Reference : Tyrosine-dependent capture of CAP-Gly domain‐containing proteins in complex mixture b...
Scientific journals : Article
Life sciences : Biochemistry, biophysics & molecular biology
Tyrosine-dependent capture of CAP-Gly domain‐containing proteins in complex mixture by EB1 C-terminal peptidic probes
Calligaris, David [Université de Liège - ULg > Département de chimie (sciences) > GIGA-R : Laboratoire de spectrométrie de masse (L.S.M.) >]
Manatschal, Cristina []
Marcellin, Marlène []
Villard, Claude []
Monsarrat, Bernard []
Burlet-Schiltz, Odile []
Steinmetz, Michel O. []
Braguer, Diane []
Lafitte, Daniel []
Verdier-Pinard, Pascal []
Journal of Proteomics
Yes (verified by ORBi)
The Netherlands
[en] 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 cytoskeletonwere 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.

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