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Raf-1 regulates Rho signaling and cell migration.

Ehrenreiter K, Piazzolla D, Velamoor V, Sobczak I, Small JV, Takeda J, Leung T, Baccarini M - J. Cell Biol. (2005)

Bottom Line: These defects are due to the hyperactivity and incorrect localization of the Rho-effector Rok-alpha to the plasma membrane.Raf-1 physically associates with Rok-alpha in wild-type (WT) cells, and reintroduction of either WT or kinase-dead Raf-1 in knockout fibroblasts rescues their defects in shape and migration.Thus, Raf-1 plays an essential, kinase-independent function as a spatial regulator of Rho downstream signaling during migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Genetics, Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, 1030 Vienna, Austria.

ABSTRACT
Raf kinases relay signals inducing proliferation, differentiation, and survival. The Raf-1 isoform has been extensively studied as the upstream kinase linking Ras activation to the MEK/ERK module. Recently, however, genetic experiments have shown that Raf-1 plays an essential role in counteracting apoptosis, and that it does so independently of its ability to activate MEK. By conditional gene ablation, we now show that Raf-1 is required for normal wound healing in vivo and for the migration of keratinocytes and fibroblasts in vitro. Raf-1-deficient cells show a symmetric, contracted appearance, characterized by cortical actin bundles and by a disordered vimentin cytoskeleton. These defects are due to the hyperactivity and incorrect localization of the Rho-effector Rok-alpha to the plasma membrane. Raf-1 physically associates with Rok-alpha in wild-type (WT) cells, and reintroduction of either WT or kinase-dead Raf-1 in knockout fibroblasts rescues their defects in shape and migration. Thus, Raf-1 plays an essential, kinase-independent function as a spatial regulator of Rho downstream signaling during migration.

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Raf-1, but not its kinase activity, is required to maintain cell shape, adhesion, and motility in fibroblasts. (A) Migration stimulates Raf-1 kinase activity. Raf-1 activation was measured in an immunocomplex kinase assay. Immunoprecipitates from WT fibroblasts treated with EGF (33 nM, 10 min) were used as an activation control. The Western blot shows the amount of Raf-1 in the immunoprecipitates. (B; top) Reexpression of KC or KD Raf-1 rescues the migratory defect of Raf-1 KO fibroblasts in a wound healing assay (20 h). (Bottom) Raf-1 expression in whole cell lysates from WT and KO fibroblasts and from stable clones of KO cells expressing either KC (KC1, KC3) or KD Raf-1 (KD9, KD11). V, empty vector. Values represent the mean ± SD of triplicate samples. (C) KC and KD Raf-1 as well as Y-27632 and (D) KD Rok-α rescue the actin and vimentin cytoskeleton defects of Raf-1 KO fibroblasts plated on fibronectin. (Top) Actin visualized with rhodamine-conjugated phalloidin. (Bottom) Staining with α-vimentin antibodies. In D, expression of KD Rok-α (HA-Rok-α KD) in WT and KO cells was verified by staining with an anti-HA antibody (HA). Asterisks represent nontransfected cells. (E) Expression of KD Rok-α improves migration of KO cells. WT and KO cells transfected with KD Rok-α (HA-Rok-α KD) or with the corresponding empty vector (HA-V) were allowed to migrate for 2.5 h in a modified Boyden chamber assay using 10% FCS as a chemoattractant. Unt, untransfected cells. The results represent the mean of four independent values and are expressed as percentage of cells that migrated through the pores of the polycarbonate membrane. For transfected cells, expression of the construct was visualized by immunofluorescence and the result were expressed as percentage of transfected cells migrated. Values represent the mean ± SD of quadruplicate samples.
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fig5: Raf-1, but not its kinase activity, is required to maintain cell shape, adhesion, and motility in fibroblasts. (A) Migration stimulates Raf-1 kinase activity. Raf-1 activation was measured in an immunocomplex kinase assay. Immunoprecipitates from WT fibroblasts treated with EGF (33 nM, 10 min) were used as an activation control. The Western blot shows the amount of Raf-1 in the immunoprecipitates. (B; top) Reexpression of KC or KD Raf-1 rescues the migratory defect of Raf-1 KO fibroblasts in a wound healing assay (20 h). (Bottom) Raf-1 expression in whole cell lysates from WT and KO fibroblasts and from stable clones of KO cells expressing either KC (KC1, KC3) or KD Raf-1 (KD9, KD11). V, empty vector. Values represent the mean ± SD of triplicate samples. (C) KC and KD Raf-1 as well as Y-27632 and (D) KD Rok-α rescue the actin and vimentin cytoskeleton defects of Raf-1 KO fibroblasts plated on fibronectin. (Top) Actin visualized with rhodamine-conjugated phalloidin. (Bottom) Staining with α-vimentin antibodies. In D, expression of KD Rok-α (HA-Rok-α KD) in WT and KO cells was verified by staining with an anti-HA antibody (HA). Asterisks represent nontransfected cells. (E) Expression of KD Rok-α improves migration of KO cells. WT and KO cells transfected with KD Rok-α (HA-Rok-α KD) or with the corresponding empty vector (HA-V) were allowed to migrate for 2.5 h in a modified Boyden chamber assay using 10% FCS as a chemoattractant. Unt, untransfected cells. The results represent the mean of four independent values and are expressed as percentage of cells that migrated through the pores of the polycarbonate membrane. For transfected cells, expression of the construct was visualized by immunofluorescence and the result were expressed as percentage of transfected cells migrated. Values represent the mean ± SD of quadruplicate samples.

