Plx1 is the 3F3/2 kinase responsible for targeting spindle checkpoint proteins to kinetochores.

Dynamic attachment of microtubules to kinetochores during mitosis generates pulling force, or tension, required for the high fidelity of chromosome separation. A lack of tension activates the spindle checkpoint and delays the anaphase onset. A key step in the tension-response pathway involves the phosphorylation of the 3F3/2 epitope by an unknown kinase on untensed kinetochores. Using a rephosphorylation assay in Xenopus laevis extracts, we identified the kinetochore-associated Polo-like kinase Plx1 as the kinase both necessary and sufficient for this phosphorylation. Indeed, Plx1 is the physiological 3F3/2 kinase involved in checkpoint response, as immunodepletion of Plx1 from checkpoint extracts abolished the 3F3/2 signal and blocked association of xMad2, xBubR1, xNdc80, and xNuf2 with kinetochores. Interestingly, the kinetochore localization of Plx1 is under the control of the checkpoint protein xMps1, as immunodepletion of xMps1 prevents binding of Plx1 to kinetochores. Thus, Plx1 couples the tension signal to cellular responses through phosphorylating the 3F3/2 epitope and targeting structural and checkpoint proteins to kinetochores.