Motif switches: decision-making in cell regulation

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Motif switches: decision-making in cell regulation Kim Van Roey1, Toby J Gibson1 and Norman E Davey1,2

Addresses 1 Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, D-69117 Heidelberg, Germany 2 Chemical Biology Core Facility, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, D-69117 Heidelberg, Germany

modules that act as molecular switches [11]. The majority of disordered interfaces characterised to date are associated with a subset of compact, degenerate and convergently evolvable interaction modules known as short linear motifs (SLiMs) [9]. SLiMs mediate diverse regulatory functionality, such as directing ligand binding, providing docking sites for modifying enzymes, controlling protein stability, and targeting proteins to specific subcellular locations [12]. In this paper, we discuss the intrinsic properties of motifs that enable SLiMs to act as switchable modules that can consider multiple input signals and cooperatively make regulatory decisions [1,13–15]. How, for example, do local protein abundance and post-translational modification (PTM) modulate SLiM-mediated interactions by controlling specificity and affinity? What role do IDRs play in unravelling the hairball interaction networks in a context-dependent manner to correctly make decisions? The answers to these questions will illuminate the fundamental role of IDRs in cell regulation.

Corresponding author: Davey, Norman E ([email protected])

Regulatory properties of short linear motifs

Tight regulation of gene products from transcription to protein degradation is required for reliable and robust control of eukaryotic cell physiology. Many of the mechanisms directing cell regulation rely on proteins detecting the state of the cell through context-dependent, tuneable interactions. These interactions underlie the ability of proteins to make decisions by combining regulatory information encoded in a protein’s expression level, localisation and modification state. This raises the question, how do proteins integrate available information to correctly make decisions? Over the past decade pioneering work on the nature and function of intrinsically disordered protein regions has revealed many elegant switching mechanisms that underlie cell signalling and regulation, prompting a reevaluation of their role in cooperative decisionmaking.

Current Opinion in Structural Biology 2012, 22:378–385 This review comes from a themed issue on Sequences and topology Edited by Christine Orengo and James Whisstock Available online 3rd April 2012 0959-440X/$ – see front matter # 2012 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2012.03.004

Introduction Although the concept of molecular switching has been established for quite some time, its use has been mainly restricted to describe the action of allosteric proteins such as G proteins, which control diverse biological functions by switching between their ‘off’ and ‘on’ states in a nucleotide-dependent manner [1]. However, the molecular switching mechanisms that are involved in mediating cell regulation are not limited to this paradigm. Recent experimental and bioinformatic advances have substantiated the functional and regulatory importance of intrinsically disordered regions (IDRs) of higher eukaryotic proteomes [2,3,4,5–7]. Accumulated evidence has given insight into the disordered protein–protein interaction interfaces that determine the function of these regions [8–10] and has revealed numerous regulatory Current Opinion in Structural Biology 2012, 22:378–385

The majority of the specificity and affinity of a SLiM is on average determined by three to four residues. This keydefining attribute of motifs underlies three major factors that render disordered interfaces ideal regulatory modules [16]. Firstly, as a result of the limited number of residues directly contacting their interaction partner, motifmediated interactions are relatively weak, with an affinity often in the low micromolar range, and are thus transient and reversible [16]. Consequently, these interactions can be easily modulated by PTM, whose bulk and charge have a significant impact on the structural and physicochemical compatibility of the motif with its interaction partner [2,17]. Furthermore, motif specificity is highly sensitive, determined by both the intrinsic affinity of the motif, which can be modulated by PTM, and the local abundance of the different interactors, which is determined by cell state-dependent expression levels, scaffolding and subcellular localisation [13,18]. As a result, to abrogate off-target effects, gene dosage of motif-containing proteins must be carefully controlled to ensure appropriate protein localisation and stoichiometry [4,18–20]. Secondly, the small interface footprint of SLiMs facilitates high functional density, an attribute evidenced by the observed promiscuity of IDRs [21]. The transient nature of SLiM-mediated interactions in association with mutually exclusive interfaces promotes competitive binding. Alternatively, cooperative use of proximal SLiMs www.sciencedirect.com

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allows dynamic yet high-avidity interactions to be built from multiple low-affinity interactions. These mechanisms enable integration of multiple inputs, a key property crucial for cooperative decision-making. Finally, SLiMs can be gained or lost by few or even single point mutations [22], which facilitates convergent evolution of motifs, thereby expediting motif proliferation [8]. This plasticity gives evolution the potential to easily evolve compact and switchable interaction modules for many important, widely applicable regulatory tasks, such as directing protein localisation or marking proteins for degradation. Consequently, the gain and loss of ligand motifs can allow evolution to rapidly rewire interaction networks, for example, to tune signal transduction [8,23].

