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10.1146/annurev.cellbio.18.031502.133614
Annu. Rev. Cell Dev. Biol. 2002. 18:421–62
doi: 10.1146/annurev.cellbio.18.031502.133614
c 2002 by Annual Reviews. All rights reserved
Copyright °
First published online as a Review in Advance on June 28, 2002
AUTOINHIBITORY DOMAINS: Modular Effectors
of Cellular Regulation
Annu. Rev. Cell Dev. Biol. 2002.18:421-462. Downloaded from www.annualreviews.org
by University of Utah - Marriot Library on 05/08/14. For personal use only.
Miles A. Pufall and Barbara J. Graves
Huntsman Cancer Institute, Department of Oncological Sciences, University of Utah,
2000 Circle of Hope, Salt Lake City, Utah 84112-5550;
e-mail: [email protected]; [email protected]
Key Words repression, intramolecular, masking, allosteric, autoinhibition
■ Abstract Autoinhibitory domains are regions of proteins that negatively regulate
the function of other domains via intramolecular interactions. Autoinhibition is a potent
regulatory mechanism that provides tight “on-site” repression. The discovery of autoinhibition generates valuable clues to how a protein is regulated within a biological
context. Mechanisms that counteract the autoinhibition, including proteolysis, posttranslational modifications, as well as addition of proteins or small molecules in trans,
often represent central regulatory pathways. In this review, we document the diversity
of instances in which autoinhibition acts in cell regulation. Seven well-characterized
examples (e.g., σ 70, Ets-1, ERM, SNARE and WASP proteins, SREBP, Src) are covered in detail. Over thirty additional examples are listed. We present experimental
approaches to characterize autoinhibitory domains and discuss the implications of this
widespread phenomenon for biological regulation in both the normal and diseased
states.
CONTENTS
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Definition of Autoinhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Autoinhibition as a Regulatory Strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Experimental Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
INHIBITION OF LIGAND BINDING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sigma 70 (σ 70 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ets-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
SNARE Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Wiscott-Aldrich Syndrome Protein (WASP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
INHIBITION OF SUBCELLULAR LOCALIZATION . . . . . . . . . . . . . . . . . . . . . . . .
ERM Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
SREBP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
INHIBITION OF ENZYMATIC ACTIVITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Src Kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conformational Change is a Common Feature
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of Autoinhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Autoinhibition Adds a Layer of Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A Second Role for the Autoinhibitory Domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Implications for Human Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
451
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452
453
INTRODUCTION
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Definition of Autoinhibition
Autoinhibition is a widespread phenomenon that plays a key role in the regulation
of proteins by facilitating the response to signaling pathways. The precise regulation of protein activities is essential for normal growth and development as well
as homeostasis. A picture is emerging from a diverse set of biological phenomena that intramolecular interactions between separable elements within a single
polypeptide provide a common regulatory strategy to modulate protein function.
Specifically, one region of a protein interacts with another to negatively regulate its
activity (Figure 1). In the clearest examples, the controlling elements make up a defined domain, the autoinhibitory domain, which mediates inhibition. The simplest
route to discovery of this regulatory pathway is the characterization of the activity
of fragments of a protein relative to the activity of the full-length species. The
enhancement of an activity of a particular domain, such as kinase activity or DNA
binding, in the absence of some other region of the protein indicates autoinhibition.
With such data in hand, the deleted region can be implicated as the autoinhibitory
domain. However, this observation is just the beginning. Defining the mechanism
of inhibition and elucidation of how the autoinhibition is counteracted or reinforced
requires extensive additional experimental investigation. Importantly, delineation
of an autoinhibitory mechanism is a guide to the discovery of crucial regulatory
schemes.
To illustrate the widespread use of autoinhibition as a regulatory strategy, this
review covers examples in which a variety of activities are inhibited. Inhibition
of ligand binding is the most common class of autoinhibition. This scenario is
well represented by inhibition of DNA-protein interactions, as in the case of transcription factors (e.g., σ 70 and Ets-1). Inhibition of protein-protein interactions
is also frequently observed (e.g., WASP, SNARE, and ERM proteins). Cellular
compartmentalization is another target of inhibition, as reported for the transcription factors SREBP and ATF6, which are sequestered in membranes, and the
ERM proteins whose membrane attachment is inhibited. Src kinase illustrates
autoinhibition of enzymatic activity. Within this diversity there is a common
thread in the mechanism of autoinhibition: an intramolecular interaction that either
directly or allosterically interferes with the function of a “targeted” domain
(Figure 1A). The functional domain could be directly occluded from a necessary ligand interaction or constrained in a nonfunctional conformation by a more
indirect mechanism.
The modular organization of proteins facilitates regulation by autoinhibition.
Individual proteins often perform a variety of functions, and separable regions of
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Figure 1 Autoinhibition is a regulatory mechanism. (A) An autoinhibitory domain
modulates the activity of a second, separable domain (center). Autoinhibition can be
counteracted and reinforced by modification (left) or by association with a second
molecule, partner protein (right). Autoinhibition is often identified experimentally by
deletion of the autoinhibitory domain. Proteolysis is also a regulatory strategy in vivo
(lower). (B) Modularity of proteins facilitates autoinhibition. A transcription factor with
two functional domains (one for DNA binding and one for transactivation) illustrates
the specificity of an autoinhibitory domain for a second, separable target domain.
Alternatively, a specialized autoinhibitory domain mediates inhibition (top); a dual
function domain mediates both inhibition and transactivation (center); or two domains
are mutually inhibitory and carry out other functions (lower).
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proteins can carry out each function. Well-defined structural domains of a protein
retain their folded state as a protein fragment and can function in isolation from
other regions. Transcription factors illustrate this concept (Figure 1B). A discretely
folded structural domain can often be identified that binds DNA independently of
the remainder of the protein. Likewise regions within transcription factors that
activate or repress transcription are functional when separated from their cognate
DNA-binding domains. The modular design of proteins has several implications
for the autoinhibitory phenomenon. First, autoinhibitory domains are distinct from
domains that are the target of the inhibition. However, the relationship of an autoinhibitory domain to other domains varies (Figure 1B). The autoinhibitory domain
can be one whose sole purpose is inhibition or it can be a domain that mediates
inhibition but also performs a second activity. Alternatively, there can be mutual
inhibition between two domains that possess other activities. A second feature of
the modularity of proteins is that domains are often linked by flexible regions.
The transition from the inhibited to the activated state usually requires this flexibility (Figure 1A). Finally, a distinguishing feature for a module that functions
as an autoinhibitory domain is the set of intramolecular interactions. Although
most protein modules can function in isolation, potentially acting like beads on a
string even within the full-length protein, an autoinhibitory domain is structurally
coupled to the targeted domain. Its inhibitory function is inextricably linked to the
function of the remainder of the protein. These intramolecular interactions, which
may be composed of seemingly small surface areas and weak interactions, are
bolstered by the relatively high effective local concentration that is generated by
tethering of the interacting parts. Although analogous inhibition might be designed
to function in trans with intermolecular interactions, the affinity would need to be
stronger and the specificity would need to be more finely tuned.
Autoinhibition as a Regulatory Strategy
Discovery of autoinhibition provides valuable clues for the investigation of biological regulatory strategies for a particular protein. The autoinhibitory domain
is an on-site repressor that restrains the targeted domain in a secure off state. In
some cases this is viewed as the default state, although in others, autoinhibition
is reinforced by external factors or modifications (Figure 1A) (e.g., Ets-1, Src,
SNAREs). A corollary of autoinhibition is the existence of regulatory strategies
that counteract the inhibition. Autoinhibition of a molecule presents a reversible
barrier that prevents spurious activation of a signaling pathway. This allows a
system to be primed for response only to appropriate signals. There are diverse
mechanisms for counteracting inhibition (Figure 1A), the most common of which
is the displacement of the inhibitory domain by a second molecule, thus replacing
the intramolecular interaction with an intermolecular interaction. However, even
this simple formulation is played out with surprising diversity (e.g., Ets-1, WASP,
SNAREs, σ 70, and Src). Other mechanisms include proteolysis of the inhibitory
domain (e.g., SREBP, ATF6), post-translational modification (ERM proteins) or
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binding of small molecules that allosterically alter the inhibitory domain. Except
in the case of proteolysis, an autoinhibitory domain also provides a mechanism
to reverse activation, resetting the switch for the next cycle of activation. The
widespread use of autoinhibition in different contexts emphasizes the importance
of tight control of both the off and on state in biological regulatory pathways.
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Experimental Approaches
In spite of extremely divergent biological settings for known autoinhibitory phenomena, there are striking similarities in the experimental approaches used to decipher inhibitory mechanisms. A standard first step is to map the minimal region
necessary for inhibition as well as the minimal region that shows full de-repressed
activity. These protein fragments are often easily engineered with cDNA clones
and produced in one of a variety of expression systems. Partial proteolysis is a
useful alternative route to discovery of autoinhibition because inhibitory function
often maps to a discrete region, and this strategy generates stable fragments that
often make up structural domains (e.g., σ 70 and Ets-1). Quantitative assays that
provide a reliable measure of the activity of the inhibited and activated fragments
are necessary to establish the validity of the autoinhibitory effects. Controls for
correct folding of the inhibited molecule are advisable because the low activity of
the inhibited molecule could reflect a structural defect. One approach for testing
the inhibitory function of the delimited minimal domain can be its transfer to a
heterologous protein. However, one of the hallmarks of autoinhibitory domains is
that their inhibitory effect requires specific features within the cognate, targeted
domain. Thus this technique is not likely to be successful unless the recipient
protein is highly related to the cognate protein.
Most mechanistic models of autoinhibition predict the existence of intramolecular interactions between the inhibitory elements and the functional domain. Mutants that activate, presumably by disrupting these interactions, are informative.
This intramolecular interaction model also can be tested experimentally using the
separated domains. A variety of protein-protein interaction assays can demonstrate
intermolecular binding of an inhibitory domain to the targeted functional domain.
An interesting variant of this experiment is to test for repression in trans. The σ 70,
SNARE, and WASP examples illustrate this approach. However, this experiment
is not always successful. Although the tethering can often be mimicked experimentally by a high concentration of the inhibitory fragment, in some cases direct
structural coupling of the inhibitory elements to the targeted domain has been
found to be crucial.
Some of the most illuminating and definitive findings come from structural
approaches. High-resolution molecular data from NMR experiments and crystallography provide the most specific information about structure, intramolecular contacts, and conformational change (e.g., σ 70 Ets-1, Src, SNARE and ERM proteins).
