Phosphorylation by Cyclin-dependent Protein Kinase 5 of the Regulatory Subunit of Retinal cGMP Phosphodiesterase

• Published in: The Journal of biological chemistry Vol. 275; no. 42; pp. 32950 - 32957
• Main Authors: Matsuura, Isao, Bondarenko, Vladimir A., Maeda, Tomoko, Kachi, Shu, Yamazaki, Matsuyo, Usukura, Jiro, Hayashi, Fumio, Yamazaki, Akio
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 20.10.2000; American Society for Biochemistry and Molecular Biology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M000702200

Cyclic GMP phosphodiesterase (PDE) is an essential component in retinal phototransduction. PDE is regulated by Pγ, the regulatory subunit of PDE, and GTP/Tα, the GTP-bound α subunit of transducin. In previous studies (Tsuboi, S., Matsumoto, H., Jackson, K. W., Tsujimoto, K., Williamas, T., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15016–15023; Tsuboi, S., Matsumoto, H., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15024–15029), we showed that Pγ is phosphorylated by a previously unknown kinase (Pγ kinase) in a GTP-dependent manner in photoreceptor outer segment membranes. We also showed that phosphorylated Pγ loses its ability to interact with GTP/Tα, but gains a 10–15 times higher ability to inhibit GTP/Tα-activated PDE than that of nonphosphorylated Pγ. Thus, we propose that the Pγ phosphorylation is probably involved in the recovery phase of phototransduction through shut off of GTP/Tα-activated PDE. Here we demonstrate that all known Pγs preserve a consensus motif for cyclin-dependent protein kinase 5 (Cdk5), a protein kinase believed to be involved in neuronal cell development, and that Pγ kinase is Cdk5 complexed with p35, a neuronal Cdk5 activator. Mutational analysis of Pγ indicates that all known Pγs contain a P-X-T-P-R sequence and that this sequence is required for the Pγ phosphorylation by Pγ kinase. In three different column chromatographies of a cytosolic fraction of frog photoreceptor outer segments, the Pγ kinase activity exactly coelutes with Cdk5 and p35. The Pγ kinase activity (∼85%) is also immunoprecipitated by a Cdk5-specific antibody, and the immunoprecipitate phosphorylates Pγ. Finally, recombinant Cdk5/p35, which were expressed using clones from a bovine retina cDNA library, phosphorylates Pγ in frog outer segment membranes in a GTP-dependent manner. These observations suggest that Cdk5 is probably involved in the recovery phase of phototransduction through phosphorylation of Pγ complexed with GTP/Tα in mature vertebrate retinal photoreceptors.