosine kinase inhibitors impair metabolism and endocrine system function: a systematic updated review.

    PubMed

    Breccia, Massimo; Molica, Matteo; Alimena, Giuliana

    2014-12-01

    Tyrosine kinase inhibitors (TKIs) advent has deeply changed the outcome of chronic myeloid leukemia (CML) patients, with improved rates of response and overall survival. However, for this success some patients paid the price of a number of peculiar side effects, the so-called off-target side effects, specific for each one TKI. These effects are due to non-selective inhibition of other tyrosine kinase receptors, such as PDGFR, c-KIT, Src, VEGF. Consequences of this inhibition, some metabolic changes during the treatment with TKIs are reported. Aim of present review is to report metabolic changes and potential mechanisms involved in the pathogenesis related to imatinib, second (nilotinib and dasatinib) and third generation (bosutinib and ponatinib) TKIs. PMID:25449685

  14. Epidermal growth factor receptor tyrosine kinase inhibitors for non-small cell lung cancer

    PubMed Central

    Asami, Kazuhiro; Atagi, Shinji

    2014-01-01

    First-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), including gefitinib and erlotinib, have proven to be highly effective agents for advanced non-small cell lung cancer (NSCLC) in patients harboring an activating EGFR mutation such as the exon 19 deletion mutation and L858R. Although those reversible small molecular targeted agents provide a significant response and survival benefit, all responders eventually acquire resistance. Second-generation EGFR-targeting agents, such as irreversible EGFR/HER2 tyrosine kinase inhibitors and pan-HER TKIs, may improve survival further and be useful for patients who acquired resistance to first-generation EGFR-TKIs. This review discusses novel therapeutic strategies for EGFR-mutated advanced NSCLC using first- and second-generation EGFR-TKIs. PMID:25302168

  15. Chromosomal location of the Syk and ZAP-70 tyrosine kinase genes in mice and humans

    SciTech Connect

    Ku, G.; Malissen, B.; Mattei, M.G.

    1994-12-31

    Several protein tyrosine kinases (PTKs), which may be grouped into two structurally different families, have been implicated in antigen receptor proximal signaling. Blk, Fyn, Lck, Lyn, and Yes belong to the Src-family kinases, whereas the spleen tyrosine kinase (Syk) and the CD3-{zeta}-associated PTK (ZAP-70) define a new one, the Syk family. These kinases differ from the Src-family kinases in that they are non-myristylated cytoplasmic polypeptides composed of two N-terminal Src homology-2 (SH2) domains and a C-terminal catalytic domain. ZAP-70 appears to be expressed exclusively in T cells and NK cells, whereas Syk is preferentially expressed in B cells, T cells, and myeloid cells. 15 refs., 2 figs.

  16. Expression, purification, and characterization of human osteoclastic protein-tyrosine phosphatase catalytic domain in Escherichia coli.

    PubMed

    Jiang, Huan; Sui, Yuan; Cui, Yue; Lin, Peng; Li, Wannan; Xing, Shu; Wang, Deli; Hu, Min; Fu, Xueqi

    2015-03-01

    Osteoclastic protein tyrosine phosphatase (PTP-oc) is a structurally unique transmembrane protein tyrosine phosphatase (PTP) that contains only a relatively small intracellular PTP catalytic domain, does not have an extracellular domain, and lacks a signal peptide proximal to the NH2 terminus. The present study reports the expression, purification, and characterization of the intracellular catalytic domain of PTP-oc (ΔPTP-oc). ΔPTP-oc was expressed in Escherichia coli cells as a fusion with a six-histidine tag and was purified via nickel affinity chromatography. When with para-nitrophenylphosphate (p-NPP) as a substrate, ΔPTP-oc exhibited classical Michaelis-Menten kinetics. Its responses to temperature and ionic strength were similar to those of other PTPs. The optimal pH value of ΔPTP-oc is approximately 7.0, unlike other PTPs, whose optimal pH values are approximately 5.0. PMID:25462809

