Exploiting classical and chemical genetics to interrogate the polo kinase phospho-proteome of fission yeast.

Abstract

Commitment to mitosis is universally regulated by the activation of a protein complex Mitotic Promoting Factor (MPF). Activation of MPF activates the downstream mitotic kinases (e.g. Polo, Aurora and NIMA related kinases) that lead to a number of different mitotic events.As in other systems, the fission yeast Polo kinase, Plo1 regulates diverse mitotic events including mitotic commitment, spindle formation, the cytokinetic ring formation/contraction, and the exit from mitosis. The aim for my project was to use a genetic and biochemical approach to assess the impact of Plo1 upon the composition of the mitotic phosphoproteome.Novel temperature sensitive plo1 alleles were generated. All known Plo1 dependent events were abolished in these mutants upon incubation at the restrictive temperature. One mutant was subsequently used in analysis where cells were arrested in mitosis with and without Polo Kinase activity and phosphopeptides whose levels were differentially regulated in the two conditions were identified through exploitation of SILAC technology. A parallel SILAC experiment identified phosphopeptides whose levels were altered when Plo1 activity was increased by mutation of the spindle pole body component Cut12. The range of phosphorylation events identified, not only fits existing models for the mitotic controls exerted by Plo1 in fission yeast, but highlight candidates for novel functions for Plo1.An analogue sensitive plo1 allele was also generated. This allele was used to determine the consequences of selective inhibition of Plo1 kinase activity following the addition of an non-hydrolysable ATP analogue to the culture. This modified kinase domain was also used to specifically target and activate Plo1 to specific subcellular locations to demonstrate that localised Plo1 activity on the Spindle Pole Body (SPB) in late G2 phase triggers commitment to mitosis.In conclusion, these studies of Plo1 function have considerably extended our current insight into established Plo1 dependent events and pathways and identified important avenues that could lead to the identification of novel functions for Plo1 in future.

Date of Award	1 Aug 2012
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Iain Hagan (Supervisor)