REVIEW ARTICLE 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 altered enzymatic and⁄ or interaction properties may transmit signals in various ways. For example, protein kinases activated by phosphorylation can themselves phosphor- ylate target proteins (e.g. receptor ⁄ receptor-associated tyrosine 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 Fax: +49 6221 54 51487 Tel: +49 6221 54 51380 E-mail: t.hoefer@dkfz-heidelberg.de (Received 15 January 2009, revised 4 March 2009, accepted 27 March 2009) doi:10.1111/j.1742-4658.2009.07027.x Multisite phosphorylation is an important mechanism for fine-tuned regula- tion of protein function. Mathematical models developed over recent years have contributed to elucidation of the functional consequences of a variety of molecular mechanisms involved in processing of the phosphorylation sites. Here we review the results of such models, together with salient experimental findings on multisite protein phosphorylation. We discuss how molecular mechanisms that can be distinguished with respect to the order and processivity of phosphorylation, as well as other factors, regulate changes in the sensitivity and kinetics of the response, the synchronization of molecular events, signalling specificity, and other functional implications. 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. FEBS Journal 276 (2009) 3177–3198 ª 2009 The Authors Journal compilation ª 2009 FEBS 3177 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 phosphorylation (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 in HeLa cells), followed by threonine (12%) and tyrosine phosphorylations (2%) [7]. With respect to kinetics, tyrosine phosphorylations generally occur faster during cell signalling than serine ⁄ threonine phosphorylations. For example, upon addition of epidermal growth factor (EGF) to HeLa cells, most tyrosines become phosphorylated within 1 min, while threonine and serine phosphorylations require up to 10 min [7]. Compared to phosphorylation of a single residue, multisite phosphorylation increases the possibilities for regulating protein function very considerably. A protein with N phosphorylation sites can exist in 2 N phosphory- lation states. Each such state may have a different func- tional characteristic. For example, the Src family kinases have at least two regulatory Tyr phosphoryla- tion sites, one activating and the other inhibitory, so that there are four (2 2 ) different phosphorylation states of these residues. Accordingly, Src kinases may exist in several distinct states of enzymatic activity (additionally depending on protein–protein interactions, some of which are also governed by phosphorylation) [14]. On the other hand, for larger N, the number of possible states becomes so high that it is unlikely that each one has specific functional properties (e.g. for N = 10, there are 1024 phosphorylation states). The reduction of such high-dimensional phosphorylation state spaces to a smaller number of functional states may occur on two levels. First, the molecular mechanisms of phosphoryla- tion may realise only a subset of the possible states. For example, for a strictly sequential phosphorylation mech- anism (and reverse-order dephosphorylation), there are only N + 1 phosphorylation states instead of 2 N . Sec- ond, several individual phosphorylation sites may coop- erate in effecting a functional outcome (e.g. through a conformational change), such that it is primarily the number of phosphorylated sites that counts rather than their specific location. Both types of dimensionality- reduction mechanisms do indeed occur in protein phosphorylation, as detailed below. Nevertheless the occurrence of many phosphorylation states (especially in random phosphorylation ⁄ dephosphorylation mecha- nisms) is an important factor shaping both dose– response curves and kinetics. These rather basic considerations already make it clear that in-depth analysis of the mechanisms and functions of multisite protein phosphorylation requires mathematical modelling. Both general mathematical analyses of multisite phosphorylation [28–36] and models of specific systems [12,13,37–46] have bee pub- lished in recent years. Here we review these theoretical developments within the context of salient