Mps1-dependent phosphorylation of the kinetochore protein Ndc80 is an intrinsic step in spindle checkpoint activation

Abstract

The kinetochore is a multi-protein complex which supports chromosome segregation in mitosis by mediating the connection between centromeric DNA and the mitotic spindle. In addition, it serves as a docking platform for the spindle checkpoint proteins. The spindle checkpoint is a surveillance mechanism which monitors the proper assembly of the mitotic spindle apparatus. It allows the transition from meta- into anaphase only if all kinetochores are bipolarly attached to the spindle microtubules and if tension across the spindle is applied. The tetrameric Ndc80-complex (Ndc80, Nuf2, Spc24, Spc25) is part of the kinetochore. It is highly conserved and was shown to be required for the spindle checkpoint in S. cerevisiae (Janke et al. 2001). The kinase Mps1 has also been implicated in spindle checkpoint control (Hartwick et al. 1996). In mammalian cells, the kinetochore localisation of Mps1 depends on the presence of the Ndc80-complex (Stucke et al. 2004). Furthermore, endogenous Mps1 was found as a faint Coomassie-stained protein band in a Ndc80-complex pull down from yeast (C. Jaeger, unpublished results). Based on these observations, the goal of the present studies was to elucidate whether components of the S. cerevisiae Ndc80-complex are phosphorylated by Mps1 and if this phosphorylation has an influence on the regulation of the spindle checkpoint. The following observations were made: 1. Mps1 physically interacts with Ndc80 ∑ Mps1 weakly associates with the kinetochore by ChIP-analysis in S. cerevisiae. ∑ Mps1 physically interacts with the Ndc80-complex after spindle checkpoint activation by Mps1-overexpression. ∑ Mps1 specifically interacts with the conserved, globular N-terminus of Ndc80 in vitro. 2. Mps1 phosphorylates Ndc80 ∑ Mps1 phosphorylates the N-terminus of Ndc80 in vitro. ∑ Ndc80-phosphorylation depends on Mps1 in vivo. 3. Ndc80-phosphorylation activates the spindle assembly checkpoint ∑ Ndc80 in its non-phosphorylated state (ndc8014A) is checkpoint deficient. ∑ constitutive pseudo-phosphorylation of Ndc80 (ndc8014D) causes lethality due to a permanently activated spindle checkpoint (cell cycle arrest in mitosis). This checkpoint activation specifically depends on the checkpoint proteins Mad2 and Bub1. fi Ndc80-phosphorylation by Mps1 is an integral step in the spindle checkpoint pathway! 4. The kinetochore of ndc8014D-cells is functional ∑ The expression levels and the kinetochore localisation of the ndc8014D protein are comparable to the wild-type protein. ∑ The ndc8014D-protein is able to compete with the wild-type protein for binding-sites at the kinetochore. ∑ the spindle in ndc8014D-cells is under tension, a state which can only be achieved in the presence of an intact kinetochore. fi the observed checkpoint activation is specifically caused by the introduced point mutations rather than by a general kinetochore defect. 5. Mps1-activity is required downstream of Ndc80 ∑ The ndc8014D mutant is unable to activate the spindle checkpoint in Mps1-depleted cells. fi Mps1 has a second function in spindle checkpoint activation downstream of Ndc80-phosphorylation which might involve the phosphorylation of the checkpoint protein Mad1. The results which were obtained in the current work provide evidence that Mps1 specifically phosphorylates Ndc80 and that Ndc80-phosphorylation is a mechanism by which the spindle checkpoint is activated in the presence of unattached kinetochores in early stages of mitosis. The functional characterisation of Ndc80-phosphorylation allows for a refinement of the current model of the spindle assembly checkpoint (Figure 32).