Tài liệu Báo cáo khoa học: Multisite protein phosphorylation – from molecular mechanisms to kinetic models pdf

Given that there are approximately 500 protein kinases in the human genome [3], which are themselves regulated by and have in all likelihood at least one spe-cific target, the number of r

Multisite protein phosphorylation – from molecular

mechanisms to kinetic models

Carlos Salazar and Thomas Ho¨fer

Research Group Modeling of Biological Systems (B086), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, Heidelberg, Germany

Introduction

Signal transduction networks are formed, in large part,

by interacting protein kinases and phosphatases

Phosphorylation of proteins by kinases (or

dephosphor-ylation by phosphatases) provides docking sites for

interaction partners or triggers conformational changes

that alter a protein’s enzymatic activity or its

interactions with other proteins or DNA These alteredenzymatic and⁄ or interaction properties may transmitsignals in various ways For example, protein kinasesactivated by phosphorylation can themselves phosphor-ylate target proteins (e.g receptor⁄ receptor-associatedtyrosine kinases, mitogen-activated protein (MAP)kinase cascades) Phosphorylation status can deter-mine the subcellular localization of a protein (e.g by

Keywords

enzyme processivity; kinetic proofreading;

mathematical models; order of phospho-site

processing; ultrasensitivity

Correspondence

C Salazar, Research Group Modeling of

Biological Systems (B086), German Cancer

Research Center (DKFZ), Im Neuenheimer

Feld 280, 69120 Heidelberg, Germany

Fax: +49 6221 54 51487

Tel: +49 6221 54 51383

E-mail: c.salazar@dkfz-heidelberg.de

T Ho¨fer, Research Group Modeling of

Biological Systems (B086), German Cancer

Research Center (DKFZ), Im Neuenheimer

Feld 280, 69120 Heidelberg, Germany

regula-of molecular mechanisms involved in processing regula-of the phosphorylationsites Here we review the results of such models, together with salientexperimental findings on multisite protein phosphorylation We discusshow molecular mechanisms that can be distinguished with respect to theorder and processivity of phosphorylation, as well as other factors, regulatechanges in the sensitivity and kinetics of the response, the synchronization

of molecular events, signalling specificity, and other functionalimplications

Abbreviations

ASF⁄ SF2, alternative splicing factor; BAD, Bcl-XL ⁄ Bcl-2-associated death promoter; CDK, cyclin dependent kinase; DYRK, dual-specificity tyrosine-regulated kinase; EGF, epidermal growth factor; ERK, extracellular signal-regulated protein kinase; ITAM, immunoreceptor tyrosine- based activation; MAP kinase, mitogen-activated protein kinase; MEK, MAPK ⁄ ERK kinase; N-WASP, neuronal Wiskott–Aldrich syndrome protein; NES, nuclear export signal; NFAT, nuclear factor of activated T cells; NLS, nuclear localization signal; PDE3B, cyclic nucleotide phosphodiesterase 3B; RS, arginine-serine repeats; SH2 domain, Src homology 2 domain; SP, serine–proline repeat; SRPK, serine-arginine- rich protein kinase; SRR, serine-rich regions; TCR, T-cell receptor; ZAP-70, zeta-chain-associated protein kinase 70.

controlling nuclear import⁄ export in Janus kinase/

signal transducer and activator of transcription (Jak/

Stat) and nuclear factor jB (NFjB) pathways) In

tran-scriptional regulation, phosphorylation events control

the binding of specific transcription factors to their

regu-latory sequence elements, as well as the action of RNA

polymerase Proteins can also be targeted for

degrada-tion through multisite phosphoryladegrada-tion (e.g the yeast

cell-cycle regulator Sic1)

Phosphorylation affects a very large number of

intra-cellular proteins, and is arguably the most widely

stud-ied post-translational modification [1] An important

(and as yet not fully resolved) question in this regard is

how many of the observed protein phosphorylation sites

are specifically regulated and serve a regulatory function

[2] Given that there are approximately 500 protein

kinases in the human genome [3], which are themselves

regulated by and have in all likelihood at least one

spe-cific target, the number of regulatory phosphorylation

sites must be in the thousands or even higher It is thus

not surprising that abnormal protein phosphorylation

events have been observed in many human diseases,

including cancer, diabetes, hypertension, heart attacks

and rheumatoid arthritis [1]

Phosphorylation⁄ dephosphorylation has been

con-sidered as a fundamental on⁄ off switch for protein

function In the last decade, however, it has become

clear that many proteins harbour multiple

phosphory-lation sites, and this can considerably expand the

repertoire for combinatorial regulation or fine-tuning

of switch properties [4–6] Phosphoproteome analyses

have shown that most phosphoproteins in eukaryotic

cells contain more than one phosphorylatable site [7]

(Phospho.ELM database, http://phospho.elm.eu.org)

