A Consensus Binding Motif for the PP4 Protein Phosphatase

Abstract

Dynamic protein phosphorylation constitutes a fundamental regulatory mechanism in all organisms. Phosphoprotein phosphatase 4 (PP4) is a conserved and essential nuclear serine and threonine phosphatase. Despite the importance of PP4, general principles of substrate selection are unknown, hampering the study of signal regulation by this phosphatase. Here, we identify and thoroughly characterize a general PP4 consensus-binding motif, the FxxP motif. X-ray crystallography studies reveal that FxxP motifs bind to a conserved pocket in the PP4 regulatory subunit PPP4R3. Systems-wide in silico searches integrated with proteomic analysis of PP4 interacting proteins allow us to identify numerous FxxP motifs in proteins controlling a range of fundamental cellular processes. We identify an FxxP motif in the cohesin release factor WAPL and show that this regulates WAPL phosphorylation status and is required for efficient cohesin release. Collectively our work uncovers basic principles of PP4 specificity with broad implications for understanding phosphorylation-mediated signaling in cells.

Keywords: EVH1 domain; PP4; SLiM; WAPL; cohesin; protein phosphatase.

Figure 1.. Identification and characterization of a PP4 binding motif

A-B) Schematic of the PP4 holoenzyme complex composed of PP4C-PPP4R2-PPP4R3. C) Venn diagram summarizing ProP-PD selections. D-E) Average peptide sequences selected by ProP-PD using PPP4R3A (D), PPP4R3B (E) as baits and a human disorderome library. F) Purification of YFP-4X(FxxP) and YFP-4X(AxxA) from stable inducible HeLa cells and western blot analysis of the purifications with the indicated antibodies. G) Purifications as in F) but analyzed by label free quantitative mass spectrometry. Each identified protein is indicated by a dot and PP4 holoenzyme components are highlighted in red and proteasome subunits in green. H) Purifications of YFP-PPP4R3B and its interaction to a FxxP containing protein (MELK) and MxPP containing protein (MCE1) analyzed in the presence of RFP-tagged 4X(FxxP) or 4X(AxxA). I) Coomassie stained SDS-PAGE of the purified PP4 holoenzyme. J) Dephosphorylation of a substrate containing a FxxP motif or a AxxA motif by the PP4 holoenzyme complex. An engineered substrate containing 3TP sites was phosphorylated with radioactive ATP and incubated with the PP4 holoenzyme. Removal of radioactive phosphate was monitored over time. Representative of 2 independent experiments with data points from both experiments shown. K) Analysis of the phosphothreonine and phosphoserine preference of the PP4 holoenzyme using synthetic model peptides and monitoring phosphate release. The mean and SD are indicated by red bars. Significance was determined using an unpaired t-test. See also Figure S1, Table S1 and Table S3.

Figure 2.. Structure of the EVH1-FxxP complex

A) Overall structure of the PPP4R3A EVH1 domain in complex with the FxxP peptide with directionality of peptide indicated. B) Details of the interaction with the binding pocket for the proline and phenylalanine residues indicated. A zoom in on the FxxP binding pocket with the position of W19 indicated as well as the position of the four key residues used to define EVH1 families is shown. C-D) Details of the interaction between the FxxP motif and the EVH1 domain and the key determinants of binding shown as a closeup of the 3D structural model (C) or in a 2D-projection plot (D) E) Comparison of the different EVH1 domains and their interaction with proline rich peptides. The nature of the residues in positions 1–4 used to define the different EVH1 families is shown and compared to the residues in PPP4R3A. F) Affinity between the indicated PPP4R3A variants and a model peptide as determined by ITC. G) Analysis of the interaction between MCE1 and the PP4 holoenzyme containing either WT or W19A PPP4R3B by immunoprecipitation and Western Blotting. H) Dephosphorylation of an in vitro phosphorylated substrate containing an FxxP motif by the PP4 holoenzyme containing either WT or W19A PPP4R3A. Representative of 2 independent experiments with data points from both experiments shown. See also Figure S1 and Table S4.

