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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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EGF promotes the release of α6β4 integrin from an insoluble cytokeratin fraction and increases its association with an actin fraction. A431 cells were plated on laminin-1 for 1 h and either left untreated or stimulated with EGF (1 ng/ml) for 15 min. The cells were then sequentially extracted to obtain membrane, actin, and cytokeratin fractions (fractions 1, 2, and 3, respectively) as described in Materials and Methods. After solubilization, α6β4 was immunoprecipitated from each fraction using the GoH3 mAb, resolved by SDS-PAGE and detected by immunoblotting using a β4-specific polyclonal antibody. Actin and cytokeratins were detected in each fraction by immunoblotting using an anti-actin polyclonal and pan-cytokeratin monoclonal Abs, respectively. Note that the β4 subunit is detected as a doublet under these conditions.
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Figure 3: EGF promotes the release of α6β4 integrin from an insoluble cytokeratin fraction and increases its association with an actin fraction. A431 cells were plated on laminin-1 for 1 h and either left untreated or stimulated with EGF (1 ng/ml) for 15 min. The cells were then sequentially extracted to obtain membrane, actin, and cytokeratin fractions (fractions 1, 2, and 3, respectively) as described in Materials and Methods. After solubilization, α6β4 was immunoprecipitated from each fraction using the GoH3 mAb, resolved by SDS-PAGE and detected by immunoblotting using a β4-specific polyclonal antibody. Actin and cytokeratins were detected in each fraction by immunoblotting using an anti-actin polyclonal and pan-cytokeratin monoclonal Abs, respectively. Note that the β4 subunit is detected as a doublet under these conditions.

Mentions: Our observation that α6β4 is redistributed from hemidesmosomes to lamellipodia and membrane ruffles in response to EGF stimulation prompted us to examine its association with cytokeratins and F-actin in more detail using an in situ extraction scheme that solubilizes proteins to an extent that correlates with their cytoskeletal associations 919. Specifically, membrane, actin, and cytokeratin fractions were obtained from A431 cells that had been either left untreated or stimulated with EGF using sequentially a Triton X-100 buffer (fraction 1, membrane), a two-detergent buffer (1.0% Tween-40/0.5% deoxycholate, fraction 2, actin) that removes the bulk of the actin cytoskeleton but not cytokeratins and associated proteins, and a third buffer containing SDS that solubilizes cytokeratins and associated proteins (fraction 3, cytokeratin; references 9, 19). The relative amount of α6β4 present in each fraction was detected by immunoprecipitation and subsequent immunoblotting with β4-specific antibodies. The relative distribution of actin and cytokeratin among the three fractions was also determined to assess the efficiency of the fractionation (Fig. 3). As expected, the cytokeratins were present largely in fraction 3 and actin was distributed between fractions 1 and 2, which represent the G-actin and F-actin pools, respectively. Importantly, EGF stimulation did not alter this relative distribution of cytoskeletal proteins among the three fractions. However, as shown in Fig. 3, EGF stimulation resulted in a substantial reduction in the amount of α6β4 in fraction 3 (cytokeratin) and an increase in the amount of α6β4 in the actin fraction in comparison to unstimulated cells. Densitometric analysis of the β4-specific bands in this figure revealed an approximate 63% reduction of α6β4 in the cytokeratin fraction and a 48% increase in the actin fraction. These observations provide evidence that the mobilization of α6β4 from hemidesmosomes that we detected by indirect immunofluorescence microscopy is associated with a disruption in its association with cytokeratins and an increase in its association with F-actin.


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)

EGF promotes the release of α6β4 integrin from an insoluble cytokeratin fraction and increases its association with an actin fraction. A431 cells were plated on laminin-1 for 1 h and either left untreated or stimulated with EGF (1 ng/ml) for 15 min. The cells were then sequentially extracted to obtain membrane, actin, and cytokeratin fractions (fractions 1, 2, and 3, respectively) as described in Materials and Methods. After solubilization, α6β4 was immunoprecipitated from each fraction using the GoH3 mAb, resolved by SDS-PAGE and detected by immunoblotting using a β4-specific polyclonal antibody. Actin and cytokeratins were detected in each fraction by immunoblotting using an anti-actin polyclonal and pan-cytokeratin monoclonal Abs, respectively. Note that the β4 subunit is detected as a doublet under these conditions.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: EGF promotes the release of α6β4 integrin from an insoluble cytokeratin fraction and increases its association with an actin fraction. A431 cells were plated on laminin-1 for 1 h and either left untreated or stimulated with EGF (1 ng/ml) for 15 min. The cells were then sequentially extracted to obtain membrane, actin, and cytokeratin fractions (fractions 1, 2, and 3, respectively) as described in Materials and Methods. After solubilization, α6β4 was immunoprecipitated from each fraction using the GoH3 mAb, resolved by SDS-PAGE and detected by immunoblotting using a β4-specific polyclonal antibody. Actin and cytokeratins were detected in each fraction by immunoblotting using an anti-actin polyclonal and pan-cytokeratin monoclonal Abs, respectively. Note that the β4 subunit is detected as a doublet under these conditions.
Mentions: Our observation that α6β4 is redistributed from hemidesmosomes to lamellipodia and membrane ruffles in response to EGF stimulation prompted us to examine its association with cytokeratins and F-actin in more detail using an in situ extraction scheme that solubilizes proteins to an extent that correlates with their cytoskeletal associations 919. Specifically, membrane, actin, and cytokeratin fractions were obtained from A431 cells that had been either left untreated or stimulated with EGF using sequentially a Triton X-100 buffer (fraction 1, membrane), a two-detergent buffer (1.0% Tween-40/0.5% deoxycholate, fraction 2, actin) that removes the bulk of the actin cytoskeleton but not cytokeratins and associated proteins, and a third buffer containing SDS that solubilizes cytokeratins and associated proteins (fraction 3, cytokeratin; references 9, 19). The relative amount of α6β4 present in each fraction was detected by immunoprecipitation and subsequent immunoblotting with β4-specific antibodies. The relative distribution of actin and cytokeratin among the three fractions was also determined to assess the efficiency of the fractionation (Fig. 3). As expected, the cytokeratins were present largely in fraction 3 and actin was distributed between fractions 1 and 2, which represent the G-actin and F-actin pools, respectively. Importantly, EGF stimulation did not alter this relative distribution of cytoskeletal proteins among the three fractions. However, as shown in Fig. 3, EGF stimulation resulted in a substantial reduction in the amount of α6β4 in fraction 3 (cytokeratin) and an increase in the amount of α6β4 in the actin fraction in comparison to unstimulated cells. Densitometric analysis of the β4-specific bands in this figure revealed an approximate 63% reduction of α6β4 in the cytokeratin fraction and a 48% increase in the actin fraction. These observations provide evidence that the mobilization of α6β4 from hemidesmosomes that we detected by indirect immunofluorescence microscopy is associated with a disruption in its association with cytokeratins and an increase in its association with F-actin.

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