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Multisite Phosphorylation Disrupts Arginine-Glutamate Salt Bridge Networks Required for Binding of Cytoplasmic Linker-associated Protein 2 (CLASP2) to End-binding Protein 1 (EB1)*

Open AccessPublished:March 29, 2012DOI:https://doi.org/10.1074/jbc.M111.316661
      A group of diverse proteins reversibly binds to growing microtubule plus ends through interactions with end-binding proteins (EBs). These +TIPs control microtubule dynamics and microtubule interactions with other intracellular structures. Here, we use cytoplasmic linker-associated protein 2 (CLASP2) binding to EB1 to determine how multisite phosphorylation regulates interactions with EB1. The central, intrinsically disordered region of vertebrate CLASP proteins contains two SXIP EB1 binding motifs that are required for EB1-mediated plus-end-tracking in vitro. In cells, both EB1 binding motifs can be functional, but most of the binding free energy results from nearby electrostatic interactions. By employing molecular dynamics simulations of the EB1 interaction with a minimal CLASP2 plus-end-tracking module, we find that conserved arginine residues in CLASP2 form extensive hydrogen-bond networks with glutamate residues predominantly in the unstructured, acidic C-terminal tail of EB1. Multisite phosphorylation of glycogen synthase kinase 3 (GSK3) sites near the EB1 binding motifs disrupts this electrostatic “molecular Velcro.” Molecular dynamics simulations and 31P NMR spectroscopy indicate that phosphorylated serines participate in intramolecular interactions with and sequester arginine residues required for EB1 binding. Multisite phosphorylation of these GSK3 motifs requires priming phosphorylation by interphase or mitotic cyclin-dependent kinases (CDKs), and we find that CDK- and GSK3-dependent phosphorylation completely disrupts CLASP2 microtubule plus-end-tracking in mitosis.

      Introduction

      Dynamic regulation of the microtubule cytoskeleton is essential for many cell functions. A diverse class of plus-end-tracking proteins, +TIPs, that display a characteristic association with growing microtubule ends in cells has been implicated in the control of intracellular microtubule dynamics, signaling, chromosome segregation, and cell migration (
      • Akhmanova A.
      • Steinmetz M.O.
      Tracking the ends. A dynamic protein network controls the fate of microtubule tips.
      ,
      • Galjart N.
      Plus-end-tracking proteins and their interactions at microtubule ends.
      ,
      • Slep K.C.
      Structural and mechanistic insights into microtubule end-binding proteins.
      ). Most +TIPs do not directly bind to growing microtubule ends but rely on interactions with end-binding proteins (EBs)
      The abbreviations used are: EB
      ending binding
      CLASP2
      cytoplasmic linker-associated protein 2
      GSK3
      glycogen synthase kinase 3
      CDK
      cyclin-dependent kinase
      STLC
      S-trityl-l-cysteine
      R-E
      arginine-glutamate
      R-pS
      arginine-phosphorylated serine
      CDK
      cyclin-dependent kinase
      EGFP
      enhanced GFP
      GMPCPP
      guanylyl(α,β)methylene diphosphonate.
      that have emerged as central components of +TIP protein interaction networks. EBs are small dimeric proteins consisting of an N-terminal calponin homology domain that directly recognizes a structural feature of growing microtubule ends (
      • Hayashi I.
      • Ikura M.
      Crystal structure of the amino-terminal microtubule binding domain of end-binding protein 1 (EB1).
      ,
      • Bieling P.
      • Kandels-Lewis S.
      • Telley I.A.
      • van Dijk J.
      • Janke C.
      • Surrey T.
      CLIP-170 tracks growing microtubule ends by dynamically recognizing composite EB1/tubulin-binding sites.
      ) and a C-terminal EB homology domain that mediates binding to other +TIPs (
      • Honnappa S.
      • John C.M.
      • Kostrewa D.
      • Winkler F.K.
      • Steinmetz M.O.
      Structural insights into the EB1-APC interaction.
      ). A pocket between the two C-terminal helices in EB1 forms a hydrophobic cavity that binds to a short SXIP sequence motif present in most +TIPs (
      • Honnappa S.
      • Gouveia S.M.
      • Weisbrich A.
      • Damberger F.F.
      • Bhavesh N.S.
      • Jawhari H.
      • Grigoriev I.
      • van Rijssel F.J.
      • Buey R.M.
      • Lawera A.
      • Jelesarov I.
      • Winkler F.K.
