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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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Activation of PAK is sufficient for release from contact inhibition. (A) HUVEC were transfected with GFP alone or in combination with plasmids encoding the indicated activated signaling molecules. G0-synchronized cells were plated on FN under sparse or confluent conditions. The percentage of transfected (GFP-positive) cells entering into S phase after mitogen treatment was determined by anti-BrdU staining. The percentage of sparse cells transfected with GFP alone entering in S phase ranged between 35 and 75%, depending on the experiment. This control value was normalized to 100% rescue. (B) Cells transfected with GFP in combination with the indicated doses of plasmids encoding activated PAK (PAK-CAAX) or dominant-negative PAK (PAK-CAAX-KD) were synchronized in G0 and plated on FN under either sparse or confluent conditions. The picture shows GFP-positive (green) PAK-CAAX transfectants that have incorporated BrdU (red) in spite of cell contact. (C) G0-synchronized cells were detached, kept in suspension or plated on FN under sparse or confluent conditions for 4 h, and treated with mitogens for 10 min or left untreated. Lysates were subjected to immune complex kinase assay with anti-PAK using myelin basic protein as a substrate. Error bars represent the mean ± SD.
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fig1: Activation of PAK is sufficient for release from contact inhibition. (A) HUVEC were transfected with GFP alone or in combination with plasmids encoding the indicated activated signaling molecules. G0-synchronized cells were plated on FN under sparse or confluent conditions. The percentage of transfected (GFP-positive) cells entering into S phase after mitogen treatment was determined by anti-BrdU staining. The percentage of sparse cells transfected with GFP alone entering in S phase ranged between 35 and 75%, depending on the experiment. This control value was normalized to 100% rescue. (B) Cells transfected with GFP in combination with the indicated doses of plasmids encoding activated PAK (PAK-CAAX) or dominant-negative PAK (PAK-CAAX-KD) were synchronized in G0 and plated on FN under either sparse or confluent conditions. The picture shows GFP-positive (green) PAK-CAAX transfectants that have incorporated BrdU (red) in spite of cell contact. (C) G0-synchronized cells were detached, kept in suspension or plated on FN under sparse or confluent conditions for 4 h, and treated with mitogens for 10 min or left untreated. Lysates were subjected to immune complex kinase assay with anti-PAK using myelin basic protein as a substrate. Error bars represent the mean ± SD.

Mentions: To avoid the potential effects of immortalization on growth control, we studied contact inhibition of growth in primary cultures of human umbilical vein endothelial cells (HUVEC). These cells undergo growth arrest and fail to respond to bFGF, EGF, and insulin as they become confluent and assemble vascular endothelial (VE)-cadherin–dependent junctions (Fig. S1, A–C, available at http://www.jcb.org/cgi/content/full/jcb.200503165/DC1). Assembly of tight junctions may prevent diffusion of peptide growth factors from the medium to the basolateral surface of polarized epithelial cells in vivo (Vermeer et al., 2003). However, experiments on cells plated on Transwell filters showed that contact inhibition of HUVEC is not caused by a segregation of RTKs from their cognate ligands (Fig. S1, A and C). In addition, GST pull-down assays with the Ras-binding domain of Raf (GST-Raf-RBD) provided evidence that cell–cell contact does not interfere with joint integrin–RTK signaling to Ras (Fig. S1 D), suggesting that the reported effect of VE-cadherin on growth factor receptor activation (Lampugnani et al., 2003) is not necessary for contact inhibition. Interestingly, extracellular signal-related protein kinase (ERK) was activated in a less sustained manner in confluent cells than in sparse cells (Fig. S1 E). We note that cell–cell contact may induce attenuation of Ras to ERK signaling through inhibition of PAK (Fig. 1 C), as PAK functions downstream of Ras to promote activation of ERK (King et al., 1998). These observations indicate that cell contact does not inhibit signaling to Ras but attenuates activation of ERK in HUVEC.


