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Centrosomal proteins CG-NAP and kendrin provide microtubule nucleation sites by anchoring gamma-tubulin ring complex.

Microtubule assembly is initiated by the gamma-tubulin ring complex (gamma-TuRC). In yeast, the microtubule is nucleated from gamma-TuRC anchored to the amino-terminus of the spindle pole body component Spc110p, which interacts with calmodulin (Cmd1p) at the carboxy-terminus. However, mammalian protein that anchors gamma-TuRC remains to be elucidated. A giant coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was localized to the centrosome via the carboxyl-terminal region. This region was found to interact with calmodulin by yeast two-hybrid screening, and it shares high homology with the carboxyl-terminal region of another centrosomal coiled-coil protein, kendrin. The amino-terminal region of either CG-NAP or kendrin indirectly associated with gamma-tubulin through binding with gamma-tubulin complex protein 2 (GCP2) and/or GCP3. Furthermore, endogenous CG-NAP and kendrin were coimmunoprecipitated with each other and with endogenous GCP2 and gamma-tubulin, suggesting that CG-NAP and kendrin form complexes and interact with gamma-TuRC in vivo. These proteins were localized to the center of microtubule asters nucleated from isolated centrosomes. Pretreatment of the centrosomes by antibody to CG-NAP or kendrin moderately inhibited the microtubule nucleation; moreover, the combination of these antibodies resulted in stronger inhibition. These results imply that CG-NAP and kendrin provide sites for microtubule nucleation in the mammalian centrosome by anchoring gamma-TuRC.

Pubmed ID: 12221128

Authors

  • Takahashi M
  • Yamagiwa A
  • Nishimura T
  • Mukai H
  • Ono Y

Journal

Molecular biology of the cell

Publication Data

September 10, 2002

Associated Grants

None

Mesh Terms

  • A Kinase Anchor Proteins
  • Adaptor Proteins, Signal Transducing
  • Amino Acid Sequence
  • Animals
  • CHO Cells
  • COS Cells
  • Calmodulin-Binding Proteins
  • Carrier Proteins
  • Cell Line
  • Cell Nucleus
  • Centrosome
  • Cricetinae
  • Cytoskeletal Proteins
  • DNA, Complementary
  • HeLa Cells
  • Humans
  • Immunoblotting
  • Microscopy, Fluorescence
  • Microtubule-Associated Proteins
  • Microtubules
  • Models, Genetic
  • Molecular Sequence Data
  • Precipitin Tests
  • Protein Binding
  • Protein Structure, Tertiary
  • Sequence Homology, Amino Acid
  • Transfection
  • Tubulin
  • Two-Hybrid System Techniques