Roles of tyrosine residues 845, 892 and 922 in constitutive activation of murine FLT3 kinase domain mutant

doi:doi:10.1038/sj.onc.1208957

Oncogene (2005) 24, 8144–8153. doi:10.1038/sj.onc.1208957; published online 1 August 2005

Roles of tyrosine residues 845, 892 and 922 in constitutive activation of murine FLT3 kinase domain mutant

Jun Ishiko¹, Masao Mizuki¹, Itaru Matsumura¹, Hirohiko Shibayama¹, Hiroyuki Sugahara¹, Glen Scholz², Hubert Serve³ and Yuzuru Kanakura¹

¹Department of Hematology and Oncology, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan
²Department of Medicine, University of Melbourne, The Royal Melbourne Hospital, Parkville 3050, Australia
³Department of Medicine, Hematology and Oncology, University of Münster, Albert-Schweitzer-Strasse 33, Münster 48129, Germany

Correspondence: M Mizuki, E-mail: mizuki@bldon.med.osaka-u.ac.jp

Received 7 January 2005; Revised 17 June 2005; Accepted 23 June 2005; Published online 1 August 2005.

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Abstract

FLT3 tyrosine kinase domain (TKD) mutations are detected in $approx$ 7% of acute myeloid leukemia patients, and suggested to correlate with poor prognosis and confer resistance to FLT3 inhibitors. To explore activation mechanism of FLT3 TKD mutation, we analysed critical tyrosine residues for the constitutive activation and downstream signaling of the mutant by generating a series of single Tyr Phe substitution mutant of all 22 cytoplasmic tyrosine residues of murine FLT3 TKD-mutant (mFLT3^Asp838Val). Tyr845Phe, Tyr892Phe and Tyr922Phe substitutions suppressed the phosphorylation of mFLT3^Asp838Val itself, the activation of Erk1/2, STAT3 and STAT5, and the factor-independent cell proliferation and survival. In contrast, these three Tyr Phe mutations partially suppressed but maintained the ligand-dependent activation and anti-apoptotic activity of wild-type FLT3, suggesting that these tyrosine residues were more critical for the constitutive activation and signaling of mFLT3^Asp838Val. These three Tyr Phe mutations also inhibited the constitutive activation of other FLT3 mutants bearing internal tandem duplication, Asp838Tyr or Ile839del. The suppression of mFLT3^Asp838Val activation and signaling by these substitutions was partially recovered by shifting the culture temperature from 37 to 33°C, or by the introduction of Cdc37 and Hsp90. Taken together, Tyr⁸⁴⁵, Tyr⁸⁹² and Tyr⁹²² are the critical residues in mFLT3^Asp838Val activation, possibly through stabilizing the active conformation of mFLT3^Asp838Val.