Dorsal ruffle microdomains potentiate Met receptor tyrosine kinase signaling and down-regulation

Abstract

Dorsal ruffles are apical protrusions induced in response to many growth factors, yet their function is poorly understood. Here we report that downstream from the hepatocyte growth factor (HGF) receptor tyrosine kinase (RTK), Met, dorsal ruffles function as both a localized signaling microdomain as well as a platform from which the Met RTK internalizes and traffics to a degradative compartment. In response to HGF, colonies of epithelial Madin-Darby canine kidney cells form dorsal ruffles for up to 20 min. Met is transcytosed from the basolateral membrane on Rab4 endosomes, to the apical surface where Met, as well as a Met substrate and scaffold protein, Gab1, localize to the dorsal ruffle membrane. This results in activation of downstream signaling proteins, as evidenced by localization of phospho-ERK1/2 to dorsal ruffles. As dorsal ruffles collapse, Met is internalized into EEA1- and Rab5-positive endosomes and is targeted for degradation through delivery to an Hrs-positive sorting compartment. Enhancing HGF-dependent dorsal ruffle formation, through overexpression of Gab1 or activated Pak1 kinase, promotes more efficient degradation of the Met RTK. Conversely, the ablation of dorsal ruffle formation, by pre-treatment with SITS (4-acetamido-4'-isothiocyabatostilbene-2',2-disulfonic acid) or expression of a Gab1 mutant, impairs Met degradation. Taken together, these data support a function for dorsal ruffles as a biologically relevant signaling microenvironment and a mechanism for Met receptor internalization and degradation.

FIGURE 1.

The Met receptor and downstream signaling complexes are recruited to dorsal ruffles. A, MDCK cells were pre-treated with cycloheximide for 2 h and then stimulated or not with 0.5 nm HGF for 5 min, fixed, and stained for Met (red) and F-actin (green). Arrowheads indicate dorsal ruffles. B, M-GFP-Gab1 cells were stimulated or not with 0.5 nm HGF for 5 min, fixed, and stained for phospho-Met (pY1234/35) (red) and Met (purple). As in B, M-GFP-Gab1 cells were stained for (C) phospho-Gab1 (red) or (D) phospho-Erk1/2 (red). Confocal images were taken with a ×100 objective. Bar represents 10 μm.

FIGURE 2.

Met localizes to the canonical endosomal network upon internalization from dorsal ruffles. A, three-dimensional images were generated from a live cell movie of M-GFP-Gab1 cells stimulated with HGF (1-min time intervals shown) using IMARIS software. Z-X (top panel) and Y-X (bottom panel) projections of the various stages of dorsal ruffle formation and collapse are shown. B, M-GFP-Gab1 cells were pre-treated with cycloheximide for 2 h and then stimulated with 0.5 nm HGF for 5 and 10 min, fixed, and stained for Met (red) and EEA1 (purple) and GFP (green). Two representative images of the different stages of dorsal ruffle formation are shown (top 2 images, individual cells are outlined) with the confocal Z-stack images of these fields (lower panels) of (i) circular dorsal ruffle (a, b, and c), (ii) constricting dorsal ruffle (a′, b′, and c′) and (iii) a collapsed dorsal ruffle (d, e, and f). M-HA-Gab1 cells were transfected with GFP-Rab5 (C) or GFP-Hrs (D), pre-treated with cycloheximide for 2 h, and then stimulated with 0.5 nm HGF for 15 and 30 min, respectively. E, cells were treated as in B and stimulated with 0.5 nm HGF for 15 and 30 min. Samples fixed at the indicated time points were stained for Met (red) and 4′,6-diamidino-2-phenylindole (blue). Arrows indicate re-localization of Gab1 to the plasma membrane. Confocal images were acquired with ×100 objective. All scale bars represent 10 μm.

FIGURE 3.

Dorsal ruffles mediate efficient Met receptor down-regulation. A, MDCK and M-HA-Gab1 cells were pre-treated with cycloheximide for 2 h, then stimulated or not with 0.5 nm HGF for 5 min. Cells were then fixed and stained for actin to visualize dorsal ruffles. Scale bar represents 10 μm. B, 10 fields of view of cells from A taken with a ×63 objective were scored for dorsal ruffle formation and the data expressed as the mean percentage of total cells forming dorsal ruffles ± S.E. of three independent experiments. C, cells were treated as in A and stimulated with HGF for the indicated time points after which cells were lysed and cell extracts were separated by SDS-PAGE and immunoblotted for Met (14G9), HA-Gab1, and actin. D, densitometric analysis of Met degradation as percentage of initial receptor remaining after HGF stimulation ± S.E. from three independent experiments was used to generate a best fit one-phase decay curve to determine the half-life (t½) of the receptor. E, MDCK and M-HA-Gab1 cells were pre-treated with DMSO, 10 μm lactacystin, or 0.1 μm concanamycin for 2 h, then stimulated or not with 0.5 nm HGF for 2 h. Met protein was immunoprecipitated, separated by SDS-PAGE, and immunoblotted for Met (14G9) and actin. Densitometric analysis of the remaining Met levels compared with unstimulated DMSO control are shown below.

