Restricting Cyclin degradation to mitotic stages and G1 in Drosophila

Norman Zielke and Frank Sprenger* 

Institute for Genetics, University of Cologne, Weyertal 121, D-50931 Cologne, Germany    *sprenger@uni-koeln.de

INTRODUCTION.
The degradation of mitotic cyclins by the Anaphase-Promoting-Complex/Cyclosome (APC/C) is restricted to mitotic stages and G1 in part by its association with the adapter proteins Fizzy/Cdc20 (Fzy) and Fizzy-related-Cdh1 (Fzr). The association of Fzy with the APC/C requires previous phosphorylation events on the APC/C that only occur during mitosis, thus restricting Fzy-dependent APC/C activity to mitosis. Fzr activation of the APC/C in contrast is negatively regulated by phosphorylation events on Fzr. Thus, Fzr can only activate the APC/C at stage of low Cdk activity, e.g. during later stages of mitosis and G1. However, G2 is also characterized by low Cdk-activity and we have identified the rca1 gene in Drosophila that is required for preventing APC/C activity in G2 (1).

RESULTS
In rca1 mutants, cells fail to execute the 16th mitosis during embryonic development. This is caused by a premature degradation of all mitotic cyclins during the preceding G2-phase. Genetic epistasis experiment show that the premature cyclin degradation is mediated by Fzr. A physical assiciation between Rca1 and Fzr was seen in immunprecipitations (1). Besides rca1 inhibition, APC/C activity during G2 is also inhibited by Cdk-dependent phosphorylation. Double mutants between Rca1 and Cyclin A or Cyclin E displayed a strong enhancement of the arrest phenotype and resulted in earlier arrest point during embryonic development.

Rca1 shares structural features with Emi1 from vertebrates that is an inhibitor of Fzr and Fzy dependent APC/C activity (2,3). However, no interactions (genetic or biochemical) was seen between Rca1 and Fzy. In addition, Emi1 failed to rescue rca1 mutants, indicating important differences between these proteins. Rca1 and Emi1 proteins contain a nuclear localization sequence, an F-box and share a region with conserved Cysteine spacing. This latter region is crucial for the function of Rca1 in G2, while the F-box is not required for ths function of Rca1. However, when Rca1 function is tested in later stages of development, a requirement for the F-box was seen. Overexpression of Rca1 was found to cause ectopic G1-S transitions and was able to suppress endocycles in salivary glands. These effects were dependent on the F-box in Rca1. Interaction of Rca1 with Skp-proteins from Drosophila support that the F-Box in Rca1 is indeed functional.

DISCUSSION
Restricting APC/C activity to mititoc stages and G2 is important for proprer cell cycle and development. We could show that this is accomplished by several activites that are partly redundant. Rca1 as well as inhibitory phosphorylation event on Fzr contribute to sufficient inactivation of APC/C activity during G2 but the indidividual contribution of each inhibitory mechansims might be different during development. Inhibition of Fzr in G2 by Rca1 requires the C-terminal region including the conserved cysteins but does not require the conserved F-box. Functional test that show a requirement for the F-box indicate that Rca1 could have several functions during the cell cycle and during development.

REFERENCES

  1. Grosskortenhaus, R. and F. Sprenger (2002). Dev Cell, 2, 29-40.
  2. Reimann, J. D., E. Freed, et al. (2001). Cell 105, 645-55.
  3. Reimann, J. D., B. E. Gardner, et al. (2001). Genes Dev 15, 3278-85.