      • Wüthrich K.
      • Akhmanova A.
      • Steinmetz M.O.
      An EB1 binding motif acts as a microtubule tip localization signal.
      ). In addition, less well characterized electrostatic interactions between positively charged residues near the SXIP motif and negatively charged amino acids near the C terminus of EB1 likely contribute to +TIP binding. Functional SXIP motifs are embedded in intrinsically disordered regions, and the C-terminal tail of EB1 itself is unstructured and not resolved by x-ray crystallography (
      • Honnappa S.
      • Gouveia S.M.
      • Weisbrich A.
      • Damberger F.F.
      • Bhavesh N.S.
      • Jawhari H.
      • Grigoriev I.
      • van Rijssel F.J.
      • Buey R.M.
      • Lawera A.
      • Jelesarov I.
      • Winkler F.K.
      • Wüthrich K.
      • Akhmanova A.
      • Steinmetz M.O.
      An EB1 binding motif acts as a microtubule tip localization signal.
      ). This has made direct structural investigation of the contribution and regulation of potential electrostatic interactions very difficult.
      Except for the presence of one or multiple SXIP motifs, +TIPs are functionally and structurally extremely heterogeneous, and +TIP interactions with EB1 and hence microtubule ends must be spatially and temporally regulated in cells. Phosphorylation has a large impact on net protein charge and is thus a likely regulator of electrostatic interactions (
      • Narayanan A.
      • Jacobson M.P.
      Computational studies of protein regulation by post-translational phosphorylation.
      ). Although there is currently no evidence that mammalian EBs are phosphorylated, phosphorylation has been reported for several +TIPs (
      • Kumar P.
      • Lyle K.S.
      • Gierke S.
      • Matov A.
      • Danuser G.
      • Wittmann T.
      GSK3β phosphorylation modulates CLASP-microtubule association and lamella microtubule attachment.
      ,
      • Näthke I.S.
      The adenomatous polyposis coli protein. The Achilles heel of the gut epithelium.
      ,
      • van der Vaart B.
      • Manatschal C.
      • Grigoriev I.
      • Olieric V.
      • Gouveia S.M.
      • Bjelic S.
      • Demmers J.
      • Vorobjev I.
      • Hoogenraad C.C.
      • Steinmetz M.O.
      • Akhmanova A.
      SLAIN2 links microtubule plus end-tracking proteins and controls microtubule growth in interphase.
      ,
      • Wu X.
      • Shen Q.T.
      • Oristian D.S.
      • Lu C.P.
      • Zheng Q.
      • Wang H.W.
      • Fuchs E.
      Skin stem cells orchestrate directional migration by regulating microtubule-ACF7 connections through GSK3β.
      ,
      • Zhang X.
      • Ems-McClung S.C.
      • Walczak C.E.
      Aurora A phosphorylates MCAK to control ran-dependent spindle bipolarity.
      ,
      • Watanabe T.
      • Noritake J.
      • Kakeno M.
      • Matsui T.
      • Harada T.
      • Wang S.
      • Itoh N.
      • Sato K.
      • Matsuzawa K.
      • Iwamatsu A.
      • Galjart N.
      • Kaibuchi K.
      Phosphorylation of CLASP2 by GSK-3β regulates its interaction with IQGAP1, EB1, and microtubules.
      ). Although it is unclear in most cases whether +TIP phosphorylation regulates microtubule plus-end-tracking in cells, we recently demonstrated that multisite phosphorylation of the EB1 binding region of CLASP2 by GSK3 inhibits EB1 and microtubule binding (
      • Kumar P.
      • Lyle K.S.
      • Gierke S.
      • Matov A.
      • Danuser G.
      • Wittmann T.
      GSK3β phosphorylation modulates CLASP-microtubule association and lamella microtubule attachment.
      ). An intriguingly similar regulation of microtubule binding by GSK3 was later described for the spectraplakin ACF7 (
      • Wu X.
      • Shen Q.T.
      • Oristian D.S.
      • Lu C.P.
      • Zheng Q.
      • Wang H.W.
      • Fuchs E.
      Skin stem cells orchestrate directional migration by regulating microtubule-ACF7 connections through GSK3β.
      ). Both CLASP2 and ACF7 are involved in pathways stabilizing microtubules at the leading edge of migrating cells.
      GSK3α and -β play essential roles in intracellular signaling, neuronal development, regulation of cell migration, cell division, and protein degradation pathways and are responsible for multisite phosphorylation of a large number of proteins (
      • Hur E.M.
