Microtubules are important components of cytoskeletons and function in many cellular processes such as cell division and motion. γ-Tubulin ring complex (γ-TuRC) is the principle microtubule nucleator. CDK5RAP2 is the activator of γ-TuRC-mediated microtubule nucleation which contains a conversed domain γ-TuNA. In this study, I focused on the regulatory role of phosphorylation. Phosphorylation of CDK5RAP2 S112 was found to activate CDK5RAP2 to bind with γ-TuRC. To understand how CDK5RAP2 phosphorylation regulates γ-TuRC activity, I found Nek2 was involved in CDK5RAP2 phosphorylation and functioned with two possible mechanisms. Nek2 overexpression inhibits the activity of CDK5RAP2 CM1 domain. In addition to phosphorylation, γ-TuNA also regulates γ-TuRC activity by dimerization and binding...[ Read more ]

Microtubules are important components of cytoskeletons and function in many cellular processes such as cell division and motion. γ-Tubulin ring complex (γ-TuRC) is the principle microtubule nucleator. CDK5RAP2 is the activator of γ-TuRC-mediated microtubule nucleation which contains a conversed domain γ-TuNA. In this study, I focused on the regulatory role of phosphorylation. Phosphorylation of CDK5RAP2 S112 was found to activate CDK5RAP2 to bind with γ-TuRC. To understand how CDK5RAP2 phosphorylation regulates γ-TuRC activity, I found Nek2 was involved in CDK5RAP2 phosphorylation and functioned with two possible mechanisms. Nek2 overexpression inhibits the activity of CDK5RAP2 CM1 domain. In addition to phosphorylation, γ-TuNA also regulates γ-TuRC activity by dimerization and binding to the inhibitory domain. The second half of γ-TuNA is critical for γ-TuNA dimerization. A short domain of γ-TuNA, CDK5RAP2 aa 74-81, is required for its binding to the inhibitory domain. To further investigate the regulation of γ- TuRC, I used mass spectrometry to characterize cytosolic and centrosome-located γ-TuRC. A novel potential interactor of γ-TuRC, DHB4, was identified in the proteomic analysis of cytosolic γ-TuRC. Furthermore, twelve phosphorylation sites of γ-tubulin, two of which were novel sites, were detected by mass spectrometry. The regulation of centrosome-located γ- TuRC was studied by centrosome proteomic analysis. All core components of γ-TuRC were identified in centrosome, though no phosphorylation of centrosomal γ-tubulin was detected.

In addition, a list of known centrosomal proteins were detected. In summary, this study elucidates the regulatory roles of CDK5RAP2 by phosphorylation and dimerization. Furthermore, this study also gives insights into the regulation of cytosolic and centrosome-localized γ-TuRC with proteomic analysis.