Lysosomal targeting of E-cadherin: a unique mechanism for the down-regulation of cell-cell adhesion during epithelial to mesenchymal transitions

Abstract

A hallmark characteristic of epithelial tumor progression as well as some processes of normal development is the loss of the epithelial phenotype and acquisition of a motile or mesenchymal phenotype. Such epithelial to mesenchymal transitions are accompanied by the loss of E-cadherin function by either transcriptional or posttranscriptional mechanisms. Here we demonstrate that, upon v-Src expression, a potent trigger of epithelial to mesenchymal transitions, E-cadherin is internalized and then shuttled to the lysosome instead of being recycled back to the lateral membrane. Thus, while E-cadherin internalization facilitates the dissolution of adherens junctions, its subsequent traffic to the lysosome serves as a means to ensure that cells do not reform their cell-cell contacts and remain motile. We also show that ubiquitin tagging of E-cadherin is essential for its sorting to the lysosome. The lysosomal targeting of E-cadherin is mediated by hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and v-Src-induced activation of the Rab5 and Rab7 GTPases. Our studies reveal that the lysosomal targeting of E-cadherin is an important posttranscriptional mechanism to deplete cellular E-cadherin during Src-induced epithelial to mesenchymal transitions.

FIG. 1.

v-Src expression promotes the formation of enlarged vesicles that contain E-cadherin. (A) MDCKpp60^v-Src cells at nonpermissive or permissive temperatures for 4 h and parental MDCK I cells stably expressing ARF6(Q67L) were fixed and labeled for E-cadherin. Expression of v-Src in MDCK cells induces the formation of enlarged vesicles that contain E-cadherin that are morphologically distinct from those observed in cells expressing the ARF6-GTP mutant. The overall levels of E-cadherin in v-Src-expressing cells appear to be reduced compared to those in untransfected MDCK cells. (B) MDCKpp60^v-Src cells stably expressing HA-tagged ARF6(T27N) were maintained at the permissive temperature of 35°C for 4 h and then fixed and labeled for E-cadherin (red) and HA (green). Expression of ARF6(T27N) blocks E-cadherin internalization and the formation of Src-induced enlarged vesicles.

FIG. 2.

Src activation induces the shuttling of E-cadherin to the lysosome and prevents its recycling to the basolateral plasma membrane. (A) MDCKpp60^v-Src cells transiently expressing GFP-tagged E-cadherin at the nonpermissive temperature of 41°C (top panels) or at the permissive temperature of 35°C (bottom panels) were incubated with LysoTracker to label lysosomes. v-Src activation induces the shuttling of E-cadherin to the lysosome. (B) Parental MDCK I cells or MDCKpp60^v-Src cells were incubated at 37 and 41°C, respectively (left panels), followed by incubation at 18°C for 2 h (middle panels) and then for 30 min at 37 or 35°C as indicated (right panels). Cells were fixed at each temperature and labeled for E-cadherin. In normal cells, E-cadherin gets internalized into the cytoplasm and then recycles back to the plasma membrane. Src activation blocks the recycling of E-cadherin to the plasma membrane and instead shuttles E-cadherin to lysosomes.

FIG. 3.

