Kinetochore "memory" of spindle checkpoint signaling in lysed mitotic cells

The spindle checkpoint prevents errors in mitosis. Cells respond to the pre sence of kinetochores that are improperly attached to the mitotic spindle b y delaying anaphase onset. Evidence suggests that phosphorylations recogniz ed by the 3F3/2 anti-phos-phoepitope antibody may be involved in the kineto chore signaling of the spindle checkpoint. Mitoric cells lysed in detergent in the absence of phosphatase inhibitors rapidly lose expression of the 3F 3/2 phosphoepitope. However, when ATP is added to lysed and rinsed mitotic cytoskeletons, kinetochores become rephosphsrylated by an endogenous, bound kinase. Kinetochore rephosphorylation in vitro produced the same different ial phosphorylation seen in appropriately fixed living cells. In chromosome s not yet aligned at the metaphase plate, kinetochores undergo rapid rephos phorylation, while those of fully congressed chromosomes are under-phosphor ylated. However, latent 3F3/2 kinase activity is retained at kinetochores o f cells at all stages of mitosis including anaphase. This latent activity i s revealed when rephosphorylation reactions are carried out for extended ti mes. The endogenous, kinetochore-bound kinase can be chemically inactivated . Remarkably, a soluble kinase activity extracted from mitotic cells also c aused differential rephosphorylation of kinetochores whose endogenous kinas e had been chemically inactivated. We suggest that, in vivo, microtubule at tachment alters the kinetochore 3F3/2 phosphoprotein, causing it to resist phosphorylation. This kinetochore modification is retained after cell lysis , producing a "memory" of the in vivo phosphorylation state. (C) 2000 Wiley -Liss, Inc.