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Proliferation Signaling and Activation of Shc, p21Ras, and Myc Via
Tyrosine 764 of Human Granulocyte Colony-Stimulating Factor Receptor
John P. de Koning,
Amrita A. Soede-Bobok,
Anita M. Schelen,
Louise Smith,
Daphne van Leeuwen,
Valeria Santini,
Boudewijn M.T. Burgering,
Johannes L. Bos,
Bob Löwenberg, and
Ivo P. Touw
From the Institute of Hematology, Erasmus University and Dr. Daniel
den Hoed Cancer Center, Rotterdam, The Netherlands; the Department of
Hematology, University of Florence, Firenze, Italy; and the Laboratory
of Physiological Chemistry, Utrecht University, Utrecht, The
Netherlands.
The membrane-distal region of the cytoplasmic domain of human
granulocyte colony-stimulating factor receptor (G-CSF-R) contains four
conserved tyrosine residues: Y704, Y729, Y744, and Y764. Three of these
(Y729, Y744, and Y764) are located in the C-terminal part of G-CSF-R,
previously shown to be essential for induction of neutrophilic
differentiation. To determine the role of the tyrosines in
G-CSF-mediated responses, we constructed tyrosine-to-phenylalanine (Y-to-F) substitution mutants and expressed these in a differentiation competent subclone of 32D cells that lacks endogenous G-CSF-R. We show
that all tyrosines can be substituted essentially without affecting the
differentiation signaling properties of G-CSF-R. However, substitution
of one specific tyrosine, ie, Y764, markedly influenced proliferation
signaling as well as the timing of differentiation. 32D cells
expressing wild-type (WT) G-CSF-R (or mutants Y704F, Y729F, or Y744F)
proliferated in G-CSF-containing cultures until day 8 and then
developed into mature neutrophils. In contrast, 32D/Y764F cells
arrested in the G1 phase of the cell cycle within 24 hours and showed
complete neutrophilic differentiation after 3 days of culture. This
resulted in an average 30-fold reduction of neutrophil production as
compared with the 32D/WT controls. Importantly, G-CSF-mediated
activation of Shc, p21Ras and the induction of c-myc were
severely reduced by substitution of Y764. These findings indicate that
Y764 of G-CSF-R is crucial for maintaining the
proliferation/differentiation balance during G-CSF-driven neutrophil
development and suggest a role for multiple signaling mechanisms in
maintaining this balance.
Blood, Vol. 91 No. 6 (March 15), 1998:
pp. 1924-1933
© 1998 by The American Society of Hematology.
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