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In Keywords adaptor; anchor; docking protein; kinase; multidomain protein; phosphatase; protein interaction; scaffold; signaling; structural disorder Correspondence L.. Here we discuss t

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Functional classification of scaffold proteins and related molecules

La´szlo´ Buday1,2and Pe´ter Tompa1

1 Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary

2 Department of Medical Chemistry, Semmelweis University Medical School, Budapest, Hungary

Introduction

Cells use a multitude of signaling proteins to alter

cel-lular behavior in response to changes in their external

and internal environment Because of the multiplicity

and broad substrate specificity of signaling enzymes, it

is of immense importance to understand how the cell

achieves efficiency and accuracy in signaling It is

generally accepted that the primary mechanism is to

promote the proximity of signaling enzymes by specific binding to a special class of regulatory proteins [1–4] These proteins come under a variety of names, such as scaffold, adaptor, anchor and docking, but invariably function by ‘enforced proximity’, i.e by binding at least two signaling enzymes together and directing, coordinating and regulating their action (Fig 1) In

Keywords

adaptor; anchor; docking protein; kinase;

multidomain protein; phosphatase; protein

interaction; scaffold; signaling; structural

disorder

Correspondence

L Buday and P Tompa, Institute of

Enzymology, Hungarian Academy of

Sciences, 1518 Budapest, P.O Box 7,

Hungary

Fax: +361 466 5465

Tel: +361 279 3115; +361 279 3143

E-mail: buday@enzim.hu; tompa@enzim.hu

(Received 14 May 2010, revised 3 August

2010, accepted 18 August 2010)

doi:10.1111/j.1742-4658.2010.07864.x

In this series of four minireviews the field of scaffold proteins and proteins

of similar molecular⁄ cellular functions is overviewed By binding and bring-ing into proximity two or more signalbring-ing proteins, these proteins direct the flow of information in the cell by activating, coordinating and regulating signaling events in regulatory networks Here we discuss the categories of scaffolds, anchors, docking proteins and adaptors in some detail, and using many examples we demonstrate that they cover a wide range of functional modes that appear to segregate into three practical categories, simple pro-teins binding two partners together (adaptors), larger multidomain propro-teins targeting and regulating more proteins in complex ways (scaffold⁄ anchor-ing proteins) and proteins specialized to initiate signalanchor-ing cascades by local-izing partners at the cell membrane (docking proteins) It will also be shown, however, that the categories partially overlap and often their names are used interchangeably in the literature In addition, although not usually considered as scaffolds, several other proteins, such as regulatory proteins with catalytic activity, phosphatase targeting subunits, E3 ubiquitin ligases, ESCRT proteins in endosomal sorting and DNA damage sensors also func-tion by bona fide scaffolding mechanisms Thus, the field is in a state of continuous advance and expansion, which demands that the classification scheme be regularly updated and, if needed, revised

Abbreviations

AKAP, A-kinase anchoring protein; DAPP, dual-adapter for phosphotyrosine and 3-phosphoinositides; DD, death domain; FADD,

Fas-associated protein with death domain; MAGUK, membrane-associated guanylate kinase; MYPT1, myosin phosphatase targeting subunit 1; Nck, non-catalytic region of tyrosine kinase adaptor protein; PDZ, post-synaptic density, disc large, zo-1 protein; PH, pleckstrin homology; PSD, post-synaptic density; RTK, receptor tyrosine kinase; SH2, Src homology 2; SH3, Src homology 3; SKAP, Src kinase-associated phosphoprotein.

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the literature, the names are often used

interchange-ably, which indicates that the functions of the proteins

in the four categories overlap significantly and they are

often difficult to distinguished This article, and the

following three in this minireview series provide a

con-cise functional description of scaffold proteins and

their kin, in general, and their distinct classes, in

par-ticular, encompassing scaffolds [5], docking proteins

[6], anchors [7] and adaptors (this article)

