Phosphorylation of an inhibitory subunit of cGMP phosphodiesterase in Rana catesbeiana rod photoreceptors. II. A possible mechanism for the turnoff of cGMP phosphodiesterase without GTP hydrolysis.
In amphibian rod photoreceptor membranes, P gamma, an inhibitory subunit of cGMP phosphodiesterase, is phosphorylated by a protein kinase when P gamma is complexed with the guanosine 5'-O-(3-thiotriphosphate)-bound alpha subunit of transducin (GTP gamma S.T alpha). Five different experiments support the conclusion that the phosphorylated P gamma loses its interaction with GTP gamma S.T alpha. These observations include 1) detection of the inhibitory effect of the GTP gamma S.T alpha.P gamma complex on cGMP phosphodiesterase activity after P gamma in the complex is phosphorylated in a system reconstituted from isolated components, 2) no stimulating effect of GTP gamma S.T alpha on the phosphorylated P gamma-inhibited cGMP phosphodiesterase in the reconstituted system, 3) physical release of phosphorylated P gamma from GTP gamma S.T alpha in the reconstituted system, 4) no inhibitory effect of phosphorylated P gamma on both GTP hydrolysis by T alpha and GTP gamma S/GDP exchange on T alpha in the reconstituted system, and 5) no enhanced activity of cGMP phosphodiesterase by GTP gamma S.T alpha in rod outer segment membranes after incubation of the membranes with the kinase preparation in the presence of ATP. Together with our data that P gamma released with GTP.T alpha is not phosphorylated, and that phosphorylated P gamma inhibits more effectively cGMP phosphodiesterase activity than nonphosphorylated P gamma (Tsuboi, S., Matsumoto, H., Jackson, K. W., Tsujimoto, K., Williams, T., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15016-15023), these observations suggest that, after P gamma is released with GTP.T alpha from catalytic subunits of cGMP phosphodiesterase, P gamma complexed with GTP.T alpha is phosphorylated by a kinase. Then, the phosphorylated P gamma is released from GTP.T alpha and binds to active cGMP phosphodiesterase to inhibit the cGMP hydrolysis. It is suggested that in some G-protein-dependent signal transduction systems G-protein-activated effector may be phosphorylated with a specific kinase and that phosphorylation of the effector results in the turnoff of signal transduction without GTP hydrolysis.
Tsuboi
S
S
Kresge Eye Institute, Department of Ophthalmology, Wayne State University, School of Medicine, Detroit, Michigan 48201.
Matsumoto
H
H
Yamazaki
A
A
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism; D011892:Rana catesbeiana; D017948:Retinal Rod Photoreceptor Cells / Q000201:enzymology*; D016415:Sequence Alignment
D006160:Guanosine Triphosphate; D011494:Protein Kinases; D015106:3',5'-Cyclic-GMP Phosphodiesterases