The PN2-3 domain of centrosomal P4.1-associated protein implements a novel mechanism for tubulin sequestration.
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.
Cormier
Anthony
A
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Bâtiment 34, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette, France.
Clément
Marie-Jeanne
MJ
Knossow
Marcel
M
Lachkar
Sylvie
S
Savarin
Philippe
P
Toma
Flavio
F
Sobel
André
A
Gigant
Benoît
B
Curmi
Patrick A
PA
D000818:Animals; D002502:Centrioles / Q000378:metabolism*; D002942:Circular Dichroism; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary / Q000502:physiology; D012756:Sheep; D008941:Spindle Apparatus / Q000378:metabolism*; D014404:Tubulin / Q000378:metabolism*
C414735:CENPJ protein, human; D008869:Microtubule-Associated Proteins; D014404:Tubulin