experi- Multisite protein phosphorylation C. Salazar and T. Ho ¨ fer 3178 FEBS Journal 276 (2009) 3177–3198 ª 2009 The Authors Journal compilation ª 2009 FEBS 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 by calcineurin [23]. In NFAT1, dephosphorylation of the SRR1 motif (and possibly also of the SP2 and SP3 motifs) induces exposure of a nuclear localization sequence (NLS), promoting nuclear import of NFAT. Full dephosphorylation is needed for maximal DNA binding of NFAT. Dephosphorylation of NFAT by cal- cineurin is counteracted by several kinases, among them CK1, GSK3 and dual-specificity tyrosine-regulated kinases (DYRKs). Experiments suggest the existence of a preferential order of phosphorylation and dephos- phorylation. DYRKs phosphorylate the SP3 motif, thus 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. C. Salazar and T. Ho ¨ fer Multisite protein phosphorylation FEBS Journal 276 (2009) 3177–3198 ª 2009 The Authors Journal compilation ª 2009 FEBS 3179 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 G 1 ⁄ 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 G 1 phase before binding to the SCF Cdc4 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 SCF Cdc4 binding. Any combination of six phosphorylated sites is suffi- cient for Sic1 degradation. While singly phosphory- lated Sic1 binds to SCF Cdc4 very weakly, multiply phosphorylated Sic1 can bind efficiently, presumably by increasing the local concentration of interaction sites around the SCF Cdc4 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 anti- bodies to known phosphorylation sites. Radionucleo- tide incorporation experiments may also provide accurate information about phosphorylation kinetics [27], but are time-consuming to perform. Mass spec- trometry allows both large-scale analysis and the identification of novel phosphorylation sites and phos- phoproteins not previously known to be involved in cellular signalling [7,8,57]. Information about phos- phorylation sites obtained in large-scale screens has been incorporated into searchable databases such as Phosphosite (http://www.phosphosite.org), Swiss-Prot (http://us.expasy.org/sprot) and Phospho.ELM (http:// phospho.elm.eu.org). Mass spectrometric data for protein phosphorylation may be very useful for kinetic analysis and modelling, although rather few applica- tions exist to date (e.g. [7, 23]). Time-resolved high- resolution NMR spectroscopy has been used recently to study mechanistic questions regarding multisite pro- tein phosphorylation [58,59]. We discuss below which type of data are required to establish kinetic models. Molecular mechanisms of multisite phosphorylation The presence of multiple phosphorylation sites raises new mechanistic questions compared to the case of sin- gle phosphorylation. These pertain to (a) the order in which individual sites are phosphorylated and (b) the number of enzyme binding events required. A third mechanistic aspect, which is relevant both for single- and multisite phosphorylation, is whether the counteracting kinase(s) and phosphatase(s) compete for binding to the target protein. We also discuss how cooperativity can arise in multiply phosphorylated proteins, and the role played by subcellular compart- mentalization. Order of phospho-site processing The order in which phosphorylation sites in a protein are acted on by kinases and phosphatases determines the possible phosphorylation states (Fig. 2A). Although it has generally been difficult to obtain such information experimentally at the required resolution, inferences have been drawn regarding the order of phospho-site processing in several cases. Sequential phosphorylation has been suggested for several kinas- es, especially Ser ⁄ Thr kinases [60–68]. When dephos- phorylation also follows a fixed order, strictly sequential or cyclic mechanisms of phosphorylation arise, depending on whether the last site to be phos- phorylated is the first, or the last, to be dephosphoryl- Multisite protein phosphorylation C. Salazar and T. Ho ¨ fer 3180 FEBS Journal 276 (2009) 3177–3198 ª 2009 The Authors Journal compilation ª 2009 FEBS 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 mechanisms are 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 has been 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 