Mutations associated with retinopathies alter mitogen-activatedprotein kinase-induced phosphorylation of neural retina leucine-zipper

[摘要] Purpose: Neural retina leucine-zipper (NRL), a member of the basicmotif leucine zipper family of transcription factors, is preferentiallyexpressed in rod photoreceptors of the mammalian retina. Mutations inNRL are associated with retinopathies; many of these are suggested tochange phosphorylation status and alter NRL-mediated transactivation ofrhodopsin promoter. The purpose of this study was to identify potentialkinases responsible for the phosphorylation of NRL and determine if suchkinase-dependent phosphorylation is altered in disease-associated NRLmutations.Methods: Metabolic labeling with 33P-orthophosphate was usedto study phosphorylation of NRL in transfected COS-1 cells. NRL or NRLmutants were expressed as glutathione S-transferase (GST)-fusionproteins and used as substrate to screen various kinases by in vitrophosphorylation assays. CV-1 cells were co-transfected with rhodopsinpromoter-reporter construct and expression plasmids, with or withoutspecific mitogen-activated protein kinase (MAPK) inhibitors, to examinetheir effect on NRL-mediated transactivation. Expression of activatedMAPKs in postnatal mice retina was determined by immunoblot analysis.Results: Metabolic labeling of NRL produces multiple phosphorylatedprotein bands in transfected COS-1 cells. Fewer but more intenseradiolabeled bands are observed for NRL-S50T, -S50A, and -P51L mutantscompared to wild-type NRL. We show that MAPK2 and p38 induce specificphosphorylation of NRL, but this pattern is altered in NRL mutants.Immunoblot analysis of extracts from developing mouse retina revealsenhanced expression of activated MAPK2 at postnatal day 0-3, concordantwith the reported phosphorylation pattern of NRL in vivo. Inhibition ofMAPK signaling pathways decreases NRL and CRX -mediated synergisticactivation of rhodopsin promoter in transfected CV-1 cells.Conclusions: Our results suggest that multiple MAPKs canphosphorylate NRL and this phosphorylation pattern is altered bydisease-associated NRL mutations. As inhibition of MAPK signalingpathways decreases NRL-mediated transactivation of rhodopsin promoter,we propose that phosphorylation changes associated with NRL mutationsperturb gene expression in rods, leading to photoreceptor degenerationin retinopathies.

[学科分类] 生物化学/生物物理