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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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PAK releases cells from contact inhibition by phosphorylating Merlin. (A) HUVEC were transfected with HA-tagged Merlin in combination with empty vector or plasmids encoding PAK-CAAX or Pak-CAAX-KD. G0-synchronized cells were plated on FN under sparse (Sp.) or confluent (Co.) conditions. After incubation with growth factors for 20 h, the cells were lysed and subjected to high resolution SDS-PAGE followed by immunoblotting with anti-Merlin. The top band corresponds to the phosphorylated form of Merlin and the bottom band to the unphosphorylated form. (B) Total lysates prepared as in A were subjected to standard SDS-PAGE and immunoblotting with anti–phospho-Merlin (Ser518) or anti-Merlin. (C) Cells were cotransfected with GFP and empty vector or vectors encoding the indicated forms of Merlin. G0-synchronized cells were plated on FN under 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. (D) Cells were cotransfected with GFP and empty vector or vectors encoding activated PAK (PAK-CAAX) in combination with the indicated forms of Merlin. The percentage of transfected cells entering S phase was determined as in Fig. 1. PAK-CAAX and various forms of Merlin were tagged with HA and detected by immunoblotting with anti-HA antibody. Error bar represents the mean ± SD.
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fig3: PAK releases cells from contact inhibition by phosphorylating Merlin. (A) HUVEC were transfected with HA-tagged Merlin in combination with empty vector or plasmids encoding PAK-CAAX or Pak-CAAX-KD. G0-synchronized cells were plated on FN under sparse (Sp.) or confluent (Co.) conditions. After incubation with growth factors for 20 h, the cells were lysed and subjected to high resolution SDS-PAGE followed by immunoblotting with anti-Merlin. The top band corresponds to the phosphorylated form of Merlin and the bottom band to the unphosphorylated form. (B) Total lysates prepared as in A were subjected to standard SDS-PAGE and immunoblotting with anti–phospho-Merlin (Ser518) or anti-Merlin. (C) Cells were cotransfected with GFP and empty vector or vectors encoding the indicated forms of Merlin. G0-synchronized cells were plated on FN under 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. (D) Cells were cotransfected with GFP and empty vector or vectors encoding activated PAK (PAK-CAAX) in combination with the indicated forms of Merlin. The percentage of transfected cells entering S phase was determined as in Fig. 1. PAK-CAAX and various forms of Merlin were tagged with HA and detected by immunoblotting with anti-HA antibody. Error bar represents the mean ± SD.

Mentions: PAK phosphorylates Merlin at Serine 518, disrupting the interaction between the NH2-terminal protein 4.1 ERM domain and the COOH-terminal tail of the protein (Shaw et al., 2001; Kissil et al., 2002; Xiao et al., 2002). To examine the potential role of PAK's phosphorylation of Merlin in release from contact inhibition, HUVEC were transfected with HA-tagged Merlin in combination with empty vector, activated PAK (PAK-CAAX), or dominant-negative PAK (PAK-CAAX-KD) and were plated under either sparse or confluent conditions in the presence of growth factors. High resolution SDS-PAGE followed by immunoblotting with anti-HA was used to monitor the mobility shift caused by the phosphorylation of Merlin (Shaw et al., 2001). Control transfectants plated under confluent conditions contained more dephosphorylated Merlin as compared with those plated under sparse conditions (Fig. 3 A). In addition, activated PAK induced significant phosphorylation of Merlin in confluent cells, whereas dominant-negative PAK caused an almost complete dephosphorylation of the protein in sparse cells (Fig. 3 A). Immunoblotting with an antibody that specifically recognizes Merlin phosphorylated at Serine 518 showed that Merlin is significantly phosphorylated at this site in cells plated under sparse conditions, but not in those plated under confluent conditions (Fig. 3 B). In addition, activated PAK caused significant phosphorylation of Merlin at Serine 518 in confluent and in suspended cells, whereas kinase-dead PAK did not exert this effect (Fig. 3 B). These results identify a potential role for Merlin phosphorylation at Serine 518 in exit from contact inhibition.


