Limits...
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.

Show MeSH

Related in: MedlinePlus

Protein kinase C mediates the effects of EGF on β4 serine phosphorylation, release of α6β4 from a cytokeratin fraction, and chemotaxis. (A) A431 cells plated on laminin-1 were labeled with 32PO4 as described in Materials and Methods and then stimulated with either PMA or vehicle (DMSO) alone. Cells were then extracted, immunoprecipitated with 439-9B mAb, blotted with the β4-specific antibody, and exposed to x-ray film. (B) A431 cells plated on laminin-1 were labeled with 32PO4 and then incubated with either Gö6976 (1 μM) or vehicle (DMSO) alone for 30 min before stimulation with EGF (100 ng/ml) for 15 min. The cells were then extracted and processed as in A. In A and B, the lower panel is an immunoblot analysis of β4 content that verifies equal loading of the samples. (C) Inhibition of EGF-induced release of α6β4 from a cytokeratin fraction. A431 cells plated on laminin-1 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 (50 ng/ml) for 30 min. The cells were then extracted with a buffer that solubilizes the actin cytoskeleton but preserves the cytokeratin network. After solubilization, α6β4 was immunoprecipitated from this residual fraction, resolved by SDS-PAGE, and detected by immunoblotting using a β4-specific polyclonal antibody as described in Materials and Methods. (D) Gö6976 inhibits EGF-stimulated chemotactic migration; chemotaxis was assessed in a modified Boyden chamber as described in Fig. 2. Gö6976 (1 μM) or vehicle alone was added 30 min before stimulation with EGF (1 ng/ml).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169473&req=5

Figure 8: Protein kinase C mediates the effects of EGF on β4 serine phosphorylation, release of α6β4 from a cytokeratin fraction, and chemotaxis. (A) A431 cells plated on laminin-1 were labeled with 32PO4 as described in Materials and Methods and then stimulated with either PMA or vehicle (DMSO) alone. Cells were then extracted, immunoprecipitated with 439-9B mAb, blotted with the β4-specific antibody, and exposed to x-ray film. (B) A431 cells plated on laminin-1 were labeled with 32PO4 and then incubated with either Gö6976 (1 μM) or vehicle (DMSO) alone for 30 min before stimulation with EGF (100 ng/ml) for 15 min. The cells were then extracted and processed as in A. In A and B, the lower panel is an immunoblot analysis of β4 content that verifies equal loading of the samples. (C) Inhibition of EGF-induced release of α6β4 from a cytokeratin fraction. A431 cells plated on laminin-1 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 (50 ng/ml) for 30 min. The cells were then extracted with a buffer that solubilizes the actin cytoskeleton but preserves the cytokeratin network. After solubilization, α6β4 was immunoprecipitated from this residual fraction, resolved by SDS-PAGE, and detected by immunoblotting using a β4-specific polyclonal antibody as described in Materials and Methods. (D) Gö6976 inhibits EGF-stimulated chemotactic migration; chemotaxis was assessed in a modified Boyden chamber as described in Fig. 2. Gö6976 (1 μM) or vehicle alone was added 30 min before stimulation with EGF (1 ng/ml).

Mentions: The above findings indicated that EGF activates a serine protein kinase that is involved in β4 phosphorylation and that could also be involved in the redistribution of α6β4 from hemidesmosomes to lamellipodia and membrane ruffles. We hypothesized that a likely candidate for this kinase is PKC because its activation by EGF is well documented 2. As an initial test of this hypothesis, we examined the effect of PMA stimulation on α6β4 localization in A431 cells. PMA stimulation mobilized α6β4 from hemidesmosomes (Fig. 7, left panels), and increases the formation of α6β4-containing lamellipodia and ruffles (data not shown) as assessed by indirect immunofluorescence microscopy. These data were substantiated biochemically by analyzing the amount of β4 that remains associated with the cytokeratin fraction after stimulation with PMA, using the detergent extraction procedure described above. PMA stimulation markedly reduced the amount of α6β4 in the cytokeratin fraction in comparison to unstimulated cells (Fig. 8 C). Consistent with a role of PKC-dependent phosphorylation in the redistribution of α6β4, we found that PMA stimulation itself increased the phosphorylation of the β4 subunit significantly as assessed by 32P-orthophosphate labeling (Fig. 8 A). The fact that we detected no tyrosine phosphorylation of β4 in response to PMA stimulation using the phosphotyrosine-specific antibodies (data not shown) indicates that the increase in 32P-orthophosphate labeling can be attributed to serine phosphorylation.


