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Blood, 1 September 2006, Vol. 108, No. 5, pp. 1542-1550.
Prepublished online as a Blood First Edition Paper on May 9, 2006; DOI 10.1182/blood-2005-07-008896.
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Submitted July 14, 2005
Accepted April 26, 2006
Identification of Y589 and Y599 in the juxtamembrane
domain of Flt3 as ligand-induced autophosphorylation sites
involved in binding of Src family kinases and the protein
tyrosine phosphatase SHP2
Elke Heiss, Kristina Masson, Christina Sundberg, Malin Pedersen, Jianmin Sun, Susanne Bengtsson, and Lars Ronnstrand*
Experimental Clinical Chemistry, Department of Laboratory Medicine, Lund University
* Corresponding author; email: lars.ronnstrand{at}med.lu.se.
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,
32D-Y589F-Flt3 showed enhanced Erk activation and
proliferation/survival whereas 32D-Y599F- 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 Src-dependent 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.
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