Báo cáo khoa học: Scaffolds are ‘active’ regulators of signaling modules doc

MINIREVIEW Scaffolds are ‘active’ regulators of signaling modules Anita Alexa, Ja ´ nos Varga and Attila Reme ´ nyi Department of Biochemistry, Eo ¨ tvo ¨ s Lora ´ nd University, Budapest, Hungary Introduction Signaling adaptors, anchors, docking proteins and scaffolds share only one common property: they can physically bind proteins with diverse signaling-relevant activities. These diverse activities include protein and lipid phosphorylation ⁄ dephosphorylation, GTP hydro- lysis, GDP ⁄ GTP exchange, protein cleavage or mem- brane depolarization. The proteins carrying out these modifications appear to be less elusive: currently, we know a great deal about their function, structure and evolution, although there is a lot to be discovered about their recruiter molecules. All recruiter proteins possess multiple protein–protein interaction elements (e.g. modular domains or linear motifs) and they can be regarded as ‘professional recruiter proteins’ (PRPs). Signaling PRPs may currently be divided into four, somewhat distinct, group of proteins [1–3]. This mini- review focuses only on signaling scaffolds and we dem- onstrate how some well-established scaffolds (e.g. Ste5, InaD, PSD95, KSR1) influence the behavior of their recruited signaling circuits. We conclude that modifica- tion of the scaffold through its recruited partners is a key feature of scaffolded complexes. This mutual rela- tionship – which is mechanistically often mediated through post-translational modifications or by induced conformational changes in the scaffold – facilitates reg- ulation of the activities of the recruited proteins in novel ways. Discovery of scaffolds For decades, scaffolds were discovered fortuitously via experiments aimed at studying the function and Keywords cellular signaling; InaD; KSR1; MAPK module; phosphorylation of scaffold; protein kinase; PSD95; signaling cascade; signaling scaffold; Ste5 Correspondence A. Reme ´ nyi, Department of Biochemistry, Eo ¨ tvo ¨ s Lora ´ nd University, Budapest, 1117 Pa ´ zma ´ ny Pe ´ ter se ´ ta ´ ny 1 ⁄ C, Hungary Fax: +36 1 381 2172 Tel: +36 1 372 2500 E-mail: remenyi@elte.hu (Received 18 May 2010, revised 18 August 2010, accepted 24 August 2010) doi:10.1111/j.1742-4658.2010.07867.x Signaling cascades, in addition to proteins with obvious signaling-relevant activities (e.g. protein kinases or receptors), also employ dedicated ‘inactive’ proteins whose functions appear to be the organization of the former com- ponents into higher order complexes through protein–protein interactions. The core function of signaling adaptors, anchors and scaffolds is the recruitment of proteins into one macromolecular complex. Several recent studies have demonstrated that the recruiter and the recruited molecules mutually influence each other in a scaffolded complex. This yields funda- mentally novel properties for the signaling complex as a whole. Because these are not merely additive to the properties of the individual compo- nents, scaffolded signaling complexes may behave as functionally distinct modules. Abbreviations CA1–5, conserved domains or motifs in KSR proteins; CaMKII, calcium ⁄ calmodulin-dependent protein kinase II; ERK, extracellular regulated kinase; IMP, impedes mitogenic signal propagation; KSR1, kinase supressor of Ras 1; MAPK, mitogen-activated protein kinase; MEK, MAPK ⁄ ERK kinase; PDZ, PSD95-DLG1-ZO1 domain; PEST, peptide sequence rich in proline (P), glutamic acid (E), serine (S) and theronine (T); PKC, protein kinase C; PRP, professional recruiter proteins; PSD, postsynaptic density; RAF, proto-oncogene MAPK kinase kinase. 4376 FEBS Journal 277 (2010) 4376–4382 ª 2010 The Authors Journal compilation ª 2010 FEBS importance of some well-known signaling enzyme or receptor activity. To date, various scaffolds have been described that are important for T-cell receptor, mito- gen-activated protein kinase (MAPK), Wnt signaling pathways or for postsynaptic density [4–7]. Systematic efforts to identify scaffolds are still missing. The first attempt to estimate the number and abundance of these proteins in the human proteome was carried out by searching the human interactome for proteins satis- fying three basic criteria: (a) a lack of intrinsic cata- lytic activity that is relevant for signaling, (b) direct interaction with at least two signaling proteins possess- ing catalytic or receptor activity, and (c) direct or indi- rect interaction of at least two of these active signaling proteins with each other [8,9]. This analysis identified  250 potential human scaffold proteins. These criteria were chosen based on the common properties of all known scaffold proteins, and indeed these analysis correctly identified many known scaffolds. More importantly, this analysis has also suggested hundreds of proteins that may function as a signaling PRP. Naturally, this type of analysis depends heavily on the accuracy of interactome data and gene function anno- tation, and it can not distinguish between adaptor, anchor or scaffold proteins. Scaffolds: more than passive recruiters