  17. Protein tyrosine phosphatase variants in human hereditary disorders and disease susceptibilities.

    PubMed

    Hendriks, Wiljan J A J; Pulido, Rafael

    2013-10-01

    Reversible tyrosine phosphorylation of proteins is a key regulatory mechanism to steer normal development and physiological functioning of multicellular organisms. Phosphotyrosine dephosphorylation is exerted by members of the super-family of protein tyrosine phosphatase (PTP) enzymes and many play such essential roles that a wide variety of hereditary disorders and disease susceptibilities in man are caused by PTP alleles. More than two decades of PTP research has resulted in a collection of PTP genetic variants with corresponding consequences at the molecular, cellular and physiological level. Here we present a comprehensive overview of these PTP gene variants that have been linked to disease states in man. Although the findings have direct bearing for disease diagnostics and for research on disease etiology, more work is necessary to translate this into therapies that alleviate the burden of these hereditary disorders and disease susceptibilities in man. PMID:23707412

  18. Tyrosine 110 in the measles virus phosphoprotein is required to block STAT1 phosphorylation

    SciTech Connect

    Devaux, Patricia; Messling, Veronika von; Songsungthong, Warangkhana; Springfeld, Christoph; Cattaneo, Roberto . E-mail: cattaneo.roberto@mayo.edu

    2007-03-30

    The measles virus (MV) P gene encodes three proteins: P, an essential polymerase cofactor, and C and V, which have multiple functions including immune evasion. We show here that the MV P protein also contributes to immune evasion, and that tyrosine 110 is required to block nuclear translocation of the signal transducer and activator of transcription factors (STAT) after interferon type I treatment. In particular, MV P inhibits STAT1 phosphorylation. This is shown not only by transient expression but also by reverse genetic analyses based on a new functional infectious cDNA derived from a MV vaccine vial (Moraten strain). Our study also identifies a conserved sequence around P protein tyrosine 110 as a candidate interaction site with a cellular protein.

  19. Ly-6A is required for T cell receptor expression and protein tyrosine kinase fyn activity.

    PubMed Central

    Lee, S K; Su, B; Maher, S E; Bothwell, A L

    1994-01-01

    To characterize the function of the Ly-6A antigen in T cell activation, antisense Ly-6 RNA was expressed in a stably transfected antigen-specific T cell clone. Reduced Ly-6A expression results in inhibition of responses to antigen, anti-TCR (anti-T cell receptor) crosslinking and concanavalin A plus recombinant interleukin 1 and causes impairment of in vitro fyn tyrosine kinase activity. More substantial reduction of Ly-6A results in reduction of TCR expression. Analysis of mRNA species indicates that the reduction is specific for the TCR beta chain. These data demonstrate that Ly-6A may regulate TCR expression and may be involved in early events of T cell activation via regulation of fyn tyrosine kinase activity. Images PMID:8187770

  20. Participation of cysteine and cystine in inactivation of tyrosine aminotransferase in rat liver homogenates.

    PubMed Central

    Buckley, W T; Milligan, L P

    1978-01-01

    1. Inactivation of tyrosine aminotransferase was studied in rat liver homogenates. Under an O2 atmosphere with cysteine added, inactivation was rapid after a lag period of approx. 1h, whereas a N2 atmosphere extended the lag period to approx. 3h. 2. Replacement of cysteine with cystine resulted in rapid inactivation both aerobically and anaerobically. 3. Removal of the particulate fraction by centrifuging rat liver homogenates at 13,000g for 9min resulted in an aerobic lag period of 0.5h in the presence of cystine and approx. 3h in the presence of cysteine. 4. It is proposed that the stimulatory effect of cysteine on tyrosine aminotransferase inactivation occurs largely as a result of oxidation to cystine, which appears to be a more directly effective agent. PMID:33669

  1. Spatial and Temporal Regulation of Receptor Tyrosine Kinase Activation and Intracellular Signal Transduction.

    PubMed

    Bergeron, John J M; Di Guglielmo, Gianni M; Dahan, Sophie; Dominguez, Michel; Posner, Barry I

    2016-06-01

    Epidermal growth factor (EGF) and insulin receptor tyrosine kinases (RTKs) exemplify how receptor location is coupled to signal transduction. Extracellular binding of ligands to these RTKs triggers their concentration into vesicles that bud off from the cell surface to generate intracellular signaling endosomes. On the exposed cytosolic surface of these endosomes, RTK autophosphorylation selects the downstream signaling proteins and lipids to effect growth factor and polypeptide hormone action. This selection is followed by the recruitment of protein tyrosine phosphatases that inactivate the RTKs and deliver them by membrane fusion and fission to late endosomes. Coincidentally, proteinases inside the endosome cleave the EGF and insulin ligands. Subsequent inward budding of the endosomal membrane generates multivesicular endosomes. Fusion with lysosomes then results in RTK degradation and downregulation. Through the spatial positioning of RTKs in target cells for EGF and insulin action, the temporal extent of signaling, attenuation, and downregulation is regulated. PMID:27023845

