Phosphorylation of Avena phytochrome in vitro as a probe of light-induced conformational changes.
A polycation-dependent protein kinase was found to be associated with purified phytochrome preparations from etiolated Avena seedlings. This kinase and three mammalian protein kinases, the catalytic subunit of cAMP-dependent protein kinase, cGMP-dependent protein kinase, and a Ca2+-activated phospholipid-dependent protein kinase, were used to probe light-induced conformational changes in 124-kilodalton Avena phytochrome in vitro. The red absorbing form of phytochrome (Pr) was found to be a substrate for all four protein kinases. Although the far-red absorbing form of phytochrome (Pfr) was as good a substrate as Pr with the cAMP-dependent protein kinase, the Pfr form was poorly phosphorylated by the other three protein kinases. Serine is the major amino acid residue phosphorylated on phytochrome regardless of the form of phytochrome used as substrate. Peptide mapping revealed that the sites of phosphorylation catalyzed by the cAMP-dependent protein kinase differ for Pr and Pfr forms of phytochrome. For the Pr form, the preferred site(s) of phosphorylation was near the amino terminus of the 124-kilodalton subunit. Upon photo-conversion to Pfr, this site can no longer be phosphorylated easily and a new phosphorylation site in the COOH-terminal nonchromophore domain of the molecule becomes accessible to the cAMP-dependent protein kinase. These studies of the phosphorylation of phytochrome provide a new means to study the effect of light absorption by phytochrome on the molecular conformation of the protein. The potential physiological implications of differential phosphorylation of Pr and Pfr await elucidation.
D004591:Electrophoresis, Polyacrylamide Gel; D006657:Histones / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D008027:Light; D010766:Phosphorylation; D010834:Phytochrome / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D006657:Histones; D007527:Isoenzymes; D010940:Plant Proteins; D010834:Phytochrome; D011494:Protein Kinases; D014357:Trypsin