Purpose: During the course of our investigations on cyclic nucleotide dependent-phosphorylation of membrane proteins, we observed that phosducin was present in both the cytosolic and nuclear fractions. It has been suggested that phosducin might have a role in regulating transcription, but its presence in the nucleus has not been previously reported. We therefore attempted to purify nuclei from bovine retina and determine whether the purified preparation contained phosducin. Cyclic nucleotide-dependent phosphorylation of the protein was also investigated in the homogenate and subcellular fractions of bovine retina.
Methods: Freshly obtained bovine retinas were homogenized and fractionated in an isotonic buffer. The homogenate and subcellular fractions were subjected to phosphorylation in the presence of gamma-32P ATP and the presence and absence of cyclic GMP. The phosphorylated proteins were identified by 1- or 2-dimensional electrophoresis and autoradiography. Phosducin was detected in the fractions by western blotting. A nuclear preparation was obtained from the homogenate by sucrose gradient centrifugation, its purity was determined with a nuclear stain, and the presence and phosphorylation of phosducin was investigated as above.
Results: Phosducin was found in the cytosol, 100,000x g membrane fraction, as well as in the 120x g pellet of a fresh, isotonic retinal homogenate, but cyclic nucleotide-dependent phosphorylation of the protein was observed only in the cytosolic fraction. Western blotting on a highly purified nuclear fraction showed that phosducin was present in the nuclei. The protein could be phosphorylated in a cyclic GMP-dependent fashion in a nuclear preparation permeabilized by freezing and thawing.
Conclusions: It has been suggested that a C-terminal fragment of phosducin might be transported into the nucleus where it might have a role in the regulation of transcription. Phosducin localization in the nucleus was shown in COS cells cotransfected with genes of phosducin and a transcription factor. However, the presence of phosducin in the nuclei of retina has not been demonstrated hitherto. The present results show that phosducin is present in the nuclei in bovine retina. It could be phosphorylated in the nuclei in a cyclic GMP-dependent fashion. These results support a possible role for phosducin in the regulation of transcription.