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First published online 9 January 2007
doi: 10.1242/jcs.03336

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Role of EGF-induced tyrosine phosphorylation of reggie-1/flotillin-2 in cell spreading and signaling to the actin cytoskeleton

Institute of Biochemistry II, University Clinic of Frankfurt am Main and Cluster of Excellence, Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany

View larger version (43K): [in this window] [in a new window]   Fig. 1. Reggie-1 is tyrosine phosphorylated in growth-factor-stimulated cells. (A) HeLa and PC12 cells were incubated with pervanadate to inhibit dephosphorylation of tyrosines. Immunoprecipitation of phosphorylated proteins was performed with an anti-Tyr-P antibody (lane PY) or without antibody (lane C), and after stringent washing, endogenous reggie-1 was detected with a specific antibody. (B) HeLa cells were transfected with R1-EGFP or EGFP constructs and immunoprecipitated with anti-GFP. Tyrosine-phosphorylated proteins were detected by western blot with an anti-Tyr-P antibody. R1-EGFP was phosphorylated, whereas the controls (EGFP and SH-R1-EGFP) were not. Membrane association of reggie-1 is necessary for the phosphorylation, because the soluble mutant G2A-R1-EGFP did not carry Tyr-P modifications. Pervanadate was used as indicated. Lane C, nontransfected cells. (C) To demonstrate the physiological tyrosine phosphorylation of reggie-1, HeLa cells were stimulated with EGF in the absence of pervanadate. Stimulation of cells with EGF resulted in a time-dependent tyrosine phosphorylation of reggie-1. (D) EGF-stimulated tyrosine phosphorylation of R1-EGFP could be inhibited by tyrphostin AG1478, which inhibits EGFR phosphorylation (HeLa cells, experiment performed with pervanadate). Antibodies used for the detection by western blot are indicated.

View larger version (38K): [in this window] [in a new window]   Fig. 2. Src kinase activity is necessary for the tyrosine phosphorylation of reggie-1. (A) Tyrosine phosphorylation of R1-EGFP could be blocked by inhibitors of Src kinases (5 µM PP1 or PP2) by about 85%, whereas 10 µM PP3, a non-inhibiting analog, had no effect. Cells were treated with the compounds for 25 minutes and subsequently with compound and pervanadate for an additional 25 minutes. (B) EGF treatment (5 minutes) induces tyrosine phosphorylation of reggie-1 in the absence of pervanadate, and Src inhibitors PP2 (10 µM) and SU6656 (10 µM) are capable of strongly inhibiting this EGF-induced phosphorylation of R1-EGFP. (C) Coexpression of R1-EGFP with Src or constitutively active Y527F-Src, but not with kinase-dead Src, resulted in phosphorylation of R1-EGFP even in the absence of pervanadate and EGF. In addition, coexpression with Fyn kinase gave a similar result. (D) In Src, Yes and Fyn-deficient mouse fibroblasts, R1-EGFP was not phosphorylated even after EGF stimulation. Coexpression of Src but not of kinase-dead Src resulted in tyrosine phosphorylation of R1-EGFP in these cells, indicating that Src activity is necessary for reggie-1 to become tyrosine phosphorylated. Lane C, nontransfected cells. Immunoprecipitation in A-D was performed with anti-GFP. Antibodies used for western blot are indicated.

View larger version (48K): [in this window] [in a new window]   Fig. 3. Reggie-1 and Src can be coimmunoprecipitated. HeLa cells were transfected with R1-EGFP and Src, Y527F-Src or dead-Src constructs and immunoprecipitation was performed with anti-GFP antibody. Src kinase coprecipitates with R1-EGFP, but the degree of coprecipitation is higher with active forms of Src.

View larger version (57K): [in this window] [in a new window]   Fig. 4. Reggie-1 is phosphorylated at several tyrosine residues. (A) Eight tyrosine residues of reggie-1 predicted to become phosphorylated were mutated to phenylalanine and the phosphorylation of these mutants was analyzed in pervanadate-stimulated cells after immunoprecipitation with anti-GFP. All single or double mutants exhibited a similar degree of phosphorylation as the wild-type protein. Note the increase in the amount of the lower band in the Y27F mutant, probably owing to increased processing. (B) Seven Tyr residues in R1-EGFP (excluding Tyr27) were mutated into Phe within one construct (7xYF) and six residues (excluding Tyr27 and Tyr163) in another one (6xYF), and the phosphorylation was analyzed upon EGF stimulation (5 minutes) in the absence of pervanadate. The 7xYF mutant was found to be unphosphorylated, indicating that multiple phosphorylation of reggie-1 can take place. The 6xYF mutant was phosphorylated, although somewhat less than the wt protein, indicating that Y163 is indeed phosphorylated. (C) Y158F and Y163F mutants were phosphorylated in equal amounts as the wild-type protein when treated with pervanadate. A-C, HeLa cells.

