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EGF-R signaling through Fyn kinase disrupts the function of integrin alpha6beta4 at hemidesmosomes: role in epithelial cell migration and carcinoma invasion.

Mariotti A, Kedeshian PA, Dans M, Curatola AM, Gagnoux-Palacios L, Giancotti FG - J. Cell Biol. (2001)

Bottom Line: By contrast, Src and Lck do not associate with alpha6beta4 to a significant extent.These observations suggest that the EGF-R causes disassembly of hemidesmosomes by activating Fyn, which in turn phosphorylates the beta4 cytoplasmic domain.Neoplastic cells expressing dominant negative Fyn display increased hemidesmosomes and migrate poorly in vitro in response to EGF.

View Article: PubMed Central - PubMed

Affiliation: Cellular Biochemistry and Biophysics Program, Department of Surgery, Sloan-Kettering Institute for Cancer Research, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.

ABSTRACT
We have examined the mechanism and functional significance of hemidesmosome disassembly during normal epithelial cell migration and squamous carcinoma invasion. Our findings indicate that a fraction of EGF receptor (EGF-R) combines with the hemidesmosomal integrin alpha6beta4 in both normal and neoplastic keratinocytes. Activation of the EGF-R causes tyrosine phosphorylation of the beta4 cytoplasmic domain and disruption of hemidesmosomes. The Src family kinase inhibitors PP1 and PP2 prevent tyrosine phosphorylation of beta4 and disassembly of hemidesmosomes without interfering with the activation of EGF-R. Coimmunoprecipitation experiments indicate that Fyn and, to a lesser extent, Yes combine with alpha6beta4. By contrast, Src and Lck do not associate with alpha6beta4 to a significant extent. A dominant negative form of Fyn, but not Src, prevents tyrosine phosphorylation of beta4 and disassembly of hemidesmosomes. These observations suggest that the EGF-R causes disassembly of hemidesmosomes by activating Fyn, which in turn phosphorylates the beta4 cytoplasmic domain. Neoplastic cells expressing dominant negative Fyn display increased hemidesmosomes and migrate poorly in vitro in response to EGF. Furthermore, dominant negative Fyn decreases the ability of squamous carcinoma cells to invade through Matrigel in vitro and to form lung metastases following intravenous injection in nude mice. These results suggest that disruption of hemidesmosomes mediated by Fyn is a prerequisite for normal keratinocyte migration and squamous carcinoma invasion.

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Dominant negative Fyn inhibits disassembly of hemidesmosomes and suppresses cell migration. The indicated 804G clones were grown until confluent and then serum starved. After wounding, the monolayers were treated with EGF (50 ng/ml) for 5 or 10 h or left untreated for 5 h, and then either stained with Crystal violet or subjected to immunofluorescent staining with affinity purified anti–BPAG-2 antibodies. Cell migration results were quantitated as described in the Materials and methods section and plotted graphically. The graph shows the mean and SD of values from a representative experiment performed in triplicate. Results similar to those shown here were obtained with all three clones expressing dominant negative Fyn and the two clones expressing dominant negative Src. Bar, 250 μm.
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fig6: Dominant negative Fyn inhibits disassembly of hemidesmosomes and suppresses cell migration. The indicated 804G clones were grown until confluent and then serum starved. After wounding, the monolayers were treated with EGF (50 ng/ml) for 5 or 10 h or left untreated for 5 h, and then either stained with Crystal violet or subjected to immunofluorescent staining with affinity purified anti–BPAG-2 antibodies. Cell migration results were quantitated as described in the Materials and methods section and plotted graphically. The graph shows the mean and SD of values from a representative experiment performed in triplicate. Results similar to those shown here were obtained with all three clones expressing dominant negative Fyn and the two clones expressing dominant negative Src. Bar, 250 μm.

Mentions: It is reasonable to assume that a cell must disassemble stable adhesions with the extracellular matrix, such as hemidesmosomes, in order to migrate and invade (Giancotti and Mainiero, 1994). Thus, we examined if inhibition of Fyn suppressed cell migration. As shown in Fig. 6, 804G clones expressing dominant negative Fyn migrated across an artificial in vitro wound much less efficiently than control clones. The migratory ability of clones expressing dominant negative Src was also reduced, but to a lesser extent than that of clones expressing dominant negative Fyn. This effect of dominant negative Src may result from stabilization of focal adhesions (Fincham and Frame, 1998). Immunofluorescent staining with antibodies to BPAG-2 revealed disassembly of hemidesmosomes at the rear end of control cells entering the wound area. Similar results were obtained with cells expressing dominant negative Src. By contrast, these posterior hemidesmosomes were not disassembled in cells expressing dominant negative Fyn (Fig. 6). These observations suggest that dominant negative Fyn inhibits cell migration by enhancing the stability of hemidesmosomes.