Scaffold proteins Similar to adaptor and anchoring proteins, scaffold proteins are also platforms for higher order signaling protein assemblies. Furthermore, they may also be associated with certain cellular compartments [10,11]. Therefore, the distinction between these groups of proteins is not clear-cut if only their interaction pro- files or spatial distribution patterns are considered. The following examples show that the interacting part- ners of scaffolds often modify the function, spatial localization or degradation rate of their recruiter. Thus, despite scaffolds not possessing apparent signal- ing-relevant catalytic activity, they play a pivotal role in determining the characteristic behavior of a signal- ing module as a whole (Fig. 1). One of the first classical scaffolds described in the literature was Ste5 from yeast [12]. It was found that the mating pathway of Saccharomyces cerevisae required a protein with no apparent catalytic activity. This protein was later shown to bind all three kinase components of the mating MAPK module [13,14]. In a-type cells, the presence of alpha factor activates a G-protein-coupled receptor, Ste5 is then recruited to the cell membrane causing Ste11, the first kinase com- ponent of a three-tiered MAPK module (Ste11–Ste7– Fus3), to be activated. It has always been suspected that Ste5 promotes signaling by enforcing proximity between the bound kinases [15]. Further biochemical analysis revealed that Ste5 plays an important role in the mating MAPK module via at least two further mechanisms: (a) allosteric activation of Fus3 by Ste5 initiates a negative feedback on the scaffold, and (b) interaction of Fus3 with Ste5 at a different site changes the MAPK allosterically so that it can be activated by Ste7. The first mechanism downregulates basal flux through the pathway when alpha factor is not present and is necessary for the proper sensing of alpha factor gradients [16–18]. The second mechanism ensures that Ste7, which also activates another MAPK, Kss1, involved in a different pathway, activates the mating specific Fus3 MAPK only when it is bound to Ste5 [19]. InaD was originally described as an anchoring pro- tein, but it can also be regarded as a scaffold [20,21]. In the phototransduction system of Drosophila A Fig. 1. Recruiting proteins influence signaling transitions between active signaling proteins. Cellular signaling is an orchestrated series of transitions: an input (i) elicits an output (o). A simple transition could be, for example, when a kinase is phosphorylated by an upstream kinase or binding of a ligand elicits a conformational change in a receptor. In turn, adaptors, anchors and scaffolds may modify these signal- ing transitions. For example, signaling cascades can read changes in receptor activation induced by a ligand through the use of adaptors. Anchors localize signaling enzymes to certain cellular locations and modify spatial patterns of signaling enzyme activation. Scaffolds and their bound partners frequently change each other’s properties and thus may form a signaling unit capable of performing novel functions specific for a certain mix of components. This facilitates fundamentally new relationships between inputs and outputs compared with transitions mediated by the same active components without the scaffold. Active signaling components are depicted as empty circles. R denotes a receptor. A. Alexa et al. Signaling scaffolds FEBS Journal 277 (2010) 4376–4382 ª 2010 The Authors Journal compilation ª 2010 FEBS 4377 melanogaster, InaD is important for fast recovery to the resting state after light-induced activation, which in turn is pivotal for visual resolution. InaD is a multi- PSD95-DLG1-ZO1 (PDZ) domain-containing protein that gathers together a number of signaling molecules to form a signaling circuit. When light activates the rhodopsin receptor, phospholipase C is activated and generates diacylglycerol. This opens a Ca 2+ channel, Trp, causing membrane depolarization. Diacylglycerol also activates protein kinase C (PKC) bound to the scaffold. In turn, PKC phosphorylates the Trp chan- nel, which renders the channel inactive, terminating the signal. This recruitment of positive (phospholi- pase C) and feed-forward inhibitors (PKC) into a sig- naling complex accounts for the generation of ion-flux pulses separated by only a few milliseconds [22]. In addition, the presence of the scaffold enables a sophis- ticated mechanism for adaptation to high-input and low-input conditions. In low-light conditions, one of the PDZ domains of the InaD resides in an open con- formation. During repeated stimulation of the pathway (under high-light conditions) the activation of PKC also results in phosphorylation of the scaffold, which in turn suffers a conformational change, turning the PDZ domain into a closed conformation and releasing its previously bound partner – most probably phos- pholipase C. This decreases the flux through the path- way, resulting in long-term adaptation [23]. Thus, InaD as a scaffold enables the signaling circuit under- lying phototransduction to perform with good time resolution and to adapt