PROTEIN-KINASE-A-CATALYZED PHOSPHORYLATION AND ITS EFFECT ON CONFORMATION IN PHYTOCHROME-A

Phytochromes are ubiquitous red/far-red wavelength-sensitive photorece ptors in plants. Oat phytochrome A is a phosphoprotein. Phytochrome A (phyA) possesses two spatially different sites for phosphorylation wit h cAMP-dependent protein kinase (PKA) [McMichael & Lagarias (1990) Bio chemistry 29, 3872-3878]. To assess the modulation of protein conforma tion by phosphorylation/dephosphorylation and its possible implication in phytochrome-mediated signal transduction, the conformations of phy tochrome have been probed by PKA catalyzed phosphorylation. The phosph orylated species were purified and analyzed, along with untreated phyt ochrome, by limited proteolysis, circular dichroism (CD) and fluoresce nce quenching measurements. No significant changes in secondary struct ure of the phyA molecule, after its phosphorylation were observed by C D. However, a subtle topographic and/or electrostatic effect of the ph ytochrome phosphorylation was detected by the time-resolved fluorescen ce quenching of Trp residues with Cs+ ions. N-Terminal phosphorylation at Ser(17) was unique to the Pr form, but both Pr and Pfr phytochrome s were phosphorylated at the hinge region to some extent. Phosphorylat ion at the hinge region resulted in noticeable changes in the proteoly tic patterns, inhibiting cleavage near the phosphorylation site and fa voring tryptic digestion of the LYs(536)-Asn(537) peptide bond. Phosph orylation at the N-terminus did not cause observable changes in the he lical structure of this region, but had an inhibitory effect on protei nase V8 accessibility at a site near the chromophore attachment. The f unctional relevance of protein phosphorylation of phyA is also discuss ed.