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First published online June 8, 2005
doi: 10.1242/10.1242/jcs.02383

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Activation of EGF receptor endocytosis and ERK1/2 signaling by BPGAP1 requires direct interaction with EEN/endophilin II and a functional RhoGAP domain

Cell Signaling and Developmental Biology Laboratory, Department of Biological Sciences, The National University of Singapore, 14 Science Drive 4, Singapore 117543, The Republic of Singapore

View larger version (22K): [in a new window]   Fig. 1. Identification of BPGAP1-interacting partner, EEN/endophilin II via protein precipitation and MALDI-TOF. (A) Full-length BPGAP1 expressed as GST-recombinant coupled to glutathione beads were incubated with either lysis buffer (–) or 293T cell lysates (+) that had been pre-cleared with GST-beads to remove non-specific binding. Bound proteins were resolved by SDS-PAGE and revealed by silver-staining. M, molecular weight marker in kilodalton (kDa). A unique band at 46 kDa (indicated by an arrow) was subjected to trypsin digestion followed by MALDI-TOF analyses as described in Materials and Methods. (B) Protein sequence of EEN with sequence coverage of 34% (underlined) over the protein.

View larger version (27K): [in a new window]   Fig. 2. BPGAP1 interacts with EEN via its PRR. (A) BPGAP1 constructs used for identifying functional interactive domain for EEN. The NNP domain (aa 1-166), NP domain (aa 1-206), PC domain (aa 167-433) and full-length BPGAP1 were expressed as GST-recombinants in E. coli and affinity-purified with glutathione-sepharose beads. NNP, N-terminus with BNIP-2 and Cdc42GAP homology domain (BCH); NP, N-terminus with BCH domain and PRR; PC, C-terminus with GTPase-activating protein domain (GAP) and PRR. (B) BPGAP1 GST-recombinants were prepared and used for pull-down assays with 293T cell lysates as described in Materials and Methods. Blots were stripped and stained with amido black to reveal loading of GST-recombinants. Bound endogenous proteins were detected by EEN antibody. Lysates expressing Flag-EEN are used as positive controls and reference for the specificity of the EEN antibody used in detecting the endogenous EEN.

View larger version (17K): [in a new window]   Fig. 3. The specific PXXP core motif within the PRR of BPGAP1 is indispensable for targeting the SH3 domain of EEN. (A) The three deletion mutants and two site-directed point mutants of BPGAP1. P1, devoid of the entire PRR; P2, retaining sequence PPPT; P3, retaining sequence KTPPPRPPLP; PP, substitution of the proline residues at 184 and 186 with alanines (underlined); P422A, substitution of proline residue at 422 with alanine (underlined). GST-recombinants of these deletion constructs (B) and site-directed mutants (C) were prepared as sepharose beads and used for pull-down assays using 293T cell lysates expressing endogenous EEN. Beads from the pull-down experiments were washed and processed for western analysis using EEN antibody. The antibody specifically detected endogenous EEN at 46 kDa, the expected mobility as verified using the overexpressed Flag-tagged EEN in the cells. (D) Cells were transfected with either the wildtype FL or PP mutant of Flag-tagged BPGAP1 and immunoprecipitated with anti-Flag M2 beads, washed and analyzed for bound endogenous EEN using EEN antibody. (E) Purified EEN from thrombin-cleaved GST recombinant was incubated with sepharose beads conjugated with GST fusions of full-length wildtype, the PP mutant of BPGAP1, or GST control, and bound targets revealed by western blot analyses using EEN antibody. WCL, whole cell lysates. FL, full-length.

View larger version (23K): [in a new window]   Fig. 4. EEN binds to BPGAP1 via its SH3 domain. (A) EEN domains used in binding studies. NT, N-terminal (aa 1-308) and CT, C-terminal (aa 309-368). NT contains a BIN/amphiphysin/Rvsp (BAR) domain and a coiled-coil region; CT contains an SH3 domain. (B) GST-recombinants of various constructs of EEN were prepared as sepharose beads and used for pull-down assays on lysates prepared from 293T cells that expressed Flag-tagged full-length BPGAP1. Beads from the pull-down experiments were washed and processed for western analyses using the Flag antibody. The blot was stripped and stained with amido black to reveal loading of GST-recombinants. (C) Purified EEN SH3 fusion of maltose binding protein, MBP-EEN SH3 or the MBP control were incubated with sepharose beads conjugated to purified GST recombinant proteins of the full-length wildtype, the PP mutant of BPGAP1, or GST control, and bound targets revealed by western blot analyses using MBP antibody. Purified MBP and MBP-EEN SH3 were analyzed with MBP antibody and revealed intact targets used in the direct binding assays. The lower apparent molecular mass for MBP-EEN SH3 compared with the MBP alone was due to the removal of internal lacZ coding sequence upon cloning of the target insert. The blot was stripped and stained by amido black to verify loading of equal amounts of GSTs.

