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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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Related in: MedlinePlus

The defects in shape and migration of Raf-1 KO cells correlate with Rok-α deregulation. (A) Lysates collected from migrating fibroblasts were assayed for Rho activity as described in Materials and methods. The phosphorylation of MYPT1, ezrin, paxillin, FAK, ERK, and the expression of Rok-α and tubulin (loading control) was determined by immunoblotting. (B) Morphology of KO cells expressing eGFP (eG) or eGFP-ezrin1-310 (DN-Ez-eG) stained to visualize the actin cytoskeleton. (C) Rok-α kinase activity is elevated in Raf-1 KO fibroblasts. Rok-α immunoprecipitates were prepared from untreated (Unt) and serum-stimulated (FCS, 10%, 1 h) WT and KO fibroblasts. Rok-α kinase activity was assayed as the ability of the immunoprecipitates to phosphorylate recombinant MYPT1. The results plotted are the mean (±SD) of three independent experiments, normalized for the amount of Rok-α present in the immunoprecipitates and expressed as percentage of the activity present in the immunoprecipitates from unstimulated WT cells. (D) Basal Rho activity is necessary to sustain Rok-α activity in WT and KO cells. WT and KO cells were treated for 3 h with a combination of clostridial toxins to ADP-ribosylate Rho. Ezrin (pT567), Rho, and ADP-ribosylated Rho (Rho*, migrating as a smear above the Rho band) were detected by immunoblotting. (E) The Rok inhibitor Y-27632 reverts hyperphosphorylation of MYPT1 and paxillin in Raf-1 KO fibroblasts. Lysates were collected from migrating cells pretreated or not with Y-27632 (10 μM, 4 h). A Rac immunoblot is shown as a loading control. (F) Y-27632 improves morphology and migration of KO fibroblasts. Left panel, phase contrast images of WT and KO fibroblasts left untreated or treated with Y-27632 for 20 min. (Right) Migration of Y-27632–treated or –untreated WT and KO fibroblasts in a wound closure assay (20 h). Values are means ± SD of three individual samples. (G) Lysates collected from subconfluent keratinocytes. Protein expression and phosphorylation was determined as in A, except that Raf-1 was detected with an antibody specific for the Raf-1 NH2 terminus. Loading control, Keratin 5. (H) Y-27632 improves migration of KO keratinocytes. Y-27632–treated and –untreated cells were allowed to migrate for 6 h in a modified Boyden chamber assay using KGM as a chemoattractant. Values are means ± SD of three individual samples.
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fig4: The defects in shape and migration of Raf-1 KO cells correlate with Rok-α deregulation. (A) Lysates collected from migrating fibroblasts were assayed for Rho activity as described in Materials and methods. The phosphorylation of MYPT1, ezrin, paxillin, FAK, ERK, and the expression of Rok-α and tubulin (loading control) was determined by immunoblotting. (B) Morphology of KO cells expressing eGFP (eG) or eGFP-ezrin1-310 (DN-Ez-eG) stained to visualize the actin cytoskeleton. (C) Rok-α kinase activity is elevated in Raf-1 KO fibroblasts. Rok-α immunoprecipitates were prepared from untreated (Unt) and serum-stimulated (FCS, 10%, 1 h) WT and KO fibroblasts. Rok-α kinase activity was assayed as the ability of the immunoprecipitates to phosphorylate recombinant MYPT1. The results plotted are the mean (±SD) of three independent experiments, normalized for the amount of Rok-α present in the immunoprecipitates and expressed as percentage of the activity present in the immunoprecipitates from unstimulated WT cells. (D) Basal Rho activity is necessary to sustain Rok-α activity in WT and KO cells. WT and KO cells were treated for 3 h with a combination of clostridial toxins to ADP-ribosylate Rho. Ezrin (pT567), Rho, and ADP-ribosylated Rho (Rho*, migrating as a smear above the Rho band) were detected by immunoblotting. (E) The Rok inhibitor Y-27632 reverts hyperphosphorylation of MYPT1 and paxillin in Raf-1 KO fibroblasts. Lysates were collected from migrating cells pretreated or not with Y-27632 (10 μM, 4 h). A Rac immunoblot is shown as a loading control. (F) Y-27632 improves morphology and migration of KO fibroblasts. Left panel, phase contrast images of WT and KO fibroblasts left untreated or treated with Y-27632 for 20 min. (Right) Migration of Y-27632–treated or –untreated WT and KO fibroblasts in a wound closure assay (20 h). Values are means ± SD of three individual samples. (G) Lysates collected from subconfluent keratinocytes. Protein expression and phosphorylation was determined as in A, except that Raf-1 was detected with an antibody specific for the Raf-1 NH2 terminus. Loading control, Keratin 5. (H) Y-27632 improves migration of KO keratinocytes. Y-27632–treated and –untreated cells were allowed to migrate for 6 h in a modified Boyden chamber assay using KGM as a chemoattractant. Values are means ± SD of three individual samples.

