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 Ala3-Ile19 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 Cys57-Trp67 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 Ala3-Ile19,Met38-Thr48, Pro47-Phe58 and Cys57-Trp67 identified both survivin pools within the same mitotic cell. A ratio of ∼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 p34cdc2, and was phosphorylated by p34cdc2 on Thr34, in vivo. By contrast, nuclear survivin began to accumulate in S phase, was not complexed with p34cdc2 and was not phosphorylated on Thr34. 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.