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Internalization of the Granulocyte-Macrophage Colony-Stimulating Factor
Receptor Is Not Required for Induction of Protein Tyrosine Phosphorylation in
Human Myeloid Cells
By Keiko Okuda, Brian Druker, Yuzuru Kanakura, Michael Koenigsmann, and James D. Griffin
Granulocyte-macrophage colony-stimulating factor (GMCSF) exerts its biologic activities through binding to specific
high-affinity cell surface receptors. After binding, the ligand/
receptor complex is rapidly internalized in most hematopoietic cells. Using a human factor-dependent cell line, M07, and
normal human neutrophils, we found that the internalization
is exquisitely temperature-dependent, such that ligand/
receptor internalization does not detectably occur at 4°C.
Activation of the GM-CSF receptor has previously been
shown t o stimulate a number of postreceptor signal transduction pathways, including activation of a tyrosine kinase and
activation of the serine/threonine kinase, Raf-I. The GM-CSFstimulated increase in tyrosine kinase activity occurs rapidly
at both 4°C and 37°C. and therefore is likely t o be independent of receptor internalization. At 37"C, the protein tyrosine
phosphorylation was transient in M07 cells, with maximum
phosphorylation observed after 5 t o 15 minutes, followed by
a rapid decline. At 4"C, the protein tyrosine phosphorylation
of the same substrates was greater than at 37"C, and no
decline in substrate phosphorylation was observed for at
least 90 minutes. In contrast t o tyrosine phosphorylation, the
activation and hyperphosphorylation of Raf-I observed at
37°C in both M07 cells and neutrophils was markedly diminished at 4°C. These results indicate that at least one postreceptor signal transduction mechanism, activation of a tyrosine
kinase, does not require ligand/receptor internalization, and
indicate that receptor internalization may be a consequence,
rather than the initiator, of signal transduction.
o 1991by The American Society of Hematology.
G
myeloid cell^.'^^'^ A cDNA encoding a low-affinity receptor
for GM-CSF has been cloned" and a potential second
subunit of the receptor has also been identifiede2' The
predicted amino acid sequences indicate that neither subunits of the receptor are likely to be kinases, suggesting that
the receptor activates tyrosine and serine/threonine kinases indirectly.
In addition to activating cellular kinases and other
biochemical events, the binding of GM-CSF to the GMCSF receptor causes the receptor-ligand complex to be
rapidly internali~ed.~'.~~
Receptor internalization is also
observed in response to certain activating agents, such as
phorbol 12-myristate 13-acetate (PMA) or the calcium
ionophore A23187.22.23
GM-CSF receptor internalization is
more rapid and complete in neutrophils than in myeloid
leukemic cell lines, and this may reflect a difference in
receptor function that is related in some way to stage of
differentiati~n.~~
The susceptibility of the GM-CSF receptor to internalization appears to be inversely correlated
with whether IL-3 can cross-compete with GM-CSF for
binding to the GM-CSF re~eptor.~'It is not known if
receptor internalization is required for, or associated with,
signal transduction.
