Limits...
Interaction of Aurora-A and centrosomin at the microtubule-nucleating site in Drosophila and mammalian cells.

Terada Y, Uetake Y, Kuriyama R - J. Cell Biol. (2003)

Bottom Line: Aurora-A and CNN are mutually dependent for localization at spindle poles, which is required for proper targeting of gamma-tubulin and other centrosomal components to the centrosome.The NH2-terminal half of CNN interacts with gamma-tubulin, and induces cytoplasmic foci that can initiate microtubule nucleation in vivo and in vitro in both Drosophila and mammalian cells.These results suggest that Aurora-A regulates centrosome assembly by controlling the CNN's ability to targeting and/or anchoring gamma-tubulin to the centrosome and organizing microtubule-nucleating sites via its interaction with the COOH-terminal sequence of CNN.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455, USA. terad002@umn.edu

ABSTRACT
A mitosis-specific Aurora-A kinase has been implicated in microtubule organization and spindle assembly in diverse organisms. However, exactly how Aurora-A controls the microtubule nucleation onto centrosomes is unknown. Here, we show that Aurora-A specifically binds to the COOH-terminal domain of a Drosophila centrosomal protein, centrosomin (CNN), which has been shown to be important for assembly of mitotic spindles and spindle poles. Aurora-A and CNN are mutually dependent for localization at spindle poles, which is required for proper targeting of gamma-tubulin and other centrosomal components to the centrosome. The NH2-terminal half of CNN interacts with gamma-tubulin, and induces cytoplasmic foci that can initiate microtubule nucleation in vivo and in vitro in both Drosophila and mammalian cells. These results suggest that Aurora-A regulates centrosome assembly by controlling the CNN's ability to targeting and/or anchoring gamma-tubulin to the centrosome and organizing microtubule-nucleating sites via its interaction with the COOH-terminal sequence of CNN.

Show MeSH
Interaction of Aurora-A with CNN and γ-tubulin. (A) A map of CNN and its deletion constructs capable of association with Aurora-A assayed by yeast two-hybrid screening (Y2H) and immunoprecipitation (IP). Microtubule-nucleating activity (MTOC activity) and γ-tubulin interaction (γ binding) are also summarized on the right. CNN contains characteristic regions, including leucine zipper motifs (Leu zipper), a potential nuclear localization signal (NLS), a putative Aurora-A phosphorylation site (Pi-site), and a glutamine-enriched region (Gln-rich). Numbers indicate the positions of amino acids. (B) HA-tagged CNN binds to Aurora-A (lanes 3 and 4), but not Aurora-B (lanes 1 and 2), in S2 cells. Proteins in immunoprecipitated (IP) and nonprecipitated supernatant (S) fractions, prepared from HA-CNN–expressing (lanes 1 and 3) and –nonexpresssing (lanes 2 and 4) cells, were identified by blotting with HA, Aurora-A, and Aurora-B antibodies. (C) Aurora-A binds to the COOH-terminal domain of CNN. In vitro synthesized full (F), NH2-terminal (N), and COOH-terminal (C) domains of CNN (Input, lanes 1 to 4) were mixed with (IP, lanes 2′ to 4′) and without (IP, lane 1′) His-tagged Aurora-A purified from bacteria.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172831&req=5

fig1: Interaction of Aurora-A with CNN and γ-tubulin. (A) A map of CNN and its deletion constructs capable of association with Aurora-A assayed by yeast two-hybrid screening (Y2H) and immunoprecipitation (IP). Microtubule-nucleating activity (MTOC activity) and γ-tubulin interaction (γ binding) are also summarized on the right. CNN contains characteristic regions, including leucine zipper motifs (Leu zipper), a potential nuclear localization signal (NLS), a putative Aurora-A phosphorylation site (Pi-site), and a glutamine-enriched region (Gln-rich). Numbers indicate the positions of amino acids. (B) HA-tagged CNN binds to Aurora-A (lanes 3 and 4), but not Aurora-B (lanes 1 and 2), in S2 cells. Proteins in immunoprecipitated (IP) and nonprecipitated supernatant (S) fractions, prepared from HA-CNN–expressing (lanes 1 and 3) and –nonexpresssing (lanes 2 and 4) cells, were identified by blotting with HA, Aurora-A, and Aurora-B antibodies. (C) Aurora-A binds to the COOH-terminal domain of CNN. In vitro synthesized full (F), NH2-terminal (N), and COOH-terminal (C) domains of CNN (Input, lanes 1 to 4) were mixed with (IP, lanes 2′ to 4′) and without (IP, lane 1′) His-tagged Aurora-A purified from bacteria.

Mentions: By screening of a Drosophila two-hybrid library, we isolated two clones encoding a molecule capable of interaction with Aurora-A. The sequence corresponds to the COOH-terminal domain of centrosomin (CNN; Fig. 1 A, clone C), a core component of the centrosome important for assembly of mitotic centrosomes in Drosophila (Heuer et al., 1995; Megraw et al., 1999). Although the truncated polypeptide covered by clone CNN-C1 appears to be sufficient for interaction with Aurora-A, the binding intensity was weaker than CNN-C. Fig. 1 B demonstrates that endogenous Aurora-A (Fig. 1 B, lanes 3 and 4), but not Aurora-B (Fig. 1 B, lanes 1 and 2), was immunoprecipitated with HA-tagged CNN expressed in S2 cells. Specificity of the COOH-terminal domain of CNN for interaction with Aurora-A was further confirmed by in vitro binding assays as summarized in Fig. 1 C.


