PMID- 10884379
OWN - NLM
STAT- MEDLINE
DCOM- 20001120
LR  - 20210209
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 275
IP  - 42
DP  - 2000 Oct 20
TI  - 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.
PG  - 32958-65
AB  - 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.
FAU - Hayashi, F
AU  - Hayashi F
AD  - Department of Biology, Faculty of Science, Kobe University, Kobe 657, Japan. 
      fhayashi@kobe-u.ac.jp
FAU - Matsuura, I
AU  - Matsuura I
FAU - Kachi, S
AU  - Kachi S
FAU - Maeda, T
AU  - Maeda T
FAU - Yamamoto, M
AU  - Yamamoto M
FAU - Fujii, Y
AU  - Fujii Y
FAU - Liu, H
AU  - Liu H
FAU - Yamazaki, M
AU  - Yamazaki M
FAU - Usukura, J
AU  - Usukura J
FAU - Yamazaki, A
AU  - Yamazaki A
LA  - eng
GR  - EY07546/EY/NEI NIH HHS/United States
GR  - EY09631/EY/NEI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (Enzyme Inhibitors)
RN  - 0 (Protein Subunits)
RN  - 146-91-8 (Guanosine Diphosphate)
RN  - 37589-80-3 (Guanosine 5'-O-(3-Thiotriphosphate))
RN  - EC 2.7.11.1 (Cyclin-Dependent Kinase 5)
RN  - EC 2.7.11.22 (Cyclin-Dependent Kinases)
RN  - EC 3.1.4.35 (3',5'-Cyclic-GMP Phosphodiesterases)
SB  - IM
MH  - 3',5'-Cyclic-GMP Phosphodiesterases/*chemistry/*metabolism
MH  - Animals
MH  - Cyclin-Dependent Kinase 5
MH  - Cyclin-Dependent Kinases/chemistry/*metabolism
MH  - Enzyme Activation
MH  - Enzyme Inhibitors/pharmacology
MH  - Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology
MH  - Guanosine Diphosphate/metabolism
MH  - Kinetics
MH  - Light
MH  - Peptide Mapping
MH  - Phosphorylation
MH  - Protein Subunits
MH  - Ranidae
MH  - Retina/*enzymology
MH  - Rod Cell Outer Segment/*enzymology
EDAT- 2000/07/08 11:00
MHDA- 2001/02/28 10:01
CRDT- 2000/07/08 11:00
PHST- 2000/07/08 11:00 [pubmed]
PHST- 2001/02/28 10:01 [medline]
PHST- 2000/07/08 11:00 [entrez]
AID - S0021-9258(20)89187-X [pii]
AID - 10.1074/jbc.M000703200 [doi]
PST - ppublish
SO  - J Biol Chem. 2000 Oct 20;275(42):32958-65. doi: 10.1074/jbc.M000703200.