Conformational changes also can be detected by biophysical techniques, including circular dichroism (CD) spectroscopy and NMR approaches (e.g., SNAREs,
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Ets-1, and WASP), as well as lower-resolution strategies such as partial proteolysis
(e.g., Ets-1) or electron microscopy (e.g., SNAREs and σ 70). These structural data
can provide a framework for proposing a mechanism for autoinhibition, especially
in cases in which structural data are available for both the inhibited and activated
states (e.g., Ets-1, SNAREs, ERM, and WASP proteins). Structural modeling also
provides a context for interpreting mutant phenotypes and directing new mutagenesis to test specific mechanistic hypotheses. One of the most interesting features
of the structural data is the observation that there is structural fluidity in the inhibitory elements. Many cases show alternative conformations in the inhibited
versus activated state.
In contrast to experimental approaches that characterize an autoinhibition phenomenon, strategies for investigation of regulatory pathways that impinge upon
autoinhibition cannot be formulated generically. The distinctive function of the
protein within its biological context must be considered. Nevertheless, a detailed
mechanistic and structural model of autoinhibition is an invaluable tool in exploring the critical routes to regulation.
INHIBITION OF LIGAND BINDING
Ligand binding is one of the most universal properties of proteins, with a vast range
of ligands from small molecules to large macromolecules, including lipids, nucleic
acids, and other proteins. It is not surprising that the greatest number of examples
of autoinhibition include cases in which ligand binding is inhibited. In the first two
examples provided below, the ligand is DNA, and the proteins of interest play a
role in regulation of gene expression. The next two examples demonstrate a role for
autoinhibition in regulation of vesicle fusion and cell motility. These essential cell
processes require ligand binding within context of multiprotein complexes. Table 1
cites other examples of autoinhibition that work at the level of DNA binding and
assembly of multiprotein complexes within the cell.
Sigma 70 (σ 70)
DNA binding by the prokaryotic transcription factor σ 70 is repressed by autoinhibition. σ 70 only binds DNA in complex with RNA polymerase. This first example is
described in detail to illustrate the methodology for discovery and characterization
of an autoinhibitory phenomenon.
SIGMA SUBUNIT DIRECTS PROMOTER SELECTIVITY OF RNA POLYMERASE HOLOENZYME Transcriptional initiation in prokaryotes requires the direct binding of the
multisubunit RNA polymerase holoenzyme to transcriptional control elements.
Biochemical and genetic data indicate that the sigma subunit within the holoenzyme directly binds the two conserved promoter elements, known as the –35 and
–10 regions, which reflects their distance from the start site of transcription. The
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TABLE 1 Further examples of autoinhibition
Proteina
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LIGAND BINDING
NF-κB, transcription
factor
Leu3p, transcription
factor
Inhibited activity
Transcription
activation by
CBP/p300
binding
Transcription
activation
Inhibitory domain/
activation (repression)b
C-terminal region masks
N-terminal interaction
domain/PKA
phosphorylation
Internal region masks
activation/activate by
alpha-isopropylmalate
DNA binding domain/
response element
binding
Masking TPR arrays/
Brf1 association
C-terminal region of
SNAPc p190 subunit/
association with Oct-1
N-terminal region/SNAPc
binding to N-terminal
region, TFIIB association
Nuclear receptors
Transcription
activation
TFIIIc131, pol III
transcription factor
SNAPc, small nuclear
RNA-activating
complex
TBP, TATA-binding
protein
TFIIIB70 binding
Hoxb-1, Drosophila
Lab (labial)—
homeodomain
transcription factor
IRF4 (Pip),
transcription factor
DNA binding
Hexapeptide motif/
EXD protein binding
DNA binding and
transcription
activation
DNA binding
C-terminal region/PU.1
binding to C-terminal
region
C terminus/
phosphorylation
C-terminal region/
phosphorylation during
viral infection
PF/PN motif/SH3
domain
C-terminal region of
cyclinT1/ binding of
TATA-SF1 to cyclinT1
N-terminal pseudoligand/
Bcl-XL binding
N-terminal region/
acidic phospholipids
p53, transcription
factor
IRF 3, interferon
regulatory factor,
transcription factor
Esx1, homeodomain
transcription factor
PTEFb, transcription
elongation factor
Bid, BH3 interacting
domain protein
Vinculin, actinmembrane linker
DNA binding
DNA binding,
DNA bending
DNA binding,
activation, nuclear
localization
DNA binding,
nuclear entry
RNA (TAR
element) binding
Apoptosis via
protein:protein
F-actin binding
Referencec
(Zhong et al.
1998)
(Wang et al.
1999)
(Lefstin &
Yamamoto
1998)
(Moir et al.
2002)
(Mittal et al.
1999)
(Kuddus &
Schmidt 1993,
Mittal &
Hernandez
1997, Zhao &
Herr 2002)
(Chan et al.
1996)
(Brass et al.
1996)
(Ko & Prives
1996)
(Lin et al.
1999)
(Yan et al.
2000)
(Fong &
Zhou 2000)
(Tan et al. 1999)
(Bakolitsa et al.
1999, Johnson
& Craig 2000)
(Continued)
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TABLE 1 (Continued)
Proteina
Inhibitory domain/
Inhibited activity activation (repression)b
Alpha-actinin
Titin binding
ENZYMATIC ACTIVITY
PAS kinase, period,
Kinase
aryl hydrocarbon,
single-minded
homology domain
Vav, GDP-GTP
GEF activity
exchange factor
(GEF)
CKI epsilon, casein
Kinase
kinase I epsilon
GSK3beta, glycogen
Kinase
synthase kinase 3beta
MRCK, myotonic
dystrophy kinaserelated Cdc42-binding
kinase
Pak1, (S. pombe)—p21GTPase activated
protein kinase, also
MIHCK
PKA/PKG, cAMP- and
cGMP-dependent
kinases
SNF1, protein kinase
Ephb2 receptor, ephrin
B1 binding receptor
tyrosine kinase
Cystathionine betasynthase
Calcineurin, CN,
protein phosphatase
2A
Kinase
Referencec
Z-repeat motif
pseudoligand/PIP2 binds
actin-binding domain
(Young &
Gautel 2000)
PAS domain/metabolites
(proposed)
(Rutter and
McKnight
2001)
N-terminal extension binds
GTPase binding site/
tyrosine phosphorylation
C-terminal extension/
phosphatase
N-terminal phosphopseudosubstrate/(inhibited
by phosphorylation)
Distal coiled-coil domain
binds kinase domain/
phorbol ester
(Aghazadeh
et al. 2000)
(Cegielska
et al. 1998)
(Dajani
et al. 2001)
(Tan et al.
2001)
Kinase
Regulatory domain/
Cdc42 binding
(Brzeska et al.
2001, Tu &
Wigler 1999)
Kinase
Pseudosubstrate/cAMP
or cGMP
(Francis
et al. 2002)
Kinase
Regulatory domain binds
catalytic domain/SNF4
binds in low glucose
Juxtamembrane domain
binds catalytic
domain/phosphorylation
C-terminal domain/
S-adenosyl-L-methionine
binding
(Jiang &
Carlson 1997)
Kinase
Condense
homocysteine
and serine in
cysteine
biosynthesis
Phosphatase
(WybengaGroot et al.
2001)
(Janosik
et al. 2001)
N-terminal pseudosubstrate/ (Tokoyoda
calmodulin binding
et al. 2000)
(Continued )
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TABLE 1 (Continued)
Proteina
Inhibited activity
Inhibitory domain/
activation (repression)b
NADP malate
dehydrogenase
Reduction of
oxaloacetate to
L-malate
C-terminal disulfide bridge
occludes active site/
reduction of disulfide bond
(Krimm
et al. 1999)
13 phosphates constitute
NES/dephosphorylation by
calcineurin reveals NLS
Intramembrane domain
sequestration/proteolysis
IBB pseudoligand/NLS
and/or importin beta
(Okamura
et al. 2000)
SUBCELLULAR LOCALIZATION
NFAT1, nuclear factor Nuclear
of activated T-cells
localization
transcription factor
Tubby, transcription
Nuclear
factor
localization
Importin alpha
Nuclear
localization
signal binding
Notch, transcription
Nuclear
factor
localization
Relish NF-κB,
transcription factor
Nuclear
localization
Transmembrane domain
anchor/binding of
intracellular domain to DSL
ligand stimulating
intramembrane proteolysis
C terminus/proteolytic
cleavage
Referencec
(Santagata
et al. 2001)
(Catimel
et al. 2001,
Kobe 1999)
(Mumm
& Kopan
2000)
(Stoven
et al. 2000)
a
Examples are included based on personal knowledge of the authors and PubMed searches using the terms autoinhibition,
autoinhibitory, self regulating, and intrasteric inhibition. This resulting list is extensive although not comprehensive.
b
The majority of examples are stimuli that counteract autoinhibition. Those that reinforce autoinhibition are in parentheses.
c
References cited are representative of most recent work, reviews, or key publications and are not intended to be comprehensive.
–35 region is recognized by sigma factors as double-stranded DNA. The –10 region is the site of duplex melting, and the ability of sigma to bind single-stranded
DNA is critical for transcription initiation. Sigma subunits can be divided into two
general classes. The primary sigmas, including σ 70 of Escherichia coli, direct transcription of most genes during normal growth, whereas more specialized sigma
subunits, such as the heat shock responsive σ 32 of E. coli, direct RNA polymerase
to a specific subset of genes (Gross et al. 1998).
The discovery that σ 70 binds
directly to DNA provided an important breakthrough in the understanding of promoter recognition. Initial work failed to detect DNA binding activity by σ 70 subunit
when it was isolated from the holoenzyme. In 1992, proteolytic fragments of σ 70
were found to bind DNA in isolation and display the same promoter selectivity
as holoenzyme. Consistent with previous genetic analysis, quantitative binding
studies identified two DNA-binding domains within σ 70 that target the –10 and
–35 promoter elements. Most significantly for our discussion, these early studies
AUTOINHIBITION REGULATES SIGMA 70 DNA BINDING
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Figure 2 Autoinhibition of DNA binding by σ 70 is counteracted by RNA polymerase.
(A) Domain structure of σ 70. Conventional nomenclature for E. coli RNA polymerase
subunit σ 70 (regions 1.1 to 4.2) designates regions of conservation among related sigma
factors. RNA polymerase holoenzyme binds to –10 and −35 elements in prokaryotic
promoters via regions 2.1–2.4 and 4.2, respectively. Region 1.1 functions as an autoinhibitory domain (Dombroski et al. 1992). (B) Model of σ 70 autoinhibition and RNA
polymerase binding to promoter DNA. In the inhibited form of σ 70, intramolecular
interactions between regions 1.1 and the C-terminal region mask region 4.2, the −35
DNA-binding domain (left). Forming holoenzyme, σ 70 binds directly to RNA polymerase, thus making a specific set of contacts (middle). Both σ 70 and RNA polymerase
undergo structural rearrangements in the transition from free holoenzyme to a closed
complex formation (not shown) and finally open complex, (right). These changes include establishment of a new set of intramolecular and intermolecular interactions
(Burgess & Anthony 2001, Gruber et al. 2001, Mekler et al. 2002, Naryshkin et al.
