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FAK signaling is critical for ErbB-2/ErbB-3 receptor cooperation for oncogenic transformation and invasion.

Benlimame N, He Q, Jie S, Xiao D, Xu YJ, Loignon M, Schlaepfer DD, Alaoui-Jamali MA - J. Cell Biol. (2005)

Bottom Line: The overexpression of members of the ErbB tyrosine kinase receptor family has been associated with cancer progression.This colocalization requires intact FAK.In summary, distinct FAK signaling has an essential function in ErbB-induced oncogenesis and invasiveness.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Lady Davis Institute of the Sir Mortimer B. Davis Jewish General Hospital, McGill University, Montreal, Quebec H3T 1E2, Canada.

ABSTRACT
The overexpression of members of the ErbB tyrosine kinase receptor family has been associated with cancer progression. We demonstrate that focal adhesion kinase (FAK) is essential for oncogenic transformation and cell invasion that is induced by ErbB-2 and -3 receptor signaling. ErbB-2/3 overexpression in FAK-deficient cells fails to promote cell transformation and rescue chemotaxis deficiency. Restoration of FAK rescues both oncogenic transformation and invasion that is induced by ErbB-2/3 in vitro and in vivo. In contrast, the inhibition of FAK in FAK-proficient invasive cancer cells prevented cell invasion and metastasis formation. The activation of ErbB-2/3 regulates FAK phosphorylation at Tyr-397, -861, and -925. ErbB-induced oncogenic transformation correlates with the ability of FAK to restore ErbB-2/3-induced mitogen-activated protein kinase (MAPK) activation; the inhibition of MAPK prevented oncogenic transformation. In contrast, the inhibition of Src but not MAPK prevented ErbB-FAK-induced chemotaxis. In migratory cells, activated ErbB-2/3 receptors colocalize with activated FAK at cell protrusions. This colocalization requires intact FAK. In summary, distinct FAK signaling has an essential function in ErbB-induced oncogenesis and invasiveness.

