Phosphorylation of an inhibitory subunit of cGMP phosphodiesterase in Rana catesbeiana rod photoreceptors. I. Characterization of the phosphorylation.
Interaction between the inhibitory subunit (P gamma) and catalytic subunits of cGMP phosphodiesterase is essential for the regulation of cGMP phosphodiesterase in vertebrate rod photoreceptors. P gamma phosphorylation in vitro has been studied using a kinase which is extracted from amphibian rod outer segments. Various chromatographies of the kinase preparation using ionic exchange, gel filtration, and heparin-Sepharose columns indicate that a kinase with M(r) 70,000 is responsible for the P gamma phosphorylation. The kinase does not require any of the known activators for protein kinases but is inhibited by cGMP in a concentration-dependent manner. Together with analysis by laser-desorption mass spectrometry, measurement of 32P radioactivity in phosphorylated P gamma indicates that P gamma extracted with GTP-bound transducin alpha subunit is not phosphorylated and that a phosphate is incorporated into more than 80% of the P gamma by the kinase. Phosphoamino acid analysis, sequencing of phosphorylated peptides derived from phosphorylated P gamma, and phosphorylation of synthetic peptides indicate threonine 22 in P gamma is phosphorylated by the kinase. Phosphorylated P gamma has a higher inhibitory activity for active cGMP phosphodiesterase than non-phosphorylated P gamma. These data suggest that threonine 22 in P gamma is phosphorylated by a specific kinase and that the P gamma phosphorylation governs the interaction between P gamma and catalytic subunits of cGMP phosphodiesterase in vertebrate rod photoreceptors.
Tsuboi
S
S
Kresge Eye Institute, Department of Ophthalmology, Wayne State University, School of Medicine, Detroit, Michigan 48201.
Matsumoto
H
H
Jackson
K W
KW
Tsujimoto
K
K
Williams
T
T
Yamazaki
A
A
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D004789:Enzyme Activation; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011892:Rana catesbeiana; D017948:Retinal Rod Photoreceptor Cells / Q000201:enzymology*; D013379:Substrate Specificity
D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D011494:Protein Kinases; D015106:3',5'-Cyclic-GMP Phosphodiesterases