We also point out overlaps and inconsistencies in

terminology, to show that members of this entire

functional class occupy positions along a rather

contin-uous functional spectrum We also describe targeting

subunits of phosphatases, which have a function in close analogy with that of anchors In addition, we also show that there are many other proteins that function using scaffolding mechanisms, and ought to

be considered in extending the classification scheme of these regulatory proteins It will be addressed that scaffold proteins and their kin not only bring signaling proteins together, but also regulate the flow of signal-ing information ussignal-ing a variety of mechanisms, owsignal-ing

to which proteins in this broad functional class no longer emerge as passive connectors, but active regula-tory elements shaping the response put out by signal-ing networks We suggest that this active role may also

be linked with extended structural disorder of the proteins [8–10], which enables dynamic communication between their distinct binding and functional elements (Fig 1 and Table 1)

It should also be made clear that because of space limitations this minireview is not comprehensive, and the reader is directed to many excellent recent reviews [1–4,11], and the other minireviews in this series [5–7] for further details and insight into this field of continu-ous advance

Scaffold/anchoring proteins Scaffold and anchoring proteins are reviewed in two of the other minireviews in this series [5,7] Here we pro-vide a short overview of their most notable examples,

to demonstrate that the distinction between these two categories is historical, rather than structural or functional

Scaffold proteins may be considered as the founders

of this functional class, epitomizing the very essence of the action of signaling regulatory proteins [5] They are defined as proteins organizing signaling complexes

by binding at least two signaling enzymes together and promoting their communication by proximity Classi-cally, they have been regarded as passive platforms for the assembly of signalosomes, but more recently it has become clear that they play rather active regulatory roles (Fig 1 and Table 1) They may operate by four basic mechanisms, such as enforced proximity, combi-natorial use of elements, dynamic regulation and con-formational fine-tuning [4,5] As demonstrated by one

of the best characterized scaffolds, Ste5, which regu-lates the extracellular signal-regulated kinase⁄ mitogen-activated protein kinase pathway in yeast, their func-tion may be to assemble complexes, enforce restricted intracellular localization, provide allosteric feedback and feed-forward regulation, and offer protection against degradation [3] They may enable context-dependent fine-tuning of pre-existing signaling

Adaptor A

Docking

P P P P

C

C

Scaffold/anchor B

Fig 1 Mechanisms of scaffold proteins and their kin This scheme

outlines the most important aspects of the function of modular

regu-latory proteins in the four (three) closely related categories In all

cat-egories, the proteins regulate signaling pathways by binding several

of the components and targeting ⁄ regulating their action in complex

ways (regulatory interactions ⁄ modifications marked by arrows).

Within this generalized scheme, there are three distinct types of

behavior (A) Adaptors are usually small, and have two binding

regions to target the action of two bound enzymes (B)

Scaf-folds ⁄ anchors are large multidomain proteins with a lot of structural

disorder, able to bind and regulate several members of a signaling

pathway (C) Docking proteins have a similar structural and functional

outline, their distinguishing feature being their ability to localize at

the membrane next to an activating receptor, to which they bind in a

phosphorylation-dependent manner In the entire class of proteins, a

high level of structural disorder (43.3% on average; Table 1) enables

key functional attributes, such as binding of several partners, to be

combined A, B and C marks general proteins P, phosphate.

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pathways or create new pathways using novel

combi-nations of signaling elements Scaffolds are extremely

heterogeneous in structure and function, which may

significantly overlap with those of other classes A few

selected examples (Table 1; cf [5]) show that they

often have separated binding domains for

protein–pro-tein interactions, and also have a high level of

struc-tural disorder, which may provide both short binding

regions and flexibility for dynamic regulatory

rear-rangements

These principles are also apparent in scaffolds that

operate in neuronal and immune cells, which are

highly polarized and form complex structures termed

synapses Synapses contain a plethora of receptors, ion

channels and signaling proteins for sensing and pro-cessing extracellular signals, organized and connected

to the cytoskeleton to form large complexes, such as the post-synaptic density (PSD) complex The best known of the scaffolds are the membrane-associated guanylate kinase (MAGUK) proteins, which have vari-ous numbers of post-synaptic density, disc large, zo-1 protein (PDZ) domains, an Src homology (SH)3 domain and a C-terminal guanylate kinase domain [1] The best studied MAGUK in T cells is DLG1, whereas in neurons it is PSD95, with notable similari-ties in function PSD95 is the most abundant scaffold

