Posttranslational Modification of Oat Phytochrome A:  Phosphorylation of a Specific Serine in a Multiple Serine Cluster

Abstract

Phytochrome A (phyA) is a photoreceptor of higher plants which mediates a variety of biochemical and physiological processes in response to red/far-red light. By detailed structural analysis of the peptides of the total tryptic digest of oat phyA, we found that the photoreceptor isolated from red light irradiated seedlings contains only one site of phosphate attachment, in the N-terminal Ser-rich region. The N-terminal tryptic phosphopeptide (residues 1-12) contains eight serine residues, any of which may be phosphorylated. Direct fast atom bombardment mass spectrometry (FAB MS/MS) analysis of the phosphorylated peptide as well as of its phosphate-containing fragment (residues 1-9) was not successful due to their hydrophilic nature and instability of the phosphate bond. beta-Elimination of the phosphorylated tryptic peptide in the presence of ethanethiol converted the phosphoserine residue to S-ethylcysteine that is stable under FAB MS/MS. FAB MS/MS analysis of the modified peptide clearly showed that the phosphate group was attached to Ser7. The in vivo phosphorylation site at Ser7 in oat phyA is discussed for its possible regulatory role in phyA function.

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

“…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).…”

“…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).…”

OWL1: An Arabidopsis J-Domain Protein Involved in Perception of Very Low Light Fluences

“…Its protein level is rapidly reduced in response to light via transcriptional and posttranslational mechanisms (Canton and Quail, 1999;Clough et al, 1999;Seo et al, 2004). Furthermore, the phosphorylation state of phyA and therefore its activity and stability is light dependent (Lapko et al, 1997;Kim et al, 2004;Rubio and Deng, 2005;Trupkin et al, 2007). In addition, there is evidence that the phosphorylation state of the phyA dimer could lead to a preferential induction either of the FR-HIR or the VLFR pathway (Ryu et al, 2005;Trupkin et al, 2007;Kneissl et al, 2008).…”

Phytochrome-mediated light signaling in plants: emerging trends

“…Like traditional kinases, phytochrome has been demonstrated to function as a phosphoprotein (McMichael and Lagarias, 1990;Lapko et al, 1997). The in vivo studies conducted on oat phyA showed phosphorylation at Serine-7 (irrespective of Pr/Pfr) and Serine-598 (Pfr specific) residues, whereas in vitro phosphorylation has been established at Serine-17 (Pr specific) and Serine-598 (Pfr specific) sites, respectively (Lapko et al, 1997(Lapko et al, , 1999.…”