Figure 4.

CIB1 specifically stimulates PAK1 activity in vivo independently of small GTPases. (A) CIB1 specifically activates the PAK1 isoform. Endogenous PAK1 (bottom left) and PAK3 (bottom right) were immunoprecipitated from lysates prepared from vector- and CIB1-transfected REF52 cells either held in suspension or replated on FN for 20 min. Immunoprecipitated PAK was subjected to in vitro kinase assays using myelin basic protein (MBP) as substrate. The bar graph (top) depicts [³²P]MBP values after normalization for PAK immunoprecipitation. [³²P]MBP values from vector control cells were set as 1. Data represent SEM from two independent experiments (error bars). (B) GTPase-independent PAK1 activation. Serum-starved vector and CIB1-transfected REF52 cells held in suspension were treated with or without 100 ng/ml toxin B and either left in suspension or adhered to FN. Cells were lysed at the indicated times, and immunoprecipitated endogenous PAK1 was subjected to in vitro kinase assays using myelin basic protein as substrate. White lines indicate that intervening lanes have been spliced out; n = 3. (C) Efficacy of toxin B treatment was determined by assaying REF52 cell lysates for activated Rac1 and Cdc42 (see PAK1 kinase and Rac/Cdc42 activation assays). Affinity precipitates were analyzed by Western blotting for both Rac1 and Cdc42.