Phosphorylation by cyclin-dependent protein kinase 5 of the regulatory subunit of retinal cGMP phosphodiesterase II. Its role in the turnoff of phosphodiesterase in vivo

Retinal cGMP phosphodiesterase (PDE) is regulated by P gamma, the regulator y subunit of PDE, and GTP/T alpha, the GTP-bound cu subunit of transducin. In the accompanying paper (Matsuura, I., Bondarenko, V. A., Maeda, T., Kach i, S., Yamazahi, M., Usukura, J., Hayashi, F., and Yamazaki, A. (2000) J. B iol. Chem. 275, 32950-32957), we have shown that all known P gammas contain a specific phosphorylation motif for cyclin-dependent protein kinase 5 (Cd k5) and that the unknown kinase is Cdk5 complexed with its activator. Here, using frog rod photoreceptor outer segments (ROS) isolated by a new method , we show that Cdk5 is involved in light-dependent P gamma phosphorylation in vivo. Under dark conditions only negligible amounts of P gamma were phos phorylated. However, under illumination that bleached less than 0.3% of the rhodopsin, similar to4% of the total P gamma was phosphorylated in less th an 10 s. P gamma dephosphorylation occurred in less than 1 s after the ligh t was turned off. Analysis of the phosphorylated amino acid, inhibition of P gamma phosphorylation by Cdk inhibitors in vivo and in vitro, and two-dim ensional peptide map analysis of P gamma phosphorylated in vivo and in vitr o indicate that Cdk5 phosphorylates a P gamma threonine in the same manner in vivo and in vitro. These observations, together with immunological data showing the presence of Cdk5 in ROS, suggest that Cdk5 is involved in light -dependent P gamma phosphorylation in ROS and that the phosphorylation is s ignificant and reversible. In an homogenate of frog ROS, PDE activated by l ight/guanosine 5'-O-(3-thiotriphosphate) (GTP gammaS) was inhibited by P ga mma alone, but not by P gamma complexed with GDP/T alpha or GTP gammaS/T al pha. Under these conditions, Py phosphorylated by Cdk5 inhibited the light/ GTP gammaS-activated PDE even in the presence of GTP gammaS/T alpha. These observations suggest that phosphorylated P gamma interacts with and inhibit s light/GTP gammaS-activated PDE, but does not interact with GTP gammaS/T a lpha in the homogenate. Together, our results strongly suggest that after a ctivation of PDE by light/GTP, P gamma is phosphorylated by Cdk5 and the ph osphorylated P gamma inhibits GTP/T alpha -activated PDE, even in the prese nce of GTP/T alpha in ROS.