Receptor internalization appears to be a general property of the hematopoietin receptors, either in response to
binding of specific ligandz4"' or in response to activation of a
receptor for another CSF (receptor tran~modulation).~~"~
It
has previously been shown that there is a hierarchical
transmodulation of hematopoietic growth factor receptors
in murine granulocyte-macrophage cells.31Such cells simultaneously express receptors for IL-3, GM-CSF, granulocyteCSF (G-CSF), and macrophage-CSF (M-CSF). Binding of
IL-3 to its receptor induces downregulation of the receptors
for the other three factors, while GM-CSF induces downmodulation of the receptors only for G-CSF and M-CSF. It
has been suggested that receptor transmodulation is associated with receptor activation and that this may be important in mediating some of the pleiotropic effects of factors
such as GM-CSF and IL-3.31
In contrast, it is also possible that receptor internaliza-
RANULOCYTE-macrophage colony-stimulating factor (GM-CSF) is a member of a family of hematopoietic growth factors that stimulate the proliferation, differentiation, and activation of blood cells and their precursor^.'.^
These biologic effects are mediated through binding of the
factors to specific, high-affinity cell surface receptors. However, postreceptor signaling events remain largely unknown. In both human and murine myeloid cell lines,
GM-CSF has been shown to stimulate protein tyrosine
kinase activity resulting in rapid phosphorylation of several
membrane and cytoplasmic protein^.^.'' For example, we
have previously shown that both GM-CSF and interleukin-3 (IL-3) activate one or more tyrosine kinase in the
human factor-dependent megakaryoblastic cell line, M07,
resulting in phosphorylation of substrates of apparent Mr =
150,000,125,000,93,000,70,000,55,000,42,000,
and 36,000.8
Evidence that tyrosine phosphorylation is important for
proliferation comes from observations that introduction of
a known tyrosine kinase receptor, such as the epidermal
growth factor (EGF) receptor, can convert an IL-3dependent murine cell line to EGF-dependence,"B1' and
also from the finding that factor-dependent cell lines can be
converted to factor independence by oncogenes containing
tyrosine kinases."-'6 In addition to activating tyrosine kinases, both GM-CSF and IL-3 rapidly induce activation of a
serine/threonine kinase, Raf-1, in M 0 7 cells and in normal
From the Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, MA.
Submitted February 28,1991; uccepted June 6, 1991.
Supported in part by Public Health Service Grants No. CA36167
and CA34183.
Address reprint requests to James D. Grifln, MD, Division of Tumor
Immunology, Dana-Farber Cancer Institute, 44 Binney St, Boston,
M A 02115.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 I991 by The American Society of Hematology.
0006-4971I91 I 7808-0027$3.OOIO
1928
Blood, Vol78, No 8 (October 15). 1991: pp 1928-1935
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GM-CSF RECEPTOR INTERNALIZATION
tion is a regulatory mechanism to limit the response of a cell
to a specific CSF, and as such serves primarily to remove
functional receptors from the cell surface. If so, it is likely
that receptor internalization is a consequence of, rather
than the initial step in, signal transduction. To better
understand the significance of receptor internalization, we
have investigated receptor signaling under conditions in
which receptor internalization is blocked. The results indicate that receptor internalization can be separated from
some aspects of receptor activation.
MATERIALS AND METHODS
Reagents. Highly purified recombinant human (rh) GM-CSF
and IL-3 were gifts from Drs Steven Clark and Gordon Wong
(Genetics Institute, Cambridge, MA). Bovine serum albumin
(BSA, Fraction V), PMA, sodium azide, and sodium orthovanadate were purchased from Sigma (St Louis, MO). Antiphosphotyrosine monoclonal antibody (MoAb) was generated using
phosphotyramine as the
This antibody is specific for
tyrosine-phosphorylated proteins and does not cross-react with
phosphoserine, phosphothreonine, phosphohistidine, or tyrosine
sulfate. When used for immunoblotting, the addition of 1 mmol/L
phosphotyrosine completely eliminated all immunoreactive bands,
while phosphothreonine or phosphoserine had no effect! Anti-GMCSF antibody (3092) is a murine MoAb that neutralizes human
natural and recombinant GM-CSF?' In control experiments, 3092
was shown to block the increase in protein tyrosine phosphorylation induced by GM-CSF. Rabbit anti-Raf-1 antiserum was generated against a synthetic peptide (CTLTTSPRLPVF) corresponding to amino acid residues 315-326 oFy-raJ and was affinity-purified
as described." The antibody was used for immunoblotting at a
dilution of 1:4,000.
Cells. The human GM-CSF- and IL-3-dependent cell line,
M07, was obtained from Dr Steven Clark (Genetics Institute) and
was originally derived by Avanzi et a1 from the peripheral blood
(PB) cells of an infant with acute megakaryocytic leukemia?4 The
cell line was cultured in RPMI1640 medium (GIBCO, Grand
Island, NY) supplemented with 10% fetal bovine serum (FBS),
rhGM-CSF 10 ng/mL, or rhIL-3 10 ng/mL. Normal PB was
obtained from healthy adult volunteers and mononuclear cells
were removed by Ficoll-Hypaque density gradient centrifugation.
