Articles in PresS. Am J Physiol Endocrinol Metab (December 2, 2008). doi:10.1152/ajpendo.90807.2008
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Hormone Signaling Through Protein Destruction – A Lesson From Plants
Xu Tan1 & Ning Zheng1,2
1
Department of Pharmacology, Box 357280, University of Washington, Seattle, WA 98195
2
Howard Hughes Medical Institute
Running Head: Ubiquitin Ligase as Hormone Receptor
Correspondence:
Ning Zheng, Ph.D
Howard Hughes Medical Institute
Department of Pharmacology
Box 357280
University of Washington
Seattle, WA 98195
Tel: 206-616-3990
Fax: 206-685-3822
E-mail: nzheng@u.washington.edu
Copyright © 2008 by the American Physiological Society.
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Abstract
Ubiquitin-dependent protein degradation has emerged as a major pathway regulating
eukaryotic biology. By employing a variety of ubiquitin ligases to target specific cellular proteins,
the ubiquitin-proteasome system controls physiological processes in a highly regulated fashion.
Recent studies on a plant hormone auxin have unveiled a novel paradigm of signal transduction, in
which ubiquitin ligases function as hormone receptors. Perceived by the F-box protein subunit of
the SCF-TIR1 ubiquitin ligase, auxin directly promotes the recruitment of a family of transcriptional
repressors for ubiquitination, thereby activating extensive transcriptional programs. Structural
studies have revealed that auxin functions through a “molecular glue” mechanism to enhance
protein-protein interactions with the assistance of another small molecule co-factor inositol
hexakisphosphate. Given the extensive repertoire of similar ubiquitin ligases in eukaryotic cells,
this novel and widely adopted hormone signaling mechanism in plants may also exist in other
organisms.
Keywords
Auxin; phytohormone; hormone receptor; F-box protein; ubiquitin ligase; protein degradation
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In multi-cellular organisms, hormones are produced and released by one tissue and
transported to another to regulate cell growth, metabolism, and a myriad of cellular responses. Past
studies have identified several families of receptor proteins in the target cells responsible for sensing
various hormones in different chemical forms. These classic receptors include cell surface receptors
such as G-protein coupled receptors (GPCR) and receptor tyrosine kinases, which trigger second
messenger and protein phosphorylation-mediated signal transduction, and intracellular receptors
such as steroid and thyroid hormone receptors, which function as transcription factors directly
regulating gene expression via a DNA-binding domain. Recent advances in plant biology have
revealed yet another paradigm of hormone perception, in which the hormone signal is sensed by
components of the ubiquitin-proteasome system that is commonly found in all eukaryotic cells.
Structural studies of the prototypical plant hormone receptor unravel a novel mechanism of hormone
action and an unexpected small molecule co-factor.
Ubiquitin Ligases and Ubiquitin-Proteasome System
Cellular protein homeostasis is controlled by the balance between protein synthesis and
degradation. While protein synthesis based on gene transcription and translation has long been
recognized as a key process regulating every physiological function, the protein degradation process
has only gained attention in the last 20 years. We now appreciate that the ubiquitin-proteasome
system (UPS) is a major route of protein degradation in eukaryotes and actively regulating a wide
spectrum of cellular processes such as gene transcription, signal transduction, metabolism, DNA
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repair and replication (9). The UPS consists of a three enzymes cascade (E1, E2, and E3) and a
multi-component proteolytic apparatus, the 26s proteasome (17). E1, or the ubiquitin-activating
enzyme, activates ubiquitin by adenylating its C-terminus and conjugates the activated ubiquitin to
the active site cysteine. E1 then transfers ubiquitin to the E2, or ubiquitin-conjugating enzyme,
which also uses a cysteine residue to form a thiolester bond with ubiquitin. At the last step, the E2conjugated ubiquitin is transferred to a lysine residue of the substrate protein, which is brought to
the vicinity of E2 by a specific E3 or ubiquitin ligase. This process repeats by adding ubiquitin to a
lysine residue of the previously conjugated ubiquitin, thereby generating a chain of polyubiquitin
linked to the substrate protein. Proteasome recognizes the ubiquitin chain pattern and recruits the
ubiquitinated protein for proteolysis. In this complex system, the specificity and temporal control
are mainly afforded by the large repertoire of E3s, hundreds of which exist in humans. For example,
certain E3s recognize specifically phosphorylated peptide sequences of the protein substrate (9).
