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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 for correct Rok-α localization. (A and B) Rok-α is not excluded from cell protrusions in migrating Raf-1 KO fibroblasts (A) and keratinocytes (B). The defect is corrected in fibroblasts expressing KC or KD Raf-1, but not in those transfected with empty vector (V; A). Rok-α staining was visualized in permeabilized fibroblasts by confocal microscopy (A); in subconfluent keratinocytes by epifluorescence (B). The dotted lines in panel f/f represent the contours of the keratinocytes. Arrows in A and B point to Rok-α membrane staining.
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fig7: Raf-1, but not its kinase activity, is required for correct Rok-α localization. (A and B) Rok-α is not excluded from cell protrusions in migrating Raf-1 KO fibroblasts (A) and keratinocytes (B). The defect is corrected in fibroblasts expressing KC or KD Raf-1, but not in those transfected with empty vector (V; A). Rok-α staining was visualized in permeabilized fibroblasts by confocal microscopy (A); in subconfluent keratinocytes by epifluorescence (B). The dotted lines in panel f/f represent the contours of the keratinocytes. Arrows in A and B point to Rok-α membrane staining.

Mentions: Lack of Raf-1 induced profound changes in the subcellular distribution of Rok-α. In KO fibroblasts Rok-α was no longer associated with the vimentin cytoskeleton, but was rather localized at the membrane at the cell front. Re-expression of either KC or KD Raf-1 restored this defect (Fig. 7 A). Similarly, Rok-α was often observed at the membrane in KO keratinocytes, whereas in WT cells the staining was essentially excluded from this location (Fig. 7 B).


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 for correct Rok-α localization. (A and B) Rok-α is not excluded from cell protrusions in migrating Raf-1 KO fibroblasts (A) and keratinocytes (B). The defect is corrected in fibroblasts expressing KC or KD Raf-1, but not in those transfected with empty vector (V; A). Rok-α staining was visualized in permeabilized fibroblasts by confocal microscopy (A); in subconfluent keratinocytes by epifluorescence (B). The dotted lines in panel f/f represent the contours of the keratinocytes. Arrows in A and B point to Rok-α membrane staining.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171799&req=5

fig7: Raf-1, but not its kinase activity, is required for correct Rok-α localization. (A and B) Rok-α is not excluded from cell protrusions in migrating Raf-1 KO fibroblasts (A) and keratinocytes (B). The defect is corrected in fibroblasts expressing KC or KD Raf-1, but not in those transfected with empty vector (V; A). Rok-α staining was visualized in permeabilized fibroblasts by confocal microscopy (A); in subconfluent keratinocytes by epifluorescence (B). The dotted lines in panel f/f represent the contours of the keratinocytes. Arrows in A and B point to Rok-α membrane staining.
Mentions: Lack of Raf-1 induced profound changes in the subcellular distribution of Rok-α. In KO fibroblasts Rok-α was no longer associated with the vimentin cytoskeleton, but was rather localized at the membrane at the cell front. Re-expression of either KC or KD Raf-1 restored this defect (Fig. 7 A). Similarly, Rok-α was often observed at the membrane in KO keratinocytes, whereas in WT cells the staining was essentially excluded from this location (Fig. 7 B).

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