title: Transformation by oncogenic mutants and ligand-dependent activation of FLT3 wild-type requires the tyrosine residues 589 and 591.
abstract: OBJECTIVE
  Mutations in the receptor tyrosine kinase FLT3 are found in up to 30% of acute myelogenous leukemia patients and are associated with an inferior prognosis. In this study, we characterized critical tyrosine residues responsible for the transforming potential of active FLT3-receptor mutants and ligand-dependent activation of FLT3-WT.
  
  
  METHODS
  We performed a detailed structure-function analysis of putative autophosphorylation tyrosine residues in the FLT3-D835Y tyrosine kinase domain (TKD) mutant. All tyrosine residues in the juxtamembrane domain (Y566, Y572, Y589, Y591, Y597, and Y599), interkinase domain (Y726 and Y768), and COOH-terminal domain (Y955 and Y969) of the FLT3-D835Y construct were successively mutated to phenylalanine and the transforming activity of these mutants was analyzed in interleukin-3-dependent Ba/F3 cells. Tyrosine residues critical for the transforming potential of FLT3-D835Y were also analyzed in FLT3 internal tandem duplication mutants (FLT3-ITD)and the FLT3 wild-type (FLT3-WT) receptor.
  
  
  RESULTS
  The substitution of the tyrosine residues by phenylalanine in the juxtamembrane, interkinase, and COOH-terminal domains resulted in a complete loss of the transforming potential of FLT3-D835Y-expressing cells which can be attributed to a significant reduction of signal tranducer and activator of transcription 5 (STAT5) phosphorylation at the molecular level. Reintroduction of single tyrosine residues revealed the critical role of Y589 and Y591 in reconstituting interleukin-3-independent growth of FLT3-TKD-expressing cells. Combined mutation of Y589 and Y591 to phenylalanine also abrogated ligand-dependent proliferation of FLT3-WT and the transforming potential of FLT3-ITD-with a subsequent abrogation of STAT5 phosphorylation.
  
  
  CONCLUSIONS
  We identified two tyrosine residues, Y589 and Y591, in the juxtamembrane domain that are critical for the ligand-dependent activation of FLT3-WT and the transforming potential of oncogenic FLT3 mutants.
authors.affiliation: GSF-National Research Center for Environment, University of Munich-Grosshadern, Munich, Germany.
authors.forename: Sridhar
authors.initials: S
authors.lastname: Vempati
authors.forename: Carola
authors.initials: C
authors.lastname: Reindl
authors.forename: Ulla
authors.initials: U
authors.lastname: Wolf
authors.forename: Ruth
authors.initials: R
authors.lastname: Kern
authors.forename: Konstantin
authors.initials: K
authors.lastname: Petropoulos
authors.forename: Vegi M
authors.initials: VM
authors.lastname: Naidu
authors.forename: Christian
authors.initials: C
authors.lastname: Buske
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Hiddemann
authors.forename: Tobias M
authors.initials: TM
authors.lastname: Kohl
authors.forename: Karsten
authors.initials: K
authors.lastname: Spiekermann
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000737:chemistry* / Q000235:genetics; D004789:Enzyme Activation / Q000502:physiology; D005434:Flow Cytometry; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017354:Point Mutation; D050799:STAT5 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics
chemical_list: C120694:Fiz1 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D050799:STAT5 Transcription Factor; D014443:Tyrosine