Functional analysis of Survivin in spindle assembly in Xenopus egg extracts.
Survivin is a member of the inhibitor of apoptosis (IAP) protein family that serves critical roles in mitosis and cytokinesis. Many studies have suggested Survivin's involvement in spindle regulation, but direct biochemical evidence for this has been lacking. Using the cell-free system of Xenopus egg extracts, we tested whether Survivin was necessary for the assembly of metaphase spindles. Removal or inhibition of Xenopus Survivin causes the disruption in the formation of metaphase spindles. In particular, we observe the generation of microtubule (MT) asters or poorly formed shortened spindle structures. In the latter phenotype the spindle structures display a decrease pole-to-pole length and a reduction of MTs around the chromatin indicating that Survivin may promote the stabilization of MT-chromatin interactions. In addition, function analysis of Survivin's conserved phosphorylation site Thr34 (Thr43 in Xenopus) and tubulin-binding domain was also assessed in regulating spindle assembly. Treatment of Xenopus egg extracts with a recombinant Survivin mutant that contained an alanine residue substitution at Thr43 (SURT43A mutant) or that was missing the C-terminal tubulin-binding domain (SURCL mutant) produced an increased frequency of MT asters and shorten abnormal spindle structures in Xenopus egg extracts. Interestingly, a phosphomimetic mutation made at residue Thr43 of Survivin (SURT43E mutant) generated a high frequency of MT asters implying that premature 'activation' of Survivin may interfere with an early stage of spindle assembly. Taken together, we propose that Survivin is a necessary component of the mitotic spindle and its phosphorylation at residue Thr43 is important for Survivin function in spindle assembly.
Canovas
Pedro M
PM
Molecular Oncology Program, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida 33612, USA.
Guadagno
Thomas M
TM
D000818:Animals; D002457:Cell Extracts; D002843:Chromatin / Q000378:metabolism; D066298:In Vitro Techniques; D008870:Microtubules / Q000502:physiology*; D009154:Mutation; D010063:Ovum / Q000378:metabolism* / Q000648:ultrastructure; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D008941:Spindle Apparatus / Q000502:physiology*; D000077022:Survivin; D014404:Tubulin / Q000378:metabolism; D014981:Xenopus; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D002457:Cell Extracts; D002843:Chromatin; D011994:Recombinant Proteins; D000077022:Survivin; D014404:Tubulin; D029867:Xenopus Proteins; C472712:birc5.2 protein, Xenopus