Journal of Molecular Biology
Volume 415, Issue 4, 27 January 2012, Pages 768-779
Journal home page for Journal of Molecular Biology

Protein Kinase Domain of CTR1 from Arabidopsis thaliana Promotes Ethylene Receptor Cross Talk

https://doi.org/10.1016/j.jmb.2011.11.046Get rights and content

Abstract

Ethylene controls many aspects of plant growth and development. Signaling by the gaseous phytohormone is initiated by disulfide-linked membrane-bound receptors, and the formation of heteromeric receptor clusters contributes to the broad range of ethylene responsiveness. In Arabidopsis thaliana, the TCS-like ethylene receptors interact with the cytosolic serine/threonine kinase constitutive triple response 1 (CTR1), a proposed mitogen-activated protein kinase kinase kinase. In the absence of the hormone, the receptor and therefore CTR1 are active. Hence, ethylene acts as an inverse agonist of its signaling pathway. The three-dimensional structures of the active, triphosphorylated and the unphosphorylated, inactive kinase domain of CTR1 in complex with staurosporine illustrate the conformational rearrangements that form the basis of activity regulation. Additionally, in analytical ultracentrifugation experiments, active kinase domains form back-to-back dimers, while inactive and activation loop variants are monomers. Together with a front-to-front activation interface, the active protein kinase dimers thereby engage in interactions that promote CTR1-mediated cross talk between ethylene receptor clusters. This model provides a structural foundation for the observed high sensitivity of plants to ethylene.

Graphical Abstract

Highlights

► Structures of active and inactive forms of the kinase domain of CTR1 are shown. ► The activity of the CTR1 kinase domain is dimerization dependent. ► The activation interface of kinase positions the activation loop close to the active site. ► Dimer and activation interfaces provide a model for CTR1-mediated ethylene receptor cross talk.

Introduction

The function of ethylene as a phytohormone was discovered in 1901.1 Since then, the realization of its profound and multifaceted impact on plant growth and development has been continuously growing.2 Today, several components of the underlying signaling network are known, but the picture is still incomplete.3

In Arabidopsis thaliana, the many responses to ethylene are regulated by a group of five membrane-bound receptors (ETR1, ETR2, ERS1, ERS2, and EIN4), which initiate signal transduction.4 The basic functional unit of the receptors is a disulfide-linked dimer,5 which binds one copper ion and therefore one ethylene.6 Non-covalent higher-order complexes between ethylene receptors were also discovered and suggested as an explanation for the broad range of ethylene sensitivity (0.2 nL/L to 1000 μL/L) as well as the dominant nature of ethylene-insensitive mutants.7 The binding site for ethylene lies within the conserved hydrophobic N-terminal receptor domain,8 which is located in the membrane of the endoplasmic reticulum.9 The C-terminal cytosolic domains resemble classical bacterial two-component systems.10 Two-component systems, which regulate numerous signaling pathways in bacteria, comprise a histidine kinase and a response regulator element (the latter is absent in ERS1 and ERS2).11 Consistent with this discovery, the autophosphorylation activity of ETR1 from A. thaliana was demonstrated in vitro,12, 13 but this activity is not essential for in vivo signaling.14

Another fundamental member of the ethylene response pathway is constitutive triple response 1 (CTR1), a cytosolic protein kinase, which bears most resemblance to the RAF family of serine/threonine protein kinases.15, 16 RAF family kinases are ordinarily activated by receptor tyrosine kinases and belong to the family of mitogen-activated protein kinase kinase kinases (MAPKKKs), which initiate the cytosolic cascade of many eukaryotic signal transduction pathways.17 CTR1 is one of only two confirmed direct recipients of ethylene receptor activity. Thus, this signaling pathway presents an interesting case, wherein a two-component signaling system manipulates an MAPKKK and possibly an MAPKKK signaling cascade. EIN2 constitutes another essential, membrane-bound transducer of ethylene signaling.18 In the absence of ethylene, EIN2 has a short half-life, an effect that appears to depend upon the functional state of the ethylene receptor–CTR1 complex.19

Since both ethylene receptors and CTR1 are active in the absence of the hormone, ethylene acts as an inverse agonist of its own pathway. Binding of ethylene inactivates the receptor–CTR1 complex and results in the accumulation of EIN3 and EIL1 (EIN3-like 1) in the nucleus.20 Consequently, deletion of CTR1 leads to a strong ethylene response in seedlings and adult plants,17 consistent with its proposed role in the pathway.

CTR1 from A. thaliana consists of 821 amino acids (Mr = 90,306) and two domains. The ∼ 540 N-terminal amino acids share little sequence homology with the N-terminus of its closest homologue, B-RAF. This domain interacts with the histidine kinase domains of ETR1 and ERS1.21 As with other MAPKKKs, deletion of the N-terminal domain of CTR1 leads to a constitutively active kinase.22, 23 The ∼ 280-amino-acid-long C-terminal domain contains all sequence motifs found in serine/threonine kinases and carries 37% sequence identity with human B-RAF.16 Here, we present the crystallographic structures of the CTR1 kinase domain in its active, threefold phosphorylated and in its inactive, unphosphorylated form. These structures reveal the intramolecular rearrangements that discern the different activity states of CTR1. They also reveal two distinct interfaces that provide a model for CTR1-mediated receptor cross talk.

Section snippets

Protein kinase domain of CTR1

The C-terminal and catalytically active protein kinase domain of CTR1 (CTR1-kd) from A. thaliana was heterologously expressed in Escherichia coli and purified and crystallized as previously described.24 In addition to the wild-type (WT) construct, we expressed and purified a kinase dead variant of CTR1-kd and characterized both biochemically and crystallographically in order to elucidate the mechanisms, which account for the activation of CTR1-kd. Both constructs start 18 residues before the

Discussion

Our observation that heterologously expressed CTR1-kd purified from E. coli with three to six phosphorylation sites, while a catalytically dead version, CTR1-D676N, expressed unphosphorylated, indicated the presence of an active and an inactive form of the CTR1 kinase domain. Kinetic assays confirmed that CTR1-kd promotes its own activation and that CTR1-D676N is catalytically inactive (Fig. 3). Surprisingly, the self-directed activity of CTR1-kd is a dynamic process, as demonstrated by the

Crystallography

Cloning, expression, purification, and crystallization of CTR1-kd and CTR1-D676N have been described previously.24 The catalytic domain of CTR1 was solved by molecular replacement using Phaser as implemented in CCP4 6.1.131 with B-RAF (PDB ID: 3C4C, 37% identity) as a search model. Coordinates of CTR1-kd were refined using REFMAC 5.632 alternating with rounds of manual model building using the program Coot.33 This model was subsequently used as a starting model for refinement of CTR1-D676N.

Acknowledgements

We would like to thank Dr. Vladimir Rubin at the European Molecular Biology Laboratory core facility for the execution of the AUC experiments and the proteomics core facility at the European Molecular Biology Laboratory, Heidelberg, for assistance with the MS analysis. We acknowledge the European Synchrotron Radiation Facility for provision of beam time at beamlines ID23-1 and ID29.

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