Fig. 4. Phosphorylation by PAK did not affect the exchange activities of PIX. (A) PAK phosphorylates PIX between residues 496 and 555 of ß1PIX. Various Flag-tagged PIX isoforms and mutants were transfected into COS-7 cells and immunoprecipitated. The precipitated proteins were subjected to in vitro phosphorylation by GST-PAK with [
-33P]ATP in the reaction buffer. The phosphorylated product was analysed on SDS-PAGE gel and exposed to X-ray film. Top, the Coomassie-Blue-stained gel. Bottom, the autoradiograph. The asterisks mark the bands of interest. The Flag-tagged proteins are: lane 1, ß2PIX; lane 2, ß1PIX; lane 3, ß1PIX1-459; lane 4, ß1PIX1-555; lane 5, 
N80ß1PIX; lane 6, ß1PIXpro (deletion of amino acids 460-495 of ß1PIX); lane 7, GST-ß1PIX-C-ter; lane 8, GST-PAK alone. (B) Various Flag-tagged isoforms of PIX were transiently transfected into COS-7 cells. The immunoprecipitated PIX was assayed for exchange activity using GST-Rac1 as substrate with and without prior in vitro phosphorylation by GST-PAK (the kinase prepared as a GST fusion protein from E. coli is constitutively active). Each exchange assay was repeated with four determinants for each time point and the average is shown. (C) GST-PAK was transfected with wild-type Cdc42 and various PIX DNA constructs into COS-7 cells. GST-PAK fusion protein from the different transfection cell lysates was isolated by glutathione-Sepharose beads. Kinase activity was then measured by in vitro kinase assay using [-33P]ATP and myelin basic protein as substrates. The figure shows results from three different sets of transfection experiments and kinase assays. Top, western blot probed with anti-GST antibody to show the amount of GST-PAK from the different transfection cell lysates isolated by glutathione beads. Middle, autoradiograph showing the corresponding MBP phosphorylation by the GST-PAK pulled down in top panel. Bottom, normalized kinase activity of the GST-PAK from the corresponding transfection cell lysates. There was mobility shift of the GST-PAK band when PAK was transfected with wild-type Cdc42 and active Cdc42.
