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Merlin/NF-2 mediates contact inhibition of growth by suppressing recruitment of Rac to the plasma membrane.

Okada T, Lopez-Lago M, Giancotti FG - J. Cell Biol. (2005)

Bottom Line: PAK's ability to release human umbilical vein endothelial cells from contact inhibition is blocked by an unphosphorylatable form of its target Merlin, suggesting that PAK promotes mitogenesis by phosphorylating, and thus inactivating, Merlin.Small interference RNA-mediated knockdown of Merlin exerts the same effects.Dominant-negative Rac blocks PAK-mediated release from contact inhibition, implying that PAK functions upstream of Rac in this signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. t-okada@ski.mskcc.org

ABSTRACT
Introduction of activated p21-activated kinase (PAK) is sufficient to release primary endothelial cells from contact inhibition of growth. Confluent cells display deficient activation of PAK and translocation of Rac to the plasma membrane at matrix adhesions. Targeting Rac to the plasma membrane rescues these cells from contact inhibition. PAK's ability to release human umbilical vein endothelial cells from contact inhibition is blocked by an unphosphorylatable form of its target Merlin, suggesting that PAK promotes mitogenesis by phosphorylating, and thus inactivating, Merlin. Merlin mutants, which are presumed to exert a dominant-negative effect, enable recruitment of Rac to matrix adhesions and promote mitogenesis in confluent cells. Small interference RNA-mediated knockdown of Merlin exerts the same effects. Dominant-negative Rac blocks PAK-mediated release from contact inhibition, implying that PAK functions upstream of Rac in this signaling pathway. These results provide a framework for understanding the tumor suppressor function of Merlin and indicate that Merlin mediates contact inhibition of growth by suppressing recruitment of Rac to matrix adhesions.

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Contact inhibition proceeds through suppression of the recruitment of Rac to the plasma membrane. (A) G0-synchronized HUVEC were detached and plated on FN under either sparse or confluent conditions. 4 h later, they were stimulated with mitogens for the indicated times and subjected to subcellular fractionation. The cytosolic (C) and crude membrane (M) fractions were isolated as described in Materials and methods. 2 μg of proteins from each fraction were analyzed by immunoblotting with the indicated antibodies. Asterisks point to the integrin pre-β1 subunit. (B) Cells transfected with GFP-Rac were synchronized in G0 and plated on FN under either sparse or confluent conditions. 4 h later they were treated with FN-coated beads for 25 min, fixed, and stained with DAPI (blue). Arrows point to FN-coated beads that had induced recruitment of GFP-Rac. The graph shows the percentage of cell-bound FN or PL beads that had caused recruitment of Rac under the indicated conditions. (C) G0-synchronized cells were plated on FN under sparse or confluent conditions or kept in suspension and treated with mitogens for 10 min or left untreated. GTP-Rac was measured by pull-down with GST-PAK-PBD. (D) Cells were cotransfected with GFP and empty vector or two different doses of HA-tagged myristoylated Rac (Myr-Rac) or with HA-Rac-L61. G0-synchronized cells were plated on FN under sparse or confluent conditions and incubated with mitogens and BrdU for 20 h. The percentage of transfected cells entering S phase was determined as described in Fig. 1 A. Expression levels were determined by immunoblotting with anti-HA (insets). Error bars represent the mean ± SD.
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fig2: Contact inhibition proceeds through suppression of the recruitment of Rac to the plasma membrane. (A) G0-synchronized HUVEC were detached and plated on FN under either sparse or confluent conditions. 4 h later, they were stimulated with mitogens for the indicated times and subjected to subcellular fractionation. The cytosolic (C) and crude membrane (M) fractions were isolated as described in Materials and methods. 2 μg of proteins from each fraction were analyzed by immunoblotting with the indicated antibodies. Asterisks point to the integrin pre-β1 subunit. (B) Cells transfected with GFP-Rac were synchronized in G0 and plated on FN under either sparse or confluent conditions. 4 h later they were treated with FN-coated beads for 25 min, fixed, and stained with DAPI (blue). Arrows point to FN-coated beads that had induced recruitment of GFP-Rac. The graph shows the percentage of cell-bound FN or PL beads that had caused recruitment of Rac under the indicated conditions. (C) G0-synchronized cells were plated on FN under sparse or confluent conditions or kept in suspension and treated with mitogens for 10 min or left untreated. GTP-Rac was measured by pull-down with GST-PAK-PBD. (D) Cells were cotransfected with GFP and empty vector or two different doses of HA-tagged myristoylated Rac (Myr-Rac) or with HA-Rac-L61. G0-synchronized cells were plated on FN under sparse or confluent conditions and incubated with mitogens and BrdU for 20 h. The percentage of transfected cells entering S phase was determined as described in Fig. 1 A. Expression levels were determined by immunoblotting with anti-HA (insets). Error bars represent the mean ± SD.