in the PSD, it has three PDZ domains, and binds

a variety of receptors⁄ channels, cytoskeletal and

Table 1 Scaffold proteins and their kin Representatives of the four categories of scaffold proteins and their relatives are presented The length of the human (unless indicated otherwise) isoform is given, along with its typical domains, binding partners and percent structural disorder, as predicted by the IUPred algorithm [42] Protein–protein interaction domains and interaction partners have been taken from the literature [1–7,22] and the list is not intended to be exhaustive AKAP, A-kinase anchoring protein; Caskin 1, CASK-interacting protein 1; DED, death effector domain; DLG(1), discs, large homolog (1); Dok1, docking protein 1; ERK, extracellular signal-regulated kinase; FADD, Fas-associated protein with death domain; FRS, FGF receptor substrate; Gab,Grb2-associated binder; Grb2, growth factor receptor-bound protein 2; IRS, insulin receptor substrate; KSR, kinase suppressor of Ras; MYPT1, myosin phosphatase targeting subunit 1; Nck1, non-cata-lytic region of tyrosine kinase adaptor protein 1; PDZ, post-synaptic density, disc large, zo-1 protein (domain); PH, pleckstrin homology domain; PI3-kinase, phosphoinositide-3-kinase; PKA, protein kinase A; PKC, protein kinase C; PP1, protein phosphatase 1; PSD, post-synaptic density; PTB, phosphotyrosine binding domain; RACK, receptor for activated C kinase; SARA, SMAD-anchor for receptor activation; SH2, Src homology 2 domain; SH3, Src homology 3 domain; Ste5, sterile 5; TGFb, transforming growth factor beta.

Protein

Length

(number of

Scaffold

Serpin

K+ channels, nNOS, NGL-2, SALM2, ADAM22, SYNGAP

95 (13.1)

Anchoring

Docking

Adaptor

SAM68, SLP-76, Sos, Synapsin, Vav

0 (0)

PAK, PRK2, Sos

48 (12.7)

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signaling proteins (Table 1) Because of its multiplicity

of binding partners, PSD95 functions in receptor

clustering, targeting receptor action on Ras and Rho

signaling, and regulating receptor modification

under-scoring dynamics of synaptic function A similar logic

applies to DLG1 (synapse-associated protein-97),

which regulates immunological synapses by physically

linking T-cell receptor signaling to cytoskeletal

rear-rangements Table 1 lists a few further scaffolds, all of

which are modular with several protein–protein

interaction domains and motifs embedded in and⁄ or

connected by long disordered regions, such as Shank1

and Caskin1 in neuronal PSD or SARA in

transform-ing growth factor beta signaltransform-ing

Scaffolds epitomize the very essence of the entire

family of signaling proteins, first appreciated in

tyro-sine kinase signaling where adaptors, scaffolds or

enzymes are recruited to the autophosphorylated

receptor tyrosine kinases (RTKs) Cytosolic kinases

and phosphatases, however, also require strict control

of their activity and subcellular localization, which is

achieved through anchoring proteins, originally defined

as proteins targeting the action of protein kinase A

(A-kinase anchoring proteins; AKAPs), on specific

substrates [7] They are usually long proteins with a lot

of structural disorder (Fig 1 and Table 1), with

func-tional attributes difficult to clearly distinguish from

that of scaffolds (described in detail in ref [7]) In

addition to AKAPs, protein kinase C also interacts

with a family of anchor proteins called receptors for

activated C kinase

Cytosolic Ser⁄ Thr phosphatases are also directed

into signaling networks by mechanisms similar to that

of protein kinase A and protein kinase C For

exam-ple, functional protein phosphatase 1 consists of a

cat-alytic subunit and a regulatory subunit The regulatory

subunits target the catalytic subunit to specific cellular

compartments and modulate substrate specificity [12]

One of the best characterized regulatory subunits is the

myosin phosphatase target subunit (MYPT1) A

num-ber of regulatory proteins, as well as Ser⁄ Thr kinases,

can associate with MYPT1, suggesting that similarly

to AKAPs and receptors for activated C kinase, the

function of MYPT1 is much broader than simply

tar-geting protein phosphatase 1 to myosin II [13]