Molecular switching mechanisms Many different mechanisms are employed by intrinsically disordered interaction interfaces to mediate switching between the different functional states of a protein. These mechanisms underlie a protein’s ability to make decisions in a context-dependent manner, allowing it to relay signals and generate appropriate cellular responses [1,13,14,24]. In the following section we discuss motifbased switching mechanisms that are prevalent in eukaryotic cell regulation. Since regulation of motif-mediated interactions is not restricted to the motif interface, we also briefly consider analogous mechanisms that affect the domain interface of these interactions (Box 1). When considering the examples provided, one should bear in mind that these are isolated from their biological context in order to illustrate each mechanism separately, while in the context of the cell these definitions partially overlap and occur in various combinations acting as complex multi-SLiM switches.

Box 1 Two sides to every story. The majority of known motif-mediated interactions involve binding of a motif to a globular domain, allowing tight control of these interactions not only by regulatory mechanisms acting at the motifside of the interaction interface, but also by modulating the domainside of the interface.  The strength of an interaction can be tuned by PTM of the domain, for instance phosphorylation of AP-2m enhances its affinity for Yxxf motif-containing proteins [54].  Mutually exclusive binding sites for distinct motifs on a domain, for example the WRPW and EH1 motif binding sites on the WD40 domain of TLE, allow competitive binding of the motif-containing proteins. The balance of the competition can again be shifted by modulating the local expression levels of the competitors, the subcellular localisation of the interactors, or the specificity for the different motifs by means of PTM of the domain [55].  Mutually exclusive interactions also allow hiding of a domain for a particular motif when the difference in affinity between interactors prevents competition, as shown for the inhibition of cyclin–CDK substrate recognition by blocking of the substrate recruitment site on cyclin by the large disordered interface of the high-affinity inhibitor p21Cip1 [56].  The domain-side of an interaction can induce high-avidity interactions, as illustrated by the dimerisation of 14-3-3 proteins for tight binding to multisite ligands [57].  In some cases binding of a motif depends on pre-assembly of a binding site. An illustrative example of such a system involves ubiquitylation of p27Kip1 by the SCFSkp2 complex, which requires dimerisation of Skp2 with the accessory protein Cks1, since these two proteins form a composite binding site for p27Kip1 [58].  Long-range allosteric effects can also affect, either positively or negatively, the binding properties of a domain, as is observed for calcium-dependent binding of Alix to ALG-2 [59].

covalently attached myristoyl moiety available to mediate recruitment to the plasma membrane (Figure 1a) [30].

Binary on–off switch

Specificity switch

Simple binary switches are either ‘on’ or ‘off’ and switching between the active and inactive state of a protein’s interaction interface can be mediated by different mechanisms. Interactions can be induced by PTM of a specific residue in a motif, which creates a functional binding site for specific protein interaction domains, including SH2, PTB and Bromo domains (Figure 1a) [17,25,26,27]. Alternatively, modification of less specific residues in or near a motif results in structural or physicochemical incompatibility with the interaction partner and thus inactivates the motif. Such a mechanism is used for instance to block entry of NFATc1 into the nucleus by phosphorylation of residues flanking its nuclear localisation signal (NLS) (Figure 1a) [28]. In contrast, binding of an effector or addition of a PTM to a site that is distinct from the interaction interface can switch a motif indirectly by modulating its binding properties through allosteric effects [29]. Such allosteric regulation is observed when Recoverin binds calcium, which results in a conformational change in the protein that makes the

The occurrence of proximal or overlapping, mutually exclusive interaction interfaces provides the means for a more subtle regulation of IDR function by switching its specificity for different binding partners [2]. In contrast to binary switching, this type of mechanism is characterised by two or more distinct functional ‘on’ states. The balance of the competition can be tipped in favour of one of the interactors by different mechanisms, providing the cell a range of solutions to fine-tune regulatory interactions.

