Specificity of affinity-purified anti-xSurvivin, anti-xAurora B, and anti-xINCENP antibodies in Xenopus interphase extracts and XTC cells. (A) Affinity-purified anti-xSurvivin antibody recognizes a band of 18 kDa in an interphase extract. (B) Affinity-purified anti-xAurora B antibody recognizes a band of 41 kDa in an interphase extract and in XTC cells. (C) Affinity-purified anti-xINCENP antibody recognizes a band of 130 kDa in an interphase extract and in XTC cells. (D-AA) Immunofluorescence localization of xAurora B (D-O) and xINCENP (P-AA) in XTC cells. (D-I) Immunolocalization of xAurora B during interphase (D) and throughout mitosis (E-I). (P-U) Immunolocalization of xINCENP during interphase (P) and throughout mitosis (Q-U). Colocalization of either xAurora B (J-O) or xINCENP (V-AA) with DNA and microtubules. Blue, DAPI; green, microtubules; red, xAurora B in (K-M), xINCENP in (W-Y); yellow and orange, overlap of xAurora B or xINCENP with microtubules.

xSurvivin, xAurora B, and xINCENP are physically associated in vivo during both interphase and mitosis. (A) xAurora B (xAurB), xINCENP, and xSurvivin were immunoprecipitated from interphase and mitotic Xenopus extracts. To determine if the chromosomal passenger proteins are physically associated, the immunoprecipitated samples were loaded onto 8 and 15% gels and were immunoblotted for xINCENP, xAurora B, and xSurvivin. Note that xINCENP is phosphorylated during mitosis and therefore exhibits retarded mobility. (B) In Xenopus extracts, the majority of xINCENP, xAurora B, and xSurvivin is physically associated. Interphase and mitotic extracts were immunodepleted with anti-xAurora B antibodies. Samples of the immunodepleted extract (xAurB Depleted Extract), beads, and interphase and mitotic whole cell extracts (WCE) were run on 8 and 15% gels and were immunoblotted for xINCENP, xAurora B, and xSurvivin. xINCENP and xSurvivin were depleted below detection levels (<1 ng) and were detected on the beads.

The activity of xAurora B is stimulated by stoichiometric xSurvivin binding and mitotic phosphorylation. (A) Recombinant GST-Survivin can stimulate an xAurora B IP kinase assay. xAurora B IP kinase assay from either interphase or mitotic extracts was conducted in the presence or absence of 10 ng of recombinant GST-Survivin. The top panel is an autoradiogram of [³²P]O₄ incorporation into a MBP substrate; the bottom panel is a Coomassie-stained gel showing similar amounts of xAurora B in each reaction. (B) Titration of the amount of xSurvivin needed to activate the xAurora B kinase immunoprecipitated from interphase and mitotic extracts. The top panel is a graph of the kinase activity of the immunoprecipitated xAurora B in the presence of the indicated amount of GST-Survivin. The bottom panel is an xAurora B immunoblot analysis demonstrating similar amounts of xAurora B kinase in the xAurora B IP kinase assay. (C) xAurora B kinase was immunopurified from either interphase or mitotic Xenopus egg extracts; Pre-I was used as a negative control. The IPs were washed extensively and either incubated in phosphatase buffer (−) or phosphatase buffer and lambda phosphatase (+), and were then washed again extensively and the kinase activity was assayed on MBP. A Coomassie-stained gel of the IP kinase assay demonstrates that there are similar amounts of xAurora B kinase in each reaction (bottom panel). (D) The phosphorylation state of xAurora B directly regulates its kinase activity. The xAurora B IPs were washed extensively, incubated in either phosphatase buffer (−) or phosphatase buffer and lambda phosphatase (+), washed again, and assayed for kinase activity on MBP substrate. Samples were also immunoblotted for xINCENP, xAurora B, and xSurvivin to determine relative amounts of the proteins.

Structural and sequence homology of Xenopus Survivin. (A) Clustal-W alignment of the amino acids encoding sequenced vertebrate Survivins. Significant residues from the crystal structure of human Survivin are conserved and are indicated by letters or symbols above the alignment: P-cdc2 phosphorylation site; star-residues that coordinate the Zn²⁺ in the Bir domain. (B-E) Comparison of solved human Survivin structure and a structural prediction of Xenopus Survivin protein produced by the SWISS-MODEL server (Peitsch, 1996; Guex and Peitsch, 1997). Ribbon diagram of Xenopus Survivin (B) and human Survivin (C). Charge distribution in Xenopus (D) and human (E) space-filled Survivin models where acidic charged residues are shown in red and basic residues are shown in blue. Note that the long C-terminal α helix is present in the Xenopus sequence; however, the homology is too low to fit in the modeled structure.

