PgmNr W460: PP1β controls ZYG-1 levels to ensure precise centrosome doubling.

Authors:
J. Iyer 1 ; N. Peel 2 ; A. Naik 2 ; M. Dougherty 1 ; M. Dekker 3 ; K. F. O'Connell 1


Institutes
1) NIH, Bethesda, MD; 2) The College of New Jersey, Ewing, NJ; 3) Max Planck Institute of Molecular Biology and Genetics, Dresden, Germany.


Keyword: Other ( Centrosome )

Abstract:

Centrosome duplication (CD) is a highly regulated process that occurs only once during each cell cycle. Deregulation of this process yields an abnormal centrosome number. This can result in aneuploidy, a hallmark of cancer cells. The nematode C. elegans is an excellent model system to study the process of CD because the core components of the CD pathway in C. elegans are conserved in humans. One such protein is the master CD kinase ZYG-1 that is absolutely essential for CD. Depleting ZYG-1 prevents CD while increasing its levels or activity causes centrosome over-duplication. Our laboratory is interested in identifying novel regulators of CD. Through our studies, we have identified the phosphatase PP1β as a biologically important and novel inhibitor of CD. PP1β acts as a molecular brake for CD as decreasing its activity causes an over-duplication of centrosomes. Western blot analysis revealed that in mutant worms with low PP1β activity, ZYG-1 protein levels are elevated. Thus, PP1β normally functions to down-regulate ZYG-1. To determine if this regulation occurs at the transcriptional, translational or post-translational level, we monitored zyg-1 transcription and translation in mutants with low PP1β activity. In spite of an observed increase in ZYG-1 protein levels, we found that zyg-1 transcription and translation were unaltered in worms with reduced PP1β activity. Therefore, we conclude that PP1β decreases ZYG-1 levels post-translationally by promoting its proteasomal degradation. We hypothesize that PP1β physically interacts with ZYG-1 and dephosphorylates it to promote its degradation. ZYG-1 being a low abundance protein is very difficult to immunoprecipitate from worm extracts. Therefore, we utilized an innovative approach to determine if ZYG-1 and PP1β interact in vivo. We tagged endogenous ZYG-1 with a Biotin Acceptor Tag (BAT) and endogenous PP1β with the E. coli biotin ligase (BirA) using the CRISPR technique. Indeed, we found that BAT-ZYG-1 became biotinylated only in the presence of PP1β-BirA. This suggests that ZYG-1 and PP1β physically interact in vivo. Thus, we have identified the master CD kinase ZYG-1 as a novel candidate substrate of PP1β. In summary, by controlling ZYG-1 levels, PP1β ensures that only one daughter centriole forms adjacent to each mother centriole during CD. Future studies will involve determining which amino acid residues of ZYG-1 are de-phosphorylated by PP1β and the mechanism by which this leads to ZYG-1 degradation.



Wormbase Genetic Index
1. zyg-1
2. gsp-1