Browsing by Subject "Tyrosine"

Now showing 1 - 9 of 9
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    CD4+ T cell mediated tumor immunity following transplantation of TRP-1 TCR gene modified hematopoietic stem cells
    (2013-12-10) Ha, Sung Pil; Touloukian, Christopher E.; Broxmeyer, Hal E.; Gardner, Thomas A.; Harrington, Maureen A.; He, Johnny J.
    Immunotherapy for cancer has held much promise as a potent modality of cancer treatment. The ability to selectively destroy diseased cells and leave healthy cells unharmed has been the goal of cancer immunotherapy for the past thirty years. However, the full capabilities of cancer immunotherapies have been elusive. Cancer immunotherapies have been consistently hampered by limited immune reactivity, a diminishing immune response over time, and a failure to overcome self-tolerance. Many of these deficiencies have been borne-out by immunotherapies that have focused on the adoptive transfer of activated or genetically modified mature CD8+ T cells. The limitations inherent in therapies involving terminally differentiated mature lymphocytes include limited duration, lack of involvement of other components of the immune system, and limited clinical efficacy. We sought to overcome these limitations by altering and enhancing long-term host immunity by genetically modifying then transplanting HSCs. To study these questions and test the efficiency of gene transfer, we cloned a tumor reactive HLA-DR4-restricted CD4+ TCR specific for the melanocyte differentiation antigen TRP-1, then constructed both a high expression lentiviral delivery system and a TCR Tg expressing the same TCR genes. We demonstrate with both mouse and human HSCs durable, high-efficiency TCR gene transfer, following long-term transplantation. We demonstrate the induction of spontaneous autoimmune vitiligo and a TCR-specific TH1 polarized memory effector CD4+ T cell population. Most importantly, we demonstrate the destruction of subcutaneous melanoma without the aid of vaccination, immune modulation, or cytokine administration. Overall, these results demonstrate the creation of a novel translational model of durable lentiviral gene transfer, the induction of spontaneous CD4+ T cell immunity, the breaking of self-tolerance, and the induction of anti-tumor immunity.
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    Monitoring focal adhesion kinase phosphorylation dynamics in live cells
    (Royal Society of Chemistry, 2017-07-24) Damayanti, Nur P.; Buno, Kevin; Narayanan, Nagarajan; Harbin, Sherry L Voytik; Deng, Meng; Irudayaraj, Joseph M.K.; Medicine, School of Medicine
    Focal adhesion kinase (FAK) is a cytoplasmic non-receptor tyrosine kinase essential for a diverse set of cellular functions. Current methods for monitoring FAK activity in response to an extracellular stimulus lack spatiotemporal resolution and/or the ability to perform multiplex detection. Here we report on a novel approach to monitor the real-time kinase phosphorylation activity of FAK in live single cells by fluorescence lifetime imaging.
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    Role of intracellular tyrosines in activating KIT induced myeloproliferative disease
    (Nature Publishing Group, 2012-07) Ma, Peilin; Mali, Raghuveer Singh; Martin, Holly; Ramdas, Baskar; Sims, Emily; Kapur, Reuben; Department of Pediatrics, IU School of Medicine
    Gain-of-function mutations in KIT receptor in humans are associated with gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM), and acute myelogenous leukemia (AML). The intracellular signals that contribute to oncogenic KIT induced myeloproliferative disease (MPD) are poorly understood. Here, we show that oncogenic KITD814V induced MPD occurs in the absence of ligand stimulation. The intracellular tyrosine residues are important for KITD814V induced MPD, albeit to varying degrees. Among the seven intracellular tyrosines examined, tyrosine 719 alone plays a unique role in regulating KITD814V induced proliferation and survival in vitro, and MPD in vivo. Importantly, the extent to which AKT, ERK and Stat5 signaling pathways are activated via the seven intracellular tyrosines in KITD814V impacts the latency of MPD and severity of the disease. Our results identify critical signaling molecules involved in regulating KITD814V induced MPD, which might be useful for developing novel therapeutic targets for hematologic malignancies involving this mutation.
