Abstract
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.
Highlights
A polycation-dependent protein kinase wasfound to far-red absorbing Pfrform, phytochrome initiates responses be associated with purifiedphytochrome preparations which are characteristicallypromoted by red light and inhibfrom etiolatedAvena seedlings
Considerable mammalian protein kinases, the catalytic subunit of progress has been made regarding the characterization of the CAMP-dependent proteinkinase, cGMP-dependent structural and spectroscopic properties of this large, soluble protein kinase, and a Ca2+-activatedphospholipid-de- chromoprotein (2, 3), but the initial biochemical response to pendent protein kinase, were used to probe light-in- phytochrome photoconversion is not yet known
To facilitate structural characconversion to P*, this site can no longer be phospho- terization of these regions of conformational change, it would rylated and a new phosphorylation site in the be of value to label sites involved in the light
Summary
Procedures.” In lanes 1-3, the protein band of 66 kDa is bovine serum albumin. In lanes 11-12, the faintlabeled band (80 kDa) corresponds to autophosphorylation of kinase G. 8 pg of total protein was loaded per well. Spectral reversibility measurements were performed to confirm that this differential modification of Pf,by kinase C and kinase G was not a result of irreversible denaturation of phytochrome under standard assay conditions. These experimentsindicated less than 5% loss of photoreversibility when either P, or Pfr was incubated for 30 min a t 30 “C under standard assay conditions for both kinases (data notshown). All values represent molar incorporations after 30-min incubations at 30 “C under standard assay conditions described under “Experimental Procedures,” and are corrected for background radiation. The average background radiation levels was determined to be 74 cpm. 1% molar incorporation represents 320 cpm/8 pgof phytochrome loaded per well after background was subtracted
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