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Ju Zhang, Judy M Ogilvie; Identification of PKG Substrates in Developing Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1356. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Protein Kinase G (PKG) signaling plays important roles in regulating diverse biological functions including tissue contractility, cell motility, and cell differentiation. The signaling is mediated via phosphorylation of various specific PKG substrates. Although more than sixty PKG substrates have been identified in mouse, only limited studies on the function or identity of PKG substrates in mouse retina have been reported. Furthermore, PKG signaling has been indicated to be involved in mouse retinal degeneration caused by mutations in Phosphodiesterase 6β (PDE6β), which normally functions to break down the PKG activator, cGMP. However, the participant PKG substrates in this process are currently unknown. Therefore, we used mass spectrometry (MS) combined with immunoprecipitation (IP) to identify PKG substrates in mouse retina.
Mouse retinas, harvested at P8, were treated with cGMP analogs: 8-CPT-cGMP, a PKG activator, or Rp-8-PET-cGMP, a PKG inhibitor. After homogenization of the treated retinas, the protein lysate was IPed using an antibody against the PKG substrate consensus sequence. The eluted protein was then loaded to SDS-PAGE gel. After electrophoresis, the gel was stained with Pro-Q Diamond to visualize phosphorylated proteins and subsequently Sypro Ruby to visualize total proteins. Bands showing differential phosphorylation were cut out and proteins identified by MS. Analysis of MS results was performed by BLAST and GPS software and confirmed by Western blotting using IP elutes with specific antibodies against MS identified proteins.
One band showing distinct difference between PKG inhibitor-treated and activator-treated mouse retinas was selected for analysis by MS. Eighty-nine proteins were identified from that band. Among these proteins, 54 of them show a high probability of being phosphorylated by PKG, 15 of which were represented by at least twice as many spectra in the PKG activator-treated retinas compared to those treated with the inhibitor. Western Blotting confirmed identified proteins to be the eluted products from IP.
We demonstrate that the drug treatment method is a useful tool for the identification of PKG substrates that may play critical roles in retinal development and/or visual function.
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