2000). In addition, σ 70 binds the –35 and –10 promoter elements and aids in the melting
of DNA for open complex formation (Wilson & Dombroski 1997).
suggested an autoinhibitory phenomenon and mapped the inhibitory function to
the N-terminal region of σ 70 (Dombroski et al. 1992) (Figure 2A). These initial
investigations led to a model of autoinhibition in which repressive interactions
between autoinhibitory domain and the two DNA-binding domains prevent σ 70
DNA binding. Furthermore, the relief of autoinhibition was modeled as a switch
from these repressive intramolecular interactions to a set of intermolecular interactions between σ 70 and RNA polymerase. A decade of additional investigation
have validated this model and led to new mechanistic insights (Figure 2B).
The autoinhibitory domain was mapped by experiments in which N-terminal
fragments were added in trans to the C-terminal fragments that are active in
DNA binding. The region necessary for trans inhibition spans the first 100 amino
acids. As expected, inhibition in trans requires a large molar excess of the inhibitory fragment. The trans experiments also delineated targets of inhibition. The
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autoinhibitory domain inhibits binding to the –35 promoter element, whereas a
fragment bearing only the DNA-binding domain for the –10 promoter region is
not inhibited in trans. It is proposed that inhibition of the –10 binding region
may require structural coupling to the autoinhibitory domain (Dombroski et al.
1993).
RNA POLYMERASE COUNTERACTS AUTOINHIBITION Interaction with RNA polymerase relieves the autoinhibition of σ 70, thus enabling DNA binding. A variety
of genetic and biochemical studies indicate that a large region of σ 70, encompassing both DNA-binding domains, contacts RNA polymerase (Lesley & Burgess
1989, Sharp et al. 1999). Most significantly, the intermolecular contacts of the
autoinhibitory domain map to a specific structural feature on RNA polymerase.
Furthermore, a subset of these polymerase-sigma interactions is also negatively
regulated by the intramolecular contacts of the autoinhibitory domain (Gruber et al.
2001). This observation led to a two-step model of binding in which an initial contact with RNA polymerase induces a conformational change in σ 70 that facilitates
access to additional contact surfaces (Figure 2B).
The conformational changes in σ 70 are a critical component of the mechanism
that activates DNA binding. Changes were monitored initially by chemical reactivity of cysteines engineered individually in both the inhibitory domain and the
DNA-binding domain (Callaci et al. 1998). A second biophysical approach utilized
luminescence resonance energy transfer to measure changes in intramolecular distances (Callaci et al. 1999). These studies identified conformational changes in
both the inhibitory domain and the two DNA-binding domains and suggest that
polymerase induces a change in the distance between the two DNA-binding regions. In summary, both genetic and biochemical observations indicate that, in
addition to a simple unmasking step, a more complex set of conformation changes
in σ 70 plays a role in polymerase-induced DNA binding.
In addition to its role in autoinhibition, the N-terminal region of σ 70 plays a
role in transcription initiation. Promoter recognition can be detected with RNA
polymerase containing a sigma that lacks the autoinhibitory domain; however,
addition of the autoinhibitory domain in trans stimulates initiation (Vuthoori et al.
2001, Wilson & Dombroski 1997). Such a role is consistent with the extensive set of
sigma-polymerase interactions that are revealed during the transition from closed to
open complex formation (Gruber et al. 2001). Additional high-resolution modeling
has lent insight into the role of σ 70 within the RNA polymerase holoenzyme.
Experiments performed with fluorescence resonance energy transfer technology
have deduced the position of σ 70 in holoenzyme. Surprisingly, the autoinhibitory
domain of σ 70 is positioned near the polymerase active site. This segment of σ 70 is
proposed to be a molecular mimic of double-stranded DNA because it is displaced
from this position by DNA in the open complex. Initial contacts made between
σ 70 and RNA polymerase are rearranged to form a stable complex similar to the
open complex (Mekler et al. 2002).
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AUTOINHIBITION PROVIDES ADDED REGULATION OF PROMOTER SELECTIVITY Autoinhibition appears to be a common control mechanism among various bacterial
sigma factors. In addition to σ 70, the heat shock factor σ 32 of E. coli also has an
N-terminal inhibitory domain (Dombroski et al. 1993). Also, the developmental
sigma factor of Bacillus subtilis, σ K, is autoinhibited (Kroos et al. 1989). In each
case, autoinhibition tightly links DNA binding of the sigma factor to its association with RNA polymerase; isolated sigma factors cannot occupy promoters alone.
Importantly, autoinhibition provides a route to activation of specialized sigmas.
For example, σ K of B. subtilis is activated by proteolysis that is tightly coupled to
signaling (Cutting et al. 1991, Rudner & Losick 2002). Exchangeable sigma factors provide specificity for gene expression in bacteria. The use of autoinhibition
in this setting helps guarantee tight control.
Ets-1
DNA binding by the eukaryotic transcription factor Ets-1 is repressed by autoinhibitory elements. A detailed structural model for the autoinhibition mechanism
illustrates the central role of conformational change. Autoinhibition of Ets-1 is
counteracted by a protein partnership and reinforced by phosphorylation. This
regulatory strategy provides Ets-1 a degree of specificity within the ets family of
transcription factors.
ETS-1 REGULATES TRANSCRIPTION BY SEQUENCE-SPECIFIC DNA BINDING In eukaryotes, transcription factors that display sequence-specific DNA binding are often used to selectively direct the transcriptional machinery to promoters. These
proteins orchestrate differential gene expression in response to physiological cues
and developmental events. Although each factor is required to direct transcriptional
control of a subset of specific genes, there is a remarkable level of conservation
among subclasses of these proteins. Specifically, the DNA-binding domains of
many regulatory transcription factors are conserved, which has resulted in similar DNA-binding properties. Specificity of action among these factors requires
strategies that distinguish individual family members. Autoinhibition represents a
strategy to regulate DNA binding and provide such specificity.
The ets gene family encodes regulatory transcription factors that illustrate this
specificity conundrum. The human genome encodes 25 ets genes. Each ETS protein bears a highly conserved DNA-binding domain (ETS domain) that displays a
winged helix-turn-helix motif and recognizes a core sequence motif 50 GGAA/T 30 .
The proteins show significant diversity outside of the ETS domain. Furthermore,
genetic studies show that individual ets genes can display unique biological properties. The founding member of the ets gene family, Ets-1, illustrates the many
layers of regulation that generate specificity for its DNA-binding activity (Graves
& Petersen 1998, Sharrocks 2001).
AUTOINHIBITION OF ETS-1 DNA BINDING Ets-1 is regulated by an intramolecular conformational switch that forms the basis of an autoinhibitory mechanism.
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Autoinhibitory elements were discovered in Ets-1 by the observation that proteolysis of Ets-1 or deletion of regions flanking the ETS domain enhanced DNAbinding activity (Graves et al. 1998, Hagman & Grosschedl 1992, Lim et al.
1992). Quantitative analyses showed that deletion of either the N-terminal or Cterminal flanking regions fully activated DNA binding, suggesting that two regions function together to mediate autoinhibition (Jonsen et al. 1996) (Figure 3A).
Structural studies using NMR spectroscopy identified three inhibitory helices
within these regions (Skalicky et al. 1996). These NMR studies also detected
structural coupling between inhibitory helices, as well as a connection between
the inhibitory elements and helix H1 of the ETS domain (Figure 3B). These initial studies established the existence of an autoinhibitory module with interacting
elements but did not provide a clear mechanism of inhibition. Most strikingly,
the data did not support a simple model in which the inhibitory elements would
mask the major DNA-binding surface of the ETS domain, the helix-turn-helix
motif.
Additional structural studies provided further insight into the mechanism of
autoinhibition. Protease sensitivity measurements and CD spectroscopy analysis of alpha helicity showed that the inhibitory elements undergo a change upon
DNA binding that involves the dramatic unfolding of one of the inhibitory helices
(Petersen et al. 1995) (Figure 3B). The energy required for this conformational
change is likely the basis of the inhibitory mechanism. High-resolution molecular models of the ETS domain in contact with DNA provided further insight
(Batchelor et al. 1998; Garvie et al. 2001; Kodandapani et al. 1996; Mo et al.
1998, 2000). Helix H1 of the ETS domain directly binds DNA via a set of phosphate contacts. Based on this structural information and mutational analysis of the
DNA-protein interaction, a key role has been proposed for helix H1. The model
suggests that helix H1 can exist in one of two conformations. In the absence
of DNA, this helix packs with the inhibitory elements to form a metastable inhibitory module. In the presence of DNA, the N terminus of helix H1 contacts
the phosphate backbone via a precisely positioned hydrogen bond. This contact
is dependent on the macrodipole of the helix and relies on the strict positioning
of the entire structural element. Interconversions between the proposed alternative states would require conformational change (Graves et al. 1998, Wang et al.
2001).
REGULATORY PATHWAYS ACT THROUGH AUTOINHIBITION The autoinhibitory mechanism functions as a rheostat-type switch. DNA binding is either enhanced by
regulatory pathways that counteract inhibition or further inhibited by regulatory
pathways that reinforce the inhibition. Ets-1 binds with a variety of partner proteins to form ternary complexes on DNA (Dickinson et al. 1999, Garvie et al.
2001, Sheridan et al. 1995, Sieweke et al. 1998). These partnerships provide additional specificity because the appropriate promoter provides an Ets-1 binding site
as well as a properly positioned binding site for the partner protein. In the case
of RUNX1 (previously CBFα2 and AML1), cooperative DNA binding has been
characterized (Goetz et al. 2000, Gu et al. 2000, Kim et al. 1999). RUNX1 enhances
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Figure 3 Autoinhibition of Ets-1 DNA binding is reinforced by phosphorylation
and counteracted by a protein partnership. (A) Domain structure of eukaryotic transcription factor Ets-1. Pointed (PNT) domain functions in protein interactions. ETS
domain bears a winged helix-turn-helix motif and displays sequence-specific DNA
binding. Autoinhibitory elements flank the ETS domain. The serine-rich region is the
site of inhibitory phosphorylation. Secondary structure of ETS domain and inhibitory
elements are provided by NMR (Donaldson et al. 1996, Skalicky et al. 1996) and
crystallographic analyses (Garvie et al. 2001). (B) Model of Ets-1 autoinhibition and
regulation of DNA binding. The autoinhibitory module structure, including the position of helix HI-1, is modeled from genetic and biochemical analyses (crystallographic
data for HI-2, H4, and H5; C.W. Garvie, personal communication) (center). Two inhibitory alpha helices, HI-1 and HI-2, cooperate to inhibit DNA binding by making
intramolecular contacts with the ETS domain, specifically helix H1. DNA binding
disrupts intramolecular contacts between the ETS domain and the autoinhibitory module, characterized by the unfolding of HI-1 (left). Phosphorylation of the serine-rich
region in response to calcium release in activated T cells further inhibits DNA binding
by stabilizing the autoinhibitory module (upper right) (Cowley & Graves 2000). A
protein partnership with RUNX1 counteracts the inhibitory mechanism by interacting
with the autoinhibitory module (Goetz et al. 2000) (lower right). The DNA-binding
domain of RUNX1 is adapted from Bravo et al. (2001). Hatched oval represents the
N-terminal region necessary for cooperative DNA binding for which there is no structural information.
