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Protein kinase C-dependent mobilization of the alpha6beta4 integrin from hemidesmosomes and its association with actin-rich cell protrusions drive the chemotactic migration of carcinoma cells.

Rabinovitz I, Toker A, Mercurio AM - J. Cell Biol. (1999)

Bottom Line: Using function-blocking antibodies, we show that the alpha6beta4 integrin participates in EGF-stimulated chemotaxis and is required for lamellae formation on laminin-1.At concentrations of EGF that stimulate A431 chemotaxis ( approximately 1 ng/ml), the alpha6beta4 integrin is mobilized from hemidesmosomes as evidenced by indirect immunofluorescence microscopy using mAbs specific for this integrin and hemidesmosomal components and its loss from a cytokeratin fraction obtained by detergent extraction.Importantly, we demonstrate that this mobilization of alpha6beta4 from hemidesmosomes and its redistribution to cell protrusions occurs by a mechanism that involves activation of protein kinase C-alpha and that it is associated with the phosphorylation of the beta4 integrin subunit on serine residues.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.

ABSTRACT
We explored the hypothesis that the chemotactic migration of carcinoma cells that assemble hemidesmosomes involves the activation of a signaling pathway that releases the alpha6beta4 integrin from these stable adhesion complexes and promotes its association with F-actin in cell protrusions enabling it to function in migration. Squamous carcinoma-derived A431 cells were used because they express alpha6beta4 and migrate in response to EGF stimulation. Using function-blocking antibodies, we show that the alpha6beta4 integrin participates in EGF-stimulated chemotaxis and is required for lamellae formation on laminin-1. At concentrations of EGF that stimulate A431 chemotaxis ( approximately 1 ng/ml), the alpha6beta4 integrin is mobilized from hemidesmosomes as evidenced by indirect immunofluorescence microscopy using mAbs specific for this integrin and hemidesmosomal components and its loss from a cytokeratin fraction obtained by detergent extraction. EGF stimulation also increased the formation of lamellipodia and membrane ruffles that contained alpha6beta4 in association with F-actin. Importantly, we demonstrate that this mobilization of alpha6beta4 from hemidesmosomes and its redistribution to cell protrusions occurs by a mechanism that involves activation of protein kinase C-alpha and that it is associated with the phosphorylation of the beta4 integrin subunit on serine residues. Thus, the chemotactic migration of A431 cells on laminin-1 requires not only the formation of F-actin-rich cell protrusions that mediate alpha6beta4-dependent cell movement but also the disruption of alpha6beta4-containing hemidesmosomes by protein kinase C.

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Inhibition of conventional PKCs blocks the EGF/PMA-induced redistribution of α6β4 from hemidesmosomes. A431 cells plated on laminin-1 for 2 h were incubated with Gö6976 (1 μM) or vehicle (DMSO) alone for 30 min before stimulating with EGF (1 ng/ml) for 15 min or PMA (25 ng/ml) for 30 min. The cells were extracted with Triton X-100 buffer before fixation to enhance visualization of hemidesmosomes. The fixed cells were stained with the α6-specific antibody GoH3 and a rhodamine-conjugated anti–rat IgG, and then analyzed using indirect immunofluorescence. Confocal images shown were taken at the basal surface of the cells. Bar, 10 μm.
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Figure 7: Inhibition of conventional PKCs blocks the EGF/PMA-induced redistribution of α6β4 from hemidesmosomes. A431 cells plated on laminin-1 for 2 h were incubated with Gö6976 (1 μM) or vehicle (DMSO) alone for 30 min before stimulating with EGF (1 ng/ml) for 15 min or PMA (25 ng/ml) for 30 min. The cells were extracted with Triton X-100 buffer before fixation to enhance visualization of hemidesmosomes. The fixed cells were stained with the α6-specific antibody GoH3 and a rhodamine-conjugated anti–rat IgG, and then analyzed using indirect immunofluorescence. Confocal images shown were taken at the basal surface of the cells. Bar, 10 μm.

Mentions: The findings that α6β4 is localized in hemidesmosomes in adherent A431 cells and that EGF stimulated their α6β4-dependent migration raised the possibility that EGF also altered the localization and cytoskeletal interactions of this integrin. Under these conditions of EGF stimulation, a striking change in the localization of α6β4 was apparent. Specifically, this integrin was substantially reduced in hemidesmosomes on the ventral surface (Fig. 1D and Fig. E, and Fig. 7, left panels), but it was readily apparent in the lamellipodia and ruffles that are formed in response to EGF stimulation (Fig. 1 E). We observed also that EGF stimulation results in a reduction of HD1/plectin staining in hemidesmosomes, indicating a disassembly of hemidesmosome structure (Fig. 1 F).


