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Blood, Vol. 91 No. 6 (March 15), 1998:
pp. 1924-1933
By
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.
GRANULOCYTE colony-stimulating factor
(G-CSF) is a 20- to 25-kD glycoprotein secreted by bone marrow stroma
cells, macrophages, fibroblasts, and endothelial cells. G-CSF
stimulates the proliferation, survival, and differentiation of myeloid
progenitor cells towards neutrophilic granulocytes.1,2
G-CSF-deficient mice show chronic neutropenia and a reduced
granulopoietic response to infections, indicating that G-CSF plays an
essential role in the regulation of granulopoiesis in both steady-state
and stress conditions.3 The biological effects of G-CSF are
mediated through a cell-surface receptor that is a member of the
hematopoietin or class I cytokine receptor superfamily and that forms
homodimeric complexes upon ligand binding.4,5
Like other members of the hematopoietin receptor superfamily, the G-CSF
receptor (G-CSF-R) lacks intrinsic tyrosine kinase activity but
activates cytoplasmic tyrosine kinases, in particular of the Jak
family.2,5,6 Jaks associate with the membrane-proximal cytoplasmic region of the hematopoietin receptors and become activated on ligand-induced receptor dimerization.7,8 Jak activation leads to tyrosine phosphorylation of the STAT (signal transducer and
activator of transcription) proteins, which form homodimers and/or heterodimers, translocate to the nucleus, and activate target genes by interaction with specific DNA sequences. G-CSF stimulation results in the activation of Jak1, Jak2, STAT1, STAT3, and
STAT5.8-11
Tyrosine kinase activity induced by G-CSF also results in the rapid
phosphorylation of four conserved cytoplasmic tyrosines (Y) of the
G-CSF-R protein (Y704, Y729, Y744, and Y764) located in the region
distal to the conserved box 2 sequence.9,12 These
phosphotyrosines form potential binding sites for signaling molecules
that contain src homology 2 (SH2) domains.13 For instance, Y704 of G-CSF-R, fitting the YXXQ consensus sequence for SH2-STAT3 binding, is involved in the recruitment and activation of
STAT3.14,15 Activation of Shc and SHP-2 (Syp),
SH2-containing signaling intermediates of the p21Ras pathway, also
requires recruitment via tyrosine residues of G-CSF-R.16
Notably, this depends on binding and activation of Jak kinases via the
membrane-proximal region.17
To accomplish neutrophilic differentiation in murine myeloid cell lines
(32D, L-GM, or FDCP1), signals provided by the C-terminal region of
G-CSF-R, spanning approximately 100 amino acids, are indispensible.18,19 This so-called differentiation domain
of G-CSF-R contains three of the four cytoplasmic tyrosines (Y729, Y744, and Y764). To what extent the cytoplasmic tyrosines of G-CSF-R contribute to G-CSF-mediated proliferation and differentiation induction in myeloid cells has not been established.
In this study, we examined the consequences of
tyrosine-to-phenylalanine (Y-to-F) substitutions in the cytoplasmic
domain of G-CSF-R for the transduction of proliferation and
differentiation signals in differentiation competent 32D cells. We show
that all tyrosines can be replaced without affecting G-CSF-induced
differentiation. Substitution of Y704, Y729, or Y744 had no effect on
proliferation signaling. In contrast, mutant Y764F failed to support
G-CSF-induced cell cycle progression from the G1 to the S phase,
resulting in accelerated differentiation and significantly reduced net
production of mature neutrophils. Strikingly, we found that activation
of Shc, p21Ras and induction of c-myc are all mediated via Y764
of G-CSF-R, indicative of a potential role of these signaling molecules in maintaining the proliferation/differentiation balance in neutrophil development.
G-CSF-R constructs and transfectants.
