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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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Rok-α associates with Raf-1. (A) WT fibroblast monolayers were induced to migrate and lysates were collected at different times after wounding. Endogenous Raf-1 immunoprecipitates obtained with a pAb against a COOH-terminal Raf-1 epitope (CTLTTSPRLPVF) were analyzed by immunoblotting to visualize the presence of Rok-α and Raf-1. (B) COS-1 cells were cotransfected with HA-Rok-α and full-length Raf-1 as well as the Raf-1 NH2-terminal (ΔC) or COOH-terminal (ΔN) domain. HA immunoprecipitates were prepared from subconfluent cells 48 h after transfection and analyzed by immunoblotting. (C and D) Raf-1 and Rok-α colocalize in nonmigrating (C) and migrating (D) fibroblasts. (Bottom; left) Raf-1; right, Rok-α. (Top) merge.
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fig6: Rok-α associates with Raf-1. (A) WT fibroblast monolayers were induced to migrate and lysates were collected at different times after wounding. Endogenous Raf-1 immunoprecipitates obtained with a pAb against a COOH-terminal Raf-1 epitope (CTLTTSPRLPVF) were analyzed by immunoblotting to visualize the presence of Rok-α and Raf-1. (B) COS-1 cells were cotransfected with HA-Rok-α and full-length Raf-1 as well as the Raf-1 NH2-terminal (ΔC) or COOH-terminal (ΔN) domain. HA immunoprecipitates were prepared from subconfluent cells 48 h after transfection and analyzed by immunoblotting. (C and D) Raf-1 and Rok-α colocalize in nonmigrating (C) and migrating (D) fibroblasts. (Bottom; left) Raf-1; right, Rok-α. (Top) merge.

Mentions: We next tested whether Raf-1 associates physically with Rok-α. Indeed, Rok-α, but not the closely related kinase Rok-β (not depicted), was present in increasing amounts in endogenous Raf-1 immunoprecipitates obtained from migrating fibroblasts (Fig. 6 A). Rok-α could be coimmunoprecipitated with an antibody recognizing the COOH terminus of Raf-1, but not its NH2 terminus, suggesting that the latter may be involved in the interaction with Rok-α. To confirm this and to test whether the association could be detected by using an antibody directed against Rok-α instead of Raf-1, we coexpressed full-length Raf-1 as well as the truncated NH2-terminal (ΔC) and COOH-terminal (ΔN) domain of Raf-1 with HA-tagged Rok-α in COS-1 cells. HA immunoprecipitates from subconfluent COS-1 cells contained full-length and ΔC Raf-1, but not the form lacking the NH2 terminus (Fig. 6 B). Thus, Raf-1 associates physically with Rok-α via its NH2 terminus. Whether Raf-1 binds Rok-α directly, or whether a bridging molecule is involved in the interaction, is presently unknown. Raf-1 and Rok-α decorated elongated structures previously identified as vimentin filaments (not depicted; Sin et al., 1998; Janosch et al., 2000). Raf-1 colocalized with Rok-α in both nonmigrating (Fig. 6 C) and migrating WT cells (Fig. 6 D), although Raf-1 distribution was not restricted to the filaments. In migrating cells, both kinases displayed a polarized pattern, being mainly concentrated around the nucleus, in the trailing edge and along the lateral margins of the cells, and essentially excluded from the protrusions.


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)

Rok-α associates with Raf-1. (A) WT fibroblast monolayers were induced to migrate and lysates were collected at different times after wounding. Endogenous Raf-1 immunoprecipitates obtained with a pAb against a COOH-terminal Raf-1 epitope (CTLTTSPRLPVF) were analyzed by immunoblotting to visualize the presence of Rok-α and Raf-1. (B) COS-1 cells were cotransfected with HA-Rok-α and full-length Raf-1 as well as the Raf-1 NH2-terminal (ΔC) or COOH-terminal (ΔN) domain. HA immunoprecipitates were prepared from subconfluent cells 48 h after transfection and analyzed by immunoblotting. (C and D) Raf-1 and Rok-α colocalize in nonmigrating (C) and migrating (D) fibroblasts. (Bottom; left) Raf-1; right, Rok-α. (Top) merge.
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Related In: Results  -  Collection

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fig6: Rok-α associates with Raf-1. (A) WT fibroblast monolayers were induced to migrate and lysates were collected at different times after wounding. Endogenous Raf-1 immunoprecipitates obtained with a pAb against a COOH-terminal Raf-1 epitope (CTLTTSPRLPVF) were analyzed by immunoblotting to visualize the presence of Rok-α and Raf-1. (B) COS-1 cells were cotransfected with HA-Rok-α and full-length Raf-1 as well as the Raf-1 NH2-terminal (ΔC) or COOH-terminal (ΔN) domain. HA immunoprecipitates were prepared from subconfluent cells 48 h after transfection and analyzed by immunoblotting. (C and D) Raf-1 and Rok-α colocalize in nonmigrating (C) and migrating (D) fibroblasts. (Bottom; left) Raf-1; right, Rok-α. (Top) merge.
Mentions: We next tested whether Raf-1 associates physically with Rok-α. Indeed, Rok-α, but not the closely related kinase Rok-β (not depicted), was present in increasing amounts in endogenous Raf-1 immunoprecipitates obtained from migrating fibroblasts (Fig. 6 A). Rok-α could be coimmunoprecipitated with an antibody recognizing the COOH terminus of Raf-1, but not its NH2 terminus, suggesting that the latter may be involved in the interaction with Rok-α. To confirm this and to test whether the association could be detected by using an antibody directed against Rok-α instead of Raf-1, we coexpressed full-length Raf-1 as well as the truncated NH2-terminal (ΔC) and COOH-terminal (ΔN) domain of Raf-1 with HA-tagged Rok-α in COS-1 cells. HA immunoprecipitates from subconfluent COS-1 cells contained full-length and ΔC Raf-1, but not the form lacking the NH2 terminus (Fig. 6 B). Thus, Raf-1 associates physically with Rok-α via its NH2 terminus. Whether Raf-1 binds Rok-α directly, or whether a bridging molecule is involved in the interaction, is presently unknown. Raf-1 and Rok-α decorated elongated structures previously identified as vimentin filaments (not depicted; Sin et al., 1998; Janosch et al., 2000). Raf-1 colocalized with Rok-α in both nonmigrating (Fig. 6 C) and migrating WT cells (Fig. 6 D), although Raf-1 distribution was not restricted to the filaments. In migrating cells, both kinases displayed a polarized pattern, being mainly concentrated around the nucleus, in the trailing edge and along the lateral margins of the cells, and essentially excluded from the protrusions.

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