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

EGF stimulates serine phosphorylation of the β4 subunit. (A) Analysis of tyrosine phosphorylation using phosphotyrosine-specific antibodies. A431 cells were plated on laminin-1 for 1 h and then either stimulated with EGF (1–100 ng/ml) for 15 min or left untreated. Detergent (RIPA) extracts were immunoprecipitated using the β4 antibody 439-9B, resolved by SDS-PAGE, and immunoblotted using a combination of the PY20 and 4G10 phosphotyrosine-specific antibodies as described in Materials and Methods. Positive controls shown for tyrosine phosphorylation are the EGF receptor immunoprecipitated from EGF-stimulated A431 cells and α6β4 immunoprecipitated from A431 cells that had been pretreated with pervanadate before extraction. Both immunoprecipitates were blotted with PY20 and 4G10 as described. (B) 32PO4 metabolic labeling and analysis of EGF-stimulated A431 cells. A431 cells plated on laminin-1 were labeled with 32PO4 as described in Materials and Methods, and stimulated with EGF for 15 min at the concentrations indicated. Cells were then extracted, immunoprecipitated with 439-9B, blotted with the β4-specific polyclonal antibody, and exposed to x-ray film (left panel). The relative intensities of the radiolabeled β4 bands were quantified using an electronic counter as shown in the bar graph in the right panel. The data shown were obtained from three separate experiments (± SE). After exposure, the membranes were probed with the β4-specific antibody to corroborate equal loading of the samples (left panel). (C) Phosphoamino-acid analysis of 32PO4-labeled β4. The radiolabeled band that corresponded to the β4 subunit obtained from cells stimulated with either 0 or 100 ng/ml EGF was excised from the PVDF membrane, acid hydrolyzed, and then separated by two-dimensional thin-layer electrophoresis as described in Materials and Methods. The plate was then exposed to x-ray film. S, phosphoserine; T, phosphothreonine; Y, phosphotyrosine.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169473&req=5

Figure 6: EGF stimulates serine phosphorylation of the β4 subunit. (A) Analysis of tyrosine phosphorylation using phosphotyrosine-specific antibodies. A431 cells were plated on laminin-1 for 1 h and then either stimulated with EGF (1–100 ng/ml) for 15 min or left untreated. Detergent (RIPA) extracts were immunoprecipitated using the β4 antibody 439-9B, resolved by SDS-PAGE, and immunoblotted using a combination of the PY20 and 4G10 phosphotyrosine-specific antibodies as described in Materials and Methods. Positive controls shown for tyrosine phosphorylation are the EGF receptor immunoprecipitated from EGF-stimulated A431 cells and α6β4 immunoprecipitated from A431 cells that had been pretreated with pervanadate before extraction. Both immunoprecipitates were blotted with PY20 and 4G10 as described. (B) 32PO4 metabolic labeling and analysis of EGF-stimulated A431 cells. A431 cells plated on laminin-1 were labeled with 32PO4 as described in Materials and Methods, and stimulated with EGF for 15 min at the concentrations indicated. Cells were then extracted, immunoprecipitated with 439-9B, blotted with the β4-specific polyclonal antibody, and exposed to x-ray film (left panel). The relative intensities of the radiolabeled β4 bands were quantified using an electronic counter as shown in the bar graph in the right panel. The data shown were obtained from three separate experiments (± SE). After exposure, the membranes were probed with the β4-specific antibody to corroborate equal loading of the samples (left panel). (C) Phosphoamino-acid analysis of 32PO4-labeled β4. The radiolabeled band that corresponded to the β4 subunit obtained from cells stimulated with either 0 or 100 ng/ml EGF was excised from the PVDF membrane, acid hydrolyzed, and then separated by two-dimensional thin-layer electrophoresis as described in Materials and Methods. The plate was then exposed to x-ray film. S, phosphoserine; T, phosphothreonine; Y, phosphotyrosine.

Mentions: The colocalization of α6β4 with phosphotyrosine in the lamellipodia and ruffles of EGF-stimulated A431 cells prompted us to examine the phosphorylation of α6β4 induced by EGF. For this purpose, α6β4 was immunoprecipitated from stimulated A431 cells with the 439-9B mAb and the immunoprecipitates were blotted with two phosphotyrosine-specific Abs (PY20 and 4G10). In these experiments, the cells were extracted with RIPA buffer because we observed a nonspecific interaction between α6β4 and the EGFR using a Triton X-100 buffer (data not shown). Under these conditions, we detected no phosphotyrosine in the β4 subunit in response to the concentration of EGF (1 ng/ml) that induced α6β4 redistribution to lamellipodia and ruffles, and that stimulated optimal A431 chemotaxis (Fig. 6 A). Moreover, even high concentrations of EGF (100 ng/ml) that induced a rapid rounding-up of adherent A431 cells and did not stimulate chemotaxis (Fig. 2 A) induced only a marginal increase, at best, in the phosphotyrosine content of the β4 subunit as detected by these antibodies (Fig. 6 A). Similar results were obtained with other α6 and β4-specific mAbs including GoH3, 2B7, A9, as well as a β4-specific polyclonal antibody (data not shown). These findings are in contrast to the report that EGF stimulation induces a substantial increase in the tyrosine phosphorylation of the β4 subunit in A431 cells 31.


