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Ju Zhang, Judy Ogilvie; Pattern of Protein Phosphorylation in Wild Type and Rd1 Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5139.
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© ARVO (1962-2015); The Authors (2016-present)
The rd1 mouse retina is a model of retinitis pigmentosa in which rod photoreceptors begin to show abnormality by postnatal day (P) 6 and undergo apoptosis around P11 as a result of a mutation in the Pde6b gene. Pde6 plays a central role in phototransduction in the adult photoreceptor outer segment. Upon light-stimulated activation of PDE6, cGMP levels decrease, resulting in closure of the cyclic nucleotide gated channels (CNGC). In the rd1 retina, a mutation in the Pde6b gene leads to an increase in cGMP levels by P6. Protein Kinase G (PKG) signaling, which is also a downstream of cGMP, has been indicated to be involved in retina degeneration. Here we determined the pattern of protein phosphorylation in wild type (wt) and rd1 mouse retina at P6 and P11.
The pattern of protein phosphorylation in wt and rd1 mice was examined by phosphoprotein enrichment followed by SDS-PAGE and mass spectrometry (MS) analysis. Protein extract from whole retina went through phosphoprotein enrichment column by which phosphorylated protein was retained and then eluted out. The obtained phosphoproteins underwent SDS-PAGE followed by Sypro Ruby and Pro-Q Diamond staining for visualizing total proteins and phosphoproteins, respectively. Bands of interest were cut out and analyzed by MS.
At both P6 and P11, only a few bands show visible differences between wt and rd1 retina. At P6, an apparent shifting occurs on one band around 90KD. MS analysis of this band demonstrates that HSP90α and HSP90β are the most abundant proteins. At P11, the most apparent difference shows on two bands around 16KD and 14KD. MS analysis identified histone protein H3 as the most abundant protein in the16KD band of rd1 . Stathmin 1 protein phosphorylation was also found at this developmental stage.
The downstream signal receptor of cGMP in photoreceptors include CNG channel, PDE6 and PKG. After activated by cGMP, PKG can phosphorylate its downstream substrates and induce further effects. This data here suggests that the HSP90 undergoing possible modification and Stathmin 1 of excessive phosphorylation may be involved in the rd1 retina degeneration process.
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