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 pho totransduction. PDE is regulated by P gamma, the regulatory subunit of PDE, and GTP/T alpha, the GTP-bound cu subunit of transducin. In previous studi es (Tsuboi, S., Matsumoto, H., Jackson, K. W., Tsujimoto, K., Williamas, T. , and Yamazaki, A. (1994) J. Biol. Chem. 269, 15016-15023; Tsuboi, S., Mats umoto, H., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15024-15029), we sho wed that P gamma is phosphorylated by a previously unknown kinase (Py kinas e) in a GTP-dependent manner in photoreceptor outer segment membranes. We a lso showed that phosphorylated P gamma loses its ability to interact with G TP/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 d emonstrate that all known P gammas preserve a consensus motif for cyclin-de pendent protein kinase 5 (Cdk5), a protein kinase believed to be involved i n neuronal cell development, and that P gamma kinase is Cdk5 complexed with p35, a neuronal Cdk5 activator. Mutational analysis of P gamma indicates t hat 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 dif ferent column chromatographies of a cytosolic fraction of frog photorecepto r outer segments, the P gamma kinase activity exactly coelutes with Cdk5 an d p35. The P gamma kinase activity (similar to 85%) is also immunoprecipita ted by a Cdk5-specific antibody, and the immunoprecipitate phosphorylates P gamma. Finally, recombinant Cdk5/p35, which were expressed using clones fr om a bovine retina cDNA library, phosphorylates P gamma in frog outer segme nt membranes in a GTP-dependent manner. These observations suggest that Cdk 5 is probably involved in the recovery phase of phototransduction through p hosphorylation of P gamma complexed with GTP/T alpha in mature vertebrate r etinal photoreceptors.