Identification of Y589 and Y599 in the juxamembrane domain of Flt3 as ligand-induced autophosphorylation sites involved in binding of Src family kinases and the protein tyrosine phosphatase SHP2
Author
Summary, in English
Early signal relay steps upon ligand binding to the receptor tyrosine kinase Flt3, i.e. sites of Flt3 autophosphorylation and subsequent docking partners, are mainly unresolved. By immunoprecipitation of specific tryptic peptides contained in the juxtamembrane region
of human Flt3 and subsequent radiosequencing we identified the tyrosine residues 572, 589, 591 and 599 as in vivo autophosphorylation sites. Focusing on Y589 and Y599, we
examined Flt3-ligand-mediated responses in WT-Flt3-, Y589F-Flt3- and Y599F-Flt3-expressing 32D cells. Compared to WT-Flt3-32D cells upon ligand-stimulation, 32DY589F-
Flt3 showed enhanced Erk activation and proliferation/survival whereas 32DY599F-Flt3 cells hereby displayed substantially diminished responses. Both pY589 and pY599 were identified as association sites for signal relay molecules including Src family kinases and SHP2. Consistently, 32D-Y589F-Flt3 and 32D-Y599F-Flt3 showed
decreased FL-triggered activation of Src family kinases. Interference with the Srcdependent negative regulation of Flt3 signaling may account for the enhanced mitogenic
response of Y589F-Flt3. Y599 was additionally found to interact with the protein tyrosine phosphatase SHP2 in a phosphorylation dependent manner. As Y599F-Flt3-32D
was unable to associate with and to phosporylate SHP2 and since silencing of SHP2 in WT-Flt3-expressing cells mimicked the Y599F-Flt3-phenotype we hypothesize that
recruitment of SHP2 to pY599 contributes to FL-mediated Erk activation and proliferation.
of human Flt3 and subsequent radiosequencing we identified the tyrosine residues 572, 589, 591 and 599 as in vivo autophosphorylation sites. Focusing on Y589 and Y599, we
examined Flt3-ligand-mediated responses in WT-Flt3-, Y589F-Flt3- and Y599F-Flt3-expressing 32D cells. Compared to WT-Flt3-32D cells upon ligand-stimulation, 32DY589F-
Flt3 showed enhanced Erk activation and proliferation/survival whereas 32DY599F-Flt3 cells hereby displayed substantially diminished responses. Both pY589 and pY599 were identified as association sites for signal relay molecules including Src family kinases and SHP2. Consistently, 32D-Y589F-Flt3 and 32D-Y599F-Flt3 showed
decreased FL-triggered activation of Src family kinases. Interference with the Srcdependent negative regulation of Flt3 signaling may account for the enhanced mitogenic
response of Y589F-Flt3. Y599 was additionally found to interact with the protein tyrosine phosphatase SHP2 in a phosphorylation dependent manner. As Y599F-Flt3-32D
was unable to associate with and to phosporylate SHP2 and since silencing of SHP2 in WT-Flt3-expressing cells mimicked the Y599F-Flt3-phenotype we hypothesize that
recruitment of SHP2 to pY599 contributes to FL-mediated Erk activation and proliferation.
Department/s
Publishing year
2006
Language
English
Pages
1542-1550
Publication/Series
Blood
Volume
108
Issue
5
Document type
Journal article
Publisher
American Society of Hematology
Topic
- Hematology
Status
Published
ISBN/ISSN/Other
- ISSN: 1528-0020