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)

Activation of PAK is sufficient for release from contact inhibition. (A) HUVEC were transfected with GFP alone or in combination with plasmids encoding the indicated activated signaling molecules. G0-synchronized cells were plated on FN under sparse or confluent conditions. The percentage of transfected (GFP-positive) cells entering into S phase after mitogen treatment was determined by anti-BrdU staining. The percentage of sparse cells transfected with GFP alone entering in S phase ranged between 35 and 75%, depending on the experiment. This control value was normalized to 100% rescue. (B) Cells transfected with GFP in combination with the indicated doses of plasmids encoding activated PAK (PAK-CAAX) or dominant-negative PAK (PAK-CAAX-KD) were synchronized in G0 and plated on FN under either sparse or confluent conditions. The picture shows GFP-positive (green) PAK-CAAX transfectants that have incorporated BrdU (red) in spite of cell contact. (C) G0-synchronized cells were detached, kept in suspension or plated on FN under sparse or confluent conditions for 4 h, and treated with mitogens for 10 min or left untreated. Lysates were subjected to immune complex kinase assay with anti-PAK using myelin basic protein as a substrate. Error bars represent the mean ± SD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171182&req=5

fig1: Activation of PAK is sufficient for release from contact inhibition. (A) HUVEC were transfected with GFP alone or in combination with plasmids encoding the indicated activated signaling molecules. G0-synchronized cells were plated on FN under sparse or confluent conditions. The percentage of transfected (GFP-positive) cells entering into S phase after mitogen treatment was determined by anti-BrdU staining. The percentage of sparse cells transfected with GFP alone entering in S phase ranged between 35 and 75%, depending on the experiment. This control value was normalized to 100% rescue. (B) Cells transfected with GFP in combination with the indicated doses of plasmids encoding activated PAK (PAK-CAAX) or dominant-negative PAK (PAK-CAAX-KD) were synchronized in G0 and plated on FN under either sparse or confluent conditions. The picture shows GFP-positive (green) PAK-CAAX transfectants that have incorporated BrdU (red) in spite of cell contact. (C) G0-synchronized cells were detached, kept in suspension or plated on FN under sparse or confluent conditions for 4 h, and treated with mitogens for 10 min or left untreated. Lysates were subjected to immune complex kinase assay with anti-PAK using myelin basic protein as a substrate. Error bars represent the mean ± SD.
Mentions: To avoid the potential effects of immortalization on growth control, we studied contact inhibition of growth in primary cultures of human umbilical vein endothelial cells (HUVEC). These cells undergo growth arrest and fail to respond to bFGF, EGF, and insulin as they become confluent and assemble vascular endothelial (VE)-cadherin–dependent junctions (Fig. S1, A–C, available at http://www.jcb.org/cgi/content/full/jcb.200503165/DC1). Assembly of tight junctions may prevent diffusion of peptide growth factors from the medium to the basolateral surface of polarized epithelial cells in vivo (Vermeer et al., 2003). However, experiments on cells plated on Transwell filters showed that contact inhibition of HUVEC is not caused by a segregation of RTKs from their cognate ligands (Fig. S1, A and C). In addition, GST pull-down assays with the Ras-binding domain of Raf (GST-Raf-RBD) provided evidence that cell–cell contact does not interfere with joint integrin–RTK signaling to Ras (Fig. S1 D), suggesting that the reported effect of VE-cadherin on growth factor receptor activation (Lampugnani et al., 2003) is not necessary for contact inhibition. Interestingly, extracellular signal-related protein kinase (ERK) was activated in a less sustained manner in confluent cells than in sparse cells (Fig. S1 E). We note that cell–cell contact may induce attenuation of Ras to ERK signaling through inhibition of PAK (Fig. 1 C), as PAK functions downstream of Ras to promote activation of ERK (King et al., 1998). These observations indicate that cell contact does not inhibit signaling to Ras but attenuates activation of ERK in HUVEC.

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