xSurvivin, xAurora B, and xINCENP cofractionate on gel filtration columns and sucrose density gradients during interphase and mitosis. (A) Interphase and mitotic extracts were separated by Superose 6 gel filtration chromatography and equal volumes of alternate fractions were run on SDS-PAGE gels. The fractions were then immunoblotted for xINCENP, xAurora B, and xSurvivin to determine the molecular weight of the passenger protein complex. The molecular weights of the void (2000 kDa), thyroglobulin (670 kDa), bovine γ globulin (158 kDa), chicken ovalbumin (44 kDa), and equine myoglobin (17 kDa) are shown. (B) xINCENP, xAurora B, and xSurvivin cofractionate on a sucrose density gradient, but the interphase complex migrates faster than the mitotic complex. Interphase and mitotic extracts were sedimented on 5–30% sucrose density gradients. Equal volumes of each fraction were run on SDS-PAGE gels and immunoblotted for xINCENP, xAurora B, xAurora A (Eg2), and xSurvivin. Thyroglobulin (19S), bovine γ globulin (7S), chicken ovalbumin (3.5S), and equine myoglobin (2S) were sedimented in a parallel gradient as markers.

xAurora B, xINCENP and xSurvivin complex interactions. (A) Schematic of an assay used to study xAurora B, xINCENP, and xSurvivin interaction. Plasmids encoding full-length xINCENP as well as xINCENP deletion constructs (pictured in C) were in vitro translated in the presence of [³⁵S]methionine and were subsequently incubated with Xenopus interphase and mitotic extracts. Anti-xAurora B antibodies, anti-xSurvivin antibodies, and Pre-I were used to immunoprecipitate the immunogenic protein and interacting xINCENP fragments from the extract. (B) An example of the assay where full-length xINCENP was immunoprecipitated with anti-xAurora B antibodies, but not with Pre-I. (C) The xINCENP constructs pictured were tested for their ability to coimmunoprecipitate with xAurora B and xSurvivin. Precipitation greater than fivefold above the amount detected by Pre-I controls is indicated by a + whereas less than fivefold was indicated by a −. Full-length xINCENP includes the following domains (N-terminal to C-terminal): the centromere-interacting domain, the chromosome-binding domain, the coiled-coil/microtubule-binding domain, and the IN-box (see Figure 7). (D) The interaction of xSurvivin with the complex is NaCl sensitive. xAurora B was immunoprecipitated from a mitotic extract and was washed in XB buffer containing the indicated amount of additional NaCl. After washing, the concentration of xAurora B and xSurvivin in the IPs was determined by quantitative immunoblot. Densitometric analysis of this experiment indicates that after washing with 300 mM NaCl, there is 10-fold more xAurora B kinase than xSurvivin. (E) The N terminus of Aurora B is required to interact with xINCENP. Full-length xAurora B (circles) and xAurora B (99–384) (triangles) were in vitro translated in the presence of [³⁵S]methionine and were subsequently incubated with Xenopus interphase extracts. Anti-xINCENP antibodies, anti-xSurvivin antibodies, and Pre-I were used to immunoprecipitate the resulting complex from the extract, and the IPs were washed with XB + 0.1% Triton-X 100 and the indicated extra NaCl (mM). The presence of either full-length or kinase domain xAurora was detected by SDS-PAGE and subsequent phosphorimager analysis.

Model of xAurora B, xINCENP, and xSurvivin interactions and regulation. (A) Interactions within the passenger protein complex. xSurvivin binds the N terminus of xINCENP, whereas the N terminus of xAurora B binds the C terminus of xINCENP. (B) Cell cycle regulation of the passenger protein complex. The interphase complex is hypophosphorylated and its migration in a sucrose gradient is consistent with either additional subunits (X) or a dimerization of the complex. A weak interaction between xSurvivin and xAurora may also exist. In mitosis, all three proteins are both present and phosphorylated, but the complex is smaller. Both the mitotic phosphorylation of the complex and the presence of the xSurvivin subunit are required for high kinase activity (On). (C) We present a highly speculative model in which the interaction between xSurvivin and xAurora is regulated within mitosis to control kinase activity. We propose that during mitosis, xAurora B kinase activity is inactivated by disrupting the interaction of xAurora B with xSurvivin (Off), perhaps through a mechanical force. It is also possible that Survivin binding to the complex could be spatially controlled. Such a mechanism could permit spatial regulation within parts of the same mitotic cell, allowing for pools of active Aurora kinase with Survivin-bound (On) versus inactive Aurora kinase with no Survivin interaction (Off).