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Regulation of cell contraction and membrane ruffling by distinct signals in migratory cells.

Cheresh DA, Leng J, Klemke RL - J. Cell Biol. (1999)

Bottom Line: Evidence is provided that actin-myosin assembly and membrane ruffling are regulated by distinct signaling pathways in the migratory cell.Interaction of cells with extracellular matrix proteins or cytokines promote cell migration through activation of the MAP kinases ERK1 and ERK2 as well as the molecular coupling of the adaptor proteins p130CAS and c-CrkII.Thus, during cell migration ERK and CAS/Crk coupling operate as components of distinct signaling pathways that control actin assembly into myosin motors and membrane ruffles, respectively.

View Article: PubMed Central - PubMed

Affiliation: Departments of Immunology and Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
Cell migration and wound contraction requires assembly of actin into a functional myosin motor unit capable of generating force. However, cell migration also involves formation of actin-containing membrane ruffles. Evidence is provided that actin-myosin assembly and membrane ruffling are regulated by distinct signaling pathways in the migratory cell. Interaction of cells with extracellular matrix proteins or cytokines promote cell migration through activation of the MAP kinases ERK1 and ERK2 as well as the molecular coupling of the adaptor proteins p130CAS and c-CrkII. ERK signaling is independent of CAS/Crk coupling and regulates myosin light chain phosphorylation leading to actin-myosin assembly during cell migration and cell-mediated contraction of a collagen matrix. In contrast, membrane ruffling, but not cell contraction, requires Rac GTPase activity and the formation of a CAS/Crk complex that functions in the context of the Rac activating protein DOCK180. Thus, during cell migration ERK and CAS/Crk coupling operate as components of distinct signaling pathways that control actin assembly into myosin motors and membrane ruffles, respectively.

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ERK-induced cell migration requires CAS/Crk and Rac activity. (A) Upper panel, serum-starved COS-7 cells were allowed to migrate for 3 h on vitronectin-coated membranes after transient transfection with a β-gal reporter construct, along with either the empty expression vector or with expression vectors encoding mutationally activated MEK, or MEK cotransfected with dominant negative CAS (CAS-SD). The number of transfected cells migrating were enumerated by counting cells on the underside of the membrane that coexpress the β-gal vector as described in Materials and Methods. Each bar represents the mean ± SEM of at least three independent experiments. Lower panels, cells treated as described for the migration experiment above were lysed in detergent and immunoblotted with antibodies to the activated form of ERK1/ERK2 (middle panel) or ERK2 (lower panel). The upper band recognized by the ERK2 antibody represents the phosphorylated/activated form of this protein (ERK2-P) that has reduced mobility as a result of being phosphorylated. Similar results were obtained with ERK1 protein (data not shown). (B) Serum-starved COS-7 cells were allowed to migrate for 3 h on vitronectin-coated membranes after transient transfection with a β-gal reporter construct, along with either the empty expression vector or with expression vectors encoding mutationally activated MEK, or MEK together with dominant negative myc-tagged Rac (RacN17) in the presence or absence of insulin (10 μg/ml) in the lower chamber. An aliquot of cells transfected with RacN17 and lysed and immunoblotted with an antibody to Rac is shown (top right). Note that RacN17 migrates slower as the result of the myc tag compared with endogenous wild-type Rac (Rac-Wt). Each bar represents the mean ± SEM of at least three independent experiments.
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Figure 3: ERK-induced cell migration requires CAS/Crk and Rac activity. (A) Upper panel, serum-starved COS-7 cells were allowed to migrate for 3 h on vitronectin-coated membranes after transient transfection with a β-gal reporter construct, along with either the empty expression vector or with expression vectors encoding mutationally activated MEK, or MEK cotransfected with dominant negative CAS (CAS-SD). The number of transfected cells migrating were enumerated by counting cells on the underside of the membrane that coexpress the β-gal vector as described in Materials and Methods. Each bar represents the mean ± SEM of at least three independent experiments. Lower panels, cells treated as described for the migration experiment above were lysed in detergent and immunoblotted with antibodies to the activated form of ERK1/ERK2 (middle panel) or ERK2 (lower panel). The upper band recognized by the ERK2 antibody represents the phosphorylated/activated form of this protein (ERK2-P) that has reduced mobility as a result of being phosphorylated. Similar results were obtained with ERK1 protein (data not shown). (B) Serum-starved COS-7 cells were allowed to migrate for 3 h on vitronectin-coated membranes after transient transfection with a β-gal reporter construct, along with either the empty expression vector or with expression vectors encoding mutationally activated MEK, or MEK together with dominant negative myc-tagged Rac (RacN17) in the presence or absence of insulin (10 μg/ml) in the lower chamber. An aliquot of cells transfected with RacN17 and lysed and immunoblotted with an antibody to Rac is shown (top right). Note that RacN17 migrates slower as the result of the myc tag compared with endogenous wild-type Rac (Rac-Wt). Each bar represents the mean ± SEM of at least three independent experiments.

