title: 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.
abstract: Cyclic GMP phosphodiesterase (PDE) is an essential component in retinal phototransduction. PDE is regulated by Pgamma, the regulatory subunit of PDE, and GTP/Talpha, the GTP-bound alpha 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 Pgamma is phosphorylated by a previously unknown kinase (Pgamma kinase) in a GTP-dependent manner in photoreceptor outer segment membranes. We also showed that phosphorylated Pgamma loses its ability to interact with GTP/Talpha, but gains a 10-15 times higher ability to inhibit GTP/Talpha-activated PDE than that of nonphosphorylated Pgamma. Thus, we propose that the Pgamma phosphorylation is probably involved in the recovery phase of phototransduction through shut off of GTP/Talpha-activated PDE. Here we demonstrate that all known Pgammas preserve a consensus motif for cyclin-dependent protein kinase 5 (Cdk5), a protein kinase believed to be involved in neuronal cell development, and that Pgamma kinase is Cdk5 complexed with p35, a neuronal Cdk5 activator. Mutational analysis of Pgamma indicates that all known Pgammas contain a P-X-T-P-R sequence and that this sequence is required for the Pgamma phosphorylation by Pgamma kinase. In three different column chromatographies of a cytosolic fraction of frog photoreceptor outer segments, the Pgamma kinase activity exactly coelutes with Cdk5 and p35. The Pgamma kinase activity ( approximately 85%) is also immunoprecipitated by a Cdk5-specific antibody, and the immunoprecipitate phosphorylates Pgamma. Finally, recombinant Cdk5/p35, which were expressed using clones from a bovine retina cDNA library, phosphorylates Pgamma 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 Pgamma complexed with GTP/Talpha in mature vertebrate retinal photoreceptors.
authors.affiliation: Departments of Ophthalmology and Pharmacology, the Kresge Eye Institute, Wayne State University, School of Medicine, Detroit, Michigan 48201, USA.
authors.forename: I
authors.initials: I
authors.lastname: Matsuura
authors.forename: V A
authors.initials: VA
authors.lastname: Bondarenko
authors.forename: T
authors.initials: T
authors.lastname: Maeda
authors.forename: S
authors.initials: S
authors.lastname: Kachi
authors.forename: M
authors.initials: M
authors.lastname: Yamazaki
authors.forename: J
authors.initials: J
authors.lastname: Usukura
authors.forename: F
authors.initials: F
authors.lastname: Hayashi
authors.forename: A
authors.initials: A
authors.lastname: Yamazaki
mesh_terms: D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D021122:Protein Subunits; D011898:Ranidae; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012160:Retina / Q000201:enzymology*; D017949:Retinal Cone Photoreceptor Cells / Q000201:enzymology; D017948:Retinal Rod Photoreceptor Cells / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015293:Transducin / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D051360:Cyclin-Dependent Kinase 5; C495905:CDK5 protein, human; C495906:Cdk5 protein, mouse; D018844:Cyclin-Dependent Kinases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D015293:Transducin