Mentions: To assess whether Rok-α deregulation caused the defects in shape and migration, Raf-1 KO cells were treated with the Rok inhibitor Y-27632. The inhibitor corrected MYPT1 and paxillin hyperphosphorylation (Fig. 4 E) as well as the redistribution of vimentin to a perinuclear location in KO fibroblasts (Fig. 5 C), and significantly improved the migratory ability and shape of Raf-1 KO fibroblasts (Fig. 4 F and Fig. 5 C) and keratinocytes (Fig. 4 H; and not depicted). Transfection with KD Rok-α had an even more profound impact than Y-27632 on both WT and KO fibroblasts, ruling out possible Rok-α–independent effects of the inhibitor. Raf-1 KO fibroblasts transfected with KD Rok-α lost their characteristic tight cortical actin bundles and contracted shape, formed stress fibers and vimentin filaments similar to those of untreated WT fibroblasts (Fig. 5 D), and migrated as efficiently as untransfected WT cells in a transwell assay (Fig. 5 E). In contrast, the expression of KD Rok-α had a deleterious effect on both the shape (Fig. 5 D) and the migratory ability (Fig. 5 E) of WT cells. This is in line with a requirement for Rok-α in the maintenance of cell shape and migration of normal fibroblasts. The opposite effects of KD Rok-α on WT and KO cells strongly argue in favor of Rok-α hyperactivity as the molecular basis of the defects of Raf-1 KO cells.


Raf-1 regulates Rho signaling and cell migration.

Ehrenreiter K, Piazzolla D, Velamoor V, Sobczak I, Small JV, Takeda J, Leung T, Baccarini M - J. Cell Biol. (2005)

Raf-1, but not its kinase activity, is required to maintain cell shape, adhesion, and motility in fibroblasts. (A) Migration stimulates Raf-1 kinase activity. Raf-1 activation was measured in an immunocomplex kinase assay. Immunoprecipitates from WT fibroblasts treated with EGF (33 nM, 10 min) were used as an activation control. The Western blot shows the amount of Raf-1 in the immunoprecipitates. (B; top) Reexpression of KC or KD Raf-1 rescues the migratory defect of Raf-1 KO fibroblasts in a wound healing assay (20 h). (Bottom) Raf-1 expression in whole cell lysates from WT and KO fibroblasts and from stable clones of KO cells expressing either KC (KC1, KC3) or KD Raf-1 (KD9, KD11). V, empty vector. Values represent the mean ± SD of triplicate samples. (C) KC and KD Raf-1 as well as Y-27632 and (D) KD Rok-α rescue the actin and vimentin cytoskeleton defects of Raf-1 KO fibroblasts plated on fibronectin. (Top) Actin visualized with rhodamine-conjugated phalloidin. (Bottom) Staining with α-vimentin antibodies. In D, expression of KD Rok-α (HA-Rok-α KD) in WT and KO cells was verified by staining with an anti-HA antibody (HA). Asterisks represent nontransfected cells. (E) Expression of KD Rok-α improves migration of KO cells. WT and KO cells transfected with KD Rok-α (HA-Rok-α KD) or with the corresponding empty vector (HA-V) were allowed to migrate for 2.5 h in a modified Boyden chamber assay using 10% FCS as a chemoattractant. Unt, untransfected cells. The results represent the mean of four independent values and are expressed as percentage of cells that migrated through the pores of the polycarbonate membrane. For transfected cells, expression of the construct was visualized by immunofluorescence and the result were expressed as percentage of transfected cells migrated. Values represent the mean ± SD of quadruplicate samples.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171799&req=5