Mentions: PAK is a well established target effector of Rac (Etienne-Manneville and Hall, 2002). However, constitutively active Rac-L61 did not fully rescue cell cycle progression in confluent HUVEC, whereas activated PAK did (Fig. 1 A). We considered the possibility that Rac-L61 did not exert a proproliferative effect in confluent cells because of incorrect targeting. To examine whether cell–cell contact interferes with the recruitment of Rac to the plasma membrane, we used biochemical fractionation and optical imaging methods. HUVEC were plated on FN under either sparse or confluent conditions for 4 h and treated with growth factors. At various times after mitogenic stimulation the cells were subjected to biochemical fractionation and immunoblotting. As shown in Fig. 2 A, the crude membrane fraction of cells plated on FN under sparse conditions contained a significant amount of Rac (∼25% of the total). Mitogenic stimulation did not modify the proportion of Rac in the crude membrane fraction of these cells. These observations are in agreement with the model that RTKs induce GTP-loading on Rac, whereas integrins mediate its recruitment to the membrane and coupling to target effectors (del Pozo et al., 2000, 2002). Parenthetically, prolonged mitogenic stimulation led to increased levels of Rac in both the cytosolic and the crude membrane fraction of sparse cells, suggesting that Rac levels increase in mid-to-late G1 in HUVEC. Interestingly, most of the total Rac (>95%) remained in the cytosolic fraction in confluent cells, indicating that the translocation of Rac to the membrane fraction is impaired in confluent cells (Fig. 2 A). This result indicates that cell contact suppresses recruitment of Rac to the plasma membrane.


Merlin/NF-2 mediates contact inhibition of growth by suppressing recruitment of Rac to the plasma membrane.

Okada T, Lopez-Lago M, Giancotti FG - J. Cell Biol. (2005)

Contact inhibition proceeds through suppression of the recruitment of Rac to the plasma membrane. (A) G0-synchronized HUVEC were detached and plated on FN under either sparse or confluent conditions. 4 h later, they were stimulated with mitogens for the indicated times and subjected to subcellular fractionation. The cytosolic (C) and crude membrane (M) fractions were isolated as described in Materials and methods. 2 μg of proteins from each fraction were analyzed by immunoblotting with the indicated antibodies. Asterisks point to the integrin pre-β1 subunit. (B) Cells transfected with GFP-Rac were synchronized in G0 and plated on FN under either sparse or confluent conditions. 4 h later they were treated with FN-coated beads for 25 min, fixed, and stained with DAPI (blue). Arrows point to FN-coated beads that had induced recruitment of GFP-Rac. The graph shows the percentage of cell-bound FN or PL beads that had caused recruitment of Rac under the indicated conditions. (C) G0-synchronized cells were plated on FN under sparse or confluent conditions or kept in suspension and treated with mitogens for 10 min or left untreated. GTP-Rac was measured by pull-down with GST-PAK-PBD. (D) Cells were cotransfected with GFP and empty vector or two different doses of HA-tagged myristoylated Rac (Myr-Rac) or with HA-Rac-L61. G0-synchronized cells were plated on FN under sparse or confluent conditions and incubated with mitogens and BrdU for 20 h. The percentage of transfected cells entering S phase was determined as described in Fig. 1 A. Expression levels were determined by immunoblotting with anti-HA (insets). Error bars represent the mean ± SD.
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Related In: Results  -  Collection