Indeed, substrate phosphorylation has emerged as a major regulatory mechanism of protein
ubiquitination, integrating protein degradation with diverse signaling pathways mediated by protein
kinases.
Since the protein synthesis process, especially transcription, is under the control of various
hormones, one is prompted to ask whether the opposite process – protein degradation is also
associated with hormone signaling. An obvious place to look for this connection is the hormone
receptor protein. Not surprisingly, a number of receptors, such as thyroid hormone receptor and
growth hormone receptor, are directly regulated by ubiquitination and proteasomal degradation (1,
25). In other cases, such as epidermal growth factor receptor and growth hormone receptor, the
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availability of the membrane-associated receptors is fine tuned by ubiquitin-mediated endocytosis
and lysosomal break down (11). In these two scenarios, the receptors are the substrates of the
ubiquitination reaction. The net result of ubiquitination is down-regulation of the receptors and thus
the hormone responses. In these cases, hormones are not directly involved in the ubiquitination
events.
More recently, a surprising new paradigm was revealed in the signaling pathway of an
important plant hormone auxin, in which the hormone takes direct control of an E3 ligase and
signals by promoting the ubiquitination and degradation of a set of transcriptional repressors (2, 13).
In other words, the hormone receptor is a ubiquitin ligase, whose activity is dependent on the
presence of the hormone molecule. This review focuses on the mechanism of this new signaling
paradigm and its implications.
TIR1 Ubiquitin Ligase as An Auxin Receptor
The natural auxin, indole-3-acetic acid, is the best studied plant hormone, which regulates
virtually every aspect of plant growth and development, from embryo patterning, growth responses
to fruit development (Fig. 1A)(26). Similar to many animal hormones, auxin achieves its multitude
of physiological effects through transcriptional regulation. In response to auxin, hundreds of genes
are activated by a family of transcriptional factors called ARFs (Auxin Response Factors), which
recognize specific promoter element characteristic of auxin response genes (7, 24). In the absence
of auxin, the protein products of a family of genes named AUX/IAAs (AUXin/Indole-3-Acetic
Acids) directly interact with ARFs and inhibit their transcriptional activity (Fig. 1B)(19, 22). In the
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presence of auxin, AUX/IAAs are rapidly removed, abrogating their inhibition of ARFs and the
expressions of auxin response genes (28). Interestingly, the transcription of some AUX/IAAs are
also activated by auxin, providing a negative feedback mechanism to modulate auxin activity (14).
The plant UPS plays a central role in auxin-induced degradation of AUX/IAA. An Arabidopsis
gene called TIR1 (Transport Inhibitor Response protein 1), which encodes a subunit of a ubiquitin
ligase complex, was shown to be a positive regulator of auxin response (20). TIR1 belongs to the Fbox family of proteins that function as interchangeable substrate receptor subunits of the SCF (Skp1Cullin1-F-box protein) ubiquitin ligase complexes, which belong to the largest superfamily of multisubunit cullin-RING E3s in eukaryotes (16). Mutations in TIR1 cause decreased auxin sensitivity in
plants, suggesting that its polyubiquitinated substrates are negative regulators of auxin signaling.
AUX/IAAs, which can directly interact with TIR1 through a short stretch of conserved region
(domain II), are the obvious substrate candidates (18). In fact, auxin stimulates AUX/IAAs-TIR1
binding and accelerates SCFTIR1-catalyzed ubiquitination and degradation of AUX/IAAs (6).
