Browsing by Subject "Phosphorylation"

Now showing 1 - 6 of 6
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    Elucidating the structural dynamics of SERCA-PLB regulation by electron paramagnetic resonance
    (2014-08) McCaffrey, Jesse Earl
    Muscle contraction and relaxation is initiated by changes in intracellular calcium, making adequate calcium transport essential for proper muscle function. A primary calcium transporter is the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA), located within the muscle cell and embedded in an organelle called the sarcoplasmic reticulum (SR). To facilitate muscle relaxation, calcium is sequestered from the cellular cytosol into the SR by SERCA. In cardiac muscle, enhanced regulation of calcium transport is needed to accommodate β-adrenergic stimulation (adrenaline demand), which is provided by the regulatory protein phospholamban (PLB). PLB binds to SERCA and inhibits calcium transport through reduction in calcium affinity. However, a phosphate group can be attached to PLB during adrenaline response (phosphorylation) which relieves PLB's inhibitory effect. The structural mechanisms for SERCA regulation by PLB, particularly with respect to phosphorylation, are not well-resolved. Under the Dissociation Model, PLB phosphorylation relieves SERCA inhibition by dissociating the SERCA-PLB complex. In contrast, the Subunit Model proposes that SERCA inhibition is relieved by a subtle structural change, where the SERCA-PLB complex is preserved. The primary goal of my thesis work is to elucidate the structural mechanisms of the SERCA-PLB complex using electron paramagnetic resonance (EPR) spectroscopy. The first study (Chapter 3) aims to discriminate between the Dissociation and Subunit models by measuring changes in the rotational diffusion of EPR spin labels rigidly coupled to PLB and SERCA. The second study (Chapter 4) further develops an anisotropic membrane system called bicelles for EPR orientation measurements on PLB and SERCA. The third study (Chapter 5) uses a combination of oriented bicelles and a novel rigid spin label (bifunctional spin label, or BSL) to measure PLB topology in the lipid membrane, with comparison to previous structural measurements by NMR and x-ray crystallography. Ongoing studies (Chapter 6) reconstitute both spin-labeled PLB and SERCA in bicelles to make PLB orientation measurements by EPR, as affected by PLB phosphorylation and binding of SERCA.
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    Examining the Role of Phospholamban Phosphorylation on Interaction with SERCA Using Fluorescence Microscopy
    (2018-07) Haydon, Suzanne
    Regulation of the Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) by Phospholamban (PLB) plays a crucial role in normal cardiomyocyte function through controlling the speed and extent of myocyte relaxation. The interaction between PLB and SERCA is altered in many forms of heart failure (HF), making these proteins potential targets for the treatment of HF. Both proteins have been extensively studied in vitro, where their basic structure and function were determined, and in animal models, where their role in disease was examined. However, key information connecting the in vitro experiments and animal models is needed to better understand the PLB-SERCA interaction and to design effective HF treatment strategies. In particular, we wanted to examine two conflicting in vitro models of how the PLB-SERCA interaction changes after PLB phosphorylation: the dissociation model and the structural model. In the dissociation model, phosphorylation causes PLB to dissociate from SERCA, while in the structural model, phosphorylation causes a shift in the PLB binding position along SERCA. In order to determine the correct model of PLB-SERCA interaction in live cells, we expressed cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to the N-termini of SERCA and PLB respectively, in HEK293 cells for fluorescence resonance energy transfer (FRET) microscopy experiments. We were able to use the native beta-adrenergic signaling system in the cells to control the state of PLB phosphorylation in a time-dependent manner. For the dissociation model to be true, we expected to see a significant reduction in FRET between CFP-SERCA and YFP-PLB after PLB phosphorylation. While significant increases in PLB phosphorylation were produced in the cells, FRET did not decrease. Instead, FRET increased with PLB phosphorylation at serine 16, indicating either a shorter distance between PLB and SERCA, or higher binding of PLB to SERCA. As the beta-adrenergic signal progressed through the cells, causing phosphorylation of PLB at threonine 17, FRET returned towards basal levels, but did not show the decrease below basal FRET levels that would indicate PLB dissociation from SERCA. Thus, we determined that there is a subtle change in the PLB-SERCA interaction due to PLB phosphorylation rather than a large scale dissociation. In order to differentiate changes in distance from changes in binding time-resolved (TR)-FRET experiments were required. Fluorescence lifetime imaging microscopy (FLIM) is a variant of TR-FRET that measures fluorescence decay curves with a fast-pulsed laser and photon counting board attached to a confocal microscope. These fluorescence decay curves provide more information than intensity measurements since they can be fit to multiple exponentials to test different interaction models. FLIM is a relatively new technique, thus we worked on developing appropriate experimental conditions for acquiring fluorescence decays that contained enough photons for multi-exponential fitting while still measuring individual cells. We were able to use FLIM to measure FRET values similar to those acquired on standard fluorescence microscopes and confirmed that phosphorylation of PLB did not cause dissociation from SERCA. However, further improvements to FLIM acquisition and analysis are needed for the multi-exponential fitting to provide a better model of PLB-SERCA interaction in live cells.
