Chian R, Young S, Danilkovitch-Miagkova A, Rönnstrand L, Leonard E, Ferrao P, Ashman L, Linnekin D
Basic Research Laboratory and the Laboratory of Immunobiology, Division of Basic Sciences, National Cancer Institute-Frederick, MD 21702, USA.
Blood. 2001 Sep 1;98(5):1365-73. doi: 10.1182/blood.v98.5.1365.
Stem cell factor (SCF) binds the receptor tyrosine kinase c-Kit and is critical for normal hematopoiesis. Substitution of valine for aspartic acid 816 (D816V) constitutively actives human c-Kit, and this mutation is found in patients with mastocytosis, leukemia, and germ cell tumors. Immortalized murine progenitor cells (MIHCs) transduced with wild-type c-Kit proliferate in response to SCF, whereas cells expressing D816V c-Kit (MIHC-D816V) are factor-independent and tumorigenic. However, the mechanisms mediating transformation by D816V c-Kit are unknown. The objective of this study was to identify signaling components that contribute to D816V c-Kit-mediated transformation. SCF stimulates association of p85PI3K with phosphorylated tyrosine 721 of wild-type c-Kit. Phosphatidylinositol 3 kinase (PI3K) subsequently contributes to the activation of Akt and Jnks. In contrast, these studies demonstrated that the D816V c-Kit mutant was constitutively associated with phosphorylated p85PI3K, and, downstream of PI3K, Jnk 1 and Jnk 2 were activated but Akt was not. Interestingly, Erks 1 and 2 were not constitutively activated by D816V c-Kit. Thus, D816V c-Kit maintains the activity of PI3K but not of all signaling pathways activated by wild-type c-Kit. Further, all pathways downstream of PI3K are not constitutively active in MIHC-D816V cells. Studies with a PI3K inhibitor and D816V/Y721F c-Kit, a mutant incapable of recruiting PI3K, indicate that constitutive activation of PI3K through direct recruitment by D816V c-Kit plays a role in factor-independent growth of MIHC and is critical for tumorigenicity.
干细胞因子（SCF）与受体酪氨酸激酶c-Kit结合，对正常造血至关重要。用缬氨酸替代天冬氨酸816（D816V）可组成性激活人c-Kit，且该突变见于肥大细胞增多症、白血病和生殖细胞肿瘤患者。用野生型c-Kit转导的永生化小鼠祖细胞（MIHCs）对SCF有反应而增殖，而表达D816V c-Kit的细胞（MIHC-D816V）不依赖因子且具有致瘤性。然而，D816V c-Kit介导转化的机制尚不清楚。本研究的目的是确定促成D816V c-Kit介导转化的信号成分。SCF刺激p85PI3K与野生型c-Kit磷酸化酪氨酸721的结合。磷脂酰肌醇3激酶（PI3K）随后促成Akt和Jnks的激活。相比之下，这些研究表明D816V c-Kit突变体与磷酸化p85PI3K组成性结合，并且在PI3K下游，Jnk 1和Jnk 2被激活但Akt未被激活。有趣的是，Erks 1和2未被D816V c-Kit组成性激活。因此，D816V c-Kit维持PI3K的活性，但不维持野生型c-Kit激活的所有信号通路的活性。此外，PI3K下游的所有通路在MIHC-D816V细胞中并非组成性激活。用PI3K抑制剂和D816V/Y721F c-Kit（一种无法募集PI3K的突变体）进行的研究表明，D816V c-Kit通过直接募集对PI3K的组成性激活在MIHC的不依赖因子生长中起作用，并且对致瘤性至关重要。
