Limits...
Essential role of the A'α/Aβ gap in the N-terminal upstream of LOV2 for the blue light signaling from LOV2 to kinase in Arabidopsis photototropin1, a plant blue light receptor.

Kashojiya S, Okajima K, Shimada T, Tokutomi S - PLoS ONE (2015)

Bottom Line: Using LOV2-STK polypeptides from Arabidopsis thaliana phot1, we found that truncation of the A'α-helix and amino acid substitutions at Glu474 and Lys475 in the gap between the A'α and the Aβ strand of LOV2 (A'α/Aβ gap) to Ala impaired the BL-induced activation of the STK, although they did not affect S390 formation.These BL-induced structural changes were observed with the Glu474Ala and the Lys475Ala substitutes, indicating that the BL signal reached the Jα-helix as well as the A'α/Aβ gap but could not activate STK.The amino acid residues, Glu474 and Lys475, in the gap are conserved among the phots of higher plants and may act as a joint to connect the structural changes in the Jα-helix with the activation of STK.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Osaka Prefecture University, Sakai, Osaka, Japan.

ABSTRACT
Phototropin (phot) is a blue light (BL) receptor in plants and is involved in phototropism, chloroplast movement, stomata opening, etc. A phot molecule has two photo-receptive domains named LOV (Light-Oxygen-Voltage) 1 and 2 in its N-terminal region and a serine/threonine kinase (STK) in its C-terminal region. STK activity is regulated mainly by LOV2, which has a cyclic photoreaction, including the transient formation of a flavin mononucleotide (FMN)-cysteinyl adduct (S390). One of the key events for the propagation of the BL signal from LOV2 to STK is conformational changes in a Jα-helix residing downstream of the LOV2 C-terminus. In contrast, we focused on the role of the A'α-helix, which is located upstream of the LOV2 N-terminus and interacts with the Jα-helix. Using LOV2-STK polypeptides from Arabidopsis thaliana phot1, we found that truncation of the A'α-helix and amino acid substitutions at Glu474 and Lys475 in the gap between the A'α and the Aβ strand of LOV2 (A'α/Aβ gap) to Ala impaired the BL-induced activation of the STK, although they did not affect S390 formation. Trypsin digested the LOV2-STK at Lys603 and Lys475 in a light-dependent manner indicating BL-induced structural changes in both the Jα-helix and the gap. The digestion at Lys603 is faster than at Lys475. These BL-induced structural changes were observed with the Glu474Ala and the Lys475Ala substitutes, indicating that the BL signal reached the Jα-helix as well as the A'α/Aβ gap but could not activate STK. The amino acid residues, Glu474 and Lys475, in the gap are conserved among the phots of higher plants and may act as a joint to connect the structural changes in the Jα-helix with the activation of STK.

No MeSH data available.


Kinase activity of At phot1 LOV2-STK E474W, (A), and K475K, (B) on P1Nt. 0, D and L indicates before incubation, in the dark and under BL irradiation, respectively.The upper and lower panels indicate autoradiogram and CBB staining of SDS-PAGE gels, respectively. The arrow and the arrowhead indicate the position of LOV2-STK and P1Nt, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4401697&req=5

pone.0124284.g007: Kinase activity of At phot1 LOV2-STK E474W, (A), and K475K, (B) on P1Nt. 0, D and L indicates before incubation, in the dark and under BL irradiation, respectively.The upper and lower panels indicate autoradiogram and CBB staining of SDS-PAGE gels, respectively. The arrow and the arrowhead indicate the position of LOV2-STK and P1Nt, respectively.

Mentions: The impairment of kinase activation without affecting conformational changes detected by peptide mapping and the photochemical properties implies that Glu474 and Lys475 play special roles in kinase activation. According to the crystal structure of As phot1 LOV2-Jα, Lys413 corresponding to the Lys475 in At phot1 is supposed to interact with Thr535, which corresponds to Thr604 in At phot1, through hydrogen-bonding [28]. In contrast, Glu412 corresponding to Glu474 in At phot1 is not involved in hydrogen bonding [28], and Glu474 faces the aqueous phase [36]. This suggests a possible interaction(s) with an unidentified partner(s) in the LOV2-STK that might be involved in signaling kinase activation. To obtain the information regarding this possible interaction, Glu474 was substituted by Trp and Lys. The E474W substitution abolished the BL-induced STK activity but E474K substitution did not (Fig 7). This suggests a neutral hydrophilic amino acid residue is an interacting partner of Glu474 because of the effect of the hydrophobicity and the lack of charge specificity in the amino acid substitution experiments.