Several proteins with 10, 20 or even more (regulatory)

phosphorylation sites are known [6,8] Multiply

phos-phorylated proteins are found in a great variety of

cellular processes; they include membrane receptors

(e.g growth-factor receptors [9] and the T-cell receptor

complex [10]), ion channels (e.g the Kv2.1 potassium

channel in mammalian neurons [11]), protein kinases

(e.g MAP kinases [12,13] and Src family kinases [14]),

adaptor proteins (e.g SH2-domain containing

leuko-cyte protein of 76 kDa [15], Vav [16] and LAT linker

of activated T cells [17] in hematopoetic cells),

cell-cycle regulators (e.g Sic1 [18], Cdc25 [19] and Sld2

[20] in budding yeast), circadian clock proteins (e.g

frequency protein, FRQ [21] in the bread mold

Neuro-spora), transcription factors (e.g Pho-4 in budding

yeast [22] and nuclear factor of activated T cells

(NFAT) in mammalian cells [23]), transcriptional

coac-tivators (e.g PC4 [24]), RNA polymerase II [25],

histones [26], splicing factors [27], and others Overall,

serine phosphorylations are the most abundant(approximately 86% of all phosphorylation sites inHeLa cells), followed by threonine (12%) and tyrosinephosphorylations (2%) [7] With respect to kinetics,tyrosine phosphorylations generally occur faster duringcell signalling than serine⁄ threonine phosphorylations.For example, upon addition of epidermal growthfactor (EGF) to HeLa cells, most tyrosines becomephosphorylated within 1 min, while threonine andserine phosphorylations require up to 10 min [7].Compared to phosphorylation of a single residue,multisite phosphorylation increases the possibilities forregulating protein function very considerably A proteinwith N phosphorylation sites can exist in 2Nphosphory-lation states Each such state may have a different func-tional characteristic For example, the Src familykinases have at least two regulatory Tyr phosphoryla-tion sites, one activating and the other inhibitory, sothat there are four (22) different phosphorylation states

of these residues Accordingly, Src kinases may exist inseveral distinct states of enzymatic activity (additionallydepending on protein–protein interactions, some ofwhich are also governed by phosphorylation) [14] Onthe other hand, for larger N, the number of possiblestates becomes so high that it is unlikely that each onehas specific functional properties (e.g for N = 10, thereare 1024 phosphorylation states) The reduction of suchhigh-dimensional phosphorylation state spaces to asmaller number of functional states may occur on twolevels First, the molecular mechanisms of phosphoryla-tion may realise only a subset of the possible states Forexample, for a strictly sequential phosphorylation mech-anism (and reverse-order dephosphorylation), there areonly N + 1 phosphorylation states instead of 2N Sec-ond, several individual phosphorylation sites may coop-erate in effecting a functional outcome (e.g through aconformational change), such that it is primarily thenumber of phosphorylated sites that counts rather thantheir specific location Both types of dimensionality-reduction mechanisms do indeed occur in proteinphosphorylation, as detailed below Nevertheless theoccurrence of many phosphorylation states (especially

in random phosphorylation⁄ dephosphorylation nisms) is an important factor shaping both dose–response curves and kinetics

mecha-These rather basic considerations already make itclear that in-depth analysis of the mechanisms andfunctions of multisite protein phosphorylation requiresmathematical modelling Both general mathematicalanalyses of multisite phosphorylation [28–36] andmodels of specific systems [12,13,37–46] have bee pub-lished in recent years Here we review these theoreticaldevelopments within the context of salient experi-

mental findings on the molecular mechanisms of protein

regulation by phosphorylation This comparison

high-lights several questions for further modelling as well as

experiments required for progress in the quantitative

understanding of multisite protein phosphorylation

Biological model systems

To provide a background for the theoretical section,

we briefly introduce three experimental model systems

that highlight various mechanistic and functional

aspects of multisite phosphorylation

Recruitment and activation of signalling proteins

at plasma membrane receptors

In response to extracellular stimuli, many plasma

membrane receptors are phosphorylated at multiple

tyrosine residues that provide docking sites for

signal-ling proteins A particularly intriguing example is

signalling through the T-cell receptor (TCR) complex

The subunits of the TCR together contain 20

regula-tory tyrosine residues located pairwise in ten

immuno-receptor tyrosine-based activation (ITAM) motifs [10]

Following binding of a cognate ligand (an antigen–

major histocompatibility complex), these tyrosine

resi-dues become phosphorylated by the Src kinase Lck,

and in turn another tyrosine kinase,

zeta-chain-asso-ciated protein kinase 70 (ZAP-70), binds strongly to

ITAMs containing two phosphotyrosines (Fig 1A)

The recruited ZAP-70 adopts an open conformation,

and becomes activated by several tyrosine

phosphory-lations (catalysed by Lck and by ZAP-70

trans-auto-phosphorylation) These events form the beginning of

a cascade of phosphorylation events that are thought

to be critical for a T cell’s ability to discriminate

between a cognate antigen (triggering an immune

response) and self-peptides (for which a response

would be detrimental) [10,47]

