Phosphorylation by cyclin-dependent protein kinase 5 of the regulatory subunit of retinal cGMP phosphodiesterase. II. Its role in the turnoff of phosphodiesterase in vivo.
Retinal cGMP phosphodiesterase (PDE) is regulated by Pgamma, the regulatory subunit of PDE, and GTP/Talpha, the GTP-bound alpha subunit of transducin. In the accompanying paper (Matsuura, I., Bondarenko, V. A., Maeda, T., Kachi, S., Yamazaki, M., Usukura, J., Hayashi, F., and Yamazaki, A. (2000) J. Biol. Chem. 275, 32950-32957), we have shown that all known Pgammas contain a specific phosphorylation motif for cyclin-dependent protein kinase 5 (Cdk5) and that the unknown kinase is Cdk5 complexed with its activator. Here, using frog rod photoreceptor outer segments (ROS) isolated by a new method, we show that Cdk5 is involved in light-dependent Pgamma phosphorylation in vivo. Under dark conditions only negligible amounts of Pgamma were phosphorylated. However, under illumination that bleached less than 0.3% of the rhodopsin, approximately 4% of the total Pgamma was phosphorylated in less than 10 s. Pgamma dephosphorylation occurred in less than 1 s after the light was turned off. Analysis of the phosphorylated amino acid, inhibition of Pgamma phosphorylation by Cdk inhibitors in vivo and in vitro, and two-dimensional peptide map analysis of Pgamma phosphorylated in vivo and in vitro indicate that Cdk5 phosphorylates a Pgamma threonine in the same manner in vivo and in vitro. These observations, together with immunological data showing the presence of Cdk5 in ROS, suggest that Cdk5 is involved in light-dependent Pgamma phosphorylation in ROS and that the phosphorylation is significant and reversible. In an homogenate of frog ROS, PDE activated by light/guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) was inhibited by Pgamma alone, but not by Pgamma complexed with GDP/Talpha or GTPgammaS/Talpha. Under these conditions, Pgamma phosphorylated by Cdk5 inhibited the light/GTPgammaS-activated PDE even in the presence of GTPgammaS/Talpha. These observations suggest that phosphorylated Pgamma interacts with and inhibits light/GTPgammaS-activated PDE, but does not interact with GTPgammaS/Talpha in the homogenate. Together, our results strongly suggest that after activation of PDE by light/GTP, Pgamma is phosphorylated by Cdk5 and the phosphorylated Pgamma inhibits GTP/Talpha-activated PDE, even in the presence of GTP/Talpha in ROS.
Hayashi
F
F
Department of Biology, Faculty of Science, Kobe University, Kobe 657, Japan. fhayashi@kobe-u.ac.jp
Matsuura
I
I
Kachi
S
S
Maeda
T
T
Yamamoto
M
M
Fujii
Y
Y
Liu
H
H
Yamazaki
M
M
Usukura
J
J
Yamazaki
A
A
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000378:metabolism; D007700:Kinetics; D008027:Light; D010449:Peptide Mapping; D010766:Phosphorylation; D021122:Protein Subunits; D011898:Ranidae; D012160:Retina / Q000201:enzymology*; D012374:Rod Cell Outer Segment / Q000201:enzymology*
D004791:Enzyme Inhibitors; D021122:Protein Subunits; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases; D015106:3',5'-Cyclic-GMP Phosphodiesterases