Identification of cyclic nucleotide-regulated phosphoproteins, including phosducin, in motile rod inner-outer segments of teleosts.
In teleost retinas, rods elongate in the light and shorten in the dark. Rod motility is mediated by the actin cytoskeleton of the inner segment and is regulated by cyclic AMP- or cyclic GMP-stimulated phosphorylation of target proteins. In this study, we have identified the target proteins of cyclic nucleotide-dependent kinases in rods, using preparations of isolated, motile rod inner-outer segments (RIS-ROS). Five proteins found in Percoll-purified RIS-ROS were phosphorylated in the presence of cAMP (> 10 nM), cGMP (> or = 10 microM) and exogenous catalytic subunit of cAMP-dependent protein kinase (PKA). The PKA inhibitor, PKI, blocked stimulation of phosphorylation by both cAMP and cGMP. Three cAMP-stimulated phosphoproteins were detected in cytoskeletal fractions of light- and dark-adapted RIS-ROS. One of these, PP33, appears to be a fish homologue of mammalian phosducin, based on immunolabeling by two different antibodies against mammalian phosducin and on electrophoretic characteristics in 2-D gels. Two additional phosducin immunoreactive bands were detected in Western blots. One, at 35 kDa, comigrated with a second cAMP-stimulated RIS-ROS phosphoprotein, PP35, which was also detected in the cytoskeleton. The other, at 37 kDa, was present in whole teleost retinas but not in purified RIS-ROS. Our results suggest that the effects of both cAMP and cGMP on teleost rod motility are mediated through PKA modulation of target phosphoproteins. These phosphoproteins include a cytoskeleton-associated phosducin homologue.
Pagh-Roehl
K
K
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Han
E
E
Burnside
B
B
D000818:Animals; D046508:Culture Techniques; D000242:Cyclic AMP / Q000502:physiology*; D006152:Cyclic GMP / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000378:metabolism; D020555:GTP-Binding Protein Regulators; D015151:Immunoblotting; D010473:Perciformes / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism
D005136:Eye Proteins; D020555:GTP-Binding Protein Regulators; D010750:Phosphoproteins; C065631:phosducin; D000242:Cyclic AMP; D006152:Cyclic GMP