Phosphorylation of the γ Subunit of the Retinal Photoreceptor cGMP Phosphodiesterase by the cAMP-Dependent Protein Kinase and Its Effect on the γ Subunit Interaction with Other Proteins

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Abstract
Cyclic GMP phosphodiesterase, a key enzyme in phototransduction, is composed of Pαβ and two Pγ subunits. Interaction of Pγ with Pαβ or with the α subunit (Tα) of transducin is crucial for the regulation of cGMP phosphodiesterase in retinal photoreceptors. Here we have investigated phosphorylation of Pγ by cAMP-dependent protein kinase and its functional effect on the Pγ interaction with Pαβ or Tα in vitro. Pγ, but not Pγ complexed with Tα (both GTP and GDP forms), is phosphorylated. Measurement of 32P radioactivity in phosphorylated Pγ, analysis of phosphorylated Pγ by laser mass spectrometry, identification of phosphoamino acid, and phosphorylation of mutant forms of Pγ indicate that only threonine 35 in Pγ is phosphorylated. Phosphorylation of Pγ mutants also reveals that the C and N terminals of Pγ which are required for the regulation of Pαβ functions are not involved in the Pγ phosphorylation but that arginine 33, which is ADP-ribosylated by an endogenous ADP-ribosyltransferase, is required for the phosphorylation. Phosphorylated Pγ has a higher inhibitory activity for trypsin-activated cGMP phosphodiesterase than nonphosphorylated Pγ, indicating that the Pγ-Pαβ interaction is affected by Pγ phosphorylation. Nonphosphorylated Pγ inhibits both the GTPase activity of Tα and the binding of a hydrolysis-resistant GTP analogue to Tα, while Pγ phosphorylation reduces these inhibitory activities. These observations suggest that a Pγ domain containing threonine 35 is involved in the Pγ-Tα interaction, and Pγ phosphorylation regulates the Pγ-Tα interaction. Our observation suggests that Pγ phosphorylation by cAMP-dependent protein kinase may function for the regulation of phototransduction in vertebrate rod photoreceptors.