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One of the mechanisms that govern switching of binding partners is modification-dependent modulation of the intrinsic affinity of the motif. The NPxY motif in the disordered tail of the integrin b3 subunit preferentially interacts with talin. However, phosphorylation of the motif switches the specificity to Dok1 (Figure 1b) [31]. Alternatively, competitive binding can be affected by modulation of local target protein abundance, which can be achieved by changing the expression level or subcellular localisation, or by scaffolding, of the competitors. Current Opinion in Structural Biology 2012, 22:378–385

Q1

380 Sequences and topology

Figure 1 (a) Binary switch PTM-induced binding

PTM-induced incompatibility

P LAT

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P

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Competition switch Rb Dok 1

talin

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KKLxF

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(c) Motif hiding PTM-independent

PTM-dependent G-actin

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P

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P LT-AG

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NLS

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NUCLEUS

NUCLEUS

Figure legend Protein

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Motif (Name / Abbreviation) Current Opinion in Structural Biology

Simple switches. (a) Binary on–off switches. PTM-induced binding: interaction of the phospho-tyrosine motif (YxxL) of the LAT protein with the SH2 domain of PLCg1 only occurs upon tyrosine phosphorylation (left). PTM-induced incompatibility: translocation of NFATc1 to the nucleus is blocked by phosphorylation of residues flanking its NLS (middle). Allostery: binding of calcium to Recoverin results in a conformational change that relieves an auto-inhibitory interaction and thereby makes the N terminal myristoyl moiety available to mediate translocation of Recoverin to the plasma membrane (right). (b) Specificity switches. Intrinsic affinity switch: phosphorylation of the NPxY motif in integrin b3 switches the binding specificity from talin to Dok1 (left). Competition switch: cyclin–CDK (CCN-CDK) and PP1 bind competitively to overlapping docking sites on the Rb protein, which makes the interactions tuneable by regulating local enzyme abundance (middle). Localisation switch: ligand-induced translocation of CD2 from the non-raft membrane fraction to lipid rafts results in CD2 switching binding partners from CD2BP2 to Fyn (right). (c) Motif hiding. PTM-independent: binding of Gactin to the RPEL motifs in MRTF masks the NLS of MRTF and blocks its translocation to the nucleus (left). PTM-dependent: phosphorylation-induced binding of BRAP2 to the SV40 LT-AG protein masks the NLS of LT-AG and blocks its translocation to the nucleus (right).

Cyclin–CDK kinase and PP1 phosphatase use overlapping docking sites on the Rb protein, which makes their association with Rb mutually exclusive and the phosphorylation state of Rb tuneable by regulating local enzyme abundance (Figure 1b) [32]. While the response of such a system to changing cyclin–CDK/PP1 levels alone would be rheostatic, in the case of Rb the inhibition of PP1 by cyclin–CDK establishes a feed-forward loop that results in a more sensitive switch-like response [33]. It should be noted that when antagonistic enzymes target the same modification site, their docking sites do not need to be overlapping for this type of regulation to occur. Modulating the activity, expression level or subcellular localisation of the enzymes can disrupt the homeostasis between the two interdependent Current Opinion in Structural Biology 2012, 22:378–385

modification activities. For instance, the phosphorylation state of multiple residues of Dzip1, which governs its function in Gli protein turnover, is determined by the antagonistic actions of CK2 kinase and PP2A phosphatase [34]. Changing the subcellular localisation of a protein is another mechanism to alter the local abundance of its binding partners. Although CD2BP2 and Fyn use the same binding site on CD2, their localisation in T cells is distinct. While the cytoplasmic CD2BP2 is the main binding partner of non-raft membrane localised CD2, it is replaced by Fyn upon ligand-induced translocation of CD2 to lipid rafts (Figure 1b) [35]. In the previous examples switching the binding specificity results in functional bifurcation and propagation of www.sciencedirect.com

