Toggle Main Menu Toggle Search

Open Access padlockePrints

A deficiency of the Cdk1 phosphorylation of fizzy increases the binding affinity to cyclin B and reduces the affinities to BubR1 and Mad2 in Drosophila melanogaster

Lookup NU author(s): Dr Jun-yong Huang, Dr Ashleigh Herriott


Full text for this publication is not currently held within this repository. Alternative links are provided below where available.


Cdk1 is the major mitotic cyclin dependent kinase, and in Human and Xenopus have been shown to phosphorylate Cdc20 and stabilise Cdc20-Mad2 interactions in order to reduce Cdc20-APC/C binding. Three of these can be identified in Drosophila Fizzy (Cdc20) at T63, T76 and T82. We have mutated these three Cdk1 phosphorylation sites and raised transformants that express GFP-Fizzy and GFP-FizzyTPD (Triple phosphorylation deficiency) proteins to determine: 1. Whether Cdk1 phosphorylation of Fizzy at these three sites is involved in its interaction with the MCC (Mitotic Checkpoint Complex). 2. Whether FizzyTPD still localises to kinetochores in mitosis. 3. Whether the mitotic checkpoint is rendered inactive. The results of in vitro Cdk1 kinase assays show that approximately 70% of the phosphorylation of Drosophila Fizzy occurs at T63, T76 and T82. In the presence of endogenous Fizzy, the GFP-FizzyTPD fusion protein follows normal wild-type GFP-Fizzy localisation patterns throughout mitosis, which includes the kinetochore localisation. In comparison to the GFP-Fizzy, GFP-FizzyTPD fusion protein has a lower binding affinity with Mad2 and BubR1. This may suggest that Cdk1 phosphorylation is required for MCC or MCC sub-complex formation. The binding affinity to Cdc27 is not significantly affected in the mutant line, suggesting that Cdk1 phosphorylation is not required for Fizzy to bind to the APC/C. Surprisingly; the binding affinity to cyclin B is dramatically increased. This suggests that GFP-FizzyTPD might be constitutively active so continuously target Cyclin B for destruction, or alternatively attempted Cdk1 phosphorylation of the FizzyTPD enhances the interaction with Cyclin B.

Publication metadata

Author(s): Huang J-Y, Herriott A, Morley G, Deak P, Pal M

Publication type: Conference Proceedings (inc. Abstract)

Publication status: Published

Conference Name: 23rd European Drosophila Research Conference

Year of Conference: 2013

Pages: 260-260

Publisher: EDRC