Centromeric binding and activity of Protein Phosphatase 4
The cell division cycle requires tight coupling between protein phosphorylation and dephosphorylation. However, understanding the cell cycle roles of multimeric protein phosphatases has been limited by the lack of knowledge of how their diverse regulatory subunits target highly conserved catalytic subunits to their sites of action. Phosphoprotein phosphatase 4 (PP4) has been recently shown to participate in the regulation of cell cycle progression. We now find that the EVH1 domain of the regulatory subunit 3 of Drosophila PP4, Falafel (Flfl), directly interacts with the centromeric protein C (CENP-C). Unlike other EVH1 domains that interact with proline-rich ligands, the crystal structure of the Flfl amino-terminal EVH1 domain bound to a CENP-C peptide reveals a new target-recognition mode for the phosphatase subunit. We also show that binding of Flfl to CENP-C is required to bring PP4 activity to centromeres to maintain CENP-C and attached core kinetochore proteins at chromosomes during mitosis.
© 2015 Macmillan Publishers Limited. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Received 21 August 2014. Accepted 18 November 2014. Published 06 January 2015. We are thankful to Magda Richter (Institute of Biochemistry and Biophysics (IBB PAS), Warsaw, Poland) and Renier Heijkants (Leiden University Medical Center, Netherlands) for their help with making DNA constructs, the Drosophila Genomics Resource Centre (supported by NIH grant 2P40OD010949-10A1) for the complementary DNA clones and Andor Udvardy (Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences (HAS-BRC)) for the critical reading of the manuscript. We also wish to thank the London Research Institute Peptide Chemistry facility, and Andrew Purkiss for assistance with data collection, and Janusz Debski and Michal Dadlez from the MS Lab (IBB PAS, Poland) for their assistance with proteomic analyses. Z.L. is on leave from HAS-BRC (Institute of Biochemistry) and was supported by the Long-Term Fellowship of the Federation of European Biochemical Societies (FEBS). This work was supported financially by grants from Cancer Research UK (C3/A11431) and the Medical Research Council (G1001696) to D.M.G. Author Contributions: Z.L. performed interaction and in vitro assays, protein purifications from cultured cells and embryos, proteomic analysis, mutagenesis and antibody production. S.L. and M.R.S. undertook crystallographic experiments. M.S.S. contributed to the phenotypic analysis of cultured cells. M.R.P. performed protein purifications from cultured cells, proteomic analysis and cell biology experiments. Z.L., M.R.P., M.R.S. and D.M.G. planned the experiments and wrote the paper that was discussed by all authors. Accession code: The atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession code 4WSF. The authors declare no competing financial interests.
Supplemental Material - 41467_2015_BFncomms6894_MOESM107_ESM.pdf
Published - ncomms6894.pdf