Browsing by Subject "phosphorylation"
Now showing 1 - 4 of 4
Results Per Page
Sort Options
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 Role of BCL-XL in cell death after spinal cord injury(2006-12-11) DIANA M. CITTELLY; J.Regino Perez-Polo; Olivera Nesic-Taylor; Jacqueline Bresnahan; Golda Leonard; Giulio TaglialatelaLong term functional impairment after rat spinal cord injury (SCI) results from secondary apoptosis regulated in part, by SCI-induced decreases in protein levels of the anti-apoptotic protein Bcl-xL. In this dissertation, I assessed the role that Bcl-xL subcellular re-routing and post-translational phosphorylation play in SCI-induced Bcl-xL decreases, and evaluated the therapeutic potential of Bcl-xL-administration after SCI. Immunohistochemical analysis showed non-phosphorylated Bcl-xL in neurons and oligodendrocytes, but not in astrocytes and microglia. Bcl-xL levels decreased in mitochondria, endoplasmic reticulum, nuclei and cytosolic extracts during the first 24h after SCI, but with a different time course for each organelle; suggesting an independent regulation of Bcl-xL shuttling from the cytosol to each compartment in the injured spinal cords. A membrane-bound phosphorylated form of Bcl-xL (P-ser62-Bcl-xL) was found in neurons in the uninjured SC. SCI did not affect P-ser62-Bcl-xL levels in organelles; however, P-ser62-Bcl-xL appeared in the cytosol early after SCI, suggesting a role for phosphorylation in SCI-induced decreases of Bcl-xL levels. Vinblastine-induced apoptosis of neuronal PC12 cells, showed that cytosolic phosphorylated Bcl-xL correlated with apoptotic cell death of neurons, suggestive of Bcl-xL-phosphorylation as a pro-apoptotic event. I found that activated microglia/macrophages robustly expressed Bcl-xL, 7 days after SCI, and a fraction of this population undergoing apoptosis, expressed P-ser62-Bcl-xL. Therefore, phosphorylation of Bcl-xL may have two opposite effects in injured spinal cords: (a) it may decrease levels of the anti-apoptotic Bcl-xL in neurons and therefore contribute to their death and, (b) it may regulate apoptosis in activated microglia/macrophages, thus curtailing the inflammatory cascades associated with SCI.\r\nTo counteract SCI-induced decreases in Bcl-xL and resulting apoptosis, I used a fusion protein made up of the TAT protein transduction domain and the Bcl-xL protein (Tat-Bcl-xL), or to its anti-apoptotic domain BH4 (Tat-BH4). Intrathecal delivery of Tat-Bcl-xL, or Tat-BH4 for 24h or 7 days after SCI, resulted in a significant decrease in apoptosis at the site of injury. However, the 7 day delivery of Tat-Bcl-xL or Tat-BH4 impaired locomotor recovery beyond the drug delivery time. Here I show that the 7 day application of Tat-Bcl-xL or Tat-BH4 increased microglia/macrophage activation and/or survival associated with an increase in neuronal losses. These results suggest that the anti-apoptotic treatment may shift neuronal apoptosis to necrosis, and initiate an inflammatory response (microglial activation) in SCI rats. As a result, Tat-Bcl-xL/Tat-BH4-induced increases in proinflammatory reactions may amplify SCI-induced neuronal cell death and additionally impair functional recovery. Given that microglial activation and inflammation are main players in shaping pathological outcomes after SCI, these results suggest that the therapeutic potential of Tat-Bcl-xL or Tat-BH4 in injured spinal cords may be limited. Moreover, chronic treatment of SCI with Tat-Bcl-xL or other anti-apoptotic treatments targeting Bcl-xL could be detrimental.\r\nItem Studies on the nucleocapsid protein of infectious bronchitis virus(Texas A&M University, 2005-08-29) Jayaram, JyothiBecause phosphorylation of the infectious bronchitis virus (IBV) nucleocapsid (N) protein may regulate its multiple roles in viral replication, the dynamics of N phosphorylation were examined. In the infected cell, N was the only viral protein that was phosphorylated as shown by 32P-orthophosphate labeling and Western blot analysis and with IBV specific polyclonal chicken antibody. Using pulse-labeling with 32Porthophosphate, the IBV N protein was found to be phosphorylated in the virion, as well as at all times during infection of Vero cells. One-hour pulse-chase analysis followed by immunoprecipitation of IBV N using rabbit anti-IBV N polyclonal antibody showed that the phosphate on the protein did not fall below 70% of the maximum and remained stable. The small but reproducible drop in phosphorylation could modulate the various functions of the N protein in the infected cell. Simultaneous labeling with 32Porthophosphate and 3H-leucine of infected CEK cells indicated a 3.5-fold increase in the ratio of the 32P:3H counts per minute (cpm) on the virion N protein as compared to the 32P:3H cpm ratio of the N protein from lysates at 7 h p.i. The 32P:3H cpm ratio of the N protein from virion from infected-Vero cell lysates was 10.5X more than the 32P:3H cpm ratio of the N protein obtained at 7 h p.i. It has been shown that the N proteins from the measles and rabies viruses form helical nucleocapsid-like structures when expressed in bacteria (Schoehn et al., 2001; Warnes et al., 1995). The ability of E. coli expressed IBV N protein to form helical-nucleocapsid-like structures was investigated using transmission electron microscopy. Full-length, purified histidine-tagged IBV N protein formed nucleocapsid-like structures when expressed in bacteria. Because E. coli -expressed histidine-tagged fragments of the IBV N protein did not form helical nucleocapsid-like structures, the full-length protein is probably required for assembly of these structures. The highly conserved IBV N protein was also used as a diagnostic tool in an ELISA for detecting anti-IBV antibody in chicken serum using a specialized microwave called the BIOWAVE. The BIOWAVE improves the processing time for an ELISA.Item The subunit exchange rate of the cyanobacterial circadian clock component kaic is independent of phosphorylation state(2009-05-15) Ihms, Elihu CarlThe study of the in vitro circadian oscillator of the cyanobacterium Synechococcus elongatus has uncovered a complex interplay of its three protein components. Synchronization of the clock's central oscillatory component, KaiC, has been thought to be achieved through subunit shuffling at specific intervals during the clock?s period. By utilizing an established fluorescence-based analysis on completely phosphorylated and dephosphorylated mutants as well as wild-type KaiC, this study has shown that shuffling rates are largely unaffected by phosphorylation state. These findings conflict with previous reports and hence revise our understanding of this oscillator.