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CDK5RAP2 stimulates microtubule nucleation by the gamma-tubulin ring complex

Yuk-Kwan Choi1, Pengfei Liu, Siu Kwan Sze

  • 1Department of Biochemistry, Hong Kong University of Science and Technology, Hong Kong, China.

The Journal of Cell Biology
|December 8, 2010
PubMed

Insights

CDK5RAP2 protein contains a domain that activates microtubule nucleation by the gamma-tubulin ring complex (γ-TuRC). This gamma-tubulin ring complex-mediated nucleation activator (γ-TuNA) is crucial for microtubule organization.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Cytoskeleton Dynamics

Background:

  • CDK5RAP2 is a human microcephaly protein.
  • It possesses a domain homologous to known gamma-tubulin complex (γ-TuC)-tethering proteins in other species.
  • The gamma-tubulin ring complex (γ-TuRC) is essential for microtubule nucleation.

Purpose of the Study:

  • To investigate the function of the conserved domain in CDK5RAP2.
  • To determine if this domain interacts with the γ-TuRC and influences its activity.
  • To characterize the role of CDK5RAP2 in microtubule nucleation.

Main Methods:

  • Biochemical assays using purified γ-TuRC and a γ-TuNA mutant.
  • Microtubule regrowth assays to assess nucleation.
  • RNA interference to deplete CDK5RAP2 and observe effects on microtubule nucleation.
  • Co-immunoprecipitation to identify proteins associated with γ-TuRC bound to γ-TuNA.

Main Results:

  • The γ-TuRC-mediated nucleation activator (γ-TuNA) domain of CDK5RAP2 directly binds to and stimulates the microtubule-nucleating activity of purified γ-TuRC in vitro.
  • γ-TuNA induces extensive, γ-TuRC-dependent microtubule nucleation in regrowth assays.
  • Depletion of CDK5RAP2 impairs both centrosomal and acentrosomal microtubule nucleation, while γ-TuRC assembly remains unaffected.
  • The γ-TuRC complex bound to γ-TuNA includes NME7, FAM128A/B, and actin.

Conclusions:

  • The γ-TuNA domain of CDK5RAP2 plays a regulatory role in γ-TuRC-mediated microtubule nucleation.
  • CDK5RAP2 is essential for microtubule nucleation, independent of its effect on γ-TuRC assembly.
  • These findings reveal a novel mechanism for regulating microtubule organization through CDK5RAP2-γ-TuRC interaction.

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