Identification of a Novel Microtubule-destabilizing Motif in CPAP That Binds to Tubulin Heterodimers and Inhibits Microtubule Assembly

Abstract

We have previously identified a new centrosomal protein, centrosomal protein 4.1-associated protein (CPAP), which is associated with the gamma-tubulin complex. Here, we report that CPAP carries a novel microtubule-destabilizing motif that not only inhibits microtubule nucleation from the centrosome but also depolymerizes taxol-stabilized microtubules. Deletion mapping and functional analyses have defined a 112-residue CPAP that is necessary and sufficient for microtubule destabilization. This 112-residue CPAP directly recognizes the plus end of a microtubule and inhibits microtubule nucleation from the centrosome. Biochemical and functional analyses revealed that this 112-residue CPAP also binds to tubulin dimers, resulting in the destabilization of microtubules. Using the tetracycline-controlled system (tet-off), we observed that overexpression of this 112-residue CPAP inhibits cell proliferation and induces apoptosis after G2/M arrest. The possible mechanisms of how this 112-residue motif in CPAP that inhibits microtubule nucleation from the centrosome and disassembles preformed microtubules are discussed.

Centrobin-mediated Regulation of the Centrosomal Protein 4.1-associated Protein (CPAP) Level Limits Centriole Length during Elongation Stage

“…Hence, it is believed that degradation of CPAP is a trigger to stop further elongation of the centrioles. Importantly, both centrobin and CPAP directly bind to tubulin and have microtubule-stabilizing property (49,52,63), suggesting that these proteins contribute to the actual polymerization of microtubules. Future studies will address how the tubulin binding property of centrobin and CPAP coordinates and contributes toward centriole assembly and regulation of centriole length during biogenesis.…”

Serum- and Glucocorticoid-Inducible Kinase 1 (SGK1) Increases Neurite Formation through Microtubule Depolymerization by SGK1 and by SGK1 Phosphorylation of tau

“…Although no conserved MTbinding domain was found in SGK1, SGK1 may interact with MT via other atypical domains, such as in the cases of CPAP (centrosomal protein 4.1-associated protein) and casein kinase II. CPAP, a centrosomal protein, carries a novel MT-destabilizing motif that inhibits MT nucleation from the centrosome and depolymerizes Taxol-stabilized MT (25). Another example is casein kinase II.…”

“…Primary neurite is a neurite that arises from the cell body. The number of neurites was determined by counting the number of primary neurites that were longer than 5 m. HeLa cells were fixed with 3.7% paraformaldehyde for 10 min at RT and permeabilized with 0.1% Triton X-100 for 5 min (25). Monoclonal anti-␤ III tubulin Ab (Sigma-Aldrich) and Cy3 donkey antimouse Abs were used for immunostaining as described above.…”

“…HeLa cells were transfected with pEGFP-human sgk1 fragment constructs by Lipofectamine 2000 reagent. Twenty-four hours later, transfected cells were incubated for 1 h at 4°C and immediately replaced by warm medium for 10 min at 37°C (25). Cells were then fixed with 3.7% paraformaldehyde for 10 min at RT and permeabilized with 0.1% Triton X-100 for 5 min.…”

“…DMSO, dimethyl sulfoxide. (24,25), in this experiment, we further examined whether SGK1 disassembles Taxol-induced prepolymerized MT and whether this effect is also ATP independent. Similarly, AKT and MBP were used to examine the specificity of SGK1.…”