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-

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´
zma
´
ny Pe
´
ter se
´
ta
´
ny 1 ⁄ C, Hungary
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(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

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

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

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

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

Acknowledgements
AR is supported by a Wellcome Trust International
Senior Fellowship (081665 ⁄ Z ⁄ 06 ⁄ Z), a Marie Curie
International Reintegration Grant (205436) within the
7th European Community Framework Programme,
and by the NKTH-OTKA H07-A 74216 grant. We are
grateful to Andra
´
s Zeke for useful discussions and for
critically reading the manuscript.
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