Mutational analysis of phototropin 1 provides insights into the mechanism underlying LOV2 signal transmission.
Phototropins (phot1 and phot2) are blue light-activated serine/threonine protein kinases that elicit a variety of photoresponses in plants. Light sensing by the phototropins is mediated by two flavin mononucleotide (FMN)-binding domains, designated LOV1 and LOV2, located in the N-terminal region of the protein. Exposure to light results in the formation of a covalent adduct between the FMN chromophore and a conserved cysteine residue within the LOV domain. LOV2 photoexcitation is essential for phot1 function in Arabidopsis and is necessary to activate phot1 kinase activity through light-induced structural changes within a conserved alpha-helix situated C-terminal to LOV2. Here we have used site-directed mutagenesis to identify further amino acid residues that are important for phot1 activation by light. Mutagenesis of bacterially expressed LOV2 and full-length phot1 expressed in insect cells indicates that perturbation of the conserved salt bridge on the surface of LOV2 does not play a role in receptor activation. However, mutation of a conserved glutamine residue (Gln(575)) within LOV2, reported previously to be required to propagate structural changes at the LOV2 surface, attenuates light-induced autophosphorylation of phot1 expressed in insect cells without compromising FMN binding. These findings, in combination with double mutant analyses, indicate that Gln(575) plays an important role in coupling light-driven cysteinyl adduct formation from within LOV2 to structural changes at the LOV2 surface that lead to activation of the C-terminal kinase domain.
Jones
Matthew A
MA
Plant Science Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
Feeney
Kevin A
KA
Kelly
Sharon M
SM
Christie
John M
JM
D000818:Animals; D017360:Arabidopsis / Q000502:physiology*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000502:physiology*; D001683:Biological Clocks; D002460:Cell Line; D056931:Cryptochromes; D003545:Cysteine; D004252:DNA Mutational Analysis; D005486:Flavin Mononucleotide; D005420:Flavoproteins / Q000235:genetics / Q000378:metabolism*; D005973:Glutamine; D055537:Light Signal Transduction; D016297:Mutagenesis, Site-Directed; D014162:Transfection
D029681:Arabidopsis Proteins; C538962:CRY1 protein, Arabidopsis; D056931:Cryptochromes; D005420:Flavoproteins; C450148:LKP2 protein, Arabidopsis; D005973:Glutamine; D005486:Flavin Mononucleotide; D003545:Cysteine