Formation of stable attachments between kinetochores and microtubules depends on the B56-PP2A phosphatase

Error-free chromosome segregation depends on the precise regulation of phosphorylation to stabilize kinetochore-microtubule attachments (K-fibers) on sister chromatids that have attached to opposite spindle poles (bi-oriented)(1). In many instances, phosphorylation correlates with K-fiber destabilization(2–7). Consistent with this, multiple kinases, including Aurora B and Plk1, are enriched at kinetochores of mal-oriented chromosomes compared to bi-oriented chromosomes, which have stable attachments(2, 8). Paradoxically, however, these kinases also target to prometaphase chromosomes that have not yet established spindle attachments and it is therefore unclear how kinetochore-microtubule interactions can be stabilized when kinase levels are high. Here we show that generation of stable K-fibers depends on the B56-PP2A phosphatase, which is enriched at centromeres/kinetochores of unattached chromosomes. When B56-PP2A is depleted, K-fibers are destabilized and chromosomes fail to align at the spindle equator. Strikingly, B56-PP2A depletion increases the phosphorylation of Aurora B and Plk1 kinetochore substrates as well as Plk1 recruitment to kinetochores. Consistent with increased substrate phosphorylation, we find that chemical inhibition of Aurora or Plk1 restores K-fibers in B56-PP2A depleted cells. Our findings reveal that PP2A, an essential tumor suppressor(9), tunes the balance of phosphorylation to promote chromosome-spindle interactions during cell division.