How is the auxin hormone signal perceived and transduced to the TIR1 ubiquitin ligase? Since
phosphorylation or other types of covalent posttranslational modifications of the substrate by an
upstream signaling molecule is often required for substrate-ubiquitin ligase interactions, it was
thought that identification of the AUX/IAA modification prerequisite for TIR1 interaction could
lead to the upstream modifying enzyme, the auxin signaling pathway, and eventually the auxin
receptor, whose identity had been elusive for decades. However, phosphorylation of AUX/IAAs
was soon ruled out as the determinant for auxin-induced interactions between TIR1 and AUX/IAAs
in a cell free system (3, 12). In 2005, two groups simultaneously reported their surprising findings
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that there is nothing else in between the plant hormone and the TIR1 ubiquitin ligase — TIR1 is the
long-sought auxin receptor (2, 13). Auxin itself provides the determining “modification” for the
AUX/IAA substrates to interact with the E3 ligase. Upon interacting with TIR1, auxin promotes the
binding of AUX/IAAs to the SCFTIR1 ubiquitin ligase complex and accelerates the
polyubiquitination of AUX/IAAs (Fig. 1B). The subsequent degradation of AUX/IAAs alleviates
their inhibition on ARFs, which then activate the auxin-induced transcription programs.
The surprising mechanism by which the plant hormone auxin signals provides the first example
of hormone perception by a ubiquitin ligase and establishes the first case of a naturally occurring
small molecule controlling the E3 activity of an SCF ubiquitin ligase complex. How does the auxin
molecule modulate the interactions between the SCFTIR1 ubiquitin ligase and its substrate
AUX/IAAs? This question becomes particularly puzzling given the fact that there are more than
two hundreds auxin analogues with different chemical structures known to have the common auxin
activity. The only common features shared by these compounds are an unsaturated planar ring
structure and a carboxyl side chain (Fig. 1A). How could they all be recognized by the auxin
receptor TIR1 and in turn regulate the ubiquitin ligase activity of the E3 enzyme?
Mechanism of Auxin Action
The recently reported X-ray crystal structures of TIR1 in different functional states reveal the
mechanisms of auxin action and auxin perception by TIR1 (21). In this study, TIR1 was cocrystallized with its binding partner ASK1 (Arabidopsis SKP1) protein, which is the adaptor subunit
of SCF, linking TIR1 to the E3 complex. TIR1 and ASK1 overall adopt a mushroom shape, with
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the F-box domain of TIR1 together with ASK1 forming the stem of the mushroom and the Cterminal 18 leucine-rich repeats (LRRs) of TIR1 forming the mushroom cap in a horse-shoe like
solenoid fold (Fig. 2A). Near the center space of the TIR1 horse shoe-shaped solenoid, three long
loops of the TIR1 LRRs, together with the inner wall of the solenoid, give rise to a large single
surface pocket for both hormone and substrate binding. Occupying the bottom portion of the TIR1
surface pocket, auxin uses its conserved carboxyl group to anchor itself to the pocket floor via a salt
bridge and hydrogen bonds with an arginine residue of TIR1. Meanwhile, the aromatic ring
structure of auxin is accommodated by the hydrophobic “wall” of the pocket through hydrophobic
packing and van der Waals interactions. The substrate peptide, corresponding to the conserved
domain II sequence of AUX/IAAs, sits on top of auxin and covers up the surface pocket. It directly
interacts with both the plant hormone and the hormone receptor. In the same study, structures of
TIR1 with different auxin analogs were also determined. Two synthetic auxin analogs, 2,4dichlorophenoxyacetic acid and 1-naphthalene acetic acid (Fig. 1A), bind to TIR1 in a similar mode
as auxin, albeit with different potencies due to their different aromatic ring structures. This partial
promiscuity of the auxin-binding site offers the structural basis for the recognition of diverse auxin
analogues by TIR1.
The action mode of auxin is in drastic contrast to the common mechanism of hormone action.