View larger version (126K): [in a new window]   Fig. 5. Effects of BPGAP1 and EEN on EGF receptor endocytosis. HeLa cells were untransfected (A) or transfected with different epitope-tagged expression plasmids coding for wildtype BPGAP1, PP, R232A and/or wildtype EEN and NT, either alone (B) or in combination (C) as indicated. Cells were then made quiescent by serum-removal for 2 hours, followed by treatment with 100 ng/ml EGF for 10 minutes in the presence of monensin. Cells were permeabilized, stained and visualized under confocal fluorescent microcopy as described in Materials and Methods. Untransfected cells in A were labeled with rhodamine-phalloidin to mark the shape of cells and the intracellular vesicles (A-C) indicate uptake of EGF receptor (grey). Cells expressing different constructs of BPGAP1 (red) and EEN (blue) were detected by appropriate anti-Flag, anti-HA or anti-Myc antibodies followed by fluorophore-conjugated secondary antibodies. Images from cells transfected with multiple constructs were merged (Merged1) to show their colocalization (purple). The second merged images (Merged2) illustrate the endocytosed EGF receptor in transfected cells. For the triple transfectants (asterisk), the transfection was optimized to express the wildtype BPGAP1 in all the cells that expressed PP mutant, validated by parallel experiments (data not shown). The intensities of images were enhanced to capture changes in the cell peripheries, including their cell protrusions. Bars, 10 µm.

View larger version (30K): [in a new window]   Fig. 6. BPGAP1 and EEN stimulate EGF receptor endocytosis and ERK1/2 phosphorylation. (A) 293T cells were transfected with vector control or expression plasmids coding for the tagged wildtype or mutant proteins, either alone or in combinations as indicated. They were made quiescent by serum depletion for 2 hours followed by stimulation with 100 ng/ml Oregon-Green-labeled EGF for the times indicated and processed for EGF uptake in the presence of monensin as described in the Materials and Methods. The total amounts of EGF-bound EGF receptor being internalized within certain populations of cells that had expressed specific tagged proteins were measured by fluorescence-based detection and calculated as mean intensity per cell, as described in Materials and Methods. Data are means of two to three independent experiments, each counted in triplicate. Variations for each specific time point and sample were typically less than 10% and the error bars are omitted for clarity. The control uptake exhibits a linear uptake rate up to 30 minutes. (B) 293T cells were transfected with vector control or expression plasmids coding for the tagged wildtype or mutant proteins, either alone or in combination as indicated. They were made quiescent by serum depletion for 18 hours followed by the stimulation with 100 ng/ml EGF for the periods indicated. Activation of the EGF receptor and ERK1/2 were analyzed by western blotting of equal amount of whole-cell lysates using anti-phospho EGF receptor and anti-phospho ERK1/2, respectively. To show equal loading of whole-cell lysates and expression of the appropriate tagged proteins, the blots were analyzed with anti-EGF receptor (EGFR), anti-ERK1/2 or anti-Flag (for EEN and NT in single or double transfectants, and for BPGAP1, PP and R232A in single transfectants), or anti-HA (for BPGAP1, PP and R232A in double-transfectants). For triple transfectants, anti-Myc (for EEN), anti-HA (for PP) and anti-Flag (for BPGAP1) were used. The ERK phosphorylation signals were expressed as number of fold over the maximal level seen by the control cells after 2 minutes. These results are representative of three independent sets of assays, each time with the control-stimulated cell lysates prepared concurrently and analyzed alongside the test constructs. This serves as an internal control for gel variations and also for normalizing the film exposures. One of the representative set is shown as Fig. 6C.

View larger version (28K): [in a new window]   Fig. 7. A model for the stimulatory effects by BPGAP1 and EEN on EGF-stimulated EGF receptor endocytosis and ERK1/2 phosphorylation. Stimulation of cells with EGF triggers the internalization of the EGF-bound EGF receptor in the form of vesicles, leading to the phosphorylation of ERK1/2. This process is enhanced by BPGAP1 or EEN alone and augmented through their interaction via their PRR and SH3 domain, respectively. However, the presence of NT mutant of EEN, which lacks the SH3 domain, completely prevents the effects of BPGAP1 in both endocytosis and ERK1/2 phosphorylation, thus acting as a dominant negative mutant in this pathway. This could involve displacing and sequestering functionally active EEN or other endophilins from BPGAP1, by forming homodimer or heterodimer complexes via their coiled-coil regions. Similarly, the GAP mutant R232A devoid of the catalytic arginine finger motif also reduces basal and EEN-stimulated EGF receptor endocytosis, suggesting that functional RhoGAP activity is required to promote endocytosis and ERK1/2 phosphorylation. Furthermore, despite failing to augment the EEN effects further and its inability to enhance endocytosis, the PP mutant (devoid of the interaction with EEN) or the GAP mutant (R232A) can also stimulate ERK1/2 phosphorylation, which suggests a distinct regulatory pathway independent of BPGAP1's stimulatory role in endocytosis. This could involve a different protein X possibly via its interaction with the BCH domain, but not linked to its direct interaction with EEN. However, this pathway still functions downstream of the control that requires an intact EEN function, as the NT mutant completely abolishes all stimulatory effect by BPGAP1, perhaps through a feedback or crosstalk from the endocytosis-dependent mechanism. BAR, BIN/amphiphysin/Rvsp domain; BCH, BNIP-2 and Cdc42GAP homology domain; GAP, GTPase-activating protein domain; PRR, proline-rich region; R232A, GAP mutant devoid of the catalytic arginine finger motif; NT, EEN mutant without the SH3 domain; ERK, extracellular signal-regulated kinase. Plus sign in circle denotes stimulatory effect while T-bars denote inhibitory effect. P, phosphorylation.

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