Phosphorylation of the gamma subunit of the retinal photoreceptor cGMP phosphodiesterase by the cAMP-dependent protein kinase and its effect on the gamma subunit interaction with other proteins.
Cyclic GMP phosphodiesterase, a key enzyme in phototransduction, is composed of P alpha beta and two P gamma subunits. Interaction of P gamma with P alpha beta or with the alpha subunit (T alpha) of transducin is crucial for the regulation of cGMP phosphodiesterase in retinal photoreceptors. Here we have investigated phosphorylation of P gamma by cAMP-dependent protein kinase and its functional effect on the P gamma interaction with P alpha beta or T alpha in vitro. P gamma, but not P gamma complexed with T alpha (both GTP and GDP forms), is phosphorylated. Measurement of 32P radioactivity in phosphorylated P gamma, analysis of phosphorylated P gamma by laser mass spectrometry, identification of phosphoamino acid, and phosphorylation of mutant forms of P gamma indicate that only threonine 35 in P gamma is phosphorylated. Phosphorylation of P gamma mutants also reveals that the C and N terminals of P gamma which are required for the regulation of P alpha beta functions are not involved in the P gamma phosphorylation but that arginine 33, which is ADP-ribosylated by an endogenous ADP-ribosyltransferase, is required for the phosphorylation. Phosphorylated P gamma has a higher inhibitory activity for trypsin-activated cGMP phosphodiesterase than nonphosphorylated P gamma, indicating that the P gamma-P alpha beta interaction is affected by P gamma phosphorylation. Nonphosphorylated P gamma inhibits both the GTPase activity of T alpha and the binding of a hydrolysis-resistant GTP analogue to T alpha, while P gamma phosphorylation reduces these inhibitory activities. These observations suggest that a P gamma domain containing threonine 35 is involved in the P gamma-T alpha interaction, and P gamma phosphorylation regulates the P gamma-T alpha interaction. Our observation suggests that P gamma phosphorylation by cAMP-dependent protein kinase may function for the regulation of phototransduction in vertebrate rod photoreceptors.
Xu
L X
LX
Department of Ophthalmology, Kresge Eye Institute, Wayne State University, School of Medicine, Detroit, Michigan 48201, USA.
Tanaka
Y
Y
Bonderenko
V A
VA
Matsuura
I
I
Matsumoto
H
H
Yamazaki
A
A
Hayashi
F
F
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000378:metabolism; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D010766:Phosphorylation; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D013912:Threonine / Q000378:metabolism; D015293:Transducin / Q000378:metabolism
D000596:Amino Acids; D013912:Threonine; D001120:Arginine; D017868:Cyclic AMP-Dependent Protein Kinases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D015293:Transducin