to different conditions. Postsynaptic density protein (PSD) 95 is another example of a scaffold whose modification by its bound partners is important for a signaling circuit as a whole. PSD95, in conjunction with other scaffolds such as Shank1–3, Homer, PSD93 and guanylate kinase-asso- ciated protein, contributes to the organization of a multiprotein complex at the postsynaptic contact zone comprised of N-methyl-d-aspartate receptor and a-amino-3-hydroxy-4-isoxazole propionic acid (AMPA) receptor, small G-protein regulators (e.g. synaptic Ras GTPase-activating protein), cell adhesion molecules, cytoskeletal elements (actin, tubulin) and enzymes (e.g. calcium ⁄ calmodulin-dependent protein kinase II [CaM- KII]) [24]. CaMKII is one of the most important regu- latory proteins in the PSD and it phosphorylates other postsynaptic proteins clustered into the complex, including PSD95 itself. For example, PSD95 is phos- phorylated at Ser73 of its PDZ1 domain by CaMKII, which causes the dissociation of one subunit (NR2A) of the N-methyl-d-aspartate receptor from PSD95 [25]. Moreover, there are now several documented examples in which phosphorylation events on the scaffold ultimately influence the composition of the PSD [26–33] (Fig. 2). Although the exact details are largely unknown, it is conceivable that these dynamic Fig. 2. Phosphorylation of PSD95 on specific sites influence its postsynaptic protein clustering ability. PSD95 is substrate to numerous protein kinases under physiological conditions. Phosphorylation can either inhibit (upper) or enhance (lower) the postsynaptic clustering ability of the PSD95 scaffold by several mechanisms (e.g. influencing protein degradation, occlusion of binding sites or induced conformational changes). For example, phosphorylation by cyclin-dependent kinase 5 in the peptide sequence rich in proline (P), glutamic acid (E), serine (S), and theronine (T) (PEST) sequence may influence the degradation of PSD95 [26], whereas phosphorylation by CAMKII is known to inhibit long-term potentiation and may interfere with ligand binding of the PDZ1 domain [27]. Phosphorylation events in a region between the PDZ2 and PDZ3 domains by p38c or JNK1 may cause internal conformational changes that influence binding to other proteins; more specifically the scaffold’s association with the cytoskeleton [28,29] or the internalization of a-amino-3-hydroxy-4-isoxazole propionic acid (AMPA) receptor [30,31], respectively. Finally, phosphorylation by Abl in the regulatory hinge region between the Src-homology (SH)3 and guanylate kinase (GuKc) domains may influence the interaction pattern of the SH3–GuKc supramodule [32,33]. PSD95 is a multidomain protein containing an L27 domain, a PEST sequence, PDZ1–3 domains, an SH3 domain and an inactive GuKc. L27 domain facilitates oligomerization, the PEST sequence is required for ubiquitinylation and hence for regulated degradation, PDZ domains bind to N-methyl- D-aspartate receptor, a-amino- 3-hydroxy-4-isoxazole propionic acid (AMPA) receptor and synaptic Ras GTPase-activating protein; SH3 and GuKc forms a supra-module mediating self-association or binding to adaptor proteins.) Signaling scaffolds A. Alexa et al. 4378 FEBS Journal 277 (2010) 4376–4382 ª 2010 The Authors Journal compilation ª 2010 FEBS post-translational modifications drive, or at least con- tribute to, the physiological mechanisms underlying learning in various neuronal types. Kinase supressor of Ras 1 (KSR1) is a further exam- ple of a multiprotein complex organizator that behaves as a dynamic scaffold. KSR proteins are scaffolds in the extracellular-regulated kinase (ERK) ⁄ MAPK pathway [34,35]. Upon Ras activation, KSR1 brings MAPK kinases (MEK1 ⁄ 2) to the plasma membrane in close proximity to the proto-oncogene MAPK kinase kinase (RAF) [36]. The localization of KSR1 in mam- malian cells is regulated by a variety of protein interac- tions and phosphorylation ⁄ dephosphorylation events. In quiescent cells, KSR1 is highly phosphorylated on AB C Fig. 3. Conformational changes of KSR1 during MAPK signaling and its regulation by phosphorylation. (A) In unstimulated cells, KSR1 exists in an inhibited state in the cytoplasm, with its C1 domain masked by IMP [38] and 14-3-3 proteins [37]. Dephosphorylation of 14-3-3 binding sites and degradation of IMP is needed to enable KSR1 activation. (B) After the receipt of a growth signal, KSR1 and b-RAF translocate to the membrane with the help of their C1 domains. The G-protein-binding domain of b-RAF also contributes to its activation, probably by reliev- ing the kinase from autoinhibition, whereas the role of the homologous domain in KSR1 is unknown. Once at the cell membrane, the two proteins dimerize with their kinase domains, leading to b-RAF kinase activation [44]. Several additional binding partners (e.g. CNK, Hyp, KSR1 CA1 domain) [45] and phosphorylation events, for example, in the activation loop, contribute to the stabilization and activation of the MEK1 ⁄ 2 phosphorylating complex. (C) Prolonged exposure to mitogenic stimuli leads to desensitization by feedback. ERK1 ⁄ 2, the MAPK ultimately activated by