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Ets-1 binding affinity for DNA with the same magnitude as autoinhibition (approximately tenfold). Furthermore, this cooperativity requires an intact inhibitory
module. These results suggest that DNA binding cooperativity functions by counteracting the autoinhibitory mechanism (Figure 3B). On the other hand, a Ca2+dependent phosphorylation of Ets-1 dramatically reduces DNA binding (Cowley
& Graves 2000, Rabault & Ghysdael 1994). These sites of phosphorylation lie
near the inhibitory elements, and an intact autoinhibitory domain is required for the
added repression. Protease sensitivity measurements and CD spectroscopy indicate
that the added phosphates stabilize the inhibitory module (Cowley & Graves 2000)
(M. Pufall, unpublished observations). This reduction in binding affinity is thought
either to abrogate DNA binding altogether or to alter the specificity of DNA binding by rendering Ets-1 more selective for promoters that provide a partner binding
site.
Regulation of gene expression requires that highly related DNA binding proteins
execute target site specificity. Within the ets family, autoinhibition appears to be
one strategy for distinguishing different family members. Autoinhibition has been
detected in several other ETS proteins, including Elk-1 (Price et al. 1995) and
PEA3 (Bojovic & Hassell 2001, Greenall et al. 2001); however, the autoinhibitory
elements are different in each of these proteins. Interestingly, helix H1 of the ETS
domain is highly conserved in all of these family members. It is possible that the
ETS domain is a conserved target of inhibition via the helix H1-DNA contact,
whereas the mechanism that affects this DNA contact may have diverged among
the various family members during evolution.
SNARE Proteins
The SNARE complex is a heterotrimeric assemblage that mediates membrane
fusion. Assembly of this complex is modulated by an autoinhibitory domain present
in the syntaxin1a component. This example is distinguished by a structural model
that shows two conformations of the syntaxin component, one in the inhibited and
one in the activated state.
SNARE PROTEINS MEDIATE MEMBRANE FUSION Membrane fusion is required for
a variety of cellular functions including transport of molecules between membranebound organelles, organelle growth, and inheritance during mitosis. Membrane fusion also is a central step in synaptic transmission in the nervous system. Preceding
fusion, vesicles must be docked to the recipient membrane, a process mediated
by a set of membrane-associated proteins, termed SNAREs (soluble NSF attachment protein receptor where NSF is N-ethyl-maleimide-sensitive fusion protein)
(Chen & Scheller 2001, Misura et al. 2000a). There are many different SNARE
proteins distributed in specific membrane locations within the cell, and both the
SNAREs and other membrane-associated proteins appear to contribute to specificity of membrane fusion (Pelham 2001, Waters & Pfeffer 1999).
Representatives from the three major families of SNARE proteins form the
SNARE complex (Figure 4). The incoming vesicle brings a v-SNARE of the
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Figure 4 Autoinhibition of syntaxin and binding of n-Sec1 regulates SNARE core
assembly. (A) Domain structure of syntaxin1a. The N-terminal inhibitory module of
syntaxin1a autonomously folds into three alpha helices. The SNARE motif forms three
alpha helices in the inhibited form but only one extended alpha helix in the activated
state within the SNARE core complex. SNARE, soluble N-ethylmaleimide-sensitive
fusion attachment protein receptor; TM, transmembrane region. (B) Model of syntaxin1a autoinhibition and SNARE core complex assembly (adapted from Chen &
Scheller 2001). The N-terminal inhibitory module of syntaxin1a interacts intramolecularly with the three alpha helices in the C-terminal region, inhibiting assembly of the
SNARE complex. The n-Sec1 protein reinforces this autoinhibition by binding both
the N and C domains (left) (Misura et al. 2000b). A structural rearrangement leads
to association of one helix from syntaxin1a, two helices of SNAP-25, and one helix
from VAMP. SNAP-25 and VAMP acquire structure in the complex and syntaxin1a
changes structure as intramolecular contacts are displaced by intermolecular contacts
(right) (Sutton et al. 1998). Syntaxin family and SNAP-25 family are t-SNARES. Yeast
homologs include Sso1p and Sec9p, respectively. The v-SNAREs includes VAMPs,
neuronal synaptobrevin, Sec22b and yeast Ykt6p. n-Sec1/munc18 homologs include
yeast Sec1, Drosophila ROP, C. elegans UNC-13. SNAP-25, 25 kDa synatopsomeassociated protein; VAMP, vesicle associated membrane protein.
VAMP family, whereas, the target membrane has two t-SNARE proteins, one of the
syntaxin family and one of the SNAP-25 family. Each SNARE protein has a membrane attachment domain, and each contributes one or two (in case of SNAP-25)
alpha helices to the SNARE core complex. Crystal structure of a SNARE core
with synaptobrevin-II, syntaxin1a, and SNAP-25B shows a four-helix bundle that
can be described generally as a coiled-coil (Sutton et al. 1998). The formation
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of the heterotrimer brings the two membranes into close proximity to facilitate
membrane fusion.
AUTOINHIBITION REGULATES SNARE COMPLEX ASSEMBLY Autoinhibition of the
syntaxin class of t-SNAREs regulates the assembly of the SNARE complex. Proteins of the syntaxin family are composed of two structural domains (Figure 4A).
The C-terminal domain binds the SNARE core by contributing a single alpha
helix to the four-helix bundle. The N-terminal domain is not necessary for SNARE
core formation. Instead, this domain impairs formation of a binary complex between syntaxin and SNAP25, as well as binding of VAMP. Early studies reported
that adding the N terminus of syntaxin1a in trans inhibited the participation of
the C terminus in SNARE core formation (Calakos et al. 1994). More recently,
kinetic studies on the yeast syntaxin homolog, Sso1p, demonstrated that assembly is regulated. The binary complex of Sso1p and the yeast SNAP25 homolog,
Sec9p, shows equal stability with either the full length or the C-terminal fragment
of Sso1p; however, the association rate constant of binary complex formation is
∼1000-fold higher with the isolated C-terminal fragment (Nicholson et al. 1998).
These studies established the N-terminal region as an autoinhibitory domain that
regulates SNARE complex assembly.
The working model of autoinhibition suggests that intramolecular interactions
between the N- and C-terminal domains of syntaxin compete for intermolecular interactions with the SNARE core (Figure 4B). Structural data and a variety
of biochemical approaches show that intramolecular interactions link the N- and
C-terminal domains. In addition to straightforward binding assays, NMR experiments suggest that interactions between N- and C-terminal domains of syntaxin1a
induce folding of the C-terminal fragment, which appears largely unstructured
in isolation. Furthermore, chemical shift changes in the N terminus are observed
in the presence of the C terminus (Dulubova et al. 1999). The full picture of intramolecular interactions comes into focus from crystallographic studies of the
yeast syntaxin Sso1p. A three-helix bundle within the terminal domain interacts
with a set of short helical segments within the C-terminal domain (Munson et al.
2000) (Figure 4B, left). The conformation of the C terminus differs from the single
helix that participates in SNARE core (Figure 4B, right). Thus SNARE complex
formation, which overcomes autoinhibition, requires both a conformational change
in the syntaxin and a switch from intramolecular to intermolecular interactions.
AUTOINHIBITION AS A REGULATORY STRATEGY A non-SNARE protein, n-Sec1
(also called munc18) provides regulation of complex assembly. A crystallographic
analysis of a complex of syntaxin1a and n-Sec1 shows both the N- and C-terminal
domains of syntaxin1a interact with n-Sec1 (Figure 4B). The structure is similar to that observed in the absence of n-Sec1, with the N- and C-terminal domains retaining the inhibitory conformation that occludes the binding of other
SNAREs to syntaxin (Misura et al. 2000b). These structural data suggest a role
for n-Sec1 in repression, possibly stabilizing the inhibitory conformation. In contrast, genetic analyses in Caenorhabditis elegans and Drosophila suggest roles
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for n-Sec1 homologs in both negative and positive regulation of membrane fusion
(Richmond et al. 2001, Wu et al. 1998). To accommodate these apparently
confusing observations, n-Sec1 is proposed to prime the conformational change in
the syntaxin C terminus or to provide a structural platform for SNARE assembly
(Misura et al. 2000b, Munson et al. 2000).
The N-terminal regions of v-SNAREs may also provide a regulatory device.
Structural studies of the N-terminal domain of two VAMPs show a profilin fold that
is entirely different from that observed in syntaxin (Gonzalez et al. 2001, Tochio
et al. 2001). Nevertheless, preliminary analysis of the yeast VAMP Ykt6p suggests
that this domain negatively regulates SNARE complex formation (Tochio et al.
2001). Thus multiple SNARE proteins may contribute to regulation of SNARE
complex assembly by an autoinhibitory mechanism.
Autoinhibition provides an important level of control for SNARE assembly.
There is considerable controversy concerning the promiscuity of SNARE assembly; however, it is likely that some degree of specificity is required for orderly
membrane fusion events. On-site repression of assembly by autoinhibition could
favor specific interactions. Future work is needed to ascertain whether regulatory
routes that counteract the inhibitory mechanism connect membrane fusion and
signaling.
Wiscott-Aldrich Syndrome Protein (WASP)
WASP proteins regulate actin assembly via activation of the Arp2/3 complex.
The activation function is masked by autoinhibition. This example illustrates how
autoinhibition can be synergistically counteracted by several signaling pathways.
WASP FUNCTIONS IN ACTIN DYNAMICS The dynamic assembly and disassembly
of actin filaments controls the shape of much of the membrane within a cell. In
addition to providing a structural scaffold, actin filaments are involved in changing
membrane shape in cell division, vesicular transport, and motility. Each of these
processes requires characteristic networks of actin filaments (Chen et al. 2000).
The WASP family of proteins serves as a paradigm for the translation of cellular
signals into directed actin polymerization (Wear et al. 2000).
The WASP family of proteins catalyzes actin nucleation and polymerization
at the membrane by activating the Arp2/3 complex, which is composed of seven
polypeptides that activate actin filament growth by increasing the rate of actin
nucleation, branching, and polymerization. The complex alone, however, nucleates actin poorly and requires the presence of an activator, including members of
the WASP family of proteins (Higgs & Pollard 2000; Zalevsky et al. 2001a,b).