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)

Protein kinase C mediates the effects of EGF on β4 serine phosphorylation, release of α6β4 from a cytokeratin fraction, and chemotaxis. (A) A431 cells plated on laminin-1 were labeled with 32PO4 as described in Materials and Methods and then stimulated with either PMA or vehicle (DMSO) alone. Cells were then extracted, immunoprecipitated with 439-9B mAb, blotted with the β4-specific antibody, and exposed to x-ray film. (B) A431 cells plated on laminin-1 were labeled with 32PO4 and then incubated with either Gö6976 (1 μM) or vehicle (DMSO) alone for 30 min before stimulation with EGF (100 ng/ml) for 15 min. The cells were then extracted and processed as in A. In A and B, the lower panel is an immunoblot analysis of β4 content that verifies equal loading of the samples. (C) Inhibition of EGF-induced release of α6β4 from a cytokeratin fraction. A431 cells plated on laminin-1 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 (50 ng/ml) for 30 min. The cells were then extracted with a buffer that solubilizes the actin cytoskeleton but preserves the cytokeratin network. After solubilization, α6β4 was immunoprecipitated from this residual fraction, resolved by SDS-PAGE, and detected by immunoblotting using a β4-specific polyclonal antibody as described in Materials and Methods. (D) Gö6976 inhibits EGF-stimulated chemotactic migration; chemotaxis was assessed in a modified Boyden chamber as described in Fig. 2. Gö6976 (1 μM) or vehicle alone was added 30 min before stimulation with EGF (1 ng/ml).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Protein kinase C mediates the effects of EGF on β4 serine phosphorylation, release of α6β4 from a cytokeratin fraction, and chemotaxis. (A) A431 cells plated on laminin-1 were labeled with 32PO4 as described in Materials and Methods and then stimulated with either PMA or vehicle (DMSO) alone. Cells were then extracted, immunoprecipitated with 439-9B mAb, blotted with the β4-specific antibody, and exposed to x-ray film. (B) A431 cells plated on laminin-1 were labeled with 32PO4 and then incubated with either Gö6976 (1 μM) or vehicle (DMSO) alone for 30 min before stimulation with EGF (100 ng/ml) for 15 min. The cells were then extracted and processed as in A. In A and B, the lower panel is an immunoblot analysis of β4 content that verifies equal loading of the samples. (C) Inhibition of EGF-induced release of α6β4 from a cytokeratin fraction. A431 cells plated on laminin-1 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 (50 ng/ml) for 30 min. The cells were then extracted with a buffer that solubilizes the actin cytoskeleton but preserves the cytokeratin network. After solubilization, α6β4 was immunoprecipitated from this residual fraction, resolved by SDS-PAGE, and detected by immunoblotting using a β4-specific polyclonal antibody as described in Materials and Methods. (D) Gö6976 inhibits EGF-stimulated chemotactic migration; chemotaxis was assessed in a modified Boyden chamber as described in Fig. 2. Gö6976 (1 μM) or vehicle alone was added 30 min before stimulation with EGF (1 ng/ml).
Mentions: The above findings indicated that EGF activates a serine protein kinase that is involved in β4 phosphorylation and that could also be involved in the redistribution of α6β4 from hemidesmosomes to lamellipodia and membrane ruffles. We hypothesized that a likely candidate for this kinase is PKC because its activation by EGF is well documented 2. As an initial test of this hypothesis, we examined the effect of PMA stimulation on α6β4 localization in A431 cells. PMA stimulation mobilized α6β4 from hemidesmosomes (Fig. 7, left panels), and increases the formation of α6β4-containing lamellipodia and ruffles (data not shown) as assessed by indirect immunofluorescence microscopy. These data were substantiated biochemically by analyzing the amount of β4 that remains associated with the cytokeratin fraction after stimulation with PMA, using the detergent extraction procedure described above. PMA stimulation markedly reduced the amount of α6β4 in the cytokeratin fraction in comparison to unstimulated cells (Fig. 8 C). Consistent with a role of PKC-dependent phosphorylation in the redistribution of α6β4, we found that PMA stimulation itself increased the phosphorylation of the β4 subunit significantly as assessed by 32P-orthophosphate labeling (Fig. 8 A). The fact that we detected no tyrosine phosphorylation of β4 in response to PMA stimulation using the phosphotyrosine-specific antibodies (data not shown) indicates that the increase in 32P-orthophosphate labeling can be attributed to serine phosphorylation.

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