  2. Mushroom tyrosinase oxidizes tyrosine-rich sequences to allow selective protein functionalization.

    PubMed

    Long, Marcus J C; Hedstrom, Lizbeth

    2012-08-13

    We show that mushroom tyrosinase catalyzes the formation of reactive o-quinones on unstructured, tyrosine-rich sequences such as hemagglutinin (HA) tags (YPYDVPDYA). In the absence of exogenous nucleophiles and at low protein concentrations, the o-quinone decomposes with fragmentation of the HA tag. At higher protein concentrations (>5 mg mL⁻¹), crosslinking is observed. Besthorn's reagent intercepts the o-quinone to give a characteristic pink complex that can be observed directly on a denaturing SDS-PAGE gel. Similar labeled species can be formed by using other nucleophiles such as Cy5-hydrazide. These reactions are selective for proteins bearing HA and other unstructured poly-tyrosine-containing tags and can be performed in lysates to create specifically tagged proteins. PMID:22807021

  3. Analysis of the Tyrosine Kinome in Melanoma Reveals Recurrent Mutations in ERBB4

    PubMed Central

    Prickett, Todd D.; Agrawal, Neena S.; Wei, Xiaomu; Yates, Kristin E.; Lin, Jimmy C.; Wunderlich, John; Cronin, Julia C.; Cruz, Pedro; Rosenberg, Steven A.; Samuels, Yardena

    2010-01-01

    Tyrosine phosphorylation is important in signaling pathways underlying tumorigenesis. A mutational analysis of the Protein Tyrosine Kinase (PTK) gene family in cutaneous metastatic melanoma identified 30 somatic mutations in the kinase domain of 19 PTKs. The whole of the coding region of these 19 PTKs was further evaluated for somatic mutations in a total of 79 melanoma samples. This analysis revealed novel ERBB4 mutations in 19% of melanoma patients and that an additional two kinases (FLT1 and PTK2B) are mutated in 10% of melanomas. Seven missense mutations in the most commonly altered PTK (ERBB4) were examined and found to increase kinase activity and transformation ability. Melanoma cells expressing mutant ERBB4 had reduced cell growth after shRNA–mediated knockdown of ERBB4 or treatment with the ERBB inhibitor lapatinib. These studies might lead to personalized therapeutics specifically targeting the kinases that are mutationally altered in individual melanomas. PMID:19718025

  4. Tyrosine administration enhances dopamine synthesis and release in light-activated rat retina

    NASA Technical Reports Server (NTRS)

    Gibson, C. J.; Watkins, C. J.; Wurtman, R. J.

    1983-01-01

    Exposure of dark-adapted albino rats to light (350 lux) significantly elevated retinal levels of the dopamine metabolite dihydroxyphenyl acetic acid during the next hour; their return to a dark environment caused dihydroxyphenyl acetic acid levels to fall. Retinal dopamine levels were increased slightly by light exposure, suggesting that the increase in dihydroxyphenyl acetic acid reflected accelerated dopamine synthesis. Administration of tyrosine (100 mg/kg, i.p.) further elevated retinal dihydroxyphenyl acetic acid among light-exposed animals, but failed to affect dopamine release among animals in the dark. These observations show that a physiological stimulus - light exposure - can cause catecholaminergic neurons to become tyrosine-dependent; they also suggest that food consumption may affect neurotransmitter release within the retina.

  5. Tyrosine Kinase Inhibitors and Vascular Toxicity: Impetus for a Classification System?

    PubMed

    Herrmann, Joerg

    2016-06-01

    The introduction of molecularly targeted therapies with tyrosine kinase inhibitors has revolutionized cancer therapy and has contributed to a steady decline in cancer-related mortality since the late 1990s. However, not only cardiac but also vascular toxicity has been reported for these agents, some as expected on-target effects (e.g., VEGF receptor inhibitors) and others as unanticipated events (e.g., BCR-Abl inhibitors). A sound understanding of these cardiovascular toxic effects is critical to advance mechanistic insight into vascular disease and clinical care. From a conceptual standpoint, there might be value in defining type I (permanent) and type II (transient) vascular toxicity. This review will focus on the tyrosine kinase inhibitors in current clinical use and their associated vascular side effects. PMID:27099141

  6. Disruption of striatal-enriched protein tyrosine phosphatase (STEP) function in neuropsychiatric disorders

    PubMed Central

    Karasawa, Takatoshi; Lombroso, Paul J.

