Necessity of tyrosine 719 and phosphatidylinositol 3′-kinase-mediated signal pathway in constitutive activation and oncogenic potential of c-kit receptor tyrosine kinase with the Asp814Val mutation

Substitution of valine (Val) for aspartic acid (Asp) at codon 814 constitutively activates murine c-kit receptor tyrosine kinase (KIT), and Asp816Val mutation, corresponding to murine Asp814Val mutation, is found in patients with mastocytosis and acute myelocytic leukemia. However, the signal transduction pathways responsible for oncogenesis by the Asp814Val mutant (KlT(Val814)) are not fully understood. To examine the oncogenic signal transduction of KITVal814, we converted 20 tyrosine (Tyr) residues to phenylalanine (Phe) in the cytoplasmic domain of KITVal814 or deleted the C-terminal region containing 2 other tyrosine residues (Del). Among various KITVal814_ derived mutants, KITVal814-Tyr719Phe and KITVal814-Del severely impaired receptor tyrosine phosphorylation and association with the p85 subunit of phosphatidylinositol 3'-kinase (p85 (P13-K)). Moreover, KITVal814-Tyr719Phe and KITVal814-Del failed to induce ligand-independent growth in Ba/F3 cells, indicating that Tyr719, the binding site for p85(PI3-K), and the C-terminal region are indispensable for factor-independent growth by KITVal814. Although the C-terminal region was also required for ligand-dependent growth by wild-type KIT (KITWT), the Tyr719Phe substitution had negligible effects on ligand-dependent growth by KITWT. Furthermore, dominant-negative P13-K significantly inhibited ligand-independent growth by KITVal814. These results demonstrate that Tyr719 is crucial for constitutive activation of KITVal814, but not for the ligand-induced activation of KIT (WT), and that the downstream signaling of P13-K plays an important role in ligand-independent growth and tumorigenicity by KITVal814, thereby suggesting that KITVal814 is a unique activating mutation that leads to a distinguishable function from the effects of KITWT. (C) 2003 by The American Society of Hematology.