In most, if not all, previously known cases, a hormone binds to an allosteric site in the receptor and
causes conformational change of the receptor to elicit downstream effects. Comparison of the
structures of free and auxin-bound TIR1 shows that auxin binding does not change the conformation
of TIR1. Rather auxin alters the protein-protein interaction by filling a hydrophobic gap between
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the two proteins (Fig. 2B). In other words, it sticks two proteins together like a “molecular glue”
instead of a conformational switch. This mechanism is consistent with the partial promiscuity of the
auxin-binding pocket, because any hydrophobic moiety that can fit in the pocket without causing
steric hindrance to substrate binding would be able to increase the affinity between TIR1 and the
substrate. In addition, this mechanism bears a certain similarity to the widespread phosphorylationdependent recruitment of ubiquitination substrates to their specific ubiquitin ligases. In those cases,
phosphate groups also do not change the conformation of the modified proteins (8, 27). Rather, they
enhance protein-protein association by providing additional interactions and extending the interface,
just like auxin does. The only difference is that phosphorylation involves covalently linked
phosphate groups catalyzed by kinases, while auxin functions through non-covalent hydrophobic
interactions, which might have an advantage of rapid response to environmental stimuli.
Inositol hexakisphosphate as A Cofactor of the Auxin Receptor TIR1
In addition to the novel mechanism of auxin action, another surprise from the structure is a
previously unknown small molecule, inositol hexakisphosphate (IP6) co-purified with TIR1 (21).
The crystal structures reveal that IP6 binds in the core of the TIR1 LRR domain, right beneath the
auxin-binding site (Fig. 2A and 2B). The strict conservation of eight out of the ten IP6 interacting
TIR1 residues in plants implies that the IP6 molecule is not a non-specifically bound contaminating
factor. Close examination of the IP6-binding site further shows that two of the key residues that
form the floor of auxin-binding site are buttressed by the IP6 molecule, suggesting an important
structural role of IP6 in supporting TIR1’s function as an auxin receptor. Although it remains
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unclear whether IP6 has any signaling function, its close connection to other inositol phosphates
indicates that TIR1-mediated auxin signaling might be controlled by additional pathways. Overall,
the ability of TIR1 to employ two small molecule ligands, auxin and IP6, showcases the remarkable
potential of a ubiquitin ligase to sense and integrate signals in signal transduction.
Ubiquitin Ligases as Hormone Receptors
How pervasive is this new paradigm of ubiquitin ligase-mediated hormone perception and
signal transduction? At least in the plant kingdom, this is not a singular case. TIR1 is found in
almost all plant species, from moss to higher plants. In Arabidopsis thaliana, five paralogues of
TIR1 protein (AFB1-5) have been shown to collectively function as auxin receptors (4). Among the
six receptors, there might be some specificity in terms of recognition of different auxin analogs and
possibly different substrates, but their general mechanisms of action are conserved. Interestingly,
COI1, a distant homolog of TIR1, has recently been shown to perceive a very different plant
hormone, jasmonate and its amino acid derivatives (Fig. 1A), and induce the degradation of another
family of transcriptional repressors (10). Although the molecular mechanism of jasmonate action
has not been determined, its action in promoting the binding between COI1 and its ubiquitination
substrate polypeptides and the subsequent transcriptional activation is highly similar to auxin. In
addition to auxin and jasmonate, gibberellin is yet another plant hormone whose perception involves
an SCF ubiquitin ligase complex. Different from auxin and jasmonate, gibberellin is not directly
sensed by an F-box protein, but instead by an extension molecule of the SCF substrate receptor
subunit (23). Upon perceiving gibberellin, the plant protein GID1 recruits a family of transcription
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regulators named DELLA proteins to the GID2/SLY1 F-box protein for ubiquitination. Given the
seven hundreds F-box proteins and the great complexity of chemicals in plants (5), new examples of
small molecule sensors like TIR1 will almost certainly emerge in the future.
Are there ubiquitin ligases functioning as hormone receptors in organisms other than plants? At
molecular level, there is no obvious reason why this is not possible. With common precursors and
synthetic pathways, many hormones in plants and animals share strikingly similar overall structures.
