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H Mou, RH Cote; Regulation of Rod Photoreceptor Phosphodiesterase (PDE6) by Phosphorylation of Its Catalytic Subunits . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1418.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To examine the hypothesis that phosphorylation of the α and ß catalytic subunits of rod PDE6 (Pαß) alters its catalytic or regulatory properties, as has been demonstrated for other members of the PDE super-family. Method: Bovine rod PDE6 was prepared and purified as described previously [Mou et al., JBC 274:18813 (1999)]. Rod outer segments (ROS) were purified on a sucrose gradient [McDowell, Meth. Neurosci. 15:123 (1993)]. Phosphorylation reactions catalyzed by commercial (PKA, MAP kinase) or endogenous ROS protein kinases were performed using [γ32P]ATP in the presence of phosphatase inhibitors. Hydrolytic and cGMP binding assays were performed as described previously [Cote, Meth. Enzymol. 315:646 (2000)]. Results: The catalytic dimer of rod PDE6 can be phosphorylated in vitro by both PKA or MAP kinase. PKA preferentially phosphorylates the ß subunit at a single site, while MAP kinase phosphorylates both α and ß. An unidentified protein kinase localized to ROS membranes («PDE6 kinase») binds to PDE6 with moderate affinity, and preferentially phosphorylates the α subunit. When the cGMP-binding GAF domains are empty, phosphorylation of PDE6 by PDE6 kinase is enhanced, in contrast to the stimulation of PDE5 phosphorylation by occupancy of its GAF domains by cGMP. Unlike PDE5, phosphorylation of PDE6 fails to significantly alter its catalytic activity. However, when ROS homogenates are pre-incubated with ATP, a low affinity class of cGMP binding sites is converted to a high affinity state; the stoichiometry of cGMP binding doubles to 4 cGMP bound per PDE6 holoenzyme. As PDE6 is progressively purified, the ability of ATP to stimulate cGMP binding is reduced, suggesting the involvement of another protein in this process. Conclusions: These results suggest that an endogenous ROS kinase may regulate rod PDE6 function by a negative feedback mechanism during visual transduction. Whether the ATP-dependent stimulation of cGMP binding allosterically regulates PDE6 or serves to alter the buffering capacity of cGMP in the outer segment remains to be determined.
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