Mapping of Low- and High-Fluence Autophosphorylation Sites in Phototropin 1
Autor: | Michael Salomon, Tibor von Zeppelin, Wolfhart Rüdiger, Elke Knieb |
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Rok vydání: | 2003 |
Předmět: |
Phosphopeptides
Phototropin Avena Molecular Sequence Data Biology Chloroplast relocation Peptide Mapping Biochemistry Receptors G-Protein-Coupled Dephosphorylation Cryptochrome Drosophila Proteins Amino Acid Sequence Phosphorylation Eye Proteins Protein kinase A Phototropism Flavoproteins Sequence Homology Amino Acid Autophosphorylation food and beverages Cyclic AMP-Dependent Protein Kinases Cryptochromes Mutagenesis Site-Directed Biophysics Photoreceptor Cells Invertebrate sense organs |
Zdroj: | Biochemistry. 42:4217-4225 |
ISSN: | 1520-4995 0006-2960 |
Popis: | Phototropins, originally detected by their blue light-dependent autophosphorylation, are plant photoreceptors involved in several blue light responses such as phototropism, chloroplast relocation, leaf expansion, rapid inhibition of hypocotyl growth, and stomatal opening. Three domains have been identified in phototropin sequences, two chromophore binding domains (LOV1 and LOV2) and a kinase domain. We describe here two additional domains, the N-terminus upstream of LOV1 and the hinge region between LOV1 and LOV2, as the regions for autophosphorylation; the phosphorylation sites were identified by site-directed mutagenesis as S27, S30, S274, S300, S317, S325, S332, and S349 of the PHOT1a sequence of Avena sativa. Investigation of the autophosphorylation in vivo revealed that serines close to the LOV1 domain are phosphorylated at lower fluence of blue light than the serines close to the LOV2 domain. Recovery of phosphorylation in vivo during a dark period after saturating irradiation is caused by dephosphorylation rather than by degradation of the phosphorylated form and new synthesis of nonphosphorylated phototropin. The results were obtained by a combination of autophosphorylation of phototropin with phosphorylation of recombinant domains by protein kinase A, which turned out to have the same site specificity as the phototropin kinase, followed by proteolysis and separation of phosphopeptides. With the knowledge of the phosphorylation sites, the physiological and biochemical consequences of autophosphorylation can now be approached by site-directed mutagenesis of phototropins. |
Databáze: | OpenAIRE |
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