Src activation promotes the lysosome-mediated degradation of E-cadherin. (A) MDCKpp60^v-Src cells grown at nonpermissive temperatures were switched to 35°C for times indicated to allow expression of v-Src. Cells were then lysed in RIPA buffer and examined for E-cadherin degradation by resolving proteins by SDS-PAGE and then labeling with anti-E-cadherin antibodies by Western blotting procedures followed by autoradiography. At low exposure a major degradation product of 35 kDa was observed. At an increased time (5 min) of autoradiography another degradation product of approximately 20 kDa became apparent. Cell lysates were probed for γ-tubulin expression as a control for equal loading of protein on SDS gels. Numbers at left are molecular masses in kilodaltons. (B) From left to right in each panel, respectively, MDCKpp60^v-Src cells were grown at the nonpermissive temperature of 41°C and then switched to 35°C for 4 h to allow expression of v-Src in medium alone or medium containing herbimycin A, chloroquine, or lactacystin. E-cadherin degradation was monitored as described above. The major degradation product of 35 kDa was quantitated by densitometry as a measure of E-cadherin degradation (right). Herbimycin A and chloroquine blocked the degradation of E-cadherin, but lactacystin had no effect. Numbers at left are molecular masses in kilodaltons. (C) MDCKpp60^v-Src cells were grown at the nonpermissive temperature of 41°C (lane 1) and then switched to 35°C for 6 h to allow expression of v-Src. In experiments done in parallel, cells were treated with type IV trypsin prior to collection of growth medium. The 90-kDa fragment released by trypsin treatment was quantitated by densitometry. E-cadherin fragments were not detected in the growth medium upon Src activation; however, 90-kDa fragments of the E-cadherin extracellular domain were observed in the growth medium upon trypsin treatment. (D) MDCKpp60^v-Src cells were grown at the nonpermissive temperature of 41°C (lane 1) and then switched to 35°C as indicated to allow expression of v-Src. The cell surface was biotinylated, and surface levels of biotinylated E-cadherin were determined as previously described (32). Surface E-cadherin is significantly decreased upon v-Src expression. (E) Epithelial cell colonies at nonpermissive temperatures, with and without treatment with chloroquine for 1 h, were switched to permissive temperatures for times indicated. Cells were fixed, labeled with phalloidin and examined by confocal immunofluorescence microscopy. Chloroquine-treated cells were significantly hampered in their ability to scatter relative to untreated cells.

FIG. 3.

Src activation promotes the lysosome-mediated degradation of E-cadherin. (A) MDCKpp60^v-Src cells grown at nonpermissive temperatures were switched to 35°C for times indicated to allow expression of v-Src. Cells were then lysed in RIPA buffer and examined for E-cadherin degradation by resolving proteins by SDS-PAGE and then labeling with anti-E-cadherin antibodies by Western blotting procedures followed by autoradiography. At low exposure a major degradation product of 35 kDa was observed. At an increased time (5 min) of autoradiography another degradation product of approximately 20 kDa became apparent. Cell lysates were probed for γ-tubulin expression as a control for equal loading of protein on SDS gels. Numbers at left are molecular masses in kilodaltons. (B) From left to right in each panel, respectively, MDCKpp60^v-Src cells were grown at the nonpermissive temperature of 41°C and then switched to 35°C for 4 h to allow expression of v-Src in medium alone or medium containing herbimycin A, chloroquine, or lactacystin. E-cadherin degradation was monitored as described above. The major degradation product of 35 kDa was quantitated by densitometry as a measure of E-cadherin degradation (right). Herbimycin A and chloroquine blocked the degradation of E-cadherin, but lactacystin had no effect. Numbers at left are molecular masses in kilodaltons. (C) MDCKpp60^v-Src cells were grown at the nonpermissive temperature of 41°C (lane 1) and then switched to 35°C for 6 h to allow expression of v-Src. In experiments done in parallel, cells were treated with type IV trypsin prior to collection of growth medium. The 90-kDa fragment released by trypsin treatment was quantitated by densitometry. E-cadherin fragments were not detected in the growth medium upon Src activation; however, 90-kDa fragments of the E-cadherin extracellular domain were observed in the growth medium upon trypsin treatment. (D) MDCKpp60^v-Src cells were grown at the nonpermissive temperature of 41°C (lane 1) and then switched to 35°C as indicated to allow expression of v-Src. The cell surface was biotinylated, and surface levels of biotinylated E-cadherin were determined as previously described (32). Surface E-cadherin is significantly decreased upon v-Src expression. (E) Epithelial cell colonies at nonpermissive temperatures, with and without treatment with chloroquine for 1 h, were switched to permissive temperatures for times indicated. Cells were fixed, labeled with phalloidin and examined by confocal immunofluorescence microscopy. Chloroquine-treated cells were significantly hampered in their ability to scatter relative to untreated cells.

