Roles of tyrosine residues 845, 892 and 922 in constitutive activation of murine FLT3 kinase domain mutant
Roles of tyrosine residues 845, 892 and 922 in constitutive activation of murine FLT3 kinase domain mutant
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DOI：
10.1038/sj.onc.1208957
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发表时间：
2005-12-01
期刊：
影响因子：
8
通讯作者：
Kanakura, Y
中科院分区：
文献类型：
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作者：
Ishiko, J;Mizuki, M;Kanakura, Y
FLT3酪氨酸激酶结构域（TKD）突变在大约7%的急性髓性白血病患者中被检测到，并被认为与不良预后相关，并导致对FLT3抑制剂的耐药性。为了探索FLT3 TKD突变的激活机制，我们通过对小鼠FLT3 TKD突变体（mFLT3(Asp838Val)）的所有22个细胞质酪氨酸残基产生一系列单一Tyr - b> Phe替代突变体，分析了FLT3 TKD突变体组成激活和下游信号传导的关键酪氨酸残基。Tyr845Phe、Tyr892Phe和Tyr922Phe的替换抑制了mFLT3（Asp838Val）自身的磷酸化，抑制了Erk1/2、STAT3和STAT5的激活，抑制了不依赖因子的细胞增殖和存活。相比之下，这三个Tyr -> Phe突变部分抑制但维持了野生型FLT3的配体依赖性激活和抗凋亡活性，这表明这些酪氨酸残基对mFLT3的组成性激活和信号传导更为关键（Asp838Val）。这三个Tyr - b> Phe突变也抑制了其他携带内部串联重复的FLT3突变体Asp838Tyr或Ile839del的组成性激活。这些取代对mFLT3（Asp838Val）激活和信号传导的抑制可以通过将培养温度从37℃转移到33℃，或通过引入Cdc37和Hsp90来部分恢复。综上所述，Tyr（845）、Tyr（892）和Tyr（922）是mFLT3（Asp838Val）激活的关键残基，可能是通过稳定mFLT3（Asp838Val）的活性构象。
FLT3 tyrosine kinase domain ( TKD) mutations are detected in similar to 7% of acute myeloid leukemia patients, and suggested to correlate with poor prognosis and confer resistance to FLT3 inhibitors. To explore activation mechanism of FLT3 TKD mutation, we analysed critical tyrosine residues for the constitutive activation and downstream signaling of the mutant by generating a series of single Tyr -> Phe substitution mutant of all 22 cytoplasmic tyrosine residues of murine FLT3 TKD-mutant (mFLT3(Asp838Val)). Tyr845Phe, Tyr892Phe and Tyr922Phe substitutions suppressed the phosphorylation of mFLT3(Asp838Val) itself, the activation of Erk1/2, STAT3 and STAT5, and the factor-independent cell proliferation and survival. In contrast, these three Tyr -> Phe mutations partially suppressed but maintained the ligand-dependent activation and anti-apoptotic activity of wild-type FLT3, suggesting that these tyrosine residues were more critical for the constitutive activation and signaling of mFLT3(Asp838Val). These three Tyr -> Phe mutations also inhibited the constitutive activation of other FLT3 mutants bearing internal tandem duplication, Asp838Tyr or Ile839del. The suppression of mFLT3(Asp838Val) activation and signaling by these substitutions was partially recovered by shifting the culture temperature from 37 to 33 degrees C, or by the introduction of Cdc37 and Hsp90. Taken together, Tyr(845), Tyr(892) and Tyr(922) are the critical residues in mFLT3(Asp838Val) activation, possibly through stabilizing the active conformation of mFLT3(Asp838Val).