Nuclear transport and DNA binding of

transcription factors

Multisite phosphorylation regulates the activity of

tran-scription factors at several levels, such as subcellular

localization, DNA binding affinity and transcriptional

activity (reviewed in Ref [6]) An example of such

multi-level regulation is provided by the transcription factors

of the NFAT family, NFAT1–4, which reside in the

cytoplasm of unstimulated cells in a highly

phosphory-lated state (Fig 1B) [48,49] In response to

calcium-mobilizing stimuli, several conserved serine residues (13

in NFAT1), located in serine-rich regions (SRR) and

serine–proline repeats (SP), are dephosphorylated bycalcineurin [23] In NFAT1, dephosphorylation of theSRR1 motif (and possibly also of the SP2 and SP3motifs) induces exposure of a nuclear localizationsequence (NLS), promoting nuclear import of NFAT.Full dephosphorylation is needed for maximal DNAbinding of NFAT Dephosphorylation of NFAT by cal-cineurin is counteracted by several kinases, among themCK1, GSK3 and dual-specificity tyrosine-regulatedkinases (DYRKs) Experiments suggest the existence of

a preferential order of phosphorylation and phorylation DYRKs phosphorylate the SP3 motif, thus

dephos-Fig 1 Prototypical examples of multisite phosphorylation in signal transduction and cell-cycle regulation (A) Receptor proteins Bind- ing of a high-affinity ligand to the T-cell receptor (TCR) leads to phosphorylation of ITAM motifs at two tyrosine sites, to which the kinase ZAP-70 binds via its tandem Src homology 2 (SH2) domains (B) Transcription factors Dephosphorylation of the transcription fac- tor NFAT (nuclear factor of activated T cells) by calcineurin (CaN) at several Ser residues induces a conformational change that exposes

a nuclear localization signal (NLS), leading to nuclear localization of NFAT, its binding to DNA, and maximal transcriptional activity NES, nuclear export signal (C) Cell-cycle inhibitors The cell-cycle inhibitor Sic1 requires phosphorylation by the cyclin-dependent kinase Cdc28 on at least six sites before it can be ubiquitinated by the Cdc4 ⁄ SCF complex and degraded by the 26S proteasome.

priming further phosphorylation of the SP2 and SRR1

motifs by GSK3 and CK1, respectively [50]

Dephos-phorylation of the SRR1 motif appears to increase the

accessibility of the SP motifs to calcineurin [23] NFAT

kinases are activated by distinct signalling pathways,

and may be differentially regulated in the cytoplasmic

and nuclear compartments

Cell-cycle regulation

Multisite phosphorylation is prominent in regulation

of the cell cycle, in particular at the G1⁄ S transition

In yeast, the cyclin kinase inhibitor Sic1 must be

phos-phorylated on at least six of nine Ser⁄ Thr residues by

a cyclin-CDK complex during G1phase before binding

to the SCFCdc4 ubiquitin ligase [18,51,52] This, in

turn, leads to ubiquitination of Sic1, its degradation

by the proteasome, release of the S-phase

cyclin-depen-dent kinase from inhibition, and, finally, the onset of

DNA synthesis (Fig 1C) The number of

phosphory-lated sites appears to be more important than the

iden-tities of the individual residues for SCFCdc4 binding

Any combination of six phosphorylated sites is

suffi-cient for Sic1 degradation While singly

phosphory-lated Sic1 binds to SCFCdc4 very weakly, multiply

phosphorylated Sic1 can bind efficiently, presumably

by increasing the local concentration of interaction

sites around the SCFCdc4 binding surface It has been

suggested that multisite phosphorylation can act as a

counting mechanism that ensures the proper timing of

critical cell-cycle transitions [51] Interestingly, another

multiple protein modification, multi-ubiquitination,

also plays a central role in the cell cycle [53]

Quantitative data

Experimental data on the dynamics of key

phosphory-lation events in signal transduction and other cellular

processes are essential for the development of accurate

quantitative models and therefore for a mechanistic

understanding of cellular behaviour Biochemical

approaches, such as immunoblotting with

phospho-specific antibodies, are routinely used for monitoring

(previously identified) phosphorylation sites, and many

studies based on this technique have yielded valuable

mechanistic insight (e.g [54]) Mathematical modelling

frequently requires quantitative information (e.g what

fraction of a given protein is phosphorylated) that is

cumbersome to obtain in this way Higher throughput

can be achieved with antibody microarrays [55], while

flow cytometric analysis of intracellular

phosphopro-teins provides single-cell resolution and high sensitivity

that cannot be achieved with immunoblotting [56]

