title: Tyrosine 842 in the activation loop is required for full transformation by the oncogenic mutant FLT3-ITD.
abstract: The type III receptor tyrosine kinase FLT3 is frequently mutated in acute myeloid leukemia. Oncogenic FLT3 mutants display constitutive activity leading to aberrant cell proliferation and survival. Phosphorylation on several critical tyrosine residues is known to be essential for FLT3 signaling. Among these tyrosine residues, Y842 is located in the so-called activation loop. The position of this tyrosine residue is well conserved in all receptor tyrosine kinases. It has been reported that phosphorylation of the activation loop tyrosine is critical for catalytic activity for some but not all receptor tyrosine kinases. The role of Y842 residue in FLT3 signaling has not yet been studied. In this report, we show that Y842 is not important for FLT3 activation or ubiquitination but plays a critical role in regulating signaling downstream of the receptor as well as controlling receptor stability. We found that mutation of Y842 in the FLT3-ITD oncogenic mutant background reduced cell viability and increased apoptosis. Furthermore, the introduction of the Y842 mutation in the FLT3-ITD background led to a dramatic reduction in in vitro colony forming capacity. Additionally, mice injected with cells expressing FLT3-ITD/Y842F displayed a significant delay in tumor formation, compared to FLT3-ITD expressing cells. Microarray analysis comparing gene expression regulated by FLT3-ITD versus FLT3-ITD/Y842F demonstrated that mutation of Y842 causes suppression of anti-apoptotic genes. Furthermore, we showed that cells expressing FLT3-ITD/Y842F display impaired activity of the RAS/ERK pathway due to reduced interaction between FLT3 and SHP2 leading to reduced SHP2 activation. Thus, we suggest that Y842 is critical for FLT3-mediated RAS/ERK signaling and cellular transformation.
authors.affiliation: Division of Translational Cancer Research, Department of Laboratory Medicine, Lund University, Medicon Village, Lund, Sweden.|Lund Stem Cell Center, Department of Laboratory Medicine, Lund University, Lund, Sweden.
authors.forename: Julhash U
authors.initials: JU
authors.lastname: Kazi
authors.affiliation: Division of Translational Cancer Research, Department of Laboratory Medicine, Lund University, Medicon Village, Lund, Sweden.|Lund Stem Cell Center, Department of Laboratory Medicine, Lund University, Lund, Sweden.
authors.forename: Rohit A
authors.initials: RA
authors.lastname: Chougule
authors.affiliation: School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong.
authors.forename: Tianfeng
authors.initials: T
authors.lastname: Li
authors.affiliation: School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong.|Department of Surgery, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong.
authors.forename: Xianwei
authors.initials: X
authors.lastname: Su
authors.affiliation: Division of Translational Cancer Research, Department of Laboratory Medicine, Lund University, Medicon Village, Lund, Sweden.|Lund Stem Cell Center, Department of Laboratory Medicine, Lund University, Lund, Sweden.
authors.forename: Sausan A
authors.initials: SA
authors.lastname: Moharram
authors.affiliation: Division of Translational Cancer Research, Department of Laboratory Medicine, Lund University, Medicon Village, Lund, Sweden.|Lund Stem Cell Center, Department of Laboratory Medicine, Lund University, Lund, Sweden.
authors.forename: Kaja
authors.initials: K
authors.lastname: Rupar
authors.affiliation: Division of Translational Cancer Research, Department of Laboratory Medicine, Lund University, Medicon Village, Lund, Sweden.|Lund Stem Cell Center, Department of Laboratory Medicine, Lund University, Lund, Sweden.
authors.forename: Alissa
authors.initials: A
authors.lastname: Marhäll
authors.affiliation: Division of Translational Cancer Research, Department of Laboratory Medicine, Lund University, Medicon Village, Lund, Sweden.|Lund Stem Cell Center, Department of Laboratory Medicine, Lund University, Lund, Sweden.
authors.forename: Mohiuddin
authors.initials: M
authors.lastname: Gazi
authors.affiliation: Division of Translational Cancer Research, Department of Laboratory Medicine, Lund University, Medicon Village, Lund, Sweden.|Lund Stem Cell Center, Department of Laboratory Medicine, Lund University, Lund, Sweden.|Department of Pathogen Biology and Immunology, School of Basic Medical Sciences, Ningxia Medical University, Yinchuan, People's Republic of China.
authors.forename: Jianmin
authors.initials: J
authors.lastname: Sun
authors.affiliation: School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Division of Translational Cancer Research, Department of Laboratory Medicine, Lund University, Medicon Village, Lund, Sweden. lars.ronnstrand@med.lu.se.|Lund Stem Cell Center, Department of Laboratory Medicine, Lund University, Lund, Sweden. lars.ronnstrand@med.lu.se.|Department of Oncology, Skåne University Hospital, Lund, Sweden. lars.ronnstrand@med.lu.se.
authors.forename: Lars
authors.identifier: 0000-0003-1275-5809
authors.initials: L
authors.lastname: Rönnstrand
mesh_terms: D000818:Animals; D017209:Apoptosis; D002460:Cell Line; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic / Q000378:metabolism* / Q000473:pathology*; D015536:Down-Regulation; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D020440:Gene Duplication; D006801:Humans; D008297:Male; D008807:Mice, Inbred BALB C; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D022423:Myeloid Cells / Q000378:metabolism; D009857:Oncogenes; D010766:Phosphorylation; D055550:Protein Stability; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11 / Q000378:metabolism; D059748:Proteolysis; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism*; D054875:Ubiquitination; D051941:fms-Like Tyrosine Kinase 3 / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D014443:Tyrosine; C497970:FLT3 protein, human; D051941:fms-Like Tyrosine Kinase 3; D048049:Extracellular Signal-Regulated MAP Kinases; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11
keywords: Activation loop; Acute myeloid leukemia; FLT3; FLT3-ITD; Microarray; SHP2; Survival; Transformation