Docking proteins

As outlined in the accompanying minireview by

Brum-mer et al [6], docking proteins were originally defined

as accessory proteins in RTK signaling with a

mem-brane-targeting region, a protein–protein interaction

site for receptor binding and an extended region with

several Tyr residues for receptor-dependent phosphory-lation (Fig 1 and Table 1) This outline applies to all four major families of classical docking protein, such

as the Grb2-associated binder⁄ daughter of sevenless (Gab⁄ DOS), insulin receptor substrate, FGF receptor substrate and docking protein families [6] Docking proteins are recruited to the site of RTK activation at the plasma membrane, they reinforce binding by virtue

of a receptor-binding domain (often a phosphotyro-sine-binding domain) and undergo multiple Tyr-phos-phorylation (there are more than five Tyr phosphorylation sites in at least one of the family members) Phosphorylation of Tyr residues is rather specific to certain RTKs, but may also proceed by cytoplasmic tyrosine kinases Tyr-phosphorylated docking proteins recruit SH2-domain-containing sig-naling components to initiate specific signal cascades, and they coordinate and regulate Tyr kinase signaling events, and also display dynamic regulatory phenom-ena described in more detail for scaffold proteins (for details see ref [6])

There are also other, atypical docking proteins [6], which lack the lipid-binding domain but have N-termi-nal domains⁄ regions that help them localize at the plasma membrane next to activating receptors, and also contain several Tyr residues that undergo phos-phorylation and recruitment of signaling proteins Although the function of these proteins (linker for activated T cells, Crk-associated substrate, SLP65) is closely related to other docking proteins, they appear

to be better classified as scaffolds

Adaptor proteins The term adaptor protein is generally used for low molecular mass molecules that serve to link two func-tional members of a catalytic pathway (Fig 1 and Table 1) Adaptors either possess two domains involved

in protein–protein interactions or use two regions com-posed of two to three domains The first group of adap-tor proteins identified was the family of SH2⁄ SH3 domain-containing proteins, including growth factor receptor-bound protein 2, Crk, CrkL and non-catalytic region of tyrosine kinase adaptor protein 1 (Nck) [14] Their SH2 domain binds specific phosphotyrosine resi-dues on activated receptors or their substrates, whereas their SH3 domains bind proline-rich motifs on down-stream target proteins Interestingly, this family of adaptor proteins contains only one SH2 domain, whereas they usually possess two or more SH3 domains In theory, more than one SH3 domains may allow the adaptor to recruit several ligands separately; however, it seems from earlier studies that cooperation

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exists between the SH3 domains for ligand binding For

example, Nck adaptor contains one SH2 domain and

three SH3 domains Although individual SH3 domains

of Nck were reported to be able to bind partners, such

as p21 protein (Cdc42⁄ Rac)-activated kinase or PRK2,

experimental data clearly showed that Nck constructs

containing all three SH3 domains bind the protein

part-ners with much higher affinity than the single SH3

domains [15,16] Therefore, it is highly likely that even

those adaptors which contain two or three tandem SH3

domains actually link only two members of a catalytic

pathway

SH2⁄ SH3 domain-containing adaptors link

down-stream target molecules to the membrane-bound

recep-tor In some cases, the adaptor may recruit binding

partners directly to the plasma membrane through its

lipid-binding domain In hematopoietic cells, pleckstrin

homology domain (PH)-containing adaptor molecules

provide important links between

phosphoinositide-3 kinase and lymphocyte function For example,

recruitment of Src kinase-associated phosphoprotein

(SKAP) and B-lymphocyte adapter molecule of 32 kDa

(Bam32)⁄ dual-adapter for phosphotyrosine and

3-phos-phoinositides (DAPP) to the plasma membrane of

acti-vated lymphocytes is driven by lipid products generated

through the action of phosphoinositide-3 kinase [17]

Whereas SKAP contains a PH domain on the

N-termi-nus and an SH3 domain on the C-termiN-termi-nus, Bam32⁄

DAPP1 possesses an SH2 domain and a PH domain

Therefore, although both adaptors could bind

phospho-inositide in the plasma membrane, SKAP recruits

pro-line-rich target molecules, whereas Bam32⁄ DAPP1 may

associate with phosphotyrosine-containing proteins

Possessing a PH domain for membrane association, this

subfamily of adaptors, including SKAP, Bam32 and

SH2B proteins, performs a bona fide adaptor function,

they nevertheless link downstream targets directly to

the plasma membrane DAPP [17]

