Browsing by Subject "Phosphorylation"
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Item Involvement of c-Jun N-terminal kinase 2 (JNK2) in Endothelin-1 (ET-1) Mediated Neurodegeneration of Retinal Ganglion Cells(ARVO Journals, 2021-05-03) Kodati, Bindu; Stankowska, Dorota L.; Krishnamoorthy, Vignesh R.; Krishnamoorthy, Raghu R.Purpose: The goal of this study was to determine whether JNK2 played a causative role in endothelin-mediated loss of RGCs in mice. Methods: JNK2-/- and wild type (C57BL/6) mice were intravitreally injected in one eye with 1 nmole of ET-1, whereas the contralateral eye was injected with the vehicle. At two time points (two hours and 24 hours) after the intravitreal injections, mice were euthanized, and phosphorylated c-Jun was assessed in retinal sections. In a separate set of experiments, JNK2-/- and wild type mice were intravitreally injected with either 1 nmole of ET-1 or its vehicle and euthanized seven days after injection. Retinal flat mounts were stained with antibodies to the RGC marker, Brn3a, and surviving RGCs were quantified. Axonal degeneration was assessed in paraphenylenediamine stained optic nerve sections. Results: Intravitreal ET-1 administration produced a significant increase in immunostaining for phospho c-Jun in wild type mice, which was appreciably lower in the JNK2 -/- mice. A significant (P < 0.05) 26% loss of RGCs was found in wild type mice, seven days after injection with ET-1. JNK2-/- mice showed a significant protection from RGC loss following ET-1 administration, compared to wild type mice injected with ET-1. A significant decrease in axonal counts and an increase in the collapsed axons was found in ET-1 injected wild type mice eyes. Conclusions: JNK2 appears to play a major role in ET-1 mediated loss of RGCs in mice. Neuroprotective effects in JNK2-/- mice following ET-1 administration occur mainly in the soma and not in the axons of RGCs.Item Phosphorlyation of Annexin A2 is essential for its association with exosomes and for migration, invasion and proliferation in triple negative breast cancer(2018-12) Desai, Priyanka P.; Vishwanatha, Jamboor K.; Basha, Riyaz; Chaudhary, PankajExosomes are membrane enclosed small vesicles that range from 40-120 nm in size and participate in cell-cell communication by transferring proteins to other cells. Annexin A2 (AnxA2), a calcium-dependent phospholipid binding protein, is present on the surface of the exosomes. AnxA2 phosphorylation plays an essential role in many physiological conditions by forming a heterotetrameric complex with p11 or S100A10 on the cell surface. We demonstrate here that the phosphorylation at Tyrosine (Tyr)-23 in the N-terminal region of AnxA2 is consequential for its association with the cell surface. This association increases the migratory, invasive and proliferative capacity of MDA-MB-231 triple negative breast cancer (TNBC) cells. An increase in cell surface AnxA2 further leads to a stronger association of AnxA2 with the exosomal surface. We also demonstrate that AnxA2 enriched exosomes promote proliferative and invasive characteristics of a different recipient cell [CAL (Centre Antoine Lacassagne) - 148]. These results demonstrate that Tyr23 phosphorylation of AnxA2 is pivotal for its association with exosomes and for imparting more malignant characteristics to the other breast cancer cells. Thus, AnxA2 could be used as a targeting approach for developing a treatment of TNBC.Item PHOSPHORYLATION OF THE REGULATORY LIGHT CHAIN PLAYS A DECISIVE ROLE IN CHOOSING BETWEEN ORDER AND CHAOS OF CROSS-BRIDGES IN MICE TIBIALIS MUSCLE(2013-04-12) Duggal, DivyaPurpose: Myosin consists of two heavy chains and four light chains. Two of the light chains, so called Regulatory Light Chains (RLC), get phosphorylated during muscle contraction. The role of this phosphorylation is currently unknown. In this work we describe our attempts to clarify this matter. Methods: A transgenic mouse was produced in which myosin RLC was devoid of serines that are normally necessary for phosphorylation. Glycerinated muscle bundles were dissected from the tibialis muscle of the mouse, and homogenized to produce myofibrils. Phosphorylation status in W.T muscle was preserved by adding phosphatase inhibitors. Essential light chain (the other two light chains of myosin, ELC1) was specifically labeled with SeTau fluorescent dye and exchanged with native ELC1. Cross-bridges of contracting myofibrils were analyzed for an error of the mean orientation. Results: Number of occurrences (histograms) of a given orientation were plotted vs. the mean orientation. The Full Width at Half Maximum (FWHM) of the mean