    2014-01-01

    Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific tyrosine phosphatase that plays a major role in the development of synaptic plasticity. Recent findings have implicated STEP in several psychiatric and neurological disorders, including Alzheimer’s disease, schizophrenia, fragile X syndrome, Huntington’s disease, stroke/ischemia, and stress-related psychiatric disorders. In these disorders, STEP protein expression levels and activity are dysregulated, contributing to the cognitive deficits that are present. In this review, we focus on the most recent findings on STEP, discuss how STEP expression and activity are maintained during normal cognitive function, and how disruptions in STEP activity contribute to a number of illnesses. PMID:25218562

  7. Family of receptor-linked protein tyrosine phosphatases in humans and Drosophila

    SciTech Connect

    Streuli, M.; Krueger, N.X.; Saito, H. ); Tsai, A.Y.M. )

    1989-11-01

    To understand the regulation of cell proliferation by tyrosine phosphorylation, characterization of protein tyrosine phosphatases is essential. The human genes LCA (leukocyte common antigen) and LAR encode putative receptor-linked PTPases. By using consensus sequence probes, two additional receptor-linked PTPase genes, DLAR and DPTP, were isolated from Drosophila melanogaster. The extra-cellular segments of both DLAR and DPTP are composed of multiple immunoglobulin-like domains and fibronectin type III-like domains. The cytoplasmic region of DLAR and DPTP, as well as human LCA and LAR, are composed of two tandemly repeated PTPase domains. PTPase activities of immunoprecipitated LCA and LAR were demonstrated by measuring the release of phosphate from a {sup 32}P-labeled (Tyr(P))peptide. Furthermore, the cytoplasmic domains of LCA, LAR, DLAR, and DPTP, expressed in Escherichia coli, have PTPase activity. Site-directed mutagenesis showed that a conserved cysteine residue is essential for PTPase activity.

  8. Mushroom Tyrosinase Oxidizes Tyrosine-rich Sequences, Allowing Selective Protein Functionalization

    PubMed Central

    Long, Marcus J. C.

    2012-01-01

    We show that mushroom tyrosinase catalyzes formation of reactive o-quinones on unstructured, tyrosine-rich sequences such as hemagglutinin (HA)-tags (YPYDVPDYA). In the absence of exogenous nucleophiles and at low protein concentrations, the o-quinone decomposes with fragmentation of the HA-tag. At higher protein concentrations (>5 mg/ml), cross-linking is observed. Besthorn’s reagent intercepts the o-quinone to give a characteristic pink complex, which can be observed directly on a denaturing SDS-PAGE gel. Similar labeled species can be formed using other nucleophiles such as Cy5-hydrazide. These reactions are selective for proteins bearing HA- and other unstructured poly-tyrosine-containing tags and can be performed in lysates to create specifically tagged proteins. PMID:22807021

  9. Design and Characterization of Mechanism-Based Inhibitors for the Tyrosine Aminomutase SgTAM

    SciTech Connect

    Montavon,T.; Christianson, C.; Festin, G.; Shen, B.; Bruner, S.

    2008-01-01

    The synthesis and evaluation of two classes of inhibitors for SgTAM, a 4-methylideneimidazole-5-one (MIO) containing tyrosine aminomutase, are described. A mechanism-based strategy was used to design analogs that mimic the substrate or product of the reaction and form covalent interactions with the enzyme through the MIO prosthetic group. The analogs were characterized by measuring inhibition constants and X-ray crystallographic structural analysis of the co-complexes bound to the aminomutase, SgTAM.