Human G-CSF-R cDNA was cloned in the eukaryotic expression
vector LNCX.20 Polymerase chain reaction techniques were
used to generate the Y-to-F mutants Y704F, Y729F, Y744F, and Y764F, as
described previously.16 A subline of the interleukin-3
(IL-3)-dependent murine myeloid cell line 32D,21 called
32D.C10,22 was maintained in RPMI 1640 medium supplemented
with 10% fetal calf serum (FCS) and 10 ng/mL of murine IL-3. The LNCX
expression constructs were linearized by Pvu I digestion and
transfected into 32D.C10 cells by electroporation. After 48 hours of
incubation, cells were selected with G418 (GIBCO-BRL, Breda, The
Netherlands) at a concentration of 0.8 mg/mL. Multiple clones were
expanded for further analysis. To determine G-CSF-R expression levels,
cells were incubated at 4°C for 60 minutes with 10 µg/mL of
biotinylated mouse antihuman G-CSF-R monoclonal antibody LMM741
(PharMingen, San Diego, CA). After washing, cells were treated at
4°C for 60 minutes with 5 µg/mL of phycoerythrin (PE)-conjugated
streptavidin. Samples were analyzed by flow cytometry using a FACSCAN
(Becton Dickinson, San Jose, CA).
Cell proliferation and morphological analysis.
To determine proliferation, cells were incubated at an initial density
of 2 × 105 cells/mL in 10% FCS/RPMI medium
supplemented with 100 ng/mL of human G-CSF, 10 ng/mL of murine IL-3, or
without growth factors. The medium was replenished every 2 to 4 days,
and the cell densities were adjusted to 2 to 4 × 105
cells/mL. Viable cells were counted on the basis of trypan blue exclusion. To analyze the morphologic features, cells were spun onto
glass slides and examined after May-Grünwald-Giemsa staining.
Cell cycle analysis.
For flow cytometric analysis of DNA content, cells were collected by
centrifugation and resuspended in 0.1% sodium citrate containing 50 µg/mL of propidium iodide. The fluorescence of the stained cells was
measured using a FACSCAN (Becton Dickinson). The Cell Fit program was
used to determine the percentages of cells in the different phases of
the cell cycle.
Shc immunoprecipitation.
Preparation of cell lysates, immunoprecipitation, and Western blotting
were performed as described.16 Anti-Shc
antibodies23 and antiphosphotyrosine antibodies 4G10
(Upstate Biotechnology Inc, Lake Placid, NY) were used.
p21Ras activation assay.
Cells (1 × 107) were deprived of serum and growth
factors and labeled by incubation for 3 hours in phosphate-free
Dulbecco's modified Eagle's medium containing 100 µCi/mL of
carrier-free [32P]orthophosphate. Subsequently, the cells
were stimulated for 5 minutes at 37°C with human G-CSF (1 µg/mL),
murine IL-3 (1 µg/mL), or without factors (control). Cell lysis,
immunoprecipitation of p21Ras with monoclonal antibody Y13-259, and
thin-layer chromatography were then performed as previously
described.24 GTP binding to p21Ras was expressed as a
percentage of total p21Ras-bound guanine nucleotide (GTP + GDP)
determined with a phosphorimager.
Analysis of c-myc expression.
Cells were deprived of serum and growth factors for 4 hours and
subsequently stimulated with human G-CSF (1 µg/mL) or murine IL-3 (1 µg/mL). At several time points, RNA was extracted from the cells
using the Ultraspec-II RNA isolation system (Biotecx Laboratories Inc,
Houston, TX). Agarose-formaldehyde gel electrophoresis and transfer to
filters (Hybond; Amersham Life Sciences, Amersham, UK) was performed
using standard procedures. As probes, a 1.4-kb EcoRI-HindIII fragment comprising the entire coding
sequence of murine c-myc and a 777-bp
HindIII-EcoRI human GAPDH fragment (control) were 32P-labeled by random priming (Boehringer, Mannheim,
Germany).
Experimental model.
Tyrosine-to-phenylalanine substitution mutants of G-CSF-R are depicted
in Fig 1A.
Expression vectors encoding the various G-CSF-R cDNAs were
introduced into a subline of the IL-3-dependent murine myeloid cell
line 32D, called 32D.C10, that do not express endogenous G-CSF-R. In
32D.C10 cells transfected with the wild-type (WT) G-CSF-R cDNA,
G-CSF induces transient proliferation followed by terminal neutrophilic
differentiation after 8 to 11 days of culture.22 Expression
levels of the different G-CSF-R proteins in the transfected 32D.C10
cells were determined by flow cytometry using G-CSF-R antibodies (Fig
1B). Several independent clones of each mutant with approximately
equivalent G-CSF-R levels were selected for further analysis.