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)

PAK releases cells from contact inhibition by phosphorylating Merlin. (A) HUVEC were transfected with HA-tagged Merlin in combination with empty vector or plasmids encoding PAK-CAAX or Pak-CAAX-KD. G0-synchronized cells were plated on FN under sparse (Sp.) or confluent (Co.) conditions. After incubation with growth factors for 20 h, the cells were lysed and subjected to high resolution SDS-PAGE followed by immunoblotting with anti-Merlin. The top band corresponds to the phosphorylated form of Merlin and the bottom band to the unphosphorylated form. (B) Total lysates prepared as in A were subjected to standard SDS-PAGE and immunoblotting with anti–phospho-Merlin (Ser518) or anti-Merlin. (C) Cells were cotransfected with GFP and empty vector or vectors encoding the indicated forms of Merlin. G0-synchronized cells were plated on FN under 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. (D) Cells were cotransfected with GFP and empty vector or vectors encoding activated PAK (PAK-CAAX) in combination with the indicated forms of Merlin. The percentage of transfected cells entering S phase was determined as in Fig. 1. PAK-CAAX and various forms of Merlin were tagged with HA and detected by immunoblotting with anti-HA antibody. Error bar represents the mean ± SD.
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Related In: Results  -  Collection

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fig3: PAK releases cells from contact inhibition by phosphorylating Merlin. (A) HUVEC were transfected with HA-tagged Merlin in combination with empty vector or plasmids encoding PAK-CAAX or Pak-CAAX-KD. G0-synchronized cells were plated on FN under sparse (Sp.) or confluent (Co.) conditions. After incubation with growth factors for 20 h, the cells were lysed and subjected to high resolution SDS-PAGE followed by immunoblotting with anti-Merlin. The top band corresponds to the phosphorylated form of Merlin and the bottom band to the unphosphorylated form. (B) Total lysates prepared as in A were subjected to standard SDS-PAGE and immunoblotting with anti–phospho-Merlin (Ser518) or anti-Merlin. (C) Cells were cotransfected with GFP and empty vector or vectors encoding the indicated forms of Merlin. G0-synchronized cells were plated on FN under 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. (D) Cells were cotransfected with GFP and empty vector or vectors encoding activated PAK (PAK-CAAX) in combination with the indicated forms of Merlin. The percentage of transfected cells entering S phase was determined as in Fig. 1. PAK-CAAX and various forms of Merlin were tagged with HA and detected by immunoblotting with anti-HA antibody. Error bar represents the mean ± SD.
Mentions: PAK phosphorylates Merlin at Serine 518, disrupting the interaction between the NH2-terminal protein 4.1 ERM domain and the COOH-terminal tail of the protein (Shaw et al., 2001; Kissil et al., 2002; Xiao et al., 2002). To examine the potential role of PAK's phosphorylation of Merlin in release from contact inhibition, HUVEC were transfected with HA-tagged Merlin in combination with empty vector, activated PAK (PAK-CAAX), or dominant-negative PAK (PAK-CAAX-KD) and were plated under either sparse or confluent conditions in the presence of growth factors. High resolution SDS-PAGE followed by immunoblotting with anti-HA was used to monitor the mobility shift caused by the phosphorylation of Merlin (Shaw et al., 2001). Control transfectants plated under confluent conditions contained more dephosphorylated Merlin as compared with those plated under sparse conditions (Fig. 3 A). In addition, activated PAK induced significant phosphorylation of Merlin in confluent cells, whereas dominant-negative PAK caused an almost complete dephosphorylation of the protein in sparse cells (Fig. 3 A). Immunoblotting with an antibody that specifically recognizes Merlin phosphorylated at Serine 518 showed that Merlin is significantly phosphorylated at this site in cells plated under sparse conditions, but not in those plated under confluent conditions (Fig. 3 B). In addition, activated PAK caused significant phosphorylation of Merlin at Serine 518 in confluent and in suspended cells, whereas kinase-dead PAK did not exert this effect (Fig. 3 B). These results identify a potential role for Merlin phosphorylation at Serine 518 in exit from contact inhibition.

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