Blue light-induced autophosphorylation of phototropin is a primary step for signaling

Abstract

Phototropins are autophosphorylating protein kinases of plantspecific blue light receptors. They regulate various blue light responses, including phototropism, chloroplast movements, hypocotyl growth inhibition, leaf flattening, and stomatal opening. However, the physiological role of autophosphorylation remains unknown. Here, we identified phosphorylation sites of Ser or Thr in the N terminus, Hinge1 region, kinase domain, and C terminus in Arabidopsis phototropin1 (phot1) by liquid chromatographytandem mass spectrometry in vivo. We substituted these Ser or Thr residues with Ala in phot1 and analyzed their functions by inspecting the phot1-mediated responses of stomatal opening, phototropism, chloroplast accumulation, and leaf flattening after the transformation of the phot1 phot2 double mutant. Among these sites, we found that autophosphorylation of Ser-851 in the activation loop of the kinase domain was required for the responses mentioned above, whereas the phosphorylation of the other Ser and Thr, except those in the activation loop, was not. Ser-849 in the loop may have an additional role in the responses. Immunological analysis revealed that Ser-851 was phosphorylated rapidly by blue light in a fluence-dependent manner and dephosphorylated gradually upon darkness. We conclude that autophosphorylation of Ser-851 is a primary step that mediates signaling between photochemical reaction and physiological events.protein kinase ͉ stomata ͉ photoreceptor ͉ light signaling P hototropins (phot1 and phot2) mediate multiple blue light responses in Arabidopsis, including phototropism, chloroplast movements, leaf flattening, leaf positioning, stomatal opening, and rapid inhibition of hypocotyl growth (1-5). These responses enhance photosynthesis and optimize plant growth, particularly under weak light (6). Under strong light, phot2 induces a chloroplast-avoidance response to prevent photodamage to photosynthetic machinery (7). Phototropins, therefore, are essential proteins for the survival of higher plants and the extension of their living areas, particularly under ever-changing environments of light.Phototropins are blue light receptor protein kinases with two light, oxygen, voltage (LOV) domains in the N terminus and a Ser/Thr protein kinase in the C terminus (8). The LOV domains possess noncovalent binding sites for the chromophore flavin mononucleotide (FMN) and cysteine residues (2, 9-11) and produce a covalent cysteinyl adduct with FMN when LOVs are illuminated with blue light (12, 13). The cysteinyl adduct formation is the primary photochemical process; the adduct formation induces conformational changes in the LOV2 domain (14, 15) and J␣-helix (16) and leads to phototropin phosphorylation and subsequent physiological processes (17, 18). Phototropin phosphorylation by blue light is demonstrated to be autophosphorylation through the use of recombinant proteins of PHOT1 and PHOT2 expressed in insect cells (2, 9, 17).Phototropin was initially found as a plasma membraneassociated phosphorylated protein in etiolate...

Higher plants use LOV to perceive blue light

“…This demonstrates for the first time that phosphorylation of phot1 is an essential step of signaling [16]. This residue of the kinase-domain activation loop is well conserved among members of the PKA family [4,16]. Phosphorylation of the equivalent site in PKA enhances kinase activity and substrate recognition [4].…”

“…Four phosphorylation sites were identified upstream of the LOV2 domain, similar regions of phot1 are phosphorylated in vitro [10]. A second study identified two additional sites in the photosensory domain, and three phosphorylation sites in the kinase domain of phot1 [16] (Figure 1). The characterization of point mutants showed that autophosphorylation of Ser851 (but none of the other residues) is necessary to trigger phototropin-dependent responses [16].…”

“…The characterization of point mutants showed that autophosphorylation of Ser851 (but none of the other residues) is necessary to trigger phototropin-dependent responses [16]. This demonstrates for the first time that phosphorylation of phot1 is an essential step of signaling [16]. This residue of the kinase-domain activation loop is well conserved among members of the PKA family [4,16].…”

“…A second study identified two additional sites in the photosensory domain, and three phosphorylation sites in the kinase domain of phot1 [16] (Figure 1). The characterization of point mutants showed that autophosphorylation of Ser851 (but none of the other residues) is necessary to trigger phototropin-dependent responses [16]. This demonstrates for the first time that phosphorylation of phot1 is an essential step of signaling [16].…”

“…However, no direct substrate for kinase activity in planta has been identified so far except the phototropins themselves [1]. In vivo mapping of phot1 phosphorylation sites provides new insights into the physiological relevance of phototropin phosphorylation [10,16]. Four phosphorylation sites were identified upstream of the LOV2 domain, similar regions of phot1 are phosphorylated in vitro [10].…”