Mutation of Y764 of G-CSF-R inhibits proliferation but accelerates
neutrophilic differentiation.
To determine the abilities of WT and mutant G-CSF-R to induce
proliferation and neutrophilic differentiation, 32D.C10 transfectants were switched from IL-3- to G-CSF-containing medium after extensive washing to remove residual IL-3. The experiments described below were
performed and repeated on at least three independent clones of each
mutant. Without IL-3 or G-CSF, all transfectants died within 1 day and
showed no signs of neutrophilic differentiation. Parental 32D.C10 cells
and cells transfected with empty LNCX vector also died within 1 day in
G-CSF-containing medium. The 32D.C10 cells expressing WT G-CSF-R
(32D/WT) proliferated in response to G-CSF for 6 to 8 days
(Fig 2). After 8 to 11 days, 32D/WT cells developed into terminally differentiated neutrophils, showing an
enlarged cytoplasm-to-nucleus ratio, neutrophilic cytoplasm, lobulated
nuclei, granules, and expression of the murine neutrophil-specific surface antigen GR-1 (Fig 3A and B and data
not shown). Similar results were obtained with 32D/Y704F, 32D/Y729F,
and 32D/Y744F cells. In contrast, 32D/Y764F cells did not proliferate
in G-CSF-containing medium and showed terminal differentiation after 2 to 4 days, instead of 8 to 11 days. On average, this inappropriate
balance of proliferation/differentiation resulted in a 30-fold reduced production of neutrophils as compared with 32D/WT cells (Fig 3C). Furthermore, 3H-thymidine uptake assays after G-CSF
stimulation showed that induction of DNA synthesis by mutant Y764F was
severely reduced on day 1 and absent on day 2 of culture (data not
shown). Stimulation of 32D/WT and 32D/Y764F cells with the combination
of IL-3 and G-CSF resulted in proliferation rates similar to those
obtained with IL-3 alone (Fig 4). In the
presence of IL-3, G-CSF did not induce neutrophilic differentiation,
indicating that IL-3-induced proliferation completely overrules the
differentiation signaling by WT G-CSF-R and by mutant Y764F.
Y764 of G-CSF-R is essential for G-CSF-induced cell cycle
progression from G1 to S phase.
Cell cycle profiles of 32D/WT and 32D/Y764F cells were analyzed by flow
cytometry (Fig 5). In the presence of IL-3,
the majority of 32D/WT and 32D/Y764F cells were in S phase. After
transfer to G-CSF-containing medium, the cell cycle distribution of
32D/WT cells changed only slightly during the first 48 hours, in
agreement with the observation that the cells continued to proliferate
at this stage of culture (Fig 2). In contrast, the number of 32D/Y764F cells in S phase had already significantly decreased 8 hours after the
switch to G-CSF. After 48 hours, 87% of cells were in G1 and only 11%
in S phase, with 35% of the total showing terminal neutrophilic differentiation. These results indicate that mutation of Y764 abrogates
G-CSF-mediated cell cycle progression from the G1 to the S phase.
The WT G-CSF-R also induces accelerated differentiation in
G1-arrested cells.
To investigate whether accelerated neutrophilic differentiation in
G-CSF-stimulated 32D/Y764F cells is the direct consequence of the lack
of proliferation, we cultured 32D/WT cells in the presence of the cell
cycle inhibitor cytosine arabinoside (Ara-C). Concentrations of Ara-C
required to inhibit G-CSF- and IL-3-mediated proliferation with
minimal cytotoxicity were 10
Activation of Shc and p21Ras and expression of c-myc are mediated
via Y764 of G-CSF-R.
We previously showed in lymphoid BAF/B03 cells that Shc/Grb2 and
SHP-2/Grb2 complexes, implicated in activation of p21Ras by a variety
of receptor systems, are both activated by G-CSF-R.16 Multiple tyrosines in G-CSF-R mediate the formation of SHP-2/Grb2 complexes, but Shc activation and Shc/Grb2 association critically depend on Y764 of G-CSF-R.16 We first confirmed that Y764
is also essential for G-CSF-induced Shc activation in myeloid 32D.C10 transfectants (Fig 7). Ras-loading assays
indicated that activation of WT G-CSF-R resulted in an approximately
eightfold increase of p21Ras-GTP as compared with nontreated controls
(Fig 8, middle panel). In contrast,
activation of mutant Y764F induced a marginal increase of p21Ras-GTP
over background levels (Fig 8, right panel). Control cells transfected
with empty LNCX vector showed no activation of p21Ras in response to
G-CSF (Fig 8, left panel).