FIG. 3.

Src activation promotes the lysosome-mediated degradation of E-cadherin. (A) MDCKpp60^v-Src cells grown at nonpermissive temperatures were switched to 35°C for times indicated to allow expression of v-Src. Cells were then lysed in RIPA buffer and examined for E-cadherin degradation by resolving proteins by SDS-PAGE and then labeling with anti-E-cadherin antibodies by Western blotting procedures followed by autoradiography. At low exposure a major degradation product of 35 kDa was observed. At an increased time (5 min) of autoradiography another degradation product of approximately 20 kDa became apparent. Cell lysates were probed for γ-tubulin expression as a control for equal loading of protein on SDS gels. Numbers at left are molecular masses in kilodaltons. (B) From left to right in each panel, respectively, MDCKpp60^v-Src cells were grown at the nonpermissive temperature of 41°C and then switched to 35°C for 4 h to allow expression of v-Src in medium alone or medium containing herbimycin A, chloroquine, or lactacystin. E-cadherin degradation was monitored as described above. The major degradation product of 35 kDa was quantitated by densitometry as a measure of E-cadherin degradation (right). Herbimycin A and chloroquine blocked the degradation of E-cadherin, but lactacystin had no effect. Numbers at left are molecular masses in kilodaltons. (C) MDCKpp60^v-Src cells were grown at the nonpermissive temperature of 41°C (lane 1) and then switched to 35°C for 6 h to allow expression of v-Src. In experiments done in parallel, cells were treated with type IV trypsin prior to collection of growth medium. The 90-kDa fragment released by trypsin treatment was quantitated by densitometry. E-cadherin fragments were not detected in the growth medium upon Src activation; however, 90-kDa fragments of the E-cadherin extracellular domain were observed in the growth medium upon trypsin treatment. (D) MDCKpp60^v-Src cells were grown at the nonpermissive temperature of 41°C (lane 1) and then switched to 35°C as indicated to allow expression of v-Src. The cell surface was biotinylated, and surface levels of biotinylated E-cadherin were determined as previously described (32). Surface E-cadherin is significantly decreased upon v-Src expression. (E) Epithelial cell colonies at nonpermissive temperatures, with and without treatment with chloroquine for 1 h, were switched to permissive temperatures for times indicated. Cells were fixed, labeled with phalloidin and examined by confocal immunofluorescence microscopy. Chloroquine-treated cells were significantly hampered in their ability to scatter relative to untreated cells.

FIG. 3.