fol- 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. C. Salazar and T. Ho ¨ fer Multisite protein phosphorylation FEBS Journal 276 (2009) 3177–3198 ª 2009 The Authors Journal compilation ª 2009 FEBS 3181 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 (2 N ) 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 mecha- 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 be enhanced by) nearby residues phosphorylated by another kinase (priming kinase). Phosphorylation of the serine S or threonine T in the (S/T)XXX(Sp ⁄ Tp) motif by the kinase GSK3 requires priming by another kinase that phosphorylates the Sp ⁄ Tp site [60–62]. In a sequence of appropriately spaced serines, only the first may need to be primed, while the remaining are then sequentially phosphorylated by GSK3. Priming phosphorylation facilitates the binding of a second kinase either by creating specific docking sites, chang- ing the substrate conformation, or dislodging the sub- strate from the cell membrane [65–69]. An interesting example of such a dual-enzyme mechanism is found in the canonical Wnt ⁄ b-catenin pathway, where sequen- tial phosphorylations of the Wnt co-receptor lipo- protein receptor-related protein 6 (LRP6) and the transcriptional cofactor b-catenin by the kinases GSK3 and 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. Enzyme Consensus sequences Docking motifs Other characteristics Ser ⁄ Thr kinases Calmodulin-dependent protein kinase II (CaMKII) RXX(S ⁄ T)– – Casein kinase 1 (CK1) (Sp/Tp)XX(S ⁄ T) – Primed substrate (D ⁄ E)XX(S ⁄ T)– – Casein kinase 2 (CK2) (S ⁄ T)XX(Sp ⁄ Tp) – Primed substrate (S ⁄ T)XX(D/E) – – Glycogen synthase kinase 3 (GSK3) (S/T)XXX(Sp ⁄ Tp) – Primed substrate Protein kinase B (PKB ⁄ Akt) RXRXX(S ⁄ T)– – Protein kinase C (PKC) (S ⁄ T)X(K ⁄ R) – – Tyr kinases EGF receptor kinase X(D ⁄ E)YX– – Abl tyrosine kinase (I ⁄ V ⁄ L)YXX(P ⁄ F) – SH2 domain Ser ⁄ Thr phosphatases Dual-specificity protein phosphatase 6 (DUSP6) TpXYp – – PP1 – RVXF FXXRXR – PP2A, PP2C RRA(Sp ⁄ Tp)VA – – Calcineurin (PP2B) – PXIXIT – Tyr phosphatases PTP1B E(Y ⁄ F ⁄ D)Yp RDXYXTDYYpR –– SHP2 YpASI YpIDL – SH2 domain Amino acids are indicated by the one-letter code; X indicates any amino acid; Sp, Tp and Yp indicate phosphoserine, phosphothreonine and phosphotyrosine, respectively. Interchangeable residues at a given position are grouped within parentheses, and separated by forward slashes. The target residues are in bold. Multisite protein phosphorylation C. Salazar and T. Ho ¨ fer 3182 FEBS Journal 276 (2009) 3177–3198 ª 2009 The Authors Journal compilation ª 2009 FEBS 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 catalytic 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 2. Enzyme processivity and order of phospho-site processing for some substrates. ASF/SF2, alternative splicing factor; ATF2, activating transcription factor 2; CDK, cyclin dependent kinase; MEK, MAPK/ERK kinase; MKP3, mitogen-activated protein kinase phosphatase 3; SRPK, serine-arginine-rich protein kinase. Substrate name Type of substrate Enzyme name (phosphorylated sites) Type of enzyme Order of phospho-site processing Enzyme processivity Other characteristics Reference b-catenin Transcription cofactor CK1 (Ser45) GSK3 (Thr41, Ser37, Ser33) Ser ⁄ Thr kinases Sequential phosphorylation (dual-kinase) ? – [130,131] ERK2 MAP kinase MEK (Thr183,Tyr185) Thr ⁄ Tyr kinase Random phosphorylation Distributive phosphorylation – [41,70] MKP3 (Thr183,Tyr185) Dual specificity (Thr ⁄ Tyr) phosphatase Sequential dephosphorylation Distributive dephosphorylation – [71] ATF2 Transcription factor p38 (Thr69, Thr71) Ser ⁄ Thr kinase Random phosphorylation Distributive phosphorylation – [46] ASF ⁄ SF2 Splicing factor SRPK1 (10 Ser sites) Clk ⁄ Sty (20 Ser sites) Ser kinase Sequential phosphorylation Processive phosphorylation Stable kinase- substrate complex [27,64,87] p130Cas Focal adhesion protein Scr (15 repeats of YXXP motif) Tyr kinase Random phosphorylation Processive phosphorylation SH3 domain [7,73] RNA polymerase II – Abl (25–52 repeats of YSPTSPS motif) Tyr kinase ? Processive phosphorylation SH2 domain [25,132,133] Pho4 Transcription factor a Pho80 ⁄ Pho85 (five Ser sites) Ser/Thr kinase Sequential phosphorylation Semi-processive