this pathway is capable of binding to the KSR ⁄ RAF complex and it can phosphorylate both partners at multiple sites. These sites promote the disassembly of the MEK1 ⁄ 2 phosphorylating complex [41], thus attenuating the mitogenic signal. Phosphorylation sites on the scaffold important for the different conformational transitions are highlighted in red, phosphorylation sites on b-RAF with gray. Position of the FxFP linear MAPK docking motif is shown in orange in KSR1 on (C). KSR1 is a multidomain protein containing five distinct regions that are conserved across various species: CA1, N-terminal region, contributing to binding of KSR1 to b-RAF; CA2: G-protein-binding- like domain with unknown function; CA3, C1 domain that binds lipids; CA4, hinge region with serine-rich sequence, possibly important for allowing various conformational transitions; and CA5, pseudokinase domain similar to the kinase domain of b-RAF, it allows dimerization with b-RAF. Note that the domain architecture of b-RAF is similar to that of KSR1, indicating their possible common origin. A. Alexa et al. Signaling scaffolds FEBS Journal 277 (2010) 4376–4382 ª 2010 The Authors Journal compilation ª 2010 FEBS 4379 two residues (Ser297 and Ser392 in mouse KSR1) by kinases bound to the scaffold. 14-3-3 dimers binding to these phosphorylated sites retain the KSR1 complex in the cytosol [37]. An E3 ubiquitin ligase, impedes mitogenic signal propagation (IMP), also contributes to sequestration of the scaffold [38]. IMP masks the C1 domain of KSR1, which is responsible for plasma- membrane targeting. Activated Ras causes many conformation changes in the preassembled complex: Ras binds IMP, which promotes its degradation. Ras binding also facilitates the formation of an active serine ⁄ threonine protein phosphatase 2A holoenzyme on the KSR1-platform, which in turn dephosphory- lates one of the 14-3-3 binding sites. These processes make the C1 domain of KSR1 accessible and help make it to move to the plasma membrane. Interest- ingly, release of 14-3-3 dimer opens the scaffold’s FXFP docking site for activated ERK [39], so all the active components of the RAF–MEK–ERK signaling module are colocalized at the site of action through KSR1. Moreover, the phosphorylation state of the molecule also influences the stability of KSR1: phos- phorylation at Ser392 and Thr274 decreases KSR1 sta- bility [40]. Phosphorylation also has an impact on the termination of KSR1-scaffolded signaling events; after binding of activated ERK2 to the FXFP motif, the MAPK is able to phosphorylate KSR1, which leads to the dissociation of the KSR1-b–RAF complex releas- ing it from the plasma membrane [41] (Fig. 3). In all the above cases, the recruited proteins influ- ence the behavior of the recruiter in an important, signaling-relevant manner. The recruited active compo- nents change the localization, protein level or interac- tion pattern of their scaffold. This allows for recruited signaling activities to be regulated in novel ways: the yeast mating MAPK module may be better suited to respond to gradients of its stimulant in yeast, or the phototransduction system may obtain better resolution and adaptability in the fruit fly. In the case of PSD95 and KSR1, they play an important role in generating localized and highly dynamic activation patterns of their involved signaling circuits. Are scaffolds derived from passive tethering elements or from inactivated active components? Passive recruitment appears to be a simpler task com- pared with the more active role that scaffolds may play. It is an interesting possibility that the highly sophisticated mutual influence of recruiter and recruited, which is a hallmark of this group of proteins, may have evolved from passively tethering proteins through evolutionary finetuning. Conversely, scaffolds may have also originated from former enzy- matically active components that lost their catalytic activity, but kept some of the connections and the regulatory features (e.g. localization, degradation or phosphorylation patterns) of their ancestors [8]. Conclusion In the past, newly discovered proteins binding multiple signaling enzymes or receptors were casually termed scaffolds – if they did not show resemblance to already known adaptors or anchors. Although recruitment of multiple signaling enzymes is indeed the core function of all PRPs, recent studies have demonstrated that scaffolds do a lot more beyond this core function. In addition to integrating signaling proteins into higher order circuits, they efficiently change the dose– response and dynamic properties of signaling cascades [42,43]. During the last decade, we have learned that scaffolds are rather ‘active’ PRPs, although not in an enzymatic, but more in a regulatory sense. 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MINIREVIEW Scaffolds are ‘active’ regulators of signaling modules Anita Alexa, Ja ´ nos Varga and Attila Reme ´ nyi Department of Biochemistry, Eo ¨ tvo ¨ s. part- ners of scaffolds often modify the function, spatial localization or degradation rate of their recruiter. Thus, despite scaffolds not possessing apparent