Src activation promotes the lysosome-mediated degradation of E-cadherin. (A) MDCKpp60^v-Src cells grown at nonpermissive temperatures were switched to 35°C for times indicated to allow expression of v-Src. Cells were then lysed in RIPA buffer and examined for E-cadherin degradation by resolving proteins by SDS-PAGE and then labeling with anti-E-cadherin antibodies by Western blotting procedures followed by autoradiography. At low exposure a major degradation product of 35 kDa was observed. At an increased time (5 min) of autoradiography another degradation product of approximately 20 kDa became apparent. Cell lysates were probed for γ-tubulin expression as a control for equal loading of protein on SDS gels. Numbers at left are molecular masses in kilodaltons. (B) From left to right in each panel, respectively, MDCKpp60^v-Src cells were grown at the nonpermissive temperature of 41°C and then switched to 35°C for 4 h to allow expression of v-Src in medium alone or medium containing herbimycin A, chloroquine, or lactacystin. E-cadherin degradation was monitored as described above. The major degradation product of 35 kDa was quantitated by densitometry as a measure of E-cadherin degradation (right). Herbimycin A and chloroquine blocked the degradation of E-cadherin, but lactacystin had no effect. Numbers at left are molecular masses in kilodaltons. (C) MDCKpp60^v-Src cells were grown at the nonpermissive temperature of 41°C (lane 1) and then switched to 35°C for 6 h to allow expression of v-Src. In experiments done in parallel, cells were treated with type IV trypsin prior to collection of growth medium. The 90-kDa fragment released by trypsin treatment was quantitated by densitometry. E-cadherin fragments were not detected in the growth medium upon Src activation; however, 90-kDa fragments of the E-cadherin extracellular domain were observed in the growth medium upon trypsin treatment. (D) MDCKpp60^v-Src cells were grown at the nonpermissive temperature of 41°C (lane 1) and then switched to 35°C as indicated to allow expression of v-Src. The cell surface was biotinylated, and surface levels of biotinylated E-cadherin were determined as previously described (32). Surface E-cadherin is significantly decreased upon v-Src expression. (E) Epithelial cell colonies at nonpermissive temperatures, with and without treatment with chloroquine for 1 h, were switched to permissive temperatures for times indicated. Cells were fixed, labeled with phalloidin and examined by confocal immunofluorescence microscopy. Chloroquine-treated cells were significantly hampered in their ability to scatter relative to untreated cells.

FIG. 4.

Ubiquitination-deficient E-cadherin is internalized but not trafficked to the lysosomes for degradation. (A) MDCKpp60^v-Src cells transiently expressing a Myc-tagged ubiquitination-deficient mutant of E-cadherin, E-cadherin(Y755F-Y756F), at nonpermissive temperatures or at 35°C for 4 h were labeled for actin (red) and Myc (green). The ubiquitination-deficient mutant of E-cadherin localizes to cell-cell contacts and to cytoplasmic vesicles. (B) Cells were transiently transfected with retrovirus encoding ARF6(T27N) and GFP for 24 h followed by transient transfection with the Myc-tagged ubiquitination-deficient mutant of E-cadherin. Cells were fixed and labeled for Myc and with Draq5, a nuclear stain. Expression of ARF6(T27N) prevents the cytoplasmic distribution of mutant E-cadherin, suggesting that internalized mutant E-cadherin was derived by endocytosis from the cell surface. (C) MDCKpp60^v-Src cells transiently expressing Myc-tagged wild-type E-cadherin or E-cadherin(Y755F-Y756F) at the permissive temperature of 35°C for 4 h were lysed in RIPA buffer, and equal amounts of the cell lysates were probed with antibodies to Myc, by Western blotting procedures. Myc-tagged E-cadherin(Y755F-Y756F) is not degraded upon expression of v-Src.

FIG. 5.

Hrs regulates the traffic of ubiquitinated E-cadherin to the lysosome. (A to D) MDCKpp60^v-Src cells transiently transfected with Myc-tagged wild-type Hrs or Hrs(S270E) as indicated were grown at the nonpermissive temperature of 41°C and then switched to 35°C for 8 h. Cells were fixed and labeled for Myc (green) and E-cadherin (A and C, red) or LAMP-1 (B and D, red). Coincident staining appears yellow in the merged images. At nonpermissive temperatures, wild-type Hrs colocalizes with E-cadherin on vesicles that do not label for LAMP-1. Upon expression of v-Src, wild-type Hrs and E-cadherin are shuttled toward the late endosomal-lysosomal compartments as seen by the colocalization with LAMP-1 antibodies. However, Hrs(S270E) and E-cadherin are retained at vesicular compartments that do not colocalize with LAMP-1. Thus, expression of Hrs(S270E) blocks the shuttling of E-cadherin to the lysosome upon Src activation. (E) Lysates of MDCKpp60^v-Src cells expressing Myc-tagged wild-type Hrs or Hrs(S270E) at permissive temperatures were immunoprecipitated with anti-Myc monoclonal antibody. Immunoprecipitates were resolved on SDS gels and probed with anti-E-cadherin antibody. Only wild-type Hrs, not the Hrs mutant lacking a functional UIM domain, coprecipitates with E-cadherin upon Src activation.

