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Open Access Publications from the University of California

Scholarly Works (8 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2013)

Discs large homolog 1 (Dlgh1) is a scaffold protein that couples T cell receptor (TCR) engagement to signal transduction and cytoskeletal reorganization. Specifically, Dlgh1 directs Lck and ZAP70 kinase activity toward the selective activation of mitogen-activated protein kinase p38 and transcription factor NFAT. In addition, Dlgh1 controls actin polymerization, TCR and lipid raft clustering, and MTOC positioning during TCR engagement. The ability to selectively activate, integrate and tune these pathways may allow Dlgh1 to promote discrete cellular responses including: T cell differentiation, proliferation, cytokine production, cell-mediated cytotoxicity and memory generation. Therefore, understanding how Dlgh1 channels proximal TCR signals to specific downstream pathways to trigger distinct cellular responses in T cells requires 1) an understanding of how Dlgh1 is regulated by post-transcriptional and post-translational modifications, and 2) the development of a suitable Dlgh1 knockout (Dlgh1 KO) mouse model to assess Dlgh1 function in vivo.

Alternative splicing and phosphorylation of Dlgh1 are post-transcriptional and post-translational modifications that regulate Dlgh1 localization, expression and function. We found that alternative splicing of Dlgh1 in CD8+ effector T cells resulted in the expression of two distinct Dlgh1 protein variants: Dlgh1 AB and Dlgh1 B. Further, these two Dlgh1 variants served as molecular conduits to drive distinct cytotoxic T lymphocyte (CTL) responses. While both Dlgh1 AB and Dlgh1 B promoted p38-independent lytic factor degranulation, which required actin polymerization and the cytoskeletal regulator WASp, only Dlgh1 AB coupled Lck to p38-dependent production of proinflammatory cytokines. In addition, this CTL effector function was found to require Lck-mediated Dlgh1 AB tyrosine phosphorylation at tyrosine 222, suggesting that Dlgh1 AB tyrosine phosphorylation may provide a unique mechanism to specifically regulate p38-dependent functions in T cells. Lastly, we found that acute inducible knockout of Dlgh1 in mature peripheral T cells prevented optimal T cell activation and effector function in CD8+ T cells, while knockout of Dlgh1 in the germline or developing T cells had little or no effect on T cell function. Together, these data suggests that the developmental timing of Dlgh1 ablation may be critical for observing Dlgh1-dependent functional effects. We propose that T lymphocytes construct functionally unique Dlgh1 AB/Lck/p38 and Dlgh1/WASp complexes as a mechanism to specifically direct proximal TCR signals towards distinct cellular responses.

    Cover page: Discs Large Homolog 1: Molecular Scaffolds Guiding T Cell Activation and Effector Function
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2015)
    Functionally diverse CD8+ T cells develop in response to antigenic stimulation with differing capacities to couple TCR engagement to downstream signals and functions. However, mechanisms of diversifying TCR signaling are largely uncharacterized. Here we identified two alternative splice variants of scaffold protein Dlg1, Dlg1AB and Dlg1B, that diversify signaling to regulate p38-dependent and -independent effector functions in CD8+ T cells. Dlg1AB, but not Dlg1B associated with Lck, coupling TCR stimulation to p38 activation and proinflammatory cytokine production. Conversely, both Dlg1AB and Dlg1B mediated p38-independent degranulation. Degranulation depended on a Dlg1 fragment containing an intact Dlg1SH3-domain and required the SH3-ligand WASp. Further, Dlg1 controlled WASp activation by promoting TCR-triggered conformational opening of WASp. Collectively, our data support a model where Dlg1 regulates p38-dependent proinflammatory cytokine production and p38-independent cytotoxic granule release through the utilization of alternative splice variants, providing a mechanism whereby TCR engagement couples downstream signals to unique effector functions in CD8+ T cells.
      Cover page: Discs Large Homolog 1 Splice Variants Regulate p38 –Dependent and –Independent Effector Functions in CD8+ T Cells
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2014)
      CD8(+) T cells respond to TCR stimulation by producing proinflammatory cytokines, and destroying infected or malignant cells through the production and release of cytotoxic granules. Scaffold protein Discs large homolog 1 (Dlg1) specifies TCR-dependent functions by channeling proximal signals toward the activation of p38-dependent proinflammatory cytokine gene expression and/or p38-independent cytotoxic granule release. Two Dlg1 variants are expressed in CD8(+) T cells via alternative splicing, Dlg1AB and Dlg1B, which have differing abilities coordinate TCR-dependent functions. Although both variants facilitate p38-independent cytotoxicity, only Dlg1AB coordinates p38-dependent proinflammatory cytokine expression. In this study, we identify TCR-induced Dlg1 tyrosine phosphorylation as a key regulatory step required for Dlg1AB-mediated p38-dependent functions, including proinflammatory cytokine expression. We find that Dlg1AB but not Dlg1B is tyrosine phosphorylated by proximal tyrosine kinase Lck in response to TCR stimulation. Furthermore, we identify Dlg1 tyrosine 222 (Y222) as a major site of Dlg1 phosphorylation required for TCR-triggered p38 activation and NFAT-dependent expression of proinflammatory cytokines, but not for p38-independent cytotoxicity. Taken together, our data support a model where TCR-induced phosphorylation of Dlg1 Y222 is a key point of control that endows Dlg1AB with the ability to coordinate p38 activation and proinflammatory cytokine production. We propose blocking Dlg1AB phosphorylation as a novel therapeutic target to specifically block proinflammatory cytokine production but not cytotoxicity.
        Cover page: Selective Phosphorylation of the Dlg1AB Variant Is Critical for TCR-Induced p38 Activation and Induction of Proinflammatory Cytokines in CD8+ T Cells
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2022)
        Despite recent advances in therapeutic approaches, patients with MLL-rearranged leukemia still have poor outcomes. Here, we find that the RNA-binding protein IGF2BP3, which is overexpressed in MLL-translocated leukemia, strongly amplifies MLL-Af4-mediated leukemogenesis. Deletion of Igf2bp3 significantly increases the survival of mice with MLL-Af4-driven leukemia and greatly attenuates disease, with a minimal impact on baseline hematopoiesis. At the cellular level, MLL-Af4 leukemia-initiating cells require Igf2bp3 for their function in leukemogenesis. At the molecular level, IGF2BP3 regulates a complex posttranscriptional operon governing leukemia cell survival and proliferation. IGF2BP3-targeted mRNA transcripts include important MLL-Af4-induced genes, such as those in the Hoxa locus, and the Ras signaling pathway. Targeting of transcripts by IGF2BP3 regulates both steady-state mRNA levels and, unexpectedly, pre-mRNA splicing. Together, our findings show that IGF2BP3 represents an attractive therapeutic target in this disease, providing important insights into mechanisms of posttranscriptional regulation in leukemia.
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2012)

