Phosphorylation by cyclin-dependent protein kinase 5 of the regulatory subunit of retinal cGMP phosphodiesterase I.: Identification of the kinase and its role in the turnoff of phosphodiesterase in vitro

Cyclic GMP phosphodiesterase (PDE) is an essential component in retinal phototransduction. PDE is regulated by P gamma, the regulatory subunit of PDE, and GTP/T alpha, the GTP-bound cu 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 P gamma is phosphorylated by a previously unknown kinase (Py kinase) in a GTP-dependent manner in photoreceptor outer segment membranes. We also showed that phosphorylated P gamma loses its ability to interact with GTP/T alpha, but gains a 10-15 times higher ability to inhibit GTP/T alpha -activated PDE than that of nonphosphorylated P gamma. Thus, we propose that the P gamma phosphorylation is probably involved in the recovery phase of phototransduction through shut off of GTP/T alpha -activated PDE. Here we demonstrate that all known P gammas preserve a consensus motif for cyclin-dependent protein kinase 5 (Cdk5), a protein kinase believed to be involved in neuronal cell development, and that P gamma kinase is Cdk5 complexed with p35, a neuronal Cdk5 activator. Mutational analysis of P gamma indicates that all known P gammas contain a P-X-T-P-R sequence and that this sequence is required for the P gamma phosphorylation by P gamma kinase. In three different column chromatographies of a cytosolic fraction of frog photoreceptor outer segments, the P gamma kinase activity exactly coelutes with Cdk5 and p35. The P gamma kinase activity (similar to 85%) is also immunoprecipitated by a Cdk5-specific antibody, and the immunoprecipitate phosphorylates P gamma. Finally, recombinant Cdk5/p35, which were expressed using clones from a bovine retina cDNA library, phosphorylates P gamma 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 P gamma complexed with GTP/T alpha in mature vertebrate retinal photoreceptors.