View larger version (32K): [in this window] [in a new window]   Fig. 5. Specific Tyr residues influence the localization and function of R1-EGFP. In steady-state HeLa cells, R1-EGFP was localized to the plasma membrane and endosomes. An increase in the soluble fraction was seen with the mutant Y27F-R1-EGFP. Both Y163F and 7xYF-R1-EGFP exhibited only plasma membrane localization without endosomal staining. In addition, the induction of changes in the actin cytoskeleton (e.g. filopodia-like protrusions) by these mutants was even stronger than with wild-type R1-EGFP, as demonstrated by means of phalloidin staining (R1-EGFP and Y163F R1-EGFP, bottom row). The two upper rows represent GFP fluorescence, whereas the bottom row shows phalloidin staining of the cells.

View larger version (36K): [in this window] [in a new window]   Fig. 6. EGF stimulation results in translocation of reggie-1 from plasma membrane into endosomes, and knockdown of reggie-1 interferes with EGF-induced actin remodeling. (A-F) Transfected HeLa cells were starved without serum for 16 hours and then stimulated with 100 ng/ml EGF for 15 minutes (B,D,F). Both wild-type R1-EGFP (B) and R1-myc (F) underwent an EGF-dependent translocation into an endosomal compartment, whereas the Y163F mutant (D) remained associated with the plasma membrane. (G,H) HeLa cells transfected with siRNA duplex oligoribonucleotides were starved for 16 hours and then stimulated with EGF for 5 minutes. Staining of the actin cytoskeleton with phalloidin revealed that EGF-induced changes in the actin cytoskeleton were impaired in reggie knockdown cells (H), which displayed fewer protrusions than the control cells (G). Panels A-D represent GFP fluorescence, whereas R1-myc was visualized in E,F by staining with an anti-myc antibody. Panels G,H show staining of the cells with fluorochrome-coupled phalloidin.

View larger version (103K): [in this window] [in a new window]   Fig. 7. Reggie-1 colocalizes with the late endosomal marker LAMP-3 and with the EGF receptor. HeLa cells transfected with R1-EGFP were starved for 16 hours and then stimulated with EGF (100 ng/ml) for 15 minutes. Staining for LAMP-3 revealed a strong colocalization with reggie-1 (A). A partial colocalization of reggie-1 with EGF receptor could also be detected (B). By contrast, no significant colocalization could be observed between R1-EGFP and the early endosomal marker EEA1 (C) or transferrin receptor (D), which serves as a marker for recycling endosomes. Images in the right-hand column show magnifications of the colocalization.

View larger version (36K): [in this window] [in a new window]   Fig. 8. Reggie-1 influences cell spreading in a tyrosine-dependent manner. HeLa cells were transfected with various R1-EGFP constructs or reggie-1-specific siRNA duplex oligoribonucleotides and allowed to spread on fibronectin-coated glass chambers for 25 minutes. After washing, cells were fixed, stained with fluorochrome-coupled phalloidin and scored as non-spread (merely attached and round), half-spread (non-flattened with filopodial protrusions) or spread (flattened with lamellipodia). (A) Representative cells of the three spreading categories (phalloidin staining). (B) R1-EGFP and most of the Tyr mutants, including 7xYF, were capable of enhancing cell spreading compared with the EGFP control, whereas the soluble G2A and partly soluble Y27F mutants strongly inhibited spreading. However, Y24F, Y124F and Y163F mutants showed no enhancing or inhibiting effect on cell spreading and were comparable with EGFP, indicating a role for these phosphorylation sites of reggie-1 in cell spreading. (C) HeLa cells transfected either with reggie-1-specific siRNA duplex oligoribonucleotides or control were subjected to the same spreading assay. Compared with the control cells, the reggie-1 knockdown cells spread less efficiently on fibronectin. (D) A representative western blot shows the significant knockdown of reggie-1 (85% for R1S3 and 89% for R1S1 oligos) compared with cells treated with control siRNA. GAPDH was used as a loading control for the lysates.