In vivo phosphorylation site mapping and functional characterization of Arabidopsis phototropin 1.
Phototropins (phot1 and phot2) are blue-light receptor kinases controlling a range of responses that optimize the photosynthetic efficiency of plants. Light sensing is mediated by two flavin-binding motifs, known as LOV1 and LOV2, located within the N-terminal region of the protein. Photoexcitation via LOV2 leads to activation of the C-terminal kinase domain and consequently receptor autophosphorylation. However, knowledge of the in-vivo phosphorylation sites for Arabidopsis phototropins is lacking and has impeded progress in elucidating the functional significance of receptor phosphorylation. We have purified phot1 from Arabidopsis and identified the in-vivo sites of receptor phosphorylation by liquid chromatography tandem mass spectrometry. Arabidopsis-derived phot1 binds flavin mononucleotide as chromophore and is phosphorylated at four major sites located upstream of LOV2 (Ser(58), Ser(85), Ser(350), and Ser(410)), three of which are induced by blue light. Nevertheless, structure-function analysis indicates that the biological activity of phot1 can be attributed to a modular unit comprising the LOV2-kinase region of the protein. Thus, peptide regions upstream of LOV2, including the sites of receptor phosphorylation identified here, do not appear to be important for receptor signaling. By contrast, these regions may be necessary for maximizing stomatal performance and possibly light-induced relocalization of phot1.
Sullivan
Stuart
S
Plant Science Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, University Avenue, Glasgow, Scotland, UK.
Thomson
Catriona E
CE
Lamont
Douglas J
DJ
Jones
Matthew A
MA
Christie
John M
JM
D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism* / Q000528:radiation effects; D002874:Chromosome Mapping / Q000379:methods*; D004268:DNA-Binding Proteins / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000378:metabolism; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008027:Light; D013058:Mass Spectrometry; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism* / Q000528:radiation effects; D010766:Phosphorylation; D010788:Photosynthesis / Q000235:genetics / Q000528:radiation effects*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000528:radiation effects
D029681:Arabidopsis Proteins; D004268:DNA-Binding Proteins; C527342:LOV1 protein, Arabidopsis; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005486:Flavin Mononucleotide; C103646:NPH1 protein, Arabidopsis; D017346:Protein Serine-Threonine Kinases