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Related in: MedlinePlus

ErbB-induced oncogenic transformation and invasion are mediated via distinct FAK signaling. (A) Differential regulation of FAK phosphorylation by ErbB-2 and -3 receptors. Serum-starved cells were stimulated with 20 ng/ml EGF (control cells) or HRG (ErbB-2, -3, and -2/3–overexpressing cells) for the indicated times. Proteins were immunoprecipitated with anti-FAK and probed with antiphosphotyrosine antibody, antiphospho-FAK antibodies specific to different residues, and anti-FAK antibody. (B) Differential regulation of MAPK phosphorylation by ErbB-2 and -3. Cell lysates from HRG-stimulated adherent cells were blotted with P-MAPK and reprobed with MAPK antibody as indicated in Materials and methods. The figure shows an increase in P-MAPK after 5 min of stimulation with HRG in FAK+/+-2/3 cells and FAK-reconstituted FAK−/−-2/3 cells compared with control cells. Exposure to UO126 strongly inhibited P-MAPK in FAK-proficient cells. (C) Inhibition of Src reduced HRG-induced FAK phosphorylation at Tyr-861 and -925. FAK+/+-2/3 cells were serum starved for 24 h and pretreated with PP2 at 100 nM for 60 min followed by treatment with 20 ng/ml HRG for 30 min. (a) Whole cell lysates were immunoprecipitated with anti-FAK and probed with antiphosphotyrosine antibody. Membranes were subsequently reprobed with antiphospho-FAK antibodies specific to different residues and total FAK. (b) FAK+/+-2/3 cells were coimmunostained for ErbB-2 and the indicated phospho-FAK in the absence or presence of PP2. Bar, 40 μm. (D) Inhibition of colony formation on agar by Src and MAPK inhibition. Cells were cultured in medium containing soft agarose either in the absence and presence of PP2 or UO126 or were transfected with dominant mutants for MEK1 or Src. Colony formation was determined 4 wk later by counting the number of cell foci. (E) Inhibition of cell invasion by Src but not MAPK inhibition. Cells were cultured in the upper chamber of the Boyden chamber in the absence and presence of PP2 or UO126 or after being transfected with dominant mutants for MEK1 or Src. HRG was used as a chemoattractant in the lower chamber. Each bar of the graphs represents the mean ± SD (error bars) of invading cells from three independent experiments.
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fig4: ErbB-induced oncogenic transformation and invasion are mediated via distinct FAK signaling. (A) Differential regulation of FAK phosphorylation by ErbB-2 and -3 receptors. Serum-starved cells were stimulated with 20 ng/ml EGF (control cells) or HRG (ErbB-2, -3, and -2/3–overexpressing cells) for the indicated times. Proteins were immunoprecipitated with anti-FAK and probed with antiphosphotyrosine antibody, antiphospho-FAK antibodies specific to different residues, and anti-FAK antibody. (B) Differential regulation of MAPK phosphorylation by ErbB-2 and -3. Cell lysates from HRG-stimulated adherent cells were blotted with P-MAPK and reprobed with MAPK antibody as indicated in Materials and methods. The figure shows an increase in P-MAPK after 5 min of stimulation with HRG in FAK+/+-2/3 cells and FAK-reconstituted FAK−/−-2/3 cells compared with control cells. Exposure to UO126 strongly inhibited P-MAPK in FAK-proficient cells. (C) Inhibition of Src reduced HRG-induced FAK phosphorylation at Tyr-861 and -925. FAK+/+-2/3 cells were serum starved for 24 h and pretreated with PP2 at 100 nM for 60 min followed by treatment with 20 ng/ml HRG for 30 min. (a) Whole cell lysates were immunoprecipitated with anti-FAK and probed with antiphosphotyrosine antibody. Membranes were subsequently reprobed with antiphospho-FAK antibodies specific to different residues and total FAK. (b) FAK+/+-2/3 cells were coimmunostained for ErbB-2 and the indicated phospho-FAK in the absence or presence of PP2. Bar, 40 μm. (D) Inhibition of colony formation on agar by Src and MAPK inhibition. Cells were cultured in medium containing soft agarose either in the absence and presence of PP2 or UO126 or were transfected with dominant mutants for MEK1 or Src. Colony formation was determined 4 wk later by counting the number of cell foci. (E) Inhibition of cell invasion by Src but not MAPK inhibition. Cells were cultured in the upper chamber of the Boyden chamber in the absence and presence of PP2 or UO126 or after being transfected with dominant mutants for MEK1 or Src. HRG was used as a chemoattractant in the lower chamber. Each bar of the graphs represents the mean ± SD (error bars) of invading cells from three independent experiments.

Mentions: To dissect the FAK-dependent signaling involved in ErbB-induced oncogenesis and invasion, we examined the impact of ErbB on FAK phosphorylation and its interaction with downstream signaling partners. FAK phosphorylation occurs at several tyrosine sites, including Tyr-397, -861, and -925; these sites are selectively regulated after ErbB activation (Sieg et al., 2000; Lu et al., 2001; Vadlamudi et al., 2002, 2003). In FAK+/+ control cells, the stimulation of ErbB by EGF resulted in a small increase in the level of FAK phosphorylation over time (Fig. 4 A, top left), whereas HRG had no effect on basal FAK phosphorylation (not depicted). The stimulation of cells overexpressing ErbB-2 with EGF increased total FAK phosphorylation as well as phosphorylation at Tyr-397, -861, and -925. This increase is likely a result of ErbB-2 transactivation by the low endogenous ErbB-1 present in these cells. FAK activation was weak in cells overexpressing kinase-deficient ErbB-3 that was stimulated with HRG (Fig. 4 A). In cells overexpressing ErbB-2 and -2/3, a clear increase in total FAK phosphorylation was seen, which was associated with the increased phosphorylation of Tyr-397, -861, and -925; the most pronounced increase was seen on Tyr-861 and -925 phosphorylation sites (Fig. 4 A).