EGF-R signaling through Fyn kinase disrupts the function of integrin alpha6beta4 at hemidesmosomes: role in epithelial cell migration and carcinoma invasion.

Mariotti A, Kedeshian PA, Dans M, Curatola AM, Gagnoux-Palacios L, Giancotti FG - J. Cell Biol. (2001)

Dominant negative Fyn inhibits disassembly of hemidesmosomes and suppresses cell migration. The indicated 804G clones were grown until confluent and then serum starved. After wounding, the monolayers were treated with EGF (50 ng/ml) for 5 or 10 h or left untreated for 5 h, and then either stained with Crystal violet or subjected to immunofluorescent staining with affinity purified anti–BPAG-2 antibodies. Cell migration results were quantitated as described in the Materials and methods section and plotted graphically. The graph shows the mean and SD of values from a representative experiment performed in triplicate. Results similar to those shown here were obtained with all three clones expressing dominant negative Fyn and the two clones expressing dominant negative Src. Bar, 250 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2150849&req=5

fig6: Dominant negative Fyn inhibits disassembly of hemidesmosomes and suppresses cell migration. The indicated 804G clones were grown until confluent and then serum starved. After wounding, the monolayers were treated with EGF (50 ng/ml) for 5 or 10 h or left untreated for 5 h, and then either stained with Crystal violet or subjected to immunofluorescent staining with affinity purified anti–BPAG-2 antibodies. Cell migration results were quantitated as described in the Materials and methods section and plotted graphically. The graph shows the mean and SD of values from a representative experiment performed in triplicate. Results similar to those shown here were obtained with all three clones expressing dominant negative Fyn and the two clones expressing dominant negative Src. Bar, 250 μm.
Mentions: It is reasonable to assume that a cell must disassemble stable adhesions with the extracellular matrix, such as hemidesmosomes, in order to migrate and invade (Giancotti and Mainiero, 1994). Thus, we examined if inhibition of Fyn suppressed cell migration. As shown in Fig. 6, 804G clones expressing dominant negative Fyn migrated across an artificial in vitro wound much less efficiently than control clones. The migratory ability of clones expressing dominant negative Src was also reduced, but to a lesser extent than that of clones expressing dominant negative Fyn. This effect of dominant negative Src may result from stabilization of focal adhesions (Fincham and Frame, 1998). Immunofluorescent staining with antibodies to BPAG-2 revealed disassembly of hemidesmosomes at the rear end of control cells entering the wound area. Similar results were obtained with cells expressing dominant negative Src. By contrast, these posterior hemidesmosomes were not disassembled in cells expressing dominant negative Fyn (Fig. 6). These observations suggest that dominant negative Fyn inhibits cell migration by enhancing the stability of hemidesmosomes.

Bottom Line: By contrast, Src and Lck do not associate with alpha6beta4 to a significant extent.These observations suggest that the EGF-R causes disassembly of hemidesmosomes by activating Fyn, which in turn phosphorylates the beta4 cytoplasmic domain.Neoplastic cells expressing dominant negative Fyn display increased hemidesmosomes and migrate poorly in vitro in response to EGF.

View Article: PubMed Central - PubMed

Affiliation: Cellular Biochemistry and Biophysics Program, Department of Surgery, Sloan-Kettering Institute for Cancer Research, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.

ABSTRACT
We have examined the mechanism and functional significance of hemidesmosome disassembly during normal epithelial cell migration and squamous carcinoma invasion. Our findings indicate that a fraction of EGF receptor (EGF-R) combines with the hemidesmosomal integrin alpha6beta4 in both normal and neoplastic keratinocytes. Activation of the EGF-R causes tyrosine phosphorylation of the beta4 cytoplasmic domain and disruption of hemidesmosomes. The Src family kinase inhibitors PP1 and PP2 prevent tyrosine phosphorylation of beta4 and disassembly of hemidesmosomes without interfering with the activation of EGF-R. Coimmunoprecipitation experiments indicate that Fyn and, to a lesser extent, Yes combine with alpha6beta4. By contrast, Src and Lck do not associate with alpha6beta4 to a significant extent. A dominant negative form of Fyn, but not Src, prevents tyrosine phosphorylation of beta4 and disassembly of hemidesmosomes. These observations suggest that the EGF-R causes disassembly of hemidesmosomes by activating Fyn, which in turn phosphorylates the beta4 cytoplasmic domain. Neoplastic cells expressing dominant negative Fyn display increased hemidesmosomes and migrate poorly in vitro in response to EGF. Furthermore, dominant negative Fyn decreases the ability of squamous carcinoma cells to invade through Matrigel in vitro and to form lung metastases following intravenous injection in nude mice. These results suggest that disruption of hemidesmosomes mediated by Fyn is a prerequisite for normal keratinocyte migration and squamous carcinoma invasion.

Show MeSH
Related in: MedlinePlus