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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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Open forms of Merlin promote recruitment of Rac to matrix adhesions. (A) HUVEC were transfected with GFP-Rac in combination with empty vector or vectors encoding HA-tagged Merlin-S518D, Merlin-ΔBB (BB), Merlin-WT, and Merlin-S518A. FN-coated beads were applied for 25 min to G0-synchronized cells plated on FN under confluent conditions. The cells were then fixed and stained with anti-HA to detect HA-Merlin (red) and DAPI (blue). The graph shows the percentage of GFP-Rac–positive beads under the indicated conditions. Arrows point to FN-coated beads that had induced recruitment of GFP-Rac. (B) Cells were transfected with vectors encoding HA-tagged Merlin-S518D or Merlin-S518A. G0-synchronized cells were plated on FN under confluent conditions and subjected to double staining with antibodies to β-catenin (red) and HA (green). The inset shows that Merlin-S518D localizes in part to adherens junctions, but does not disrupt them. Error bar represents the mean ± SD.
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fig4: Open forms of Merlin promote recruitment of Rac to matrix adhesions. (A) HUVEC were transfected with GFP-Rac in combination with empty vector or vectors encoding HA-tagged Merlin-S518D, Merlin-ΔBB (BB), Merlin-WT, and Merlin-S518A. FN-coated beads were applied for 25 min to G0-synchronized cells plated on FN under confluent conditions. The cells were then fixed and stained with anti-HA to detect HA-Merlin (red) and DAPI (blue). The graph shows the percentage of GFP-Rac–positive beads under the indicated conditions. Arrows point to FN-coated beads that had induced recruitment of GFP-Rac. (B) Cells were transfected with vectors encoding HA-tagged Merlin-S518D or Merlin-S518A. G0-synchronized cells were plated on FN under confluent conditions and subjected to double staining with antibodies to β-catenin (red) and HA (green). The inset shows that Merlin-S518D localizes in part to adherens junctions, but does not disrupt them. Error bar represents the mean ± SD.

Mentions: Previous studies have suggested that the “closed,” dephosphorylated form of Merlin suppresses Rac signaling (Shaw et al., 2001; Kissil et al., 2002), raising the possibility that PAK functions upstream of Rac during release from contact inhibition. We thus examined if Merlin suppresses recruitment of Rac to matrix adhesions in confluent cells. HUVEC were transfected with GFP-Rac, alone or in combination with HA-tagged Merlin-S518D, Merlin-ΔBB, Merlin-WT, Merlin-S518A, or empty vector. The cells were plated under confluent conditions and incubated with FN-coated beads. Confocal microscopy indicated that expression of Merlin-ΔBB or Merlin-S518D restores recruitment of GFP-Rac to FN-coated beads in confluent HUVEC (Fig. 4 A). Notably, anti-HA staining indicated that both mutant forms of Merlin localize underneath FN-coated beads (Fig. 4 A and not depicted), in agreement with the hypothesis that they interfere with the ability of endogenous Merlin to suppress integrin-mediated recruitment of Rac. These observations are consistent with the hypothesis that Merlin suppresses recruitment of Rac to matrix adhesions in contact-inhibited cells.


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)

Open forms of Merlin promote recruitment of Rac to matrix adhesions. (A) HUVEC were transfected with GFP-Rac in combination with empty vector or vectors encoding HA-tagged Merlin-S518D, Merlin-ΔBB (BB), Merlin-WT, and Merlin-S518A. FN-coated beads were applied for 25 min to G0-synchronized cells plated on FN under confluent conditions. The cells were then fixed and stained with anti-HA to detect HA-Merlin (red) and DAPI (blue). The graph shows the percentage of GFP-Rac–positive beads under the indicated conditions. Arrows point to FN-coated beads that had induced recruitment of GFP-Rac. (B) Cells were transfected with vectors encoding HA-tagged Merlin-S518D or Merlin-S518A. G0-synchronized cells were plated on FN under confluent conditions and subjected to double staining with antibodies to β-catenin (red) and HA (green). The inset shows that Merlin-S518D localizes in part to adherens junctions, but does not disrupt them. Error bar represents the mean ± SD.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171182&req=5

fig4: Open forms of Merlin promote recruitment of Rac to matrix adhesions. (A) HUVEC were transfected with GFP-Rac in combination with empty vector or vectors encoding HA-tagged Merlin-S518D, Merlin-ΔBB (BB), Merlin-WT, and Merlin-S518A. FN-coated beads were applied for 25 min to G0-synchronized cells plated on FN under confluent conditions. The cells were then fixed and stained with anti-HA to detect HA-Merlin (red) and DAPI (blue). The graph shows the percentage of GFP-Rac–positive beads under the indicated conditions. Arrows point to FN-coated beads that had induced recruitment of GFP-Rac. (B) Cells were transfected with vectors encoding HA-tagged Merlin-S518D or Merlin-S518A. G0-synchronized cells were plated on FN under confluent conditions and subjected to double staining with antibodies to β-catenin (red) and HA (green). The inset shows that Merlin-S518D localizes in part to adherens junctions, but does not disrupt them. Error bar represents the mean ± SD.
Mentions: Previous studies have suggested that the “closed,” dephosphorylated form of Merlin suppresses Rac signaling (Shaw et al., 2001; Kissil et al., 2002), raising the possibility that PAK functions upstream of Rac during release from contact inhibition. We thus examined if Merlin suppresses recruitment of Rac to matrix adhesions in confluent cells. HUVEC were transfected with GFP-Rac, alone or in combination with HA-tagged Merlin-S518D, Merlin-ΔBB, Merlin-WT, Merlin-S518A, or empty vector. The cells were plated under confluent conditions and incubated with FN-coated beads. Confocal microscopy indicated that expression of Merlin-ΔBB or Merlin-S518D restores recruitment of GFP-Rac to FN-coated beads in confluent HUVEC (Fig. 4 A). Notably, anti-HA staining indicated that both mutant forms of Merlin localize underneath FN-coated beads (Fig. 4 A and not depicted), in agreement with the hypothesis that they interfere with the ability of endogenous Merlin to suppress integrin-mediated recruitment of Rac. These observations are consistent with the hypothesis that Merlin suppresses recruitment of Rac to matrix adhesions in contact-inhibited cells.

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