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distinct signals. However, an interaction at one motif can sterically mask an overlapping or adjacent motif, thereby precluding it from binding. In certain cases this has no functional consequences other than hiding of the motif for its binding partners, thus acting as a binary switch with respect to the functional outcome. Binding of the blocking protein can be PTM-independent, as is the case for hiding of the NLS of MRTF transcription factors by binding of G-actin [36], or PTM-dependent, for instance blocking of the NLS of the SV40 LT-AG protein by phosphorylation-induced binding of BRAP2 (Figure 1c) [37]. Cumulative switch

The regulatory potential of PTM-dependent motif-based switching mechanisms can be drastically increased when

they are controlled by multisite modification. The increased contribution of enzyme kinetics, potential interdependency between modification sites, and possible involvement of distinct modifying enzymes allow fine-tuned modulation of these mechanisms. Multisite modification enables signal integration by combining multiple inputs, and allows proteins to quantify time and temporally control regulatory processes, or alternatively, to quantify enzyme activity and probe the strength of a signal [38,39,40]. Furthermore, cumulative switches can mediate rheostatic regulation by gradually altering the affinity of a motif for an interaction partner by successive addition of multiple PTMs. For example, binding of motifs within the intrinsically disordered transactivation region of p53 to CBP/p300 is enhanced gradually and additively by successive phosphorylation of residues in

Figure 2

(a) Cumulative switch Positive rheostat

Negative rheostat P

p53

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P

P

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TAD region

P

P

P

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EGFR

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YSSxP

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P

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P

Affinity of EGFR for c-CbI

(b) Avidity-sensing switch PTM-independent

PTM-dependent

Syk P P FcRγ

ITAM

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Cargo Protein

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Cargo Cargo Cargo Cargo Protein Protein Protein Protein

Cargo Protein

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Eps15

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DPF

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AP2

AP2

AP2

AP2

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(c) Sequential switch Sequential specificity switch

Priming PTM UBC9 P HSF1

P

φKxExxS

SUMO

φKxExxS

SUMO P

Smad3

PY box

SPxLSP

Pin1

GSK3

P

P

φKxExxS

P

PY box

Nedd4L

SPxLSP

P P

PY box

P

P

SPxLSP

Figure legend Protein

Protein

Small molecule

Post-translational modification

Motif (Regular expression)

Motif (Name / Abbreviation) Current Opinion in Structural Biology

Complex switches. (a) Cumulative switches. Positive rheostat: successive phosphorylation of residues in the intrinsically disordered transactivation (TAD) region of p53 gradually and additively increases the affinity of p53 for CBP/p300 (left). Negative rheostat: while tyrosine phosphorylation of the YSSxP motif in EGFR is crucial for binding to c-Cbl, additional serine phosphorylations in this motif gradually decrease the affinity of EGFR for c-Cbl (right). (b) Avidity-sensing switch. PTM-dependent: full ligand-induced activation of the FcRg immunoreceptor results in double phosphorylation of its ITAM motif, which binds with high avidity to the tandem SH2 domains of Syk kinase (left). PTM-independent: the presence of multiple DPF motifs in Eps15 has been hypothesised to allow high-avidity binding to AP2-cargo protein complexes (right). (c) Sequential switches. Priming PTM: sumoylation of the lysine residue in the composite fKxExxS motif of HSF1 is promoted by prior phosphorylation of the serine residue, which enhances recruitment of the SUMO ligase UBC9 (left). Sequential specificity switch: Smad3 activation is coupled to Smad3 destruction through consecutive phosphorylation events. Phosphorylation of the PY box creates a binding site for Pin1, while phosphorylation of the SPxLSP motif creates a docking site for GSK3. Subsequent phosphorylation by GSK3 creates a binding site for the third WW domain of Nedd4L, whose second WW domain displaces Pin1 (right). www.sciencedirect.com

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382 Sequences and topology

this region. This allows p53 to compete more efficiently for the limited amount of CBP/p300 expressed in cells as additional sites are phosphorylated in response to prolonged or enhanced stress-induced signalling (Figure 2a) [41]. The same mechanism can also be applied to gradually decrease binding strength, as is observed for the interaction between EGFR and the E3 ubiquitin ligase c-Cbl (Figure 2a) [42]. Avidity switch

