Autophosphorylation desensitizes phytochrome signal transduction

Abstract

Plant red/far-red photoreceptor phytochromes are known as autophosphorylating serine/threonine kinases. However, the functional roles of autophosphorylation and kinase activity of phytochromes are largely unknown. We recently reported that the autophosphorylation of phytochrome A (phyA) plays an important role in regulating plant phytochrome signaling by controlling phyA protein stability. Two serine residues in the N-terminal extension (NTE) region were identified as autophosphorylation sites, and phyA mutant proteins with serine-to-alanine mutations were degraded in plants at a significantly slower rate than the wild-type under light conditions, resulting in transgenic plants with hypersensitive light responses. In addition, the autophosphorylation site phyA mutants had normal protein kinase activities. Collectively, our results suggest that phytochrome autophosphorylation provides a mechanism for signal desensitization in phytochrome-mediated light signaling by accelerating the degradation of phytochrome A.

Keywords: autophosphorylation; light signaling; phosphorylation; phytochrome; protein degradation; protein kinase; signal desensitization.

Figure 1

(A and B) Multiple sequence alignments of monocot (A) and dicot (B) phyA proteins. PhyA sequences of nine monocots or five dicots were used for multiple sequence alignment using the CLC Sequence Viewer 5 program (www.clcbio.com). Ser8 and Ser18 of oat phyA are indicated with arrowheads. Phosphorylatable serines predicted by the NetPhos 2.0 server are indicated with an asterisk beneath the sequence alignments. (C and D) Far-red fluence-rate response curves for inhibition of hypocotyl growth of transgenic Arabidopsis seedlings expressing the autophosphorylation site mutants. phyA-201, a phyA-deficient Arabidopsis (Ler ecotype); Ler, wild-type Arabidopsis; Wt-OX6, a transgenic Arabidopsis transformed with wild-type oat phyA; Δ6–12 (#26-2), Δ6–12/S18A (#20-2), S8-12A (#2-2), and S8-12A/S18A (#28-4) transgenic Arabidopsis plants transformed with the corresponding autophosphorylation mutants. Δ6–12, a mutant with a deletion of seven amino acid residues (⁶PASSSSS¹²); Δ6–12/S18A, a combination mutant of Δ6–12 and S18a; S8-12A, a mutant of with five serine residues (8–12aa) substituted to alanines; S8-12A/S18A, a combination mutant of S8-12A and S18A. Each hypocotyl length was normalized to the hypocotyl lengths of dark-grown controls. Data are means (n ≥ 30) ± s.d.

Figure 2

In vitro phospho-transfer analysis of site-specific mutants of three known phosphorylation sites. Zinc fluorescence (Zinc blot), SDS-PAGE gel, and Autoradiogram (BAS) are shown. Zinc fluorescence shows chromophore-assembled phyA. PIF3 was used as the substrate protein in this analysis.

Figure 3

An explanatory model for the functional role of phytochrome autophosphorylation. Phy-Pr, the Pr form of phytochrome; Phy-Pfr, the Pfr form of phytochrome; Phy-Pr-P, the phosphorylated Pr form; Phy-Pfr-P, the phosphorylated Pfr form; R, red light; FR, far-red light; PPase, protein phosphatase; AutoP, autophosphorylation. In the dark, phytochrome is synthesized and accumulates as the Pr form in the cytosol (Phy-Pr). Upon illumination, the Pr form is photoactivated to the Pfr form (Phy-Pfr), which regulates gene expression for photomorphogenesis and is degraded via the ubiquitin/26S proteasome pathway. At this time, phosphorylated phytochromes (Phy-Pfr-P) are rapidly degraded for the efficient desensitization of the phyA signal, which is necessary for responses to subsequent changes in fluctuating light environments.