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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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Analysis of wild-type and myristoylated PKC-α kinase activity. 293T cells were transfected with either the pCMV5 vector alone, the PKC-α-FLAG cDNA, or the myristoylated PKC-α-FLAG cDNA as described in Materials and Methods. These PKC proteins were immunoprecipitated using a FLAG-specific Ab. Expression of PKC-α was confirmed by immunoblotting these precipitates with a PKC-α specific Ab and this information was used to normalize PKC-α expression for the kinase assays (data not shown). Immune complex kinase assays were performed using MBP as the substrate as described in Materials and Methods.
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Figure 9: Analysis of wild-type and myristoylated PKC-α kinase activity. 293T cells were transfected with either the pCMV5 vector alone, the PKC-α-FLAG cDNA, or the myristoylated PKC-α-FLAG cDNA as described in Materials and Methods. These PKC proteins were immunoprecipitated using a FLAG-specific Ab. Expression of PKC-α was confirmed by immunoblotting these precipitates with a PKC-α specific Ab and this information was used to normalize PKC-α expression for the kinase assays (data not shown). Immune complex kinase assays were performed using MBP as the substrate as described in Materials and Methods.

Mentions: The above data suggested the participation of a conventional PKC isoform in the disassembly of the hemidesmosome and mobilization of α6β4 integrin. To obtain additional evidence for PKC involvement, we examined the possibility that activation of PKC-α, a widely distributed conventional PKC isoform, was sufficient to disassemble hemidesmosomes in the absence of EGF stimulation. For this purpose, we constructed a constitutively active PKC-α cDNA that contained the Src myristoylation site at its amino terminus. This myristoylated PKC-α exhibited a high level of in vitro kinase activity relative to the wild-type enzyme (Fig. 9). Subsequently, we expressed both the myristoylated and wild-type PKC-α cDNAs in A431 cells and analyzed the effect of these cDNAs on hemidesmosome structure. As shown in Fig. 10A431 cells that expressed myristoylated PKC-α, as evidenced by expression of the epitope tag (FLAG), showed a striking reduction in hemidesmosomes. More specifically, expression of both HD-1 and α6β4 was markedly reduced on the basal surface of these cells. In contrast, the cells that expressed the wild-type PKC-α, showed little change in the formation of hemidesmosomes. These results suggest that activation of PKC-α is sufficient to cause redistribution of the α6β4 integrin and other components of the hemidesmosome.


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)

Analysis of wild-type and myristoylated PKC-α kinase activity. 293T cells were transfected with either the pCMV5 vector alone, the PKC-α-FLAG cDNA, or the myristoylated PKC-α-FLAG cDNA as described in Materials and Methods. These PKC proteins were immunoprecipitated using a FLAG-specific Ab. Expression of PKC-α was confirmed by immunoblotting these precipitates with a PKC-α specific Ab and this information was used to normalize PKC-α expression for the kinase assays (data not shown). Immune complex kinase assays were performed using MBP as the substrate as described in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 9: Analysis of wild-type and myristoylated PKC-α kinase activity. 293T cells were transfected with either the pCMV5 vector alone, the PKC-α-FLAG cDNA, or the myristoylated PKC-α-FLAG cDNA as described in Materials and Methods. These PKC proteins were immunoprecipitated using a FLAG-specific Ab. Expression of PKC-α was confirmed by immunoblotting these precipitates with a PKC-α specific Ab and this information was used to normalize PKC-α expression for the kinase assays (data not shown). Immune complex kinase assays were performed using MBP as the substrate as described in Materials and Methods.
Mentions: The above data suggested the participation of a conventional PKC isoform in the disassembly of the hemidesmosome and mobilization of α6β4 integrin. To obtain additional evidence for PKC involvement, we examined the possibility that activation of PKC-α, a widely distributed conventional PKC isoform, was sufficient to disassemble hemidesmosomes in the absence of EGF stimulation. For this purpose, we constructed a constitutively active PKC-α cDNA that contained the Src myristoylation site at its amino terminus. This myristoylated PKC-α exhibited a high level of in vitro kinase activity relative to the wild-type enzyme (Fig. 9). Subsequently, we expressed both the myristoylated and wild-type PKC-α cDNAs in A431 cells and analyzed the effect of these cDNAs on hemidesmosome structure. As shown in Fig. 10A431 cells that expressed myristoylated PKC-α, as evidenced by expression of the epitope tag (FLAG), showed a striking reduction in hemidesmosomes. More specifically, expression of both HD-1 and α6β4 was markedly reduced on the basal surface of these cells. In contrast, the cells that expressed the wild-type PKC-α, showed little change in the formation of hemidesmosomes. These results suggest that activation of PKC-α is sufficient to cause redistribution of the α6β4 integrin and other components of the hemidesmosome.

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