phosphorylation – [22,76] Sic1 CDK inhibitor a Cdc28–Cln1,2 (nine Ser ⁄ Thr sites) Ser ⁄ Thr kinase Random phosphorylation Distributive phosphorylation – [18,51] a cyclin-CDK complex. C. Salazar and T. Ho ¨ fer Multisite protein phosphorylation FEBS Journal 276 (2009) 3177–3198 ª 2009 The Authors Journal compilation ª 2009 FEBS 3183 next site before it dissociates is k cat ⁄ (k cat + k off ), where k off and k cat are 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 P processive ¼ k cat k cat þ k off  N ð1Þ (assuming, for simplicity, that all the sites have the same k cat and are modified sequentially). Indeed, k cat 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 k cat can 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 K d 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 phos- phorylation, while distributive enzymes may process the phosphorylation sites in a random manner. For example, the intermolecular autophosphorylation of several Tyr residues in the fibroblast growth factor receptor 1 kinase apparently proceeds in a sequential and processive manner [80]. Dual phosphorylation of extracellular regulated kinase (ERK) by MEK in the MAP kinase cascade was reported to occur via a ran- dom and distributive mechanism [41,70]. However, a processive kinase can also catalyse random phosphory- lations, as recently proposed for phosphorylation of the focal adhesion protein p130Cas by Scr kinase [81]. Conversely, sequential DUSP6 dephosphorylation of ERK2 at Thr and Tyr was shown to occur distribu- tively [71]. Thus there appears to be no strict link between the degree of processivity of a kinase and random or sequential phosphorylation of its multiple target sites. The phosphorylation order and enzyme processivity of some relevant proteins are listed in Table 2. Competition mechanisms The interactions between the target protein and its modifying enzymes can lead to two distinct types of competition effects (Fig. 2C). The binding affinities of kinases and phosphatases may change with the phos- phorylation state of the target protein. For example, the fully phosphorylated target may lose (or retain) its affinity for the kinase. Such affinity changes may lead to interesting effects when the concentration of the kinase is much smaller than that of the target protein [28–30,82,83]. In this case, target proteins of various phosphorylation states compete for the kinase (or, equally, for the phosphatase). When the kinase remains associated with the higher or fully phosphory- lated forms of its target protein, product inhibition will result, because the bound kinase is not available to act on unphosphorylated target molecules. Conversely, when the concentrations of the modify- ing enzymes [kinase(s) and phosphatase(s)] are large compared to their target protein, as may be the case in signal transduction, the enzymes can compete for bind- ing to the target. Phosphorylation is then inhibited by the phosphatase and dephosphorylation by the kinase. In particular, when the kinase has a high affinity for the phosphorylated target, the latter is sequestered and is not available for dephosphorylation. The structural basis for such competition may involve overlapping binding sites for kinases and phosphatases on the tar- get, such that they are unable to bind to the target at the same time [84]. Multisite protein phosphorylation C. Salazar and T. Ho ¨ fer 3184 FEBS Journal 276 (2009) 3177–3198 ª 2009 The Authors Journal compilation ª 2009 FEBS 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 the splicing factor ASF ⁄ SF2 [27,87,88]. A docking motif in ASF ⁄ SF2 restricts its phosphorylation by SRPK1 to the N-terminal half (approximately 10 sites) of the RS domain, mediating nuclear import of ASF ⁄ SF2 and localization 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 the plasma membrane to the nucleus. For example, in rest- ing cells, the NFAT phosphatase calcineurin resides predominantly in the cytoplasm, but upon cell stimula- tion may be imported into the nucleus together with NFAT to maintain NFAT dephosphorylation and nuclear localization [89,90]. The NFAT kinases GSK3 and CK1, which phosphorylate the SP2 and SRR1 motifs, respectively, are present in both subcellular compartments. However, DYRK2 and DYRK1A, which