Browsing by Subject "Phosphoproteins"
Now showing 1 - 10 of 11
Results Per Page
Sort Options
Item 14-3-3σ regulation of and interaction with YAP1 in acquired gemcitabine resistance via promoting ribonucleotide reductase expression(Impact Journals, LLC, 2016-04-05) Qin, Li; Dong, Zizheng; Zhang, Jian-Ting; Department of Pharmacology and Toxicology, IU School of MedicineGemcitabine is an important anticancer therapeutics approved for treatment of several human cancers including locally advanced or metastatic pancreatic ductal adenocarcinoma (PDAC). Its clinical effectiveness, however, is hindered by existence of intrinsic and development of acquired resistances. Previously, it was found that 14-3-3σ expression associates with poor clinical outcome of PDAC patients. It was also found that 14-3-3σ expression is up-regulated in gemcitabine resistant PDAC cells and contributes to the acquired gemcitabine resistance. In this study, we investigated the molecular mechanism of 14-3-3σ function in gemcitabine resistance and found that 14-3-3σ up-regulates YAP1 expression and then binds to YAP1 to inhibit gemcitabine-induced caspase 8 activation and apoptosis. 14-3-3σ association with YAP1 up-regulates the expression of ribonucleotide reductase M1 and M2, which may mediate 14-3-3σ/YAP1 function in the acquired gemcitabine resistance. These findings suggest a possible role of YAP1 signaling in gemcitabine resistance.Item Autophagy is a gatekeeper of hepatic differentiation and carcinogenesis by controlling the degradation of Yap(Nature Research, 2018-11-23) Lee, Youngmin A.; Noon, Luke A.; Akat, Kemal M.; Ybanez, Maria D.; Lee, Ting-Fang; Berres, Marie-Luise; Fujiwara, Naoto; Goossens, Nicolas; Chou, Hsin-I; Parvin-Nejad, Fatemeh P.; Khambu, Bilon; Kramer, Elisabeth G.M.; Gordon, Ronald; Pfleger, Cathie; Germain, Doris; John, Gareth R.; Campbell, Kirk N.; Yue, Zhenyu; Yin, Xiao-Ming; Cuervo, Ana Maria; Czaja, Mark J.; Fiel, M. Isabel; Hoshida, Yujin; Friedman, Scott L.; Pathology and Laboratory Medicine, School of MedicineActivation of the Hippo pathway effector Yap underlies many liver cancers, however no germline or somatic mutations have been identified. Autophagy maintains essential metabolic functions of the liver, and autophagy-deficient murine models develop benign adenomas and hepatomegaly, which have been attributed to activation of the p62/Sqstm1-Nrf2 axis. Here, we show that Yap is an autophagy substrate and mediator of tissue remodeling and hepatocarcinogenesis independent of the p62/Sqstm1-Nrf2 axis. Hepatocyte-specific deletion of Atg7 promotes liver size, fibrosis, progenitor cell expansion, and hepatocarcinogenesis, which is rescued by concurrent deletion of Yap. Our results shed new light on mechanisms of Yap degradation and the sequence of events that follow disruption of autophagy, which is impaired in chronic liver disease.Item Designing Visible Light-Cured Thiol-Acrylate Hydrogels for Studying the HIPPO Pathway Activation in Hepatocellular Carcinoma Cells(Wiley Blackwell (John Wiley & Sons), 2016-04) Lin, Tsai-Yu; Bragg, John C.; Lin, Chien-Chi; Department of Biomedical Engineering, School of Engineering and TechnologyVarious polymerization mechanisms have been developed to prepare peptide-immobilized poly(ethylene glycol) (PEG) hydrogels, a class of biomaterials suitable for studying cell biology in vitro. Here, a visible light mediated thiol-acrylate photopolymerization scheme is reported to synthesize dually degradable PEG-peptide hydrogels with controllable crosslinking and degradability. The influence of immobilized monothiol pendant peptide is systematically evaluated on the crosslinking of these hydrogels. Further, methods are proposed to modulate hydrogel crosslinking, including adjusting concentration of comonomer or altering the design of multifunctional peptide crosslinker. Due to the formation of thioether ester bonds, these hydrogels are hydrolytically degradable. If the dithiol peptide linkers used are susceptible to protease cleavage, these thiol-acrylate hydrogels can be designed to undergo partial proteolysis. The differences between linear and multiarm PEG-acrylate (i.e., PEGDA vs PEG4A) are also evaluated. Finally, the use of the mixed-mode thiol-acrylate PEG4A-peptide hydrogels is explored for in situ encapsulation of hepatocellular carcinoma cells (Huh7). The effects of matrix stiffness and integrin binding motif (e.g., RGDS) on Huh7 cell growth and HIPPO pathway activation are