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fig2: Contact inhibition proceeds through suppression of the recruitment of Rac to the plasma membrane. (A) G0-synchronized HUVEC were detached and plated on FN under either sparse or confluent conditions. 4 h later, they were stimulated with mitogens for the indicated times and subjected to subcellular fractionation. The cytosolic (C) and crude membrane (M) fractions were isolated as described in Materials and methods. 2 μg of proteins from each fraction were analyzed by immunoblotting with the indicated antibodies. Asterisks point to the integrin pre-β1 subunit. (B) Cells transfected with GFP-Rac were synchronized in G0 and plated on FN under either sparse or confluent conditions. 4 h later they were treated with FN-coated beads for 25 min, fixed, and stained with DAPI (blue). Arrows point to FN-coated beads that had induced recruitment of GFP-Rac. The graph shows the percentage of cell-bound FN or PL beads that had caused recruitment of Rac under the indicated conditions. (C) G0-synchronized cells were plated on FN under sparse or confluent conditions or kept in suspension and treated with mitogens for 10 min or left untreated. GTP-Rac was measured by pull-down with GST-PAK-PBD. (D) Cells were cotransfected with GFP and empty vector or two different doses of HA-tagged myristoylated Rac (Myr-Rac) or with HA-Rac-L61. G0-synchronized cells were plated on FN under sparse or confluent conditions and incubated with mitogens and BrdU for 20 h. The percentage of transfected cells entering S phase was determined as described in Fig. 1 A. Expression levels were determined by immunoblotting with anti-HA (insets). Error bars represent the mean ± SD.
Mentions: PAK is a well established target effector of Rac (Etienne-Manneville and Hall, 2002). However, constitutively active Rac-L61 did not fully rescue cell cycle progression in confluent HUVEC, whereas activated PAK did (Fig. 1 A). We considered the possibility that Rac-L61 did not exert a proproliferative effect in confluent cells because of incorrect targeting. To examine whether cell–cell contact interferes with the recruitment of Rac to the plasma membrane, we used biochemical fractionation and optical imaging methods. HUVEC were plated on FN under either sparse or confluent conditions for 4 h and treated with growth factors. At various times after mitogenic stimulation the cells were subjected to biochemical fractionation and immunoblotting. As shown in Fig. 2 A, the crude membrane fraction of cells plated on FN under sparse conditions contained a significant amount of Rac (∼25% of the total). Mitogenic stimulation did not modify the proportion of Rac in the crude membrane fraction of these cells. These observations are in agreement with the model that RTKs induce GTP-loading on Rac, whereas integrins mediate its recruitment to the membrane and coupling to target effectors (del Pozo et al., 2000, 2002). Parenthetically, prolonged mitogenic stimulation led to increased levels of Rac in both the cytosolic and the crude membrane fraction of sparse cells, suggesting that Rac levels increase in mid-to-late G1 in HUVEC. Interestingly, most of the total Rac (>95%) remained in the cytosolic fraction in confluent cells, indicating that the translocation of Rac to the membrane fraction is impaired in confluent cells (Fig. 2 A). This result indicates that cell contact suppresses recruitment of Rac to the plasma membrane.

Bottom Line: PAK's ability to release human umbilical vein endothelial cells from contact inhibition is blocked by an unphosphorylatable form of its target Merlin, suggesting that PAK promotes mitogenesis by phosphorylating, and thus inactivating, Merlin.Small interference RNA-mediated knockdown of Merlin exerts the same effects.Dominant-negative Rac blocks PAK-mediated release from contact inhibition, implying that PAK functions upstream of Rac in this signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. t-okada@ski.mskcc.org

ABSTRACT
Introduction of activated p21-activated kinase (PAK) is sufficient to release primary endothelial cells from contact inhibition of growth. Confluent cells display deficient activation of PAK and translocation of Rac to the plasma membrane at matrix adhesions. Targeting Rac to the plasma membrane rescues these cells from contact inhibition. PAK's ability to release human umbilical vein endothelial cells from contact inhibition is blocked by an unphosphorylatable form of its target Merlin, suggesting that PAK promotes mitogenesis by phosphorylating, and thus inactivating, Merlin. Merlin mutants, which are presumed to exert a dominant-negative effect, enable recruitment of Rac to matrix adhesions and promote mitogenesis in confluent cells. Small interference RNA-mediated knockdown of Merlin exerts the same effects. Dominant-negative Rac blocks PAK-mediated release from contact inhibition, implying that PAK functions upstream of Rac in this signaling pathway. These results provide a framework for understanding the tumor suppressor function of Merlin and indicate that Merlin mediates contact inhibition of growth by suppressing recruitment of Rac to matrix adhesions.

Show MeSH
Related in: MedlinePlus