Browsing by Subject "Protein-Tyrosine Kinases"
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Item Head and neck cancer: current treatment and new opportunities(2010-06-18) Hughes, RandallItem Itk is a Critical Regulator of Spatiotemporal Localization at the Immunological Synapse(2010-05-14) Singleton, Kentner Leroy; Wülfing, ChristophThe activation of T cells by antigen presenting cells (APCs) is an important step in the initiation of the adaptive immune response. Itk, a member of the Tec family of non-receptor protein tyrosine kinases, is important for T cell activation – Itk-/- CD4+ T cells are hyporesponsive, displaying decreased calcium flux, proliferation, and IL2 production compared to wildtype T cells. The mechanism by which Itk mediates this effect is not fully elucidated. Here we show that Itk is a key regulator of spatiotemporal localization of receptors and proximal signaling intermediates at the T cell / APC interface. As part of this organizational regulation, we found that Itk, through the recruitment of SLAT, mediates activation of Cdc42 at the center of the interface, which is critically required for actin polymerization. We show that targeting activated Cdc42 to the center of the interface restores actin polymerization in the Itk-/- T cell while the addition of constitutively active Cdc42 to the entirety of the interface cannot. These results provide beginnings of a mechanistic explanation of how Itk both regulates the actin cytoskeleton and acts to amplify T cell signaling. These results further demonstrate that control of protein localization at the immunological synapse can be the critical determinant in protein function and that the center of the interface is a site of active signaling.Item Novel Insights into DNA Double-Strand Break Repair and Its Cancer Implications(2016-07-27) Hardebeck, Molly Catherine; Shay, Jerry W.; Brekken, Rolf A.; Bachoo, Robert; Burma, SandeepDespite the aggressive treatment with DNA damage-inducing agents, glioblastomas (GBM) inevitably develop therapy resistance, leading to relapse and patient mortality. Cancer cells that survive therapy acquire additional damage-induced oncogenic changes that likely facilitate therapy resistance and tumor recurrence. To understand which damage-induced oncogenic alterations may promote tumor recurrence, we previously irradiated brains of mice harboring deletions of key tumor suppressors frequently lost in GBM. The most significant acquired alteration was amplification of the Met tyrosine kinase. We find that Met-expressing cells display cancer stem cell properties, augmented tumorigenesis, up-regulation of numerous DNA damage response (DDR) proteins, and an extended G2/M arrest. We hypothesize that Met expression drives therapy resistance and may be a potential target for radiosensitizing GBM. An alternative sensitization approach could involve direct inhibition of key DDR proteins, specifically in the homologous recombination (HR) double-strand break (DSB) repair pathway which is implicated in radioresistance of GBM stem cells. One indispensable step of HR is DNA-end resection, primarily executed by the exonuclease EXO1. We found that an EXO1 construct lacking the C-terminus and containing only the nuclease domain does not localize to DSBs, causing severe resection and repair defects. We hypothesized that the C-terminus of EXO1 serves as a platform for proteins to regulate EXO1's function. We found that the C-terminus interacts with BLM helicase, and it contains four Ser/Thr-Pro sites that are phosphorylated by CDKs1/2 to promote resection. We are currently examining whether CDK phosphorylation of EXO1 modulates the duration of the G2/M checkpoint since proper DNA repair requires a halt in the cell cycle. We are using CRISPR technology to generate EXO1 knock-out cells that will be complemented with WT or CDK-mutant EXO1 for checkpoint studies. We hypothesize that CDK phosphorylation of EXO1 serves to regulate resection and sustain the G2/M checkpoint. To further elucidate the role of EXO1 in maintaining genomic stability, we examined a cancer-associated SNP in EXO1 and found that it causes resection and DSB repair defects which may contribute to genomic instability and cancer progression. Overall, we provide novel insights into multiple aspects of DSB repair and identify potential targets for cancer therapy.Item Pathogenic and