Regulation of a GSK3 like kinase in plant steroid signaling.

Brassinosteroids (BRs) are special polyhydroxylated steroid hormones essential for plant growth and development. BRs are perceived by a plasma membrane associated receptor, BRI1, which initiates an intracellular signaling pathway involving a glycogen synthase kinase 3 (GSK3) like kinase (BIN2) and two transcription factors BES1 and BZR1. It was proposed that BIN2 negatively regulates BR signaling through phosphorylating BES1 and BZR1 to promote their degradation and inhibit their DNA binding activity. However, the functional study of BIN2 was mainly through its gain-of-function mutants, raising the question of whether endogenous BIN2 plays a role in BR signaling. A suppressor screen of a weak bin2 gain-of-function mutant and screens for T-DNA insertional mutants resulted in the identification of nine bin2 loss-of-function mutants. Genetic, biochemical and physiological studies of a loss-of-function bin2 mutant and a triple knockout mutant of BIN2 and its two closest homologs (BIL1, BIL2) revealed that BIN2 functions redundantly with BIL1 and BIL2 to regulate BR signaling, with BIN2 playing a dominant role. Interestingly, the triple knockout plant still responds to BR treatment, suggesting the possible involvement of other GSK3s. We propose that Arabidopsis brassinosteroids utilize a group of GSK3 kinases to negatively regulate the BR signaling pathway. To investigate the BIN2 regulation mechanism, which is one of the biggest mysteries in the field, I conducted a yeast two-hybrid screen and identified five putative BIN2 binding proteins (BBPs) belonging to a plant specific gene family with 17 members in Arabidopsis. Genetic study through both gain-of-function and loss-of-function approaches revealed that BBP1, a plasma membrane localized protein, functions as a positive regulator in the BR signaling pathway. My biochemical results suggest that BBP1 regulates BR signaling through direct interaction with BIN2 at the cell membrane to prevent its phosphorylation activity. My study also revealed that the amount of BBP1 is regulated by BR. Taken together, these results suggest that the plant steroid hormone induces BBP1 to positively regulate the BR signaling pathway through its inhibitory effects on BIN2.PhDBiological SciencesMolecular biologyPlant biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/126859/2/3276333.pd