Desensitization of activated receptors is an important mechanism for terminating signal transduction. Here we show that phytochrome (phy) A, a predominant photoreceptor for seedling deetiolation, colocalizes in nuclear bodies with CONSTITUTIVELY PHOTOMORPHOGENIC (COP) 1, a RING motif-containing E3 ligase. The phyA PAS domain interacts with the COP1 WD40 domain. Both the Pr and the Pfr forms of phyA, as well as the PHYA apoprotein, are ubiquitinated by COP1 in vitro. The phyA destruction rate is decreased in cop1 mutants and by expression of a COP1 RING motif mutant. Our results indicate that COP1 acts as an E3 ligase to regulate phyA signaling by targeting elimination of the phyA photoreceptor itself.

Phototropin is a blue light photoreceptor for tropic responses, relocation of chloroplasts, and stomata opening in plants. Phototropin has two chromophoric domains named light-oxygenvoltage-sensing (LOV) 1 and 2 in the N-terminal half, and a serine͞threonine (Ser͞Thr) protein kinase motif in the C-terminal half. Concerning the kinase activity of phototropin, only autophosphorylation has been detected so far. However, we found that phototropin can phosphorylate a protein other than phototropin itself. Bacterially expressed Arabidopsis phototropin 2 kinase domain (KD) with GST-tag showed a constitutive kinase activity on casein, a common in vitro substrate of Ser͞Thr protein kinase. By using this in vitro assay system, the roles of each LOV domain were studied. Addition of LOV2 to KD (GST-L2-KD) inhibits the kinase activity that is canceled by light. This light activation of kinase disappeared on introduction of a mutation blocking photochemical reaction in the LOV2 domain. Accordingly, LOV2 domain acts as a major light-regulated molecular switch of casein phosphorylation. Interestingly, isolated LOV2 from the KD still binds to the KD in a light-dependent manner and functions in similar ways, indicating the role of LOV2 domain as an inhibitor of the kinase activity in the substrate phosphorylation. LOV1, in contrast, contributes little to the photoactivation in GST-L1-L2-KD; however, it acts as an attenuator of the light activation of the kinase by LOV2.ight in the wavelength region from UV to far-red is an important stimulus for plants that precisely regulates developmental and cell motility processes. To sense the light conditions including intensity, quality, and direction, plants have acquired three major photoreceptors (1): phytochrome, cryptochrome, and phototropin.Phototropin (2, 3), originally identified as a photoreceptor for tropic responses (4), has also been found to mediate chloroplast relocations (5-7), stomatal opening (8), leaf expansion (9), and rapid inhibition of hypocotyl elongation (10). Most plants have two isoforms of phototropin, named phot1 and phot2 (2). In Arabidopsis thaliana (At), phot1 and phot2 share tropic responses and also one of the chloroplast relocation responses, photoaccumulation, depending on fluence rate of light (6). Photoavoidance response, on the other hand, is mediated by only phot2 (5). In contrast, stomatal opening is regulated redundantly by both phot1 and phot2 (8).The phototropins are composed of 900-1,000 amino acid residues and two prosthetic flavin mononucleotide (FMN) molecules (2, 3) (Fig. 1A Top). The N-terminal half has a pair of FMN-binding domains with Ϸ100 residues designated lightoxygen-voltage-sensing (LOV) 1 and 2 (11), and the C-terminal half forms a serine͞threonine (Ser͞Thr) kinase domain connected to the LOV2 domain with a linker region (Fig. 1 A).Upon absorption of blue light, FMN in the two LOV domains undergoes a unique cyclic photochemical reaction through formation and breakage of a covalent bond (12) with a highly conserved cysteine found in ...

Conformational dynamics of LOV2 domain of phototropin, a plant blue light photoreceptor, is studied by the pulsed laser induced transient grating (TG) technique. The TG signal of LOV2 without the linker part to the kinase domain exhibits the thermal grating signal due to the heat releasing from the excited state and a weak population grating by the adduct formation. The diffusion coefficients of the adduct product after forming the chemical bond between the chromophore and Cys residue are found to be slightly smaller than that of the reactant, which implies that the core shrinks slightly on the adduct formation. After that change, no significant conformational change was observed. On the other hand, the signal of LOV2 with the linker part to the kinase domain clearly shows very different diffusion coefficients between the original and the adduct species. The large difference indicates significant global conformational change of the protein moiety upon the adduct formation. More interestingly, the diffusion coefficient is found to be time-dependent in the observation time range. The dynamics representing the global conformational change is a clear indication of a spectral silent intermediate between the excited triplet state and the signaling product. From the temporal profile analysis of the signal, the rate of the conformational change is determined to be 2 ms.

Phototropin (Phot) is a blue-light receptor in plants. The molecule has two FMN (flavin mononucleotide) binding domains named LOV (light-, oxygen-, and voltage-sensing), which is a subset of the PAS (Per-Arnt-Sim) superfamily. Illumination of the phot-LOV domains in the dark state (D447) produces a covalent C(4a) flavin-cysteinyl adduct (S390) via a triplet excited state (L660), which reverts to D447 in the dark. In this work, we studied the light-induced structural changes in the LOV2 domain of Adiantum phytochrome3 (phy3), which is a fusion protein of phot containing the phytochrome chromophoric domain, by low-temperature UV-visible and FTIR spectroscopy. UV-visible spectroscopy detected only one intermediate state, S390, in the temperature range from 77 to 295 K, indicating that the adduct is produced even at temperatures as low as 77 K, although a portion of D447 cannot be converted to S390 at low temperatures possibly because of motional freezing. In the whole temperature range, FTIR spectra in the S-H stretching frequency region showed that Cys966 of phy3-LOV2 is protonated in D447 and unprotonated on illumination, supporting adduct formation. The pK(a) of the S-H group in D447 is estimated to be >10. FTIR spectra also showed the light-induced appearance of a positive peak around 3621 cm(-1) in the whole temperature range, indicating that adduct formation accompanies rearrangement of a hydrogen bond of a water molecule(s), which can be either water25, water45, or both, near the chromophore. In contrast to the weak temperature dependence of the spectral changes in the UV-visible absorption and the FTIR of both S-H and O-H stretching bands, light-induced changes in the amide I vibration that probes protein backbone structure vary significantly with the increase in temperature. The spectral changes suggest that light excitation of FMN loosens the local structure around it, particularly in turns, in the early stages and that another change subsequently takes place to tighten it, mainly in beta-structure, but some occur in the alpha-helical structure of the protein moiety as well. Interestingly, these changes proceed without altering the shape of UV-visible spectra, suggesting the presence of multiple conformation states in S390.