Imatinib mesylate resistant c-Kit mutations are sensitive to PKC412. (A) Schematic of the c-KIT protein indicating the relative location of structural and functional domains, as well as location of 14 c-KIT mutations evaluated. The JM delVV mutation occurs at V559V560. ▪ indicates amino terminal signal peptide. Other domains of c-KIT are indicated as follows: □, extracellular domain; TM, transmembrane domain; JM, juxtamembrane domain; TK1, nucleotide binding subdomain of split kinase domain; KI, kinase insert domain; and TK2, catalytic subdomain of split kinase domain. (B) Ba/F3-transforming c-KIT mutations are constitutively phosphorylated and phosphorylate Stat3 and Stat5. Blots from top to bottom: antiphosphotyrosine Western blot of Ba/F3 lysates immunoprecipitated with anti-c-KIT; anti-c-KIT Western blot of immunoprecipitates in top blot; anti-phospho-Stat3 Western blot of whole-cell lysates (WCLs) from IP in top blot; anti-Stat3 Western blot of same WCLs; anti-phospho-Stat5 Western blot of WCLs from IP in top blot; and anti-Stat5a Western blot of the same WCLs. (C) Representative growth curves of delTYD + RG, V560G, D816Y, and D816V transduced Ba/F3 cell lines. Plotted is the percentage ± SD of ³H-thymidine incorporation of drug-treated cells relative to no-drug controls. Cells were treated with imatinib mesylate (black) or PKC412 (red). Cells were grown in the absence of IL-3 or rhSCF. (D) Dose response of PKC412 inhibition of c-KIT, Stat5, and Stat3 phosphorylation. Shown are data from delTYD + RG, V560G, D816Y, and D816V transformed Ba/F3 cells. Blots are as in panel B. Cells were grown in the absence of IL-3 and the indicated concentration of PKC412 (nM) for 4 hours. (E) PKC412 does not inhibit IL-3-induced Stat5 phosphorylation. Blots from top to bottom: anti-phospho-Stat3 Western blot of WCLs from indicated cell lines with or without IL-3 (10 ng/mL) and PKC412 (250 nM/mL), for which Ba/F3 cells were starved of IL-3 and serum for 4 hours prior to addition of IL-3 and PKC412; anti-Stat3 Western blot of WCLs in top blot; anti-phospho-Stat5 Western blot of WCLs as in top blot; and anti-Stat5a Western blot of same WCLs. (F) Dose response of Stat5 phosphorylation to PKC412 in Ba/F3 cells. Blots are as for panel E. Ba/F3 cells were starved of IL-3 and FCS for 4 hours, then incubated with or without IL-3 (10 ng/mL) in the presence of the indicated concentration of PKC412 for 4 hours.