Figure 3.

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.