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Jerome E. Roger, Keerthi Ranganath, Hannah Breit, Jessica Chang, Anand Swaroop; GSK3-Mediated Phosphorylation Regulates the Stability of NRL. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5586. doi: https://doi.org/.
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Transcription factor activity can be modulated by post-translational modifications (PTM) such as phosphorylation, SUMOylation, and acetylation. Neural Retina Leucine zipper (NRL) is a basic motif-leucine transcription factor necessary for rod photoreceptor differentiation. Our previous work demonstrated the importance of PTM in regulating NRL transcriptional activity (such as SUMOylation on K20 and phosphorylation on S50). In addition, mutations of codon 50 (S50T) and 51 (P51S) that alter phosphorylation are associated with human autosomal dominant retinitis pigmentosa (ADRP). This study was undertaken to identify the kinase(s) that control NRL phosphorylation and investigate the consequence on NRL function.
HEK293T cells were transfected with different NRL phosphorylation mutants. Cell extracts were analyzed by immunoblotting or immunocytochemistry. In some experiments, transfected cells were treated with MG132 and/or cyclohexamide. In vitro kinase assays were performed.
In silico analysis suggested sequential phosphorylation of NRL by glycogen synthase kinase 3-beta (GSK3β) on S46, T42 and S38. We demonstrated that mutations of S38 through S50 decreased the number of phosphorylated NRL isoforms, and complete phosphorylation of NRL required a priming phosphorylation of S50 by a kinase yet to be identified. We showed by in vitro kinase assay that GSK3β directly phosphorylated NRL. Co-transfection of Nrl mutants with GSK3β confirmed the sequential phosphorylation of NRL from S46 to S38. GSK3β-dependent phosphorylation triggered NRL poly-ubiquitination and degradation by the proteasome, which was blocked with S50 and S38 NRL mutants. Human mutations S50T and P51S showed a longer half-life compared to wild-type (WT) NRL, confirming GSK3β’s role in regulation of NRL protein stability. Exposure of dark-adapted WT mice to bright light (10,000 Lx) for 1 hour caused a rapid decrease in NRL protein but no change in amount of transcript.
Our study suggests that regulation of NRL stability by phosphorylation participates in preserving photoreceptor integrity in the retina under specific stress conditions.
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