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.

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.

Merlin mediates contact inhibition of growth by suppressing membrane recruitment of Rac. (A) HUVEC were electroporated with GFP-Rac and then either transfected with a siRNA oligonucleotide targeting human Merlin or mock-transfected as a control. 36 h later, cells were synchronized in G0, detached, and plated on FN under either sparse or confluent conditions, as indicated, and treated with FN beads. The graph shows the percentage of GFP-Rac–positive beads under the indicated conditions. (B) Cells were transfected with a siRNA oligonucleotide targeting human Merlin (+). G0-synchronized cells were detached, plated on FN under sparse or confluent conditions, and incubated with mitogens and BrdU for 20 h. The graph shows the percentage of cells entering S phase under the indicated conditions. Total lysates were subjected to immunoblotting with the indicated antibodies. (C) Cells were electroporated with a vector encoding Myc-tagged mouse Merlin (mMerlin) or empty vector and transfected with the anti–human siRNA oligonucleotide. After 36 h, cells were synchronized in G0, detached, and plated on FN under confluent conditions for 4 h. The cells were either incubated with mitogens and BrdU for 20 h to measure entry into S phase or treated with mitogens for 12 h and subjected to immunoblotting with the indicated antibodies. (D) siRNA-transfected cells were detached and plated on FN-coated coverslips under confluent conditions in the presence of growth factors. After 20 h, they were subjected to double immunofluorescent staining with anti–VE-cadherin (green) and anti–β-catenin (red). The bottom panels are XZ section views of VE-cadherin and DAPI staining. Error bars represent the mean ± SD.

Hypothetical model of Merlin's function. Cadherin-initiated adhesion prevents activation of Pak in contact-inhibited cells and thereby causes accumulation of dephosphorylated Merlin. This closed form of Merlin suppresses integrin-mediated recruitment of Rac, and hence mitogenic signaling. Upon release from contact inhibition, Pak phosphorylates and inactivates Merlin, allowing recruitment of Rac to the membrane and mitogenic signaling.

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.

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.

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.