was calculated. FWHM is an index of cross-bridge order/chaos. Small FWHM means order, large FWHM means chaos. During active contraction FWHM values in de-phosphorylated and phosphorylated muscle were 0.363 ± 0.049 and 0.456 ± 0.083, respectively. Two-tailed t test revealed statistically extremely significant difference (P<<0.001, t=4.83 with 63 degrees of freedom). Confidence intervals were from 0.0545 to 0.131. Conclusions: Phosphorylation of RLC causes considerable increase in the cone of angles within which myosin head interacts with thin filaments to produce force.Item POWER STROKE OF SKELETAL MUSCLE CROSS-BRIDGES CONTAINING PHOSPHORYLATED AND DE-PHOSPHORYLATED MYOSIN REGULATORY LIGHT CHAIN(2013-04-12) Midde, KrishnaPurpose: Muscle contracts by cyclic, ATP-driven, reorientations of myosin cross-bridges while they are interacting with actin. It is widely recognized that during force generating step of this interaction, referred to as a power-stroke, myosin head forms a firm attachment to actin, while the torque is provided by the rotation of myosin neck (a cross-bridge consists of a head plus a neck). During contraction, the Regulatory Light Chain of myosin (RLC), which resides on the neck, gets phosphorylated. An important goal of muscle research is to elucidate the role of phosphorylation during active contraction. We set out to measure how the rate of power stroke is affected by phosphorylation in muscle ex vivo. Methods: The rate can only be established when small number of molecules is investigated. This is accomplished by Confocal Fluorescence Correlation Spectroscopy (CFCS). Results: Phosphorylation of RLC speeds up power stroke from ~0.64/s when RLC is de-phosphorylated to ~1.5/s when it is phosphorylated. Conclusions: Using this technique, we show that the state of phosphorylation of the Regulatory Light Chain (RLC) of myosin has a profound effect on kinetics of ex vivo rabbit psoas cross-bridges during contraction.Item The Phosphorylation of Endogenous Substrates by Calcium/Calmodulin-Dependent Protein Kinase II in Pancreatic β-Cells(1998-06-01) Krueger, Kimberly A.; Richard Easom; Rafael Alvarez; S. Dan DimitrijevichKrueger, Kimberly A., The Phosphorylation of Endogenous Substrates by Calcium/Calmodulin-dependent Protein Kinase II in Pancreatic β-cells. Doctor of Philosophy (Biomedical Sciences), June, 1998, 165 pp., 35 illustrations, references, 259 titles. Increasing evidence supports a physiological role for calcium/calmodulin-dependent protein kinase II (CaM kinase II) in the secretion of insulin from pancreatic β-cell. While it has been previously demonstrated that CaM kinase II is activated by glucose in isolated rat islets implicating this enzyme in the secretion process, its cellular targets are unidentified. Potential candidates would likely exhibit strong binding to the enzyme, an association with the cytoskeleton, or an involvement in the secretion process. Based on these criteria, the following study represents an evaluation, in situ, of two proteins to function as substrates for CaM kinase II. Microtubule-associated protein, MAP-2 is one of the best substrates of CaM kinase II in vitro thought to be involved in secretion process. Synapsin I phosphorylation in the neuron by CaM kinase II is essential for neurotransmitter release. Unique to this study, both proteins were determined to be expressed in clonal mouse β-cells (βTC3) and primary rat islet β-cells. By immunoprecipation, in situ phosphorylation of MAP-2 and synapsin I was induced in permeabilized βTC3 cells within a calcium range shown to activate endogenous CaM kinase II under identical conditions. Two-dimensional tryptic phosphopeptide mapping of both proteins revealed that sites phosphorylated by CaM kinase II in vitro, while distinct from sites phosphorylated by protein kinase A in vitro, were largely homologous to those sites phosphorylated in situ upon incubation of the βTC3 cells with increased free calcium. Immunofluorescence verified expression of both proteins in βTC3 cells and pancreatic slices, however, synapsin I exhibited little co-localization with insulin containing dense core granules as demonstrated by immunogold electron microscopy. These data provide evidence that MAP-2 and synapsin I are phosphorylated by CaM kinase II in the pancreatic β-cell in situ. While the data suggest that synapsin I may not be involved in insulin secretion, an association with other known microvesicles of the β-cell, similarly secreted, may be possible. The phosphorylation of these CaM kinase II substrates may reveal an important intermediate step in the mediation of the glucose response in the pancreatic β-cell.