Essential role of the A'α/Aβ gap in the N-terminal upstream of LOV2 for the blue light signaling from LOV2 to kinase in Arabidopsis photototropin1, a plant blue light receptor.

Kashojiya S, Okajima K, Shimada T, Tokutomi S - PLoS ONE (2015)

Kinase activity of At phot1 LOV2-STK E474W, (A), and K475K, (B) on P1Nt. 0, D and L indicates before incubation, in the dark and under BL irradiation, respectively.The upper and lower panels indicate autoradiogram and CBB staining of SDS-PAGE gels, respectively. The arrow and the arrowhead indicate the position of LOV2-STK and P1Nt, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401697&req=5

pone.0124284.g007: Kinase activity of At phot1 LOV2-STK E474W, (A), and K475K, (B) on P1Nt. 0, D and L indicates before incubation, in the dark and under BL irradiation, respectively.The upper and lower panels indicate autoradiogram and CBB staining of SDS-PAGE gels, respectively. The arrow and the arrowhead indicate the position of LOV2-STK and P1Nt, respectively.
Mentions: The impairment of kinase activation without affecting conformational changes detected by peptide mapping and the photochemical properties implies that Glu474 and Lys475 play special roles in kinase activation. According to the crystal structure of As phot1 LOV2-Jα, Lys413 corresponding to the Lys475 in At phot1 is supposed to interact with Thr535, which corresponds to Thr604 in At phot1, through hydrogen-bonding [28]. In contrast, Glu412 corresponding to Glu474 in At phot1 is not involved in hydrogen bonding [28], and Glu474 faces the aqueous phase [36]. This suggests a possible interaction(s) with an unidentified partner(s) in the LOV2-STK that might be involved in signaling kinase activation. To obtain the information regarding this possible interaction, Glu474 was substituted by Trp and Lys. The E474W substitution abolished the BL-induced STK activity but E474K substitution did not (Fig 7). This suggests a neutral hydrophilic amino acid residue is an interacting partner of Glu474 because of the effect of the hydrophobicity and the lack of charge specificity in the amino acid substitution experiments.

Bottom Line: Using LOV2-STK polypeptides from Arabidopsis thaliana phot1, we found that truncation of the A'α-helix and amino acid substitutions at Glu474 and Lys475 in the gap between the A'α and the Aβ strand of LOV2 (A'α/Aβ gap) to Ala impaired the BL-induced activation of the STK, although they did not affect S390 formation.These BL-induced structural changes were observed with the Glu474Ala and the Lys475Ala substitutes, indicating that the BL signal reached the Jα-helix as well as the A'α/Aβ gap but could not activate STK.The amino acid residues, Glu474 and Lys475, in the gap are conserved among the phots of higher plants and may act as a joint to connect the structural changes in the Jα-helix with the activation of STK.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Osaka Prefecture University, Sakai, Osaka, Japan.

ABSTRACT
Phototropin (phot) is a blue light (BL) receptor in plants and is involved in phototropism, chloroplast movement, stomata opening, etc. A phot molecule has two photo-receptive domains named LOV (Light-Oxygen-Voltage) 1 and 2 in its N-terminal region and a serine/threonine kinase (STK) in its C-terminal region. STK activity is regulated mainly by LOV2, which has a cyclic photoreaction, including the transient formation of a flavin mononucleotide (FMN)-cysteinyl adduct (S390). One of the key events for the propagation of the BL signal from LOV2 to STK is conformational changes in a Jα-helix residing downstream of the LOV2 C-terminus. In contrast, we focused on the role of the A'α-helix, which is located upstream of the LOV2 N-terminus and interacts with the Jα-helix. Using LOV2-STK polypeptides from Arabidopsis thaliana phot1, we found that truncation of the A'α-helix and amino acid substitutions at Glu474 and Lys475 in the gap between the A'α and the Aβ strand of LOV2 (A'α/Aβ gap) to Ala impaired the BL-induced activation of the STK, although they did not affect S390 formation. Trypsin digested the LOV2-STK at Lys603 and Lys475 in a light-dependent manner indicating BL-induced structural changes in both the Jα-helix and the gap. The digestion at Lys603 is faster than at Lys475. These BL-induced structural changes were observed with the Glu474Ala and the Lys475Ala substitutes, indicating that the BL signal reached the Jα-helix as well as the A'α/Aβ gap but could not activate STK. The amino acid residues, Glu474 and Lys475, in the gap are conserved among the phots of higher plants and may act as a joint to connect the structural changes in the Jα-helix with the activation of STK.

No MeSH data available.