Mad2 binding by phosphorylated kinetochores links error detection and checkpoint action in mitosis

The spindle checkpoint must detect the presence of unattached or improperly attached kinetochores and must then inhibit progression through the cell c ycle until the offending condition is resolved. Detection probably involves attachment-sensitive kinetochore phosphorylation (reviewed in [1,2]). A ke y player in the checkpoint's response is the Mad2 protein, which prevents a ctivation of the anaphase-promoting complex (APC) by the Cdc20 protein [3-8 ], Microinjection of Mad2 antibodies results in premature anaphase onset [9 ,10], and excess Mad2 protein causes arrest in mitosis [5,11]. We have prev iously shown that Mad2 localizes to unattached kinetochores in vertebrate c ells, and that this localization ceases as kinetochores accumulate microtub ules [10,12,13], But how is Mad2 binding limited to unattached kinetochores ? Here, we used lysed PtK1 cells to study kinetochore phosphorylation and M ad2 binding. We found that Mad2 binds to phosphorylated kinetochores, but n ot to unphosphorylated ones. Our data suggest that it is kinetochore protei n phosphorylation that promotes Mad2 binding to unattached kinetochores. Th us, we have identified a probable molecular link between attachment-sensiti ve kinetochore phosphorylation and the inhibition of anaphase, The complete pathway for error control in mitosis can now be outlined. (C) Elsevier Sci ence Ltd ISSN 0960-9822.