Phosphorylation-dependent interaction with 14-3-3 in the regulation of bad trafficking in retinal ganglion cells.
OBJECTIVE
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.
Yang
Xiangjun
X
Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, Louisville, Kentucky 40202, USA.
Luo
Cheng
C
Cai
Jian
J
Pierce
William M
WM
Tezel
Gülgün
G
D048948:14-3-3 Proteins / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis; D001369:Axons / Q000473:pathology; D015153:Blotting, Western; D002452:Cell Count; D004195:Disease Models, Animal; D015180:Electrophoresis, Gel, Two-Dimensional; D019084:Fluorescent Antibody Technique, Indirect; D005901:Glaucoma / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D020287:In Situ Nick-End Labeling; D008297:Male; D013058:Mass Spectrometry; D009901:Optic Nerve Diseases / Q000378:metabolism* / Q000473:pathology; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D040901:Proteomics; D051381:Rats; D011914:Rats, Inbred BN; D012165:Retinal Ganglion Cells / Q000378:metabolism*; D015854:Up-Regulation; D051025:bcl-Associated Death Protein / Q000378:metabolism*
D048948:14-3-3 Proteins; C494819:BAD protein, human; C494821:Bad protein, rat; D010750:Phosphoproteins; D051025:bcl-Associated Death Protein