However, all these methods require appropriate bodies to known phosphorylation sites Radionucleo-tide incorporation experiments may also provideaccurate information about phosphorylation kinetics[27], but are time-consuming to perform Mass spec-trometry allows both large-scale analysis and theidentification of novel phosphorylation sites and phos-phoproteins not previously known to be involved incellular signalling [7,8,57] Information about phos-phorylation sites obtained in large-scale screens hasbeen incorporated into searchable databases such asPhosphosite (http://www.phosphosite.org), Swiss-Prot(http://us.expasy.org/sprot) and Phospho.ELM (http://phospho.elm.eu.org) Mass spectrometric data forprotein phosphorylation may be very useful for kineticanalysis and modelling, although rather few applica-tions exist to date (e.g [7, 23]) Time-resolved high-resolution NMR spectroscopy has been used recently

anti-to study mechanistic questions regarding multisite tein phosphorylation [58,59] We discuss below whichtype of data are required to establish kinetic models

pro-Molecular mechanisms of multisite phosphorylation

The presence of multiple phosphorylation sites raisesnew mechanistic questions compared to the case of sin-gle phosphorylation These pertain to (a) the order inwhich individual sites are phosphorylated and (b) thenumber of enzyme binding events required A thirdmechanistic aspect, which is relevant both forsingle- and multisite phosphorylation, is whether thecounteracting kinase(s) and phosphatase(s) competefor binding to the target protein We also discuss howcooperativity can arise in multiply phosphorylatedproteins, and the role played by subcellular compart-mentalization

Order of phospho-site processingThe order in which phosphorylation sites in a proteinare acted on by kinases and phosphatases determinesthe possible phosphorylation states (Fig 2A).Although it has generally been difficult to obtain suchinformation experimentally at the required resolution,inferences have been drawn regarding the order ofphospho-site processing in several cases Sequentialphosphorylation has been suggested for several kinas-

es, especially Ser⁄ Thr kinases [60–68] When phorylation also follows a fixed order, strictlysequential or cyclic mechanisms of phosphorylationarise, depending on whether the last site to be phos-phorylated is the first, or the last, to be dephosphoryl-

dephos-ated Both types of mechanism have been proposed,

one for NFAT and the other for rhodopsin [38,69]

Alternatively, a particular site may be modified

irre-spective of the phosphorylation state of the other sites,

giving rise to essentially random phosphorylation and

dephosphorylation

Combinations of random and sequential mechanismsare possible For example, it is conceivable that phos-phorylation of a protein is random while dephosphory-lation is sequential, e.g for the MAP kinase ERK2[41,70,71] A particularly interesting mixed case hasbeen suggested for the yeast cell-cycle regulator Sld2,

Fig 2 Mechanistic aspects of multisite phosphorylation (A) Order of phospho-site processing Phosphorylation sites can be modified lowing a strict order The last site to be phosphorylated may be the first (sequential mechanism) or the last (cyclic mechanism) to become dephosphorylated Alternatively, the sites can be modified in a completely random manner In some cases, multiple sites must be randomly phosphorylated before a site with a specific function becomes accessible to the kinase (hierarchical mechanism) (B) Enzyme processivity The enzyme can modify all the sites without intermediate dissociation from the substrate (processive kinetics), or, conversely, must bind and dissociate repeatedly before all residues become phosphorylated (distributive kinetics) (C) Competition effects At low enzyme concen- trations, the distinct phosphorylation forms of the substrate may compete for binding the enzyme, while counteracting enzymes may compete for binding the substrate at low substrate concentrations (D) Conformational changes and cooperativity The dynamic equilibrium between distinct functional conformations may be affected by the phosphorylation state of the protein In the example shown, phosphoryla- tion of each site increases the probability of a closed conformation with a higher affinity for the kinase, which accelerates the remaining phosphorylation steps (cooperative kinetics) (E) Compartmentalization Phosphorylation sites exerting distinct functions can be modified by kinases localized in distinct subcellular compartments In the example shown, the subcellular localization of a substrate is regulated by cytoplasmic and nuclear kinases.

fol-for which random phosphorylation of multiple

Ser⁄ Thr residues appears to allow the eventual

phos-phorylation of a critical threonine, possibly through a

conformational change (hierarchical mechanism) [20]

