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First published online April 1, 2009
doi: 10.1242/10.1242/jcs.039552

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PP2A regulates BMP signalling by interacting with BMP receptor complexes and by dephosphorylating both the C-terminus and the linker region of Smad1

Luiza Bengtsson^1,*,, Raphaela Schwappacher^1,*, Martin Roth^2,, Jan H. Boergermann¹, Sylke Hassel² and Petra Knaus^1,¶

¹ Institute of Chemistry and Biochemistry, FU Berlin, Thielallee 63, 14195 Berlin, Germany
² Department of Physiological Chemistry II, University of Wuerzburg, Am Hubland, 97074 Wuerzburg, Germany

View larger version (22K): [in this window] [in a new window]   Fig. 1. PP2A-B subunit interacts with BMP/TGFβ receptors. (A) PP2A-B interacts with BMPRII-tail and BMPRII-SF. Recombinant GST-BMPRII-tail, GST-BMPRII-SF or GST alone was incubated with C2C12 cell lysates. Bound proteins were separated by SDS-PAGE and PP2A-B was detected by immunoblotting with anti-PP2A-B antibody (upper panel). Lane 1 shows the expression of PP2A-B subunit in C2C12 cells. Lower panel shows the input of total GST fusion protein. (B) Endogenous PP2A-B subunit interacts with endogenous BMP and TGFβ receptors. C2C12 cell lysate was incubated with the indicated antibodies which were covalently bound to protein-A-Sepharose. Lane 1, the PP2A-B subunit expression level; lane 2, unrelated antibody with subsequent protein-A-Sepharose incubation; lane 3, protein-A-Sepharose alone; lane 4, anti-TGFβRI; lane 5, anti-TGFβRII; lane 6, anti-BMPRIa; lane 7, anti-BMPRII). Precipitates were analyzed using SDS-PAGE and immunoblotting with anti-PP2A-B antibody.

View larger version (38K): [in this window] [in a new window]   Fig. 2. Different PP2A-B subunits interact with BMPRII independently of receptor kinase activity and of the PP2A catalytic activity. (A) The association of PP2A-Bβ subunit with BMPRII is not inhibited by a kinase-dead mutation in BMPRII. HEK293T cells were transfected with the indicated constructs, lysed and incubated with anti-PP2A-B antibody. Co-precipitated BMPRII-LF (lane 6), BMPRII-LF-K230R (lane 7) and BMPRII-SF (lane 8) were analyzed by SDS-PAGE followed by immunoblotting with anti-HA antibody. Mock-transfected cells (lane 5) and incubation with protein-A-Sepharose beads alone (lanes 9-12) served as controls. An aliquot of each lysate was used to control for protein expression with anti-HA antibody (lanes 1-4). The lower panel shows a shorter exposure of immunoprecipitated PP2A-Bβ. The asterisk indicates an nonspecific band. (B,C) PP2A-B-BMPRII interaction is not PP2A-B isoform-specific and does not depend on phosphatase activity of PP2A. HEK293T cells were transfected with the indicated constructs. Immunoprecipitation was performed by incubation with (B) anti-FLAG antibody to precipitate PP2A-B (lanes 6 and 7) and PP2A-B-26-38 (lanes 8 and 9) or (C) anti-BMPRII antibody covalently linked to protein-A-Sepharose to precipitate BMPRII coexpressed with PP2A-B. After separation by SDS-PAGE, protein complexes were examined by immunoblotting with (B) anti-MYC antibody (lower panel) or (C) anti-HA antibody (upper panel). Expression controls in B used anti-MYC and anti-FLAG antibodies (lanes 1-5), and in C anti-HA antibody. The asterisks indicate nonspecific bands.

View larger version (27K): [in this window] [in a new window]   Fig. 3. PP2A dephosphorylates BMPRII. (A) Scheme of the in vitro phosphorylation-dephosphorylation assay of BMPRII. (B) Immunoprecipitated BMPRII-LF was subjected to in vitro phosphorylation using [-32P]ATP. The dephosphorylation assay was started by the addition of recombinant PP2A to the phosphorylated receptor. Incorporation and removal of [-32P]ATP was monitored by autoradiography (upper panel), and the amount of the bead-coupled BMPRII-LF by immunoblotting with anti-HA antibody (lower panel). (C) Quantification of the results in B. Phosphorylation of BMPRII-LF was quantified relative to the amount of BMPRII-LF protein using ImageJ. (D) Scheme of the in vivo and in vitro phosphorylation-dephosphorylation assay of BMPRIa. (E) After BMP2 stimulation, phospho-BMPRIa was immunoprecipitated and subjected to in vitro dephosphorylation by recombinant PP2A. Serine phosphorylation of the receptor was measured by immunoblotting (upper panel). Lower panel shows the amount of bead-coupled BMPRIa.

