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.
