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K. M. Janisch, H. Perry-Friedman, R. Davis, S. H. Tsang; -Subunit of cGMP-Phosphodiesterase6 (PDE6) Phosphorylation Deficient Mutants T22A and T35A Reveal Novel Regulatory Mechanisms in Photoreceptor Signaling. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1113.
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There is a controversy whether or not phosphorylation increases or decreases the ability of PDE6γ to inhibit the catalytic PDE6αß. We re-investigated this question with in vivo studies. We generated transgenic mice expressing a mutation of either one or both of the T22 and T35 sites of the PDE6γ protein, replacing the threonine (T) with alanine (A). Alanine substitution eliminates phosphorylation but is likely to be sufficiently conservative to have little effect itself on the secondary structure of the PDE6γ molecule.
The murine opsin promoter was used to direct expression of wild type, T22A, T35A and T22A/T35A PDE6γ mutant transgenes. The founder mice were then crossed with PDE6gtm1/PDE6gtm1 mice that lacked PDE6γ to generate mice that only synthesize transgenic PDE6γ. Age-matched wild-type (WT) C57BL/6 mice were used as controls. All mice were dark-adapted overnight prior to use in the experiments. Detection of the transferred PDE6γ on nitrocellulose membrane was done either with antibodies specifically recognising the phosphorylated T22 or T35 residue. The membranes were stripped and re-probed with anti-native PDE6γ antibodies for assaying of the total PDE6γ content.
This study is the first demonstration of light induced post-translational modification (i.e. phosphorylation) of PDE6γ in mice. Although different effects of T22 and T35 phosphorylation can be observed using cell-free assay systems, the function of phosphorylation on PDE6γ activity in vivo may be more dynamic and context-dependent than can be replicated in vitro. We demonstrated light-induced phosphorylation of T22 residue of PDE6γ; and probable light-induced dephosphorylation of T35 of PDE6γ in vivo. Studies of mutant mice showed that there is interdependency between the level of phosphorylation in T22 and T35 residues. Light induced post-translational modification of PDE6γ is a novel mechanism in the regulation of photoreceptor signaling.
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