Mass spectrometric characterization of oat phytochrome A: Isoforms and posttranslational modifications

Abstract

At least four mRNAs for oat phytochrome A~phyA! are present in etiolated oat tissue. The complete amino acid sequences of two phyA isoforms~A3 and A4! and the N-terminal amino acid sequence of a third isoform~A5! were deduced from cDNA sequencing~Hershey et al., 1985!. In the present study, heterogeneity of phyA on a protein level was studied by tryptic mapping using electrospray ionization mass-spectrometry~ESIMS!. The total tryptic digest of iodoacetamide-modified phyA was fractionated by gel filtration chromatography followed by reversed-phase highperformance liquid chromatography. ESIMS was used to identify peptides. Amino acid sequences of the peptides were confirmed or determined by collision-induced dissociation mass spectrometry~CID MS!, MS0MS, or by subdigestion of the tryptic peptides followed by ESIMS analysis. More than 97% of the phyA3 sequence~1,128 amino acid residues! was determined in the present study. Mass-spectrometric analysis of peptides unique to each form showed that phyA purified from etiolated oat seedling is represented by three isoforms A5, A3, and A4, with ratio 3.4:2.3:1.0. Possible light-induced changes in phytochrome in vivo phosphorylation site at Ser7~Lapko VN et al., 1997, Biochemistry 36:10595-10599! as well at Ser17 and Ser598~known as in vitro phosphorylation sites! were also analyzed. The extent of phosphorylation at Ser7 appears to be the same for phyA isolated from dark-grown and red-light illuminated seedlings. In addition to Ser7, Ser598 was identified as an in vivo phosphorylation site in oat phyA. Ser598 phosphorylation was found only in phyA from the red light-treated seedlings, suggesting that the protein phosphorylation plays a functional role in the phytochrome A-mediated light-signal transduction.

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

“…In fact, the Ser598 phosphorylation was thought to play an active role in the phyA-mediated lightsignal transduction (Lapko et al, 1999;Park et al, 2000;Kim et al, 2002b). Here, we showed that the transgenic plants of the S598A mutant were hypersensitive to light.…”

“…Ser598 phosphorylation occurs only in the Pfr form of phyA in red light-treated oat seedlings (McMichael and Lagarias, 1990;Lapko et al, 1999), whereas phosphorylation of other sites (Ser7 and Ser17) was found either Pr/Pfr indifferent or Pr specific (Lapko et al, 1996(Lapko et al, , 1997. In fact, the Ser598 phosphorylation was thought to play an active role in the phyA-mediated lightsignal transduction (Lapko et al, 1999;Park et al, 2000;Kim et al, 2002b).…”

“…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.…”

“…There are two Ser sites, Serine-7 (Ser7) and , that are phosphorylated in vivo. Phosphorylation at Ser7 in the N-terminal extension region (NTE) is similar in both Pr and Pfr forms, whereas Ser598 in the hinge region is phosphorylated in a Pfr preferential manner (Lapko et al, 1997(Lapko et al, , 1999. The in vitro phosphorylation sites of oat phyA have also been identified using protein kinase A (PKA).…”