PP2A-B' holoenzyme substrate recognition, regulation and role in cytokinesis

Abstract

Protein phosphatase 2A (PP2A) is a major Ser/Thr phosphatase; it forms diverse heterotrimeric holoenzymes that counteract kinase actions. Using a peptidome that tiles the disordered regions of the human proteome, we identified proteins containing [LMFI]xx[ILV]xEx motifs that serve as interaction sites for B'-family PP2A regulatory subunits and holoenzymes. The B'-binding motifs have important roles in substrate recognition and in competitive inhibition of substrate binding. With more than 100 novel ligands identified, we confirmed that the recently identified LxxIxEx B'α-binding motifs serve as common binding sites for B' subunits with minor variations, and that S/T phosphorylation or D/E residues at positions 2, 7, 8 and 9 of the motifs reinforce interactions. Hundreds of proteins in the human proteome harbor intrinsic or phosphorylation-responsive B'-interaction motifs, and localize at distinct cellular organelles, such as midbody, predicting kinase-facilitated recruitment of PP2A-B' holoenzymes for tight spatiotemporal control of phosphorylation at mitosis and cytokinesis. Moroever, Polo-like kinase 1-mediated phosphorylation of Cyk4/RACGAP1, a centralspindlin component at the midbody, facilitates binding of both RhoA guanine nucleotide exchange factor (epithelial cell transforming sequence 2 (Ect2)) and PP2A-B' that in turn dephosphorylates Cyk4 and disrupts Ect2 binding. This feedback signaling loop precisely controls RhoA activation and specifies a restricted region for cleavage furrow ingression. Our results provide a framework for further investigation of diverse signaling circuits formed by PP2A-B' holoenzymes in various cellular processes.

Keywords: CIP2A; PP2A-B′ holoenzyme; SLiMs; centrosome; cytokinesis; midbody.

Figure 1

Identification of SLiMs containing peptides that bind to the B′ regulatory subunits and holoenzymes by ProP-PD selection. (a) Logos and consensus sequences of binding motifs for B′ regulatory subunits and holoenzymes identified by ProP-PD. Dots indicate that no strong preference of amino acids was found at the corresponding positions. (b) Venn diagrams indicating overlaps among peptides enriched from ProP-PD selection of B′γ1, B′α, B′γ1 holoenzyme (PP2A-B’γ1) and B′ε holoenzyme (PP2A-B’ε). (c) Structural illustration of substrate motif binding and placement of phosphorylation (Pi) sites surrounding the motif to the active site of PP2A-B′ holoenzyme. The structure of a consensus motif (magenta) from BubR1 bound to B′γ1 (yellow; PDB code: 5jja) was overlapped to structure of PP2A holoenzyme (PDB code: 2NPP). The dashed lines and stars stand for peptide fragments and phosphorylation sites upstream and downstream of the B′-interaction motif. N and C stand for N-terminal and C- terminal sides of the bound motif.

Figure 2

Binding assays of PP2A B′-binding motifs with B′ subunits and PP2A holoenzymes. (a) Pull-down PP2A-B′α (left) and PP2A-B′γ1 (right) holoenzyme via GST-tagged peptides suggested as PP2A B′ ligands by ProP-PD. Proteins associated with GS4B resins were examined by SDS-polyacrylamide gel electrophoresis and visualized by Coomassie blue staining. (b) Association and dissociation curves of binding between a representative GST-tagged peptide (DENND2C) to B′α (blue), B′γ1 (red) and PP2A-B′γ1 holoenzyme (green) detected by BLI. The calculated results for DENND2C and diverse other B′-interaction motifs identified by ProP-PD are summarized in the table below. (c) Co-immunoprecipitation (co-IP) of FLAG- and V5-tagged B′γ1 substrates identified by ProP-PD or bioinformatic prediction after co-transfection of FLAG-SENP6, FLAG-USP53, V5-CIP2A and FLAG-GLI2 with YFP-tagged B′γ1 in HEK293 cells. Lanes 1 and 2 show the blotting of indicated proteins of 20% amount of cell lysates used for co-IP.