      • Zhou F.Q.
      GSK3 signaling in neural development.
      ,
      • Jope R.S.
      • Yuskaitis C.J.
      • Beurel E.
      Glycogen synthase kinase-3 (GSK3). Inflammation, diseases, and therapeutics.
      ). Despite this central importance of GSK3 and its involvement in numerous disease processes, the molecular mechanisms by which multisite phosphorylation regulates protein functions are not well understood. Highly dynamic electrostatic interactions between disordered protein regions are likely central to many intracellular processes (
      • Uversky V.N.
      Multitude of binding modes attainable by intrinsically disordered proteins. A portrait gallery of disorder-based complexes.
      ,
      • Mittag T.
      • Kay L.E.
      • Forman-Kay J.D.
      Protein dynamics and conformational disorder in molecular recognition.
      ), and here we use CLASP2 and EB1 as a model system to investigate how intrinsically disordered proteins can be regulated by multisite phosphorylation.

      DISCUSSION

      Multisite phosphorylation is a common posttranslational modification in eukaryotic cells and adds a layer of combinatorial complexity to intracellular signaling networks. However, the underlying mechanisms by which multisite phosphorylation regulates protein interactions and activity are incompletely understood. GSK3α and -β are ubiquitous protein kinases that are responsible for the majority of sequential multisite phosphorylation in eukaryotic cells (
      • Hur E.M.
      • Zhou F.Q.
      GSK3 signaling in neural development.
      ,
      • Jope R.S.
      • Yuskaitis C.J.
      • Beurel E.
      Glycogen synthase kinase-3 (GSK3). Inflammation, diseases, and therapeutics.
      ). We previously reported that CLASP2, which is involved in microtubule stabilization at the cell cortex in migrating interphase cells, is a substrate of GSK3 phosphorylation and identified two phosphorylation motifs that regulate CLASP2-microtubule interactions (
      • Kumar P.
      • Lyle K.S.
      • Gierke S.
      • Matov A.
      • Danuser G.
      • Wittmann T.
      GSK3β phosphorylation modulates CLASP-microtubule association and lamella microtubule attachment.
      ,
      • Mimori-Kiyosue Y.
      • Grigoriev I.
      • Lansbergen G.
      • Sasaki H.
      • Matsui C.
      • Severin F.
      • Galjart N.
      • Grosveld F.
      • Vorobjev I.
      • Tsukita S.
      • Akhmanova A.
      CLASP1 and CLASP2 bind to EB1 and regulate microtubule plus-end dynamics at the cell cortex.
      ). These GSK3 phosphorylation motifs are in close proximity to SXIP sequence motifs that have recently been identified to mediate +TIP interactions with EB1 (
      • Honnappa S.
      • Gouveia S.M.
      • Weisbrich A.
      • Damberger F.F.
      • Bhavesh N.S.
      • Jawhari H.
      • Grigoriev I.
      • van Rijssel F.J.
      • Buey R.M.
      • Lawera A.
      • Jelesarov I.
      • Winkler F.K.
      • Wüthrich K.
      • Akhmanova A.
      • Steinmetz M.O.
      An EB1 binding motif acts as a microtubule tip localization signal.
      ). This region is nearly identical in CLASP1 and CLASP2 and highly conserved in CLASP proteins from other species (Fig. 1B).
      Because the C terminus of EB1 is highly negatively charged and sequences surrounding SXIP motifs in CLASP2 are interspersed with positively charged arginine residues, electrostatic interactions likely contribute to binding to EB1. However, both the C-terminal tail of EB1 and the region surrounding the CLASP2 SXIP motifs are intrinsically disordered, preventing direct structural analysis of this interaction. Based on molecular dynamics simulations of an EB1-bound CLASP2 model peptide containing one SXIP motif followed by the arginine-rich GSK3 phosphorylation motif, we propose a model of how electrostatic interactions mediate CLASP2-EB1 binding and are perturbed by phosphorylation. Because of the large possible conformational space, molecular dynamics simulations of intrinsically disordered proteins are inherently incomplete (
      • Rauscher S.
      • Pomès R.
      Molecular simulations of protein disorder.
      ). Thus, the simulation outcomes likely represent possible local energy minima and may not completely represent the ensemble at thermodynamic equilibrium. Nevertheless, comparison of multiple simulation runs identified common types of interactions. In all simulations of the non-phosphorylated CLASP2 peptide bound to EB1, conserved arginine residues in CLASP2 formed bidentate salt bridges preferentially with glutamate residues near the unstructured C terminus of EB1. Such coplanar arginine-glutamate (R-E) salt bridges are geometrically and energetically highly favorable (
      • Mandell D.J.