Derived from tryptophan, the plant hormone auxin is reminiscent of animal hormones serotonin and
melatonin. With fatty acids as precursors, jasmonate in plants and prostaglandin in animals are both
cyclopentanone derivatives. On the ubiquitin ligase side, TIR1-like F-box proteins and the SCF E3
complexes are universally found in all eukaryotic cells (16). Expansion of the SCF-like cullinRING ubiquitin ligases occurred in both the plant and animal kingdoms. In fact, a mammalian SCFlike cullin-RING E3 complex has been recently shown to be a receptor for an exogenous chemical,
dioxin (15). With the nearly one thousand predicted human ubiquitin ligases, it would not be
surprising if some of them employ a similar strategy to sense and transduce endogenous hormone
signals. The fact that we have yet to discover a TIR1-like hormone receptor in humans might be to
due to the same reason that it took so long to find the auxin receptor in plants. Unlike conventional
hormones, auxin does not bind its receptor TIR1 with very high affinity. Upon docking to auxinbound TIR1, AUX/IAAs cover up auxin and contribute to the formation of the complete auxinbinding site. Although the presence of AUX/IAAs secures auxin binding, AUX/IAAs get rapidly
ubiquitinated and degraded, making it difficult to capture this co-receptor complex. In the same
vein of reasoning, traditional experiments in the past could have missed the opportunity to isolate a
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hormone-receptor complex in other systems if the high affinity hormone receptor is a ubiquitin
ligase-substrate pair. Now with the first example of this new paradigm of hormone perception
revealed in plants, we will have “the prepared mind” for potentially finding a similar case in other
organisms.
Despite the long stride made in the past years, the picture of auxin perception in plants is far
from complete. For example, it remains unclear whether TIR1 and its five paralogues AFB1–5
show any specificity toward different natural auxin compounds and some of the AUX/IAA
substrates proteins. Furthermore, when, where, and in what form are AUX/IAAs ubiquitinated by
SCFTIR1 upon auxin sensing? Is IP6 simply a constitutively bound structural co-factor of TIR1 or is
it a signaling molecule connecting auxin signaling with other cellular pathways through TIR1?
Finally, are there non-TIR1-like auxin receptors transducing the auxin signal for other outputs?
Further studies on these questions will allow us to fully recognize the multi-layers of specificity
behind this new paradigm of hormone signaling and at least partially explain the multitude of
diverse effects of auxin.
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ACKNOWLEDGEMENTS
We thank Nabiha H. Saifee for critical reading of the manuscript.
GRANTS
This work was supported by grants from the National Institute of Health and the Pew Scholar
Program. N. Z. is a Howard Hughes Medical Institute Investigator.
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Figure legends
Fig. 1. The SCFTIR1 ubiquitin ligase functions as the receptor for the plant hormone auxin. (A)
Chemical structures of the natural auxin, two auxin analogues (2,4-dichlorophenoxyacetic acid [2,4D] and 1-naphthalene acetic acid [1-NAA]), and jasmonate-isoleucine [JA-Ile]. (B) The role of
SCFTIR1 ubiquitin ligase in the auxin signaling pathway in plants. In the absence of auxin,
AUX/IAAs repress the expression of auxin response genes by binding to the transcription activator
ARFs (left). In the presence of auxin, the multi-subunit SCFTIR1 ubiquitin ligase recognizes the
hormone and promotes the polyubiquitination and proteasomal degradation of AUX/IAAs in a
hormone-dependent manner (middle). Auxin-induced degradation of AUX/IAAs frees ARFs, which
activate the transcription of auxin response genes.
Figure 2. The structure and mechanism of auxin action. (A) Crystal structure of ASK1-TIR1-IP6 in
complex with auxin and an AUX/IAA domain II peptide. Both ASK1 (magenta) and TIR1 (blue)
are shown in ribbon. Auxin is shown in space-filled model ( green and red spheres). The AUX/IAA
peptide (orange) and IP6 (red) are shown in sticks. (B) A schematic diagram showing how auxin is
sensed by TIR1 with the assistance of IP6 and how the hormone promotes TIR1-AUX/IAA
interactions.