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CSC-1: a subunit of the Aurora B kinase complex that binds to the survivin-like protein BIR-1 and the incenp-like protein ICP-1.

Romano A, Guse A, Krascenicova I, Schnabel H, Schnabel R, Glotzer M - J. Cell Biol. (2003)

Bottom Line: CSC-1 associates with ICP-1, BIR-1, and AIR-2 in vivo.ICP-1 dramatically stimulates AIR-2 kinase activity.This activity is not stimulated by CSC-1/BIR-1, suggesting that these two subunits function as targeting subunits for AIR-2 kinase.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Molecular Pathology, Dr. Bohrgasse 7, Vienna A-1030, Austria.

ABSTRACT
The Aurora B kinase complex is a critical regulator of chromosome segregation and cytokinesis. In Caenorhabditis elegans, AIR-2 (Aurora B) function requires ICP-1 (Incenp) and BIR-1 (Survivin). In various systems, Aurora B binds to orthologues of these proteins. Through genetic analysis, we have identified a new subunit of the Aurora B kinase complex, CSC-1. C. elegans embryos depleted of CSC-1, AIR-2, ICP-1, or BIR-1 have identical phenotypes. CSC-1, BIR-1, and ICP-1 are interdependent for their localization, and all are required for AIR-2 localization. In vitro, CSC-1 binds directly to BIR-1. The CSC-1/BIR-1 complex, but not the individual subunits, associates with ICP-1. CSC-1 associates with ICP-1, BIR-1, and AIR-2 in vivo. ICP-1 dramatically stimulates AIR-2 kinase activity. This activity is not stimulated by CSC-1/BIR-1, suggesting that these two subunits function as targeting subunits for AIR-2 kinase.

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Recombinant AIR-2, ICP-1, CSC-1, and BIR-1 form complexes in vitro. AIR-2, ICP-1, CSC-1, and BIR-1 were individually expressed in insect cells, and lysates were mixed as indicated, purified using the CBD affinity tag, and analyzed by Western blotting to detect protein–protein interactions among ABI complex members. The Western blots shown in A, B, and C were processed in parallel. (A) CBD-tagged ICP-1 does not bind to CSC-1 and binds weakly to BIR-1. All three proteins together form a robust complex in vitro, CBD–ICP-1 binds to AIR-2–His, and the complex containing all four subunits can be reconstituted in vitro. (B) Using CBD-tagged AIR-2, similar complexes can be reconstituted. ICP-1 binds to CBD–AIR-2. CBD–AIR-2 does not bind to CSC-1 or BIR-1 individually, nor to the CSC-1/BIR-1 complex, but it does bind to the CSC-1/BIR-1/ICP-1 complex. The bottom panel shows the corresponding kinase activity using MBP as a substrate. ICP-1 is required for AIR-2 activity, and CSC-1 and BIR-1 do not enhance the MBP phosphorylation. (C) Control binding reactions for A and B show that little binding is observed in the absence of CBD-tagged subunits. (D) Coomassie staining of SDS-PAGE of purified soluble ABI complex. Insect cells were coinfected with the indicated combinations of viruses. The subunits containing the affinity tag used for the purification are indicated by a square. Uncleaved CBD–AIR-2 is indicated by an asterisk. The lower panel shows kinase activity toward MBP. The amount of kinase activity in lanes 1 and 2 correlates with the abundance of AIR-2 and ICP-1 (see also B).
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fig5: Recombinant AIR-2, ICP-1, CSC-1, and BIR-1 form complexes in vitro. AIR-2, ICP-1, CSC-1, and BIR-1 were individually expressed in insect cells, and lysates were mixed as indicated, purified using the CBD affinity tag, and analyzed by Western blotting to detect protein–protein interactions among ABI complex members. The Western blots shown in A, B, and C were processed in parallel. (A) CBD-tagged ICP-1 does not bind to CSC-1 and binds weakly to BIR-1. All three proteins together form a robust complex in vitro, CBD–ICP-1 binds to AIR-2–His, and the complex containing all four subunits can be reconstituted in vitro. (B) Using CBD-tagged AIR-2, similar complexes can be reconstituted. ICP-1 binds to CBD–AIR-2. CBD–AIR-2 does not bind to CSC-1 or BIR-1 individually, nor to the CSC-1/BIR-1 complex, but it does bind to the CSC-1/BIR-1/ICP-1 complex. The bottom panel shows the corresponding kinase activity using MBP as a substrate. ICP-1 is required for AIR-2 activity, and CSC-1 and BIR-1 do not enhance the MBP phosphorylation. (C) Control binding reactions for A and B show that little binding is observed in the absence of CBD-tagged subunits. (D) Coomassie staining of SDS-PAGE of purified soluble ABI complex. Insect cells were coinfected with the indicated combinations of viruses. The subunits containing the affinity tag used for the purification are indicated by a square. Uncleaved CBD–AIR-2 is indicated by an asterisk. The lower panel shows kinase activity toward MBP. The amount of kinase activity in lanes 1 and 2 correlates with the abundance of AIR-2 and ICP-1 (see also B).