Protective Potential of B Cell Dysregulation in Systemic Lupus Erythematosus(2014-07-25) Mayeux, Jessica; Stüve, Olaf; Satterthwaite, Anne B.; Mohan, Chandra; Monson, Nancy L.; Davis, LaurieSystemic lupus erythematosus (SLE) is an autoimmune disease characterized by loss of tolerance to nuclear antigens. Hyperactive B cells are present in SLE patients and murine models of lupus, many of which have defects in inhibitors of B cell receptor (BCR) signaling or plasma cell differentiation. Autoantibodies against a wide range of self antigens contribute to the pathogenesis of SLE and are used to diagnose SLE, determine prognosis, and predict specific disease manifestations. Autoantibodies form immune complexes which deposit in the kidney and joints, resulting in glomerulonephritis and arthritis, respectively. Autoantibodies against antigens in the CNS can cause Neuropsychiatric SLE manifestations, such as psychosis, memory loss, seizures, strokes, and mood disorders. Human SLE patients and murine models of lupus are used here to identify novel autoantibodies in SLE and to better understand the mechanisms by which autoantibodies accumulate in SLE. Protein arrays can be used to identify autoantibodies and autoantigens that are targeted in SLE patients. Using this approach, we identified Stress Induced Phosphoprotein 1 (STIP1) as an autoantigen in a subset of SLE patients. Those patients with elevated levels of anti-STIP1 IgG autoantibodies in their serum were less likely to have parameters associated with more severe disease, suggesting a protective role for anti-STIP1 IgG. In addition, I defined a genetic interaction between the src tyrosine kinase, Lyn, and the Ets family transcription factor, Ets1, in autoimmunity. Lyn is both a positive and negative regulator of BCR signaling; however its net effect is inhibitory. Lyn deficiency results in hyperactive B cells and Lyn-/- mice serve as a murine model of SLE. Ets1 is a regulator of plasma cell differentiation, and Ets1-/- mice have a similar phenotype to Lyn-/- mice. Lyn and Ets1 are in a shared pathway in which Lyn maintains Ets1 levels, thus limiting plasma cell accumulation. Compound heterozygotes of Lyn and Ets1 were used to determine whether partial loss of Lyn and Ets1 results in accelerated autoimmunity. Lyn and Ets1 were found to synergize in limiting the accumulation of activated and memory T cells, myeloid dendritic cells, age associated B cells, and IgM, but not IgG autoantibodies.Item Role of Bruton’s Tyrosine Kinase and Interleukin-6 in Plasma Cell Accumulation and Autoantibody Production in Lyn-Deficient Mice, A Model of Lupus(2011-08-10) Gutierrez, Maria Antonietta; Satterthwaite, Anne B.Systemic lupus erythematosus (SLE) is characterized by loss of tolerance to nuclear antigens such as DNA and chromatin, resulting in autoantibody production, immune complex deposition, inflammation, and end organ damage such as glomerulonephritis (GN). Currently, only non-specific, immunosuppressive therapies are approved for use in lupus patients. These have undesirable side effects and risks. The development of more targeted therapies is necessary and requires a better understanding of the mechanisms that contribute to the production of autoantibodies. Mice deficient in Lyn, a gene associated with human lupus, develop several features characteristic of SLE, including peripheral plasma cell accumulation, anti-dsDNA antibodies, and GN. Lyn is a Src-family tyrosine kinase that, in general, inhibits B cell and myeloid cell activity. Loss of Lyn results in cellular hyperactivity associated with autoantibody production. Bruton’s tyrosine kinase (Btk), which is critical to B cell receptor (BCR) signaling, mediates BCR hypersensitivity and autoantibody production in lyn-/- mice. B cell hyperresponsiveness is not, however, sufficient for the autoimmune phenotype; additional Btk-dependent events are required. Btk also contributes to myeloid cell function, and generally opposes Lyn action in these cells as in B cells. However, the relative contribution of myeloid hyperactivity to autoantibody production in lyn-/- mice is unknown. Lyn-deficient mice expressing reduced Btk dosage in B cells and no Btk in myeloid cells were utilized to better define how Lyn and Btk regulate and mediate, respectively, the progression from tolerance to autoimmunity. Two major checkpoints regulating autoantibody