FIGURE 4.

Gab1 induced dorsal ruffle formation in HeLa cells promotes more rapid Met RTK degradation. A, HeLa cells, plated on glass coverslips were transiently transfected with vector or HA-Gab1 and 48 h later, pre-treated with cycloheximide for 2 h, stimulated or not with 0.5 nm HGF for 5 min, fixed, and stained for HA (red) and actin (green). B, cells treated as in A were scored for dorsal ruffle formation, the data are expressed as the mean percentage of total cells forming dorsal ruffles ± S.E. of three independent experiments. C, HeLa cells transfected with vector or HA-Gab1 were stimulated or not with 0.5 nm HGF, fixed, and stained for Met (red) and HA (green). D, HeLa cells transfected as in C were stimulated with 0.5 nm HGF for the indicated time points. Total cell extracts were separated by SDS-PAGE and immunoblotted for Met (147), HA, and actin. E, densitometric analysis of Met degradation from D presented as percentage of the initial receptor remaining after HGF stimulation, ± S.E. from three independent experiments. F, HeLa cells transiently transfected with vector or increasing amounts of HA-Gab1 were stimulated or not with 0.5 nm HGF for 5 min and lysed in RIPA buffer. Met protein was immunoprecipitated (IP), separated by SDS-PAGE, and immunoblotted for ubiquitin, stripped, and reprobed for Met (147). Total cell extracts were immunoblotted for HA. Scale bars represent 10 μm.

FIGURE 5.

Pak1-mediated dorsal ruffles enhances Met receptor degradation. HeLa cells transiently transfected with myc-Pak1 His⁸³-Leu⁸⁶ were pre-treated with cycloheximide for 2 h and stimulated or not with 0.5 nm HGF for 5 min, fixed, and stained for (A) actin (green) and myc (red) or (B) Met (green) and myc (red). Scale bar represents 10 μm. C, HeLa cells transiently transfected with vector or myc-Pak1 His⁸³-Leu⁸⁶, were stimulated with 0.5 nm HGF for the indicated times. Total cell lysates were separated by SDS-PAGE and immunoblotted for endogenous Met receptor (147), myc-Pak1, and actin. D, densitometric analysis of Met degradation from C presented as a percentage of initial receptor remaining after HGF stimulation ± S.E. from three independent experiments.

FIGURE 6.

Disruption of dorsal ruffles delays Met receptor down-regulation. A, M-GFP-Gab1 cells were pre-treated with cycloheximide for 2 h and then 20 min prior to stimulation with 0.5 nm HGF, were also incubated with DMSO or 0.5 mm SITS. Cells were then fixed and stained for 4′,6-diamidino-2-phenylindole (blue). B, the number of cells forming dorsal ruffles was scored from 10 fields of view from A and represented as the percentage of cells forming dorsal ruffles in the presence of DMSO (■) or SITS (□) treatment ± S.E. from three independent experiments. C, M-GFP-Gab1 cells were pre-treated with DMSO or 0.5 mm SITS and stimulated with 0.5 nm HGF for the indicated times. Proteins from cell extracts were separated by SDS-PAGE and immunoblotted for endogenous Met receptor, pY1234/35 Met, and actin. D, densitometric analysis of Met degradation from C presented as percentage of initial receptor remaining after HGF stimulation, ± S.E. from three independent experiments. E, M-GFP-Gab1 cells plated on coverslips were treated as in A and stimulated with 0.5 nm HGF for the indicated time points, fixed, and stained for Met (red) and EEA1 (green). Dorsal ruffles are indicated by arrows and peripheral ruffles by arrowheads. F, M-HA-Gab1 and 3 HA-Gab1ΔDR cell lines were pre-treated with cycloheximide for 2 h, stimulated or not with 0.5 nm HGF for 5 min, fixed, and stained for HA. Representative images of M-HA-Gab1 and M-HA-Gab1ΔDR clone D are shown. G, the percentage of cells from F forming dorsal ruffles was calculated and represented as the mean ± S.E. of three independent experiments. H, cells from F were stimulated with 0.5 nm HGF in the presence of cycloheximide for the indicated time points. Densitometric analysis of Met levels (see immunoblots in supplemental Fig. S6F) are presented as percentage of initial receptor remaining after HGF stimulation ± S.E. from three independent experiments. All scale bars represent 10 μm.

FIGURE 7.

Model of Met RTK down-regulation through dorsal ruffles. Prior to HGF stimulation, the Met RTK is localized to the basolateral membrane. HGF stimulation promotes Met tyrosine phosphorylation and internalization through clathrin-coated pits. Met RTKs are then transcytosed on Rab4-positive vesicles to the apical membrane where Met can bind to and phosphorylate Gab1, leading to the formation of a dorsal ruffle. Met-Gab1 signaling complexes go on to induce activation of ERK1/2 within the dorsal ruffle microdomain. Upon collapse of the dorsal ruffle, Met and Gab1 are bulk internalized into the cell. Although Met RTKs continue down the endocytic pathway (Rab5, Hrs-positive endosomes) for eventual lysosomal degradation, Gab1 proteins re-localize to basolateral membranes.