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Domain swapping to assess the mechanistic basis of Arabidopsis phototropin 1 receptor kinase activation and endocytosis by blue light.
- Source :
-
The Plant cell [Plant Cell] 2009 Oct; Vol. 21 (10), pp. 3226-44. Date of Electronic Publication: 2009 Oct 30. - Publication Year :
- 2009
-
Abstract
- Phototropins (phot1 and phot2) are plasma membrane-associated receptor kinases that respond specifically to blue and UV wavelengths. In addition to a C-terminal Ser/Thr kinase domain, phototropins contain two N-terminal chromophore binding LOV domains that function as photoswitches to regulate a wide range of enzymatic activities in prokaryotes and eukaryotes. Through domain swapping, we show that the photochemical properties of Arabidopsis thaliana phot1 rely on interactions between LOV1 and LOV2, which are facilitated by their intervening linker sequence. Functional analysis of domain-swap proteins supports a mechanism whereby LOV2 acts as a dark-state repressor of phot1 activity both in vitro and in vivo. Moreover, we find a photoactive role for LOV1 in arresting chloroplast accumulation at high light intensities. Unlike LOV2, LOV1 cannot operate as a dark-state repressor, resulting in constitutive receptor autophosphorylation and accelerated internalization from the plasma membrane. Coexpression of active and inactive forms of phot1 demonstrates that autophosphorylation can occur intermolecularly, independent of LOV1, via light-dependent receptor dimerization in vivo. Indeed, transphosphorylation is sufficient to promote phot1 internalization through a clathrin-dependent endocytic pathway triggered primarily by phosphorylation of Ser-851 within the kinase activation loop. The mechanistic implications of these findings in regard to light-driven receptor activation and trafficking are discussed.
- Subjects :
- Arabidopsis genetics
Arabidopsis Proteins chemistry
Arabidopsis Proteins genetics
Chloroplasts metabolism
Chromatography, Liquid
Clathrin metabolism
DNA-Binding Proteins chemistry
DNA-Binding Proteins genetics
DNA-Binding Proteins metabolism
Endocytosis physiology
Immunoprecipitation
Microscopy, Confocal
Molecular Sequence Data
Mutagenesis
Phosphorylation radiation effects
Phototropins chemistry
Phototropins genetics
Plants, Genetically Modified genetics
Plants, Genetically Modified metabolism
Plants, Genetically Modified radiation effects
Protein Binding radiation effects
Reverse Transcriptase Polymerase Chain Reaction
Tandem Mass Spectrometry
Arabidopsis metabolism
Arabidopsis radiation effects
Arabidopsis Proteins metabolism
Endocytosis radiation effects
Light
Phototropins metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1532-298X
- Volume :
- 21
- Issue :
- 10
- Database :
- MEDLINE
- Journal :
- The Plant cell
- Publication Type :
- Academic Journal
- Accession number :
- 19880798
- Full Text :
- https://doi.org/10.1105/tpc.109.067876