Functional Analysis of an Anchoring Protein CG-NAP in the Centrosome

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Microtubule assembly is initiated by γ-tubulin ring complex (γ-TuRC). In yeast, microtubule is nucleated from γ-TuRC anchored to the amino terminus of the spindle pole body component SpcllOp, which interacts with Cmd1p (calmodulin) at the carboxyl terminus. However, mammalian protein that anchors γ-TuRC remains to be elucidated. A giant coiled-coil protein CG-NAP (c___entrosome and G___olgi localized PKN___-a___ssociated p___rotein) was localized to the centrosome via the carboxyl-terminal region. This region was found to interact with calmodulin by yeast two-hybrid screening, and shares high homology with the caroboxyl-terminal region of another centrosomal coiled-coil protein, kendrin. The amino-terminal region of either CG-NAP or kendrin indirectly associated with γ-tubulin through binding with γ-tubulin complex protein 2 (GCP2) and/or GCP3. Furthermore, endogenous CG-NAP and kendrin were coimmunoprecipitated each other, and with endogenous GCP2 and γ-tubulin, suggesting that CG-NAP and kendrin form complex and interact with γ-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 aster formation, moreover, combination of these antibodies resulted in stronger inhibition. These results imply that CG-NAP and kendrin provide sites for microtubule nucleation in mammalian centrosome by anchoring γ-TuRC.