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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 RIS Download

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

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