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The transcription factor AP-1 is required for EGF-induced activation of rho-like GTPases, cytoskeletal rearrangements, motility, and in vitro invasion of A431 cells.

Malliri A, Symons M, Hennigan RF, Hurlstone AF, Lamb RF, Wheeler T, Ozanne BW - J. Cell Biol. (1998)

Bottom Line: Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression.However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells.Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion.

View Article: PubMed Central - PubMed

Affiliation: Beatson Institute for Cancer Research, Bearsden, Glasgow, G61 1BD, United Kingdom.

ABSTRACT
Human squamous cell carcinomas (SCC) frequently express elevated levels of epidermal growth factor receptor (EGFR). EGFR overexpression in SCC-derived cell lines correlates with their ability to invade in an in vitro invasion assay in response to EGF, whereas benign epidermal cells, which express low levels of EGFR, do not invade. EGF-induced invasion of SCC-derived A431 cells is inhibited by sustained expression of the dominant negative mutant of c-Jun, TAM67, suggesting a role for the transcription factor AP-1 (activator protein-1) in regulating invasion. Significantly, we establish that sustained TAM67 expression inhibits growth factor-induced cell motility and the reorganization of the cytoskeleton and cell-shape changes essential for this process: TAM67 expression inhibits EGF-induced membrane ruffling, lamellipodia formation, cortical actin polymerization and cell rounding. Introduction of a dominant negative mutant of Rac and of the Rho inhibitor C3 transferase into A431 cells indicates that EGF-induced membrane ruffling and lamellipodia formation are regulated by Rac, whereas EGF-induced cortical actin polymerization and cell rounding are controlled by Rho. Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression. However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells. Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion.

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(a) NA and TA clones express similar levels of EGFR.  Western blot analysis of EGFR in A431 cells, two NA, and three  TA clones using a sheep anti–human EGFR polyclonal antibody.  (b) EGF-induced EGFR autophosphorylation is equivalent in  A431, NA, and TA clones. The top panel represents a Western  blot for phoshotyrosine, using an anti-phosphotyrosine–specific  mouse mAb, after EGF treatment of A431 cells, NA, and TA  clones. The bottom panel represents a similar Western blot  probed with sheep polyclonal anti-EGFR antiserum to show  equivalent loading as well as similar levels of EGFR expression  between the different clones. (c) EGF stimulates phosphorylation of MAPK in A431, NA, and TA clones. The top panel represents MAPK phosphorylation detected using an antibody specific  for the phosphorylated forms of MAPK. The bottom panel represents a similar Western blot probed with an anti-ERK2–specific  antibody to demonstrate equal loading.
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Figure 3: (a) NA and TA clones express similar levels of EGFR. Western blot analysis of EGFR in A431 cells, two NA, and three TA clones using a sheep anti–human EGFR polyclonal antibody. (b) EGF-induced EGFR autophosphorylation is equivalent in A431, NA, and TA clones. The top panel represents a Western blot for phoshotyrosine, using an anti-phosphotyrosine–specific mouse mAb, after EGF treatment of A431 cells, NA, and TA clones. The bottom panel represents a similar Western blot probed with sheep polyclonal anti-EGFR antiserum to show equivalent loading as well as similar levels of EGFR expression between the different clones. (c) EGF stimulates phosphorylation of MAPK in A431, NA, and TA clones. The top panel represents MAPK phosphorylation detected using an antibody specific for the phosphorylated forms of MAPK. The bottom panel represents a similar Western blot probed with an anti-ERK2–specific antibody to demonstrate equal loading.

Mentions: All the SCC-derived cell lines that invade in vitro in response to EGF, including A431 cells, have high levels of EGFR (Stanton et al., 1994). We tested whether the impaired invasiveness of TA cells resulted from decreased EGFR expression and/or signaling activity. Quantitation of the level of EGFR expression for A431 and NA and TA clones by Western blotting (Fig. 3 a) or by ligand binding assays (data not shown) revealed that the level of EGFR expression was equivalent in TA, NA, and A431 cells. Furthermore, EGF-induced EGFR autophosphorylation was equivalent in A431, NA, and TA cells, as determined by Western blot analysis using an anti-phoshotyrosine antibody (Fig. 3 b), and by an EGFR in vitro kinase assay (data not shown). The ability of EGF to stimulate phosphorylation of MAPK was also equivalent in TA, NA, and A431 cells (Fig. 3 c). Thus, TAM67 expression inhibits chemotaxis and invasion of A431 cells without directly interfering with EGFR expression, ligand activation of its tyrosine kinase activity, or its ability to activate MAPK.