studied using PEG4A-peptide hydrogels. This visible light poly-merized thiol-acrylate hydrogel system represents an alternative to existing light-cured hydrogel platforms and shall be useful in many biomedical applications.Item Differential RNA splicing as a potentially important driver mechanism in multiple myeloma(Ferrata Storti Foundation, 2021-03-01) Bauer, Michael A.; Ashby, Cody; Wardell, Christopher; Boyle, Eileen M.; Ortiz, Maria; Flynt, Erin; Thakurta, Anjan; Morgan, Gareth; Walker, Brian A.; Medicine, School of MedicineDisruption of the normal splicing patterns of RNA is a major factor in the pathogenesis of a number of diseases. Increasingly research has shown the strong influence that splicing patterns can have on cancer progression. Multiple Myeloma is a molecularly heterogeneous disease classified by the presence of key translocations, gene expression profiles and mutations but the splicing patterns in MM remains largely unexplored. We take a multifaceted approach to define the extent and impact of alternative splicing in MM. We look at the spliceosome component, SF3B1, with hotspot mutations (K700E and K666T/Q) shown to result in an increase in alternative splicing in other cancers. We discovered a number of differentially spliced genes in comparison of the SF3B1 mutant and wild type samples that included, MZB1, DYNLL1, TMEM14C and splicing related genes DHX9, CLASRP, and SNRPE. We identified a broader role for abnormal splicing showing clear differences in the extent of novel splice variants in the different translocation groups. We show that a high number of novel splice loci is associated with adverse survival and an ultra-high risk group. The enumeration of patterns of alternative splicing has the potential to refine MM classification and to aid in the risk stratification of patients.Item Dual regulation of voltage- and ligand-gated calcium channels by collapsin response mediator protein 2(2013-10-07) Brittain, Joel Matthew; Khanna, Rajesh; Cummins, Theodore R.; Oxford, G. S.; Quilliam, Lawrence; Thurmond, Debbie C.Synaptic transmission is coordinated by a litany of protein-protein interactions that rely on the proper localization and function of pre- and post-synaptic Ca2+ channels. The axonal guidance/specification collapsin response mediator protein-2 (CRMP-2) was identified as a potential partner of the pre-synaptic N-type voltage-gated Ca2+ channel (CaV2.2). CRMP-2 bound directly to CaV2.2 in two regions; the channel domain I-II intracellular loop and the distal C-terminus. Both proteins co-localized within presynaptic sites in hippocampal neurons. Overexpression in hippocampal neurons of a CRMP-2 protein fused to EGFP caused a significant increase in Ca2+ channel current density whereas lentivirus-mediated CRMP-2 knockdown abolished this effect. Cell surface biotinylation studies showed an increased number of CaV2.2 at the cell surface in CRMP-2–overexpressing neurons. Both activity- and CRMP-2-phosphoryation altered the interaction between CaV2.2 and CRMP-2. I identified a CRMP-2-derived peptide (called CBD3) that bound CaV2.2 and effectively disrupted the interaction between CaV2.2 and CRMP-2. CBD3 peptide fused to the HIV TAT protein (TAT-CBD3) decreased neuropeptide release from sensory neurons and excitatory synaptic transmission in dorsal horn neurons, and reversed neuropathic hypersensitivity produced by an antiretroviral drug. Unchecked Ca2+ influx via N-methyl-D-aspartate receptors (NMDARs) has been linked to activation of neurotoxic cascades culminating in cell death (i.e. excitotoxicity). CRMP-2 was suggested to affect NMDAR trafficking and possibly involved in neuronal survival following excitotoxicity. Based upon these studies, I hypothesized that a peptide from CRMP2 could preserve neurons in the face of excitotoxic challenges. Lentiviral–mediated CRMP2 knockdown or treatment with TAT-CBD3 blocked neuronal death following glutamate exposure likely via blunting toxicity from NMDAR-mediated delayed calcium deregulation. TAT-CBD3 induced internalization of the NMDAR subunit NR2B in dendritic spines without altering somal surface expression. TAT-CBD3 reduced NMDA-mediated Ca2+-influx and currents in cultured neurons. The presented work validates CRMP-2 as a novel modulator of pre- and post-synaptic Ca2+ channels and provides evidence that the TAT-CBD3 peptide could be useful as a potential therapeutic for both chronic neuropathic pain and excitotoxicity following stroke or other