Abstract
Phototropin (phot) is a light-regulated protein kinase that mediates a variety of photoresponses in plants, such as phototropism, chloroplast positioning, and stomata opening. Arabidopsis has two homologues, phot1 and phot2, that share physiological functions depending on light intensity. A phot molecule has two photoreceptive light oxygen voltage-sensing domains, LOV1 and LOV2, and a Ser/Thr kinase domain. The LOV domains undergo a photocycle upon blue light (BL) stimulation, including transient adduct formation between the chromophore and a conserved cysteine (S390 intermediate) that leads to activation of the kinase. To uncover the mechanism underlying the photoactivation of the kinase, we have introduced a kinase assay system composed of a phot1 LOV2-linker-kinase polypeptide as a light-regulated kinase and its N-terminal polypeptide as an artificial substrate (Okajima, K., Matsuoka, D., and Tokutomi, S. (2011) LOV2-linker-kinase phosphorylates LOV1-containing N-terminal polypeptide substrate via photoreaction of LOV2 in Arabidopsis phototropin1. FEBS Lett. 585, 3391-3395). In the present study, we extended the assay system to phot2 and compared the photochemistry and kinase activation by BL between phot1 and phot2 to gain insight into the molecular basis for the different photosensitivities of phot1 and phot2. Photosensitivity of kinase activation by BL and the lifetime of S390 of phot1 were 10 times higher and longer, respectively, than those of phot2. This correlation was confirmed by an amino acid substitution experiment with phot1 to shorten the lifetime of S390. The present results demonstrated that the photosensitivity of kinase activation in phot involves the lifetime of S390 in LOV2, suggesting that the lifetime is one of the key factors for the different photosensitivities observed for phot1 and phot2.
Highlights
The plant photoreceptor “phototropin” is a light-regulated kinase containing the LOV photosensory domains
The LOV domains undergo a photocycle upon blue light (BL) stimulation, including transient adduct formation between the chromophore and a conserved cysteine (S390 intermediate) that leads to activation of the kinase
To uncover the mechanism underlying the photoactivation of the kinase, we have introduced a kinase assay system composed of a phot1 LOV2-linkerkinase polypeptide as a light-regulated kinase and its N-terminal polypeptide as an artificial substrate (Okajima, K., Matsuoka, D., and Tokutomi, S. (2011) LOV2-linker-kinase phosphorylates LOV1-containing N-terminal polypeptide substrate via photoreaction of LOV2 in Arabidopsis phototropin1
Summary
The plant photoreceptor “phototropin” is a light-regulated kinase containing the LOV photosensory domains. Because LOV2 plays a major role in the photoregulation of the kinase [22, 25, 30, 31], we recently introduced a new in vitro assay system consisting of the At phot LOV2-linker-kinase polypeptide as a light-regulated kinase and its N-terminal polypeptide including six of nine autophosphorylation sites as a substrate [26]. The LOV2-linker-kinase showed a photoreaction similar to that of the LOV2 domain and phosphorylated the N-terminal polypeptide in a light-dependent manner, indicating that the in vitro system could serve as a useful tool in elucidating the molecular basis for photoregulation of the kinase. Phot, a high light sensor, showed much shorter half-lives of LOV2 than phot, which can sense low light This raised an interesting question regarding whether the lifetime of S390 was involved in the light-induced
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