Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis.
CTR1 encodes a negative regulator of the ethylene response pathway in Arabidopsis 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 least in vitro, this domain does not autoinhibit kinase function. Molecular analysis of loss-of-function ctr1 alleles indicated that several mutations disrupt the kinase catalytic domain, and in vitro studies 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 has no detectable effect on the kinase activity of CTR1 in 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 transgenic Arabidopsis. These results suggest that CTR1 interacts with ETR1 in vivo, and that this association is required to turn off the ethylene-signaling pathway.
Huang
Yafan
Y
Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60607, USA.
Li
Hui
H
Hutchison
Claire E
CE
Laskey
James
J
Kieber
Joseph J
JJ
D000483:Alleles; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D005030:Ethylenes / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010940:Plant Proteins / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*
D005030:Ethylenes; D010940:Plant Proteins; D011956:Receptors, Cell Surface; C083750:ethylene receptors, plant; C036216:ethylene; D011494:Protein Kinases; C477753:CTR1 protein, Arabidopsis; D017346:Protein Serine-Threonine Kinases