      • Chorny I.
      • Groban E.S.
      • Wong S.E.
      • Levine E.
      • Rapp C.S.
      • Jacobson M.P.
      Strengths of hydrogen bonds involving phosphorylated amino acid side chains.
      ). We find that lysine residues cannot substitute for arginines likely because of the less favorable lysine-glutamate hydrogen bond geometry (
      • Mandell D.J.
      • Chorny I.
      • Groban E.S.
      • Wong S.E.
      • Levine E.
      • Rapp C.S.
      • Jacobson M.P.
      Strengths of hydrogen bonds involving phosphorylated amino acid side chains.
      ), which may explain the strong conservation of these arginines in CLASPs (Fig. 1B). Thus, although hydrophobic interactions of the SXIP motif itself determine the specificity of binding to EB1, molecular Velcro formed by multiple coplanar R-E salt bridges likely contributes significantly to binding strength. Although our molecular dynamics analysis focused on the N-terminal SKIP motif, the sequence around the more C-terminal SRIP motif is very similar, and we obtained qualitatively similar results in molecular dynamics simulations.
      M. Chimenti and T. Wittmann, unpublished results.
      In simulations of EB1-bound phosphorylated CLASP2 peptides, these intermolecular R-E interactions were mostly replaced by intramolecular salt bridges between the arginine residues and phosphorylated serines (R-pS). Such coplanar R-pS salt bridges are predicted to be significantly more favorable than even the bidentate R-E side chain interaction (
      • Mandell D.J.
      • Chorny I.
      • Groban E.S.
      • Wong S.E.
      • Levine E.
      • Rapp C.S.
      • Jacobson M.P.
      Strengths of hydrogen bonds involving phosphorylated amino acid side chains.
      ), and we propose that phosphoserines outcompete R-E interactions. Because our simulations are short on a molecular time-scale and start from an artificial situation in which the phosphorylated CLASP2 peptide is bound to EB1, we did not observe complete dissociation of the interaction. In reality, CLASP2 phosphorylation likely occurs when it is not bound to EB1. We expect that pre-existing intramolecular R-pS salt bridges prevent binding to EB1, which is supported both by our cell and in vitro experiments (FIGURE 2, FIGURE 3). Intramolecular R-pS bridges also rapidly formed in simulations of the phosphorylated CLASP2 peptide alone and are supported by 31P NMR spectroscopy data (Fig. 4). In addition, the formation of similar stable R-pS salt bridges has been proposed in hyperphosphorylated RS dipeptide repeat motifs (
      • Hamelberg D.
      • Shen T.
      • McCammon J.A.
      A proposed signaling motif for nuclear import in mRNA processing via the formation of arginine claw.
      ).
      Both GSK3 kinases recognize a characteristic (S/T)XXX(pS/pT) repeat sequence in which the C-terminal serine or threonine residue has already been phosphorylated by a different priming kinase or by GSK3 itself. Interestingly, our data show that GSK3β phosphorylation motifs do not always conform to this consensus sequence. At least in vitro, additional phosphorylation occurs most likely at Ser-728 because it is N-terminal to the GSK3β phosphorylation motif even though the distance between Ser-733 and Ser-728 does not conform to the consensus. In addition, the absence of phosphorylation intermediates in in vitro GSK3β kinase reactions strongly suggests that phosphorylation by GSK3β proceeds processively, which would indicate a switch-like response of GSK3β-mediated CLASP2 regulation (
      • Salazar C.
      • Höfer T.
      Multisite protein phosphorylation. From molecular mechanisms to kinetic models.
      ).
      The priming sites of the GSK3 phosphorylation motifs in CLASP2 are highly conserved cyclin-dependent kinase consensus motifs. Although phosphorylation of Ser-775 by CDK5 has been reported earlier (
      • Watanabe T.
      • Noritake J.
      • Kakeno M.
      • Matsui T.
      • Harada T.
      • Wang S.
      • Itoh N.
      • Sato K.
      • Matsuzawa K.
      • Iwamatsu A.
      • Galjart N.
      • Kaibuchi K.
      Phosphorylation of CLASP2 by GSK-3β regulates its interaction with IQGAP1, EB1, and microtubules.
