Phototropin Receptor Kinase Activation by Blue Light

Abstract

Phototropins (phot1 and phot2) are blue light-activated serine/ threonine protein kinases that function to mediate a variety of adaptive processes that serve to optimize the photosynthetic efficiency of plants and thereby promote their growth. Light sensing by the phototropins is mediated by a repeated motif located within the N-terminal region of the protein designated the LOV domain. Although phototropins possess two LOV photosensors (LOV1 and LOV2), recent biophysical and structure-function analyses clearly indicate that the LOV2 domain plays a predominant role in regulating phototropin kinase activity owing to specific protein changes that occur in response to LOV2 photoexcitation. In particular, the central b-sheet scaffold plays a role in propagating the photochemical signal generated from within LOV2 to protein changes at the surface that are necessary for kinase activation. Light-Induced Activation of Phot1The primary amino acid structures of plant phototropins (phot1 and phot2) can be separated into two distinct regions: a N-terminal photosensory region linked to a C-terminal effector domain that includes a classic serine/threonine kinase motif. [1][2][3] The N-terminal photosensory region comprises two LOV domains each of which binds the blue light absorbing cofactor flavin mononucleotide (FMN) as chromophore. 4,5 LOV domains exhibit significant homology to motifs found in a diverse range of eukaryotic and prokaryotic proteins involved in sensing Light, Oxygen or Voltage, hence the acronym LOV. 6 Bacterially expressed LOV domains are photochemically reactive as monitored by absorbance spectroscopy. 7 In the dark, LOV domains bind FMN non-covalently forming a spectral species, LOV 447 , which absorbs maximally near 447 nm. 7,8 Irradiation of the domain induces the formation of a covalent bond between the C(4a) carbon of the FMN and the sulfur atom of a nearby, conserved cysteine residue within the domain. Cysteinyl adduct formation occurs within microseconds of illumination and produces a spectral species, LOV 390 , that absorbs maximally near 390 nm. 8 Mutation of the afore-mentioned active-site cysteine to either alanine or serine results in a loss of photochemical reactivity of the LOV domain. 7,8 Moreover, this mutation has been used successfully to probe the roles of LOV1 and LOV2 in regulating phototropin activity and function. [9][10][11][12] As reported previously (ref. 13), the Cys → Ala mutation within LOV2 of Arabidopsis thaliana phot1 (C517A) results in a loss of light-induced receptor autophosphorylation when expressed in insect cells (Fig. 1A), demonstrating that LOV2 is the principle photosensor regulating phot1 kinase activity. A similar mode of action has been reported for the LOV2 domain of Arabidopsis phot2. 12,14,15 The LOV1 domain, by contrast, is proposed to mediate receptor dimerization 16 and/or modulate the photoreactivity of LOV2. 14 Substitution of the conserved active-site cysteine with methionine has been shown to result in the formation of a unique photoproduct species abso...

Phototropin Encoded by a Single-Copy Gene Mediates Chloroplast Photorelocation Movements in the LiverwortMarchantia polymorpha

“…Thus, Mpphot is a blue-light-regulated kinase, similar to other phototropins. Mpphot was slightly autophosphorylated in darkness in vitro, as reported for other phototropins (Matsuoka and Tokutomi, 2005;Jones et al, 2007;Jones and Christie, 2008;Aihara et al, 2012). Autophosphorylation activity may be involved in chloroplast dark positioning, which is mediated by Acphot2 (Tsuboi et al, 2007), Atphot2 (Suetsugu et al, 2005a), and Mpphot.…”

Mutations in N-terminal Flanking Region of Blue Light-sensing Light-Oxygen and Voltage 2 (LOV2) Domain Disrupt Its Repressive Activity on Kinase Domain in the Chlamydomonas Phototropin

“…4C). It should be noted that the background activity in darkness was relatively high, which has been reported for other phototropin samples as well (40,41).…”

Phototropism: Growing towards an Understanding of Plant Movement

“…While the FMN molecule is noncovalently associated with the LOV domain in darkness, upon absorption of BL, a reversible photocycle is initiated such that the activated FMN forms a covalent adduct with a nearby Cys residue in the LOV domain (Christie et al, , 2002Salomon et al, 2000). Although their photocycles are similar, the LOV1 domain is thought primarily to regulate receptor di/multimerization (Salomon et al, 2004;Nakasako et al, 2008;Nakasone et al, 2013), whereas LOV2 appears to regulate the C-terminal PKD of phots through a novel BL-induced derepression mechanism (Christie et al, 2002;Harper et al, 2003Harper et al, , 2004Jones et al, 2007;Jones and Christie, 2008;Nakasako et al, 2008;Tokutomi et al, 2008). In the absence of light, the LOV2 domain is folded in a way that causes steric inhibition of the PKD (Figure 2A).…”