Model of the ternary complex between PDGFR, NHERF1 and PTEN. NHERF1 oligomerizes through PDZ domain interactions and is presented dimerized in this scheme. NHERF1 interacts through its PDZ1 domain with the PDZ motifs of both PDGFR and PTEN. PTEN dephosphorylates the second messenger PIP₃ that is produced by PI3K. PI3K p85 regulatory subunit in complex with the p110 catalytic subunit is directly recruited to the phospho-tyrosine 751 of PDGFR upon PDGF stimulation. Other proteins recruited to activated PDGFR are not shown.

siRNA depletion of NHERF2 increases the activation of the PI3K pathway. (A) Efficient depletion of NHERF2 in NHERF1(−/−) MEFs by specific siRNA treatment as compared to control scrambled siRNA. (B) Time-course experiment showing the activation of Akt in NHERF1(−/−) MEFs pretreated for 96 h with NHERF2 siRNA or control siRNA. The graphs show the normalized activation of Akt in one representative experiment (left), and the fold difference of normalized Akt activation in NHERF2-depleted and control (Ctr) siRNA-treated cells from similar experiments in three different NHERF1(−/−) MEF lines (right). Asterisks mark statistically significant differences (P<0.005).

Complex formation between PTEN, NHERF proteins and PDGFR. (A) Pull-down of endogenous PTEN and PDGFRβ from mouse brain total lysate (TL) with GST-NHERF1 and NHERF2 mouse (m) and human (h) fusion proteins. (B) Bridging experiments in which the GST-PTEN-CT and GST-PTEN-351 filter-immobilized proteins shown on top of the gels were overlaid in a first step with GST-NHERF1, NHERF2 or buffer, and in a second step with GST-Myc-PDGFR-CT. The binding of PDGFR-CT was revealed by further incubation with Myc antibody, followed by the corresponding secondary antibody. (C) Same experiment as in (B), except that the full-length NHERF1 and NHERF2 used for the first-step overlay were replaced by their corresponding domains schematically shown in Figure 1A. Note bridging of a complex between PTEN and PDGFR by full-length or both PDZ domains of NHERF proteins.

Enhanced ruffle formation and migration of NHERF1(−/−) MEFs stimulated with PDGF. (A) Immunofluorescence analysis of NHERF1(+/+) and (−/−) MEFs showing actin reorganization after treatment with control serum-free medium or 5 ng/ml PDGF-BB for 10 min. Images (× 400) were taken with an Axioskop 40 Zeiss microscope equipped with AxioVision Rel.4.2 software. Solid and open arrowheads indicate peripheral ruffles and circular dorsal ruffles, respectively. (B) Chemotactic migration of NHERF1(+/+) and (−/−) MEFs toward PDGF-BB (concentrations indicated) or EGF (20 ng/ml) gradients. On ordinate, the number of migratory cells, processed as described in Materials and methods, is represented as the photometric intensity of the hematoxylin–eosin staining. The means±standard deviations were calculated from triplicates within the same experiment and asterisks mark statistically significant differences (P<0.05). This experiment was repeated three times with similar results.

In vivo binding between PTEN and NHERF2. (A) FLAG-tagged PTEN-124/3A or pCMV vector was coexpressed in 293T cells with GST-tagged human (h) and mouse (m) NHERF2 or pEBG vector control (that expresses 29 kDa GST protein), as shown. Upper panels show co-immunoprecipitation of FLAG-PTEN with GST-NHERF2 pulled with glutathione (Gt) beads directly from the cell lysates. Lower panels show the reciprocal co-immunoprecipitation where the human and mouse NHERF2 proteins detected by anti-GST antibodies are indicated by lines. (B) Co-immunoprecipitation of endogenous NHERF2 (arrowheads) with PTEN only in the LN18 glioblastoma (GBM) cells that express endogenous PTEN and not in the LN308 PTEN-deficient cells. Both cell lines express PDGFR and were treated (+) or not (−) for 20 min with 50 ng/ml PDGF-BB. (C) Co-immunoprecipitation of endogenous NHERF2 with PTEN in normal mouse lung tissue. TL, total lysate; HC, heavy chain; gIg, matched goat Ig control.

