scholarly journals Identification and Specific Localization of Tyrosine-Phosphorylated Proteins in Trypanosoma brucei

2009 ◽  
Vol 8 (4) ◽  
pp. 617-626 ◽  
Author(s):  
Isabelle R. E. Nett ◽  
Lindsay Davidson ◽  
Douglas Lamont ◽  
Michael A. J. Ferguson
Keyword(s):  
Trypanosoma Brucei ◽  
Tyrosine Kinases ◽  
Immunofluorescence Microscopy ◽  
Significant Proportion ◽  
Tyrosine Residues ◽  
Phosphorylated Proteins ◽  
Cell Extracts ◽  
Proteomics Approach ◽  
Specific Localization ◽  
Multicellular Organisms

ABSTRACT Phosphorylation on tyrosine residues is a key signal transduction mechanism known to regulate intercellular and intracellular communication in multicellular organisms. Despite the lack of conventional tyrosine kinases in the genome of the single cell organism Trypanosoma brucei, phosphorylation on trypanosomal protein tyrosine residues has been reported for this parasite. However, the identities of most of the tyrosine-phosphorylated proteins and their precise site(s) of phosphorylation were unknown. Here, we have applied a phosphotyrosine-specific proteomics approach to identify 34 phosphotyrosine-containing proteins from whole-cell extracts of procyclic form T. brucei. A significant proportion of the phosphotyrosine-containing proteins identified in this study were protein kinases of the CMGC kinase group as well as some proteins of unknown function and proteins involved in energy metabolism, protein synthesis, and RNA metabolism. Interestingly, immunofluorescence microscopy using anti-phosphotyrosine antibodies suggests that there is a concentration of tyrosine-phosphorylated proteins associated with cytoskeletal structures (basal body and flagellum) and in the nucleolus of the parasite. This localization of tyrosine-phosphorylated proteins supports the idea that the function of signaling molecules is controlled by their precise location in T. brucei, a principle well known from higher eukaryotes.

After chronic myelogenous leukemia: tyrosine kinase inhibitors in other hematologic malignancies

Blood ◽  
2005 ◽  
Vol 105 (1) ◽  
pp. 22-30 ◽  
Author(s):  
Martha Wadleigh ◽  
Daniel J. DeAngelo ◽  
James D. Griffin ◽  
Richard M. Stone
Keyword(s):  
Cell Growth ◽  
Small Molecules ◽  
Chronic Myelogenous Leukemia ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Hematologic Malignancies ◽  
Myelogenous Leukemia ◽  
Pathologic Feature ◽  
Growth And Survival ◽  
Tyrosine Residues

AbstractTyrosine kinases phosphorylate proteins on tyrosine residues, producing a biologic signal that influences many aspects of cellular function including cell growth, proliferation, differentiation, and death. Constitutive or unregulated activity through mutation or overexpression of these enzymes is a common pathologic feature in many acute and chronic leukemias. Inhibition of tyrosine kinases represents a strategy to disrupt signaling pathways that promote neoplastic growth and survival in hematologic malignancies and likely in other neoplasias as well. This review focuses on tyrosine kinases that have been implicated in the pathogenesis of hematologic diseases other than chronic myelogenous leukemia and discusses the evidence for the use of small molecules to target these kinases.

Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies

1989 ◽  
Vol 93 (3) ◽  
pp. 491-500 ◽  
Author(s):  
A. Woods ◽  
T. Sherwin ◽  
R. Sasse ◽  
T.H. MacRae ◽  
A.J. Baines ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibodies ◽  
Trypanosoma Brucei ◽  
Immunofluorescence Microscopy ◽  
Complex Mixtures ◽  
Immunogold Electron Microscopy ◽  
Basal Bodies ◽  
Tubulin Isotypes ◽  
Definition Of ◽  
Attachment Zone

The detergent-insoluble T. brucei cytoskeleton consists of several morphologically distinct regions and organelles, many of which are detectable only by electron microscopy. We have produced a set of monoclonal antibodies that define each structural component of this highly ordered cytoskeleton. The monoclonal antibodies were selected by cloning of hybridomas produced from mice injected with complex mixtures of proteins of either the cytoskeleton itself or salt extracts thereof. Four antibodies define particular tubulin isotypes and locate the microtubules of the axoneme and sub-pellicular array; two antibodies recognize the flagellum attachment zone; one recognizes the paraflagellar rod and another the basal bodies. Finally, one antibody defines a detergent-insoluble component of the nucleus. The antigens detected by each monoclonal antibody have been analysed by immunofluorescence microscopy, immunogold electron microscopy and Western blotting.

scholarly journals A monoclonal antibody to a mitotic microtubule-associated protein blocks mitotic progression.