The various mechanisms differ considerably in the

number of phosphorylation states they generate

Sequential mechanisms have a linear dependence on

the number (N) of phosphorylation sites (strictly

sequential: N + 1; cyclic: 2N), while the number of

states grows exponentially (2N) for random

mecha-nisms The difference is considerable: for 13 regulatory

sites (as in NFAT1 [23]), there would be 8192 possible

phosphorylation states in the case of a random

nism but only 14 states for a strictly sequential

mecha-nism Below we analyse the consequences of such

differences for the regulatory properties of the protein

The amino acid sequence can determine the order of

phosphorylation (see Table 1) In particular, a

consen-sus sequence for a kinase may occur repetitively, thus

establishing a hierarchy in the phosphorylation For

example, yeast kinase SRPK family kinases, which are

implicated in RNA processing, sequentially

phosphory-late Ser residues in consecutive arginine-serine (RS)dipeptide repeats [63,64] Moreover, the substrate spec-ificity of certain kinases may depend on (or beenhanced by) nearby residues phosphorylated byanother kinase (priming kinase) Phosphorylation ofthe serine S or threonine T in the (S/T)XXX(Sp⁄ Tp)motif by the kinase GSK3 requires priming by anotherkinase that phosphorylates the Sp⁄ Tp site [60–62] In asequence of appropriately spaced serines, only the firstmay need to be primed, while the remaining are thensequentially phosphorylated by GSK3 Primingphosphorylation facilitates the binding of a secondkinase either by creating specific docking sites, chang-ing the substrate conformation, or dislodging the sub-strate from the cell membrane [65–69] An interestingexample of such a dual-enzyme mechanism is found inthe canonical Wnt⁄ b-catenin pathway, where sequen-tial phosphorylations of the Wnt co-receptor lipo-protein receptor-related protein 6 (LRP6) and thetranscriptional cofactor b-catenin by the kinases GSK3and CK1 mirror each other Sequential phosphoryla-tion of b-catenin by CK1 and cytosolic GSK3 anta-

Table 1 Consensus sequences and docking motifs for some kinases and phosphatases PP1, protein phosphatase; PTP1B, protein tyrosine phosphatase 1B; SHP2, Src homology domain-containing protein tyrosine phosphatase 2.

Tyr kinases

gonizes Wnt⁄ b-catenin signalling, whereas plasma

mem-brane-associated GSK3 primes further LRP6

phos-phorylation by CK1 in response to Wnt stimulation

and activates Wnt⁄ b-catenin signalling [65]

To achieve high specificity, many protein kinases

and phosphatases recognize their targets through

inter-actions that occur outside of the active site [72]

Tyro-sine kinases and phosphatases often utilize dedicated

interaction domains, such as SH2 and SH3 domains,

that are distinct from the catalytic domain [14,73,74]

Specific docking interactions may also occur in the

cat-alytic domain but outside of the catcat-alytic site, as found

for many serine⁄ threonine kinases and phosphatases

[72] These mechanisms appear to contribute in some

cases to sequential processing of the phosphorylation

sites

The three-dimensional structure of the substrate

may also affect the order of (de)phosphorylation

Random phosphorylation may be linked to the

adoption of a flexible or unfolded structure by the

target protein so that several residues become equally

accessible to the kinase In some cases, the order of

phosphorylation is not determined by structural

factors but rather by the activation kinetics of the

participating kinases For example, Ser⁄ Thr

phos-phorylation of the EGF receptor by several

down-stream kinases such as the MAP kinases ERK1/2

and p38 shows delayed kinetics compared to

auto-phosphorylation of the EGF receptor on multiple

tyrosine residues [7]

Processivity of phosphorylation

Kinases (or phosphatases) may differ in the number of

binding events required to phosphorylate (or

dephos-phorylate) all target sites on a protein (reviewed in

Ref [75]) A kinase may bind to the substrate and

phosphorylate all the sites while staying bound

(pro-cessive mechanism) (Fig 2B) Conversely, the kinase

may bind, phosphorylate one residue and dissociate, so

that next phosphorylation first requires re-binding of a

kinase molecule (distributive mechanism)

Although some proteins clearly follow one of these

two models (see Table 2), the processive and

distribu-tive mechanisms are the extremes of a continuous

spectrum For example, the cyclin-CDK complex

Pho80⁄ Pho85 phosphorylates the yeast transcription

factor Pho4 on five serines, with a mean of

approxi-mately two phosphorylation events per

enzyme–sub-strate binding [76] The degree of processivity depends

on the relative time scales of enzyme dissociation and

catalytic reaction [77], and can be quantified as follows:

the probability that an enzyme proceeds to modify the Table

next site before it dissociates is kcat⁄ (kcat+ koff), where

koff and kcatare the dissociation rate constant and the

catalytic rate constant, respectively, of a

substrate-bound enzyme molecule The probability of a fully

processive modification of N sites is then

kcatþ koff

ð1Þ

(assuming, for simplicity, that all the sites have the

same kcatand are modified sequentially)

Indeed, kcat values as fast as 10Æs)1 have been

reported for protein kinases, while dissociation rate

constants may be much lower (0.01Æs)1 and below)

However, phosphorylation rates in the minute range

have been reported for a processive substrate,

indicat-ing that kcatcan also be much lower [78], as required

for distributive phosphorylation mechanisms For

example, the splicing factor ASF⁄ SF2 is fully

phos-phorylated during a single encounter with its kinase

SRPK1 due to the high-affinity interaction between

the proteins (equilibrium dissociation constant Kd

approximately 50 nm) [27] By contrast, the

dissocia-tion rate of the MEK:pERK2 complex is at least five

times as fast as the phosphorylation rate of the second

site in ERK2 [77] Enzyme processivity may be

enhanced by the presence of protein–protein

interac-tion domains such as SH2 and SH3 that recognize

newly phosphorylated products, allowing repositioning

of the enzyme and substrate [73,74] Tethering a

sub-strate to its modifying enzymes through a scaffold

pro-tein can also increase the degree of processivity [79]

