Greenebaum Cancer Center, University of Maryland, Baltimore, Maryland, United States of America.
PLoS One. 2013 Sep 5;8(9):e74653. doi: 10.1371/journal.pone.0074653. eCollection 2013.
The type III receptor tyrosine kinase fms-like tyrosine kinase 3 (FLT3) is expressed on both normal hematopoietic stem cells and acute myeloid leukemia (AML) cells and regulates their proliferation. Internal tandem duplication (ITD) mutation of FLT3 is present in a third of AML cases, results in constitutive activation and aberrant signaling of FLT3, and is associated with adverse treatment outcomes. While wild-type (WT) FLT3 is predominantly a 150 kDa complex glycosylated cell surface protein, FLT3-ITD is partially retained in the endoplasmic reticulum as a 130 kDa underglycosylated species associated with the chaperones calnexin and heat shock protein (HSP) 90, and mediates aberrant STAT5 signaling, which upregulates the oncogenic serine/threonine kinase Pim-1. FLT3 contains a Pim-1 substrate consensus serine phosphorylation site, and we hypothesized that it might be a Pim-1 substrate. Pim-1 was indeed found to directly interact with and serine-phosphorylate FLT3. Pim-1 inhibition decreased the expression and half-life of 130 kDa FLT3, with partial abrogation by proteasome inhibition, in association with decreased FLT3 binding to calnexin and HSP90, and increased 150 kDa FLT3 expression and half-life, with abrogation by inhibition of glycosylation. These findings were consistent with Pim-1 stabilizing FLT3-ITD as a 130 kDa species associated with calnexin and HSP90 and inhibiting its glycosylation to form the 150 kDa species. Pim-1 knockdown effects were similar. Pim-1 inhibition also decreased phosphorylation of FLT3 at tyrosine 591 and of STAT5, and expression of Pim-1 itself, consistent with inhibition of the FLT3-ITD-STAT5 signaling pathway. Finally, Pim-1 inhibition synergized with FLT3 inhibition in inducing apoptosis of FLT3-ITD cells. This is, to our knowledge, the first demonstration of a role of Pim-1 in a positive feedback loop promoting aberrant signaling in malignant cells.
III 型受体酪氨酸激酶样酪氨酸激酶 3（FLT3）在正常造血干细胞和急性髓系白血病（AML）细胞上均有表达，并调节其增殖。FLT3 的内部串联重复（ITD）突变存在于三分之一的 AML 病例中，导致 FLT3 的组成性激活和异常信号转导，并与不良治疗结果相关。虽然野生型（WT）FLT3 主要是 150 kDa 的复合糖基化细胞表面蛋白，但 FLT3-ITD 部分保留在内质网中，作为 130 kDa 的低聚糖种，与伴侣蛋白 calnexin 和热休克蛋白（HSP）90 相关，并介导异常的 STAT5 信号转导，上调致癌丝氨酸/苏氨酸激酶 Pim-1。FLT3 包含 Pim-1 底物的丝氨酸磷酸化共识位点，我们假设它可能是 Pim-1 的底物。事实证明，Pim-1 确实与 FLT3 直接相互作用并使其丝氨酸磷酸化。Pim-1 抑制降低了 130 kDa FLT3 的表达和半衰期，通过蛋白酶体抑制部分阻断，与 FLT3 与 calnexin 和 HSP90 的结合减少以及 150 kDa FLT3 的表达和半衰期增加相关，通过抑制糖基化来阻断。这些发现与 Pim-1 稳定 FLT3-ITD 作为与 calnexin 和 HSP90 相关的 130 kDa 物种以及抑制其糖基化形成 150 kDa 物种的作用一致。Pim-1 敲低的作用也相似。Pim-1 抑制还降低了 FLT3 酪氨酸 591 位点和 STAT5 的磷酸化以及 Pim-1 自身的表达，与抑制 FLT3-ITD-STAT5 信号通路一致。最后，Pim-1 抑制与 FLT3 抑制协同诱导 FLT3-ITD 细胞凋亡。这是，据我们所知，首次证明 Pim-1 在促进恶性细胞异常信号的正反馈回路中发挥作用。