FIG. 6.

Expression of v-Src induces the activation of Rab5 and Rab7 to facilitate the shuttling of E-cadherin to the lysosome. (A) MDCKpp60^v-Src cells transiently transfected with GFP-tagged wild-type Rab5 (lanes 1 and 2) and GFP-tagged wild-type Rab7 (lanes 3 and 4) were incubated at either 41 or 35°C for 4 h as indicated, and the nucleotide bound to Rab5 and Rab7 was determined. Upon Src activation the cellular levels of GTP-bound Rab5 and Rab7 are markedly enhanced. (B and C) MDCKpp60^v-Src cells transiently transfected with GFP-tagged wild-type Rab5 at the nonpermissive temperature of 41°C (B) or at the permissive temperature of 35°C (C) were fixed and labeled for E-cadherin (red). Coincident staining appears yellow in the merged images. Upon Src activation, wild-type Rab5 localizes to enlarged endosomes that also contain E-cadherin. Some Rab5-positive endosomes did not contain E-cadherin; these vesicles could represent endosomes that are derived from the apical membrane, which also contain Rab5 (7). (D) MDCKpp60^v-Src cells expressing ARF6(T27N) were transiently transfected with GFP-tagged wild-type Rab5, incubated at 35°C for 4 h, and labeled for E-cadherin (red). Coexpression of dominant-negative ARF6, ARF6(T27N), with wild-type Rab5 blocked the internalization of E-cadherin but did not prevent the fusion of Rab5-positive endosomes in response to Src activation. (E) Parental MDCK cells transiently transfected with HA-tagged Rab5(Q79L) were labeled for E-cadherin. Activated Rab5 expression had no effect on the adherens junctions. (F) MDCKpp60^v-Src cells transiently transfected with GFP-tagged Rab5(S34N) were grown at 35°C for 4 h and labeled for E-cadherin (red). Expression of dominant-negative Rab5, Rab5(S34N), blocks the formation of enlarged endosomes upon Src activation. The arrow points to an E-cadherin-positive enlarged endosome formed by expression of v-Src in an untransfected cell adjacent to a Rab5(S34N)-transfected cell where enlarged endosomes are not observed. (G and H) MDCKpp60^v-Src cells transiently transfected with GFP-tagged wild-type Rab7 at 41 or 35°C were fixed and labeled for E-cadherin (red). In the absence of Src activation, wild-type Rab7 localizes to late endosomes. Upon Src activation, wild-type Rab7 localizes to enlarged vesicles that also contain E-cadherin and are likely formed by the fusion of early and late endosomes. (I) MDCKpp60^v-Src cells transiently transfected with GFP-tagged Rab7(T22N) at 41°C were incubated at the permissive temperature of 35°C for 4 h and labeled for E-cadherin (blue) and LysoTracker (red) to visualize lysosomes. Expression of dominant-negative Rab7, Rab7(T22N), prevents the trafficking of E-cadherin to the lysosome. (J) Untransfected MDCKpp60^v-Src cells (lanes 1 and 2) and those transiently transfected with GFP-tagged Rab5(S34N) (lane 3) and GFP-tagged Rab7(T22N) (lane 4) were maintained at 41 or 35°C for 4 h as indicated. Cells were then lysed in RIPA buffer, and the cell lysates were analyzed by SDS-PAGE and blotted with antibodies against E-cadherin. The 35-kDa E-cadherin degradation product was quantified using a densitometer and plotted in the graph shown below. Transient expression of Rab5(S34N) and Rab7(T22N) blocked the degradation of E-cadherin in response to Src activation.

FIG. 6.