View larger version (40K): [in this window] [in a new window]   Fig. 4. PP2A dephosphorylates BMP-Smads. (A) BMP2-induced C-terminal Smad1/5/8 phosphorylation is attenuated by PP2A-Bβ. C2C12 cells were transfected with HA-tagged PP2A-Bβ (lanes 1 and 2) or empty vector (lanes 3 and 4) and stimulated with BMP2 (lanes 2 and 4) or left untreated (lanes 1 and 3). Cell lysates were analyzed by immunoblotting for phospho-Smad1/5/8 levels. β-actin was used as a loading control (lower panel) and anti-HA antibodies as a control for PP2A-Bβ expression (middle panel). The asterisk indicates a nonspecific band. Numbers below the lanes are the ratio of phospho-Smad to actin signal quantified by ImageJ (also in B,C and E). (B) BMP2-induced Smad1/5/8 phosphorylation is enhanced by inhibition of PP2A. C2C12 cells were stimulated with BMP2 (lane 2) or BMP2 and okadaic acid (lane 4) or okadaic acid (lane 3) or left untreated (lane 1). After SDS-PAGE, cell lysates were immunoblotted with anti-p-Smad1/5/8 (C-term.) antibody (upper panels). Equal loading was monitored by anti-β-actin antibody (lower panel). (C) PP2A-Bβ overexpression induces dephosphorylation of the linker region of Smad1. C2C12 cells overexpressing HA-tagged PP2A-Bβ (lanes 5-8) or empty vector (lanes 1-4) were starved and treated without ligand (lanes 1 and 5), with BMP2 (lanes 2 and 6), with BMP2 and okadaic acid (lanes 3 and 7) or okadaic acid alone (lanes 4 and 8). The lysates were analyzed by SDS-PAGE and immunoblotting for the phosphorylated linker region of BMP-Smads using an anti-MAPK substrate antibody (anti-p linker; panel a). Panel b shows a longer exposure of the anti-p-linker blot. Expression and loading was controlled for using anti-HA antibody (panel c), anti-β-actin antibody (panel d), and anti-Smad1 antibody (panel e). (D) Control of downregulation of endogenous PP2A-A using RNAi. The mRNA levels of PP2A in C2C12 cells transfected with either control non-silencing or PP2A-A-specific shRNAs were examined by RT-PCR using PP2A-A subunit-specific oligonucleotides. β-actin served as control. (E) Downregulation of A subunit enhances Smad linker phosphorylation. C2C12 cells were transfected with a plasmid encoding shRNA against PP2A-A. Level of Smad linker phosphorylation was monitored as in C. (F) CDK1 and ERK2 phosphorylate Smad1 in vitro. The upper panel shows the autoradiography of phosphorylated Smad1 after treatment with CamKII (lane 1), CDK1 (lane 2) and ERK2 (lane 3). The lower panel shows the amount of Smad1 in the sample. (G) PP2A dephosphorylates Erk2-mediated linker phosphorylation of Smad1 in vitro. MBP-Smad1 immobilized to amylose resin was in vitro phosphorylated. MBP alone served as control for nonspecific phosphorylation (lanes 7-9). PP2A-mediated dephosporylation was started by the addition of PP2A to the samples (lanes 4-6). Following SDS-PAGE, Smad linker phosphorylation was analyzed using immunoblot anti-p linker (panels a and b). Anti-Smad1 (panel c) and anti-MBP antibody (panel d) show the amount of MBP-Smad1 and MBP in each sample. (H) PP2A dephosphorylates BMP2-mediated C-terminally and linker-phosphorylated Smad1 in vitro. After BMP2 treatment, endogenous Smad1 was immunoprecipitated and subjected to in vitro dephosphorylation using recombinant PP2A. The precipitates were analyzed by SDS-PAGE and immunoblotting with anti-P-C-terminal and anti-p-linker antibodies. Anti-Smad1 immunoblotting monitors the amount of Smad1 in each sample. The percentages of the PP2A-mediated reduction in phosphorylation are given on the right. The intensities of the phospho signals and the Smad1 signals of a representative experiment were measured with ImageJ.

View larger version (73K): [in this window] [in a new window]   Fig. 5. Inhibition of PP2A represses Smad nuclear translocation. C2C12 cells were either left untreated, stimulated with BMP2, BMP2 and okadaic acid or okadaic acid alone, as indicated. After cell fixation and permeabilization, localization of endogenous Smad1 and co-Smad4 was detected using anti-Smad1 (upper panels) and anti-Smad4 antibodies (lower panels).

View larger version (34K): [in this window] [in a new window]   Fig. 6. PP2A is a BMP-Smad linker phosphatase. (A) Different PP2A-B subunits together with PP2A catalytic subunit enhance the activity of a BMP2-regulated promoter. C2C12 cells were transfected with the indicated constructs (empty vector, lanes 1 and 2; PP2A-Bβ-HA, lanes 3 and 4; PP2A-B-FLAG, lanes 5 and 6; PP2A-B-26-38-FLAG, lanes 7 and 8) and co-transfected with BRE-luc and a reference reporter. Cells were starved and stimulated with BMP2 (black columns) or left untreated (grey columns) and the luciferase activity was measured. An aliquot of each lysate was taken to monitor expression of PP2A-B subunits using anti-HA and anti-FLAG antibodies. Standard deviations result from the mean of two measurements, the result was reproduced in three independent experiments. (B) Inhibition of PP2A activity partially reverses the positive regulative effect of the B-subunits. The experiment was carried out as in A. Cells were starved and stimulated with BMP2 (black columns), BMP2 and okadaic acid (light grey columns), okadaic acid alone (white columns) or left untreated (dark grey columns). Luciferase activities were measured as described above and samples overexpressing empty vector or PP2A-Bβ-HA were pooled to determine PP2A-Bβ expression by anti-PP2A-B antibody. Standard deviations result from the mean of six measurements from two experiments. (C) Downregulation of A subunit enhances the activity of a BMP2-regulated promoter. Cells expressing shRNAs against PP2A-A were assayed as in A. (D) PP2A has different signalling outcomes on Smad1 linker and C-terminal mutants. Cells expressing the indicated constructs were analyzed as in A.

View larger version (25K): [in this window] [in a new window]   Fig. 7. Model for mechanism of action of PP2A in the regulation of BMP signalling. (A) Schematic summary of our results. (B) Upon BMP2 stimulation, both MAPK and BMPRI become activated and phosphorylate R-Smads in the linker region and at the C-terminus, respectively. PP2A targeted to receptor complexes and Smads via its regulatory subunit causes slight dephosphorylation of the C-terminus and a more thorough one of the linker region. Only the linker-dephosphorylated Smad complexes can translocate into the nucleus and activate BMP target genes.

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