Granulocytes were prepared from Ficoll-Hypaque pellets by dextran sedimentation as previously described,n and were greater than
99% granulocytes by morphologic analysis and immunofluorescent
staining with an MoAb reactive with a granulocyte-specific epitope
of Fc receptor I11 (FcRIII) (CD16; J. Griffin, 1988, unpublished).
Stimulation with factors and cell lysis. Exponentially growing
M 0 7 cells were washed free of serum and growth factors and
incubated in serum-free RPMI1640 containing 0.5% BSA for 6 to
18 hours at 37°C to factor-deprive the cells. The cells were then
equilibrated at 37°C or on wet ice in a 4°C cold room, and then
further exposed to GM-CSF or IL-3 (10 ng/mL unless otherwise
indicated) for 0 to 120 minutes. After stimulation, cells were lysed
on ice without centrifugation or washing by adding 1 volume of
boiling 2X sample buffer (80 mmol/L Tris-HC1, pH 6.7,8% sodium
dodecyl sulphate, 20% glycerol, 10 mmol/L 2-mercaptothanol, 6
mmol/L EGTA, 3 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride [PMSF], 0.15 U/mL aprotonin, 10 pg/mL leupeptin,
and 1 mmol/L sodium orthovanadate [reagents from Sigma]).
Insoluble material was removed by centrifugation at 4°C for 15
minutes at 10,OOOg. Neutrophils were pretreated with 1 mmol/L
diisopropylfluorophosphate (Sigma) for 30 minutes at 4°C to
inhibit proteases before stimulation with GM-CSF (10 ng/mL) at
either 37°C or 4°C. After stimulation, the neutrophils were washed
1929
with cold phosphate-buffered saline (PBS) and lysed in lysis buffer
(20 mmol/L Tris-HC1, pH 8.0, 137 mmol/L NaCI, 10% glycerol,
1% Nonidet P-40) containing 1 mmol/L PMSF (Sigma), 0.15
U/mL aprotonin (Sigma), 10 mmol/L EDTA, 10 pg/mL leupeptin
(Sigma), 100 mmol/L sodium fluoride, and 2 mmol/L sodium
orthovanadate at 4°C for 20 minutes. Insoluble material was
removed by centrifugation at 4°C for 15 minutes at 10,OOOg.
Centrifugations were performed in a refrigerated microfuge (Eppendorf 5402, Hamburg, Germany) to eliminate any possible
sample warming during centrifugation.
Gel electrophoresis and immunoblotting. Lysates ( - 150 kg for
13 x 12 cm gels) were electropheresed on one-dimensional sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
with 7.5% to 12% polyacrylamide. After electrophoresis, proteins
were electrophoretically transferred from the gel onto a 0.2-mm
nitrocellulose filter (Schleicher & Schuell, Keene, Nq) in a buffer
containing 25 mmol/L Tris, 192 mmol/L glycine, and 20% methanol at 0.4 A for 4 hours at 4°C. Residual binding sites on the filter
were blocked by incubating the nitrocellulose in TBS (10 mmol/L
Tris, pH 8.0,150 mmol/L NaCl) containing 1% gelatin for 1hour at
25°C. The blots were then washed in TBST (TBS with 0.05%
Tween 20) and incubated overnight with either anti-P-Tyr MoAb
(1.5 kg/mL in TBST) or anti-Raf-1 polyclonal antibody (1:4,000 in
TBST). The primary antibody was removed and the blots were
washed four times in TBST. To detect antibody reactions, the blots
were incubated for 2 hours with alkaline phosphatase-conjugated
antimouse or antirabbit IgG (Promega Biotec, Madison, WI)
diluted 1:2,000 in TBST, washed three times in TBST, and then
placed in a buffer containing 100 mmol/L Tris-HCI, pH 9.5, 100
mmol/L NaCl, 5 mmol/L MgCl,, 330 pg of Nitro blue tetrazolium
(NBT) per milliliter, and 150 mg of 5-bromo-4 chloro-3-indolyl
phosphate (BICP) per milliliter for 10 to 30 minutes. Enzymatic
color development was stopped by rinsing the filters in deionized
water. Prestained protein molecular weight standards were run on
each gel and were obtained from BRL (Gaithersburg, MD).