Mentions: We next investigated whether BIR-1, CSC-1, or the BIR-1/CSC-1 complex biochemically interacts with ICP-1 and AIR-2 kinase. Cell lysates were prepared from insect cells infected with baculoviruses expressing the individual proteins. Using chitin-binding domain (CBD)–tagged ICP-1, we examined whether a complex could be formed between ICP-1 and CSC-1 or BIR-1 by mixing these lysates and recovering the tagged ICP-1 on chitin beads. In these binary binding assays, no detectable interactions were found between ICP-1 and CSC-1; a weak but reproducible interaction was observed between ICP-1 and BIR-1. However when lysates containing all three subunits were mixed, robust formation of an ICP-1/BIR-1/CSC-1 complex was observed (Fig. 5 A). These data, combined with the previous observation of a robust BIR-1/CSC-1 complex, suggest that ICP-1 binds strongly to this subcomplex, but not to the individual subunits.


CSC-1: a subunit of the Aurora B kinase complex that binds to the survivin-like protein BIR-1 and the incenp-like protein ICP-1.

Romano A, Guse A, Krascenicova I, Schnabel H, Schnabel R, Glotzer M - J. Cell Biol. (2003)

Recombinant AIR-2, ICP-1, CSC-1, and BIR-1 form complexes in vitro. AIR-2, ICP-1, CSC-1, and BIR-1 were individually expressed in insect cells, and lysates were mixed as indicated, purified using the CBD affinity tag, and analyzed by Western blotting to detect protein–protein interactions among ABI complex members. The Western blots shown in A, B, and C were processed in parallel. (A) CBD-tagged ICP-1 does not bind to CSC-1 and binds weakly to BIR-1. All three proteins together form a robust complex in vitro, CBD–ICP-1 binds to AIR-2–His, and the complex containing all four subunits can be reconstituted in vitro. (B) Using CBD-tagged AIR-2, similar complexes can be reconstituted. ICP-1 binds to CBD–AIR-2. CBD–AIR-2 does not bind to CSC-1 or BIR-1 individually, nor to the CSC-1/BIR-1 complex, but it does bind to the CSC-1/BIR-1/ICP-1 complex. The bottom panel shows the corresponding kinase activity using MBP as a substrate. ICP-1 is required for AIR-2 activity, and CSC-1 and BIR-1 do not enhance the MBP phosphorylation. (C) Control binding reactions for A and B show that little binding is observed in the absence of CBD-tagged subunits. (D) Coomassie staining of SDS-PAGE of purified soluble ABI complex. Insect cells were coinfected with the indicated combinations of viruses. The subunits containing the affinity tag used for the purification are indicated by a square. Uncleaved CBD–AIR-2 is indicated by an asterisk. The lower panel shows kinase activity toward MBP. The amount of kinase activity in lanes 1 and 2 correlates with the abundance of AIR-2 and ICP-1 (see also B).
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getmorefigures.php?uid=PMC2172917&req=5