In vitro direct binding between PTEN and NHERF proteins. (A) Diagram of NHERF1 (358 residues) and PTEN (403 residues) constructs. I5, isoform 5; FL, full length; CT, carboxy terminus. (B) PTEN with intact PDZ motif binds to NHERF1-I5. GST pull-down assay with GST, GST-PTEN and GST-PTEN-401 of proteins (0.3 mg) from lysates of 293T cells transfected with vector (pCMV), FLAG-tagged NHERF1-I5 or Myc-tagged ZO-1 protein. TL, control total lysate (30 μg). The filter was re-probed with anti-GST antibody to confirm that equal amounts of GST-fusion proteins were used (not shown). (C) NHERF1-PDZ1 domain binds preferentially to PTEN-3A phosphorylation-deficient mutant. Proteins (0.3 mg) from lysates of 293T cells transfected with FLAG-tagged PTEN wild type or PTEN-3A mutant were incubated with GST-NHERF1 fusion proteins (5 μg) as indicated. The filter was probed with the anti-FLAG M2 antibody. TL, control total lysate (30 μg). (D) Direct binding of PTEN and PDGFR to NHERFs. A 0.5 μg portion of GST-NHERF1 or NHERF2 fusion proteins was separated on gel, transferred to filter and overlaid with 10 μg/ml of either GST-PTEN-CT or GST-Myc-PDGFR-CT. The filters were subsequently probed with PTEN and Myc antibodies, respectively, and then stripped and re-probed with GST antibody.

Association of PTEN, PDGFRβ and NHERF2 in a high MW complex in normal brain. (A) Gel filtration of mouse brain total lysate (TL) from NHERF1(+/+) mice. Shown on top are the MWs of eluted fractions (shown with dots) that were calculated using standard proteins (Amersham). Western blotting was performed using antibodies indicated on the left. Bracket and stars indicate high MW peak and monomeric protein peaks, respectively. (B) Flow chart showing the immunodepletion (Dep) of PTEN from brain TL. In a first step (1), half of the TL was depleted with an antibody against PTEN's CT and half with a matched mouse Ig control (Ctr:mIg). The resulting supernatants (Sn) were depleted in a second step (2) with an antibody against PTEN's NT or with matched goat Ig control (Ctr:gIg). (C) Gel filtration PTEN profile after CT antibody depletion or sequential CT followed by NT (CT → NT) antibody depletion. Same amount of brain total lysate (TL) was loaded on each membrane and matched PTEN antibody and control Ig membranes were processed concomitantly. Note partial depletion of PTEN from the high MW peak (bracket) only by NT antibody. (D) Western analysis with antibodies indicated on the right of proteins immunoprecipitated on beads (IP) or remaining in the supernatants (Sn) after brain TL immunodepletion with the antibodies indicated on top. Note co-immunoprecipitation of NHERF2 and PDGFR with PTEN NT antibody.

Activation of the PI3K pathway in NHERF1(−/−) MEFs. (A) Time-course experiment showing activation of Akt and p70 S6 kinase by stimulation with PDGF-BB (0.5 ng/ml) of NHERF1(+/+) and (−/−) MEFs. The activation shown with phospho-specific antibodies P-Akt and P-p70^S6K was normalized to the corresponding Akt and p70^S6K total protein levels, respectively. The upper graph shows the normalized activation of Akt in NHERF1(+/+) and (−/−) MEFs from one representative experiment. The lower graph shows the fold difference of normalized Akt activation in NHERF1(−/−) (Ko) versus NHERF1(+/+) (wt) cells from three similar experiments. Asterisks mark statistically significant differences (P<0.01) between the fold-difference activation at various time points and the basal fold-difference activation at time 0. (B) Reintroduction of NHERF1 in NHERF1(−/−) MEFs reduces Akt phosphorylation compared to vector control. The graph quantifies the fold difference of normalized Akt activation in control versus NHERF1-reconstituted cells. Mean values±standard deviations are computed from four experiments in three different NHERF1(−/−) cell lines (P<0.0001). (C) Expression of NHERF1-PDZ1 in NHERF1(+/+) MEFs increases Akt activation. The arrowhead indicates endogenous NHERF1. The fold difference of normalized Akt activation in NHERF1-PDZ1-expressing versus control cells is shown as means±standard deviations from three experiments in two different NHERF1(+/+) cell lines (P<0.01). (D) Membrane–cytoplasm fractionation showing increased cytoplasmic PTEN in NHERF1(−/−) MEFs. The graph shows the percent increase of PTEN cytoplasmic (cyt.) levels normalized to actin in three different matched pairs of NHERF1(−/−) (ko) and (+/+) (wt) cells. (E) PDGF stimulation of the same cells as in (A) does not reveal significant differences in the activation of Erk, as detected by phospho-specific P-Erk antibody. (F) Stimulation of NHERF1(+/+) and (−/−) MEFs with 20 ng/ml EGF shows similar activation of the PI3K and Erk pathways in both cell types. The normalized activation of Erk in (E) and Akt in (F) is shown as mean±standard deviation of values from three similar experiments.