      ), we now demonstrate that both priming sites are efficiently phosphorylated by mitotic (cycB/CDK1) and interphase CDKs (p25/CDK5). CLASP2 was also phosphorylated by cycA/CDK2 that is active during the S/G2 transition but not by cycD1/CDK4, which is active in late G1 (
      • Morgan D.O.
      Cyclin-dependent kinases. Engines, clocks, and microprocessors.
      ), indicating potentially complex cell cycle regulation of CLASP2.
      P. Kumar and T. Wittmann, unpublished results.
      Phosphorylation of the same GSK3 motifs regulates CLASP2-microtubule interactions in both interphase and mitosis. In interphase, CLASPs are only partially phosphorylated (
      • Kumar P.
      • Lyle K.S.
      • Gierke S.
      • Matov A.
      • Danuser G.
      • Wittmann T.
      GSK3β phosphorylation modulates CLASP-microtubule association and lamella microtubule attachment.
      ), yet we find that CDK5 phosphorylates both priming sites equally well. CDK5 is thought to be mostly functional in neuronal cells (
      • Jessberger S.
      • Gage F.H.
      • Eisch A.J.
      • Lagace D.C.
      Making a neuron. Cdk5 in embryonic and adult neurogenesis.
      ). Thus, a different kinase may phosphorylate Ser-775 in other interphase cells, or competing phosphatase activity could result in partial dephosphorylation of the GSK3 motifs.
      In contrast, in mitosis CDK1- and GSK3-dependent phosphorylation globally switches off CLASP2 plus-end-tracking. In mammalian cells, CLASPs are localized to kinetochores independent of microtubules. CLASPs may regulate the dynamics of kinetochore microtubules, and CLASP depletion results in mitotic defects (
      • Maiato H.
      • Fairley E.A.
      • Rieder C.L.
      • Swedlow J.R.
      • Sunkel C.E.
      • Earnshaw W.C.
      Human CLASP1 is an outer kinetochore component that regulates spindle microtubule dynamics.
      ,
      • Pereira A.L.
      • Pereira A.J.
      • Maia A.R.
      • Drabek K.
      • Sayas C.L.
      • Hergert P.J.
      • Lince-Faria M.
      • Matos I.
      • Duque C.
      • Stepanova T.
      • Rieder C.L.
      • Earnshaw W.C.
      • Galjart N.
      • Maiato H.
      Mammalian CLASP1 and CLASP2 cooperate to ensure mitotic fidelity by regulating spindle and kinetochore function.
      ,
      • Mimori-Kiyosue Y.
      • Grigoriev I.
      • Sasaki H.
      • Matsui C.
      • Akhmanova A.
      • Tsukita S.
      • Vorobjev I.
      Mammalian CLASPs are required for mitotic spindle organization and kinetochore alignment.
      ,
      • Hannak E.
      • Heald R.
      Xorbit/CLASP links dynamic microtubules to chromosomes in the Xenopus meiotic spindle.
      ,
      • Maffini S.
      • Maia A.R.
      • Manning A.L.
      • Maliga Z.
      • Pereira A.L.
      • Junqueira M.
      • Shevchenko A.
      • Hyman A.
      • Yates 3rd, J.R.
      • Galjart N.
      • Compton D.A.
      • Maiato H.
      Motor-independent targeting of CLASPs to kinetochores by CENP-E promotes microtubule turnover and poleward flux.
      ). GSK3 activity is also required for accurate chromosome segregation (
      • Tighe A.
      • Ray-Sinha A.
      • Staples O.D.
      • Taylor S.S.
      GSK-3 inhibitors induce chromosome instability.
      ), and it is thus likely that mitotic CLASP function depends on highly localized regulation of kinetochore-associated CLASP phosphorylation as CLASPs would need to be dephosphorylated to mediate kinetochore-microtubule interactions (
      • Welburn J.P.
      • Vleugel M.
      • Liu D.
      • Yates 3rd, J.R.
      • Lampson M.A.
      • Fukagawa T.
      • Cheeseman I.M.
      Aurora B phosphorylates spatially distinct targets to differentially regulate the kinetochore-microtubule interface.
      ). Mitotic inhibition of microtubule plus-end-tracking has also been observed for other +TIPs such as SLAIN2 and Kebab, although phosphorylation sites involved have not been identified (
      • van der Vaart B.
      • Manatschal C.
      • Grigoriev I.
      • Olieric V.
      • Gouveia S.M.
      • Bjelic S.
