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.


Absorption spectra and of the amino acid substitutes of At phot1 LOV2-STK in a solution containing 20 mM Tris-HCl, pH 7.8, 100 mM NaCl, 10% (w/v) glycerol, and 1 mM Na2EGTA at 20°C.The black and the gray line were measured after dark adaptation and BL irradiation, respectively. The insets show the light minus dark absorption difference spectra.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124284.g004: Absorption spectra and of the amino acid substitutes of At phot1 LOV2-STK in a solution containing 20 mM Tris-HCl, pH 7.8, 100 mM NaCl, 10% (w/v) glycerol, and 1 mM Na2EGTA at 20°C.The black and the gray line were measured after dark adaptation and BL irradiation, respectively. The insets show the light minus dark absorption difference spectra.

Mentions: To find key amino acid residues for the signal transduction from LOV2 to STK in the A’α-helix as well as the A’α/Aβ gap, charged amino acids (highlighted in Fig 1) were substituted with alanine and their kinase activities (Fig 3) and photochemistry (Fig 4) were studied. Among the substitutes, D465A in the A’α-helix and N476A in the A’α/Aβ gap preserved similar levels of phosphorylation as WT in a light dependent manner indicating that these amino acid residues do not contribute significantly to the photoactivation of STK. E471A and R472A also exhibited BL-induced phosphorylation; however, their activation levels were lower than of the activation levels of D465A and N476A. A comparison of R472A and E471A demonstrated that R472A has a slightly higher kinase activity than E471A under BL. In contrast, E474A and K475A impaired the BL-induced phosphorylation.


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)

Absorption spectra and of the amino acid substitutes of At phot1 LOV2-STK in a solution containing 20 mM Tris-HCl, pH 7.8, 100 mM NaCl, 10% (w/v) glycerol, and 1 mM Na2EGTA at 20°C.The black and the gray line were measured after dark adaptation and BL irradiation, respectively. The insets show the light minus dark absorption difference spectra.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124284.g004: Absorption spectra and of the amino acid substitutes of At phot1 LOV2-STK in a solution containing 20 mM Tris-HCl, pH 7.8, 100 mM NaCl, 10% (w/v) glycerol, and 1 mM Na2EGTA at 20°C.The black and the gray line were measured after dark adaptation and BL irradiation, respectively. The insets show the light minus dark absorption difference spectra.
Mentions: To find key amino acid residues for the signal transduction from LOV2 to STK in the A’α-helix as well as the A’α/Aβ gap, charged amino acids (highlighted in Fig 1) were substituted with alanine and their kinase activities (Fig 3) and photochemistry (Fig 4) were studied. Among the substitutes, D465A in the A’α-helix and N476A in the A’α/Aβ gap preserved similar levels of phosphorylation as WT in a light dependent manner indicating that these amino acid residues do not contribute significantly to the photoactivation of STK. E471A and R472A also exhibited BL-induced phosphorylation; however, their activation levels were lower than of the activation levels of D465A and N476A. A comparison of R472A and E471A demonstrated that R472A has a slightly higher kinase activity than E471A under BL. In contrast, E474A and K475A impaired the BL-induced phosphorylation.

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.