Ras, Raf-1, MEK, ERK-2, and PI-3K regulate

CD13/APN induction. KS1767 cells were incubated in 1% serum 24 hours before transient transfection with 1 μg reporter plasmids containing the CD13/APN promoter and 2 μg of the indicated expression plasmids (for details, see “Materials and methods”), followed by stimulation with bFGF and/or addition of chemical inhibitors of specific pathway intermediates at concentrations detailed in “Materials and methods.” The MAP1-SEAP plasmid (1 μg) was cotransfected in each condition to normalize for transfection efficiency. Results are shown as fold activation at 48 hours after transfection over the activity of the promoterless vector plasmid transfected in parallel. (A) Manumycin A, Ras inhibitor; Ras-17, dominant-negative expression plasmid; and Ras-61, constitutively active Ras expression plasmid. (B) Raf-1–wt, wild-type Raf expression plasmid; Raf-1–C4B, dominant-negative Raf expression plasmid; and Raf-1–BXB, constitutively activated Raf-1 expression plasmid. (C) PD98059, MEK inhibitor; SB203580, p38 kinase inhibitor; MEK-DN, dominant-negative expression plasmid; and MEK-ACT, constitutively active MEK expression plasmid. (D) ERK2-B3, ERK2-C3–dominant-negative ERK2 expression plasmids. (E) Wortmannin and LY290042, PI-3K inhibitors. Error bars indicate standard deviations of at least 3 replicates.