Expression of v-Src induces the activation of Rab5 and Rab7 to facilitate the shuttling of E-cadherin to the lysosome. (A) MDCKpp60^v-Src cells transiently transfected with GFP-tagged wild-type Rab5 (lanes 1 and 2) and GFP-tagged wild-type Rab7 (lanes 3 and 4) were incubated at either 41 or 35°C for 4 h as indicated, and the nucleotide bound to Rab5 and Rab7 was determined. Upon Src activation the cellular levels of GTP-bound Rab5 and Rab7 are markedly enhanced. (B and C) MDCKpp60^v-Src cells transiently transfected with GFP-tagged wild-type Rab5 at the nonpermissive temperature of 41°C (B) or at the permissive temperature of 35°C (C) were fixed and labeled for E-cadherin (red). Coincident staining appears yellow in the merged images. Upon Src activation, wild-type Rab5 localizes to enlarged endosomes that also contain E-cadherin. Some Rab5-positive endosomes did not contain E-cadherin; these vesicles could represent endosomes that are derived from the apical membrane, which also contain Rab5 (7). (D) MDCKpp60^v-Src cells expressing ARF6(T27N) were transiently transfected with GFP-tagged wild-type Rab5, incubated at 35°C for 4 h, and labeled for E-cadherin (red). Coexpression of dominant-negative ARF6, ARF6(T27N), with wild-type Rab5 blocked the internalization of E-cadherin but did not prevent the fusion of Rab5-positive endosomes in response to Src activation. (E) Parental MDCK cells transiently transfected with HA-tagged Rab5(Q79L) were labeled for E-cadherin. Activated Rab5 expression had no effect on the adherens junctions. (F) MDCKpp60^v-Src cells transiently transfected with GFP-tagged Rab5(S34N) were grown at 35°C for 4 h and labeled for E-cadherin (red). Expression of dominant-negative Rab5, Rab5(S34N), blocks the formation of enlarged endosomes upon Src activation. The arrow points to an E-cadherin-positive enlarged endosome formed by expression of v-Src in an untransfected cell adjacent to a Rab5(S34N)-transfected cell where enlarged endosomes are not observed. (G and H) MDCKpp60^v-Src cells transiently transfected with GFP-tagged wild-type Rab7 at 41 or 35°C were fixed and labeled for E-cadherin (red). In the absence of Src activation, wild-type Rab7 localizes to late endosomes. Upon Src activation, wild-type Rab7 localizes to enlarged vesicles that also contain E-cadherin and are likely formed by the fusion of early and late endosomes. (I) MDCKpp60^v-Src cells transiently transfected with GFP-tagged Rab7(T22N) at 41°C were incubated at the permissive temperature of 35°C for 4 h and labeled for E-cadherin (blue) and LysoTracker (red) to visualize lysosomes. Expression of dominant-negative Rab7, Rab7(T22N), prevents the trafficking of E-cadherin to the lysosome. (J) Untransfected MDCKpp60^v-Src cells (lanes 1 and 2) and those transiently transfected with GFP-tagged Rab5(S34N) (lane 3) and GFP-tagged Rab7(T22N) (lane 4) were maintained at 41 or 35°C for 4 h as indicated. Cells were then lysed in RIPA buffer, and the cell lysates were analyzed by SDS-PAGE and blotted with antibodies against E-cadherin. The 35-kDa E-cadherin degradation product was quantified using a densitometer and plotted in the graph shown below. Transient expression of Rab5(S34N) and Rab7(T22N) blocked the degradation of E-cadherin in response to Src activation.

FIG. 6.