neuronal insults.Item Hypoxic conditions differentially regulate TAZ and YAP in cancer cells(Elsevier, 2014-11-15) Yan, Libo; Cai, Qingchun; Xu, Yan; Department of Obstetrics & Gynecology, IU School of MedicineThe Hippo-YAP pathway is altered and implicated as an oncogenic signaling pathway in many human cancers. Hypoxia is an important microenvironmental factor that promotes tumorigenesis. However, the effects of hypoxia on the two most important Hippo-YAP effectors, YAP (Yes-associated protein) and TAZ (transcriptional co-activator with PDZ-binding motif), have not been reported. In this work, we demonstrated that TAZ was functionally involved in cell proliferation and/or migration in epithelial ovarian cancer (EOC) or human ovarian surface epithelial (HOSE) cells. Hypoxic conditions (1% O2 or hypoxia mimics) induced a reduction of YAP phosphorylation (S127) and total YAP expression in EOC cell lines OVCAR5 and SKOV3. However, these conditions up-regulated levels of S69 phosphorylated TAZ in EOC cells. The known TAZ kinases, Lats1 and Akt, were unlikely to be involved in up-regulated pTAZ by hypoxic conditions. Together, our data revealed new and differential regulating mechanisms of TAZ and YAP in cancer cells by hypoxia conditions.Item Interplay between collapsin response mediator protein 2 (CRMP2) phosphorylation and sumoylation modulates NaV1.7 trafficking(2015-07-06) Dustrude, Erik Thomas; Brustovetsky, Nickolay; Khanna, Rajesh; Cummins, Theodore R.; Jerde, Travis; Obukhov, AlexanderThe voltage-gated sodium channel Nav1.7 has gained traction as a pain target with recognition that loss-of-function mutations in SCN9A, the gene encoding Nav1.7, are associated with congenital insensitivity to pain, whereas gain-of-function mutations produce distinct pain syndromes due to increased Nav1.7 activity. Selective inhibition of Nav1.7 is fundamental to modulating pain via this channel. Understanding the regulation of Nav1.7 at the cellular and molecular level is critical for advancing better therapeutics for pain. Although trafficking of Nav1.7 remains poorly understood, recent studies have begun to investigate post-translational modifications of Navs and/or auxiliary subunits as well as protein-protein interactions as Nav-trafficking mechanisms. Here, I tested if post-translational modifications of a novel Nav1.7-interacting protein, the axonal collapsin response mediator protein 2 (CRMP2) by small ubiquitin-like modifier (SUMO) and phosphorylation could affect Nav trafficking and function. Expression of a CRMP2 SUMOylation incompetent mutant (CRMP2-K374A) in neuronal model CAD cells, which express predominantly Nav1.7 currents, led to a significant reduction in huwentoxin-IV-sensitive Nav1.7 currents. Increasing deSUMOylation with sentrin/SUMO-specific protease SENP1 or SENP2 in wildtype CRMP2-expressing CAD cells decreased Nav1.7 currents. Consistent with reduced current density, biotinylation revealed significant reduction in surface Nav1.7 levels of CAD cells expressing CRMP2-K374A or SENP proteins. Diminution of Nav1.7 sodium current was recapitulated in sensory neurons expressing CRMP2-K374A. Because CRMP2 functions are regulated by its phosphorylation state, I next investigated possible interplay between phosphorylation and SUMOylation of CRMP2 on Nav1.7. Phosphorylation of CRMP2 by cyclin dependent kinase 5 (Cdk5) was necessary for maintaining Nav1.7 surface expression and current density whereas phosphorylation by Fyn kinase reduced CRMP2 SUMOylation and Nav1.7 current density. Binding to Nav1.7 was decreased following (i) loss of CRMP2 SUMOylation, (ii) loss of CRMP2 phosphorylation by Cdk5, or (iii) gain of CRMP2 phosphorylation by Fyn. Altering CRMP2 modification events simultaneously was not synergistic in reducing Nav1.7 currents, suggesting that Nav1.7 co-opts multiple CRMP2 modifications for regulatory control of this channel. Loss of either CRMP2 SUMOylation or Cdk5 phosphorylation triggered Nav1.7 internalization involving E3 ubiquitin ligase Nedd4-2 as well as endocytosis adaptor proteins Numb and Eps15. Collectively, my findings identify a novel mechanism for regulation of Nav1.7.Item Investigating the molecular mechanism of phospholamban regulation of the Ca²-pump of cardiac sarcoplasmic reticulum(2010-12) Akin, Brandy Lee; Jones, Larry R.; Field, Loren J.; Hudmon, Andrew; Hurley, Thomas D., 1961-; Roach, Peter J.The Ca2+ pump or Ca2+-ATPase of cardiac sarcoplasmic reticulum, SERCA2a, is regulated