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    Phosphorylated and SUMO-deficient progesterone receptors drive proliferative gene signatures during breast cancer progression.
    (2012-07) Knutson, Todd Philip
    Introduction: Progesterone receptors (PR) are emerging as important breast cancer drivers. Phosphorylation events common to breast cancer cells impact PR transcriptional activity, in part by direct phosphorylation. PR-B but not PR-A isoforms are phosphorylated on Ser294 by MAPK and CDK2. Phospho-Ser294 PRs are resistant to ligand-dependent Lys388 SUMOylation (i.e. a repressive modification). Antagonism of PR SUMOylation by mitogenic protein kinases provides a mechanism for derepression (i.e. transcriptional activation) of target genes. As a broad range of PR protein expression is observed clinically, a PR gene signature would provide a valuable marker of PR contribution to early breast cancer progression. Methods: Global gene expression patterns were measured in T47D and MCF-7 breast cancer cells expressing either wild-type (SUMOylation-capable) or K388R (SUMOylation-deficient) PRs and subjected to pathway analysis. Gene sets were validated by RT-qPCR. Recruitment of coregulators and histone methylation levels were determined by chromatin immunoprecipitation. Changes in cell proliferation and survival were determined by MTT and western blotting. Finally, human breast tumor cohort datasets were probed to identify PR-associated gene signatures; metagene analysis was employed to define survival rates in patients whose tumors express a PR gene signature. Results: “SUMO-sensitive” PR target genes (i.e. repressed by PR SUMOylation) primarily include genes required for proliferative and pro-survival signaling. DeSUMOylated K388R receptors are preferentially recruited to enhancer regions of derepressed genes (i.e. MSX2, RGS2, MAP1A, and PDK4) along with the steroid receptor coactivator, CBP, and MLL2, a histone methyltransferase mediator of nucleosome remodeling. PR SUMOylation blocks these events, suggesting that SUMO modification of PR prevents interactions with mediators of early chromatin remodeling at “closed” enhancer regions. SUMO-deficient (phospho-Ser294) PR gene signatures are significantly associated with ERBB2-positive luminal breast tumors and predictive of early metastasis and shortened survival. Treatment with antiprogestin or MEK inhibitor abrogated expression of SUMO-sensitive PR targetgenes and inhibited proliferation in BT-474 (ER+/PR+/ERBB2+) breast cancer cells. Conclusions: We conclude that reversible PR SUMOylation/deSUMOylation profoundly alters target gene selection in breast cancer cells. Phosphorylation-induced PR deSUMOylation favors a permissive chromatin environment via recruitment of CBP and MLL2. Patients whose ER+/PR+ tumors are driven by hyperactive (i.e. derepressed) phospho-PRs may benefit from endocrine (antiestrogen) therapies that contain an antiprogestin. Supplementary files: The supplementary files presented in this dissertation are fully described in the appendices. They include: (A) Antibodies used in this study, (B) PCR primer sets used in this study, (C) Genes differentially regulated by wild-type and SUMO-deficient PR, (D) Overlapping lists of PR-dependent target genes from previously described gene expression microarrays, (E) The PR ligand-dependent (LD) and ligand-independent (LI) KR>WT gene signatures, (F) Breast tumor Oncomine concepts associated with the LD KR>WT gene signature.
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    Phosphorylation-induced structural changes in smooth muscle myosin.