fig5: Raf-1, but not its kinase activity, is required to maintain cell shape, adhesion, and motility in fibroblasts. (A) Migration stimulates Raf-1 kinase activity. Raf-1 activation was measured in an immunocomplex kinase assay. Immunoprecipitates from WT fibroblasts treated with EGF (33 nM, 10 min) were used as an activation control. The Western blot shows the amount of Raf-1 in the immunoprecipitates. (B; top) Reexpression of KC or KD Raf-1 rescues the migratory defect of Raf-1 KO fibroblasts in a wound healing assay (20 h). (Bottom) Raf-1 expression in whole cell lysates from WT and KO fibroblasts and from stable clones of KO cells expressing either KC (KC1, KC3) or KD Raf-1 (KD9, KD11). V, empty vector. Values represent the mean ± SD of triplicate samples. (C) KC and KD Raf-1 as well as Y-27632 and (D) KD Rok-α rescue the actin and vimentin cytoskeleton defects of Raf-1 KO fibroblasts plated on fibronectin. (Top) Actin visualized with rhodamine-conjugated phalloidin. (Bottom) Staining with α-vimentin antibodies. In D, expression of KD Rok-α (HA-Rok-α KD) in WT and KO cells was verified by staining with an anti-HA antibody (HA). Asterisks represent nontransfected cells. (E) Expression of KD Rok-α improves migration of KO cells. WT and KO cells transfected with KD Rok-α (HA-Rok-α KD) or with the corresponding empty vector (HA-V) were allowed to migrate for 2.5 h in a modified Boyden chamber assay using 10% FCS as a chemoattractant. Unt, untransfected cells. The results represent the mean of four independent values and are expressed as percentage of cells that migrated through the pores of the polycarbonate membrane. For transfected cells, expression of the construct was visualized by immunofluorescence and the result were expressed as percentage of transfected cells migrated. Values represent the mean ± SD of quadruplicate samples.
Mentions: To assess whether Rok-α deregulation caused the defects in shape and migration, Raf-1 KO cells were treated with the Rok inhibitor Y-27632. The inhibitor corrected MYPT1 and paxillin hyperphosphorylation (Fig. 4 E) as well as the redistribution of vimentin to a perinuclear location in KO fibroblasts (Fig. 5 C), and significantly improved the migratory ability and shape of Raf-1 KO fibroblasts (Fig. 4 F and Fig. 5 C) and keratinocytes (Fig. 4 H; and not depicted). Transfection with KD Rok-α had an even more profound impact than Y-27632 on both WT and KO fibroblasts, ruling out possible Rok-α–independent effects of the inhibitor. Raf-1 KO fibroblasts transfected with KD Rok-α lost their characteristic tight cortical actin bundles and contracted shape, formed stress fibers and vimentin filaments similar to those of untreated WT fibroblasts (Fig. 5 D), and migrated as efficiently as untransfected WT cells in a transwell assay (Fig. 5 E). In contrast, the expression of KD Rok-α had a deleterious effect on both the shape (Fig. 5 D) and the migratory ability (Fig. 5 E) of WT cells. This is in line with a requirement for Rok-α in the maintenance of cell shape and migration of normal fibroblasts. The opposite effects of KD Rok-α on WT and KO cells strongly argue in favor of Rok-α hyperactivity as the molecular basis of the defects of Raf-1 KO cells.

Bottom Line: These defects are due to the hyperactivity and incorrect localization of the Rho-effector Rok-alpha to the plasma membrane.Raf-1 physically associates with Rok-alpha in wild-type (WT) cells, and reintroduction of either WT or kinase-dead Raf-1 in knockout fibroblasts rescues their defects in shape and migration.Thus, Raf-1 plays an essential, kinase-independent function as a spatial regulator of Rho downstream signaling during migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Genetics, Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, 1030 Vienna, Austria.

ABSTRACT
Raf kinases relay signals inducing proliferation, differentiation, and survival. The Raf-1 isoform has been extensively studied as the upstream kinase linking Ras activation to the MEK/ERK module. Recently, however, genetic experiments have shown that Raf-1 plays an essential role in counteracting apoptosis, and that it does so independently of its ability to activate MEK. By conditional gene ablation, we now show that Raf-1 is required for normal wound healing in vivo and for the migration of keratinocytes and fibroblasts in vitro. Raf-1-deficient cells show a symmetric, contracted appearance, characterized by cortical actin bundles and by a disordered vimentin cytoskeleton. These defects are due to the hyperactivity and incorrect localization of the Rho-effector Rok-alpha to the plasma membrane. Raf-1 physically associates with Rok-alpha in wild-type (WT) cells, and reintroduction of either WT or kinase-dead Raf-1 in knockout fibroblasts rescues their defects in shape and migration. Thus, Raf-1 plays an essential, kinase-independent function as a spatial regulator of Rho downstream signaling during migration.

Show MeSH
Related in: MedlinePlus