Previously, we have investigated the contribution of the cytoplasmic
tyrosine residues of G-CSF-R to signaling using BAF/B03 cell
transfectants expressing Y-to-F substitution mutants.15,16 These studies provided information on the specific involvement of these
tyrosines in the activation of signaling substrates of the Jak/STAT and
p21Ras signaling pathways. However, replacement of the tyrosines did
not affect the proliferation signaling abilities of G-CSF-R. This was
not unexpected, because earlier work had shown that truncated forms of
G-CSF-R, which lack all tyrosine residues and which fail to mediate
proliferation in myeloid 32D or L-GM cells, still efficiently
transduced proliferation signals in BAF/B03 cells.18,27
Apparently, proliferation control mechanisms mediated via regions
C-terminal of the box 2 consensus domain are bypassed in BAF/B03 cells.
This, combined with their inability to differentiate towards the
myeloid lineage, makes BAF/B03 cells not suitable for studying the
coupled proliferation/differentiation response to G-CSF.
Submitted June 24, 1997;
accepted November 3, 1997.
The authors thank Dr Kevin P. Foley (Fred Hutchinson Cancer Research
Center, Seattle, WA) for providing murine c-myc cDNA, Karola
van Rooyen for preparation of figures, and Drs Alister Ward and Marieke
von Lindern for manuscript reading.
1.
Nicola NA:
Granulocyte colony-stimulating factor and differentiation-induction in myeloid leukemia cells.
Int J Cell Cloning
5:1,
1987[Abstract]
2.
Demetri GD,
Griffin JD:
Granulocyte colony-stimulating factor and its receptor.
Blood
78:2791,
1991
3.
Lieschke GJ,
Grail D,
Hodgson G,
Metcalf D,
Stanley E,
Cheers C,
Fowler KJ,
Basu S,
Zhan YF,
Dunn AR:
Mice lacking granulocyte colony-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization.
Blood
84:1737,
1994
4.
Bazan JF:
Structural design and molecular evolution of a cytokine receptor superfamily.
Proc Natl Acad Sci USA
87:6934,
1990
5.
Fukunaga R,
Seto Y,
Mizushima S,
Nagata S:
Three different mRNAs encoding human granulocyte colony-stimulating factor receptor.
Proc Natl Acad Sci USA
87:8702,
1990
6.
Isfort RJ,
Ihle JN:
Multiple haematopoietic growth factors signal through tyrosine phosphorylation.
Growth Factors
2:213,
1990[Medline]
[Order article via Infotrieve]
7.
Quelle FW,
Sato N,
Witthuhn BA,
Inhorn RC,
Eder M,
Miyajima A,
Griffin JD,
Ihle JN:
Jak2 associates with the
8.
Dong F,
Van Paassen M,
Van Buitenen C,
Hoefsloot LH,
Löwenberg B,
Touw IP:
A point mutation in the granulocyte colony-stimulating factor receptor (G-CSF-R) gene in a case of acute myeloid leukemia results in the overexpression of a novel G-CSF-R isoform.
Blood
85:902,
1995
9.
Nicholson SE,
Oates AC,
Harpur AG,
Ziemiecki A,
Wilks AF,
Layton JE:
Tyrosine kinase Jak1 is associated with the granulocyte-colony-stimulating factor receptor and both become tyrosine-phosphorylated after receptor activation.
Proc Natl Acad Sci USA
91:2985,
1994
10.
Tian S-S,
Lamb P,
Seidel HM,
Stein RB,
Rosen J:
Rapid activation of the STAT3 transcription factor by granulocyte colony-stimulating factor.
Blood
84:1760,
1994
11.
Tian S-S,
Tapley P,
Sincich C,
Stein RB,
Rosen J,
Lamb P:
Multiple signaling pathways induced by granulocyte colony-stimulating factor involving activation of JAKs, STAT5, and/or STAT3 are required for regulation of three distinct classes of immediate early genes.
Blood
88:4435,
1996
12.