The conserved C terminus of all WASP family members, termed the VCA domain (verprolin homology, cofilin homology, acidic region), is necessary for this
activation (Figure 5A). The VCA domain has been shown to bind directly to the
Arp2/3 complex, inducing a conformational change that converts it into an active form (Volkmann et al. 2001, Zalevsky et al. 2001b). Although fully active
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Figure 5 Autoinhibition of WASP regulates its activation of the Arp2/3 complex and
actin polymerization. (A) Domain structure of WASP/N-WASP [adapted from (Higgs
& Pollard 2001)]. The enabled/VASP homology (EVH1), basic region (BR), Rho
GTPase-binding domain, (GBD), and proline-rich region each bind ligands that activate WASP/N-WASP. The BR and GBD comprise the minimal autoinhibitory domain
regulating the VCA domain. The VCA domain stimulates actin polymerization and
branching by activating the Arp2/3 complex. The hematopoietic-restricted WASP and
more ubiquitously expressed N-WASP are ∼50% identical and share a similar gene
structure. The differences in N-WASP, including extended proline-rich and acidic regions, as well as an extra V domain, influence the activity of the protein in vitro;
however, the in vivo significance is not known. V, verprolin homology domain; C,
cofilin homology domain, also known as the central or connector region; A, acidic
domain. Note: Sometimes V and C together are referred to as WASP homology domain (W or WH1) (Higgs & Pollard 2001). (B) Model of autoinhibition and activation
by chemotactic stimuli [adapted from (Prehoda et al. 2000, Rohatgi et al. 2000)]. Intramolecular interactions between inhibitory elements, BR-GBD, and the VCA domain
mask the activating function of N-WASP (left). Chemotactic agents stimulate exchange
of GDP to GTP in the Rho GTPase Cdc42 and up-regulate phosphatidylinositol-4,5bisphosphate (PIP2) levels in the membrane. Activated GTP-Cdc42 and PIP2 displace
the inhibitory elements GBD and BR, allowing the VCA domain to activate the Arp2/3
complex, which induces both de novo actin polymerization and actin branching, both
necessary for the formation of filopodia (Miki et al. 1998). Trimerization of Arp2/3
(black circles) and V domain–bound actin (gray circles) is proposed to stimulate actin
polymerization (right) (Robinson et al. 2001).
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in isolation, the activity of the VCA domain is inhibited in the context of the
full-length protein by the N terminus, suggesting an autoinhibitory mechanism
(Higgs & Pollard 2001) (Figure 5B). The N terminus, in turn, makes contacts with
Cdc42 and phosphatidyl inositol-4,5-biphosphate (PIP2), which are anchored in
the membrane. Much work has been done on many WASP family members, but the
member that most clearly illustrates autoinhibition is N-WASP, which is discussed
below.
MULTIPLE SIGNALING PATHWAYS COUNTERACT AUTOINHIBITION Inhibition of the
activity of N-WASP by the N-terminal region can be counteracted by a wide variety
of cellular inputs. The N terminus is composed of four domains: the Enabled/VASP
homology 1 domain (EVH1), a basic region (BR), the GTPase-binding domain
(GBD), and the proline-rich domain (sometimes referred to as the PRD) (Higgs
& Pollard 2001) (Figure 5A). Each of these domains has been implicated in serving as a binding surface for activating molecules: EVH1 is related to pleckstrin
homology domains and binds proline-rich regions, the BR binds PIP2, the GBD
binds Rho GTPases such as Cdc42, and the proline-rich region binds SH3 domains
(Abdul-Manan et al. 1999; Fedorov et al. 1999; Fukuoka et al. 2001; Prehoda et al.
2000; Rohatgi et al. 2000, 2001; Rudolph et al. 1998). Membrane-bound PIP2
activates WASP alone or synergistically with either an SH3 domain–containing
protein or Cdc42 (Fukuoka et al. 2001; Prehoda et al. 2000; Rohatgi et al. 2000,
2001) (Figure 5B). Thus N-WASP is a control point for a variety of cell signals
directing targeted actin polymerization at the membrane.
MECHANISTIC MODELING Despite the variety of possible activating inputs, deletion analysis of N-WASP revealed a minimally sized autoinhibitory region
(Prehoda et al. 2000, Rohatgi et al. 2000). Fragments of the N terminus were
added in trans to gauge their inhibitory effect on the VCA domain. Although the
GDB partially inhibits VCA activity, fragments containing both the GDB and the
BR are necessary to fully inhibit the VCA domain in actin polymerization assays in
vitro. Surprisingly, although both regions are necessary for full inhibition, only the
GBD binds directly to the VCA domain (Figure 5B). This binding is not enhanced
by the addition of other domains, nor do other domains interact with the VCA
domain independently. These results suggested an inhibitory mechanism. GDB is
hypothesized to mediate direct contact with the VCA, an interaction that provides
only partial inhibition. The BR is then postulated to enhance the inhibitory effect
of the GBD, without directly binding the VCA or affecting the affinity of the GBD
for the VCA.
The inhibitory module may function by occluding Arp2/3 binding; however,
support of this mechanism is controversial. One group showed that N-terminal
fragments containing the GBD-BR prevent binding of Arp2/3 to the VCA domain
(Rohatgi et al. 2000), whereas another group showed the opposite (Prehoda et al.
2000). Strikingly, the latter group also demonstrated an interaction between the
BR and Arp2/3 bound to the VCA region. This interaction suggests a different
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model in which the Arp2/3 module could in fact bind to the VCA region but could
not be activated owing to the direct interaction with the BR (Prehoda et al. 2000).
In this alternative model, the BR either masks the activity of the Arp2/3 complex,
or prevents it from undergoing the structural transition necessary for activation.
The exact role of the GBD in this model is not yet clear.
Structural studies provide more detail of this intramolecular interaction and further highlight the metastable nature of the inhibitory elements. NMR spectroscopy
revealed that stabilization of the GDB is critical to both autoinhibition and activation by Cdc42. The GBD alone was shown by both CD and NMR spectroscopy to
be unstructured in the absence of binding partners. Addition of alcoholic solvents,
which stabilize protein folding, induced the formation of two anti-parallel beta
sheets and an alpha helix. Strikingly, highly similar secondary structural elements
were induced in the presence of either the activating ligand, Cdc42, or the inhibited
target, the VCA domain. Biochemical studies further indicated that the binding of
the GBD to Cdc42 and the VCA are mutually exclusive. Together, these studies
indicate that the GBD is stabilized either intramolecularly by the VCA domain
to create an inhibited WASP or intermolecularly by Cdc42 to create an activated
WASP. Importantly, GBD and the VCA contacts only involve the coffilin (C)
homology region (Kim et al. 2000). This leaves open the possibility that either
model for Arp2/3 binding could be correct. Arp2/3 could bind to WASP through
part of the C domain and A domain even in its inhibited form, or it could clash
sterically in the C domain (Abdul-Manan et al. 1999, Kim et al. 2000). Amide
exchange experiments using a GBD-VCA fragment indicate that the GBD is only
partially displaced by Cdc42, which again suggests that multiple inputs are necessary to relieve autoinhibition (Buck et al. 2001). The mechanistic and structural
work provide a valuable model of counteracting autoinhibition, although the exact
mechanism of inhibition of Arp2/3 activation has not yet been resolved.
ROUTE TO SPECIFICITY The variable N termini of WASP family members might
provide a route to specificity in directing actin polymerization. WASP, N-WASP,
and Scar proteins are all homologous in their VCA and proline-rich regions, but
they diverge significantly in their inhibitory N termini (Higgs & Pollard 2001,
Machesky et al. 1999). Interestingly, however, Scar proteins, similar to WASP
and N-WASP, also localize to the leading edge of cells (Hahne et al. 2001). An
attractive hypothesis that highlights the potential of autoinhibitory modules is that
Scar, WASP, and N-WASP have similar functions but are regulated differently by
cellular signals due to differences in their N termini. This possibility awaits further
study.
INHIBITION OF SUBCELLULAR LOCALIZATION
The compartmentalization of proteins within a cell is essential for the regulation
and execution of a variety of activities. Our two examples illustrate opposite scenarios. In the first example, membrane attachment is inhibited, thus preventing
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localization to the plasma membrane. In the second example, a transcription factor
is sequestered in the ER membrane, thereby preventing its nuclear localization.
Table 1 lists additional examples of regulation of subcellular compartmentalization. In particular, regulation of nuclear trafficking is a common target for autoinhibition.
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ERM Proteins
ERM proteins link the plasma membrane to the actin cytoskeleton. The domains
for membrane interaction and actin binding are autoinhibited by reciprocal intramolecular interactions. This example has a high-resolution structural model of
a fully inhibited molecule dramatically illustrating intramolecular masking as an
autoinhibition mechanism.
ERM PROTEINS LINK PLASMA MEMBRANE TO ACTIN CYTOSKELETON The function
of the cytoskeleton requires attachment to a variety of cellular anchors. A key linkage is the physical connection between cytoskeletal actin and the plasma membrane. Actin-membrane linkages are critical for cell adhesion, organization of
cell surface receptors, cell cortical resilience, and the formation of specialized
membrane structures including microvilli and nerve growth cones. The ERM proteins, ezrin, radixin, and moesin crosslink the membrane to the actin cytoskeleton
(Bretscher 1999, Bretscher et al. 2000, Mangeat et al. 1999). These proteins display an autoinhibitory mechanism that regulates both actin binding and membrane
attachment.
The ERM proteins are characterized by two conserved domains that provide
the link between the plasma membrane and the actin cytoskeleton. The N-terminal
domain, termed N-ERM associate domain (N-ERMAD), displays a FERM domain that functions in membrane binding (Figure 6A). The N-ERMAD binds
directly either to integral membrane proteins such as CD44 and I-CAM-1,-2, or
to an adaptor protein such as EBP50, which binds membrane proteins (Bretscher
1999). The FERM domain is well structured with three connected subdomains, as
determined by crystallographic studies of moesin and radixin (Edwards & Keep
2001, Hamada et al. 2000, Pearson et al. 2000). The C-terminal domain, termed
C-ERMAD, binds F-actin at its extreme terminus (Pestonjamasp et al. 1995,
Turunen et al. 1994) (Figure 6A).