Interaction of Aurora-A and centrosomin at the microtubule-nucleating site in Drosophila and mammalian cells.

Terada Y, Uetake Y, Kuriyama R - J. Cell Biol. (2003)

Interaction of Aurora-A with CNN and γ-tubulin. (A) A map of CNN and its deletion constructs capable of association with Aurora-A assayed by yeast two-hybrid screening (Y2H) and immunoprecipitation (IP). Microtubule-nucleating activity (MTOC activity) and γ-tubulin interaction (γ binding) are also summarized on the right. CNN contains characteristic regions, including leucine zipper motifs (Leu zipper), a potential nuclear localization signal (NLS), a putative Aurora-A phosphorylation site (Pi-site), and a glutamine-enriched region (Gln-rich). Numbers indicate the positions of amino acids. (B) HA-tagged CNN binds to Aurora-A (lanes 3 and 4), but not Aurora-B (lanes 1 and 2), in S2 cells. Proteins in immunoprecipitated (IP) and nonprecipitated supernatant (S) fractions, prepared from HA-CNN–expressing (lanes 1 and 3) and –nonexpresssing (lanes 2 and 4) cells, were identified by blotting with HA, Aurora-A, and Aurora-B antibodies. (C) Aurora-A binds to the COOH-terminal domain of CNN. In vitro synthesized full (F), NH2-terminal (N), and COOH-terminal (C) domains of CNN (Input, lanes 1 to 4) were mixed with (IP, lanes 2′ to 4′) and without (IP, lane 1′) His-tagged Aurora-A purified from bacteria.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172831&req=5

fig1: Interaction of Aurora-A with CNN and γ-tubulin. (A) A map of CNN and its deletion constructs capable of association with Aurora-A assayed by yeast two-hybrid screening (Y2H) and immunoprecipitation (IP). Microtubule-nucleating activity (MTOC activity) and γ-tubulin interaction (γ binding) are also summarized on the right. CNN contains characteristic regions, including leucine zipper motifs (Leu zipper), a potential nuclear localization signal (NLS), a putative Aurora-A phosphorylation site (Pi-site), and a glutamine-enriched region (Gln-rich). Numbers indicate the positions of amino acids. (B) HA-tagged CNN binds to Aurora-A (lanes 3 and 4), but not Aurora-B (lanes 1 and 2), in S2 cells. Proteins in immunoprecipitated (IP) and nonprecipitated supernatant (S) fractions, prepared from HA-CNN–expressing (lanes 1 and 3) and –nonexpresssing (lanes 2 and 4) cells, were identified by blotting with HA, Aurora-A, and Aurora-B antibodies. (C) Aurora-A binds to the COOH-terminal domain of CNN. In vitro synthesized full (F), NH2-terminal (N), and COOH-terminal (C) domains of CNN (Input, lanes 1 to 4) were mixed with (IP, lanes 2′ to 4′) and without (IP, lane 1′) His-tagged Aurora-A purified from bacteria.
Mentions: By screening of a Drosophila two-hybrid library, we isolated two clones encoding a molecule capable of interaction with Aurora-A. The sequence corresponds to the COOH-terminal domain of centrosomin (CNN; Fig. 1 A, clone C), a core component of the centrosome important for assembly of mitotic centrosomes in Drosophila (Heuer et al., 1995; Megraw et al., 1999). Although the truncated polypeptide covered by clone CNN-C1 appears to be sufficient for interaction with Aurora-A, the binding intensity was weaker than CNN-C. Fig. 1 B demonstrates that endogenous Aurora-A (Fig. 1 B, lanes 3 and 4), but not Aurora-B (Fig. 1 B, lanes 1 and 2), was immunoprecipitated with HA-tagged CNN expressed in S2 cells. Specificity of the COOH-terminal domain of CNN for interaction with Aurora-A was further confirmed by in vitro binding assays as summarized in Fig. 1 C.

Bottom Line: Aurora-A and CNN are mutually dependent for localization at spindle poles, which is required for proper targeting of gamma-tubulin and other centrosomal components to the centrosome.The NH2-terminal half of CNN interacts with gamma-tubulin, and induces cytoplasmic foci that can initiate microtubule nucleation in vivo and in vitro in both Drosophila and mammalian cells.These results suggest that Aurora-A regulates centrosome assembly by controlling the CNN's ability to targeting and/or anchoring gamma-tubulin to the centrosome and organizing microtubule-nucleating sites via its interaction with the COOH-terminal sequence of CNN.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455, USA. terad002@umn.edu

ABSTRACT
A mitosis-specific Aurora-A kinase has been implicated in microtubule organization and spindle assembly in diverse organisms. However, exactly how Aurora-A controls the microtubule nucleation onto centrosomes is unknown. Here, we show that Aurora-A specifically binds to the COOH-terminal domain of a Drosophila centrosomal protein, centrosomin (CNN), which has been shown to be important for assembly of mitotic spindles and spindle poles. Aurora-A and CNN are mutually dependent for localization at spindle poles, which is required for proper targeting of gamma-tubulin and other centrosomal components to the centrosome. The NH2-terminal half of CNN interacts with gamma-tubulin, and induces cytoplasmic foci that can initiate microtubule nucleation in vivo and in vitro in both Drosophila and mammalian cells. These results suggest that Aurora-A regulates centrosome assembly by controlling the CNN's ability to targeting and/or anchoring gamma-tubulin to the centrosome and organizing microtubule-nucleating sites via its interaction with the COOH-terminal sequence of CNN.

Show MeSH