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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 activation and CAS/Crk coupling are separate signaling events necessary for cytokine-induced cell migration. (A) Serum-starved COS-7 cells were allowed to migrate for 3 h on vitronectin-coated membranes in the presence or absence of insulin (10 μg/ml) after transient transfection with a β-gal reporter construct, along with either the empty expression vector (Mock) or with expression vectors encoding gst-tagged CAS without its substrate domain (CAS-SD), or myc-tagged Crk with a mutated SH2 domain (Crk-SH2). CAS-SD and Crk-SH2 have been shown to prevent CAS/Crk coupling and downstream signals (Matsuda et al. 1993; Feller et al. 1994; Klemke et al. 1998). 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. An aliquot of cells treated as described for the migration experiment above was lysed in detergent and immunoblotted with antibodies to either the phosphorylated/activated form of ERK1/ERK2 (lower panel), Crk, or CAS (top right). Note that Crk-SH2 shows reduced mobility compared with wild-type endogenous Crk (Crk-WT) as the result of the molecular tag. (B) Serum-starved COS-7 cells were allowed to migrate in the presence or absence of the MEK inhibitor PD98059 (25 μM) with or without insulin (10 μg/ml). Cell migration and ERK1/ERK2 activity in these cells were determined as described above. Similar findings were obtained with fibronectin and collagen type I–coated membranes (data not shown). Each bar represents the mean ± SEM of at least three independent experiments. (C) Serum-starved COS-7 cells pretreated with or without the MEK inhibitor PD98059 (50 μM) for 2 h were exposed to insulin (10 μg/ml) for 5 min before being lysed in detergent. CAS was immunoprecipitated and then immunoblotted with antibodies to either phosphotyrosine, Crk, or CAS. The detergent lysates from these cells were also examined for changes in ERK1/ERK2 activity as described above. The result shown is representative from at least three independent experiments.
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Figure 1: ERK activation and CAS/Crk coupling are separate signaling events necessary for cytokine-induced cell migration. (A) Serum-starved COS-7 cells were allowed to migrate for 3 h on vitronectin-coated membranes in the presence or absence of insulin (10 μg/ml) after transient transfection with a β-gal reporter construct, along with either the empty expression vector (Mock) or with expression vectors encoding gst-tagged CAS without its substrate domain (CAS-SD), or myc-tagged Crk with a mutated SH2 domain (Crk-SH2). CAS-SD and Crk-SH2 have been shown to prevent CAS/Crk coupling and downstream signals (Matsuda et al. 1993; Feller et al. 1994; Klemke et al. 1998). 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. An aliquot of cells treated as described for the migration experiment above was lysed in detergent and immunoblotted with antibodies to either the phosphorylated/activated form of ERK1/ERK2 (lower panel), Crk, or CAS (top right). Note that Crk-SH2 shows reduced mobility compared with wild-type endogenous Crk (Crk-WT) as the result of the molecular tag. (B) Serum-starved COS-7 cells were allowed to migrate in the presence or absence of the MEK inhibitor PD98059 (25 μM) with or without insulin (10 μg/ml). Cell migration and ERK1/ERK2 activity in these cells were determined as described above. Similar findings were obtained with fibronectin and collagen type I–coated membranes (data not shown). Each bar represents the mean ± SEM of at least three independent experiments. (C) Serum-starved COS-7 cells pretreated with or without the MEK inhibitor PD98059 (50 μM) for 2 h were exposed to insulin (10 μg/ml) for 5 min before being lysed in detergent. CAS was immunoprecipitated and then immunoblotted with antibodies to either phosphotyrosine, Crk, or CAS. The detergent lysates from these cells were also examined for changes in ERK1/ERK2 activity as described above. The result shown is representative from at least three independent experiments.

Mentions: Recently, we reported that CAS/Crk coupling as well as ERK activation facilitate cell migration (Klemke et al. 1997, Klemke et al. 1998). Each of these events were shown to be required for cell migration, yet it remains unclear whether these signals represent the same or parallel pathways involved in regulation of this process. To address this issue, cells were transfected with CAS lacking its substrate domain (CAS-SD) or Crk with a mutated SH2 domain (CRK-SH2), either of which are capable of preventing CAS/Crk coupling and downstream signaling events (Feller et al. 1994; Matsuda et al. 1994; Klemke et al. 1998). Cells containing these cDNAs were treated with the cytokine insulin and examined for migration as well as ERK activity. Expression of either CAS-SD or Crk-SH2 blocked insulin-induced cell migration, yet had no effect on ERK activity in these cells (Fig. 1A). Similar findings were observed in cells expressing Crk with a mutated amino-terminal SH3 domain that retains its ability to couple to CAS, but is unable to link to downstream effector molecules such as DOCK180 or C3G (data not shown; Matsuda et al. 1994; Kiyokawa et al. 1998a,Kiyokawa et al. 1998b). These findings reveal that disruption of CAS/Crk coupling or its binding to downstream effectors can suppress cell migration without influencing ERK activity.