Expression of v-Src induces the activation of Rab5 and Rab7 to facilitate the shuttling of E-cadherin to the lysosome. (A) MDCKpp60^v-Src cells transiently transfected with GFP-tagged wild-type Rab5 (lanes 1 and 2) and GFP-tagged wild-type Rab7 (lanes 3 and 4) were incubated at either 41 or 35°C for 4 h as indicated, and the nucleotide bound to Rab5 and Rab7 was determined. Upon Src activation the cellular levels of GTP-bound Rab5 and Rab7 are markedly enhanced. (B and C) MDCKpp60^v-Src cells transiently transfected with GFP-tagged wild-type Rab5 at the nonpermissive temperature of 41°C (B) or at the permissive temperature of 35°C (C) were fixed and labeled for E-cadherin (red). Coincident staining appears yellow in the merged images. Upon Src activation, wild-type Rab5 localizes to enlarged endosomes that also contain E-cadherin. Some Rab5-positive endosomes did not contain E-cadherin; these vesicles could represent endosomes that are derived from the apical membrane, which also contain Rab5 (7). (D) MDCKpp60^v-Src cells expressing ARF6(T27N) were transiently transfected with GFP-tagged wild-type Rab5, incubated at 35°C for 4 h, and labeled for E-cadherin (red). Coexpression of dominant-negative ARF6, ARF6(T27N), with wild-type Rab5 blocked the internalization of E-cadherin but did not prevent the fusion of Rab5-positive endosomes in response to Src activation. (E) Parental MDCK cells transiently transfected with HA-tagged Rab5(Q79L) were labeled for E-cadherin. Activated Rab5 expression had no effect on the adherens junctions. (F) MDCKpp60^v-Src cells transiently transfected with GFP-tagged Rab5(S34N) were grown at 35°C for 4 h and labeled for E-cadherin (red). Expression of dominant-negative Rab5, Rab5(S34N), blocks the formation of enlarged endosomes upon Src activation. The arrow points to an E-cadherin-positive enlarged endosome formed by expression of v-Src in an untransfected cell adjacent to a Rab5(S34N)-transfected cell where enlarged endosomes are not observed. (G and H) MDCKpp60^v-Src cells transiently transfected with GFP-tagged wild-type Rab7 at 41 or 35°C were fixed and labeled for E-cadherin (red). In the absence of Src activation, wild-type Rab7 localizes to late endosomes. Upon Src activation, wild-type Rab7 localizes to enlarged vesicles that also contain E-cadherin and are likely formed by the fusion of early and late endosomes. (I) MDCKpp60^v-Src cells transiently transfected with GFP-tagged Rab7(T22N) at 41°C were incubated at the permissive temperature of 35°C for 4 h and labeled for E-cadherin (blue) and LysoTracker (red) to visualize lysosomes. Expression of dominant-negative Rab7, Rab7(T22N), prevents the trafficking of E-cadherin to the lysosome. (J) Untransfected MDCKpp60^v-Src cells (lanes 1 and 2) and those transiently transfected with GFP-tagged Rab5(S34N) (lane 3) and GFP-tagged Rab7(T22N) (lane 4) were maintained at 41 or 35°C for 4 h as indicated. Cells were then lysed in RIPA buffer, and the cell lysates were analyzed by SDS-PAGE and blotted with antibodies against E-cadherin. The 35-kDa E-cadherin degradation product was quantified using a densitometer and plotted in the graph shown below. Transient expression of Rab5(S34N) and Rab7(T22N) blocked the degradation of E-cadherin in response to Src activation.

FIG. 6.