AUTOINHIBITION REGULATES BOTH MEMBRANE AND ACTIN BINDING ERM proteins display intramolecular interactions that mask both the FERM domain interaction with membrane and the interaction of the C terminus with F-actin. Initially discovered by binding assays with protein fragments (Gary & Bretscher
1995, Henry et al. 1995, Martin et al. 1995), autoinhibition is now best understood from a high-resolution structural study of a complex between N-ERMAD
and C-ERMAD fragments of moesin (Pearson et al. 2000). The C-ERMAD forms
an extended structure that contacts a large surface area of the FERM domain in
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Figure 6 Autoinhibition of ERM proteins regulates membrane attachment to actin
cytoskeleton. (A) Domain structure of ERM proteins (moesin shown). The N-ERMAD
is composed of a FERM domain (F1, F2, and F3 subdomains), which binds membraneassociated proteins. The C-ERMAD includes an actin-binding site (hatched ). These
two halves are linked by a putative coiled-coil motif denoted the helical domain. (B)
Model of ERM protein autoinhibition and activation (adapted from Pearson et al. 2000).
Structural information for the inhibited form is derived from crystal structure of moesin
N-ERMAD and C-ERMAD fragments in complex without helical region linkage (Pearson et al. 2000). With the modeled intact protein (shown), the C-ERMAD interacts
intramolecularly with the N-ERMAD, masking both membrane attachment and actin
binding activities (left). The binding of PIP2 to the FERM domain and phosphorylation
of the C-ERMAD activates ERM proteins to bind membrane-associated protein and
F-actin, thus crosslinking the plasma membrane and the actin cytoskeleton (right).
ERM, ezrin, radixin, moesin: three proteins encoded by paralogous genes; FERM, 4.1
(four point one), ezrin, radixin, moesin homology domain; ERMAD, ERM-association
domain.
a meandering manner (Figure 6B). The interface is composed of residues that
are highly conserved among the ERM proteins. Most importantly, the C-terminal
part of the interface overlaps with the F-actin-binding site, thereby, masking actin
association. The transition from the inhibited to activated state involves the switch
from intramolecular to intermolecular interactions with F-actin (Figure 6B). The
conformation of the C-terminal region may change in the presence of actin
(Pearson et al. 2000). The intramolecular interactions that inhibit actin binding
also inhibit EBP50 binding (Reczek & Bretscher 1998). In this case, the FERM
domain also switches from intramolecular to intermolecular interactions to attach
to the membrane. Thus the ERM proteins have two masked activities. Interestingly,
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intermolecular interactions between N-ERMADs and C-ERMADs from different
ERM proteins are also detected in cells and in vitro, and the resulting head-to-tail
oligomers may be functionally important (Gary & Bretscher 1995, Gautreau et al.
2000, Nguyen et al. 2001).
ACTIVATION OF ERM PROTEINS Both post-translational modifications and ligand
binding counteract autoinhibition of ERM proteins. Phosphorylation of a specific
threonine within the C-ERMAD weakens the intramolecular interaction (Matsui
et al. 1998, Simons et al. 1998). Consistent with this role, this threonine, as mapped
by crystallography, is located within the intramolecular interface, but with some
solvent exposure (Pearson et al. 2000). In addition to affecting intramolecular interactions, the phosphorylation of this threonine may play a role in the transition
from oligomeric to active monomeric forms of ezrin (Gautreau et al. 2000). Binding of phospholipids is another route to activation. Phospholipids bind the FERM
domain; PIP2 specifically enhances binding of ERM proteins to CD44 (Hirao
et al. 1996, Niggli et al. 1995) and synergizes with phosphorylation to stimulate
actin binding (Nakamura et al. 1999). A crystal structure of the radixin FERM
domain in complex with IP3 (the head group of PIP2) suggests phospholipid binding might allosterically disrupt intramolecular interactions (Hamada et al. 2000).
The diversity of subdomains in the FERM domain and the modular nature of the
C-ERMAD suggest that other mechanisms of activation of ERM proteins remain
to be elucidated (Gautreau et al. 2002).
Microfilament attachment to the plasma membrane plays an important role in
dynamic membrane events, such as adhesion, motility, and establishment of polarity. The ERM proteins have emerged as central players in many of these processes,
having both a structural and regulatory role. It is interesting that both actin binding
and membrane binding are targets of autoinhibition. Thus the subcellular localization of the ERM proteins is regulated by autoinhibition. Finally, the dormant
monomeric form of the ERM proteins facilitates the cellular response to changing
conditions and supports the dynamic nature of the cortical cytoskeleton and the
cellular membrane.
SREBP
The eukaryotic transcription factor SREBP (sterol response element-binding protein) is sequestered in the ER membrane. Proteolyis within the membrane releases
SREBP and allows nuclear localization. Coincidentally, the membrane-localized
protease that releases SREBP shows similarity to the protease that cleaves the
B. subtilis sigma factor σ K to counteract its autoinhibition (Brown et al. 2000).
SREBP REGULATES CHOLESTEROL HOMEOSTASIS Homeostatic levels of substances
in the cell must be strictly maintained. Many of the substances, such as oxygen,
metals, and cholesterol, are necessary at certain levels but are detrimental at higher
levels. The regulation of cholesterol in particular has been carefully studied in this
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regard. Cholesterol is necessary for membrane fluidity, assembly of signaling rafts,
and as a biosynthetic precursor for hormones. Excess levels of cholesterol form
crystals that are extremely toxic to cells (Brown & Goldstein 1997).
Cholesterol levels are regulated by SREBP, whose activity is in turn repressed
by an autoinhibitory mechanism. SREBP, a transcription factor that binds DNA
via a basic helix-loop-helix leucine zipper (bHLH-ZIP) motif, is maintained in
an inactive form as a two-pass transmembrane protein (Brown & Goldstein 1997,
1999) (Figure 7). As opposed to other autoinhibitory mechanisms described in this
review, this transmembrane domain does not function by an intramolecular masking mechanism but by sequestering the transcription factor in the endoplasmic
reticulum (ER), away from its site of action. Proteolysis triggered by low cholesterol liberates SREBP from its membrane tether, allowing it to translocate to the
nucleus, where it regulates genes encoding enzymes essential for the cholesterol
biosynthetic pathway (Brown & Goldstein 1997).
REGULATED INTRAMEMBRANE PROTEOLYSIS COUNTERACTS THE AUTOINHIBITION
OF SREBP Liberation of SREBP and other membrane-tethered proteins is exe-
cuted by regulated intramembrane proteolysis (RIP), which involves the sequential
cleavage of a transmembrane protein. The first cleavage shortens the portion of
the protein projecting into the extracytosolic space. The second cleavage then cuts
within the transmembrane domain, which disrupts anchoring (Brown et al. 2000).
In the case of SREBP, conditions of low cholesterol are sensed by SCAP (SREBP
cleavage-activating protein), which binds the C-terminal domain of SREBP and
transports it from the ER to the Golgi (Figure 7B, left). In the lumen of the cis-Golgi,
site 1 protease (S1P) cleaves SREBP between the two transmembrane domains
(DeBose-Boyd et al. 1999, Nohturfft et al. 1999). The N terminus of the protein,
which contains the transcription factor and a single-pass type 2 transmembrane
domain, is subsequently cleaved by site 2 protease (S2P) within the transmembrane
region (Rawson et al. 1997). Next, the SREBP C-terminal fragment diffuses into
the cytoplasm and translocates to the nucleus to regulate transcription (Brown &
Goldstein 1997).
RIP REGULATES OTHER MEMBRANE-BOUND PROTEINS Other targets of RIP include
apolipoprotein (APP), NOTCH, Ire1, and ATF6 (Brown et al. 2000). Of these, the
RIP of the transcription factor ATF6 has striking similarity to SREBP. ATF6 is
sequestered in the ER by a type 2, single-pass transmembrane domain and released
after sequential cleavage by S1P and S2P (Ye et al. 2000) (Figure 7B, right). The
transcriptionally competent N-terminal fragment translocates to the nucleus and
activates genes regulated by ER stress response elements (Haze et al. 1999, Yoshida
et al. 1998). The sensor for detecting unfolded proteins has not been identified.
Although different from the autoinhibitory mechanisms described elsewhere
that require intramolecular interactions, this autoinhibitory mechanism is otherwise very similar. First, if their transmembrane autoinhibitory domains are deleted,
these transcription factors are constitutively active. Second, autoinhibition via the
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Figure 7 Autoinhibition of nuclear localization of transcription factors SREBP and
ATF6 is counteracted by regulated intramembrane proteolysis, RIP. (A) Domain structure of ATF6/SREBP [adapted from (Brown & Goldstein 1997, Brown et al. 2000,
Li et al. 2000)]. SREBP is a two-pass transmembrane protein. The portion of the protein N terminal of the transmembrane domain is a transcription factor (TF ) that binds
DNA via a bHLH-ZIP domain at sterol responsive elements. The C terminus is necessary for sterol-responsive cleavage (Brown & Goldstein 1997). Activating transcription
factor (ATF6) is a type 2 transmembrane domain protein. Like SREBP, the part of the
protein N terminal to the transmembrane domain is a transcription factor, in this case
binding endoplasmic reticulum stress response elements via a bZIP domain (Yoshida
et al. 2001). TM, transmembrane domain; bHLH-ZIP, basic helix-loop-helix zipper
motif; b-ZIP, basic leucine zipper motif. (B) Model of autoinhibition of SREBP and
ATF6 and activation by RIP (adapted from Brown et al. 2000). SREBP is sequestered
in the ER until the sterol cleavage-activating protein (SCAP) senses low cholesterol
levels. SCAP then chaperones SREBP to the cis-Golgi for cleavage by site 1 protease
(S1P). After S1P cleavage, the N terminus is cleaved by site 2 protease (S2P) within the
transmembrane domain, liberating the transcription factor from the membrane. ATF6
is cleaved by S1P in response to the ER stress response induced by increased levels
of unfolded protein. ATF6 also is subsequently cleaved by S2P in the transmembrane
domain, releasing the transcription factor from autoinhibition.
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transmembrane domain is overcome in response to cell signals: sterol starvation
or excess unfolded protein levels. In addition, these examples show a particularly
exquisite level of specificity. SREBP and ATF6 are cleaved by the same two proteases, yet the cellular signals are different, and there appears to be no cross talk
(Haze et al. 1999, Ye et al. 2000).
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INHIBITION OF ENZYMATIC ACTIVITY
The vast literature on enzymes presents a plethora of strategies for regulation.
Many are regulated by autoinhibition as defined in this review. The classic zymogen is an excellent example (Khan & James 1998). In this case, an autoinhibitory
domain inhibits protease activity, and activation is mediated by proteolysis of the
inhibitory elements. In several examples presented in Table 1, small molecules
or post-translational modifications counteract autoinhibition of enzymes. Examples also illustrate the autoinhibitory domain as a pseudosubstrate that binds the
catalytic site and is displaced during activation. We have chosen to highlight the
classic Src kinase story in which the autoinhibitory regions act allosterically to
block kinase activity and for which there is a high-resolution structure of the
inhibited state.
Src Kinase
Kinase activity of Src is repressed by two inhibitory regions. The replacement
of these inhibitory intramolecular interactions with analogous intermolecular interactions leads to de-repression. This example illustrates how relatively weak
interactions function in cis-acting autoinhibition, whereas stronger intermolecular
interactions are necessary for activation in trans.