1990 ◽  
Vol 111 (2) ◽  
pp. 511-522 ◽  
Author(s):  
C Nislow ◽  
C Sellitto ◽  
R Kuriyama ◽  
J R McIntosh
Keyword(s):  
Monoclonal Antibody ◽  
Protein S ◽  
Dependent Manner ◽  
Mitotic Spindles ◽  
Mitotic Progression ◽  
Microtubule Organization ◽  
Cell Extracts ◽  
Mitotic Inhibition ◽  
Specific Localization ◽  
Dose Dependent

A monoclonal antibody raised against mitotic spindles isolated from CHO cells ([CHO1], Sellitto, C., and R. Kuriyama. 1988. J. Cell Biol. 106:431-439) identifies an epitope that resides on polypeptides of 95 and 105 kD and is localized in the spindles of diverse organisms. The antigen is distributed throughout the spindle at metaphase but becomes concentrated in a progressively narrower zone on either side of the spindle midplane as anaphase progresses. Microinjection of CHO1, either as an ascites fluid or as purified IgM, results in mitotic inhibition in a stage-specific and dose-dependent manner. Parallel control injections with nonimmune IgMs do not yield significant mitotic inhibition. Immunofluorescence analysis of injected cells reveals that those which complete mitosis display normal localization of CHO1, whereas arrested cells show no specific localization of the CHO1 antigen within the spindle. Immunoelectron microscopic images of such arrested cells indicate aberrant microtubule organization. The CHO1 antigen in HeLa cell extracts copurifies with taxol-stabilized microtubules. Neither of the polypeptides bearing the antigen is extracted from microtubules by ATP or GTP, but both are approximately 60% extracted with 0.5 M NaCl. Sucrose gradient analysis reveals that the antigens sediment at approximately 11S. The CHO 1 antigen appears to be a novel mitotic MAP whose proper distribution within the spindle is required for mitosis. The properties of the antigen(s) suggest that the corresponding protein(s) are part of the mechanism that holds the antiparallel microtubules of the two interdigitating half spindles together during anaphase.

scholarly journals Ku Heterodimer-Independent End Joining in Trypanosoma brucei Cell Extracts Relies upon Sequence Microhomology

2007 ◽  
Vol 6 (10) ◽  
pp. 1773-1781 ◽  
Author(s):  
Peter Burton ◽  
David J. McBride ◽  
Jonathan M. Wilkes ◽  
J. David Barry ◽  
Richard McCulloch
Keyword(s):  
Homologous Recombination ◽  
Trypanosoma Brucei ◽  
Antigenic Variation ◽  
Bacterial Species ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Cell Extracts ◽  
Ligase Iv

ABSTRACT DNA double-strand breaks (DSBs) are repaired primarily by two distinct pathways: homologous recombination and nonhomologous end joining (NHEJ). NHEJ has been found in all eukaryotes examined to date and has been described recently for some bacterial species, illustrating its ancestry. Trypanosoma brucei is a divergent eukaryotic protist that evades host immunity by antigenic variation, a process in which homologous recombination plays a crucial function. While homologous recombination has been examined in some detail in T. brucei, little work has been done to examine what other DSB repair pathways the parasite utilizes. Here we show that T. brucei cell extracts support the end joining of linear DNA molecules. These reactions are independent of the Ku heterodimer, indicating that they are distinct from NHEJ, and are guided by sequence microhomology. We also demonstrate bioinformatically that T. brucei, in common with other kinetoplastids, does not encode recognizable homologues of DNA ligase IV or XRCC4, suggesting that NHEJ is either absent or mechanistically diverged in these pathogens.

