title: Multisite phosphorylation disrupts arginine-glutamate salt bridge networks required for binding of cytoplasmic linker-associated protein 2 (CLASP2) to end-binding protein 1 (EB1).
abstract: 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 (31)P 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.
authors.affiliation: Department of Cell and Tissue Biology, University of California, San Francisco, California 94143.
authors.forename: Praveen
authors.initials: P
authors.lastname: Kumar
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Chimenti
authors.affiliation: Department of Cell and Tissue Biology, University of California, San Francisco, California 94143.
authors.forename: Hayley
authors.initials: H
authors.lastname: Pemble
authors.affiliation: Department of Cell and Tissue Biology, University of California, San Francisco, California 94143.
authors.forename: André
authors.initials: A
authors.lastname: Schönichen
authors.affiliation: Department of Cell and Tissue Biology, University of California, San Francisco, California 94143.
authors.forename: Oliver
authors.initials: O
authors.lastname: Thompson
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Jacobson
authors.affiliation: Department of Cell and Tissue Biology, University of California, San Francisco, California 94143. Electronic address: torsten.wittmann@ucsf.edu.
authors.forename: Torsten
authors.initials: T
authors.lastname: Wittmann
mesh_terms: D020816:Amino Acid Motifs; D038362:Glycogen Synthase Kinase 3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008938:Mitosis / Q000502:physiology; D056004:Molecular Dynamics Simulation; D010766:Phosphorylation / Q000502:physiology; D017434:Protein Structure, Tertiary; D055672:Static Electricity
chemical_list: C497851:CLASP2 protein, human; C500985:MAPRE1 protein, human; D008869:Microtubule-Associated Proteins; D038362:Glycogen Synthase Kinase 3