phosphorylate the SP3 motif, are cytoplasmic and nuclear, respectively [50]. DYRK2 probably helps to maintain the phosphorylated state of cytoplasmic NFAT in resting cells, whereas DYRK1A re-phospho- rylates nuclear NFAT and promotes its export from the nucleus. Such compartmentalization of kinases or phosphatases confers different functions, and, in turn, may expand the repertoire for regulating signal trans- duction networks. Kinetic modelling of multisite phosphorylation General framework Kinetic models of multisite protein phosphorylation are quite distinct from those of traditional enzyme kinetics [91,92] for several reasons. First, the number of molecular states to be accounted for is usually larger (including partially phosphorylated states, both enzyme-bound and free, and, where appropriate, vari- ous conformations of the protein due to its phosphory- lation state). Second, and more importantly, the simultaneous presence of kinases and phosphatases needs to be considered in a physiological context, so that there are at least two counteracting enzymes in the system (although consideration of a single enzyme acting on the target may be relevant for in vitro experi- ments). Indeed, we show below that, in general, no explicit enzymatic rate laws can be derived for phos- phorylation and dephosphorylation reactions. Third, there are usually no strict concentration hierarchies in phosphorylation modules [i.e. target protein, kinase(s) and phosphatase(s)], so that enzymes and their C. Salazar and T. Ho ¨ fer Multisite protein phosphorylation FEBS Journal 276 (2009) 3177–3198 ª 2009 The Authors Journal compilation ª 2009 FEBS 3185 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 (X n,0 ) or in a complex with the kinase (X n,K ) or phosphatase (X n,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 and phosphorylation states can be described by a set of kinetic equations. For example, the balance for the unphosphorylated substrate in a binary complex with the kinase is dX 0;K dt ¼ d k K L 0 X 0;0 À X 0;K  reversible binding of kinase À a 1 X 0;K phosphorylation ð2Þ where d k and L 0 denote the dissociation rate constant and equilibrium dissociation constant for the binding of the kinase, a 1 is the phosphorylation rate constant of the first phosphorylation site, and K is the concen- tration of free kinase. A model of this type can easily be solved numerically, but contains a rather large number of parameters that need to be specified (6N + 4 when the kinase and phosphatase are assumed to have different binding, dissociation and catalytic rate constants for each phosphorylation state). The model can be simplified by exploiting time-scale hierarchies. Perhaps the simplest assumption is that enzyme–target binding interactions occur more rapidly than the addition and cleavage of phosphoryl groups, and thus a rapid-equilibrium approximation for kinase and phosphatase binding can be applied [29,82]. This approximation models a distributive mechanism of (de)phosphorylation whereby the enzymes have to bind and dissociate many times before the target protein is fully (de)phosphorylated. The system dynamics can be formulated in terms of the total concentration Y n = X n,0 + X n,K + X n,P attained by the various phosphorylated forms. Moreover, the number of parameters is reduced considerably as only the equilib- rium dissociation constants (and no longer the binding and dissociation rate constants) are needed (Fig. 3, lower box). The total concentrations of the phospho- forms Y n are governed by the algebro-differential equation system dY n dt ¼ a n Y nÀ1 phosphorylation of Y nÀ1 Àða nþ1 þ b n ÞY n phosphorylation and dephosphorylation of Y n þ b nþ1 Y nþ1 dephosphorylation of Y nþ1 ; for 0 n N ð3Þ with effective rates of phosphorylation and dephos- phorylation of a n ¼ a n K=L nÀ1 1 þ K=L nÀ1 þ P=Q nÀ1 and b n ¼ b n P=Q n 1 þ K=L n þ P=Q n ; ð4Þ 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. Multisite protein phosphorylation C. Salazar and T. Ho ¨ fer 3186 FEBS Journal 276 (2009) 3177–3198 ª 2009 The Authors Journal compilation ª 2009 FEBS [...]