Binding of 'zs~-labeledGM-CSF to myeloid cells. M 0 7 cells or
neutrophils were washed and briefly treated with mild acid
conditions to remove prebound GM-CSF (1.5 x lo* cells in 5 mL
of cold PBS titrated to pH 3.0 with HCI for 2 minutes at 4"C),
followed by immediate washing in 50 mL of cold PBS.8" We have
previously shown that this treatment removes greater than 80% of
receptor-bound GM-CSF without damaging the GM-CSF recept ~ r . ~Aliquots
,'~
of 5 x lo6cells were then cultured at either 4°C or
37°C in 300 pL of binding buffer (BB) consisting of RPMI 1640
(GIBCO) with BSA (2 mg/mL) and 25 mmol/L HEPES, pH 7.5,
with 0.028 to 6.9 ng/mL (455 pmol/L) '*'I-GM-CSF (New England
Nuclear Dupont, Boston, MA, specific activity, 70 to 100 mCi/mg)
in the presence or absence of an approximately 100-fold excess of
cold ligand to assess specifically bound counts, with a final
incubation volume of 500 pL in 1.5-mL polypropylene tubes
(Sarstedt Inc, Princeton, NJ). Binding was performed for 0 to 90
minutes. Under these conditions, nonspecific binding was typically
in the range of 5% to 20%. After incubation, the cells were
resuspended, layered over a cushion of 0.75 mL of 75% FBS,and
spun for 90 seconds in a microfuge. After aspirating the supernatant, the cell pellets were cut off and counted with an efficiency of
approximately 70% (LKB Gammamaster 1277; Turku, Finland).
Specific binding (SB) of GM-CSF was determined from the mean
of duplicate samples and was defined as follows: SB = (binding
observed in the absence of cold competitor) - (nonspecific binding
observed in the presence of a 100-fold excess of GM-CSF [NSB]).
To measure the internalization of ligand, aliquots of cells were
treated with 1mL of cold acid solution (0.5 mol/L NaCU0.2 mol/L
acetic acid, pH 2.5) for 5 minutes, followed by washing twice in cold
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1930
OKUDA ET AL
PBS. As previously reported, this acid treatment removes greater
than 90% of cell surface bound GM-CSF."
Raf-1 immune complex kinase assay. Raf-1 was immunoprecipitated from control (factor-starved) or CSF-treated M07 cells."
The immune complexes were collected on protein A Sepharose
beads, washed, and incubated in a kinase buffer (25 mmol/L
HEPES, pH 7.4, 1 mmol/L dithiothreitol [DTT], 10 mmol/L
MgCI,, 15 pmol/L ATP) containing 10 pCi y3'P-ATP and histone
H1 (5 pg/assay; Boehringer Mannheim, Indianapolis, IN) as an
exogenous kinase ~ubstrate".'~
for 10 minutes at 20°C. The immune
complexes were then separated by SDS-PAGE and the dried gel
exposed to x-ray film to visualize incorporation of 32Pinto histone
H1.
""V"
.