Mentions: The small GTPase Rho controls cell shape and motility by increasing intracellular tension and contractility (Etienne-Manneville and Hall, 2002). The shape of Raf-1 KO cells, the defects in spreading, and their failure to migrate all suggested a possible hyperactivation of Rho. Rho downstream targets such as the regulatory subunit of the myosin light chain phosphatase (MYPT1; pT696), ezrin (pT567), paxillin (pY118), and FAK (pY397; Fig. 4 A) were hyperphosphorylated in KO cells. Yet, basal Rho activity was not elevated in Raf-1 KO cells, and migration induced Rho-activation was in fact delayed (Fig. 4 A). Activated paxillin (Tsubouchi et al., 2002) and ezrin (Speck et al., 2003) can reportedly inhibit Rho activity, and are likely responsible for the decreased Rho stimulation. Phosphorylation (and inactivation) of MYPT1 results in the phosphorylation of myosin light chain (Kimura et al., 1996) and ezrin (Fukata et al., 1998). Ezrin phosphorylation, in turn, relieves intramolecular inhibition and enables ezrin to act as an actin filament/plasma membrane cross-linker (Bretscher et al., 2002). Together, MYPT1 and ezrin phosphorylation explain the contracted appearance and the tight cortical actin network observed in KO cells. Indeed, expression of a dominant-negative ezrin mutant (DN-Ez-eG, ezrin1-310 fused to eGFP) corrects these defects (Fig. 4 B, compare transfected and untransfected cell). The Rho effector Rok-α phosphorylates MYPT1 directly (Kawano et al., 1999) and regulates ezrin both by direct phosphorylation and by inactivation of MYPT1 (Fukata et al., 1998; Matsui et al., 1998), and further induces paxillin and FAK hyperphosphorylation (Sinnett-Smith et al., 2001; Tsuji et al., 2002). It also phosphorylates vimentin, thereby causing the collapse of the vimentin filaments into a juxtanuclear aggregate (Sin et al., 1998), an additional characteristic feature of Raf-1 KO fibroblasts (see Fig. 5 C). Thus, the defects observed in KO cells were consistent with deregulation of Rok-α enzymatic activity.


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)

The defects in shape and migration of Raf-1 KO cells correlate with Rok-α deregulation. (A) Lysates collected from migrating fibroblasts were assayed for Rho activity as described in Materials and methods. The phosphorylation of MYPT1, ezrin, paxillin, FAK, ERK, and the expression of Rok-α and tubulin (loading control) was determined by immunoblotting. (B) Morphology of KO cells expressing eGFP (eG) or eGFP-ezrin1-310 (DN-Ez-eG) stained to visualize the actin cytoskeleton. (C) Rok-α kinase activity is elevated in Raf-1 KO fibroblasts. Rok-α immunoprecipitates were prepared from untreated (Unt) and serum-stimulated (FCS, 10%, 1 h) WT and KO fibroblasts. Rok-α kinase activity was assayed as the ability of the immunoprecipitates to phosphorylate recombinant MYPT1. The results plotted are the mean (±SD) of three independent experiments, normalized for the amount of Rok-α present in the immunoprecipitates and expressed as percentage of the activity present in the immunoprecipitates from unstimulated WT cells. (D) Basal Rho activity is necessary to sustain Rok-α activity in WT and KO cells. WT and KO cells were treated for 3 h with a combination of clostridial toxins to ADP-ribosylate Rho. Ezrin (pT567), Rho, and ADP-ribosylated Rho (Rho*, migrating as a smear above the Rho band) were detected by immunoblotting. (E) The Rok inhibitor Y-27632 reverts hyperphosphorylation of MYPT1 and paxillin in Raf-1 KO fibroblasts. Lysates were collected from migrating cells pretreated or not with Y-27632 (10 μM, 4 h). A Rac immunoblot is shown as a loading control. (F) Y-27632 improves morphology and migration of KO fibroblasts. Left panel, phase contrast images of WT and KO fibroblasts left untreated or treated with Y-27632 for 20 min. (Right) Migration of Y-27632–treated or –untreated WT and KO fibroblasts in a wound closure assay (20 h). Values are means ± SD of three individual samples. (G) Lysates collected from subconfluent keratinocytes. Protein expression and phosphorylation was determined as in A, except that Raf-1 was detected with an antibody specific for the Raf-1 NH2 terminus. Loading control, Keratin 5. (H) Y-27632 improves migration of KO keratinocytes. Y-27632–treated and –untreated cells were allowed to migrate for 6 h in a modified Boyden chamber assay using KGM as a chemoattractant. Values are means ± SD of three individual samples.
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Related In: Results  -  Collection

