title: Survivin exists in immunochemically distinct subcellular pools and is involved in spindle microtubule function.
abstract: Survivin is a member of the inhibitor of apoptosis gene family that has been implicated in both apoptosis inhibition and regulation of mitosis. However, the subcellular distribution of survivin has been controversial and variously described as a microtubule-associated protein or chromosomal passenger protein. Here, we show that antibodies directed to the survivin sequence Ala(3)-Ile(19) exclusively recognized a nuclear pool of survivin that segregated with nucleoplasmic proteins, but not with outer nuclear matrix or nuclear matrix proteins. By immunofluorescence, nuclear survivin localized to kinetochores of metaphase chromosomes, and to the central spindle midzone at anaphase. However, antibodies to Cys(57)-Trp(67) identified a cytosolic pool of survivin, which associated with interphase microtubules, centrosomes, spindle poles and mitotic spindle microtubules at metaphase and anaphase. Polyclonal antibodies recognizing survivin epitopes Ala(3)-Ile(19), Met(38)-Thr(48), Pro(47)-Phe(58) and Cys(57)-Trp(67) identified both survivin pools within the same mitotic cell. A ratio of approximately 1:6 for nuclear versus cytosolic survivin was obtained by quantitative subcellular fractionation. In synchronized cultures, cytosolic survivin abruptly increased at mitosis, physically associated with p34(cdc2), and was phosphorylated by p34(cdc2) on Thr(34), in vivo. By contrast, nuclear survivin began to accumulate in S phase, was not complexed with p34(cdc2) and was not phosphorylated on Thr(34). Intracellular loading of a polyclonal antibody to survivin caused microtubule defects and resulted in formation of multipolar mitotic spindles, but did not interfere with cytokinesis. These data demonstrate that although both reported localizations of survivin exist in mitotic cells, the preponderant survivin pool is associated with microtubules and participates in the assembly of a bipolar mitotic spindle.
authors.affiliation: Boyer Center for Molecular Medicine, Department of Pathology, Yale University School of Medicine, 295 Congress Avenue, New Haven, CT 06536, USA.
authors.forename: Paola
authors.initials: P
authors.lastname: Fortugno
authors.forename: Nathan R
authors.initials: NR
authors.lastname: Wall
authors.forename: Alessandra
authors.initials: A
authors.lastname: Giodini
authors.forename: Daniel S
authors.initials: DS
authors.lastname: O'Connor
authors.forename: Janet
authors.initials: J
authors.lastname: Plescia
authors.forename: Karen M
authors.initials: KM
authors.lastname: Padgett
authors.forename: Simona
authors.initials: S
authors.lastname: Tognin
authors.forename: Pier Carlo
authors.initials: PC
authors.lastname: Marchisio
authors.forename: Dario C
authors.initials: DC
authors.lastname: Altieri
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins / Q000276:immunology / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008898:Mimosine / Q000378:metabolism; D009363:Neoplasm Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism*; D000077022:Survivin
chemical_list: D000911:Antibodies, Monoclonal; C107489:BIRC5 protein, human; D018797:Cell Cycle Proteins; D015853:Cysteine Proteinase Inhibitors; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D000077022:Survivin; D008898:Mimosine; D016203:CDC2 Protein Kinase