Plant Kelch phosphatases are Ser/Thr phosphatases involved in cell cycle regulation

Abstract

Brassinosteroids (BRs) are plant steroid hormones sensed by the membrane receptor kinase BRI1. Activation of BRI1 leads to the dephosphorylation of BZR1/BES1 transcription factors. Overexpression of the Kelch phosphatase BRI1 SUPPRESSOR 1 (BSU1) rescued the growth defects of bri1 mutants. Subsequent studies identified BSU1 as a protein tyrosine phosphatase, which promotes BR signaling by dephosphorylating a phosphotyrosine in the glycogen synthase kinase 3 BIN2. Crystal structures of the BSU1 phosphatase domain now reveal a high degree of structural similarity to protein phosphatase 1 (PP1), a eukaryotic serine/threonine phosphatase. Consistently, BSU1 efficiently dephosphorylated phosphothreonine- and phosphoserine-containing substrate peptides, but showed no detectable activity toward BIN2 and other phosphotyrosine substrates. A catalytically inactive BSU1 phosphatase domain suppresses the growth phenotypes of the Arabidopsis bri1-5 mutant and binds the BSU1 homologs BSL1-3. bsu1 and bsu1 bsl1 bsl2/3 loss-of-function mutants display wild-type-like BR responses, but exhibit stomatal patterning and fertility defects. Importantly, the PP1-like C-terminal tail of BSU1 is phosphorylated at Thr785 by a cyclin-dependent kinase complex. The phosphorylated tail binds to the BSU1 substrate-binding grooves, blocking access to the active site. Mutation of Thr785 to alanine activates BSU1, suggesting that Kelch phosphatases and PP1 share a common regulatory mechanism. Deletion of the Marchantia polymorpha Kelch phosphatase MpBSLM results in an undifferentiated cell mass phenotype, associated with the over-activation of a cell cycle reporter. Taken together, our experiments suggest that plant Kelch phosphatases act as PP1-like cell cycle regulators, rather than as tyrosine phosphatases in brassinosteroid signaling. (243 words) Significance StatementBrassinosteroid hormones initiate a well-characterized signaling pathways in plants. While the early signaling events at the plasma membrane have been thoroughly examined, the precise sequence of molecular events that comprise the cytoplasmic signaling cascade is less well-understood. Here, the molecular function of the Kelch protein phosphatase BSU1 is re-evaluated. Previous reports have indicated that BSU1 dephosphorylates the GSK3 kinase BIN2 on a central tyrosine residue. However, structural, quantitative enzyme kinetic, and genetic analyses now suggest that BSU1 is a PP1-like serine/threonine phosphatase involved in cell cycle control rather than a tyrosine phosphatase involved in BIN2 de-phosphorylation. The regulation of BSU1 by a cyclin-dependent kinase complex and the role of Kelch phosphatases in cell cycle progression in Marchantia polymorpha are experimentally characterized. (121 words)