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. 2008 Oct 24;283(43):29273-84.
doi: 10.1074/jbc.M803443200. Epub 2008 Aug 20.

PP4R4/KIAA1622 Forms a Novel Stable Cytosolic Complex With Phosphoprotein Phosphatase 4

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Free PMC article

PP4R4/KIAA1622 Forms a Novel Stable Cytosolic Complex With Phosphoprotein Phosphatase 4

Ginny I Chen et al. J Biol Chem. .
Free PMC article

Abstract

Protein serine/threonine phosphatase 4 (PP4c) is an essential polypeptide involved in critical cellular processes such as microtubule growth and organization, DNA damage checkpoint recovery, apoptosis, and tumor necrosis factor alpha signaling. Like other phosphatases of the PP2A family, PP4c interacts with regulatory proteins, which specify substrate targeting and intracellular localization. The identification of these regulatory proteins is, therefore, key to fully understanding the function of this enzyme class. Here, using a sensitive affinity purification/mass spectrometry approach, we identify a novel, stable cytosolic PP4c interacting partner, KIAA1622, which we have renamed PP4R4. PP4R4 displays weak sequence homology with the A (scaffolding) subunit of the PP2A holoenzyme and specifically associates with PP4c (and not with the related PP2Ac or PP6c phosphatases). The PP4c.PP4R4 interaction is disrupted by mutations analogous to those abrogating the association of PP2Ac with PP2A A subunit. However, unlike the PP2A A subunit, which plays a scaffolding role, PP4R4 does not bridge PP4c with previously characterized PP4 regulatory subunits. PP4c.PP4R4 complexes exhibit phosphatase activity toward a fluorogenic substrate and gammaH2AX, but this activity is lower than that associated with the PP4c.PP4R2.PP4R3 complex, which itself is less active than the free PP4c catalytic subunit. Our data demonstrate that PP4R4 forms a novel cytosolic complex with PP4c, independent from the complexes containing PP4R1, PP4R2.PP4R3, and alpha4, and that the regulatory subunits of PP4c have evolved different modes of interaction with the catalytic subunit.

