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. 2015 Jan 6;6:5894.
doi: 10.1038/ncomms6894.

Centromeric Binding and Activity of Protein Phosphatase 4

Free PMC article

Centromeric Binding and Activity of Protein Phosphatase 4

Zoltan Lipinszki et al. Nat Commun. .
Free PMC article


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.


Figure 1
Figure 1. Interacting domains of Falafel and CENP-C.
(a) Schematic representation of the common structural elements of PP4R3 proteins compared with Drosophila R3, Falafel. R3s contain a conserved PH/EVH1-like domain and Smk-1 domain with unknown function in their N-termini, followed by Armadillo/HEAT repeats (ARM) in the middle and a variable length (dotted line, according to HomoloGene R3 orthologues can be 767 to 1,136 amino acid-long) low complexity region (LCR) at the C-termini. Falafel fragments used in this study are indicated below. (b) In vitro binding of GST-tagged CENP-C with IVTT-expressed 35S-Met-labelled Falafel but not with labelled R2 or PP4c. (c) Confocal microscopy of interphase D.Mel-2 cells showing nuclear and centromeric enrichment of Falafel. Magnified images below show co-localization of Falafel with the centromeric protein CENP-A/CID. Scale bar, 5 μm. (d) In vitro binding of GST-tagged N-terminal (FlflN, see a), (but not Middle and C-terminal FlflM and FlflC, see a) fragment of Falafel with 35S-Met-labelled full-length (fl) and C-terminal part (C′) but not with N-terminal part (N′) of CENP-C. (e) Identification of the Falafel Binding Domain (FBD/fragment C9), a 92-amino acid-long region of the C-terminal part of CENP-C that can bind FlflN in vitro. CEN indicates the centromeric localization motif. ‘*’ indicates globin from the reticulocyte lysate. (f) The EVH1 domain-containing GST-tagged FlflN (aa 1–361) and its truncated form Flfl1–168 (aa 1–168) specifically bind to 35S-Met-labelled CENP-CFBD in vitro. In contrast Flfl169–361 (aa 169–361), the EVH1-lacking part of FlflN, which includes only an Smk-1 domain, cannot interact with 35S-CENP-CFBD. ‘*’ indicates globin from the reticulocyte lysate. (g) Western blots revealing that the entire PP4 complex is co-precipitated with FLAG::CENP-CFBD from cultured cells or syncytial embryos; anti-αTubulin provides a loading and negative control. Left hand panels show Western blots of cultured cells expressing FLAG only (1) or FLAG::CENP-CFBD (2). Right hand panels show Western blots of syncytial embryo proteins purified on immobilized GST::CENP-CFBD. GST only serves as a negative control. Coomassie-stained gels demonstrate the loading of the bait proteins.
Figure 2
Figure 2. CENP-C′s FIM motif is crucial for Falafel binding and recruitment to the centromere.
(a) Peptide array: Flfl1–168 binds to two 20-mer peptides in CENP-CFBD identifying the 19-amino acid-long Falafel-interacting motif (FIM). (b) 35S-Met-labelled Flfl1–168 is able to show in vitro binding to GST-tagged wild-type FBD (GST::FBD) but not to the FIM-deleted FBD (GST::FBDΔFIM). (c) GFP–Trap purification of full-length Falafel (GFP::Flfl) or its 1–168 amino acid fragment (GFP::Flf1-168) co-precipitates FLAG-tagged CENP-C (FLAG::CENP-C). This interaction is lost following deletion of the FIM (ΔFIM). GFP::Flfl169–973 provides a negative control. Ponceau S staining assesses loading. (d) D.Mel-2 cells depleted of endogenous CENP-C and co-expressing FLAG::Flfl1–168 with either GFP::CENP-CWT or GFP::CENP-CΔFIM. In the absence of FIM, Flfl1–168 no longer localizes to the centromere. Arrows indicate centromeric FLAG::Flfl1–168 signals. Scale bars, 5 μm. See also Supplementary Fig. 1e. (e) Peptide substitution array indicating that the sequence 1057Phe–Lys–Lys–Pro1060 within CENP-C is critical for Flfl1–168 binding in vitro.
Figure 3
Figure 3. Structure of the Flfl1–122–CENP-CFIM complex.