Characterization of a coiled coil protein present in the basal body of Trypanosoma brucei

1999 ◽  
Vol 112 (24) ◽  
pp. 4687-4694 ◽  
Author(s):  
V. Dilbeck ◽  
M. Berberof ◽  
A. Van Cauwenberge ◽  
H. Alexandre ◽  
E. Pays
Keyword(s):  
Trypanosoma Brucei ◽  
Basal Body ◽  
Coiled Coil ◽  
Western Blots ◽  
Cell Extracts ◽  
Acid Protein ◽  
Coil Structure ◽  
Tubulin Antibodies ◽  
Body Component ◽  
Gamma Tubulin

TBBC (for Trypanosoma brucei basal body component) is a unique gene transcribed in a 4.8 kb mRNA encoding a 1,410 amino acid protein that consists almost entirely of a coiled coil structure. This protein appeared to localize in the basal body, with an accessory presence at the posterior end of the cell, the nucleus and over the flagellum. Since the two other known components of the trypanosome basal body are (gamma)-tubulin and an uncharacterized component termed BBA4 we performed double immunofluorescence experiments with anti-TBBC and either anti-BBA4 or anti-(gamma)-tubulin antibodies. These three components did not colocalize but were very closely associated, BBA4 being the most proximal to the kinetoplast DNA. Anti-TBBC antibodies detected a 170 kDa protein in western blots of total HeLa cell extracts. Moreover, these antibodies stained the centriole of HeLa and COS cells as well as the centriole of mouse spermatozoa, indicating that a TBBC-like centriolar component has been conserved during the evolution of eukaryotes.

scholarly journals Phosphorylation of Tyrosine Residues in the Kinase Domain and Juxtamembrane Region Regulates the Biological and Catalytic Activities of Eph Receptors

2000 ◽  
Vol 20 (13) ◽  
pp. 4791-4805 ◽  
Author(s):  
Kathleen L. Binns ◽  
Paul P. Taylor ◽  
Frank Sicheri ◽  
Tony Pawson ◽  
Sacha J. Holland
Keyword(s):  
Tyrosine Kinases ◽  
Biological Activities ◽  
Phosphorylation Site ◽  
Kinase Domain ◽  
Kinase Assay ◽  
Eph Receptors ◽  
Amino Acid Homology ◽  
Tyrosine Residues ◽  
Juxtamembrane Region ◽  
Activation Segment

ABSTRACT Members of the Eph family of receptor tyrosine kinases exhibit a striking degree of amino acid homology, particularly notable in the kinase and membrane-proximal regions. A mutagenesis approach was taken to address the functions of specific conserved tyrosine residues within these catalytic and juxtamembrane domains. Ligand stimulation of wild-type EphB2 in neuronal NG108-15 cells resulted in an upregulation of catalytic activity and an increase in cellular tyrosine phosphorylation, accompanied by a retraction of neuritic processes. Tyrosine-to-phenylalanine substitutions within the conserved juxtamembrane motif abolished these responses. The mechanistic basis for these observations was examined using the highly related EphA4 receptor in a continuous coupled kinase assay. Tandem mass spectrometry experiments confirmed autophosphorylation of the two juxtamembrane tyrosine residues and also identified a tyrosine within the kinase domain activation segment as a phosphorylation site. Kinetic analysis revealed a decreased affinity for peptide substrate upon substitution of activation segment or juxtamembrane tyrosines. Together, our data suggest that the catalytic and therefore biological activities of Eph receptors are controlled by a two-component inhibitory mechanism, which is released by phosphorylation of the juxtamembrane and activation segment tyrosine residues.

scholarly journals The role of cytochrome c in caspase activation in Drosophila melanogaster cells

2002 ◽  
Vol 156 (6) ◽  
pp. 1089-1098 ◽  
Author(s):  
Loretta Dorstyn ◽  
Stuart Read ◽  
Dimitrios Cakouros ◽  
Jun R. Huh ◽  
Bruce A. Hay ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Mammalian Cells ◽  
Significant Proportion ◽  
Ectopic Expression ◽  
Caspase Activation ◽  
Cytochrome C Release ◽  
Cell Extracts ◽  
Effector Caspase ◽  
Drosophila Cells

The release of cytochrome c from mitochondria is necessary for the formation of the Apaf-1 apoptosome and subsequent activation of caspase-9 in mammalian cells. However, the role of cytochrome c in caspase activation in Drosophila cells is not well understood. We demonstrate here that cytochrome c remains associated with mitochondria during apoptosis of Drosophila cells and that the initiator caspase DRONC and effector caspase DRICE are activated after various death stimuli without any significant release of cytochrome c in the cytosol. Ectopic expression of the proapoptotic Bcl-2 protein, DEBCL, also fails to show any cytochrome c release from mitochondria. A significant proportion of cellular DRONC and DRICE appears to localize near mitochondria, suggesting that an apoptosome may form in the vicinity of mitochondria in the absence of cytochrome c release. In vitro, DRONC was recruited to a >700-kD complex, similar to the mammalian apoptosome in cell extracts supplemented with cytochrome c and dATP. These results suggest that caspase activation in insects follows a more primitive mechanism that may be the precursor to the caspase activation pathways in mammals.

scholarly journals Tyrosine phosphorylation of CD45 phosphotyrosine phosphatase by p50csk kinase creates a binding site for p56lck tyrosine kinase and activates the phosphatase.

