The PN2-3 Domain of Centrosomal P4.1-associated Protein Implements a Novel Mechanism for Tubulin Sequestration

• DOI: 10.1074/jbc.m808249200
• Publication Date: 2009-01-09T01:13:59Z
• Authors: Anthony Cormier; Marie-Jeanne Clément; Marcel Knossow; Sylvie Lachkar; Philippe Savarin; Flavio Toma; André Sobel; Benoît Gigant; Patrick A. Curmi
• Keywords: 0301 basic medicine; 570; [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM]; Molecular Biology/Biochemistry [q-bio.BM]; [SDV.BBM.BP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics; [SDV.BC]Life Sciences [q-bio]/Cellular Biology; Spindle Apparatus; [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry; 03 medical and health sciences; Tubulin; Animals; Humans; [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM]; [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM]; [SDV.BC] Life Sciences [q-bio]/Cellular Biology; Nuclear Magnetic Resonance, Biomolecular; Centrioles; Sheep; [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM]; Molecular Biology/Structural Biology [q-bio.BM]; Circular Dichroism; [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry; 540; [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM]; Protein Structure, Tertiary; [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics; [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM]; [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry; Molecular Biology/Biophysics; Microtubule-Associated Proteins

Microtubules are cytoskeletal components involved in multiple cell functions such as mitosis, motility, or intracellular traffic. In vivo, these polymers made of alphabeta-tubulin nucleate mostly from the centrosome to establish the interphasic microtubule network or, during mitosis, the mitotic spindle. Centrosomal P4.1-associated protein (CPAP; also named CENPJ) is a centrosomal protein involved in the assembly of centrioles and important for the centrosome function. This protein contains a microtubule-destabilizing region referred to as PN2-3. Here we decrypt the microtubule destabilization activity of PN2-3 at the molecular level and show that it results from the sequestration of tubulin by PN2-3 in a non-polymerizable 1:1 complex. We also map the tubulin/PN2-3 interaction both on the PN2-3 sequence and on the tubulin surface. NMR and CD data on free PN2-3 in solution show that this is an intrinsically unstructured protein that comprises a 23-amino acid residue alpha-helix. This helix is embedded in a 76-residue region that interacts strongly with tubulin. The interference of PN2-3 with well characterized tubulin properties, namely GTPase activity, nucleotide exchange, vinblastine-induced self-assembly, and stathmin family protein binding, highlights the beta subunit surface located at the intermolecular longitudinal interface when tubulin is embedded in a microtubule as a tubulin/PN2-3 interaction area. These findings characterize the PN2-3 fragment of CPAP as a protein with an unprecedented tubulin sequestering mechanism distinct from that of stathmin family proteins.