Limits...
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.

Show MeSH

Related in: MedlinePlus

PAK promotes recruitment of Rac to matrix adhesions. (A) Cells were cotransfected with GFP and empty vector, or vector encoding activated PAK (PAK-CAAX) or Pak-CAAX-H83/86L, in combination or not with dominant-negative Rac (Rac-N17). 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. 2. PAK-CAAX was tagged with HA and hence detected by immunoblotting with anti-HA, whereas Rac-N17 was tagged with Myc and thus detected with anti-Myc. (B) HUVEC were transfected with GFP-Rac in combination with empty vector or vector encoding activated PAK (PAK-CAAX). They were then synchronized in G0 and plated on FN under sparse or confluent conditions. FN-coated beads were applied for 25 min. The cells were fixed and stained with anti-HA to detect PAK-CAAX (red) and DAPI to stain nuclei (blue). Arrows point to FN-coated beads that induced recruitment of GFP-Rac. The graph shows the percentage of GFP-Rac–positive beads under the indicated conditions. Error bars represent the mean ± SD.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171182&req=5

fig6: PAK promotes recruitment of Rac to matrix adhesions. (A) Cells were cotransfected with GFP and empty vector, or vector encoding activated PAK (PAK-CAAX) or Pak-CAAX-H83/86L, in combination or not with dominant-negative Rac (Rac-N17). 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. 2. PAK-CAAX was tagged with HA and hence detected by immunoblotting with anti-HA, whereas Rac-N17 was tagged with Myc and thus detected with anti-Myc. (B) HUVEC were transfected with GFP-Rac in combination with empty vector or vector encoding activated PAK (PAK-CAAX). They were then synchronized in G0 and plated on FN under sparse or confluent conditions. FN-coated beads were applied for 25 min. The cells were fixed and stained with anti-HA to detect PAK-CAAX (red) and DAPI to stain nuclei (blue). Arrows point to FN-coated beads that induced recruitment of GFP-Rac. The graph shows the percentage of GFP-Rac–positive beads under the indicated conditions. Error bars represent the mean ± SD.

Mentions: PAK is a key target effector of Rac, but it can also function upstream of Rac (Bokoch, 2003), raising the possibility that PAK functions in proliferative signaling through Rac-mediated activation of target effectors other than PAK. This data supports the hypothesis that PAK regulates Rac translocation to the membrane through phosphorylation and inactivation of Merlin. To confirm that PAK functions upstream of Rac during release from contact inhibition, we first tested if PAK rescues cells from contact inhibition through Rac. HUVEC were transfected with activated PAK, alone or in combination with dominant-negative Rac. As shown in Fig. 6 A, dominant-negative Rac (Rac-N17) suppressed PAK's ability to release confluent HUVEC from contact inhibition, suggesting that PAK functions upstream of Rac in this process. A mutation crippling the CRIB domain (H83/86L) did not impair the ability of activated PAK to rescue HUVEC from contact inhibition, indicating that this effect does not require Rac binding to PAK. As expected, dominant-negative Rac also interfered with the ability of the CRIB domain mutant form of activated PAK to release cells from contact inhibition (Fig. 6 A). These findings indicate that PAK functions upstream of Rac in release 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 promotes recruitment of Rac to matrix adhesions. (A) Cells were cotransfected with GFP and empty vector, or vector encoding activated PAK (PAK-CAAX) or Pak-CAAX-H83/86L, in combination or not with dominant-negative Rac (Rac-N17). 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. 2. PAK-CAAX was tagged with HA and hence detected by immunoblotting with anti-HA, whereas Rac-N17 was tagged with Myc and thus detected with anti-Myc. (B) HUVEC were transfected with GFP-Rac in combination with empty vector or vector encoding activated PAK (PAK-CAAX). They were then synchronized in G0 and plated on FN under sparse or confluent conditions. FN-coated beads were applied for 25 min. The cells were fixed and stained with anti-HA to detect PAK-CAAX (red) and DAPI to stain nuclei (blue). Arrows point to FN-coated beads that induced recruitment of GFP-Rac. The graph shows the percentage of GFP-Rac–positive beads under the indicated conditions. Error bars represent the mean ± SD.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: PAK promotes recruitment of Rac to matrix adhesions. (A) Cells were cotransfected with GFP and empty vector, or vector encoding activated PAK (PAK-CAAX) or Pak-CAAX-H83/86L, in combination or not with dominant-negative Rac (Rac-N17). 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. 2. PAK-CAAX was tagged with HA and hence detected by immunoblotting with anti-HA, whereas Rac-N17 was tagged with Myc and thus detected with anti-Myc. (B) HUVEC were transfected with GFP-Rac in combination with empty vector or vector encoding activated PAK (PAK-CAAX). They were then synchronized in G0 and plated on FN under sparse or confluent conditions. FN-coated beads were applied for 25 min. The cells were fixed and stained with anti-HA to detect PAK-CAAX (red) and DAPI to stain nuclei (blue). Arrows point to FN-coated beads that induced recruitment of GFP-Rac. The graph shows the percentage of GFP-Rac–positive beads under the indicated conditions. Error bars represent the mean ± SD.
Mentions: PAK is a key target effector of Rac, but it can also function upstream of Rac (Bokoch, 2003), raising the possibility that PAK functions in proliferative signaling through Rac-mediated activation of target effectors other than PAK. This data supports the hypothesis that PAK regulates Rac translocation to the membrane through phosphorylation and inactivation of Merlin. To confirm that PAK functions upstream of Rac during release from contact inhibition, we first tested if PAK rescues cells from contact inhibition through Rac. HUVEC were transfected with activated PAK, alone or in combination with dominant-negative Rac. As shown in Fig. 6 A, dominant-negative Rac (Rac-N17) suppressed PAK's ability to release confluent HUVEC from contact inhibition, suggesting that PAK functions upstream of Rac in this process. A mutation crippling the CRIB domain (H83/86L) did not impair the ability of activated PAK to rescue HUVEC from contact inhibition, indicating that this effect does not require Rac binding to PAK. As expected, dominant-negative Rac also interfered with the ability of the CRIB domain mutant form of activated PAK to release cells from contact inhibition (Fig. 6 A). These findings indicate that PAK functions upstream of Rac in release 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