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 stimulates serine phosphorylation of the β4 subunit. (A) Analysis of tyrosine phosphorylation using phosphotyrosine-specific antibodies. A431 cells were plated on laminin-1 for 1 h and then either stimulated with EGF (1–100 ng/ml) for 15 min or left untreated. Detergent (RIPA) extracts were immunoprecipitated using the β4 antibody 439-9B, resolved by SDS-PAGE, and immunoblotted using a combination of the PY20 and 4G10 phosphotyrosine-specific antibodies as described in Materials and Methods. Positive controls shown for tyrosine phosphorylation are the EGF receptor immunoprecipitated from EGF-stimulated A431 cells and α6β4 immunoprecipitated from A431 cells that had been pretreated with pervanadate before extraction. Both immunoprecipitates were blotted with PY20 and 4G10 as described. (B) 32PO4 metabolic labeling and analysis of EGF-stimulated A431 cells. A431 cells plated on laminin-1 were labeled with 32PO4 as described in Materials and Methods, and stimulated with EGF for 15 min at the concentrations indicated. Cells were then extracted, immunoprecipitated with 439-9B, blotted with the β4-specific polyclonal antibody, and exposed to x-ray film (left panel). The relative intensities of the radiolabeled β4 bands were quantified using an electronic counter as shown in the bar graph in the right panel. The data shown were obtained from three separate experiments (± SE). After exposure, the membranes were probed with the β4-specific antibody to corroborate equal loading of the samples (left panel). (C) Phosphoamino-acid analysis of 32PO4-labeled β4. The radiolabeled band that corresponded to the β4 subunit obtained from cells stimulated with either 0 or 100 ng/ml EGF was excised from the PVDF membrane, acid hydrolyzed, and then separated by two-dimensional thin-layer electrophoresis as described in Materials and Methods. The plate was then exposed to x-ray film. S, phosphoserine; T, phosphothreonine; Y, phosphotyrosine.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: EGF stimulates serine phosphorylation of the β4 subunit. (A) Analysis of tyrosine phosphorylation using phosphotyrosine-specific antibodies. A431 cells were plated on laminin-1 for 1 h and then either stimulated with EGF (1–100 ng/ml) for 15 min or left untreated. Detergent (RIPA) extracts were immunoprecipitated using the β4 antibody 439-9B, resolved by SDS-PAGE, and immunoblotted using a combination of the PY20 and 4G10 phosphotyrosine-specific antibodies as described in Materials and Methods. Positive controls shown for tyrosine phosphorylation are the EGF receptor immunoprecipitated from EGF-stimulated A431 cells and α6β4 immunoprecipitated from A431 cells that had been pretreated with pervanadate before extraction. Both immunoprecipitates were blotted with PY20 and 4G10 as described. (B) 32PO4 metabolic labeling and analysis of EGF-stimulated A431 cells. A431 cells plated on laminin-1 were labeled with 32PO4 as described in Materials and Methods, and stimulated with EGF for 15 min at the concentrations indicated. Cells were then extracted, immunoprecipitated with 439-9B, blotted with the β4-specific polyclonal antibody, and exposed to x-ray film (left panel). The relative intensities of the radiolabeled β4 bands were quantified using an electronic counter as shown in the bar graph in the right panel. The data shown were obtained from three separate experiments (± SE). After exposure, the membranes were probed with the β4-specific antibody to corroborate equal loading of the samples (left panel). (C) Phosphoamino-acid analysis of 32PO4-labeled β4. The radiolabeled band that corresponded to the β4 subunit obtained from cells stimulated with either 0 or 100 ng/ml EGF was excised from the PVDF membrane, acid hydrolyzed, and then separated by two-dimensional thin-layer electrophoresis as described in Materials and Methods. The plate was then exposed to x-ray film. S, phosphoserine; T, phosphothreonine; Y, phosphotyrosine.
Mentions: The colocalization of α6β4 with phosphotyrosine in the lamellipodia and ruffles of EGF-stimulated A431 cells prompted us to examine the phosphorylation of α6β4 induced by EGF. For this purpose, α6β4 was immunoprecipitated from stimulated A431 cells with the 439-9B mAb and the immunoprecipitates were blotted with two phosphotyrosine-specific Abs (PY20 and 4G10). In these experiments, the cells were extracted with RIPA buffer because we observed a nonspecific interaction between α6β4 and the EGFR using a Triton X-100 buffer (data not shown). Under these conditions, we detected no phosphotyrosine in the β4 subunit in response to the concentration of EGF (1 ng/ml) that induced α6β4 redistribution to lamellipodia and ruffles, and that stimulated optimal A431 chemotaxis (Fig. 6 A). Moreover, even high concentrations of EGF (100 ng/ml) that induced a rapid rounding-up of adherent A431 cells and did not stimulate chemotaxis (Fig. 2 A) induced only a marginal increase, at best, in the phosphotyrosine content of the β4 subunit as detected by these antibodies (Fig. 6 A). Similar results were obtained with other α6 and β4-specific mAbs including GoH3, 2B7, A9, as well as a β4-specific polyclonal antibody (data not shown). These findings are in contrast to the report that EGF stimulation induces a substantial increase in the tyrosine phosphorylation of the β4 subunit in A431 cells 31.

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