  10. Phosphatase of Regenerating Liver 3 (PRL3) Provokes a Tyrosine Phosphoproteome to Drive Prometastatic Signal Transduction*

    PubMed Central

    Walls, Chad D.; Iliuk, Anton; Bai, Yunpeng; Wang, Mu; Tao, W. Andy; Zhang, Zhong-Yin

    2013-01-01

    Phosphatase of regenerating liver 3 (PRL3) is suspected to be a causative factor toward cellular metastasis when in excess. To date, the molecular basis for PRL3 function remains an enigma, making efforts at distilling a concerted mechanism for PRL3-mediated metastatic dissemination very difficult. We previously discovered that PRL3 expressing cells exhibit a pronounced increase in protein tyrosine phosphorylation. Here we take an unbiased mass spectrometry-based approach toward identifying the phosphoproteins exhibiting enhanced levels of tyrosine phosphorylation with a goal to define the “PRL3-mediated signaling network.” Phosphoproteomic data support intracellular activation of an extensive signaling network normally governed by extracellular ligand-activated transmembrane growth factor, cytokine, and integrin receptors in the PRL3 cells. Additionally, data implicate the Src tyrosine kinase as the major intracellular kinase responsible for “hijacking” this network and provide strong evidence that aberrant Src activation is a major consequence of PRL3 overexpression. Importantly, the data support a PDGF(α/β)-, Eph (A2/B3/B4)-, and Integrin (β1/β5)-receptor array as being the predominant network coordinator in the PRL3 cells, corroborating a PRL3-induced mesenchymal-state. Within this network, we find that tyrosine phosphorylation is increased on a multitude of signaling effectors responsible for Rho-family GTPase, PI3K-Akt, STAT, and ERK activation, linking observations made by the field as a whole under Src as a primary signal transducer. Our phosphoproteomic data paint the most comprehensive picture to date of how PRL3 drives prometastatic molecular events through Src activation. PMID:24030100

  11. 13-week repeated dose toxicity study of l-tyrosine in rats by daily oral administration.

    PubMed

    Shibui, Yusuke; Manabe, Yasuhiro; Kodama, Terutaka; Gonsho, Akinori

    2016-01-01

    To evaluate the potential toxicity of l-tyrosine, 4 groups of Crl:CD(SD) rats of both sexes were administered l-tyrosine in water suspension by gavage once daily for 13 weeks at doses of 0 (vehicle), 200, 600 or 2000 mg/kg bw/day. Findings related to l-tyrosine administration were as follows. Edema of the cornified layer at the limiting ridge or forestomach was seen in 600 mg/kg bw/day female group and in both sexes of 2000 mg/kg bw/day group. In the liver, increased weight and hypertrophy of centrilobular hepatocytes were seen in both sexes at 2000 mg/kg bw/day, associated with slight increases in ALT and AST. Regarding the kidney morphology and function, increased hyaline droplets in the proximal tubules and increased urinary protein were seen in the 2000 mg/kg bw/day male group. In addition, increased kidney weight was also observed in both sexes of the 2000 mg/kg bw/day group, although the histological changes attributable to the weight increase remained unclear. As for blood chemistry, increases in triglycerides, total cholesterol, phospholipids, potassium ion, calcium, total protein, and α1 globulin were also seen in both sexes at 2000 mg/kg bw/day. Thus, in this study the no-observed-adverse-effect level (NOAEL) of l-tyrosine was considered to be 600 mg/kg bw/day for males and 200 mg/kg bw/day for females. PMID:26646752

  12. Dopamine and food reward: effects of acute tyrosine/phenylalanine depletion on appetite.

    PubMed

    Hardman, Charlotte A; Herbert, Vanessa M B; Brunstrom, Jeffrey M; Munafò, Marcus R; Rogers, Peter J

    2012-03-20

    It has been suggested that obese individuals over-eat in order to compensate for deficits in the dopaminergic reward system. The current study used acute tyrosine/phenylalanine depletion (ATPD) to investigate the effect of reduced dopamine function on appetite and the reward value of food in healthy volunteers. The compensatory-eating hypothesis would predict an increase in the reward value and consumption of food following depletion by this method. In a double-blind, counterbalanced, crossover study, 17 male participants (mean age=29.2 (SEM=2.7) years; mean body mass index=24.4 (SEM=0.6) kg/m(2)) were administered with a tyrosine/phenylalanine-free mixture (TYR/PHE-free; depletion condition) and a balanced amino acid mixture (BAL; control). Plasma amino acid levels were measured at baseline and peak depletion (300 min). Appetite, willingness to pay for food, liking, desired portion size and ad libitum food intake were also assessed. The TYR/PHE-free mixture was associated with significant decreases in tyrosine, phenylalanine, and the ratio of tyrosine+phenylalanine to the other large neutral amino acids (all p<.001). There were no effects on our measures of willingness to pay for food or liking. However, in the TYR/PHE-free condition, participants reported significantly lower levels of hunger following a fixed-test meal relative to the BAL condition. In conclusion, we found no evidence for compensatory eating following ATPD. Our results also provide support for the role of dopamine in motivational components of eating. PMID:22230253