5000 -
-0-
37degrees
4degrees
37 acid wash
4acidwash
3000
I
t
RESULTS
'251-GM-CSF
intemalizatwnis temperature-dependent. We
have previously shown that internalization of '"I-GM-CSF
after binding to neutrophils is temperature-dependent,
with no detectable internalization at 4OC." These studies
were repeated with the factor-dependent cell line, M 0 7
(Fig 1A). At 37"C, 30% to 40% of total GM-CSF was
acid-resistant (internalized) at each concentration of 9 GM-CSF. Although the binding of Iz5I-GM-CSFwas less at
4°C than at 37°C over the 90-minute time period, as
expected, no internalization was detected in multiple experiments. In similar experiments, M 0 7 cells were cultured for
0 to 90 minutes at both 4°C and 37°C with a single
concentration of lXI-GM-CSF, 1.0 ng/mL, and the amount
of internalized ligand was measured as above. Again, no
internalization of lXI-GM-CSFwas detected at 4"C, while
at 37°C up to one-third of the total cell-associated counts
were acid-resistant. These results indicate that internalization of '"1-GM-CSF does not occur in M 0 7 cells at 4"C,
consistent with our previous results in neutrophils." M 0 7
cells differ from neutrophils in that the rate and completeness of internalization is 10wer.2~
GM-CSF induces activation of protein tyrosine kinase
activity at 4°C. We have previously shown that GM-CSF
and IL-3 rapidly induce activation of a receptor-associated
tyrosine kinase in M 0 7 cells, resulting in tyrosine phosphorylation of several cytoplasmic substrates that can be readily
identified by immunoblotting with an antiphosphotyrosine
MoAb.8 To determine if GM-CSF receptor-associated activation of tyrosine kinase activity was temperature-dependent, and thereby potentially dependent on receptor internalization, antiphosphotyrosine immunoblots were
performed on M 0 7 cells treated with GM-CSF at either
4°C (wet ice) or 37°C for 5 to 90 minutes (Fig 2). Increased
tyrosine phosphorylation of multiple substrates (pp150,
pp93, pp70, and pp42 can be detected in Fig 2) was
observed at either temperature in multiple experiments,
although with slightly different kinetics. Peak tyrosine
phosphorylation of these substrates was observed after 5 to
15 minutes at 37"C, but at greater than 30 minutes at 4°C.
At both temperatures, however, onset of tyrosine phosphorylation was evident in less than 5 minutes. At 3 7 T , the
extent of tyrosine phosphorylation of substrates rapidly
declined after 15 to 30 minutes, and we have previously
shown that this decline can be eliminated by inhibiting
tyrosine phosphatases with orthovanadate. In contrast, at
4"C, no evidence of a decline in tyrosine phosphorylation
0
A
4
2
6
[GM-CSF] (ng/ml)
Total, 37C
600
400
1 /
200
0
B
0
20
40
60
80
1 0
Time (min)
Fig 1. "%GM-CSF is not internalized at 4°C. (A) M07 cells were
exposed to '251-GM-CSF(0.028 to 6.9 ng/mL) for 90 minutes at either
4°C or 37°C. and then the total cell-associated counts and the
intracellular counts ("acid wash") were determined as described in
Materials and Methods. (B) M07 cells were exposed to a single
concentration of '"I-GM-CSF (1.0 ng/mL) for up to 90 minutes, and
then total and intracellular counts determined as above.
was observed for up to 90 minutes, indicating either that
tyrosine phosphatases were not induced or that tyrosine
kinase activity remained higher than tyrosine phosphatase
activity. Several techniques were used to ensure that no
warming of samples occurred during preparation. In particular, in most experiments, cells were lysed directly in boiling
SDS sample buffer, avoiding any manipulation, including
centrifugation, that could induce warming. In other experiments, cells were first washed with cold PBS, centrifuged in
a refrigerated microfuge, and lysed in standard lysis buffer.
The results were identical with either method, indicating
that GM-CSF can induce tyrosine kinase activity in M 0 7
cells equally well at either 37°C or 4°C. Similar results were
obtained when the M 0 7 cells were stimulated with IL-3
(data not shown). In other experiments, M 0 7 cells were
pretreated with sodium azide (0.1%) for 30 minutes to
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GM-CSF RECEPTOR INTERNALlZATlON
1931
Flg 2. GM-CSF stimulates ln-
masod protein tyrosine phosphorylation at elther 4°C or 3PC
in M07 cells. Cells were factordeprived overnight and then
stimulated with GM-CSF (10 ng/
mL) on wet ice (4°C) or at 37°C.
and lysed at the indicated times
directly into boiling 2X sample
buffer. Changes in protein tyrosine phosphorylation were detected by immunoblotting. The
time after GM-CSF stimuletion
at which the cells were lysed at
either temperature is shown. Molecular weight markers on the
left are in kilodaltons.
block A” production. This treatment substantially reduced GM-CSF-stimulated tyrosine phosphorylation at
both 3PC and 4°C.