Mentions: To investigate directly whether formation of a CAS/Crk complex could activate ERK, serum-starved COS-7 cells were transiently transfected with vectors encoding CAS and Crk or mutationally activated MEK. These cells were then examined for ERK activity and migration. Expression of MEK in these cells promoted a four- to fivefold increase in cell migration and significantly increased ERK activity compared with mock-transfected control cells (Fig. 2 A). However, while CAS/Crk transfected cells showed a fourfold increase in cell migration, there was no change in ERK activity (Fig. 2 A). Similar findings were obtained in FG carcinoma cells stably transfected with c-Crk (FG-C). These cells also showed significantly enhanced migration, yet ERK activity was the same as control cells (Fig. 2b and Fig. C). Together, these findings indicate that CAS/Crk-induced cell migration does not result from increased ERK activity. However, since ERK signaling appeared to be a separate event necessary for cell migration, we investigated whether ERK activity was also necessary for CAS/Crk-induced cell movement. To investigate this possibility, FG-C and COS-7 cells transfected with CAS and Crk were exposed to PD98059 and analyzed for their ability to migrate on ECM proteins. In this case, PD98059 blocked cell migration induced by CAS/Crk (Fig. 2a and Fig. b) without affecting the formation of CAS/Crk complexes in these cells (data not shown). Conversely, MEK-induced cell migration was blocked by expression of CAS-SD or dominant negative RacN17 (Fig. 3A and Fig. B) without impacting ERK activity (data not shown). Thus, CAS/Crk/Rac signaling and ERK activation appear to be separate biochemical pathways necessary for cell migration.


Regulation of cell contraction and membrane ruffling by distinct signals in migratory cells.

Cheresh DA, Leng J, Klemke RL - J. Cell Biol. (1999)

