Phosphorylation-Dependent Interaction with 14-3-3 in the Regulation of Bad Trafficking in Retinal Ganglion Cells
Open Access
- 1 June 2008
- journal article
- glaucoma
- Published by Association for Research in Vision and Ophthalmology (ARVO) in Investigative Ophthalmology & Visual Science
- Vol. 49 (6), 2483-2494
- https://doi.org/10.1167/iovs.07-1344
Abstract
Purpose. To focus on the proteomic analysis of 14-3-3 proteins and to determine their cellular localization and functional role during glaucomatous neurodegeneration. methods. Complementary proteomic approaches were used to identify phosphorylated proteins in a chronic pressure-induced rat model of glaucoma. To detect interacting proteins, specific protein complexes were eluted using coimmunoprecipitation and recombinant protein-based affinity pull-down for subsequent mass spectrometric analysis. Western blot analysis was performed for validation of the proteomic findings, and immunohistochemical analysis of rat eyes and human donor eyes determined the cellular localization of 14-3-3 proteins. In addition, in vivo treatment experiments were conducted using JNK and protein phosphatase inhibitors. results. Findings of mass spectrometry, Western blotting, and tissue immunolabeling revealed the presence of different 14-3-3 isotopes in RGCs and their up-regulation and phosphorylation during glaucomatous neurodegeneration. Consecutive experiments through proteomic analysis identified various proteins interacting with 14-3-3, which included calmodulin and a proapoptotic member of the Bcl-2 family, Bad; 14-3-3 was found to keep phospho-Bad sequestered in the cytoplasm. However, this association was disrupted in ocular hypertensive eyes in correlation with Bad dephosphorylation and 14-3-3 phosphorylation, thereby leading to mitochondrial translocation of Bad for apoptotic function. Inhibition of JNK activity and of protein phosphatase activity complementarily secured the 14-3-3-scaffold of Bad in the cytoplasm and preserved optic nerve axons in ocular hypertensive eyes. conclusions. Findings of this in vivo study identify that an important protein family associated with checkpoint control pathways, 14-3-3, is involved in cellular signaling during glaucomatous neurodegeneration in a phosphorylation-dependent manner.This publication has 43 references indexed in Scilit:
- Proteomic Identification of Oxidatively Modified Retinal Proteins in a Chronic Pressure-Induced Rat Model of GlaucomaInvestigative Ophthalmology & Visual Science, 2005
- Effects of elevated intraocular pressure on mouse retinal ganglion cellsVision Research, 2005
- JNK promotes Bax translocation to mitochondria through phosphorylation of 14-3-3 proteinsThe EMBO Journal, 2004
- The Dimeric Versus Monomeric Status of 14-3-3ζ Is Controlled by Phosphorylation of Ser58 at the Dimer InterfaceOnline Journal of Public Health Informatics, 2003
- Cell-Permeable Peptide Inhibitors of JNKDiabetes, 2001
- The Mechanisms of hsp27 Antibody-Mediated Apoptosis in Retinal Neuronal CellsJournal of Neuroscience, 2000
- Movement of Bax from the Cytosol to Mitochondria during ApoptosisThe Journal of cell biology, 1997
- Cytosol-to-membrane redistribution of Bax and Bcl-XLduring apoptosisProceedings of the National Academy of Sciences of the United States of America, 1997
- A Rat Model of Chronic Pressure-induced Optic Nerve DamageExperimental Eye Research, 1997
- Interaction of 14-3-3 with Signaling Proteins Is Mediated by the Recognition of PhosphoserineCell, 1996