Similar studies were performed with neutrophils, but
aniiphosphotyrosine immunoblotswere of inconsistent quality when the cells were lysed directly in boiling sample
buffer. Pretreatment of neutrophils with the protease
inhibitor diisopropylfluorophosphate(DFP) before stimulation with GM-CSF, followed by lysis in either sample buffer
or standard lysis buffer (see Materials and Methods), gave
the best results (Fig 3) and showed that increased tyrosine
phosphorylation of the same substrates was evident after
GM-CSF stimulation at either 37°C or 4°C. The kinetics of
dephosphorylation were not detectably different in neutrophils at 37°C or 4”C, but it is possible that pretreatment with
DFP may have affected the phosphatases involved.
GM-CSF does not induce hyperphosphotylation or activation of Raf-I at 4°C. We havc previously shown that both
GM-CSF and IL-3 rapidly induce phosphorylation and
activation of the cytoplasmic serine/threonine kinase Raf-1
Fig 3. GM-CSF stimulates increased protein tyrosine phosphorylation at either 4% or 3PC in normal neutrophils. Neutrophils were prepared from
fresh heparinized bloodand pretmatedwith 1mmol/L
DFP for 1 hour at 4°C to inhibit proteases. The cells
were then stimulated with GM-CSF (10 ng/mL) for 0
t o 90 minutas at either 4°C or 37°C before lysis as
described in Materials and Methods. Changes in
protein tyrosine phosphorylation were detected by
immunoblotting. The time after GM-CSF stimulation
at which the cells were lysed at either temperature is
shown. Molecular weight markers on the left are in
kilodaltons. In some experiments, neutrophils were
lysed directly in boiling sample buffer with similar
results. In the absence of GM-CSF stimulation, no
changes in protein tyrosine phosphorylation were
detected (not shown).
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OKUDA ET AL
1932
in M07 cells at 37°C. The relationship of tyrosine kinase
activation to Raf-1 phosphorylation is unclear. In M07
cells, most of the Raf-1 phosphorylation is phosphoserine,
rather than phosphotyrosine. To determine if signal transduction at 4°C involved Raf-1 in addition to tyrosine kinase
activity, the extent of phosphorylation and enzyme activity
of Raf-1 were compared after GM-CSF treatment at eithcr
37°C or 4°C in both M07 cells and neutrophils (Fig 4A and
B). Increased phosphorylation of Raf-1, as shown by a shift
in mobility in SDS-PAGE, was readily detected at 37°C. but
not at 4°C. Similarly,when Raf-1-associated kinase activity
was measured using histone H1 as a substrate in an in vitro
kinase assay, a twofold to fivefold increase in Raf-lassociated kinase activity was detected after treating M07
cells with GM-CSF for 5 to 15 minutes at 37°C in multiple
experiments, as we have previously shown." In control
experiments, if anti-Raf-1 antibody was omitted from the
immunoprecipitation, the washed protein A beads precipitated only a minimal level of background kinase activity,
which was not increased by GM-CSF treatment of the cells.
In contrast, no increase was detected in four experiments
when the cells were stimulated at 4°C (data not shown).
Similar results were observed in DFP-pretreated neutrophils (data not shown). Thus, in contrast to the temperatureindependent activation of tyrosine kinase activity induced
by GM-CSF, activation and hyperphosphorylationof Raf-1
was temperature-dependent.
