The hematopoietic growth factor receptor Flt3 plays an important role in both myelopoiesis and B-cell production. Mutations of Flt3 found in approximately 30% of acute myeloid leukemias cause constitutive activation of the receptor tyrosine kinase. Cells expressing these Flt3 mutants show enhanced survival and proliferation, and, when evaluated in a mouse model, leukemia-associated Flt3 mutations resulted in fatal myeloproliferative disease (Kelly et al. Blood 2002). Despite the important roles of Flt3 in both hematopoiesis and leukemogenesis, the mechanisms that regulate Flt3 remain incompletely understood. In particular, little is known about regulation of receptor quantity. To address this issue, we examined Flt3 protein turnover in human leukemic cell lines expressing wild-type or mutant Flt3. To determine protein half-life, cells were treated with cycloheximide to block new protein synthesis, and, at specific intervals thereafter, cells were lysed and remaining Flt3 protein determined by Western blotting and densitometry. Dilution studies were performed to confirm that Western blot signals showed linear variation with amounts of Flt3 protein in the relevant concentration range. In the absence of Flt3 ligand stimulation, wild-type Flt3 had a half-life of approximately 2 hours. Cell stimulation by Flt3 ligand, however, markedly accelerated Flt3 degradation: its half-life was reduced to approximately 25 minutes. Repeat immunoblotting with an anti-Actin antibody verified equivalent sample loading, and protein assays of the cell lysates revealed no overall decrease in cellular protein within the time periods studied. We next evaluated the degradation of a constitutively-active Flt3 mutant. The MV4;11 human leukemic cell line expresses a tandem duplication mutant of Flt3, the most common type of leukemia-associated Flt3 mutation. At intervals after blocking new protein synthesis, MV4;11 cells were lysed and Flt3 protein assessed by Western blot and densitometry. We found that the half-life of mutant Flt3 was significantly shorter than that of the unstimulated wild-type receptor but very similar to that seen after ligand-stimulation. This suggested that Flt3 kinase activity, or the consequent activation of intracellular signaling proteins, might regulate receptor degradation. To identify mechanisms responsible for activation-induced Flt3 degradation, we examined the effects of protein degradation inhibitors. We found that MG132 blocked degradation of the mutant receptor, implicating the proteosome pathway in activated Flt3 degradation. The Flt3 receptor has been reported to phosphorylate Cbl, a signaling protein and ubiquitin ligase. Cbl can promote proteosomal degradation of the signaling proteins it binds. However, we found no evidence of a direct association between Flt3 and Cbl by co-immunoprecipitation. Flt3 also activates Src-family tyrosine kinases, which have been shown to regulate the internalization and degradation of related receptors such as c-Kit. However, PP1, an inhibitor selective for Src-family tyrosine kinases, had no effect on Flt3 degradation. The very rapid degradation of mutant Flt3 suggests that the high levels of Flt3 mRNA expression reported for MLL leukemias might not produce equivalent increases in mutant Flt3 protein. It is possible that leukemogenic effects of Flt3 activating mutation require co-operating abnormalities of protein degradation. Our results further raise the possibility that therapeutic use of proteosome inhibitors might exacerbate Flt3-related leukemias.

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