Many motifs, and their corresponding interacting domains, occur in tandem arrays, or even a higher number of repeats, which allows for multivalent interactions [43]. Cooperative use of multiple low-affinity binding sites results in highavidity interactions, which could serve as a sensitive switch by increasing the duration of an interaction until it reaches a biologically relevant timescale [44]. Signalling through avidity-dependent complexes is a common theme in cell regulation, including ligand-induced activation of immunoreceptors [13,27,43,45]. Full activation of these receptors results in double phosphorylation of their Immunoreceptor Tyrosine-based Activation Motif (ITAM) sequence, which then binds with high avidity to the tandem SH2 domains of, for instance, Syk kinase. This allows the kinase to bind with increased affinity and outcompete inhibitory phosphatases that bind to the monophosphorylated receptor (Figure 2b) [45,46]. Theoretically, the use of motif repeats as an avidity-sensing switch might also allow proteins to quantify stoichiometry, thus modulating signal transmission based on ligand concentration. It has been hypothesised that the endocytosis accessory protein Eps15 might probe cargo concentration through an unstructured region with no less than 15 DPF motifs, via which it can engage in multiple low-affinity interactions with multiple AP-2a proteins (Figure 2b) [47]. Sequential switch

An important contribution of motif-based switching of protein function to the reliability and robustness of cell regulatory processes is the ability to generate an appropriate output based on integration of multiple input signals in an ordered fashion via sequential switching, which mediates an ordered transition from one functional form of a protein to another. This mainly involves interdependent modification events, as is the case for the composite fKxExxS motif (f represents a bulky hydrophobic residue), where prior phosphorylation of the serine residue promotes sumoylation of the lysine residue by enhanced recruitment of the SUMO ligase UBC9 (Figure 2c) [48]. However, sequentially altering the specificity of motif-containing regions to switch from one binding partner to another in a specified order is not restricted to the generation of priming sites for other modifying enzymes. The coupling of TGFb-induced activation of Smad proteins to their destruction via sequential phosphorylation-dependent recruitment of distinct WW domain-containing proteins clearly illustrates Current Opinion in Structural Biology 2012, 22:378–385

the fine-tuned regulation that can be achieved by a sequential switch mechanism (Figure 2c) [49].

Conclusions Depending on prevailing internal and external conditions, proteins must decide whether and how downstream signalling will proceed in order to elicit appropriate responses [15]. The intrinsic properties of SLiMs make them ideal switches to engage in context-dependent and dynamic interactions, which are invaluable for reliable and robust decision-making [2,13,16]. However, SLiMs are not inherently dynamic. A motif in isolation is quite static, and this raises the question, how do these natively unstructured interfaces mediate decision-making? In the context of the cell, they do not function in isolation but instead a multiplicity of interfaces act cooperatively and competitively to ensure combinatorial decision-making in a highly regulated manner [13]. This emergent property allows simple and highly evolvable SLiMs to mediate robust and complex regulation. Until recently, with the exception of a few canonical SLiMs, much of the functional repertoire of IDRs remained to be systematically functionally characterised. Historically, the difficulty of interrogating IDRs originated from a shortage of experimental methods to extensively characterise disordered interfaces. However, this has now been largely rectified [5]. As a result, the regulatory potency of IDRs has been shown to extend far beyond the acknowledged canonical disordered regions such as the highly modified tails of histones [50] and the termini of tumour suppressor protein p53 [51], or the archetypal domain-binding motifs such as SH3, SH2 and PDZ ligands [25]. Disordered interfaces have been established as key mediators of dynamic interactions underlying large portions of the complex regulation of proteins in higher eukaryotic cells. Consequently, the interaction interfaces of IDRs should be seen as universal drivers of regulatory processes on the proteome level, analogous to the regulatory regions of genes in transcriptional regulation [52] and the UTRs of mRNA in translational regulation [53].

Acknowledgements This work was supported by an FP7 Health grant (No. 242129; SyBoSS) from the European Commission (to Kim Van Roey) and an EMBL Interdisciplinary PostDoc fellowship (EIPOD) (to Norman E. Davey). We would like to thank Holger Dinkel, Aino Ja¨rvelin and Richard Edwards for critical evaluation of the manuscript.

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