Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling inArabidopsis

Open Access
Publisher Website
Google Scholar
Abstract
Summary: CTR1encodes a negative regulator of the ethylene response pathway inArabidopsis thaliana. The C‐terminal domain of CTR1 is similar to the Raf family of protein kinases, but its first two‐thirds encodes a novel protein domain. We used a variety of approaches to investigate the function of these two CTR1 domains. Recombinant CTR1 protein was purified from a baculoviral expression system, and shown to possess intrinsic Ser/Thr protein kinase activity with enzymatic properties similar to Raf‐1. Deletion of the N‐terminal domain did not elevate the kinase activity of CTR1, indicating that, at leastin vitro, this domain does not autoinhibit kinase function. Molecular analysis of loss‐of‐functionctr1alleles indicated that several mutations disrupt the kinase catalytic domain, andin vitrostudies confirmed that at least one of these eliminates kinase activity, which indicates that kinase activity is required for CTR1 function. One missense mutation,ctr1–8, was found to result from an amino acid substitution within a new conserved motif within the N‐terminal domain.Ctr1–8 hasno detectable effect on the kinase activity of CTR1in vitro,but rather disrupts the interaction with the ethylene receptor ETR1. This mutation also disrupts the dominant negative effect that results from overexpression of the CTR1 amino‐terminal domain in transgenicArabidopsis. These results suggest that CTR1 interacts with ETR1in vivo, and that this association is required to turn off the ethylene‐signaling pathway.