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Mutations in the yeast cyclin-dependent kinase Cdc28 reveal a role in the spindle assembly checkpoint

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Abstract

Anaphase onset and mitotic exit are regulated by the spindle assembly or kinetochore checkpoint, which inhibits the anaphase-promoting complex (APC), preventing the degradation of anaphase inhibitors and mitotic cyclins. As a result, cells arrest with high cyclin-dependent kinase (CDK) activity due to the accumulation of cyclins. Aside from this, a clear-cut demonstration of a direct role for CDKs in the spindle checkpoint response has been elusive. Cdc28 is the main CDK driving the cell cycle in budding yeast. In this report, mutations in cdc28 are described that confer specific checkpoint defects, supersensitivity towards microtubule poisons and chromosome loss. Two alleles encode single mutations in the N and C terminal regions, respectively (R10G and R288G), and one allele specifies two mutations near the C terminus (F245L, I284T). These cdc28 mutants are unable to arrest or efficiently prevent sister chromatid separation during treatment with nocodazole. Genetic interactions with checkpoint and apc mutants suggest Cdc28 may regulate checkpoint arrest downstream of the MAD2 and BUB2 pathways. These studies identify a C-terminal domain of Cdc28 required for checkpoint arrest upon spindle damage that mediates chromosome stability during vegetative growth, suggesting that it has an essential surveillance function in the unperturbed cell cycle.

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Acknowledgments

We are grateful to T. Davis, P. Hieter, A. Hoyt, D. Koshland, M. Longtine, A. Murray, A. Page, F. Spencer and M. Winey and their respective labs for generously providing reagents. We acknowledge C. Lassy for help with confocal microscopy, A. Page for guidance regarding the APC, C. Dubal and F. Spencer for detailed chromosome loss assay methods, B. Tobe for criticisms of the manuscript, J. Fitz Gerald for performing the Cdc28 modeling studies, and M. Spiotto for enthusiastic assistance. We thank all members of the Kron Lab and our colleagues at the Center for Molecular Oncology for helpful discussions. These studies were funded by grants to S.J.K. from the James S. McDonnell Foundation, the Ludwig Institute for Cancer Research and NIH R01 GM60443. S.J.K. is a Leukemia and Lymphoma Society Scholar

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  1. Center for Molecular Oncology and Dept. of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA

    A. A. Kitazono, D. A. Garza & S. J. Kron

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  1. A. A. Kitazono
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  2. D. A. Garza
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  3. S. J. Kron
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Correspondence to S. J. Kron.

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Communicated by A. Aguilera

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Kitazono, A.A., Garza, D.A. & Kron, S.J. Mutations in the yeast cyclin-dependent kinase Cdc28 reveal a role in the spindle assembly checkpoint. Mol Gen Genomics 269, 672–684 (2003). https://doi.org/10.1007/s00438-003-0870-y

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