fig5: Recombinant AIR-2, ICP-1, CSC-1, and BIR-1 form complexes in vitro. AIR-2, ICP-1, CSC-1, and BIR-1 were individually expressed in insect cells, and lysates were mixed as indicated, purified using the CBD affinity tag, and analyzed by Western blotting to detect protein–protein interactions among ABI complex members. The Western blots shown in A, B, and C were processed in parallel. (A) CBD-tagged ICP-1 does not bind to CSC-1 and binds weakly to BIR-1. All three proteins together form a robust complex in vitro, CBD–ICP-1 binds to AIR-2–His, and the complex containing all four subunits can be reconstituted in vitro. (B) Using CBD-tagged AIR-2, similar complexes can be reconstituted. ICP-1 binds to CBD–AIR-2. CBD–AIR-2 does not bind to CSC-1 or BIR-1 individually, nor to the CSC-1/BIR-1 complex, but it does bind to the CSC-1/BIR-1/ICP-1 complex. The bottom panel shows the corresponding kinase activity using MBP as a substrate. ICP-1 is required for AIR-2 activity, and CSC-1 and BIR-1 do not enhance the MBP phosphorylation. (C) Control binding reactions for A and B show that little binding is observed in the absence of CBD-tagged subunits. (D) Coomassie staining of SDS-PAGE of purified soluble ABI complex. Insect cells were coinfected with the indicated combinations of viruses. The subunits containing the affinity tag used for the purification are indicated by a square. Uncleaved CBD–AIR-2 is indicated by an asterisk. The lower panel shows kinase activity toward MBP. The amount of kinase activity in lanes 1 and 2 correlates with the abundance of AIR-2 and ICP-1 (see also B).
Mentions: We next investigated whether BIR-1, CSC-1, or the BIR-1/CSC-1 complex biochemically interacts with ICP-1 and AIR-2 kinase. Cell lysates were prepared from insect cells infected with baculoviruses expressing the individual proteins. Using chitin-binding domain (CBD)–tagged ICP-1, we examined whether a complex could be formed between ICP-1 and CSC-1 or BIR-1 by mixing these lysates and recovering the tagged ICP-1 on chitin beads. In these binary binding assays, no detectable interactions were found between ICP-1 and CSC-1; a weak but reproducible interaction was observed between ICP-1 and BIR-1. However when lysates containing all three subunits were mixed, robust formation of an ICP-1/BIR-1/CSC-1 complex was observed (Fig. 5 A). These data, combined with the previous observation of a robust BIR-1/CSC-1 complex, suggest that ICP-1 binds strongly to this subcomplex, but not to the individual subunits.

Bottom Line: CSC-1 associates with ICP-1, BIR-1, and AIR-2 in vivo.ICP-1 dramatically stimulates AIR-2 kinase activity.This activity is not stimulated by CSC-1/BIR-1, suggesting that these two subunits function as targeting subunits for AIR-2 kinase.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Molecular Pathology, Dr. Bohrgasse 7, Vienna A-1030, Austria.

ABSTRACT
The Aurora B kinase complex is a critical regulator of chromosome segregation and cytokinesis. In Caenorhabditis elegans, AIR-2 (Aurora B) function requires ICP-1 (Incenp) and BIR-1 (Survivin). In various systems, Aurora B binds to orthologues of these proteins. Through genetic analysis, we have identified a new subunit of the Aurora B kinase complex, CSC-1. C. elegans embryos depleted of CSC-1, AIR-2, ICP-1, or BIR-1 have identical phenotypes. CSC-1, BIR-1, and ICP-1 are interdependent for their localization, and all are required for AIR-2 localization. In vitro, CSC-1 binds directly to BIR-1. The CSC-1/BIR-1 complex, but not the individual subunits, associates with ICP-1. CSC-1 associates with ICP-1, BIR-1, and AIR-2 in vivo. ICP-1 dramatically stimulates AIR-2 kinase activity. This activity is not stimulated by CSC-1/BIR-1, suggesting that these two subunits function as targeting subunits for AIR-2 kinase.

Show MeSH
Related in: MedlinePlus