title: Tyrosine-dependent capture of CAP-Gly domain-containing proteins in complex mixture by EB1 C-terminal peptidic probes.
abstract: Microtubule dynamics is regulated by an array of microtubule associated proteins of which the microtubule plus-end tracking proteins (+TIPs) are prominent examples. +TIPs form dynamic interaction networks at growing microtubule ends in an EB1-dependent manner. The interaction between the C-terminal domain of EB1 and the CAP-Gly domains of the +TIP CLIP-170 depends on the last tyrosine residue of EB1. In the present study, we generated peptidic probes corresponding to the C-terminal tail of EB1 to affinity-capture binding partners from cell lysates. Using an MS-based approach, we showed that the last 15 amino-acid residues of EB1, either free or immobilized on beads, bound recombinant CAP-Gly domains of CLIP-170. We further demonstrate that this binding was prevented when the C-terminal tyrosine of EB1 was absent in the peptidic probes. Western blotting in combination with a label-free quantitative proteomic analysis revealed that the peptidic probe harboring the C-terminal tyrosine of EB1 effectively pulled-down proteins with CAP-Gly domains from endothelial cell extracts. Additional proteins known to interact directly or indirectly with EB1 and the microtubule cytoskeleton were also identified. Our peptidic probes represent valuable tools to detect changes induced in EB1-dependent +TIP networks by external cues such as growth factors and small molecules.
authors.affiliation: Inserm UMR 911, Centre de Recherche en Oncologie biologique et en Oncopharmacologie 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France.
authors.forename: David
authors.initials: D
authors.lastname: Calligaris
authors.forename: Cristina
authors.initials: C
authors.lastname: Manatschal
authors.forename: Marlène
authors.initials: M
authors.lastname: Marcellin
authors.forename: Claude
authors.initials: C
authors.lastname: Villard
authors.forename: Bernard
authors.initials: B
authors.lastname: Monsarrat
authors.forename: Odile
authors.initials: O
authors.lastname: Burlet-Schiltz
authors.forename: Michel O
authors.initials: MO
authors.lastname: Steinmetz
authors.forename: Diane
authors.initials: D
authors.lastname: Braguer
authors.forename: Daniel
authors.initials: D
authors.lastname: Lafitte
authors.forename: Pascal
authors.initials: P
authors.lastname: Verdier-Pinard
mesh_terms: D001665:Binding Sites; D002460:Cell Line; D045424:Complex Mixtures; D042783:Endothelial Cells / Q000378:metabolism*; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000737:chemistry*; D015335:Molecular Probes / Q000737:chemistry*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D017434:Protein Structure, Tertiary; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D045424:Complex Mixtures; C405804:EB1 microtubule binding proteins; D008869:Microtubule-Associated Proteins; D015335:Molecular Probes; D014443:Tyrosine