Two biochemical methods have mainly been

employed to determine the processivity of substrate

phosphorylation In the ‘start-trap’ strategy, ATP is

added to the enzyme–substrate complex, together with

an inhibitor that can trap the free enzyme [27] In a

distributive mechanism, the inhibitor traps the free

enzyme, stopping the reaction before full

phosphoryla-tion is achieved By contrast, in a processive

mecha-nism, the inhibitor does not influence the rate or

extent of phosphorylation A second strategy consists

of measuring the phosphorylation rate at various

con-centrations of substrate (or enzyme) [73] For a

distrib-utive mechanism, the partially phosphorylated forms

can act as competitive inhibitors of phosphorylation,

so that increases in substrate concentration result in a

decreased formation rate of the fully phosphorylated

substrate Recently, time-resolved high-resolution

NMR spectroscopy has been used to identify the

pres-ence of free partially phosphorylated forms of the

substrate and the existence of a defined order of

phos-phorylation [58]

Processive enzymes can catalyse sequential phorylation, while distributive enzymes may processthe phosphorylation sites in a random manner Forexample, the intermolecular autophosphorylation ofseveral Tyr residues in the fibroblast growth factorreceptor 1 kinase apparently proceeds in a sequentialand processive manner [80] Dual phosphorylation ofextracellular regulated kinase (ERK) by MEK in theMAP kinase cascade was reported to occur via a ran-dom and distributive mechanism [41,70] However, aprocessive kinase can also catalyse random phosphory-lations, as recently proposed for phosphorylation ofthe focal adhesion protein p130Cas by Scr kinase [81].Conversely, sequential DUSP6 dephosphorylation ofERK2 at Thr and Tyr was shown to occur distribu-tively [71] Thus there appears to be no strict linkbetween the degree of processivity of a kinase andrandom or sequential phosphorylation of its multipletarget sites The phosphorylation order and enzymeprocessivity of some relevant proteins are listed inTable 2

phos-Competition mechanismsThe interactions between the target protein and itsmodifying enzymes can lead to two distinct types ofcompetition effects (Fig 2C) The binding affinities ofkinases and phosphatases may change with the phos-phorylation state of the target protein For example,the fully phosphorylated target may lose (or retain) itsaffinity for the kinase Such affinity changes may lead

to interesting effects when the concentration of thekinase is much smaller than that of the target protein[28–30,82,83] In this case, target proteins of variousphosphorylation states compete for the kinase (or,equally, for the phosphatase) When the kinaseremains associated with the higher or fully phosphory-lated forms of its target protein, product inhibition willresult, because the bound kinase is not available to act

on unphosphorylated target molecules

Conversely, when the concentrations of the ing enzymes [kinase(s) and phosphatase(s)] are largecompared to their target protein, as may be the case insignal transduction, the enzymes can compete for bind-ing to the target Phosphorylation is then inhibited bythe phosphatase and dephosphorylation by the kinase

modify-In particular, when the kinase has a high affinity forthe phosphorylated target, the latter is sequestered and

is not available for dephosphorylation The structuralbasis for such competition may involve overlappingbinding sites for kinases and phosphatases on the tar-get, such that they are unable to bind to the target atthe same time [84]

The phosphorylation of a particular residue can also

compete with other covalent modifications For

exam-ple, in addition to phosphorylation, Ser and Thr

resi-dues are also targets for glycoxylation, while the

hydroxyl group of Tyr residues can be phosphorylated

or sulfated [4] Intermolecular competition can occur

between substrates of similar affinity for the same

enzyme; a substrate with a lower affinity will be

phosphorylated once the preferred targets have been

saturated with the enzyme [30]

Conformational changes and cooperativity

For some proteins, phosphorylation controls their

function by creating or eliminating docking sites for

the recruitment of specific binding partners In other

cases, phosphorylation alters the local environment of

a catalytic center or a binding site For proteins with a

large number of regulatory phosphorylation sites,

phosphorylation sites distant from such functional

motifs may regulate protein activity by inducing

changes in its global conformation [23,85] (Fig 2D)

For example, extensive charge modifications caused by

multiple phosphorylations on NFAT have been

pre-dicted to alter its tertiary structure [85]

As a plausible model for the control of protein

con-formation by multisite phosphorylation, it has been

proposed that individual phosphorylation events shift

the equilibrium between two or more pre-existing

con-formations of the protein [23,38,86] For instance, the

nucleo-cytoplasmic transport of NFAT can be

accounted for by a conformational switch model, with

an active conformation that is transported from the

cytoplasm to the nucleus and an inactive conformation

that is exported back to the cytoplasm The probability

of attaining the active conformation increases with

each dephosphorylation step [23,38] Somewhat more

complicated models with four conformation states

have also been proposed [39]

The conformation of the target protein can also

affect the binding of kinases or phosphatases, and the

kinetics of the (de)phosphorylations This can induce

cooperativity among the phosphorylation states For

example, in the case of NFAT, dephosphorylation of

the SRR1 region enhances dephosphorylation of the

SP2 and SP3 motifs by calcineurin [23]

