Regulation by light of cyclic nucleotide-dependent protein kinases and their substrates in frog rod outer segments.
Cyclic nucleotides (both cAMP and cGMP) stimulate the phosphorylation of several proteins of 65-70, 50-52, 21, 13, and 12 kD in rod outer segments (ROS) of the frog retina. Subcellular fractionation showed that phosphopeptides of 67, 21, 13, and 12 kD were soluble and phosphopeptides of 69, 67, 50-52, and 12 kD were membrane associated at physiological ionic strength. Components I and II, 13 and 12 kD, respectively, are the major cyclic nucleotide-dependent phosphoproteins of ROS and have been reported to be phosphorylated in the dark and dephosphorylated in the light. Under unstimulated conditions, phosphorylated Components I and II were found in the soluble fraction. Cyclic nucleotide stimulation of phosphorylation resulted in increased phospho-Components I and II in the soluble fraction, and phospho-Component II on the membrane. Light had no effect on the phosphorylation level of soluble Components I and II, but it caused a depletion within 1 s of the membrane-bound phospho-Component II. A half-maximal decrease in membrane-bound Component II was seen at 5 x 10(5) rhodopsins bleached per outer segment. The cyclic nucleotide-dependent protein kinase(s) were found primarily in the peripheral membrane fraction of ROS proteins. 8-bromo cyclic AMP was two orders of magnitude more effective than 8-bromo cyclic GMP at stimulating Component I and II phosphorylation. An active peptide of the Walsh inhibitor of cAMP-dependent protein kinase [PKI(5-22)amide] blocked the phosphorylation with an IC50 of 10 nM. Photoaffinity labeling studies with 8-N3-cAMP and 8-N3-cGMP revealed the presence of a 52-kD band specifically labeled with 8-N3-cAMP, but no specific 8-N3-cGMP labeling. These data suggest that cyclic nucleotide-dependent protein phosphorylation in ROS occurs via the activation of a cAMP-dependent protein kinase.
Hamm
H
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Department of Physiology and Biophysics, University of Illinois College of Medicine, Chicago 60612.
D000818:Animals; D008027:Light; D009712:Nucleotides, Cyclic / Q000502:physiology*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D010786:Photoreceptor Cells / Q000201:enzymology*; D011494:Protein Kinases / Q000378:metabolism*; D011892:Rana catesbeiana; D012374:Rod Cell Outer Segment / Q000201:enzymology* / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology / Q000378:metabolism
D009712:Nucleotides, Cyclic; D010750:Phosphoproteins; D010770:Phosphotransferases; D011494:Protein Kinases