Mutations in the Kit kinase domain confer imatinib resistance to neoplastic mast cells.

(A) Schematic representations of wild-type Kit (Kit^WT) and mutant Kit showing the extracellular domain (ECD), the transmembrane domain (TM), and the kinase domain. Asp814 and Asp816, D in black; Tyr814 and Val816, Y and V in red. In addition to D816V, V560G is found in HMC-1.2’s Kit, but we did not find a difference between RCM and HMC-1.2 in Kit localization and oncogenic signalling. (B) RCM (left) and HMC-1.2 (right) were treated with vehicle (0), imatinib (Kit kinase inhibitor, closed circles), or PKC412 (inhibits type III tyrosine kinase, open circles) for 24 hours. Proliferation was assessed by [³H]-thymidine incorporation. Results (c.p.m.) are means ± SD (n = 3). (C) RCM and HMC-1.2 were treated with vehicle, 1 μM PKC412 or 5 μM imatinib (IMA) for 16 hours. Cell lysates were immunoblotted with anti-Kit, anti-phospho-Kit^Tyr721 (anti-pKit^Tyr721), anti-Akt, anti-pAkt, anti-STAT5, anti-pSTAT5, and anti-cleaved caspase-3. Note that imatinib did not affect the phosphorylation of Kit or cell proliferation.