FAK signaling is critical for ErbB-2/ErbB-3 receptor cooperation for oncogenic transformation and invasion.

Benlimame N, He Q, Jie S, Xiao D, Xu YJ, Loignon M, Schlaepfer DD, Alaoui-Jamali MA - J. Cell Biol. (2005)

ErbB-induced oncogenic transformation and invasion are mediated via distinct FAK signaling. (A) Differential regulation of FAK phosphorylation by ErbB-2 and -3 receptors. Serum-starved cells were stimulated with 20 ng/ml EGF (control cells) or HRG (ErbB-2, -3, and -2/3–overexpressing cells) for the indicated times. Proteins were immunoprecipitated with anti-FAK and probed with antiphosphotyrosine antibody, antiphospho-FAK antibodies specific to different residues, and anti-FAK antibody. (B) Differential regulation of MAPK phosphorylation by ErbB-2 and -3. Cell lysates from HRG-stimulated adherent cells were blotted with P-MAPK and reprobed with MAPK antibody as indicated in Materials and methods. The figure shows an increase in P-MAPK after 5 min of stimulation with HRG in FAK+/+-2/3 cells and FAK-reconstituted FAK−/−-2/3 cells compared with control cells. Exposure to UO126 strongly inhibited P-MAPK in FAK-proficient cells. (C) Inhibition of Src reduced HRG-induced FAK phosphorylation at Tyr-861 and -925. FAK+/+-2/3 cells were serum starved for 24 h and pretreated with PP2 at 100 nM for 60 min followed by treatment with 20 ng/ml HRG for 30 min. (a) Whole cell lysates were immunoprecipitated with anti-FAK and probed with antiphosphotyrosine antibody. Membranes were subsequently reprobed with antiphospho-FAK antibodies specific to different residues and total FAK. (b) FAK+/+-2/3 cells were coimmunostained for ErbB-2 and the indicated phospho-FAK in the absence or presence of PP2. Bar, 40 μm. (D) Inhibition of colony formation on agar by Src and MAPK inhibition. Cells were cultured in medium containing soft agarose either in the absence and presence of PP2 or UO126 or were transfected with dominant mutants for MEK1 or Src. Colony formation was determined 4 wk later by counting the number of cell foci. (E) Inhibition of cell invasion by Src but not MAPK inhibition. Cells were cultured in the upper chamber of the Boyden chamber in the absence and presence of PP2 or UO126 or after being transfected with dominant mutants for MEK1 or Src. HRG was used as a chemoattractant in the lower chamber. Each bar of the graphs represents the mean ± SD (error bars) of invading cells from three independent experiments.
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Related In: Results  -  Collection