Compartmentalization

Phosphorylation sites can be modified by two or more

kinases (or phosphatases) that are localized in distinct

subcellular compartments (Fig 2E) An example is the

interplay between the cytoplasmic kinase SRPK1 and

the nuclear kinase Clk⁄ Sty in phosphorylation of thesplicing factor ASF⁄ SF2 [27,87,88] A docking motif inASF⁄ SF2 restricts its phosphorylation by SRPK1 to theN-terminal half (approximately 10 sites) of the RSdomain, mediating nuclear import of ASF⁄ SF2 andlocalization in nuclear speckles [87] Clk⁄ Sty, however,can phosphorylate the entire RS domain (approximately

20 sites), causing release of ASF⁄ SF2 from speckles.The subcellular localization of kinases and phospha-tases is an important issue in signalling from theplasma membrane to the nucleus For example, in rest-ing cells, the NFAT phosphatase calcineurin residespredominantly in the cytoplasm, but upon cell stimula-tion may be imported into the nucleus together withNFAT to maintain NFAT dephosphorylation andnuclear localization [89,90] The NFAT kinases GSK3and CK1, which phosphorylate the SP2 and SRR1motifs, respectively, are present in both subcellularcompartments However, DYRK2 and DYRK1A,which phosphorylate the SP3 motif, are cytoplasmicand nuclear, respectively [50] DYRK2 probably helps

to maintain the phosphorylated state of cytoplasmicNFAT in resting cells, whereas DYRK1A re-phospho-rylates nuclear NFAT and promotes its export fromthe nucleus Such compartmentalization of kinases orphosphatases confers different functions, and, in turn,may expand the repertoire for regulating signal trans-duction networks

Kinetic modelling of multisite phosphorylation

General frameworkKinetic models of multisite protein phosphorylationare quite distinct from those of traditional enzymekinetics [91,92] for several reasons First, the number

of molecular states to be accounted for is usuallylarger (including partially phosphorylated states, bothenzyme-bound and free, and, where appropriate, vari-ous conformations of the protein due to its phosphory-lation state) Second, and more importantly, thesimultaneous presence of kinases and phosphatasesneeds to be considered in a physiological context, sothat there are at least two counteracting enzymes inthe system (although consideration of a single enzymeacting on the target may be relevant for in vitro experi-ments) Indeed, we show below that, in general, noexplicit enzymatic rate laws can be derived for phos-phorylation and dephosphorylation reactions Third,there are usually no strict concentration hierarchies inphosphorylation modules [i.e target protein, kinase(s)and phosphatase(s)], so that enzymes and their

subtrates may have similar concentrations The low

enzyme concentration is the chief condition for

deriva-tion of Michaelis–Menten-type enzymatic rate laws,

although this can be relaxed in certain cases [93–95]

However, as a rule of thumb, explicit enzymatic rate

laws (Michaelis–Menten or other) can generally not be

derived when the concentrations of the various

enzyme–substrate complexes are appreciable compared

to the free concentrations of substrate and product

This situation is probably common in protein

phos-phorylation networks

For these reason, Michaelis–Menten kinetics are not

an appropriate starting point for studying the kinetic

behaviour of (multisite) phosphorylation modules

[29,82,95], although some authors have used them [32]

Instead, a mathematical description based on

elemen-tary steps of enzyme–substrate binding and catalysis is

appropriate [29,33,82] As an example of how this

for-malism works, Fig 3 (upper box) shows the strictly

sequential mechanism of phosphorylation [29] For

each phosphorylation state, the substrate can occur in

a free form (Xn,0) or in a complex with the kinase

(Xn,K) or phosphatase (Xn,P), where n = 0, … N is the

number of phosphorylated residues (simultaneous

binding of kinases and phosphatases to the target

pro-tein has not been considered here but may also occur)

The dynamic behaviour of all possible complexes andphosphorylation states can be described by a set ofkinetic equations For example, the balance for theunphosphorylated substrate in a binary complex withthe kinase is

ð2Þ

where dkand L0 denote the dissociation rate constantand equilibrium dissociation constant for the binding

of the kinase, a1 is the phosphorylation rate constant

of the first phosphorylation site, and K is the tration of free kinase A model of this type can easily

concen-be solved numerically, but contains a rather largenumber of parameters that need to be specified(6N + 4 when the kinase and phosphatase areassumed to have different binding, dissociation andcatalytic rate constants for each phosphorylationstate)

The model can be simplified by exploiting time-scalehierarchies Perhaps the simplest assumption is thatenzyme–target binding interactions occur more rapidlythan the addition and cleavage of phosphoryl groups,and thus a rapid-equilibrium approximation for kinaseand phosphatase binding can be applied [29,82] Thisapproximation models a distributive mechanism of(de)phosphorylation whereby the enzymes have to bindand dissociate many times before the target protein isfully (de)phosphorylated The system dynamics can beformulated in terms of the total concentration

Yn= Xn,0+ Xn,K+ Xn,P attained by the variousphosphorylated forms Moreover, the number ofparameters is reduced considerably as only the equilib-rium dissociation constants (and no longer the bindingand dissociation rate constants) are needed (Fig 3,lower box) The total concentrations of the phospho-forms Yn are governed by the algebro-differentialequation system

dYn

 ðanþ1þ bnÞYn phosphorylation and

þ bnþ1Ynþ1 dephosphorylation

respectively, and the conservation conditions

Fig 3 Reaction scheme for a multisite protein phosphorylation

module A model based on elementary steps for the sequential

mechanism of phosphorylation is shown in the upper box In each

phosphorylation state, the substrate can occur in a free form (X n,0 )

or in a complex with the kinase (X n,K ) or phosphatase (X n,P ).

