Microtubules are one major type of cytoskeleton and play important roles in various cellular activities, such as intracellular transportation and cell proliferation. γ-Tubulin ring complex (γTuRC), the principal microtubule nucleator in mammalian cells, regulates microtubule nucleation on microtubule organizing centers, such as centrosomes and Golgi complexes. CDK5RAP2 contains a short but highly conserved motif that stimulates microtubule nucleation from γTuRC and is thus called γTuRC-mediated nucleation activator (γTuNA). Moreover, the γTuNA is located within centrosomin motif 1 (CM1) which is conserved from human to yeast in all CDK5RAP2 orthologues. Until now, the mechanisms of γTuRC activation by γTuNA and how this activation is regulated are poorly understood. Here, I identified t...[ Read more ]

Microtubules are one major type of cytoskeleton and play important roles in various cellular activities, such as intracellular transportation and cell proliferation. γ-Tubulin ring complex (γTuRC), the principal microtubule nucleator in mammalian cells, regulates microtubule nucleation on microtubule organizing centers, such as centrosomes and Golgi complexes. CDK5RAP2 contains a short but highly conserved motif that stimulates microtubule nucleation from γTuRC and is thus called γTuRC-mediated nucleation activator (γTuNA). Moreover, the γTuNA is located within centrosomin motif 1 (CM1) which is conserved from human to yeast in all CDK5RAP2 orthologues. Until now, the mechanisms of γTuRC activation by γTuNA and how this activation is regulated are poorly understood. Here, I identified the γTuRC components, γ-tubulin and γ-tubulin complex protein 2 (GCP2), were in close proximity to the γTuNA in γTuRC using a diazirine-based chemical crosslinking approach. Mutational substitutions of the conserved residues within their potential γTuNA binding domains impair the microtubule nucleation on centrosomes. Thus, based on a recent γTuRC structure, I propose a model for γTuRC activation by γTuNA. Furthermore, I have also identified a conserved segment which is close to the γTuNA within CM1 as the γTuNA inhibitor (γTuNA-In). γTuNA-In directly binds to γTuNA and blocks γTuNA-γTuRC interaction. Removal of the autoinhibition by mutational disruptions which abolish the interaction between γTuNA and γTuNA-In results in drastically increased microtubule nucleation on the major microtubule organizing centers in animal cells, which are centrosomes and the Golgi, and affects the centrosome positioning, Golgi assembly and cell polarization. Notably, Ser112 phosphorylation of CDK5RAP2, which is known to be induced by NEK2, counteracts the autoinhibition by disrupting γTuNA‒γTuNA-In interaction. However, protein factors such as NEK2 and calmodulin are also participated in the regulation of γTuNA activity. Together, these data reveal the potential mechanisms of γTuRC activation by γTuNA and the controlling mechanisms of γTuNA function.