... to a single-site target, multisite phosphorylation expands the possibilities for protein protein interactions and the phosphorylation sequence, thus FEBS Journal 276 (2009) 317 7–3 198 ª 2009 The Authors Journal compilation ª 2009 FEBS 3187 Multisite protein phosphorylation C Salazar and T Hofer ¨ Fig 4 Mechanistic effects of multisite phosphorylation on the dose–response curves and phosphorylation kinetics... Sudarsanam S (2002) The protein kinase complement of the human genome Science 298, 191 2–1 934 4 Yang XJ (2005) Multisite protein modification and intramolecular signaling Oncogene 24, 165 3–1 662 5 Cohen P (2000) The regulation of protein function by multisite phosphorylation – a 25 year update Trends Biochem Sci 25, 59 6–6 01 3194 6 Holmberg CI, Tran SEF, Eriksson JE & Sistonen L (2002) Multisite phosphorylation. .. substrate degradation Cell 124, 8 9–1 03 54 Morton S, Davis RJ, McLaren A & Cohen P (2003) A reinvestigation of the multisite phosphorylation of the transcription factor c-Jun EMBO J 22, 387 6–3 886 55 Gembitsky DS, Lawlor K, Jacovina A, Yaneva M & Tempst P (2004) A prototype antibody microarray platform to monitor changes in protein tyrosine phosphorylation Mol Cell Proteomics 3, 110 2–1 118 56 Sachs K, Perez O,... unstructured proteins and their functions Nat Rev Mol Cell Biol 6, 19 7–2 08 113 Verkhivker GM (2005) Protein conformational transitions coupled to binding in molecular recognition of unstructured proteins: deciphering the effect of intermolecular interactions on computational structure prediction of the p27Kip1 protein bound to the cyclin A–cyclin-dependent kinase 2 complex Proteins 58, 70 6–7 16 114 Gunasekaran... forms of the target protein Kinetic and functional implications of various phosphorylation mechanisms Multisite phosphorylation has been associated with signal integration, threshold responses, signalling specificity, precise timing, and other properties Based on the results of mathematical models, we discuss how these functional implications are related to the mechanisms of multisite phosphorylation presented... events by multisite phosphorylation (A) Redundance Phosphorylation at any site is sufficient for protein activation (B) Summation Phosphorylation of each site has an additive effect on the protein activity (C) Synergy Phosphorylation of both sites is required for protein activation (D) Antagonism One phosphorylation may enhance and another inhibit the protein activity stability of the substrate–kinase... of Enzyme Kinetics, 3rd edn Portland Press, London Tzafriri AR (2003) Michaelis–Menten kinetics at high enzyme concentrations Bull Math Biol 65, 111 1–1 129 Multisite protein phosphorylation 94 Schnell S & Maini PK (2000) Enzyme kinetics at high enzyme concentration Bull Math Biol 62, 48 3–4 99 95 Ciliberto A, Capuani F & Tyson JJ (2007) Modeling networks of coupled enzymatic reactions using the total quasi-steady... by the MAP kinase cascade, the phosphoinositol-3-kinase ⁄ Akt pathway and the cAMP pathway Any phosphorylation is enough to trigger dissociation of BAD from the anti-apoptotic protein Bcl-XL, inhibiting the pro-apoptotic activity of BAD [123] In other cases, the effect of multisite phosphorylation on the protein activity is additive (Fig 8B) For instance, phosphorylation of two distinct sites in cyclic... kinase scaffold protein Ste5 Multisite protein phosphorylation to the bc G -protein subunit at the plasma membrane, where it was assumed that each phosphorylation decreased the binding energy by 1.4 kcal mol)1 (Kd is increased by a factor of 10) [116] Generally, the degree of ultrasensitivity depends both on the number of phosphorylation sites and the change in binding affinity with each phosphorylation. .. (2000) Cell signaling by receptor tyrosine kinases Cell 103, 21 1–2 25 10 Acuto O, Bartolo VD & Michel F (2008) Tailoring T-cell receptor signals by proximal negative feedback mechanisms Nat Rev Immunol 8, 69 9–7 12 11 Mohapatra DP, Park KS & Trimmer JS (2002) Dynamic regulation of the voltage-gated Kv2.1 potassium channel by multisite phosphorylation Biochem Soc Trans 35, 106 4–1 068 12 Markevich NI, Hoek . REVIEW ARTICLE Multisite protein phosphorylation – from molecular mechanisms to kinetic models Carlos Salazar and Thomas Ho ¨ fer Research. (DUSP6) TpXYp – – PP1 – RVXF FXXRXR – PP2A, PP2C RRA(Sp ⁄ Tp)VA – – Calcineurin (PP2B) – PXIXIT – Tyr phosphatases PTP1B E(Y ⁄ F ⁄ D)Yp RDXYXTDYYpR – SHP2 YpASI YpIDL –