Protein Kinase A-Catalyzed Phosphorylation and Its Effect on Conformation in Phytochrome A

Abstract

Phytochromes are ubiquitous red/far-red wavelength-sensitive photoreceptors in plants. Oat phytochrome A is a phosphoprotein. Phytochrome A (phyA) possesses two spatially different sites for phosphorylation with cAMP-dependent protein kinase (PKA) [McMichael & Lagarias (1990) Biochemistry 29, 3872-3878]. To assess the modulation of protein conformation by phosphorylation/dephosphorylation and its possible implication in phytochrome-mediated signal transduction, the conformations of phytochrome have been probed by PKA catalyzed phosphorylation. The phosphorylated species were purified and analyzed, along with untreated phytochrome, by limited proteolysis, circular dichroism (CD) and fluorescence quenching measurements. No significant changes in secondary structure of the phyA molecule after its phosphorylation were observed by CD. However, a subtle topographic and/or electrostatic effect of the phytochrome phosphorylation was detected by the time-resolved fluorescence quenching of Trp residues with Cs+ ions. N-Terminal phosphorylation at Ser17 was unique to the Pr form, but both Pr and Pfr phytochromes were phosphorylated at the hinge region to some extent. Phosphorylation at the hinge region resulted in noticeable changes in the proteolytic patterns, inhibiting cleavage near the phosphorylation site and favoring tryptic digestion of the Lys536-Asn537 peptide bond. Phosphorylation at the N-terminus did not cause observable changes in the helical structure of this region, but had an inhibitory effect on proteinase V8 accessibility at a site near the chromophore attachment. The functional relevance of protein phosphorylation of phyA is also discussed.

Phytochrome Phosphorylation Modulates Light Signaling by Influencing the Protein–Protein Interaction[W]

“…We previously reported that there were no significant spectroscopic and conformational changes in phyA by PKAcatalyzed phosphorylation in vitro (Lapko et al, 1996). A circular dichroism analysis showed no significant secondary structural changes upon phyA phosphorylation.…”

“…Only proteolytic patterns with trypsin showed a subtle conformational change near the hinge region containing Ser598. The PKA-catalyzed phosphorylation of oat phyA inhibited protease accessibility at the Lys536-Asn537 bond (Lapko et al, 1996). Spectroscopic analyses of the purified S598A protein also displayed the absorbance and red/ FR difference spectra identical with those of the wild-type phyA (see supplemental data online).…”

“…Ser598 of oat phyA is phosphorylated in a Pfr preferential manner both in vivo and in vitro (Lapko et al, 1996(Lapko et al, , 1999. Because oat phyA is physiologically active in transgenic plants, including Arabidopsis thaliana (Boylan and Quail, 1991;Boylan et al, 1994), in vivo functional assays of oat phyA mutants are possible by transforming the oat phyA mutant genes into phyA-deficient Arabidopsis (phyA-201, Landsberg erecta [Ler] ecotype).…”

“…The sites of phytochrome phosphorylation in vivo and in vitro have been identified with oat (Avena sativa) phyA (McMichael and Lagarias, 1990;Lapko et al, 1996Lapko et al, , 1997Lapko et al, , 1999. There are two Ser sites, Serine-7 (Ser7) and , that are phosphorylated in vivo.…”

“…Two Ser, Serine-17 (Ser17) and Ser598, are detected as the in vitro phosphorylation sites by PKA. Ser17 is phosphorylated primarily in a Pr preferential manner, whereas Ser598 phosphorylation is preferred in the Pfr form (McMichael and Lagarias, 1990;Lapko et al, 1996). Because this Ser598 residue is phosphorylated only in the Pfr form that is considered the active form of phytochrome, it was suggested that the phosphorylation and dephosphorylation of Ser598 serves as a switch in phytochrome signaling (Park et al, 2000;Kim et al, 2002b).…”