The majority of adaptor molecules are implicated in

RTK signaling, however, other cell-surface receptors

also utilize adaptors possessing specific modular

domains Death receptors, members of the tumor

necrosis factor receptor superfamily, possess a

cytoplas-mic death domain (DD) They transmit signals through

apical protein complexes, which are nucleated by the

death domain adaptors Fas-associated protein with

death domain (FADD) and tumor necrosis factor

receptor type 1-associated death domain protein

(TRADD) FADD is a protein containing two

structur-ally similar protein motifs, the N-terminal death

effec-tor domain and the C-terminal DD The primary role

of FADD in death receptor signaling is to recruit

initia-tor procaspase 8 and procaspase 10 to death recepinitia-tors

[18] The principle by which the SH2⁄ SH3 domain-con-taining adaptors and FADD function is very similar, and the modular domains of growth factor receptor-bound protein 2 and FADD are commutable This was demonstrated by creating an artificial signaling pathway

in which the SH2 domain of growth factor receptor-bound protein 2 was fused to the death effector domain

of FADD The chimeric adaptor protein could reroute RTK signals to induce procaspase activation and cell death [19]

Classification of scaffolds and its limitations

The functions of the above four closely related catego-ries of signaling regulatory proteins show significant similarities and overlap (Fig 1 and Table 1), but appear to segregate into three broad functional catego-ries: simple proteins binding two partners together (adaptors), larger multidomain proteins targeting and regulating more proteins in complex ways (scaf-folds⁄ anchoring proteins) and proteins specialized to initiate signaling cascades by localizing partners at the cell membrane (docking proteins)

This classification has both advantages and limita-tions Its major advantage is simplification, because it enables one to think in terms of a few concepts instead

of a practically unmanageable number of individual observations This very fact, however, also makes it inherently limited, because of the necessary neglect of many details If such exceptions to the ‘rule’ of the system become too numerous, the system has to be improved⁄ refined to comply with progress in the field Here we list a few such notable exceptions that may eventually demand the extension of the classification scheme

A diagnostic mark of the limitations inherent in the scheme is that very often the names are used inter-changeably, without much attempt to clarify where the given protein belongs to For example, anchoring pro-teins are often also termed scaffolds [4,7], the term scaf-folds and adaptors are sometimes used interchangeably [1,4], the distinction between docking and adaptor is somewhat arbitrary [6], and docking proteins may also

be termed adaptors and scaffolds [2] There are many individual proteins called by different names in differ-ent articles, such as MyD88 scaffold and adaptor [3], linker for activated T cells (LAT) scaffold [3] and dock-ing protein [6], GAB dockdock-ing protein [6] and scaffold [4], mAKAP anchor protein [7] and scaffold [4],

MAG-UK proteins, which ‘anchor’ receptors at the synapse, scaffold [1] and adaptor [20], FGF receptor substrate docking protein [6] and ‘scaffold adaptor’ [21] These

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and many other examples in the literature demonstrate

that the terms cannot be unequivocally separated

A different type of limitation is the exclusion of

pro-teins with enzymatic activity Although it serves the

simple purpose of separating two basic functions in

signal transduction – enzymes that act and proteins

that orchestrate their action [22] – it is clear that it

cannot be done for many relevant proteins that have

both enzymatic domains and regulatory

protein–pro-tein interaction domains In effect, there are several

proteins considered as scaffolds that have enzymatic

activity, such as MAGUK proteins already discussed

among scaffolds [3], RNAse E [23], MEKK1 of the

JNK signaling pathway [24], RTKs themselves [3] or

integrin-linked kinase [25] Actually, there is little

func-tional distinction between a scaffold protein binding a

given enzyme partner and a scaffold that has an

enzy-matic domain of the same type

An additional complicating factor is that there are

many other signaling pathways beyond kinase cascades,

which are rather neglected in this context For example,

transfer of the small protein ubiquitin proceeds via a

cascade, from E1 ubiquitin-activating enzyme to E2

ubiquitin-conjugating enzyme to a specific substrate

with the intervention of a targeting-type of interaction

mediated by E3 ubiquitin ligases Because ubiquitinated

target proteins may be targeted for degradation or

sig-naling activation [26], the operation of E3 proteins,

such as MDM2 [27] and Siah-2 [28], can be best

ratio-nalized by the scaffold concept The assembly of

Esc-herichia coli RNA degradosome by RNAse E [23] is

also of very similar molecular logic It has been

sug-gested that there are also adaptors that are important in

pathways activated by internal signals, such as DNA

damage, e.g MDC1 in DNA doule-strand breaks [20]

