Kinetochore localization and microtubule interaction of the human spindle checkpoint kinase Mps1
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Members of the Mps1 protein kinase family have been implicated in the regulation of the kinetochore-mediated spindle assembly checkpoint in species ranging from yeast to man. However, conflicting data have been reported on the subcellular localization of vertebrate Mps1 kinases and their possible roles in centrosome duplication. Moreover, little is presently known about the regulation of Mps1 kinases during the cell cycle. Here, we have used immunofluorescence microscopy, immunoblotting and siRNA-mediated depletion of hMps1 to re-investigate the subcellular localization of this kinase. Our data confirm the kinetochore association of hMps1 but suggest that the centrosome staining produced by some anti-hMps1 antibodies could be due to cross-reactivity with other proteins. We also show that the kinetochore association of hMps1 is mediated by the amino-terminal, non-catalytic domain and specifically requires the presence of the Hec1/Ndc80-Nuf2 complex at the kinetochore. Finally, we have combined in vitro binding studies and kinase assays to explore the influence of microtubules on hMps1 activity. Our data indicate that the catalytic domain of hMps1 displays affinity for microtubules and that microtubule binding could contribute to the regulation of kinase activity.
KeywordsMyelin Basic Protein Nocodazole Spindle Pole U2OS Cell Spindle Checkpoint
Enhanced green fluorescent protein
Myelin basic protein
We are particularly grateful to Drs. Emma Lees and Mark Winey for kindly providing the Ag3 antibody and the bacterial hMps1 expression plasmids, respectively. We also thank Drs. Martin-Lluesma, H.H.W. Silljé, and S. Taylor for providing additional antibodies and plasmids, and Dr. T.U. Mayer for a generous gift of polymerized microtubules. Finally, we thank Dr. R. Neef and T.A. Kufer for helpful discussions and advice. This work was supported by the Max Planck Society and the Fonds der Chemischen Industrie.
Fig S1 Epitope mapping for anti-hMps1 N1, N2, and C1 Mabs. HeLa cells were transfected with the indicated myc-hMps1 constructs and total cell lysates were prepared 36 hours post-transfection. Proteins were separated by SDS-PAGE and probed by Western blotting with anti-myc antibodies (first panel), anti-hMps1 N1 (second panel), anti-hMps1 N2 (third panel) and anti-hMps1 C1 (fourth panel). Molecular weight markers are indicated on the right, the filled circle designates an immunoreactive protein present in all samples, presumably endogenous c-myc protein. (See comment on the numbering of hMps1 constructs in Materials and methods)
- Hauf S, Cole RW, LaTerra S, Zimmer C, Schnapp G, Walter R, Heckel A, van Meel J, Rieder CL, Peters JM (2003) The small molecule hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint. J Cell Biol 161:281–294CrossRefPubMedGoogle Scholar
- van Kreeveld S, Winey M (2004) The budding yeast spindle pole body: a centrosome analog. In: Nigg EA (ed) Centrosomes in development and disease. Wiley-VCH, Weinheim (in press)Google Scholar
- Wilkinson CJ, Andersen JS, Mann M, Nigg EA (2004) A proteomic approach to the inventory of the human centrosome. In: Nigg EA (ed) Centrosomes in development and disease. Wiley-VCH, Weinheim (in press)Google Scholar