Michael Stark Lab

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  • Charlotte Lloyd

The roles of yeast Ipll1 protein kinase in chromosome bi-orientation and the spindle checkpoint response
Accurate chromosome segregation is vital for the maintenance of genome integrity during cell division and the conserved protein kinase Ipl1/Aurora B plays critical roles in ensuring that correct segregation will occur (1, 2). We are studying Ipl1 protein kinase in the budding yeast Saccharomyces cerevisiae. One role of Ipl1 is to promote chromosome bi-orientation, ensuring that sister chromatids are attached to microtubules from opposite spindle poles so that they are pulled in opposite directions during anaphase (3). Ipl1 kinase activity is regulated by its association with other conserved proteins – Sli15 (INCENP), Bir1 (Survivin) and Nbl1 (Borealin) – and we are interested in how these regulatory subunits control Ipl1 kinase activity during chromosome bi-orientation (4). We have also recently found that Ipl1 is involved in the spindle checkpoint response that delays anaphase chromosome segregation when sister chromatids are not under tension from spindle microtubules (5). This response is likely to be important for delaying anaphase when cells contain mono-oriented chromosomes, allowing time for correction to the bi-oriented state, and we would like to understand how Mad3 phosphorylation is involved in the checkpoint mechanism.
  1. Andrews, P.D., Knatko, E., Moore, W.J., and Swedlow, J.R. (2003). Mitotic mechanics: the auroras come into view. Curr. Opin. Cell Biol. 15, 672-683.
  2. Tanaka, T., Rachidi, N., Janke, C., Pereira, G., Galova, M., Schiebel, E., Stark, M. J. R. and Nasmyth, K. (2002). Evidence that the Ipl1/Sli15 (Aurora kinase/INCENP) complex promotes chromosome bi-orientation by altering kinetochore-spindle pole connections. Cell 108, 317-329.
  3. Tanaka, T. U., Stark, M. J. R. and Tanaka, K. (2005). Kinetochore capture and bi-orientation on the mitotic spindle. Nature Rev. Mol. Cell Biol. 6, 929-942.
  4. King, E. M. J., Rachidi, N., Morrice, N., Hardwick, K. G. and Stark, M. J. R. (2007). Ipl1p-dependent phosphorylation of Mad3p is required for the spindle checkpoint response to lack of tension at kinetochores. Genes Devel. 21, 1163-1168.
  5. Makrantoni, V. and Stark, M. J. R. (2009). Efficient chromosome bi-orientation and the tension checkpoint in Saccharomyces cerevisiae both require Bir1. Mol. Cell. Biol. 29, 4552-4562.
  6. Keating, P., Rachidi, N., Tanaka, T. U. and Stark, M. J. R. (2009). Ipl1-dependent phosphorylation of Dam1 is reduced by tension applied on kinetochores J. Cell Sci. 122, 4375-4382.
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Analysis of yeast Elongator phosphorylation and its functional consequences
Elongator is a highly conserved, six-subunit protein complex that is required for proper organ development in plants (1) and mutations in the largest subunit (Elp1) of human Elongator are associated with familial dysautonomia (2), a severe neurodevelopmental disease. In the yeast Saccharomyces cerevisiae, Elongator is non-essential for growth in the laboratory, but Elongator null mutations show a number of defects compared with wild-type cells including thermosensitivity as well as reduced tolerance to chemical stressors (3). The principal and possibly only role of Elongator is to promote wobble uridine modification in a subset of tRNAs, which is required for them to function efficiently in translation (4). Despite the expectation that such an activity should be constitutive to ensure that tRNA function remains optimal at all times, yeast Elongator function appears to be regulated by phosphorylation of the Elp1 subunit (5, 6). In collaboration with the laboratory of Raffael Schaffrath in the University of Kassel, we are identifying both the protein kinases and phosphatases that regulate Elongator and the sites of phosphorylation on Elp1 and other Elongator subunits that are functionally important, so that we can determine why Elongator may be regulated in this manner.
  1. Nelissen, H., Fleury, D., Bruno, L., Robles, P., De Veylder, L., Traas, J., Micol, J. L., Van Montagu, M., Inze, D. and Van Lijsebettens, M. (2005). The elongata mutants identify a functional Elongator complex in plants with a role in cell proliferation during organ growth. Proc Natl Acad Sci USA 102, 7754-7759.
  2. Close, P., Hawkes, N., Cornez, I., Creppe, C., Lambert, C. A., Rogister, B., Siebenlist, U., Merville, M. P., Slaugenhaupt, S. A., Bours, V., Svejstrup, J. Q. and Chariot, A. (2006). Transcription impairment and cell migration defects in Elongator-depleted cells: implication for familial dysautonomia. Mol Cell 22, 521-531.
  3. Frohloff, F., Fichtner, L., Jablonowski, D., Breunig, K. D. and Schaffrath, R. (2001). Saccharomyces cerevisiae Elongator mutations confer resistance to the Kluyveromyces lactis zymocin. EMBO J 20, 1993-2003.
  4. Huang, B., Johansson, M. J. and Bystrom, A. S. (2005). An early step in wobble uridine tRNA modification requires the Elongator complex. RNA 11, 424-436.
  5. Jablonowski, D., Butler, A. R., Fichtner, L., Gardiner, D., Schaffrath, R. & Stark, M. J. R. (2001). Sit4p protein phosphatase is required for sensitivity of Saccharomyces cerevisiae to Kluyveromyces lactis zymocin. Genetics 159, 1479-1489.
  6. Mehlgarten, C., Jablonowski, D., Breunig, K. D., Stark, M. J. R. and Schaffrath, R.  Elongator function depends on antagonistic regulation by casein kinase Hrr25 and protein phosphatase Sit4. (2009). Mol Microbiol 73, 869-881.
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