Protein kinase C-dependent mobilization of the alpha6beta4 integrin from hemidesmosomes and its association with actin-rich cell protrusions drive the chemotactic migration of carcinoma cells.

Rabinovitz I, Toker A, Mercurio AM - J. Cell Biol. (1999)

Inhibition of conventional PKCs blocks the EGF/PMA-induced redistribution of α6β4 from hemidesmosomes. A431 cells plated on laminin-1 for 2 h were incubated with Gö6976 (1 μM) or vehicle (DMSO) alone for 30 min before stimulating with EGF (1 ng/ml) for 15 min or PMA (25 ng/ml) for 30 min. The cells were extracted with Triton X-100 buffer before fixation to enhance visualization of hemidesmosomes. The fixed cells were stained with the α6-specific antibody GoH3 and a rhodamine-conjugated anti–rat IgG, and then analyzed using indirect immunofluorescence. Confocal images shown were taken at the basal surface of the cells. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169473&req=5

Figure 7: Inhibition of conventional PKCs blocks the EGF/PMA-induced redistribution of α6β4 from hemidesmosomes. A431 cells plated on laminin-1 for 2 h were incubated with Gö6976 (1 μM) or vehicle (DMSO) alone for 30 min before stimulating with EGF (1 ng/ml) for 15 min or PMA (25 ng/ml) for 30 min. The cells were extracted with Triton X-100 buffer before fixation to enhance visualization of hemidesmosomes. The fixed cells were stained with the α6-specific antibody GoH3 and a rhodamine-conjugated anti–rat IgG, and then analyzed using indirect immunofluorescence. Confocal images shown were taken at the basal surface of the cells. Bar, 10 μm.
Mentions: The findings that α6β4 is localized in hemidesmosomes in adherent A431 cells and that EGF stimulated their α6β4-dependent migration raised the possibility that EGF also altered the localization and cytoskeletal interactions of this integrin. Under these conditions of EGF stimulation, a striking change in the localization of α6β4 was apparent. Specifically, this integrin was substantially reduced in hemidesmosomes on the ventral surface (Fig. 1D and Fig. E, and Fig. 7, left panels), but it was readily apparent in the lamellipodia and ruffles that are formed in response to EGF stimulation (Fig. 1 E). We observed also that EGF stimulation results in a reduction of HD1/plectin staining in hemidesmosomes, indicating a disassembly of hemidesmosome structure (Fig. 1 F).

Bottom Line: Using function-blocking antibodies, we show that the alpha6beta4 integrin participates in EGF-stimulated chemotaxis and is required for lamellae formation on laminin-1.At concentrations of EGF that stimulate A431 chemotaxis ( approximately 1 ng/ml), the alpha6beta4 integrin is mobilized from hemidesmosomes as evidenced by indirect immunofluorescence microscopy using mAbs specific for this integrin and hemidesmosomal components and its loss from a cytokeratin fraction obtained by detergent extraction.Importantly, we demonstrate that this mobilization of alpha6beta4 from hemidesmosomes and its redistribution to cell protrusions occurs by a mechanism that involves activation of protein kinase C-alpha and that it is associated with the phosphorylation of the beta4 integrin subunit on serine residues.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.

ABSTRACT
We explored the hypothesis that the chemotactic migration of carcinoma cells that assemble hemidesmosomes involves the activation of a signaling pathway that releases the alpha6beta4 integrin from these stable adhesion complexes and promotes its association with F-actin in cell protrusions enabling it to function in migration. Squamous carcinoma-derived A431 cells were used because they express alpha6beta4 and migrate in response to EGF stimulation. Using function-blocking antibodies, we show that the alpha6beta4 integrin participates in EGF-stimulated chemotaxis and is required for lamellae formation on laminin-1. At concentrations of EGF that stimulate A431 chemotaxis ( approximately 1 ng/ml), the alpha6beta4 integrin is mobilized from hemidesmosomes as evidenced by indirect immunofluorescence microscopy using mAbs specific for this integrin and hemidesmosomal components and its loss from a cytokeratin fraction obtained by detergent extraction. EGF stimulation also increased the formation of lamellipodia and membrane ruffles that contained alpha6beta4 in association with F-actin. Importantly, we demonstrate that this mobilization of alpha6beta4 from hemidesmosomes and its redistribution to cell protrusions occurs by a mechanism that involves activation of protein kinase C-alpha and that it is associated with the phosphorylation of the beta4 integrin subunit on serine residues. Thus, the chemotactic migration of A431 cells on laminin-1 requires not only the formation of F-actin-rich cell protrusions that mediate alpha6beta4-dependent cell movement but also the disruption of alpha6beta4-containing hemidesmosomes by protein kinase C.

Show MeSH
Related in: MedlinePlus