Figure 3

The interaction between CIP2A and PP2A-B′ holoenzymes reveals the mechanism of CIP2A in inhibition of PP2A-B′ holoenzyme function. (a) Schematic representation of protein domains of CIP2A and the location of its B′-binding motif. (b) Pull-down assays between GST-tagged B′-binding motif (GST-DENND2C and GST-SYT16) and PP2A-B′γ1 holoenzyme was blocked by increasing concentrations of synthetic peptides of the B′-binding motif of CIP2A. Proteins associated with GS4B resins were examined similar to Figure 2a. (c) Isothermal titration calorimetry measured the binding affinity between CIP2A synthetic peptide and B′γ1. The result showed a direct but weak interaction between CIP2A and B′γ.

Figure 4

Plk1-mediated Cyk4 phosphorylation facilitates Cyk4 interaction with PP2A-B′. (a) Schematic representation of protein domains of Cyk4, and the locations of B′-binding motif, mitotic kinesin-like protein 1 (MKLP1)-binding region, the Plk1 phosphorylation sites (red) and Ect2-binding motif. Locations of coiled-coil (CC), C1 and RhoGAP domains on Cyk4 are indicated. (b) Pull down of GST-pCyk4 (1–177) with B′ε, and PR70 via GS4B resin. Proteins associated with GS4B resins were examined similar to Figure 2a. (c) Gel filtration chromatography of B′γ1 alone and its mixture with Cyk4 (1–177; left panel) and pCyk4 (1–177; right panel). B′γ1 co-migrates with pCyk4 (1–177), but not with Cyk4 (1–177). (d) Pull down of GST-B′γ1 with titrated concentrations of Cyk4 (1–177; left) and pCyk4 (1–177; right). The results showed that B′γ1 has a very weak binding with Cyk4 (1–177) while the binding is drastically increased upon Plk1-catalyzed phosphorylation (pCyk4; 1–177). (e) Top-down MS characterized the phosphorylation sites of Cyk4. MS shows differential phosphorylation of Cyk4 (left). The tetrakisphosphorylated Cyk4 was further identified by tandem MS (right). The sequence map of Cyk4 and its fragments identified by CID (red) and ECD (blue) were indicated. The four phosphorylation sites were localized to Ser151, Ser159, Ser166 and Ser172 based on 1 CID and 1 ECD spectra as highlighted. p, 2p, 3p, 4p and 5p represent mono-, bis-, tris-, tetrakis- and pentakis-phosphorylated CYK4, respectively. (f) Isothermal titration calorimetry measured the binding affinities between B′γ1 and original (left panel) or phosphomimetic Cyk4 peptides (S149E, middle panel; S149E/S151E, right panel).

Figure 5

Plk1-dependent PP2A–Cyk4 interaction and the role of B′ in RhoA activation and cleavage furrow progression. (a) Immunostaining revealed the cellular localizations of PP2Ac, A-subunit and B′γ1 regulatory subunits at the dark zone and bulge region of the midbody, similar to Cyk4. (b) Co-immunoprecipitation (Co-IP) of FLAG-Cyk4 with PP2Ac in the presence and absence of PIk1 inhibitor (BI-2536) in the presence of recombinant V5-CIP2A. The interactions were examined at different time points (0–90 min) after removal of monastrol that synchronized the Hela cells at anaphase. PP2Ac was blotted to reflect the interactions between PP2A-B′ holoenzymes and Cyk4. (c) Pull down of pCyk4 by GST-Ect2 (left) and gel filtration chromatography of the pCyk4–Ect2 complex (right) before and after treatment by PP2A-B′ε holoenzymes. (d) Immunostaining to examine the cellular structures of RhoA activation zone, α-tubulin and dividing chromosomes after B′β/B′γ1/B′ε siRNA knockdown for cells at mitotic anaphase 24 h after siRNA transfection. Measurements of distance between separating chromosomes, diameter of cleavage furrow and the RhoA active zone for cells at mitotic anaphase 24 h after transfection of B′β/B′γ1/B′ε siRNA were shown below. Results were compared with cells transfected with control siRNA. (e) Cartoon illustrating the signaling circuit formed by Plk1 and PP2A-B′ holoenzymes in controlling Cyk4 phosphorylation and Ect2 recruitment at the midbody and subsequent control of RhoA activation and cleavage furrow ingression.