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fig4: The defects in shape and migration of Raf-1 KO cells correlate with Rok-α deregulation. (A) Lysates collected from migrating fibroblasts were assayed for Rho activity as described in Materials and methods. The phosphorylation of MYPT1, ezrin, paxillin, FAK, ERK, and the expression of Rok-α and tubulin (loading control) was determined by immunoblotting. (B) Morphology of KO cells expressing eGFP (eG) or eGFP-ezrin1-310 (DN-Ez-eG) stained to visualize the actin cytoskeleton. (C) Rok-α kinase activity is elevated in Raf-1 KO fibroblasts. Rok-α immunoprecipitates were prepared from untreated (Unt) and serum-stimulated (FCS, 10%, 1 h) WT and KO fibroblasts. Rok-α kinase activity was assayed as the ability of the immunoprecipitates to phosphorylate recombinant MYPT1. The results plotted are the mean (±SD) of three independent experiments, normalized for the amount of Rok-α present in the immunoprecipitates and expressed as percentage of the activity present in the immunoprecipitates from unstimulated WT cells. (D) Basal Rho activity is necessary to sustain Rok-α activity in WT and KO cells. WT and KO cells were treated for 3 h with a combination of clostridial toxins to ADP-ribosylate Rho. Ezrin (pT567), Rho, and ADP-ribosylated Rho (Rho*, migrating as a smear above the Rho band) were detected by immunoblotting. (E) The Rok inhibitor Y-27632 reverts hyperphosphorylation of MYPT1 and paxillin in Raf-1 KO fibroblasts. Lysates were collected from migrating cells pretreated or not with Y-27632 (10 μM, 4 h). A Rac immunoblot is shown as a loading control. (F) Y-27632 improves morphology and migration of KO fibroblasts. Left panel, phase contrast images of WT and KO fibroblasts left untreated or treated with Y-27632 for 20 min. (Right) Migration of Y-27632–treated or –untreated WT and KO fibroblasts in a wound closure assay (20 h). Values are means ± SD of three individual samples. (G) Lysates collected from subconfluent keratinocytes. Protein expression and phosphorylation was determined as in A, except that Raf-1 was detected with an antibody specific for the Raf-1 NH2 terminus. Loading control, Keratin 5. (H) Y-27632 improves migration of KO keratinocytes. Y-27632–treated and –untreated cells were allowed to migrate for 6 h in a modified Boyden chamber assay using KGM as a chemoattractant. Values are means ± SD of three individual samples.
Mentions: The small GTPase Rho controls cell shape and motility by increasing intracellular tension and contractility (Etienne-Manneville and Hall, 2002). The shape of Raf-1 KO cells, the defects in spreading, and their failure to migrate all suggested a possible hyperactivation of Rho. Rho downstream targets such as the regulatory subunit of the myosin light chain phosphatase (MYPT1; pT696), ezrin (pT567), paxillin (pY118), and FAK (pY397; Fig. 4 A) were hyperphosphorylated in KO cells. Yet, basal Rho activity was not elevated in Raf-1 KO cells, and migration induced Rho-activation was in fact delayed (Fig. 4 A). Activated paxillin (Tsubouchi et al., 2002) and ezrin (Speck et al., 2003) can reportedly inhibit Rho activity, and are likely responsible for the decreased Rho stimulation. Phosphorylation (and inactivation) of MYPT1 results in the phosphorylation of myosin light chain (Kimura et al., 1996) and ezrin (Fukata et al., 1998). Ezrin phosphorylation, in turn, relieves intramolecular inhibition and enables ezrin to act as an actin filament/plasma membrane cross-linker (Bretscher et al., 2002). Together, MYPT1 and ezrin phosphorylation explain the contracted appearance and the tight cortical actin network observed in KO cells. Indeed, expression of a dominant-negative ezrin mutant (DN-Ez-eG, ezrin1-310 fused to eGFP) corrects these defects (Fig. 4 B, compare transfected and untransfected cell). The Rho effector Rok-α phosphorylates MYPT1 directly (Kawano et al., 1999) and regulates ezrin both by direct phosphorylation and by inactivation of MYPT1 (Fukata et al., 1998; Matsui et al., 1998), and further induces paxillin and FAK hyperphosphorylation (Sinnett-Smith et al., 2001; Tsuji et al., 2002). It also phosphorylates vimentin, thereby causing the collapse of the vimentin filaments into a juxtanuclear aggregate (Sin et al., 1998), an additional characteristic feature of Raf-1 KO fibroblasts (see Fig. 5 C). Thus, the defects observed in KO cells were consistent with deregulation of Rok-α enzymatic activity.

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