Yoshikawa A,
Murakami H,
Nagata S:
Distinct signal transduction through the tyrosine-containing domains of the granulocyte colony-stimulating factor receptor.
EMBO J
14:5288,
1995[Medline]
[Order article via Infotrieve]
13.
Moran MF,
Koch CA,
Anderson D,
Ellis C,
England L,
Martin GS,
Pawson T:
Src homology region 2 domains direct protein-protein interactions in signal transduction.
Proc Natl Acad Sci USA
87:8622,
1990
14.
Stahl N,
Farruggella TJ,
Boulton TG,
Zhong Z,
Darnell JE,
Yancopoulos GD:
Choice of STATs and other substrates specified by modular tyrosine-based motifs in cytokine receptors.
Science
267:1349,
1995
15.
De Koning JP,
Dong F,
Smith L,
Schelen AM,
Barge RMY,
Van der Plas DC,
Hoefsloot LH,
Löwenberg B,
Touw IP:
The membrane-distal cytoplasmic region of human granulocyte colony-stimulating factor receptor is required for STAT3 but not STAT1 homodimer formation.
Blood
87:1335,
1996
16.
De Koning JP,
Schelen AM,
Dong F,
Van Buitenen C,
Burgering BMT,
Bos JL,
Löwenberg B,
Touw IP:
Specific involvement of tyrosine 764 of human granulocyte colony-stimulating factor receptor in signal transduction mediated by p145/Shc/GRB2 or p90/GRB2 complexes.
Blood
87:132,
1996
17.
Barge RMY,
De Koning JP,
Pouwels K,
Dong F,
Löwenberg B,
Touw IP:
Tryptophan 650 of human granulocyte colony-stimulating factor (G-CSF) receptor, implicated in the activation of JAK2, is also required for G-CSF-mediated activation of signaling complexes of the p21ras route.
Blood
87:2148,
1996
18.
Dong F,
Van Buitenen C,
Pouwels K,
Hoefsloot LH,
Löwenberg B,
Touw IP:
Distinct cytoplasmic regions of the human granulocyte colony-stimulating factor receptor involved in induction of proliferation and maturation.
Mol Cell Biol
13:7774,
1993
19.
Fukunaga R,
Ishizaka-Ikeda E,
Nagata S:
Growth and differentiation signals mediated by different regions in the cytoplasmic domain of granulocyte colony-stimulating factor receptor.
Cell
74:1079,
1993[Medline]
[Order article via Infotrieve]
20.
Miller AD,
Rosman GJ:
Improved retroviral vectors for gene transfer and expression.
Biotechniques
7:980,
1989[Medline]
[Order article via Infotrieve]
21.
Greenberger JS,
Sakakeeny MA,
Humphries RK,
Eaves CJ,
Eckner RJ:
Demonstration of permanent factor-dependent multipotential (erythroid/neutrophil/basophil) hematopoietic progenitor cell lines.
Proc Natl Acad Sci USA
80:2931,
1983
22.
Dong F,
Brynes RK,
Tidow N,
Welte K,
Löwenberg B,
Touw IP:
Mutations in the gene for the granulocyte colony-stimulating factor receptor in patients with acute myeloid leukemia preceded by severe congenital neutropenia.
N Engl J Med
333:487,
1995
23.
Pronk GJ,
De Vries-Smits AMM,
Buday L,
Downward J,
Maassen JA,
Medema RH,
Bos JL:
Involvement of Shc in insulin- and epidermal growth factor-induced activation of p21ras.
Mol Cell Biol
14:1575,
1994
24.
Burgering BMT,
Medema RH,
Maassen JA,
Van de Wetering ML,
Van der Eb AJ,
McCormick F,
Bos JL:
Insulin stimulation of gene expression mediated by p21ras activation.
EMBO J
10:1103,
1991[Medline]
[Order article via Infotrieve]
25.
Gotoh N,
Tojo A,
Shibuya M:
A novel pathway from phosphorylation of tyrosine residues 239/240 of Shc, contributing to suppress apoptosis by IL-3.
EMBO J
15:6197,
1996[Medline]
[Order article via Infotrieve]
26.
Gotoh N,
Tojo A,
Shibuya M:
Tyrosine phosphorylation sites at amino acids 239 and 240 of Shc are involved in epidermal growth factor-induced mitogenic signaling that is distinct from Ras/mitogen-activated protein kinase activation.
