Cell cycle dependent association of EBP50 with protein phosphatase 2A in endothelial cells

Abstract

Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 (EBP50) is a phosphorylatable PDZ domain-containing adaptor protein that is abundantly expressed in epithelium but was not yet studied in the endothelium. We report unusual nuclear localization of EBP50 in bovine pulmonary artery endothelial cells (BPAEC). Immunofluorescent staining and cellular fractionation demonstrated that EBP50 is present in the nuclear and perinuclear region in interphase cells. In the prophase of mitosis EBP50 redistributes to the cytoplasmic region in a phosphorylation dependent manner and during mitosis EBP50 co-localizes with protein phosphatase 2A (PP2A). Furthermore, in vitro wound healing of BPAEC expressing phospho-mimic mutant of EBP50 was accelerated indicating that EBP50 is involved in the regulation of the cell division. Cell cycle dependent specific interactions were detected between EBP50 and the subunits of PP2A (A, C, and Bα) with immunoprecipitation and pull-down experiments. The interaction of EBP50 with the Bα containing form of PP2A suggests that this holoenzyme of PP2A can be responsible for the dephosphorylation of EBP50 in cytokinesis. Moreover, the results underline the significance of EBP50 in cell division via reversible phosphorylation of the protein with cyclin dependent kinase and PP2A in normal cells.

Figure 1. Nuclear localization of EBP50 in BPAEC.

A) Immunofluorescence staining of confluent BPAEC (a–c,g–i), HUVEC (d–f), MCF7 (j–l) and pCMV-myc EBP50 transfected BPAEC (m–o), and HeLa (p–r) cells was performed using anti-EBP50 (anti-SLC9A3R1 antibody, Abgent) (a,j: red, d: green), anti-tubulin (b,k: green), anti-NHERF2 (g: green), and monoclonal anti-c-myc (m,p: green) primary antibodies. Actin microfilaments were stained with Texas Red conjugated phalloidin (e,h, n,q: red). c,f,i,l,o and r are merged images of a–b, d–e, g–h, j–k, m–n, and p–q, respectively. Representative data of at least three independent experiments are shown. Scale bars: 100 µm. B) Cellular fractionations of BPAE and MCF7 cells were made as described in Materials and Methods. The fractions were analyzed with anti-EBP50 (anti-SLC9A3R1 antibody Abgent), anti-β-tubulin as a cytoplasmic and anti-lamin A/C antibodies as a nuclear marker. CL: cell lysate, CP1: cytoplasmic fraction 1, CP2: cytoplasmic fraction 2, N: nuclear fraction.

Figure 2. Localization of EBP50 during the phases of the cell cycle.

Immunofluorescence staining of BPAE cells was performed using anti-EBP50 (red) primary antibody (anti-SLC9A3R1 antibody, Abgent). Phases of the cell cycle were identified by tubulin (green) and DAPI (blue) staining. Representative images from five independent experiments are shown. Scale bar: 100 µm.

Figure 3. Localization of EBP50 is phosphorylation-dependent in BPAEC.

pCMV-Myc EBP50 wild type (wtEBP50) and pCMV-Myc EBP50 S288D∶S310D phosphomimic mutant (muEBP50) proteins were analysed by Western blot (A) and immunofluorescence (B). Anti-myc (green) antibody was used for labeling of wild type and mutant EBP50 both in Western blot and immunofluorescent experiments. Actin microfilaments were stained with Texas Red conjugated phalloidin (red) and DAPI (blue) staining was used to visualize the nuclei. Scale bar: 100 µm. Panel C: Phosphorylation level of EBP50 in synchronized BPAE cells. Cells were arrested in G1/S phase using double thymidine block and in mitotic phase by nocodazole treatment as described in Materials and Methods. Samples of asynchronized cells (AS), 1st and 2nd thymidine block cells (G1/S), and cells after 3 or 8 h release of the thymidine block without or with addition of 80 ng/ml nocodazole (ND) were tested for EBP50 (anti-NHERF1(A310) antibody, Cell Signaling Technology) by Western blot. Representative images from at least three independent experiments are shown.

Figure 4. Phospho-mimic EBP50 supports wound healing.

BPAEC were transfected with pCMV-Myc EBP50 wild type (wt EBP50) and pCMV-Myc EBP50 S288D∶S310D phosphomimic mutant (mu EBP50) and were plated onto two 8W10E arrays 24 h post-transfection. After cells achieved monolayer density (about 1000 Ω impedance) an alternate current of 5 mA at 60 kHz frequency was applied for 30 sec duration to establish wounds in the cell layer (0 h); after that the impedance was measured for 5 h. The average results from three independent experiments each made with two or three parallel measurements are shown. Error bars represent SD.

Figure 5. PP2A subunits associate with cellular EBP50.

EBP50 (panel A) or PP2A C subunit (panel B) was immunoprecipitated from lysates of thymidine or nocodazole treated BPAEC as described in Materials and Methods. The IP complexes were probed for PP2Aa, PP2Ac, and PP1c (α and δ) (A) or EBP50 (anti-NHERF1(A310) antibody, Cell Signaling Technology) and PP2Ac subunit (B). Ø: cell lysate without immunoprecipitation, AS: asynchonized cell lysate, S: early S phase cell lysate, M: mitotic phase lysate. Additional bands at 55 kD are IgG. Representative blots from three independent experiments are shown.

Figure 6. Bα subunit of PP2A interacts with EBP50.

Panels A and B: Bacterially expressed glutathione S-transferase (GST) and GST-tagged wild-type EBP50 were loaded onto glutathione-Sepharose as described in Materials and Methods. After a washing step the resin samples were incubated with BPAEC lysate or cell lysis buffer. Non-binding proteins were washed out and the bound proteins were eluted with 10 mM glutathion. Coomassie staining (A) and Western blot probed with PP2A A, B, B′, or C specific antibodies (B) of the bacterial and endothelial cell lysates (Total) and the eluted fractions after the pull-down are shown. Panel C: BPAEC monolayers were transfected with pcDNA3.1 V5-His (vector ctr), pcDNA3.1 V5-His PP2A Bα (PP2A Bα–V5) and pcDNA3.1 V5-His B′γ (PP2A B′γ–V5) plasmids. Lysates of the transfected cells were incubated with Anti-V5 Agarose Affinity Gel as described in Materials and Methods, and the bound proteins were eluted by boiling the resin in 1× SDS sample buffer. Western blot analysis of the lysates of transfected cells (Total) and eluted samples were done using EBP50 (upper panel) and V5-tag (lower panel) specific antibodies. Additional bands at 55 kD are IgG. Representative data of three independent experiments are shown.

Figure 7. Co-localization of EBP50 and PP2Ac during mitosis in BPAEC.

(A) Immunofluorescence staining of BPAEC was performed using anti-EBP50 (anti-SLC9A3R1 antibody, Abgent) (red) and anti-PP2Ac (green) primary antibodies. Phases of the cell cycle were identified using DAPI staining (not shown). Scale bars: 100 µm. Co-localization of EBP50 (B) or NHERF2 (C) and PP2Ac was evaluated by determination of Pearson cross-correlation coefficient. The results are presented as means ± SD from 50–100 (B) or 25–30 (C) independent cells for each studied phase of the cell cycle. Statistical analysis was done with ANOVA on ranks. Significant changes compared to the interphase cells are indicated by * (P<0.05). I: interphase, P: prophase, PM: prometaphase, M: metaphase, A: anaphase, T: telophase, C: cytokinesis, LC: late cytokinesis.