THE ROLE OF SRC KINASE IN SIGNALING Responses to stimuli propogated at the
cell surface in metazoans often involves the sequential phosphorylation of proteins
within a signaling pathway. Each protein in the pathway is thus potentially able
to be regulated on two levels: kinase activity and association with its downstream
substrate. One well-studied example that involves autoinhibition of both of these
types of regulation is Src kinase (Gonfloni et al. 2000).
Src, and eight other related family members, are non-receptor tyrosine kinases
that can be activated by cell surface receptor tyrosine kinases (RTKs), as well
as by downstream targets in growth and proliferation pathways (Wybenga-Groot
et al. 2001). Each family member can be functionally divided roughly into two
components. The N-terminal component has a membrane-anchoring myristylation
site and Src homology domains 2 and 3 (SH2 and SH3) domains (Figure 8A).
The C-terminal component contains the kinase domain, as well as a C-terminal
extension, which interacts intramolecularly with the SH2 and SH3 domains to
mediate autoinhibition of the kinase domain (Figure 8B). Activation of Src kinase
is mediated by the binding of the SH2 and SH3 domains to a ligand, such as
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Figure 8 Autoinhibition of Src kinase activity is regulated by ligands for SH2 and
SH3 domains. (A) Domain structure of Src kinase (adapted from Pawson 1997). The
kinase activity is mediated by a two-part kinase domain that shows homology to other
tyrosine kinases. The Src homology domains (SH2 and SH3) and the C-terminal extension cooperate to form an autoinhibitory module that inhibits the activity of the kinase
domain. Phosphorylation of tyrosine 527 in the C-terminal extension is required for
autoinhibition. The myristylation site at the N terminus is required for membrane localization and proper signaling. (B) Model of autoinhibition and activation (adapted
from Xu et al. 1999). The SH2 and SH3 domains constrain the kinase domain in an
inactive conformation through intramolecular interactions. The SH3 domain interacts
with a pseudoligand in the linker connecting the N-terminal kinase lobe to the SH2
domain. The SH2 domain binds a pseudoligand containing the phosphotyrosine 527
in the C-terminal extension (left). These intramolecular interactions are displaced by
high-affinity intermolecular interactions between the SH2 and SH3 domains and activating ligands. Consensus motifs for ligands are noted (right). Disruption of these
intramolecular interactions allows the kinase domain to adopt an active conformation,
resulting in an activating phosphorylation at tyrosine 416 (Sicheri et al. 1997; Xu et al.
1997, 1999).
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the phospho-tyrosines in the cytoplasmic domain of RTKs (Kuriyan & Cowburn
1997, Moran et al. 1990, Wybenga-Groot et al. 2001) (Figure 8B). The activated Src
kinase then phosphorylates substrates that trigger the Ras and PI3 kinase pathways,
which induce growth and cell proliferation (Aftab et al. 1997).
IDENTIFICATION OF INHIBITORY ELEMENTS Autoinhibition of Src kinase was discovered in the context of an oncogenic version of Src. v-Src kinase, encoded
by Rous sarcoma virus, is responsible for cell transformation by this retrovirus
(Stehelin et al. 1976). The cellular homolog, c-Src, is unable to transform cells
(Parker et al. 1984). The only difference between the two proteins is a missing
C-terminal extension that is phosphorylated at tyrosine 527 in c-Src (Cooper et al.
1986) (Figure 8). Mutation of Y527 abolished phosphorylation and activated the
kinase activity as well as its transformation capacity (Superti-Furga et al. 1993).
These early studies identified this phosphorylated C-terminal extension as an autoinhibitory domain.
Additional experimental analyses elucidated the basic components of Src autoinhibition and activation. Inhibitory intramolecular interactions involving the
C-terminal extension and the SH2 and SH3 domains are replaced by intermolecular interactions with SH2 and SH3 ligands of other proteins (Figure 8B). The
involvement of these domains in autoinhibition was suggested by the observation that Src kinase could be activated by deletion or mutation of SH2 and SH3
domains (Hirai & Varmus 1990a,b,c). In addition, these domains likely worked
with the C-terminal extension in autoinhibition. However, a key to understanding
this mechanism is the ligand specificity of the SH2 and SH3 domains. These domains were initially implicated in protein-protein interactions by binding studies
with specific peptide ligands. The SH2 domains bind phospho-tyrosine-containing
peptides with a consensus Tyr-Glu-Glu-Leu, whereas SH3 domains bind peptides
with Pro-X-X-Pro-X-Arg consensus in an unusual PPII helix conformation. Binding of SH2 and SH3 peptide ligands was shown to activate Src kinase. Furthermore,
polypeptides that contained both SH2 and SH3 ligands were demonstrated to be
synergistic activators of Src kinase (Alexandropoulos & Baltimore 1996). This
synergistic activation suggested that the two domains also cooperate in autoinhibition. A working model emerged upon discovery that the phosphorylated C-terminal
extension specifies a cryptic SH2 ligand. Thus these early studies defined the inhibitory elements, implicated intramolecular interactions, and identified a route to
counteract the autoinhibition.
STRUCTURAL STUDIES PROVIDE A CLEAR PICTURE Biochemical and structural
studies led to a more detailed mechanistic model of autoinhibition (Figure 8B)
(Hubbard 1999). The phosphorylated C-terminal extension, with its SH2 cryptic
ligand sequence, was shown by binding assays to interact intramolecularly with
the SH2 domain, and disruption of this interaction by mutagenesis activates the
kinase (Liu et al. 1993). Crystal structures of c-Src and Src family member Hck
finally showed the full set of intramolecular interactions. The kinase domain of
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c-Src forms a bilobed structure with the SH2 and SH3 domains extended from the
N-terminal kinase lobe and the phospho-tyrosine-containing C-terminal extended
from the C-terminal lobe. Satisfyingly, the structures confirmed the intramolecular
interaction of the SH2 domain with the C-terminal extension. More surprisingly,
the SH3 domain was shown to interact with a cryptic binding motif in the linker
between the N-terminal kinase lobe and the SH2 domain. Although this motif contained only one of the two prolines in the consensus Pro-X-X-Pro motif, it adopted
the characteristic left handed PPII conformation. This imperfect helix formed a
weak pseudoligand for the SH3 domain (Pawson 1997; Sicheri et al. 1997; Xu et al.
1997, 1999). Constraint of the SH2 domain by the phosphorylated C-terminal extension was then shown by mutational analysis to precisely position this imperfect
helix for interaction with the SH3 domain (Young et al. 2001). The combination
of these two weak pseudoligand interactions thus locks the N- and C-terminal
lobes of the kinase domain in a closed position, thereby inhibiting kinase activity
(Gonfloni et al. 2000; Hubbard 1999; Superti-Furga & Gonfloni 1997; Xu et al.
1997, 1999; Young et al. 2001).
INHIBITION AFFECTS TARGETING The detailed structural model of the inhibited
state elucidates the mechanism of autoinhibition of the kinase activity, as well
as inhibition of kinase targeting (Figure 8B). The interaction surfaces of the SH2
and SH3 domains are occupied in the inhibited form of the kinase. Mutations
engineered in the protein that disrupt these intramolecular interactions, although
activating to the kinase activity, also disrupt intermolecular interaction with activating molecules or downstream substrates (Hirai & Varmus 1990a,b,c; Pawson
1997). Disruption of intermolecular interactions with target sequences breaks the
signal transduction chain, preventing Src from transforming cells despite the increased kinase activity (Brown & Cooper 1996). The presence of the intramolecular
pseudoligands provides exquisite control of targeting and activation. However, the
intramolecular interactions of the SH2 and SH3 domains with the non-consensus
pseudoligands are weak in the wild-type kinase, and can be disrupted in the presence of a high-affinity ligand (Liu et al. 1993). Similar intramolecular interactions
in other Src family members are hypothesized to mediate specificity. Variations
in SH2 and SH3 domains of family members bind with high affinity to different
sequences than Src, which directs targeting of family members to a different set
of substrates (Brown & Cooper 1996).
CONCLUSIONS
The seven examples articulated in this review illustrate the central role of autoinhibitory domains in the control of proteins within regulatory pathways. This
on-site negative control assures tight regulation; the latent activity can be fully
utilized only when activated within a regulatory pathway. In many cases, the autoinhibition phenomenon has led investigators to discover a mode of regulation
within the protein. The additional cases cited in Table 1 illustrate the widespread
deployment of autoinhibition. The number of entries, although not comprehensive,
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emphasizes the versatility of this strategy and suggests that autoinhibition plays a
role in the regulation of a large number of proteins.
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Conformational Change is a Common Feature
of Autoinhibition
The identification of autoinhibitory domains derives primarily from functional
studies; however, structural data are essential for mechanistic modeling. There are
three levels of structural data. The first step is defining the structure of the domain
that executes the targeted activity as well as the structure of the inhibitory elements. Even secondary structure data of these elements can be useful. Second, the
mapping of possible intramolecular interactions between inhibitory elements and
the targeted domain provides the proof of the basic phenomenon. Finally, a highresolution three-dimensional description of the inhibited and activated structure
provides a complete framework for a mechanistic model of autoinhibition. Several
of the examples discussed have substantial structural data. The SNARE protein
syntaxin1a has a complete structural model of inhibited and activated states. The
ERM proteins and Src kinase have a complete model of the inhibited state. Ets-1,
WASP, and σ 70 have structural information for both the inhibited and activated
species, although not a complete three-dimensional model.
Structural data for many proteins highlight the essential role of conformational
change in the autoinhibitory mechanism. In the simplest model, the inhibitory
domain sterically masks the active site on the targeted domain, and a conformational change unmasks the active site during activation. Most cases approximate
this simple model, showing two juxtaposed domains moving with respect to each
other. However, the conformational changes are usually more complex. In several cases we noted conformational flexibility of elements within a single domain.
For example, one helix within the inhibitory module of Ets-1 unfolds upon DNA
binding. In the case of syntaxin1a, there are two alternate conformations of the
targeted domain, one for the inhibited state and one for the SNARE complex. In
Src, the inhibitory elements repress kinase activity by allosterically constraining
the entire catalytic domain. In WASP, the structure of the inhibitory GDB domain is induced by both the intramolecular and intermolecular interactions. σ 70
shows structural changes within the DNA binding domain and the autoinhibitory
domain during activation. These well-characterized examples emphasize the dynamic nature of proteins. These rich conformational repertoires enable a variety
of regulatory strategies, autoinhibition being an excellent example.