by phospholamban (PLB), a small inhibitory phosphoprotein that decreases the apparent Ca2+ affinity of the enzyme. We propose that PLB decreases Ca2+ affinity by stabilizing the Ca2+-free, E2·ATP state of the enzyme, thus blocking the transition to E1, the high Ca2+ affinity state required for Ca2+ binding and ATP hydrolysis. The purpose of this dissertation research is to critically evaluate this idea using series of cross-linkable PLB mutants of increasing inhibitory strength (N30C-PLB < PLB3 < PLB4). Three hypotheses were tested; each specifically designed to address a fundamental point in the mechanism of PLB action. Hypothesis 1: SERCA2a with PLB bound is catalytically inactive. The catalytic activity of SERCA2a irreversibly cross-linked to PLB (PLB/SER) was assessed. Ca2+-ATPase activity, and formation of the phosphorylated intermediates were all completely inhibited. Thus, PLB/SER is entirely catalytically inactive. Hypothesis 2: PLB decreases the Ca2+ affinity of SERCA2a by competing with Ca2+ for binding to SERCA2a. The functional effects of N30C-PLB, PLB3, and PLB4 on Ca2+-ATPase activity and phosphoenzyme formation were measured, and correlated with their binding interactions with SERCA2a measured by chemical cross-linking. Successively higher Ca2+ concentrations were required to both activate the enzyme co-expressed with N30C-PLB, PLB3, and PLB4 and to dissociate N30C-PLB, PLB3, and PLB4 from SERCA2a, suggesting competition between PLB and Ca2+ for binding to SERCA2a. This was confirmed with the Ca2+ pump mutant, D351A, which is catalytically inactive but retains strong Ca2+ binding. Increasingly higher Ca2+ concentrations were also required to dissociate N30C-PLB, PLB3, and PLB4 from D351A, demonstrating directly that PLB competes with Ca2+ for binding to the Ca2+ pump. Hypothesis 3: PLB binds exclusively to the Ca2+-free E2 state with bound nucleotide (E2·ATP). Thapsigargin, vanadate, and nucleotide effects on PLB cross-linking to SERCA2a were determined. All three PLB mutants bound preferentially to E2 state with bound nucleotide (E2·ATP), and not at all to the thapsigargin or vanadate bound states. We conclude that PLB inhibits SERCA2a activity by stabilizing a unique E2·ATP conformation that cannot bind Ca2+.Item Na+, K+-Pump/Phospholemman Are Concentrated at Intercalated Discs for Conduction(American Heart Association, 2022) Chen, Zhenhui; Sung, Yen-Ling; Chen, Peng-Sheng; Li, Xiaochun; Guo, Jin; Biostatistics and Health Data Science, School of MedicineItem REGULATION OF CHOP TRANSLATION IN RESPONSE TO eIF2 PHOSPHORYLATION AND ITS ROLE IN CELL FATE(2012-05) Palam, Lakshmi Reddy; Wek, Ronald C.; Herring, Brian P.; Harris, Robert A. (Robert Allison), 1939-; Skalnik, David G.In response to different environmental stresses, phosphorylation of eukaryotic initiation factor-2 (eIF2) rapidly reduces protein synthesis, which lowers energy expenditure and facilitates reprogramming of gene expression to remediate stress damage. Central to the changes in gene expression, eIF2 phosphorylation also enhances translation of ATF4, a transcriptional activator of genes subject to the Integrated Stress Response (ISR). The ISR increases the expression of genes important for alleviating stress, or alternatively triggering apoptosis. One ISR target gene encodes the transcriptional regulator CHOP whose accumulation is critical for stress-induced apoptosis. In this dissertation research, I show that eIF2 phosphorylation induces preferential translation of CHOP by a mechanism involving a single upstream ORF (uORF) located in the 5’-leader of the CHOP mRNA. In the absence of stress and low eIF2 phosphorylation, translation of the uORF serves as a barrier that prevents translation of the downstream CHOP coding region. Enhanced eIF2 phosphorylation during stress facilitates ribosome bypass of the uORF, and instead results in the translation of CHOP. Stable cell lines were also constructed that express CHOP transcript containing the wild type uORF or deleted for the uORF and each were analyzed for expression changes in response to the different stress conditions. Increased CHOP levels due to the absence of inhibitory uORF sensitized the cells to stress-induced apoptosis when compared to the cells that express CHOP mRNA containing the wild type uORF. This new mechanism of translational control explains how expression of CHOP and the fate of cells are tightly linked to the levels of phosphorylated eIF2 and stress damage.