and BRCA1 in many cellular pathways including DNA

repair [29] In a similar vein, trafficking of signaling

proteins is itself dependent on adaptors associated with

protein sorting in endosomes, such as ESCRT [30] In

addition, both proteins and also metabolites can be

scaffolded, as demonstrated by Pmel17, a physiological

amyloid that ‘scaffolds’ and ‘sequesters’ toxic

interme-diates during the biosynthesis of the pigment melanin in

melanocytes in the skin [31]

Of further note, many fully or largely disordered

pro-teins noted for their assembly function (see below) are

not usually considered to be scaffold proteins, although

they do bind and orchestrate the action of several

sig-naling partners, such as caldesmon (Ca2+⁄ calmodulin,

F-actin, myosin, tropomyosin), SIBLING proteins

(integrin, complement factor H, CD44, fibronectin), or

RNAPII CTD (capping, splicing and polyadenylation

factors) The molecular mechanism and function of

these proteins also comply with the scaffolding princi-ples outlined in this article

Scaffold proteins and structural disorder

The decision on what is included among scaffolds can also be approached from a structural point of view, because in all categories – with the possible exception of adaptors – the proteins have a very high level of func-tion-related structural disorder (Table 1) It has recently been recognized that a significant proportion of eukary-otic genomes encodes for proteins (IDPs) or regions of proteins (IDRs) that lack a well-defined 3D structure under native, functional conditions [10,32–34] Struc-tural disorder abounds in proteins of regulatory and sig-naling function, and it is also closely correlated with disease, such as cancer and neurodegenerative disorders The molecular function of IDPs⁄ IDRs may stem either from recognition of partner molecules via short motifs [35,36] or disordered domains [37], from regulatory post-translational modification and also from providing

‘entropic-chain’ functions, such as linkers and segments contributing entropic exclusion and force generation

As a result, the molecular function of scaffolds corre-sponds to the ‘assembler’ function of IDPs, i.e they have been described to assemble complexes [9,34,37] The role of structural disorder in scaffold-type func-tions is also apparent in hub proteins, which have been found to have a large number of binding partners in high-throughput studies of protein–protein interactions

in the interactome [38] In particular, ‘party’ hubs have been defined as those being able to bind their partners simultaneously, which is the very essence of the action

of scaffolds Hub proteins have an elevated level of dis-order [39], which is also the case with the examples cited here (average disorder, 43.3%; Table 1), and also previ-ous findings that scaffold proteins constitute one of the most disordered functional categories [8,40] and the average disorder correlates with the number of subunits

of multiprotein complexes [41] In all, structural disor-der seems to be closely associated with several attributes

of scaffold function, such as the ability of binding multi-ple partners, mediating their commulti-plex and transient interactions and themselves undergoing a complex array

of regulatory post-translational modifications (Fig 1)

Conclusion: where is the field of scaffolds headed?

From all the considerations described, it seems that a useful practical functional classification of scaffold proteins and their kin can be given At first sight, all

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the proteins enlisted represent variations on a common

theme, binding signaling proteins together to direct

and control the flow of information in the cell This

basic theme segregates in a rather consistent manner

into three coherent categories Scaffold⁄ anchor

pro-teins usually bind more than two signaling components

together and regulate their activity in complex and

dynamic ways, involving activation and repression of

activity Adaptor proteins are usually smaller and their

function is simpler, connecting two partners together

Docking proteins distinguish themselves by assembling

signaling complexes at the plasma membrane in a

Tyr-phosphorylation-dependent way There are many

pro-teins excluded from this scheme, although they do act

via very closely related mechanisms Their inclusion

following careful judgment of their functional modes,

possibly leading to an extension of the classification

scheme, should be considered

Acknowledgements

This work is supported by grants OTKA K60694 and

NK71582 from the Hungarian Scientific Research

Fund and ETT 245⁄ 2006 from the Hungarian Ministry

of Health (for PT), the Miha´ly Pola´nyi Program

(Agency for Research Fund Management and

Research Exploitation, KPI) and a ‘Lendu¨let’ grant

from the Hungarian Academy of Sciences (for LB)

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