Figures

FIGURE 1.
FIGURE 1.
Identification of interacting partners for FLAG-PP4c. A, HEK293 cells were stably transfected with pcDNA3-FLAG-PP4c or pcDNA3-FLAG alone. After G418 selection, equal amounts of lysate were separated by SDS-PAGE, and immunoblots (IB) were performed using an anti-PP4c antibody. Arrows indicate the positions of endogenous and epitope-tagged PP4c. B, Venn diagram showing the overlap between the interactors identified from TAP-PP4c purifications (gray; as per Gingras et al. (18)) and the FLAG-PP4c purifications (see Table 1 for details).
FIGURE 2.
FIGURE 2.
PP4R4, a novel PP4c interactor, is a ∼100-kDa protein resembling the PP2A A subunit. A, domain architecture similarities between human PP4R4, PP2A Aα, and PP4R1. HEAT repeats are indicated by black boxes. The percentage identity between PP4R4 and the other HEAT repeats over the shaded portion of the sequence is indicated. The PP2A Aα HEAT repeats responsible for interaction with the catalytic subunit and the B′γ subunit are indicated. aa, amino acids. B, chemically synthesized siRNAs directed against PP4R4 were transfected into HEK293 cells. 72 h after transfection, cells were collected. Equal amounts of lysate from each sample were separated by SDS-PAGE, and the proteins were transferred to nitrocellulose. Crude antiserum from a rabbit injected with glutathione S-transferase-PP4R4 C terminus was used for immunoblotting (IB). The arrow indicates the position of the ∼100-kDa band, which disappears after transfection of PP4R4 siRNAs. C, anti-PP4R4 was used in immunoblotting of lysate from untransfected cells (left) or cells stably expressing FLAG-PP4R4 (SDS-7.5%-PAGE). D, effect of silencing PP4c on the expression levels of PP4R4. U2OS cells transfected with PP4c (or PP4R4, two independent siRNAs) siRNAs were collected after 40 h and processed for immunoblotting analysis as in B using antibodies to PP4c or PP4R4. The position of the proteins is indicated. E, anti-PP4R4 or preimmune serum was used to immunoprecipitate (IP) endogenous PP4R4 from mouse brain lysate; the immune complexes were separated via SDS-PAGE, transferred to nitrocellulose, and subjected to immunoblotting with the PP4R4 antiserum or with an antiserum reactive to PP4c (Bethyl A300–835A). The position of PP4c is indicated.
FIGURE 3.
FIGURE 3.
PP4R4 specifically interacts with PP4c. A, 293T cells were co-transfected with HA-PP4R4 and FLAG-tagged catalytic subunits as indicated. Cells were harvested 48 h post-transfection, and the levels of HA-PP4R4 in the lysates were monitored by immunoblotting (IB) with the anti-HA antibody. FLAG-tagged phosphatases were next isolated on anti-FLAG M2-Sepharose beads. HA-PP4R4 was detected by immunoblotting with anti-HA, whereas the FLAG-tagged phosphatases were analyzed with anti-FLAG antibody. IP, immunoprecipitate. B, anti-FLAG pulldown was performed from HEK293 cells stably expressing FLAG alone, FLAG-PP2Acα, and FLAG-PP4c, and an immunoblot for endogenous PP4R4 was performed. FLAG-PP4R4 lysate was utilized to confirm the location of the protein. C, the catalytic activity of PP4c is not required for interaction with PP4R4. PP4c wt, H56Q, or R86A were co-expressed in 293T cells with HA-PP4R4 and recovered on FLAG-agarose. Immunoblotting with anti-HA was performed. The wild type protein used for this figure, PP4c, migrates faster than the mutants (and the wild type protein used in Fig. 6), as it was cloned in our older version of the pcDNA3-FLAG vector, which has a shorter linker. This linker does not affect the binding properties of the protein. D, 293T cells were co-transfected with TAP-tagged regulatory subunits and FLAG-tagged catalytic subunits. TAP-eIF4E was used as negative control. Cells were collected 48 h post-transfection. FLAG-PP4c and FLAG-PP2Acα were isolated on anti-FLAG beads, and the protein A moiety of the TAP-tagged proteins was detected by incubation with normal rabbit serum (the rabbit IgGs bind to protein A) followed by detection using donkey anti-rabbit IgG conjugated to horseradish peroxidase.
FIGURE 4.
FIGURE 4.
PP4R4 is a cytoplasmic regulatory subunit for PP4c. A, localization of PP4R4 by indirect immunofluorescence. Vero cells were transfected with HA-tagged constructs as indicated; 24 h post transfection, cells were fixed, permeabilized, and stained with anti-HA antibody, as described under “Experimental Procedures.” B, model depicting multiple PP4c-containing stable complexes. PP4R1, PP4R2-PP4R3, and PP4R4 are all specific interactors for PP4c. α4-CCT interacts with both PP2Ac and PP4c. Although PP2A A-PP2A B′δ exhibits a stronger affinity for PP2Ac, these proteins also weakly interact with PP4c.
FIGURE 5.
FIGURE 5.
Differential activity of PP4 subcomplexes. A, Sf9 cells were infected with the indicated combinations of baculoviruses. Protein complexes were purified via FLAG immunoprecipitation (IP), resolved on SDS-PAGE, and visualized by colloidal blue. B, phosphatase activity of purified complexes in A was monitored using a DiFMUP fluorogenic substrate. RFU indicates relative fluorescence units. Levels of Strep-PP4c, FLAG-PP4R4, and FLAG-PP4R2 were determined using anti-PP4c and anti-FLAG antibodies. Note that comparable amounts of Strep-PP4c were used for the phosphatase assay. IB, immunoblot. C, increasing amounts of the purified complexes were incubated with acid-washed histones isolated after γ-irradiation. The relative levels of phosphorylation on H2AX were monitored by immunoblotting with a γH2AX antibody, whereas the levels of Strep-PP4c, FLAG-PP4R4, or FLAG-PP4R2 in the reactions were detected by immunoblotting for PP4c or the FLAG epitope. D, Sf9 cells were infected with baculovirus expressing Strep-PP4c with increasing amount of FLAG-PP4R2. The protein phosphatases were isolated via the StreptagII moiety and analyzed by SDS-PAGE and colloidal blue. E, phosphatase activity in the samples from D was determined using DiFMUP as a substrate, as described in B. Less phosphatase was used for this experiment.
FIGURE 6.
FIGURE 6.
Effect of mutation of the PP4R4 or PP4c residues corresponding to the PP2Ac·PP2A A interface on complex formation. A, 293T cells were transiently transfected with FLAG-PP4R4 wild type or point mutants. The levels of PP4c and the PP4R4 mutants in the lysates were monitored by immunoblotting (IB) with PP4c and FLAG antibodies, respectively. FLAG-tagged proteins were next precipitated (IP), and co-precipitation of endogenous PP4c was monitored using an anti-PP4c antibody. B, FLAG-tagged PP4c mutants were transiently transfected in 293T cells, and lysates were prepared 48 h post-transfection. Immunoprecipitation on anti-FLAG-Sepharose beads was performed, and the immune complexes were resolved by SDS-PAGE followed by transfer onto nitrocellulose. Co-precipitation of endogenous PP4R1, PP4R2, and PP4R4 were detected using antibodies to the endogenous proteins.

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