Expression of v-Src induces the activation of Rab5 and Rab7 to facilitate the shuttling of E-cadherin to the lysosome. (A) MDCKpp60^v-Src cells transiently transfected with GFP-tagged wild-type Rab5 (lanes 1 and 2) and GFP-tagged wild-type Rab7 (lanes 3 and 4) were incubated at either 41 or 35°C for 4 h as indicated, and the nucleotide bound to Rab5 and Rab7 was determined. Upon Src activation the cellular levels of GTP-bound Rab5 and Rab7 are markedly enhanced. (B and C) MDCKpp60^v-Src cells transiently transfected with GFP-tagged wild-type Rab5 at the nonpermissive temperature of 41°C (B) or at the permissive temperature of 35°C (C) were fixed and labeled for E-cadherin (red). Coincident staining appears yellow in the merged images. Upon Src activation, wild-type Rab5 localizes to enlarged endosomes that also contain E-cadherin. Some Rab5-positive endosomes did not contain E-cadherin; these vesicles could represent endosomes that are derived from the apical membrane, which also contain Rab5 (7). (D) MDCKpp60^v-Src cells expressing ARF6(T27N) were transiently transfected with GFP-tagged wild-type Rab5, incubated at 35°C for 4 h, and labeled for E-cadherin (red). Coexpression of dominant-negative ARF6, ARF6(T27N), with wild-type Rab5 blocked the internalization of E-cadherin but did not prevent the fusion of Rab5-positive endosomes in response to Src activation. (E) Parental MDCK cells transiently transfected with HA-tagged Rab5(Q79L) were labeled for E-cadherin. Activated Rab5 expression had no effect on the adherens junctions. (F) MDCKpp60^v-Src cells transiently transfected with GFP-tagged Rab5(S34N) were grown at 35°C for 4 h and labeled for E-cadherin (red). Expression of dominant-negative Rab5, Rab5(S34N), blocks the formation of enlarged endosomes upon Src activation. The arrow points to an E-cadherin-positive enlarged endosome formed by expression of v-Src in an untransfected cell adjacent to a Rab5(S34N)-transfected cell where enlarged endosomes are not observed. (G and H) MDCKpp60^v-Src cells transiently transfected with GFP-tagged wild-type Rab7 at 41 or 35°C were fixed and labeled for E-cadherin (red). In the absence of Src activation, wild-type Rab7 localizes to late endosomes. Upon Src activation, wild-type Rab7 localizes to enlarged vesicles that also contain E-cadherin and are likely formed by the fusion of early and late endosomes. (I) MDCKpp60^v-Src cells transiently transfected with GFP-tagged Rab7(T22N) at 41°C were incubated at the permissive temperature of 35°C for 4 h and labeled for E-cadherin (blue) and LysoTracker (red) to visualize lysosomes. Expression of dominant-negative Rab7, Rab7(T22N), prevents the trafficking of E-cadherin to the lysosome. (J) Untransfected MDCKpp60^v-Src cells (lanes 1 and 2) and those transiently transfected with GFP-tagged Rab5(S34N) (lane 3) and GFP-tagged Rab7(T22N) (lane 4) were maintained at 41 or 35°C for 4 h as indicated. Cells were then lysed in RIPA buffer, and the cell lysates were analyzed by SDS-PAGE and blotted with antibodies against E-cadherin. The 35-kDa E-cadherin degradation product was quantified using a densitometer and plotted in the graph shown below. Transient expression of Rab5(S34N) and Rab7(T22N) blocked the degradation of E-cadherin in response to Src activation.

FIG. 7.

Working model for the regulation of the E-cadherin turnover rate in response to Src activation. As previously described, the activation of ARF6 in response to extracellular stimuli and/or Src activation promotes the internalization of E-cadherin to endosomes vis a process mediated by Nm23-H1 and dynamin (32). Src also promotes the ubiquitination of E-cadherin (11), although it is unclear whether this occurs at the plasma membrane or on internal vesicular compartments. Src-mediated activation of Rab5 promotes early-endosome fusion to induce the formation of E-cadherin-positive enlarged endosomes and perhaps accelerates the rate of E-cadherin internalization. Ubiquitinated E-cadherin is sorted in early endosomes via its recognition by Hrs. Thus, while nonubiquitinated E-cadherin is recycled back to the plasma membrane, ubiquitinated E-cadherin is trafficked to the lysosome for degradation. The latter step is facilitated by Src-induced activation of Rab7. It is unclear at this time if Src induces the phosphorylation of Hrs to promote the lysosomal targeting of E-cadherin. HGF, hepatocyte growth factor.