(a) Ribbon diagram of the Falafel Pleckstrin Homology-like domain (Flfl1–122; wheat) with CENP-C FIM peptide (red) in two different orientations. (b) Representation of Flfl1–122 surface conservation coloured from red (highly conserved) to white (non-conserved). The deepest red ‘shelf’ lying in the middle of Flfl1–122 is due to the highly conserved Trp20. (c) Sequence alignment of EVH1 domains from Homer, Spred, Ena, Mena, Wasp and N-Wasp. Species are Mm (Mouse), Hs (Human) and Dm (Drosophila). Secondary structural elements of the Flfl1–122 are represented by arrows (β strands), squiggles (α helices) and T (turns). Highly conserved residues are coloured in green. The invariant tryptophan is coloured in red and residues involved in proline stacking in EVH1 but not Flfl1–122 in yellow. The relative accessibility of each residue is rendered as blue-coloured boxes located at the last line of each block. The blue scale is set as follows: blue, accessible; cyan, intermediate and white, buried. The Flfl1–122 deviates from the canonical EVH1 domain; a conserved phenylalanine (Phe77 in 1EVH) is replaced by leucine (Leu70) and the side chain of a conserved tyrosine (Tyr12 in Flfl1–122) is in a different orientation to that in 1EVH and other EVH1 family members. (d) Close up view of Flfl1–122–FIM interaction site. CENP-C residues have been labelled and three critical residues from the Flfl1–122 ligand recognition site are highlighted in yellow. Phe1057 occupies a hydrophobic pocket composed largely of the methylene groups of a series of hydrophilic amino acid side chains, while Pro1060 of CENP-C is sandwiched between Leu70 and the highly conserved Trp20.
Figure 4
Figure 4. Presence and catalytic activity of PP4 are required to regulate mitotic centromere integrity.
(a) A proportion of CENP-C is mislocalized from the centromeres and accumulated along the spindle and spindle poles in flfl-depleted mitotic cells. kan RNAi is used as a negative control. The same phenotype is observed after replacement of endogenous CENP-C with FLAG::CENP-CΔFIM or replacement of endogenous PP4c with FLAG::PP4PhD. Scale bar, 5 μm. Spd2, centrosome marker; CENP-A/CID, centromere marker. (b) Proportion of mitotic cells showing CENP-C mislocalization on kan (control), flfl or pp4c depletions. (c) FLAG::CENP-CΔFIM cannot complement centromeric function of depleted endogenous CENP-C. (d) FLAG::PP4cWT can fully rescue CENP-C displacement resulting from depletion of endogenous PP4c, whereas the phosphatase-dead form (FLAG::PP4cPhD) cannot. Bars represent s.d. in all three graphs; n>50 for each condition.
Figure 5
Figure 5. PP4 phosphoregulates Falafel and CENP-C.
(a) Treatment of D.Mel-2 cells with interfering ds flfl RNA leads to reduction of both Falafel and PP4c, suggesting that the depletion of a regulatory subunit of PP4 results in the destabilization of the catalytic subunit (as observed also for PP2A (ref. 8)). Falafel migrates as a doublet. Treatment with 50 nM okadaic acid (OA) indicates that the upper band represents a phosphorylated form. On depletion of PP4c (dsRNApp4c) but not PP2Ac (dsRNAmts) the phosphorylated form of Falafel predominates, suggesting that Falafel is a novel PP4 substrate. αTubulin is loading control. (b) The CENP-CFBD fragment is phosphorylated in vivo. FLAG::FBD undergoes shifts in its electrophoretic mobility following treatment in vitro with λ-PPase. Samples were run on Phos-tag SDS–PAGE followed by immunoblotting. (c) Phospho status of CENP-CFBD depends on the presence of PP4. Extracts of cells expressing FLAG::FBD treated with nothing (control), DMSO, 50 nM okadaic acid (OA) and interfering dsRNAs targeted against flfl (dsRNAflfl), pp4c (dsRNApp4c) and control (dsRNAkan). Phosphorylated forms are seen following okadaic acid treatment and following depletion of Falafel or PP4c, indicating that the PP4 holoenzyme is required to maintain the dephosphorylation status of FLAG::FBD in vivo. (d) Electrophoretic mobility of Falafel in extracts of cells after control RNAi (dsRNAkan) or RNAi directed against the 5′ untranslated region (UTR) of pp4c (dsRNApp4c-5UTR). Cells are expressing either the transgenic FLAG-tagged phosphatase-dead variant of PP4c (FLAG::PP4cPhD) or its wild-type counterpart (FLAG::PP4cWT). Note that Falafel migrates as its phosphorylated form in the FLAG::PP4cPhD-expressing cell line, indicating that the PP4 catalytic activity is required for Falafel dephosphorylation in vivo. (e) A hypothetic model demonstrating that PP4 activity is localized at centromeres in Drosophila cells. Regulatory subunit 3, Falafel, directly interacts with the Falafel Binding Domain of CENP-C (violet arrow), which brings the trimeric holoenzyme of PP4 to centromeres. Centromeric PP4 activity is important for the integrity of mitotic centromeres and affects the phospho status of Falafel and CENP-C (highlighted as red stars).

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