The centrosome in animal cells provides a major microtubule-nucleating site that regulates the microtubule cytoskeleton temporally and spatially throughout the cell cycle. A large coiled-coil centrosome protein identified in Drosophila can bind to calmodulin. Biochemical studies reveal that this novel centrosome protein, centrosome protein of 309 kDa (Cp309), cofractionates with the gamma-tubulin ring complex and the centrosome-complementing activity. CP309 is required for microtubule nucleation mediated by centrosomes and it interacts with the gamma-tubulin small complex. These findings suggest that the microtubule-nucleating activity of the centrosome requires the function of CP309 (Kawaguchi, 2004).
Because CP309 contains a CaM-binding motif, whether CP309 can bind to CaM was examined along with whether this binding is Ca2+ dependent. CaM-agarose beads were incubated with Drosophila embryo extracts in the presence of 2.5 mM Ca2+ or 5 mM EGTA (EGTA was used to chelate the Ca2+ in the extracts). Proteins bound to the CaM agarose beads were analyzed by Western blotting, probing with antibodies against CP309. It was found that significantly more CP309 in the extracts bound to CaM-agarose beads in the presence of Ca2+ than in the presence EGTA. Next, it was asked whether Ca2+ facilitates CaM-free CP309 binding to CaM. CaM-free CP309 was prepared by eluting CP309 from CaM-agarose beads with EGTA and then used in the same binding assay as described above. It was found that the CaM-free CP309 also binds to CaM-agarose beads more efficiently in the presence of Ca2+ than in the presence of EGTA. Therefore, although CP309 can bind to CaM in the absence of Ca2+, Ca2+ significantly enhances the binding (Kawaguchi, 2004).
Previous studies have suggested that Kendrin and CG-NAP interact with a mammalian-gammaTuSC component (Takahashi, 2002). Consistent with this, biochemical studies have shown that CP309 cofractionates with gammaTuSC and gammaTuRC in Drosophila embryo extracts. However, it was found that gammaTuRC/gammaTuSC and CP309 do not coimmunoprecipitate with each other in the embryo extracts. This suggests that the interaction between CP309 and gammaTuRC/gammaTuSC is weak. Because both CP309 and gammaTuSC could be co-expressed in Sf9 cells to levels higher than those in the embryo extracts, it was examined whether CP309 and gammaTuSC interact with each other in Sf9 cells. FLAG-tagged CP309, gamma-tubulin, and untagged Dgrips84 and 91 were expressed in Sf9 cells, and reciprocal immunoprecipitations were carried out using antibodies against CP309, Dgrip84, and gamma-tubulin. Either preimmune serum for CP309 antibody or random nonimmunized rabbit IgGs were used as control. It was found that the antibody against CP309 immunoprecipitates all three gammaTuSC subunits. Because the antibodies against gamma-tubulin and Dgrip84 also immunoprecipitate CP309, it is concluded that CP309 interacts with gammaTuSC. The above-mentioned experiments were carried out in the presence or absence of Ca2+ and CaM; they did not affect the interaction between CP309 and gammaTuSC. Therefore, Ca2+ or CaM does not seem to regulate the interaction between CP309 and gammaTuSC in vitro (Kawaguchi, 2004).
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