          Background

          The polarized reorganization of the T cell membrane and intracellular signaling molecules in response to T cell receptor (TCR) engagement has been implicated in the modulation of T cell development and effector responses. In siRNA-based studies Dlg1, a MAGUK scaffold protein and member of the Scribble polarity complex, has been shown to play a role in T cell polarity and TCR signal specificity, however the role of Dlg1 in T cell development and function in vivo remains unclear.

          Methodology/principal findings

          Here we present the combined data from three independently-derived dlg1-knockout mouse models; two germline deficient knockouts and one conditional knockout. While defects were not observed in T cell development, TCR-induced early phospho-signaling, actin-mediated events, or proliferation in any of the models, the acute knockdown of Dlg1 in Jurkat T cells diminished accumulation of actin at the IS. Further, while Th1-type cytokine production appeared unaffected in T cells derived from mice with a dlg1 germline-deficiency, altered production of TCR-dependent Th1 and Th2-type cytokines was observed in T cells derived from mice with a conditional loss of dlg1 expression and T cells with acute Dlg1 suppression, suggesting a differential requirement for Dlg1 activity in signaling events leading to Th1 versus Th2 cytokine induction. The observed inconsistencies between these and other knockout models and siRNA strategies suggest that 1) compensatory upregulation of alternate gene(s) may be masking a role for dlg1 in controlling TCR-mediated events in dlg1 deficient mice and 2) the developmental stage during which dlg1 ablation begins may control the degree to which compensatory events occur.

          Conclusions/significance

          These findings provide a potential explanation for the discrepancies observed in various studies using different dlg1-deficient T cell models and underscore the importance of acute dlg1 ablation to avoid the upregulation of compensatory mechanisms for future functional studies of the Dlg1 protein.
            Cover page: Characterization of In Vivo Dlg1 Deletion on T Cell Development and Function
            • Article
            UCLA Previously Published Works (2020)
            Despite recent advances in therapeutic approaches, patients with MLL-rearranged leukemia still have poor outcomes and a high risk of relapse. Here, we found that MLL-AF4, the most common MLL fusion protein in patients, transcriptionally induces IGF2BP3 and that IGF2BP3 strongly amplifies MLL-Af4 mediated leukemogenesis. Deletion of Igf2bp3 significantly increases the survival of mice with MLL-Af4 driven leukemia and greatly attenuates disease, with a minimal impact on baseline hematopoiesis. At the cellular level, MLL-Af4 leukemia-initiating cells require Igf2bp3 for their function in leukemogenesis. eCLIP and transcriptome analysis of MLL-Af4 transformed stem and progenitor cells and MLL-Af4 bulk leukemia cells reveals a complex IGF2BP3-regulated post-transcriptional operon governing leukemia cell survival and proliferation. Regulated mRNA targets include important leukemogenic genes such as those in the Hoxa locus and numerous genes within the Ras signaling pathway. Together, our findings show that IGF2BP3 is an essential positive regulator of MLL-AF4 mediated leukemogenesis and represents an attractive therapeutic target in this disease.
              • Article
              • Peer Reviewed
              UC San Francisco Previously Published Works (2022)
              Molecular characterization of cell types using single-cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, identification of the tissue-specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type-specific RNA splicing was discovered and analyzed across tissues within an individual.
                Cover page: The Tabula Sapiens: A multiple-organ, single-cell transcriptomic atlas of humans
                • Article
                • Peer Reviewed
                UCLA Previously Published Works (2022)
                Cell-free RNA from liquid biopsies can be analyzed to determine disease tissue of origin. We extend this concept to identify cell types of origin using the Tabula Sapiens transcriptomic cell atlas as well as individual tissue transcriptomic cell atlases in combination with the Human Protein Atlas RNA consensus dataset. We define cell type signature scores, which allow the inference of cell types that contribute to cell-free RNA for a variety of diseases.
                  Cover page: Cell types of origin of the cell-free transcriptome
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