    (2010-07) Kast, David John Edward
    Activation of smooth muscle myosin (SMM) requires phosphorylation of the myosin's regulatory light chain (RLC) to relieve autoinhibitory head-head interactions, but the structural basis of this mechanism in unknown. There are no crystal structures of any fragment of SMM, and there are no crystal structures of any RLC that contains the N-terminal 24 amino acids required for phosphorylation. Site-directed spin labeling of this N-terminal segment, referred to as the phosphorylation domain (PD), showed that phosphorylation increases α-helicity, mobility and solvent accessibility of the PD. A model emerged, where the unphosphorylated RLC is compact with a disordered PD, and phosphorylation causes the PD extend away from the RLC while inducing helical ordering. The goal of this research is to test the hypothesis that the PD functions as a structural switch that changes the structure of RLC upon phosphorylation, and to define these structural changes in atomic detail. Complementary fluorescence resonance energy transfer (FRET) experiments and molecular dynamics (MD) simulations were performed to elucidate structural changes in the phosphorylation domain (PD) of smooth muscle regulatory light chain (RLC). MD simulations on the isolated PD reveal disorder-to-order transition, where residues K11-Q15 are disordered in the unphosphorylated PD but completely α-helical in the phosphorylated PD. A salt bridge formed between R16 and the phosphorylated S19 promotes ordering by stabilizing α-helicity and reducing conformational fluctuations. Consequently, this disorder-to-order transition is regulated by delicate balance between enthalpy and entropy. To elucidate the structural changes of the PD in context with the RLC bound to smooth muscle myosin, donor-acceptor pairs of probes were attached to three site-directed di-Cys mutants of RLC, each having one Cys at position 129 in the C-terminal lobe and the other at position 2, 3, or 7 in the N-terminal PD. Labeled RLC was reconstituted onto myosin S1. Time-resolved FRET demonstrated two simultaneously resolved structural states of the RLC, closed and open, which are present in both unphosphorylated and phosphorylated biochemical states. All three FRET pairs show that phosphorylation shifts the equilibrium toward the open state, increasing its mole fraction by 23%. Molecular dynamics simulations agree with FRET data in remarkable detail, supporting the coexistence of two structural states, with phosphorylation shifting the system toward a more open and mobile structure. This agreement between experiment and simulation validates the additional structural details provided by the MD simulations: In the closed state, the PD is bent onto the surface of the C-terminal lobe, stabilized by two specific interdomain salt bridges. In the open state, the PD is more helical and straight, resides farther from the C-terminal lobe, and is stabilized by a specific intradomain salt bridge. The closed and open states are also present in phosphorylated HMM, while unphosphorylated HMM possess the closed state an intermediate distance distribution. Phosphorylation forces the PD to adopt S1-like states without increasing the mean separation of the two myosin heads. The result is a vivid atomic-resolution model of the molecular mechanism by which phosphorylation activates smooth muscle.
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    Post-translational regulation of mammalian DNA cytidine deaminases.
    (2012-06) Demorest, Zachary Lee
    Our immune system is faced with the challenge of neutralizing the daily bombardment of invading pathogens. This requires two major response mechanisms in order to achieve this task. The innate immune system acts quickly in response to nonspecific bacterial, viral and nucleic acid based antigens to prevent the onset of disease. In contrast, the adaptive immune system functions slowly but has the added advantage of providing memory and long-term immunity to specific pathogens. Two members of the AID/APOBEC family of cytidine deaminases, APOBEC3G (A3G) and activationinduced deaminase (AID), are critical components of the innate and adaptive immune responses. A3G is a potent inhibitor of Human Immunodeficiency Virus type-1 (HIV-1) as well as a number of other endogenous replicative elements that threaten our genomic integrity. This is accomplished by actively mutating cytosines to uracil in the viral genome during replication. These mutations render the virus non-functional and therefore incapable of spreading infection. AID is necessary for our bodies to generate a diverse antibody repertoire. This is achieved through the initiation of class-switch recombination and somatic hypermutation, processes that allow developing B-cells to generate antibodies of varying isotype with a high specificity for a given antigen. Both of these enzymes, AID and A3G, are DNA mutating enzymes with the potential to wreak havoc on our genome and contribute to cancer. The regulation of these enzymes is multifaceted, involving differential transcription, miRNAs, subcellular localization, interactions with regulatory proteins, and post-translational modifications.
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    Regulation and subcellular compartmentalization of ataxin-1 phosphorylation at Serine776.
    (2011-03) Lai, Shaojuan
    Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant cerebellar ataxia caused by the expansion of a CAG repeat encoding an abnormally long polyglutamine tract in Ataxin-1 protein. Although many studies demonstrate that subcellular distribution of Ataxin-1 and protein folding/degradation pathways modulate neurodegeneration, the mechanism of pathogenesis is not completely understood. Phosphorylation of Ataxin-1 at Serine776 (S776) was previously shown to regulate Ataxin-1's functions and SCA1 pathogenicity. In addition, mice expressing human wild type Ataxin-1-[30Q] with a mutation replacing S776 with a phosphomimicking aspartic acid show similar SCA1 pathology as Ataxin-1-[82Q] mice. Here I investigated the mechanism by which phosphorylation of Ataxin-1 at S776 is regulated. I found in the cerebellum a large proportion of Ataxin-1 is phosphorylated at S776 with phosphorylated S776 enriched in the nucleus. While the kinase activity for Ataxin-1 at S776 is localized to the cerebellar cytoplasm, the phosphatase activity is restricted to the nucleus. PP2A was shown to be the phosphatase for phosphorylated S776 Ataxin-1 (Ataxin-1-pS776). 14-3-3, a protein enriched in the cytoplasm, blocks dephosphorylation of Ataxin-1-pS776 by PP2A in the cytoplasm and may affect the shuttling of Ataxin-1 to the nucleus. This work suggests that Ataxin-1 after it is phosphorylated in the cytoplasm, shuttles to the nucleus where it is dephosphorylated by PP2A. The separation of phosphorylation and dephosphorylation of S776-Ataxin-1 into two subcellular compartments may suggest that they regulate different Ataxin-1 functions in different subcellular compartments.