A
37°C
Tim (mln)= 0
107
5
15
DISCUSSION
The GM-CSF receptor is widely expressed on both
hematopoietic and nonhematopoietic cells. Recent studies
suggest that the receptor is structurally complex, being
composed of at least two chains,Iqm and may also be
associated with other hematopoictic growth factor receptors in some cell^.^-'' The binding of GM-CSF to its
receptor is known to be rapidly followed by a number of
biochemical events, but thc dctails of signal transduction
rcmain largely unknown. In the neutrophil, GM-CSF induces Na2+influx, release of arachidonic acid, and gcneration lipoxygcnase products such as leukotricne l34:*=
These cvcnts are inhibited by pertussis toxin, suggcsting
that some initial aspects of signal transduction are coupled
to the receptor through a G protein." Also, in both
neutrophils and immature myeloid cells, GM-CSF has been
shown to rapidly inducc tyrosine phosphorylation of several
proteins."'" Bccausc neither of thc known chains of the
rcceptor havc a predictcd amino acid structure consistent
with intrinsic tyrosine kinase activity, it is likely that the
rcceptor activates a tyrosine kinase that is either associated
with the receptor or linked to the reccptor through another
mechanism, such as a G protein. The tyrosine kinase
activation is likely to be a very proximal event in signal
transduction bccause introduction of other known tyrosine
kinase receptors, such as the EGF receptor".l' or c-fms,.''
converts cells from IL-3- or GM-CSF-dependency to EGFor M-CSF-dependency, respectively.
4°C
90 60 120
0
5
15
3060
120
4
+
Rat-phon
>Raf
6S
8
4°C
37°C
Tim (mln)= 0
107
6S
5
15
30 60
90
0
5
15
30 60
90
4
+ Raf-phon
+Rnf
Flg 4. GM-CSF stimulates a
shHt In the electrophoreticmobllity of Raf-1at 3pC but not at 4%
In both M07 cells and normal
neutrophils. Factor-deprived
M07 cells (A) or freshly prepared
neutrophils ( 6 ) were cultured
with GM-CSF (10 ng/mL) for the
Indicated times, lysed as described in the legends to Figs 2
and 3, and Raf-1 detected by
lmmunoblotthgwitha monospecific antipeptide antibody recognizing human Raf-1. The slower
mobility of Raf-1 in SDS-PAGE
after GM-CSF treatment was
shown to be due to phosphorylation by treating immune complexes with alkaline phosphatase as previously descrlbed."
The representative experiment
shown was performed with the
same cells used to generate the
data shown in Figs 2 and 3. In the
absence of GM-CSF, no shHt in
Raf-1 mobility was detected (not
shown).
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GM-CSF RECEPTOR INTERNALIZATION
In contrast to the induction of tyrosine kinase activity, the
role of receptor internalization in GM-CSF signal transduction is unclear. In neutrophils, GM-CSF induces rapid and
complete receptor internalization, followed by degradation
of the ligand.= The intracellular fate of the receptor has not
been determined because antireceptor antibodies are not
yet available. PMA also induces receptor downmodulation
by itself and, in the presence of GM-CSF, PMA accelerates
ligand/seceptor internalization.= In early myeloid cells,
such as the M 0 7 cell line studied here, the rate of receptor
internalization in response to either ligand or PMA is
slower than in neutrophils. Interestingly, the rate of receptor internalization is inversely correlated with GM-CSFI
IL-3 cross-~ompetition.2~
It is possible that the physical
structure of the receptor of early myeloid cells is different
than that of neutrophils. If GM-CSFIIL-3 cross-competition is due to the presence of heterocomplexes of both
receptors, such complexes may not internalize readily.
We report here that at least one aspect of GM-CSF
receptor signal transduction can occur in the apparent
absence of receptor internalization in both M 0 7 cells and
neutrophils. Internalization of the GM-CSF receptor has
been followed indirectly by measuring internalization of
radiolabeled ligand complexed to the receptor. In neutrophils, we have previously shown that ligandlreceptor internalization is associated with nearly complete loss of the
receptor from the cell surface that persists for at least 4
hours.U In both M 0 7 cells and neutrophils, internalization
of the ligand/receptor complex is exquisitely temperaturedependent and does not occur at 4°C. In contrast, GM-CSF
activates tyrosine kinase activity as well, or better, at 4°C
than at 37"C, resulting in the phosphorylation of the same
substrates. Interestingly, dephosphorylation of these substrates in M 0 7 cells was inhibited at 4°C. The differences in
rate of dephosphorylation between 37°C and 4°C were less
obvious for neutrophils than for M 0 7 cells. This finding
may be simply due to differences in preparation of the cells.