ERK-induced cell migration requires CAS/Crk and Rac activity. (A) Upper panel, serum-starved COS-7 cells were allowed to migrate for 3 h on vitronectin-coated membranes after transient transfection with a β-gal reporter construct, along with either the empty expression vector or with expression vectors encoding mutationally activated MEK, or MEK cotransfected with dominant negative CAS (CAS-SD). The number of transfected cells migrating were enumerated by counting cells on the underside of the membrane that coexpress the β-gal vector as described in Materials and Methods. Each bar represents the mean ± SEM of at least three independent experiments. Lower panels, cells treated as described for the migration experiment above were lysed in detergent and immunoblotted with antibodies to the activated form of ERK1/ERK2 (middle panel) or ERK2 (lower panel). The upper band recognized by the ERK2 antibody represents the phosphorylated/activated form of this protein (ERK2-P) that has reduced mobility as a result of being phosphorylated. Similar results were obtained with ERK1 protein (data not shown). (B) Serum-starved COS-7 cells were allowed to migrate for 3 h on vitronectin-coated membranes after transient transfection with a β-gal reporter construct, along with either the empty expression vector or with expression vectors encoding mutationally activated MEK, or MEK together with dominant negative myc-tagged Rac (RacN17) in the presence or absence of insulin (10 μg/ml) in the lower chamber. An aliquot of cells transfected with RacN17 and lysed and immunoblotted with an antibody to Rac is shown (top right). Note that RacN17 migrates slower as the result of the myc tag compared with endogenous wild-type Rac (Rac-Wt). Each bar represents the mean ± SEM of at least three independent experiments.
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Figure 3: ERK-induced cell migration requires CAS/Crk and Rac activity. (A) Upper panel, serum-starved COS-7 cells were allowed to migrate for 3 h on vitronectin-coated membranes after transient transfection with a β-gal reporter construct, along with either the empty expression vector or with expression vectors encoding mutationally activated MEK, or MEK cotransfected with dominant negative CAS (CAS-SD). The number of transfected cells migrating were enumerated by counting cells on the underside of the membrane that coexpress the β-gal vector as described in Materials and Methods. Each bar represents the mean ± SEM of at least three independent experiments. Lower panels, cells treated as described for the migration experiment above were lysed in detergent and immunoblotted with antibodies to the activated form of ERK1/ERK2 (middle panel) or ERK2 (lower panel). The upper band recognized by the ERK2 antibody represents the phosphorylated/activated form of this protein (ERK2-P) that has reduced mobility as a result of being phosphorylated. Similar results were obtained with ERK1 protein (data not shown). (B) Serum-starved COS-7 cells were allowed to migrate for 3 h on vitronectin-coated membranes after transient transfection with a β-gal reporter construct, along with either the empty expression vector or with expression vectors encoding mutationally activated MEK, or MEK together with dominant negative myc-tagged Rac (RacN17) in the presence or absence of insulin (10 μg/ml) in the lower chamber. An aliquot of cells transfected with RacN17 and lysed and immunoblotted with an antibody to Rac is shown (top right). Note that RacN17 migrates slower as the result of the myc tag compared with endogenous wild-type Rac (Rac-Wt). Each bar represents the mean ± SEM of at least three independent experiments.
Mentions: To investigate directly whether formation of a CAS/Crk complex could activate ERK, serum-starved COS-7 cells were transiently transfected with vectors encoding CAS and Crk or mutationally activated MEK. These cells were then examined for ERK activity and migration. Expression of MEK in these cells promoted a four- to fivefold increase in cell migration and significantly increased ERK activity compared with mock-transfected control cells (Fig. 2 A). However, while CAS/Crk transfected cells showed a fourfold increase in cell migration, there was no change in ERK activity (Fig. 2 A). Similar findings were obtained in FG carcinoma cells stably transfected with c-Crk (FG-C). These cells also showed significantly enhanced migration, yet ERK activity was the same as control cells (Fig. 2b and Fig. C). Together, these findings indicate that CAS/Crk-induced cell migration does not result from increased ERK activity. However, since ERK signaling appeared to be a separate event necessary for cell migration, we investigated whether ERK activity was also necessary for CAS/Crk-induced cell movement. To investigate this possibility, FG-C and COS-7 cells transfected with CAS and Crk were exposed to PD98059 and analyzed for their ability to migrate on ECM proteins. In this case, PD98059 blocked cell migration induced by CAS/Crk (Fig. 2a and Fig. b) without affecting the formation of CAS/Crk complexes in these cells (data not shown). Conversely, MEK-induced cell migration was blocked by expression of CAS-SD or dominant negative RacN17 (Fig. 3A and Fig. B) without impacting ERK activity (data not shown). Thus, CAS/Crk/Rac signaling and ERK activation appear to be separate biochemical pathways necessary for cell migration.

Bottom Line: Evidence is provided that actin-myosin assembly and membrane ruffling are regulated by distinct signaling pathways in the migratory cell.Interaction of cells with extracellular matrix proteins or cytokines promote cell migration through activation of the MAP kinases ERK1 and ERK2 as well as the molecular coupling of the adaptor proteins p130CAS and c-CrkII.Thus, during cell migration ERK and CAS/Crk coupling operate as components of distinct signaling pathways that control actin assembly into myosin motors and membrane ruffles, respectively.

View Article: PubMed Central - PubMed

Affiliation: Departments of Immunology and Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
Cell migration and wound contraction requires assembly of actin into a functional myosin motor unit capable of generating force. However, cell migration also involves formation of actin-containing membrane ruffles. Evidence is provided that actin-myosin assembly and membrane ruffling are regulated by distinct signaling pathways in the migratory cell. Interaction of cells with extracellular matrix proteins or cytokines promote cell migration through activation of the MAP kinases ERK1 and ERK2 as well as the molecular coupling of the adaptor proteins p130CAS and c-CrkII. ERK signaling is independent of CAS/Crk coupling and regulates myosin light chain phosphorylation leading to actin-myosin assembly during cell migration and cell-mediated contraction of a collagen matrix. In contrast, membrane ruffling, but not cell contraction, requires Rac GTPase activity and the formation of a CAS/Crk complex that functions in the context of the Rac activating protein DOCK180. Thus, during cell migration ERK and CAS/Crk coupling operate as components of distinct signaling pathways that control actin assembly into myosin motors and membrane ruffles, respectively.

Show MeSH
Related in: MedlinePlus