Two putative BIN2 substrates are nuclear components of brassinosteroid signaling.
GSK3 is a highly conserved kinase that negatively regulates many cellular processes by phosphorylating a variety of protein substrates. BIN2 is a GSK3-like kinase in Arabidopsis that functions as a negative regulator of brassinosteroid (BR) signaling. It was proposed that BR signals, perceived by a membrane BR receptor complex that ... contains the leucine (Leu)-rich repeat receptor-like kinase BRI1, inactivate BIN2 to relieve its inhibitory effect on unknown downstream BR-signaling components. Using a yeast (Saccharomyces cerevisiae) two-hybrid approach, we discovered a potential BIN2 substrate that is identical to a recently identified BR-signaling protein, BES1. BES1 and its closest homolog, BZR1, which was also uncovered as a potential BR-signaling protein, display specific interactions with BIN2 in yeast. Both BES1 and BZR1 contain many copies of a conserved GSK3 phosphorylation site and can be phosphorylated by BIN2 in vitro via a novel GSK3 phosphorylation mechanism that is independent of a priming phosphorylation or a scaffold protein. Five independent bes1 alleles containing the same proline-233-Leu mutation were identified as semidominant suppressors of two different bri1 mutations. Over-expression of the wild-type BZR1 gene partially complemented bin2/+ mutants and resulted in a BRI1 overexpression phenotype in a BIN2(+) background, whereas overexpression of a mutated BZR1 gene containing the corresponding proline-234-Leu mutation rescued a weak bri1 mutation and led to a bes1-like phenotype. Confocal microscopic analysis indicated that both BES1 and BZR1 proteins were mainly localized in the nucleus. We propose that BES1/BZR1 are two nuclear components of BR signaling that are negatively regulated by BIN2 through a phosphorylation-initiated process.
Mesh Terms:
Amino Acid Sequence, Arabidopsis, Arabidopsis Proteins, Base Sequence, Cholestanols, Gene Expression Regulation, Plant, Microscopy, Confocal, Molecular Sequence Data, Mutation, Nuclear Proteins, Phosphorylation, Protein Kinases, Saccharomyces cerevisiae, Sequence Homology, Amino Acid, Signal Transduction, Steroids, Heterocyclic, Substrate Specificity, Two-Hybrid System Techniques
Amino Acid Sequence, Arabidopsis, Arabidopsis Proteins, Base Sequence, Cholestanols, Gene Expression Regulation, Plant, Microscopy, Confocal, Molecular Sequence Data, Mutation, Nuclear Proteins, Phosphorylation, Protein Kinases, Saccharomyces cerevisiae, Sequence Homology, Amino Acid, Signal Transduction, Steroids, Heterocyclic, Substrate Specificity, Two-Hybrid System Techniques
Plant Physiol.
Date: Nov. 01, 2002
PubMed ID: 12427989
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