Phosphorylation-dependent interaction with 14-3-3 regulates
Bad trafficking in retinal ganglion cells
Xiangjun Yang 1,
Cheng Luo 1,
Jian Cai 2,
William Pierce 2,
and
Gulgun Tezel 3*
1 Ophthalmology & Visual Sciences, University of Louisville, Louisville, Kentucky, United States
2 Pharmacology & Toxicology, University of Louisville, Louisville, Kentucky, United States
3 Ophthalmology & Visual Sciences, University of Louisville, 301 E. Muhammad Ali Blvd., Louisville, Kentucky, 40202, United States; Anatomical Sciences & Neurobiology, University of Louisville, Louisville, Kentucky, United States
* To whom correspondence should be addressed. E-mail: gulgun.tezel{at}louisville.edu.
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Abstract |
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Purpose: To focus on the proteomic analysis of 14-3-3 proteins and determine their cellular localization and functional role during glaucomatous neurodegeneration.
Methods: Complementary proteomic approaches were utilized to identify phosphorylated proteins in a chronic pressure-induced rat model of glaucoma. To detect interacting proteins, specific protein complexes were eluted using co-immunoprecipitation 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 performed 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 pro-apoptotic 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 inhibition 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.
Key Words:
glaucoma posterior segment, ganglion cell, optic nerve head, apoptosis, mitochondria, protein kinase