The transcription factor AP-1 is required for EGF-induced activation of rho-like GTPases, cytoskeletal rearrangements, motility, and in vitro invasion of A431 cells.

Malliri A, Symons M, Hennigan RF, Hurlstone AF, Lamb RF, Wheeler T, Ozanne BW - J. Cell Biol. (1998)

(a) NA and TA clones express similar levels of EGFR.  Western blot analysis of EGFR in A431 cells, two NA, and three  TA clones using a sheep anti–human EGFR polyclonal antibody.  (b) EGF-induced EGFR autophosphorylation is equivalent in  A431, NA, and TA clones. The top panel represents a Western  blot for phoshotyrosine, using an anti-phosphotyrosine–specific  mouse mAb, after EGF treatment of A431 cells, NA, and TA  clones. The bottom panel represents a similar Western blot  probed with sheep polyclonal anti-EGFR antiserum to show  equivalent loading as well as similar levels of EGFR expression  between the different clones. (c) EGF stimulates phosphorylation of MAPK in A431, NA, and TA clones. The top panel represents MAPK phosphorylation detected using an antibody specific  for the phosphorylated forms of MAPK. The bottom panel represents a similar Western blot probed with an anti-ERK2–specific  antibody to demonstrate equal loading.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132955&req=5

Figure 3: (a) NA and TA clones express similar levels of EGFR. Western blot analysis of EGFR in A431 cells, two NA, and three TA clones using a sheep anti–human EGFR polyclonal antibody. (b) EGF-induced EGFR autophosphorylation is equivalent in A431, NA, and TA clones. The top panel represents a Western blot for phoshotyrosine, using an anti-phosphotyrosine–specific mouse mAb, after EGF treatment of A431 cells, NA, and TA clones. The bottom panel represents a similar Western blot probed with sheep polyclonal anti-EGFR antiserum to show equivalent loading as well as similar levels of EGFR expression between the different clones. (c) EGF stimulates phosphorylation of MAPK in A431, NA, and TA clones. The top panel represents MAPK phosphorylation detected using an antibody specific for the phosphorylated forms of MAPK. The bottom panel represents a similar Western blot probed with an anti-ERK2–specific antibody to demonstrate equal loading.
Mentions: All the SCC-derived cell lines that invade in vitro in response to EGF, including A431 cells, have high levels of EGFR (Stanton et al., 1994). We tested whether the impaired invasiveness of TA cells resulted from decreased EGFR expression and/or signaling activity. Quantitation of the level of EGFR expression for A431 and NA and TA clones by Western blotting (Fig. 3 a) or by ligand binding assays (data not shown) revealed that the level of EGFR expression was equivalent in TA, NA, and A431 cells. Furthermore, EGF-induced EGFR autophosphorylation was equivalent in A431, NA, and TA cells, as determined by Western blot analysis using an anti-phoshotyrosine antibody (Fig. 3 b), and by an EGFR in vitro kinase assay (data not shown). The ability of EGF to stimulate phosphorylation of MAPK was also equivalent in TA, NA, and A431 cells (Fig. 3 c). Thus, TAM67 expression inhibits chemotaxis and invasion of A431 cells without directly interfering with EGFR expression, ligand activation of its tyrosine kinase activity, or its ability to activate MAPK.

Bottom Line: Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression.However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells.Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion.

View Article: PubMed Central - PubMed

Affiliation: Beatson Institute for Cancer Research, Bearsden, Glasgow, G61 1BD, United Kingdom.

ABSTRACT
Human squamous cell carcinomas (SCC) frequently express elevated levels of epidermal growth factor receptor (EGFR). EGFR overexpression in SCC-derived cell lines correlates with their ability to invade in an in vitro invasion assay in response to EGF, whereas benign epidermal cells, which express low levels of EGFR, do not invade. EGF-induced invasion of SCC-derived A431 cells is inhibited by sustained expression of the dominant negative mutant of c-Jun, TAM67, suggesting a role for the transcription factor AP-1 (activator protein-1) in regulating invasion. Significantly, we establish that sustained TAM67 expression inhibits growth factor-induced cell motility and the reorganization of the cytoskeleton and cell-shape changes essential for this process: TAM67 expression inhibits EGF-induced membrane ruffling, lamellipodia formation, cortical actin polymerization and cell rounding. Introduction of a dominant negative mutant of Rac and of the Rho inhibitor C3 transferase into A431 cells indicates that EGF-induced membrane ruffling and lamellipodia formation are regulated by Rac, whereas EGF-induced cortical actin polymerization and cell rounding are controlled by Rho. Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression. However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells. Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion.

Show MeSH
Related in: MedlinePlus