Show All Figures
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fig4: ErbB-induced oncogenic transformation and invasion are mediated via distinct FAK signaling. (A) Differential regulation of FAK phosphorylation by ErbB-2 and -3 receptors. Serum-starved cells were stimulated with 20 ng/ml EGF (control cells) or HRG (ErbB-2, -3, and -2/3–overexpressing cells) for the indicated times. Proteins were immunoprecipitated with anti-FAK and probed with antiphosphotyrosine antibody, antiphospho-FAK antibodies specific to different residues, and anti-FAK antibody. (B) Differential regulation of MAPK phosphorylation by ErbB-2 and -3. Cell lysates from HRG-stimulated adherent cells were blotted with P-MAPK and reprobed with MAPK antibody as indicated in Materials and methods. The figure shows an increase in P-MAPK after 5 min of stimulation with HRG in FAK+/+-2/3 cells and FAK-reconstituted FAK−/−-2/3 cells compared with control cells. Exposure to UO126 strongly inhibited P-MAPK in FAK-proficient cells. (C) Inhibition of Src reduced HRG-induced FAK phosphorylation at Tyr-861 and -925. FAK+/+-2/3 cells were serum starved for 24 h and pretreated with PP2 at 100 nM for 60 min followed by treatment with 20 ng/ml HRG for 30 min. (a) Whole cell lysates were immunoprecipitated with anti-FAK and probed with antiphosphotyrosine antibody. Membranes were subsequently reprobed with antiphospho-FAK antibodies specific to different residues and total FAK. (b) FAK+/+-2/3 cells were coimmunostained for ErbB-2 and the indicated phospho-FAK in the absence or presence of PP2. Bar, 40 μm. (D) Inhibition of colony formation on agar by Src and MAPK inhibition. Cells were cultured in medium containing soft agarose either in the absence and presence of PP2 or UO126 or were transfected with dominant mutants for MEK1 or Src. Colony formation was determined 4 wk later by counting the number of cell foci. (E) Inhibition of cell invasion by Src but not MAPK inhibition. Cells were cultured in the upper chamber of the Boyden chamber in the absence and presence of PP2 or UO126 or after being transfected with dominant mutants for MEK1 or Src. HRG was used as a chemoattractant in the lower chamber. Each bar of the graphs represents the mean ± SD (error bars) of invading cells from three independent experiments.
Mentions: To dissect the FAK-dependent signaling involved in ErbB-induced oncogenesis and invasion, we examined the impact of ErbB on FAK phosphorylation and its interaction with downstream signaling partners. FAK phosphorylation occurs at several tyrosine sites, including Tyr-397, -861, and -925; these sites are selectively regulated after ErbB activation (Sieg et al., 2000; Lu et al., 2001; Vadlamudi et al., 2002, 2003). In FAK+/+ control cells, the stimulation of ErbB by EGF resulted in a small increase in the level of FAK phosphorylation over time (Fig. 4 A, top left), whereas HRG had no effect on basal FAK phosphorylation (not depicted). The stimulation of cells overexpressing ErbB-2 with EGF increased total FAK phosphorylation as well as phosphorylation at Tyr-397, -861, and -925. This increase is likely a result of ErbB-2 transactivation by the low endogenous ErbB-1 present in these cells. FAK activation was weak in cells overexpressing kinase-deficient ErbB-3 that was stimulated with HRG (Fig. 4 A). In cells overexpressing ErbB-2 and -2/3, a clear increase in total FAK phosphorylation was seen, which was associated with the increased phosphorylation of Tyr-397, -861, and -925; the most pronounced increase was seen on Tyr-861 and -925 phosphorylation sites (Fig. 4 A).

Bottom Line: The overexpression of members of the ErbB tyrosine kinase receptor family has been associated with cancer progression.This colocalization requires intact FAK.In summary, distinct FAK signaling has an essential function in ErbB-induced oncogenesis and invasiveness.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Lady Davis Institute of the Sir Mortimer B. Davis Jewish General Hospital, McGill University, Montreal, Quebec H3T 1E2, Canada.

ABSTRACT
The overexpression of members of the ErbB tyrosine kinase receptor family has been associated with cancer progression. We demonstrate that focal adhesion kinase (FAK) is essential for oncogenic transformation and cell invasion that is induced by ErbB-2 and -3 receptor signaling. ErbB-2/3 overexpression in FAK-deficient cells fails to promote cell transformation and rescue chemotaxis deficiency. Restoration of FAK rescues both oncogenic transformation and invasion that is induced by ErbB-2/3 in vitro and in vivo. In contrast, the inhibition of FAK in FAK-proficient invasive cancer cells prevented cell invasion and metastasis formation. The activation of ErbB-2/3 regulates FAK phosphorylation at Tyr-397, -861, and -925. ErbB-induced oncogenic transformation correlates with the ability of FAK to restore ErbB-2/3-induced mitogen-activated protein kinase (MAPK) activation; the inhibition of MAPK prevented oncogenic transformation. In contrast, the inhibition of Src but not MAPK prevented ErbB-FAK-induced chemotaxis. In migratory cells, activated ErbB-2/3 receptors colocalize with activated FAK at cell protrusions. This colocalization requires intact FAK. In summary, distinct FAK signaling has an essential function in ErbB-induced oncogenesis and invasiveness.

Show MeSH
Related in: MedlinePlus