Mutant PILSAP acts as a dominant-negative molecule. (A) HEK293 cells were transfected with a Wt or mutant PILSAP expression plasmid and aminopeptidase activity was measured using Leu-MCA as the substrate. Error bars indicate SDs. (B) MSS31 cells were transfected with a Wt or E343A expression vector and immunoprecipitated with an anti-myc antibody. Thereafter, pull-down analysis was performed using myc-tagged Wt and E343A. (C) Mock, Wt, or E343A transfected MSS31 cells were serum starved and stimulated with or without VEGF (50 ng/mL) for 12 hours. Incorporated BrdU was measured by a chemiluminescent BrdU ELISA. Error bars indicate SDs. (D) MSS31 cells were transfected with the indicated combination of expression vectors (wild-type PILSAP, E343A, and ΔN-PDK1). S6K was immunoprecipitated and kinase activities were measured using S6 peptide as the substrate. The densitometric intensities were determined and normalized with those of the total protein level. (E) MSS31 cells were transfected with the indicated combination of expression vectors (E343A, ΔN-PDK1, and wild-type PDK1) and were stimulated with or without VEGF (50 ng/mL).Association of S6K, PDK1 including ΔN-PDK1, and PILSAP including E343 was analyzed by immunoprecipitation followed by Western blotting. Total PILSAP and PDK1 were shown by Western blotting in the bottom 2 panels.