Autoinhibition Adds a Layer of Regulation
The autoinhibitory mechanism is a constitutive damper that serves to repress an
activity, safeguarding against inappropriate function, which means that the activity will be executed only in the presence of an activating signal that is sufficiently strong to overcome the inhibition. This review discussed proteins that
carry out a wide variety of functions in a cell. In every case, activity requires
exquisite control. DNA-binding proteins must localize and bind to appropriate
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promoters; membrane fusions events must accurately connect cellular organelles
to surface membranes; and cytoskeletal elements must expand to meet the needs of
the cell while connecting to the membrane. In most cases a route to activation has
been identified. Proteolysis is the simple solution as exhibited by SREBP. Ligand
binding is the more common route, as seen in many other examples. Nevertheless, each case provides a unique picture. WASP and ERM similarly illustrate how
multiple pathways could function synergistically in activation. In ERM proteins,
two different activities are inhibited; each inhibited domain binds a ligand, either
actin or a membrane-associated protein, upon activation. The SNARE protein syntaxin1a has a clearly defined inhibitory domain, and the only activating ligand is
the SNARE complex itself. In several cases, the inhibition is reinforced. Phosphorylation of Ets-1 strengthens the negative regulation of DNA binding by stabilizing the inhibitory domain. An auxiliary protein, n-Sec1, stabilizes the inhibitory
intramolecular interactions of the SNARE protein syntaxin. The phosphorylation
of the inhibitory domain of Src is essential for autoinhibition. This reinforcement
can drive the autoinhibition to an even more secure level of control, and possibly
make autoinhibition itself subject to regulation.
Autoinhibition not only creates an on-off state but also the potential for selectivity. The need for this activity is most dramatic in cases in which families of
related proteins must be assigned to specific tasks. There are multiple types of
sigma factors in each bacterium; Ets-1 belongs to a family of related transcription factors; there are related SNARE proteins in all membrane compartments; and
ERM, WASP, and Src all bear highly conserved domains that are found in a variety
of related proteins. It is proposed that autoinhibition can help distinguish among
these related proteins. In the most general model, all factors would be repressed
such that a specific signaling mechanism would provide activation. Elucidation of
valid, undoubtedly more complex, scenarios await more comparative analyses of
related proteins.
A Second Role for the Autoinhibitory Domain
The autoinhibitory domain often acquires a new function in the activated molecule.
In the case of Ets-1, the activating protein RUNX1 interacts with the inhibitory
elements and is a transcriptional regulator. In this way, the intermolecular interactions formed by the autoinhibitory domain stabilize components that play a
role in transcriptional regulation. Likewise, in the case of σ 70, the autoinhibitory
domain contacts RNA polymerase and participates in the transcription initiation
mechanism. The inhibitory elements of WASP bind activating ligands that provide
important membrane linkages. The mutually inhibitory domains of the ERM proteins provide binding sites for both ends of the membrane-cytoskeletal crosslink.
Finally, the SH2 and SH3 domains of Src can function in the activated state by facilitating docking of Src to substrates with cognate ligands. In each case, one could
argue that the second function of the autoinhibitory domain, which is displayed
in the activated state, is also masked within the inhibited state by intramolecular
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interactions. Therefore, the identification of the inhibitory domain and the target
of repression is biased by the assay used in the initial characterization. With this
perspective one can predict that there are undoubtedly many more examples of
autoinhibition to be discovered.
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Implications for Human Disease
Autoinhibition can be disrupted in disease states. In a genetic disease, a mutated
gene could be altered within the region that encodes the inhibitory element or
the surface that interacts with the autoinhibitory domain. The mutated gene could
direct synthesis of a protein that is constitutively active, having lost the negative
control afforded by the intramolecular network. Oncogenic versions of both src
and ets-1, which were originally discovered in acutely transforming retroviruses,
have such inactivated autoinhibitory domains. In both cases, a C-terminal deletion
removes inhibitory elements leading to enhanced activity and providing the oncogenic activity of the avian retroviruses RSV and E-26 virus, respectively (Lim et al.
1992, Sefton & Hunter 1986). A more subtle disruption is implicated in the case
of a human disease and WASP. An inherited single-point mutation in the GBD
correlates with myeloid disease in the form of severe congenital neutropenia. The
position of the mutation suggests that it could result in release of VCA inhibition,
leading to constitutive activation of Arp2/3 and mis-regulated actin polymerization
(Devriendt et al. 2001).
Chromosome translocations are another route to release of autoinhibition. In
reciprocal translocations there are cases in which the two breakpoints disrupt two
genes, and the translocation creates a new genetic locus that encodes a fusion protein. Many such translocations have been associated with human leukemia (Look
1997). Retention of functional domains from two disparate proteins and their juxtaposition in a new context often leads to a gain-of-function phenotype that is
oncogenic. Most relevant to our discussion is the potential loss of the original
structural context that might have provided autoinhibition. In this scenario, the
retained functional domain would have enhanced activity. The oncogenic fusion
protein Pax3-FKHR fits this prediction (Bennicelli et al. 1999). Discovery of other
such examples should be possible with more quantitative functional assays. Irrespective of the type of mutation, the general picture within these disease contexts
is similar; the mutation alters a regulatory function that is specific to the function
of a single protein, resulting in a highly specific disease phenotype that involves
gain of function rather than loss of function.
Because an autoinhibitory mechanism is specific to the regulation of a particular activity within a unique protein, it is suitable for targeted therapeutics. An
autoinhibitory domain could be targeted for either activation or further repression
by a small molecule. The drug would be directed to a single protein to intervene
with its unique regulatory mechanism. An understanding of the full repertoire
of regulatory strategies for a particular protein, including autoinhibition, should
facilitate such pharmaceutical research efforts.
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ACKNOWLEDGMENTS
This project is supported in part by the National Institutes of Health with research funding to B.J.G. (R01 GM38663) and fellowship support to M.A.P. (T32
CA93247, GM08537). Support from the Huntsman Cancer Foundation is also
gratefully acknowledged. We thank current and past members of the Graves laboratory who have contributed to the Ets-1 autoinhibition story including Jeannine
Petersen, Matt Jonsen, Marc Gillespie, Jean Hsu, Mary Nelson and Hong Wang.
We also thank our long-standing collaborators on the Ets-1 studies, Nancy Speck
(Dartmouth College), Lawrence McIntosh (University of British Columbia), and
Tom Alber (UC, Berkeley). We thank University of Utah colleages Mary
Beckerle and Janet Shaw for advice on cell biology sections of the review and
Valerie Orlemann for assistance with manuscript preparation.
The Annual Review of Cell and Developmental Biology is online at
http://cellbio.annualreviews.org
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Annual Review of Cell and Developmental Biology
Volume 18, 2002
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CONTENTS
MEMBRANE TRAFFIC EXPLOITED BY PROTEIN TOXINS, Kirsten Sandvig and
Bo van Deurs
A CELL BIOLOGICAL PERSPECTIVE ON ALZHEIMER’S DISEASE, Wim Annaert
and Bart De Strooper
GENE CO-OPTION IN PHYSIOLOGICAL AND MORPHOLOGICAL EVOLUTION,
John R. True and Sean B. Carroll
THE MECHANISMS OF POLLINATION AND FERTILIZATION IN PLANTS,
Elizabeth M. Lord and Scott D. Russell
TYPE III PROTEIN SECRETION IN YERSINIA SPECIES,
Kumaran S. Ramamurthi and Olaf Schneewind
BREAK INS AND BREAK OUTS: VIRAL INTERACTIONS WITH THE
CYTOSKELETON OF MAMMALIAN CELLS, Gregory A. Smith and
Lynn W. Enquist
RECEPTOR KINASE SIGNALING IN PLANT DEVELOPMENT, Philip Becraft
CHROMOSOME-MICROTUBULE INTERACTIONS DURING MITOSIS,
J. Richard McIntosh, Ekaterina L. Grishchuk, and Robert R. West
THE CHLAMYDIAL INCLUSION: ESCAPE FROM THE ENDOCYTIC PATHWAY,
Kenneth A. Fields and Ted Hackstadt
CELLULAR CONTROL OF ACTIN NUCLEATION, Matthew D. Welch
and R. Dyche Mullins
MEMBRANE FUSION IN EUKARYOTIC CELLS, Andreas Mayer
BACTERIAL TOXINS THAT MODIFY THE ACTIN CYTOSKELETON,
Joseph T. Barbieri, Matthew J. Riese, and Klaus Aktories
PROTEOLYSIS AND STEROL REGULATION, Randolph Y. Hampton
1
25
53
81
107
135
163
193
221
247
289
315
345
GOLGI ARCHITECTURE AND INHERITANCE, James Shorter
and Graham Warren
AUTOINHIBITORY DOMAINS: MODULAR EFFECTORS OF CELLULAR
REGULATION, Miles A. Pufall and Barbara J. Graves
421
MOLECULAR MECHANISMS OF EPITHELIAL MORPHOGENESIS,
Frieder Schöck and Norbert Perrimon
463
379
vii
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CONTENTS
Annu. Rev. Cell Dev. Biol. 2002.18:421-462. Downloaded from www.annualreviews.org
by University of Utah - Marriot Library on 05/08/14. For personal use only.
CONTROL AND DEVELOPMENTAL TIMING BY MICRORNAS AND THEIR
TARGETS, Amy E. Pasquinelli and Gary Ruvkun
REGULATORY PRINCIPLES OF DEVELOPMENTAL SIGNALING, M. Freeman
and J. B. Gurdon
SIGNALING PATHWAYS IN VASCULAR DEVELOPMENT, Janet Rossant and
Lorraine Howard
TRANSCRIPTIONAL AND TRANSLATIONAL CONTROL IN THE MAMMALIAN
UNFOLDED PROTEIN RESPONSE, Heather P. Harding, Marcella Calfon,
Fumihiko Urano, Isabel Novoa, and David Ron
ACTIN CYTOSKELETON REGULATION IN NEURONAL MORPHOGENESIS
AND STRUCTURAL PLASTICITY, Liqun Luo
STRIATED MUSCLE CYTOARCHITECTURE: AN INTRICATE WEB OF FORM AND
FUNCTION, Kathleen A. Clark, Abigail S. McElhinny, Mary C. Beckerle,
and Carol C. Gregorio
REMEMBRANCE OF THINGS PAST: CHROMATIN REMODELING IN PLANT
DEVELOPMENT, Justin Goodrich and Susan Tweedie
MYOGENIC REGULATORY FACTORS AND THE SPECIFICATION OF MUSCLE
PROGENITORS IN VERTEBRATE EMBRYOS, Mary Elizabeth Pownall,
Marcus K. Gustafsson, and Charles P. Emerson, Jr.
INDEXES
Subject Index
Cumulative Index of Contributing Authors, Volumes 14–18
Cumulative Index of Chapter Titles, Volumes 14–18
ERRATA
An online log of corrections to Annual Review of Cell and Developmental
Biology chapters (if any, 1997 to the present) may be found
at http://cellbio.annualreviews.org/errata.shtml
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