Because protein–protein interactions generally occur more rapidly

than catalytic steps, the model can be simplified and the number of

parameters considerably reduced (lower box) See text for more

details.

In general, this system is nonlinear with respect to

the Ynvariables and has no explicit solution except for

special cases [29,82]

Thus comprehensive kinetic models of multisite

phos-phorylation require knowledge of protein

concentra-tions (kinases, phosphatases and substrate) and the

binding and dissociation rate constants for the enzymes

(or at least the Kdvalues), as well as the rate constants

of phosphorylation and dephosphorylation reactions

Large-scale measurements of cellular protein

concentra-tions have been performed (e.g in budding yeast [96]),

and binding affinities (or dissociation constants) have

been determined in some cases [27] Viscosity and

fast-mixing kinetic methods have recently been applied to

dissect the individual steps in substrate phosphorylation

such as substrate binding, product release and catalytic

steps [27,97] One way to address this difficulty may be

to design kinetic experiments that allow simultaneous

fitting of several kinetic parameters (e.g by determining

the time course of substrate phosphorylation forms

combined with dose–response curves, and possibly also

mutations of individual phosphorylation sites)

Sequential versus random phoshorylation order

Analysis of the random mechanism is, in principle,

more complex due to the larger number of

phosphory-lation states, but the same formalism as given for the

sequential scheme applies However, there is an

inter-esting connection with regard to the kinetic description

of random and sequential phosphorylation

mecha-nisms In the special case that the parameters do not

depend on the phosphorylation state of the target

pro-tein (an= a, bn= b, Ln= L, Qn= Q), the random

mechanism can be mapped exactly onto a sequential

one by grouping all n-times phosphorylated target

mol-ecules into a single class regardless of the position of

the phosphorylated residues [29] The concentrations of

these new grouped variables for the random scheme,

Yn, are given by the system of Eqns (3–5) with new

effec-tive rate constants of phosphorylation and

dephosphor-ylation, aranand bran, defined as follows:

aran¼ ðN  n þ 1Þa and bran¼ nb; ð6Þ

where a and b are as given in Eqn (5) Equation (6)

expresses the fact that the effective phosphorylation

rate decreases as the target becomes increasinglyphosphorylated because fewer residues remain avail-able for phosphorylation This is exactly the oppositefor dephosphorylation, and as a result of this rapidphosphorylation of the unphosphorylated target andrapid dephosphorylation of the phosphorylatedtarget, the random mechanism has a tendency toproduce partially phosphorylated forms of the targetprotein

Kinetic and functional implications of various phosphorylation mechanisms

Multisite phosphorylation has been associated withsignal integration, threshold responses, signallingspecificity, precise timing, and other properties Based

on the results of mathematical models, we discusshow these functional implications are related to themechanisms of multisite phosphorylation presentedabove

Graded, switch-like and bi-stable responsesPhosphorylation modules may exhibit a wide variety

of stimulus–response relationships, whereby the lus is usually translated into activity of a kinase (orphosphatase, e.g for the calcineurin⁄ NFAT pathway).Several studies have identified important parametersthat shape the stimulus–response relationship includ-ing: (a) the concentrations of the modifying enzymesrelative to the substrate, (b) the affinities of the modi-fying enzymes for the various phosphorylation states

stimu-of the target and (c) the (cooperative or tive) kinetics of the catalytic steps [28,29,33,82,83].Even when a single phosphorylatable site is involved,changes in these parameters can produce diverseresponses such as graded (or hyperbolic), ultrasensitive(or sigmoidal), and even dual thresholds [82] In partic-ular, when the substrate concentration is so large thatthe enzymes operate near saturation and the kinasereadily dissociates from the phosphorylated target(and, likewise, the phosphatase from the unphosphory-lated target), a steep threshold response, or ‘switch’, isobtained This phenomenon has been termed zero-order ultrasensitivity [98], and has been experimentallyobserved for the phosphorylation of phosphorylaseand isocitrate dehydrogenase [99,100] However, ultra-sensitivity does not occur if the kinase (or phospha-tase) remains sequestered by the phosphorylated(dephosphorylated) substrate [28,82,101]

non-coopera-Compared to a single-site target, multisite ylation expands the possibilities for protein–proteininteractions and the phosphorylation sequence, thus