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    Rtr1 is a dual specificity phosphatase that dephosphorylates Tyr1 and Ser5 on the RNA Polymerase II CTD
    (Elsevier, 2014-08-12) Hsu, Peter L.; Yang, Fan; Smith-Kinnaman, Whitney; Yang, Wen; Song, Jae-Eun; Mosley, Amber L.; Varani, Gabriele; Department of Biochemistry and Molecular Biology, IU School of Medicine
    The phosphorylation state of heptapeptide repeats within the C-terminal domain (CTD) of the largest subunit of RNA Polymerase II (PolII) controls the transcription cycle and is maintained by the competing action of kinases and phosphatases. Rtr1 was recently proposed to be the enzyme responsible for the transition of PolII into the elongation and termination phases of transcription by removing the phosphate marker on Serine 5, but this attribution was questioned by the apparent lack of enzymatic activity. Here we demonstrate that Rtr1 is a phosphatase of new structure that is auto-inhibited by its own C-terminus. The enzymatic activity of the protein in vitro is functionally important in vivo as well: a single amino acid mutation that reduces activity leads to the same phenotype in vivo as deletion of the protein-coding gene from yeast. Surprisingly, Rtr1 dephosphorylates not only Serine 5 on the CTD, but also the newly described anti-termination Tyrosine 1 marker, supporting the hypothesis that Rtr1 and its homologs promote the transition from transcription to termination.
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    Targeting the Role of Tyrosine in Amot Protein-lipid Binding Events
    (Office of the Vice Chancellor for Research, 2015-04-17) Abufares, Nawara A.; Kimble-Hill, Ann C.
    Amot proteins have been shown to control cell proliferation and differentiation and can selectively bind with high affinity to phosphoinositol containing membranes. This binding event is linked to endocytosis, changes in cellular polarity, and apical membrane sequestration of nuclear transcription factors associated with development of cancer phenotypes. Although the lipid selectivity of the protein has been well characterized, the mechanisms involved in the Amot coiled-coil homology domain (ACCH) binding these membranes are not yet known. The fluorescence properties of the ACCH domain were used to characterize the binding event, however it became clear each of the five native tyrosines proximity to membrane might differ based on fluorescence resonance energy transfer experiments with fluorescently tagged lipids. A variety of short peptides correlating to the amino acid sequence of Amot surrounding these tyrosines were assayed and observed in different membrane mimicking environments to determine if each tyrosine had the ability to bury into the hydrophobic region of the membrane (alcohol study), or simply interacted with the hydrophilic head groups (liposome study). Interactions were characterized by shifts in absorbance, excitation and emission scans peaks. A characterization of these shifts with respect to what is seen with the various tyrosine-phenalanine mutants will further our understanding of whether each tyrosine is buried within the protein or interacts with the membrane. Mentor: Ann Kimble-Hill, Department of Biochemistry, IU School of Medicine
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    Targeting the Role of Tyrosine in Amot Protein-Lipid Binding Events
    (Office of the Vice Chancellor for Research, 2016-04-08) Abufares, Nawara A.; Gebre, Haben; Ray, Bruce D.; Kimble-Hill, Ann C.
    Angiomotins (Amots) are a family of adaptor proteins that have been shown to control cell proliferation and differentiation. Amots can selectively bind with high affinity to phosphoinositol containing membranes through the Amot coiled-coil homology (ACCH) domain. This binding event is linked to endocytosis, changes in cellular polarity, and apical membrane sequestration of nuclear transcription factors associated with development of cancerous phenotypes. Although the lipid selectivity of the protein has been well characterized, the residues involved in the ACCH domain binding these membranes have not been fully described. Understanding the structure-function relationship may provide pathways to modulate protein sorting and downstream signaling events inducing cellular differentiation, cancer cell proliferation, and migration. The fluorescent properties of the ACCH domain were previously used to characterize the binding event. However, the relative proximity of the five native tyrosines to the membrane may have led to differences in perceived lipid binding affinities based on fluorescence resonance energy transfer with fluorescently tagged lipids. A variety of short peptides correlating to the amino acid sequence of Amot surrounding these tyrosines were assayed and observed in different membrane mimicking environments. This was done to determine if each tyrosine had the ability to bury into the hydrophobic region of the membrane mimicked by the carbon chain lengths (alcohol study), or simply interacted with the hydrophilic head groups of the lipid (liposome study). In addition, the full length Amot80 ACCH domains (wild-type and tyrosine-to-phenylalanine mutants) were screened for trends in the varying environments. Interactions were characterized by shifts in maximum wavelengths for absorbance, excitation and emission peaks. A characterization of these shifts with respect to what is seen with the various tyrosine and phenalanine mutants may further our understanding of whether each tyrosine is buried within the protein or interacts with the head groups of the membrane.