production were identified and found to be breached in lyn-/- mice. The first checkpoint regulates Btk-mediated accumulation of long-lived plasma cells co-incident with polyclonal IgM autoreactivity. This is due in part to impaired migration of lyn-/- plasma cells towards SDF-1 and involves a B cell intrinsic effect of Lyn-deficiency. The second checkpoint regulates the class-switching of B cells with lupus-associated autoantigen specificities and the production of pathogenic autoantibodies. This step requires IL-6, which is produced in excess by lyn-/- myeloid cells in a Btk-dependent manner. These results suggest that both B and myeloid defects contribute to autoimmunity in lyn-/- mice and identify Btk and IL-6 as potential therapeutic targets for SLE.Item Safeguard of Mitosis: The Spindle Checkpoint(2016-11-18) Ji, Zhejian; Chen, Zhijian J.; Cobb, Melanie H.; Rice, Luke M.; Yu, HongtaoIn mitosis, the kinetochore-microtubule attachment is under surveillance by the spindle checkpoint to ensure the fidelity of chromosome segregation. Defects in the checkpoint could lead to aneuploidy, which has been implicated in cancers, birth defects, and other human diseases. In presence of kinetochores that are not attached or improperly attached to microtubules, the checkpoint signals to assemble the mitotic checkpoint complex (MCC), which consists of BubR1-Bub3, Mad2, and Cdc20. The diffusible MCC inhibits the ubiquitin ligase activity of the anaphase-promoting complex or cyclosome bound to its co-activator Cdc20 (APC/C-Cdc20) to arrest cells in mitosis. Nevertheless, it remains unknown how the checkpoint monitors the status of the kinetochore-microtubule attachment. Neither is clear how MCC is assembled in an active checkpoint signaling. My graduate work has answered these two questions by revealing the critical functions of a checkpoint kinase, monopolar spindle 1 (Mps1), in both attachment sensing and checkpoint signaling. Of kinetochore proteins, the KMN network acts as both a critical microtubule receptor and a signaling platform for the spindle checkpoint. The human KMN contains the kinetochore null 1 complex (Knl1C), the minichromosome instability 12 complex (Mis12C), and the nuclear division cycle 80 complex (Ndc80C). In my first project, I have shown that the non-kinase domain of Mps1 directly binds to Ndc80C through two independent interactions. Both interactions involve the microtubule-binding surfaces of Ndc80C and are directly inhibited by microtubules. Elimination of one such interaction in human cells causes checkpoint defects expected from a failure in detecting unattached kinetochores. This competition between Mps1 and microtubules for Ndc80C binding thus constitutes a direct mechanism for unattached kinetochore detection. The next question is how the kinetochore-associated Mps1 kinase rules the checkpoint signaling. At kinetochore, Mps1 phosphorylates the scaffolding protein Knl1. Phosphorylated Knl1 (pKnl1) recruits checkpoint complexes budding uninhibited by benomyl 1-3 (Bub1-Bub3) and Bub1-related protein in complex with Bub3 (BubR1-Bub3) to kinetochores. My following work has demonstrated that Mps1 promotes the inhibition of APC/CCdc20 by MCC components in vitro through phosphorylating Bub1 and mitosis arrest deficiency 1 (Mad1). Phosphorylated Bub1 (pBub1) binds with Mad1-Mad2. Phosphorylated Mad1 (pMad1) directly interacts with Cdc20. Mutations of Mps1 phosphorylation sites in Bub1 or Mad1 abrogate the spindle checkpoint in human cells. Therefore, Mps1 promotes checkpoint activation through a pKnl1-pBub1-pMad1 phosphorylation cascade, in which phosphorylation of upstream components enables binding of downstream ones. We propose that this sequential multi-target phosphorylation cascade allows Mps1 to amplify checkpoint signals and makes the checkpoint highly responsive to Mps1, which itself is regulated by kinetochore-microtubule attachment. Taken together, my graduate work has solved two long-standing questions in spindle checkpoint regulation. Accordingly, Mps1 recognizes the unattached kinetochores via its non-kinase domain, while activates the checkpoint signaling through its kinase activity. The dual function of Mps1 couples checkpoint activation with unattached kinetochore detection, making checkpoint under the control of kinetochore-microtubule attachment.Item [Southwestern News](1996-07-12) Martinez, Emily