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.
Zhao
Jun
J
Department of Molecular and Cell Biology, University of Michigan, Ann Arbor 48109-1048, USA.
Peng
Peng
P
Schmitz
Robert J
RJ
Decker
Adria D
AD
Tax
Frans E
FE
Li
Jianming
J
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D060406:Brassinosteroids; D002777:Cholestanols / Q000378:metabolism*; D004268:DNA-Binding Proteins; D018506:Gene Expression Regulation, Plant; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D013260:Steroids, Heterocyclic / Q000378:metabolism*; D013379:Substrate Specificity; D020798:Two-Hybrid System Techniques
D029681:Arabidopsis Proteins; D060406:Brassinosteroids; D002777:Cholestanols; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011494:Protein Kinases; D013260:Steroids, Heterocyclic; C456160:BES1 protein, Arabidopsis; C454444:BZR1 protein, Arabidopsis; C448985:At4g18710 protein, Arabidopsis; C108270:BRI1 protein, Arabidopsis; C023623:brassinolide