Mol Cell Biol
17:1824,
1997[Abstract]
27.
Ziegler SF,
Bird TA,
Morella KK,
Mosley B,
Gearing DP,
Baumann H:
Distinct regions of the human granulocyte-colony-stimulating factor receptor cytoplasmic domain are required for proliferation and gene induction.
Mol Cell Biol
13:2384,
1993
28.
Okuda K,
Ernst TJ,
Griffin JD:
Inhibition of p21ras activation blocks proliferation but not differentiation of interleukin-3-dependent myeloid cells.
J Biol Chem
269:24602,
1994
29.
Cleveland JL,
Troppmair J,
Packham G,
Askew DS,
Lloyd P,
González-Garcia M,
Nuñez G,
Ihle JN,
Rapp UR:
v-raf suppresses apoptosis and promotes growth of interleukin-3-dependent myeloid cells.
Oncogene
9:2217,
1994[Medline]
[Order article via Infotrieve]
30.
Muszynski KW,
Ruscetti FW,
Heidecker G,
Rapp U,
Troppmair J,
Gooya JM,
Keller JR:
Raf-1 protein is required for growth factor-induced proliferation of hematopoietic cells.
J Exp Med
181:2189,
1995
31.
Askew DS,
Ashmun RA,
Simmons BC,
Cleveland JL:
Constitutive c-myc expression in an IL-3-dependent myeloid cell line suppresses cell cycle arrest and accelerates apoptosis.
Oncogene
6:1915,
1991[Medline]
[Order article via Infotrieve]
32.
Roussel MF,
Cleveland JL,
Shurtleff SA,
Sherr CJ:
Myc rescue of a mutant CSF-1 receptor impaired in mitogenic signalling.
Nature
353:361,
1991[Medline]
[Order article via Infotrieve]
33.
Evan GI,
Wyllie AH,
Gilbert CS,
Littlewood TD,
Land H,
Brooks M,
Waters CM,
Penn LZ,
Hancock DC:
Induction of apoptosis in fibroblasts by c-myc protein.
Cell
69:119,
1992[Medline]
[Order article via Infotrieve]
34.
Holt JT,
Redner RL,
Nienhuis AW:
An oligomer complementary to c-myc mRNA inhibits proliferation of HL-60 promyelocytic cells and induces differentiation.
Mol Cell Biol
8:963,
1988
35.
Leone G,
DeGregori J,
Sears R,
Jakoi L,
Nevins JR:
Myc and Ras collaborate in inducing accumulation of active cyclin E/Cdk2 and E2F.
Nature
38 |
Abstract
Introduction
Abstract
) WT; (
) Y704F;
(
) Y729F; (
) Y744F; (
) Y764F; (
) LNCX.
6 M and
10
) IL-3 + Ara-C. (B) Cell cycle distribution of 32D/WT cells cultured for 3 days in G-CSF- or IL-3-containing medium in the absence or
presence of Ara-C. The percentages of cells in the G1, S, and G2/M
phases of the cell cycle are given in the upper right of the panels.
(C) Morphology of 32D/WT cells cultured for 3 days in G-CSF- or
IL-3-containing medium in the presence of Ara-C
(May-Grünwald-Giemsa staining; original magnification × 630).
(D) The percentages of terminally differentiated 32D/WT cells cultured
in G-CSF- or IL-3-containing medium in the absence or presence of
Ara-C. At least 200 cells were scored at the indicated times. Symbols
are the same as in (A).
), with G-CSF, or with IL-3.
Cells were lysed and p21Ras was collected by immunoprecipitation.
Nucleotides bound to p21Ras were eluted and separated by thin-layer
chromatography. The positions of the GTP and GDP standards are
indicated. The level of p21Ras-GTP, expressed as a percentage of total
nucleotide bound to p21Ras (GTP + GDP), is given under each lane.
-chain and that site-directed mutagenesis of this tyrosine
results in reduced cellular viability of BAF/B03 transfectants in
response to GM-CSF. On the other hand, overexpression of Shc proteins
in GM-CSF-dependent TF-1 cells increases the proliferative response to
GM-CSF, in agreement with a role for Shc in mitogenic
signaling.