1994 ◽  
Vol 14 (2) ◽  
pp. 1308-1321 ◽  
Author(s):  
M Autero ◽  
J Saharinen ◽  
T Pessa-Morikawa ◽  
M Soula-Rothhut ◽  
C Oetken ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Lymphocyte Activation ◽  
Sh2 Domain ◽  
Protein Tyrosine Kinases ◽  
Phosphotyrosine Phosphatase ◽  
Intact Cells ◽  
Tyrosine Residues ◽  
Family Protein ◽  
Ptpase Activity

Src family protein tyrosine kinases (PTKs) play an essential role in antigen receptor-initiated lymphocyte activation. Their activity is largely regulated by a negative regulatory tyrosine which is a substrate for the activating action of the CD45 phosphotyrosine phosphatase (PTPase) or, conversely, the suppressing action of the cytosolic p50csk PTK. Here we report that CD45 was phosphorylated by p50csk on two tyrosine residues, one of them identified as Tyr-1193. This residue was not phosphorylated by T-cell PTKs p56lck and p59fyn. Tyr-1193 was phosphorylated in intact T cells, and phosphorylation increased upon treatment with PTPase inhibitors, indicating that this tyrosine is a target for a constitutively active PTK. Cotransfection of CD45 and csk into COS-1 cells caused tyrosine phosphorylation of CD45 in the intact cells. Tyrosine-phosphorylated CD45 bound p56lck through the SH2 domain of the kinase. Finally, p50csk-mediated phosphorylation of CD45 caused a severalfold increase in its PTPase activity. Our results show that direct tyrosine phosphorylation of CD45 can affect its activity and association with Src family PTKs and that this phosphorylation could be mediated by p50csk. If this is also true in the intact cells, it adds a new dimension to the physiological function of p50csk in T lymphocytes.

scholarly journals Interactions of p59fyn and ZAP-70 with T-cell receptor activation motifs: defining the nature of a signalling motif.

1994 ◽  
Vol 14 (6) ◽  
pp. 3729-3741 ◽  
Author(s):  
L K Gauen ◽  
Y Zhu ◽  
F Letourneur ◽  
Q Hu ◽  
J B Bolen ◽  
...  
Keyword(s):  
T Cell Receptor ◽  
Tyrosine Kinases ◽  
Molecular Basis ◽  
Kinase Activity ◽  
Cell Receptor ◽  
Receptor Activation ◽  
Single Mutation ◽  
Tyrosine Residues ◽  
Sh2 Domains ◽  
Activation Motif

The tyrosine-based activation motif is a 20- to 25-amino-acid sequence contained in the cytoplasmic domains of many hematopoietic receptors which is sufficient by itself to reconstitute signalling. This motif is characterized by two YXXL/I sequences separated by approximately 10 residues. The molecular basis of signalling by this motif is unknown. Here we demonstrate that the tyrosine-based activation motif is required and sufficient for association with the tyrosine kinases p59fyn and ZAP-70, suggesting that association with these kinases is a general feature of this motif. Focusing on the single activation motif present in epsilon, we analyzed which residues of the motif were critical for binding of p59fyn and ZAP-70. Surprisingly, we found that no single mutation of any residue of epsilon resulted in the loss of p59fyn association. In contrast, single mutations at five residues of the epsilon activating motif abrogated ZAP-70 binding. Both of the tyrosines and the leucine or isoleucine residues that follow them were critical. The spacing between the tyrosines was also important, as deletion of two residues disrupted binding of ZAP-70, although p59fyn binding was not disrupted. Most of the defined features of the tyrosine activation motif are therefore requirements for ZAP-70 binding. Interestingly, the interaction of ZAP-70 with the motif was dependent on the presence of both ZAP-70 SH2 domains and both of the tyrosine residues in the motif, suggesting that ZAP-70 interacts with two phosphotyrosine residues and that the binding of the two SH2 domains is cooperative. In addition, we demonstrate that the interaction between the tyrosine activation motif is direct and requires prior tyrosine phosphorylation of the motif. We propose that the activation of cells by the tyrosine activating motif occurs in four discrete steps: binding of p59fyn, phosphorylation of the motif, binding of ZAP-70, and activation of ZAP-70 kinase activity.

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