      • Demmers J.
      • Vorobjev I.
      • Hoogenraad C.C.
      • Steinmetz M.O.
      • Akhmanova A.
      SLAIN2 links microtubule plus end-tracking proteins and controls microtubule growth in interphase.
      ,
      • Meireles A.M.
      • Dzhindzhev N.S.
      • Ohkura H.
      Kebab. Kinetochore and EB1 associated basic protein that dynamically changes its localization during Drosophila mitosis.
      ). The functional relevance of mitotic phosphorylation of these proteins remains to be determined.
      In conclusion, phosphoregulation of +TIPs is likely common and may explain the abundance of arginine residues around SXIP motifs in CLASP proteins and other +TIPs. Spectraplakins contain GSK3 phosphorylation motifs in an arginine-rich region near a functional SXIP motif at the C terminus (
      • Honnappa S.
      • Gouveia S.M.
      • Weisbrich A.
      • Damberger F.F.
      • Bhavesh N.S.
      • Jawhari H.
      • Grigoriev I.
      • van Rijssel F.J.
      • Buey R.M.
      • Lawera A.
      • Jelesarov I.
      • Winkler F.K.
      • Wüthrich K.
      • Akhmanova A.
      • Steinmetz M.O.
      An EB1 binding motif acts as a microtubule tip localization signal.
      ,
      • Wu X.
      • Shen Q.T.
      • Oristian D.S.
      • Lu C.P.
      • Zheng Q.
      • Wang H.W.
      • Fuchs E.
      Skin stem cells orchestrate directional migration by regulating microtubule-ACF7 connections through GSK3β.
      ), and phosphorylation by Aurora B kinase in an arginine-rich region inhibits plus-end-tracking of the mitotic centromere-associated kinesin (MCAK) (
      • Moore A.T.
      • Rankin K.E.
      • von Dassow G.
      • Peris L.
      • Wagenbach M.
      • Ovechkina Y.
      • Andrieux A.
      • Job D.
      • Wordeman L.
      MCAK associates with the tips of polymerizing microtubules.
      ). Interestingly, GSK3 phosphorylation also directly inhibits EB1-independent microtubule binding of CLASP2 and ACF7 (
      • Kumar P.
      • Lyle K.S.
      • Gierke S.
      • Matov A.
      • Danuser G.
      • Wittmann T.
      GSK3β phosphorylation modulates CLASP-microtubule association and lamella microtubule attachment.
      ,
      • Wu X.
      • Shen Q.T.
      • Oristian D.S.
      • Lu C.P.
      • Zheng Q.
      • Wang H.W.
      • Fuchs E.
      Skin stem cells orchestrate directional migration by regulating microtubule-ACF7 connections through GSK3β.
      ), and the acidic C terminus of EB1 is highly similar to the C-terminal tail of most α/β tubulin isoforms (
      • Komarova Y.
      • Lansbergen G.
      • Galjart N.
      • Grosveld F.
      • Borisy G.G.
      • Akhmanova A.
      EB1 and EB3 control CLIP dissociation from the ends of growing microtubules.
      ). In addition, most microtubule-associated proteins contain large intrinsically disordered regions. Microtubule binding often depends on the acidic tubulin tail and is inhibited by phosphorylation (
      • Cassimeris L.
      • Spittle C.
      Regulation of microtubule-associated proteins.
      ). Thus, molecular mechanisms similar to that proposed here for regulation by multisite phosphorylation may also apply to other microtubule-binding proteins. Furthermore, it has become increasingly evident that a large percentage of the proteome does not form stably folded domain structures but exists in an unfolded, intrinsically disordered state, and multisite phosphorylation often occurs in such disordered regions (
      • Pearlman S.M.
      • Serber Z.
      • Ferrell Jr., J.E.
      A mechanism for the evolution of phosphorylation sites.
      ). The strong electrostatic perturbation introduced by multiple phosphate residues provides one mechanism for regulating such interactions by perturbing favorable but potentially nonspecific electrostatic interactions involving unstructured peptides.

      Acknowledgments

      We thank Peter Bieling for advice on the plus-end-tracking assay, Colin Smith and Cristina Melero for helpful discussions and assistance with fluorescence polarization assays, and Mark Kelly for assistance with NMR spectroscopy. This research was conducted in part in a facility constructed with support from Research Facilities Improvement Program Grant C06 RR16490 from the National Center for Research Resources of the National Institutes of Health.

      Supplementary Material

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