  13. Molecular cloning and characterization of a tyrosine phosphatase from Monosiga brevicollis.

    PubMed

    Zhao, Benjamin F; Zhao, Zhizhuang Joe

    2014-10-31

    Protein tyrosine phosphorylation is thought to be a unique feature of multicellular animals. Interestingly, the genome of the unicellular protist Monosiga brevicollis reveals a surprisingly high number and diversity of protein tyrosine kinases, protein tyrosine phosphatases (PTPs), and phosphotyrosine-binding domains. Our study focuses on a hypothetical SH2 domain-containing PTP (SHP), which interestingly has a predicted structure that is distinct from SHPs found in animals. In this study, we isolated cDNA of the enzyme and discovered that its actual sequence was different from the predicted sequence as a result of non-consensus RNA splicing. Contrary to the predicted structure with one SH2 domain and a disrupted phosphatase domain, Monosiga brevicollis SHP (MbSHP) contains two SH2 domains and an intact PTP domain, closely resembling SHP enzymes found in animals. We further expressed the full-length and SH2 domain-truncated forms of the enzyme in Escherichiacoli cells and characterized their enzymatic activities. The double-SH2 domain-truncated form of the enzyme effectively dephosphorylated a common PTP substrate with a specific activity among the highest in characterized PTPs, while the full-length and the N-terminal SH2 domain-truncated forms of the enzyme showed much lower activity with altered pH dependency and responses to ionic strength and common PTP inhibitors. This indicates that SH2 domains suppress the catalytic activity. SHP represents a highly conserved ancient PTP, and studying MbSHP should provide a better understanding about the evolution of tyrosine phosphorylation. PMID:25445586

  14. Nitric oxide reversibly inhibits the epidermal growth factor receptor tyrosine kinase.

    PubMed Central

    Estrada, C; Gómez, C; Martín-Nieto, J; De Frutos, T; Jiménez, A; Villalobo, A

    1997-01-01

    Although it has been demonstrated that NO inhibits the proliferation of different cell types, the mechanisms of its anti-mitotic action are not well understood. In this work we have studied the possible interaction of NO with the epidermal growth factor receptor (EGFR), using transfected fibroblasts which overexpress the human EGFR. The NO donors S-nitroso-N-acetylpenicillamine (SNAP), 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA-NO) and N-¿4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl¿propane -1, 3-diamine (DETA-NO) inhibited DNA synthesis of fibroblasts growing in the presence of fetal calf serum, epidermal growth factor (EGF) or EGF plus insulin, as assessed by [methyl-3H]thymidine incorporation. Neither 8-bromo-cGMP nor the cGMP-phosphodiesterase inhibitor zaprinast mimicked this effect, suggesting that NO is unlikely to inhibit cell proliferation via a cGMP-dependent pathway. SNAP, DEA-NO and DETA-NO also inhibited the transphosphorylation of the EGFR and its tyrosine kinase activity toward the exogenous substrate poly-l-(Glu-Tyr), as measured in permeabilized cells using [gamma-32P]ATP as phosphate donor. In contrast, 3-[morpholinosydnonimine hydrochloride] (SIN-1), a peroxynitrite-forming compound, did not significantly inhibit either DNA synthesis or the EGFR tyrosine kinase activity. The inhibitory action of DEA-NO on the EGFR tyrosine kinase was prevented by haemoglobin, an NO scavenger, but not by superoxide dismutase, and was reversed by dithiothreitol. The binding of EGF to its receptor was unaffected by DEA-NO. The inhibitory action of DEA-NO on the EGF-dependent transphosphorylation of the receptor was also demonstrated in intact cells by immunoblot analysis using an anti-phosphotyrosine antibody. Taken together, these results suggest that NO, but not peroxynitrite, inhibits in a reversible manner the EGFR tyrosine kinase activity by S-nitrosylation of the receptor. PMID:9291107

  15. Phosphorylation by the c-Abl protein tyrosine kinase inhibits parkin's ubiquitination and protective function

    PubMed Central

    Ko, Han Seok; Lee, Yunjong; Shin, Joo-Ho; Karuppagounder, Senthilkumar S.; Gadad, Bharathi Shrikanth; Koleske, Anthony