Figure 3.. PP4 interactions mediated by FxxP and MxPP motifs

A) Quantitative label free mass spectrometry analysis of PP4 holoenzyme components. PP4 components are indicated in red and proteins interacting through FxxP or MxPP motifs indicated in blue. B) Summary of in silico prediction of FxxP motifs in human proteins and in interactors. A PWM P Value cut off of 10E-5 and 10-E4 was used for the proteome and interactors, respectively C) Validated motifs by ITC or co-immunoprecipitation assays with K_D for peptides measured by ITC indicated D) Binding of myc-tagged PRP16 wild type (WT) or FxxP mutant (AxxA) (n=3), FLAG-tagged MCE1 full length or MCE1Δ585–603 (n= 5) or FLAG-tagged CDC25B WT or AxxA to YFP-tagged PPP4R3A or PPP4R3B in HeLa cells (n=3). PP4 interactors were transfected into stable cell lines expressing YFP-tagged PP4 components and purifications analyzed by western blot. Below is indicated the conservation of the FxxP and MxPP motifs analyzed. Inputs for purifications are in Supplemental Figure 2C. E) Schematic of CCDC6 and conservation of FxxP motif. F) Binding affinity for indicated peptides measured by ITC and using the EVH1 domain of PPP4R3A. G) Interaction of FLAG-tagged CCDC6 constructs with PPP4R3A-YFP in cells. The indicated FLAG-tagged CCDC6 constructs were transfected into a HeLa cell line stably expressing inducible PPP4R3A-YFP and complexes were affinity purified using a YFP affinity resin (n=3). H) The indicated YFP-CCDC6 constructs were transfected into HeLa cells and analyzed by live cell microscopy. Bar is 10 μM. The intensity of YFP signal in nucleus and cytoplasm was measured and their ratio was calculated. Representative images are shown and each dot in the plot represents a single cell analyzed. Median distribution of YFP signal is indicated by red line. Significance was determined using an unpaired t-test. See also Figure S2, Figure S3, Table S2, Table S3, Table S5 and Data S1.

Figure 4.. Direct binding of PP4 to WAPL is required for cohesin release

A) Schematic of WAPL and conservation of FxxP motif. Residues dephosphorylated in a FxxP-dependent manner are indicated in red. B) Coomassie stained SDS-PAGE gel of GST-WAPL 375–625 WT or AxxA used for ITC experiments with the EVH1 domain from PPP4R3A. ITC measurements (n=2) with black curve indicating data and fitting for GST-WAPL 375–625 and red fitting for GST WAPL 375–625 AxxA. C) Interaction between PPP4R3B and RFP-tagged WAPL WT or AxxA mutant. RFP-tagged WAPL constructs were transfected into HeLa cells stably expressing inducible YFP or YFP-PPP4R3B and then an affinity YFP resin was used to purify complexes. A representative blot from 4 independent experiments is shown. D) WAPL WT and mutant (AxxA) phosphorylation was analyzed by Phos-tag or regular SDS-PAGE in asynchronous, S-phase, and mitotic cells. A representative blot from 6 independent experiments is shown. E) Representative images of chromosome spreads from the indicated conditions. Chromosomes were scored based on four categories as indicated and fraction of cells with type I or II morphology calculated. At least 100 cells per condition per experiment were scored. Error bars represent SD. Significance was determined using an unpaired t-test. F) Analysis of anaphase bridges by immunofluorescence in the indicated conditions and using cells stably expressing YFP tagged WAPL WT or AxxA. The fraction of cells showing anaphase bridges was calculated for each condition. At least 100 cells per condition per experiment were scored. Error bars represent SD. Significance was determined using an unpaired t-test. G) Purification of YFP-tagged WAPL proteins from asynchronous cells using an YFP affinity resin and analyzing the binding to cohesin components, RAD21 and SMC3 was analyzed by using quantitative western blotting. The level of RAD21 and SMC3 was set to 1 for WAPL WT purifications. Error bars represent SEM. Significance was determined using an unpaired t-test. See also Figure S4 and Table S3.