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

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

ERK activation and CAS/Crk coupling are separate signaling events necessary for cytokine-induced cell migration. (A) Serum-starved COS-7 cells were allowed to migrate for 3 h on vitronectin-coated membranes in the presence or absence of insulin (10 μg/ml) after transient transfection with a β-gal reporter construct, along with either the empty expression vector (Mock) or with expression vectors encoding gst-tagged CAS without its substrate domain (CAS-SD), or myc-tagged Crk with a mutated SH2 domain (Crk-SH2). CAS-SD and Crk-SH2 have been shown to prevent CAS/Crk coupling and downstream signals (Matsuda et al. 1993; Feller et al. 1994; Klemke et al. 1998). 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. An aliquot of cells treated as described for the migration experiment above was lysed in detergent and immunoblotted with antibodies to either the phosphorylated/activated form of ERK1/ERK2 (lower panel), Crk, or CAS (top right). Note that Crk-SH2 shows reduced mobility compared with wild-type endogenous Crk (Crk-WT) as the result of the molecular tag. (B) Serum-starved COS-7 cells were allowed to migrate in the presence or absence of the MEK inhibitor PD98059 (25 μM) with or without insulin (10 μg/ml). Cell migration and ERK1/ERK2 activity in these cells were determined as described above. Similar findings were obtained with fibronectin and collagen type I–coated membranes (data not shown). Each bar represents the mean ± SEM of at least three independent experiments. (C) Serum-starved COS-7 cells pretreated with or without the MEK inhibitor PD98059 (50 μM) for 2 h were exposed to insulin (10 μg/ml) for 5 min before being lysed in detergent. CAS was immunoprecipitated and then immunoblotted with antibodies to either phosphotyrosine, Crk, or CAS. The detergent lysates from these cells were also examined for changes in ERK1/ERK2 activity as described above. The result shown is representative from at least three independent experiments.
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Related In: Results  -  Collection

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Figure 1: ERK activation and CAS/Crk coupling are separate signaling events necessary for cytokine-induced cell migration. (A) Serum-starved COS-7 cells were allowed to migrate for 3 h on vitronectin-coated membranes in the presence or absence of insulin (10 μg/ml) after transient transfection with a β-gal reporter construct, along with either the empty expression vector (Mock) or with expression vectors encoding gst-tagged CAS without its substrate domain (CAS-SD), or myc-tagged Crk with a mutated SH2 domain (Crk-SH2). CAS-SD and Crk-SH2 have been shown to prevent CAS/Crk coupling and downstream signals (Matsuda et al. 1993; Feller et al. 1994; Klemke et al. 1998). 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. An aliquot of cells treated as described for the migration experiment above was lysed in detergent and immunoblotted with antibodies to either the phosphorylated/activated form of ERK1/ERK2 (lower panel), Crk, or CAS (top right). Note that Crk-SH2 shows reduced mobility compared with wild-type endogenous Crk (Crk-WT) as the result of the molecular tag. (B) Serum-starved COS-7 cells were allowed to migrate in the presence or absence of the MEK inhibitor PD98059 (25 μM) with or without insulin (10 μg/ml). Cell migration and ERK1/ERK2 activity in these cells were determined as described above. Similar findings were obtained with fibronectin and collagen type I–coated membranes (data not shown). Each bar represents the mean ± SEM of at least three independent experiments. (C) Serum-starved COS-7 cells pretreated with or without the MEK inhibitor PD98059 (50 μM) for 2 h were exposed to insulin (10 μg/ml) for 5 min before being lysed in detergent. CAS was immunoprecipitated and then immunoblotted with antibodies to either phosphotyrosine, Crk, or CAS. The detergent lysates from these cells were also examined for changes in ERK1/ERK2 activity as described above. The result shown is representative from at least three independent experiments.
Mentions: Recently, we reported that CAS/Crk coupling as well as ERK activation facilitate cell migration (Klemke et al. 1997, Klemke et al. 1998). Each of these events were shown to be required for cell migration, yet it remains unclear whether these signals represent the same or parallel pathways involved in regulation of this process. To address this issue, cells were transfected with CAS lacking its substrate domain (CAS-SD) or Crk with a mutated SH2 domain (CRK-SH2), either of which are capable of preventing CAS/Crk coupling and downstream signaling events (Feller et al. 1994; Matsuda et al. 1994; Klemke et al. 1998). Cells containing these cDNAs were treated with the cytokine insulin and examined for migration as well as ERK activity. Expression of either CAS-SD or Crk-SH2 blocked insulin-induced cell migration, yet had no effect on ERK activity in these cells (Fig. 1A). Similar findings were observed in cells expressing Crk with a mutated amino-terminal SH3 domain that retains its ability to couple to CAS, but is unable to link to downstream effector molecules such as DOCK180 or C3G (data not shown; Matsuda et al. 1994; Kiyokawa et al. 1998a,Kiyokawa et al. 1998b). These findings reveal that disruption of CAS/Crk coupling or its binding to downstream effectors can suppress cell migration without influencing ERK activity.

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