Browsing by Subject "MAPK"
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Item Effects of site specific phosphorylation on the structure and functions of the glucocorticoid receptor(2008-09-04) Anna Magdalena Stwora de Garza; Raj Kumar, Ph.D.; Wlodek Bujalowski, Ph.D.; Michael Garabedian, Ph.D.; Golda Leonard, Ph.D.; E. Brad Thompson, M.D.Ligand dependant transcription factors, like nuclear hormone receptors (NHRs), are capable of exerting transcriptional regulation in the nucleus in response to various intra- and extracellular signals. Transcription factors contain segments that are intrinsically disordered (ID) under native conditions. Posttranslational modifications, such as phosphorylation, affect protein stability and activity of proteins. Conformational changes of such disordered domains have been shown to facilitate binding of one or more coregulatory proteins. The glucocorticoid receptor (GR) belongs to the NHR super family and contains such an ID domain in its N-terminal region, the AF1. This transactivation domain must interact with co-regulators for optimal activity and contains most of the conserved phosphorylation sites (S203, S211, and S226) in the human GR. Published data has linked site-specific phosphorylation of the GR to physiological functions of the GR in a leukemia cell line model (5). This project’s aims were to study how site-specific phosphorylation affects the structure and function of the glucocorticoid receptor. The aims of the project were: 1) to test the effect of site-specific phosphorylation on the conformation of the recombinant AF1 domain of the human GR, 2) to test the effects of site-specific phosphorylation on the interactions of AF1 with specific coregulatory proteins and the subsequent changes in transcriptional activity in CV-1 cells, and 3) to test if site-specific phosphorylation of the GR is controlled by MAPK activity and if this phosphorylation is sufficient to restore lost GR function in refractory hematological malignancies. \r\n We show for the first time, that ID AF1 domain of glucocorticoid receptor (GR) adopts a functionally folded conformation due to site-specific (S211) phosphorylation by p38 MAPK that we have earlier shown to be involved in the apoptotic and gene-inductive events initiated by GR. These conformational changes are important for AF1s interaction with coregulatory proteins, and subsequent GRE mediated transcriptional activity of the GR. \r\n Finally, these conformational changes are important for AF1s interaction with coregulatory proteins, and subsequent GRE mediated transcriptional activity of the GR. This activating phosphorylation, specifically S211, is controlled by balanced MAPK activity in in vitro cell line models providing and additional mechanism for resistance. Where phosphorylated p38 levels are high relative to low ERK and JNK activity levels. Further suggesting that p38 MAPK activity plays a role in structural and functional consequences of the GR. \r\nItem Heat shock-induced apoptosis(2013-12) Mahajan, Indra Maria; Wright, Casey Wyatt; Bratton, Shawn B.Apoptosis is a conserved program of cell death that promotes organism homeostasis in all stages of life. Two main pathways activate caspases, which are cysteinyl-aspartate proteases that execute apoptosis. The extrinsic pathway is initiated by cell surface death receptors, while the intrinsic pathway is initiated by intracellular signals that cause permeabilization of the outer mitochondrial membrane (MOMP). The Bcl-2 protein family regulates MOMP, which causes the release of several pro-apoptotic proteins (such as cytochrome c, Smac) into the cytosol. Bcl-2 proteins share homology in up to four "BH" domains and are subdivided into three subgroups. Pro-apoptotic Bax and Bak catalyze pore formation in the mitochondria, while anti-apoptotic members (Bcl-2, Mcl-1) inhibit MOMP. The third subgroup, termed BH3-only, promotes MOMP by either antagonizing Bcl-2 proteins or by directly activating Bax/Bak, and initiate apoptosis in response to various stressors, including heat shock (HS). Hyperthermia or acute HS reportedly induces apoptosis through caspase-2-mediated cleavage of BID, engaging the intrinsic pathway. However, additional evidence suggests that this pathway could represent an amplification loop. Thus we hypothesized that during HS, another BH3-only protein such as BIM, that does not require cleavage, could engage MOMP. Herein, we report that BIM mediates an alternative HS-induced apoptosis pathway. Cells lacking BIM are resistant to HS and exhibit better short and long-term survival than either Bid[superscript -/-] or Bax[superscript -/-]Bak[superscript -/-]. Moreover, caspase-2 induces apoptosis in Bim[superscript -/-] but not Bid[superscript -/-] cells, implying that caspase-2 kills exclusively through BID. Interestingly, Bim[superscript -/-] and Bax[superscript -/-]Bak[superscript -/-] cells are entirely resistant to MOMP, but the Bax[superscript -/-]Bak[superscript -/-] cells still undergo caspase-3 activation and remain partially sensitive to HS, indicating that BIM triggers caspase-3 activation upstream of mitochondria. Thus, BIM plays an important role in HS-induced apoptosis. Hyperthermia has clinical applications for the treatment of solid tumors. Unfortunately, a practical limitation is the development of thermotolerance, which confers resistance not only to subsequent HS but also to radiotherapy and chemotherapy. Therefore, a better understanding of the molecular mechanisms involved both in heat-induced apoptosis and thermotolerance could lead to new therapeutic interventions. Here we also show evidence for a putative role for the stress kinase JNK signaling pathway in the regulation of thermotolerance.Item Regulation of the p38 MAPK Signaling Pathway by the Circadian Clock(2013-08-12) Goldsmith, Charles SidneyMitogen activated protein kinase (MAPK) pathways are conserved biochemical signal transduction pathways in eukaryotic organisms. These signaling pathways demonstrate great versatility in their ability to detect various environmental stimuli and direct an appropriate cellular response. The circadian clock is a timekeeping mechanism that temporally coordinates diverse biological functions in an organism with the environment. Thus, it is not surprising that MAPK pathways have been utilized by the circadian clock to regulate many essential functions. Due to the conserved nature of circadian clocks and MAPK signaling pathways in eukaryotes, it is possible to develop hypotheses in simple model organisms, such as the fungus Neurospora, that are relevant to more complex organisms. The OS-2 MAPK pathway in the filamentous fungus Neurospora is rhythmically activated by the circadian clock. In order to generate this rhythmic signal, the circadian oscillator directly regulates the rhythmic transcription of the os-4 MAPKKK and histidine phosphotransferase hpt-1, which are upstream regulators of the OS-2 MAPK. Also, the circadian rhythm in MAPK activation produces a more robust stress response during the time of the day that stress is most likely to be encountered. Based on these data, a model for the clock regulation of MAPK activation is presented, and a biological significance is assigned to the rhythms in this pathway. Informed by these findings in Neurospora, the related p38 MAPK pathway was studied in mammalian cell lines that represent functionally distinct tissues in regards to clock function. A rhythm in p38 MAPK activation was observed in cells derived from the suprachiasmatic nucleus and fibroblasts of a mouse, the master pacemaker and a peripheral tissue, respectively. In cells that lacked a functional circadian oscillator, the rhythm in p38 activation was absent, and overall levels of p38 protein were lower. These data demonstrate a circadian clock-dependent oscillation in p38 activity. These studies provide a basis to understand how the circadian clock generates endogenous rhythms in MAPK signal transduction pathways. Also, the characterization of clock-regulated stress response pathways provides an understanding of the adaptive advantage of the circadian clock.