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 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.
Matsuura
I
I
Departments of Ophthalmology and Pharmacology, the Kresge Eye Institute, Wayne State University, School of Medicine, Detroit, Michigan 48201, USA.
Bondarenko
V A
VA
Maeda
T
T
Kachi
S
S
Yamazaki
M
M
Usukura
J
J
Hayashi
F
F
Yamazaki
A
A
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
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