Neutrophils were pretreated with DFP to block protease
activity before stimulation, and it is possible that DFP
might reduce the rate of dephosphorylation of substrates.
Due to a lower level of protease activity in M 0 7 cells, DFP
pretreatment is not necessary. We have previously shown
that GM-CSF-induced tyrosine phosphorylation in M 0 7
cells is controlled by the opposing actions of a receptoractivated tyrosine kinase and a tyrosine
Inhibition of tyrosine phosphatase activity with sodium
orthovanadate enhances and prolongs GM-CSF-induced
tyrasine phosphorylation of most of the substrates, and also
enhances factor-dependent cell proliferation.' The kinetics
of tyrosine phosphorylation observed at 4°C were remarkably similar to those observed at 37°C in the presence of
sodium orthovanadate. It is possible that the tyrosine
phosphatase that serves to regulate GM-CSF receptor
signal transduction is relatively inactive at 4°C. Alternatively, the tyrosine kinase activity may exceed tyrosine
phosphatase activity for a more prolonged period of time at
4°C than at 37°C. Overall, these results show that receptor
internalization can be at least partially separated from
1933
receptor activation. Further, the results indicate that balance of tyrosine kinase activity and tyrosine phosphatase
activity is kinetically different at 4°C than at 37"C, and this
may facilitate investigations into the identity of the various
substrates that are phosphorylated on tyrosine.
Methods to chemically block receptor internalization
would be useful to further investigate receptor function.
We found that sodium azide, which is commonly used to
inhibit internalization of many types of receptors for
binding studies, inhibits GM-CSF-stimulated protein tyrosine phosphorylation at both 37°C and 4°C. However,
because sodium azide acts through depletion of intracellular ATP, it is quite possible that the kinases are nonfunctional due to lack of ATP. Azide has previously been
reported to inhibit the insulin receptor tyrosine kinase
through a similar mechanism.41
Our observation that the GM-CSF receptor-associated
tyrosine kinase is active at 4°C is not unanticipated. Although many types of enzymatic activity are diminished at
4"C, tyrosine kinases are typically only minimally sensitive
to temperature. For example, tyrosine kinase activity associated with the platelet-derived growth factor
the
epidermal growth factor receptor,"3 the insulin receptor,@
the insulin-like growth factor I1 receptor,4' and the M-CSF
receptor46are all active at 4°C. The activation of tyrosine
kinase activity at 4°C by the GM-CSF receptor is, however,
the first example of such activity by a growth factor receptor
that is not itself a tyrosine kinase.
In contrast to activation of tyrosine kinase activity,
hyperphosphorylation and activation of the serinelthreonine kinase Raf-1 was greatly diminished at 4°C in the M 0 7
cell line or in neutrophils. This result may occur because
phosphorylation of Raf-1 requires receptor internalization,
but is more likely to be due to the possibility that the kinase
that phosphorylates and actives Raf-1 in myeloid cells is
inactive at 4°C. Raf-1 is known to be activated by mitogens
in many different cell lineages and may be a common link in
signal transduction pathways between various growth factor
receptors and the nucleu~.~'
Our results indirectly show that
phosphorylation of Raf-1 is either distal to, or unrekated to,
activation of tyrosine kinases by GM-CSF in myeloid cells,
and directly show that activation of tyrosine kinases does
not require prior activation of Raf-1. This latter conclusion
is consistent with those obtained from several previously
studied cell systems in which Raf-1 is activated, because the
growth factor receptors themselves have been tyrosine
kinases.
The studies presented here do not address whether or
not receptor internalization is required for proliferation,
because the cells do not proliferate at 4°C. It is possible that
receptor internalization is required for proliferation, and
that the activation of tyrosine kinase activity is unrelated to
cell growth. Again, however, this possibility is unlikely
because of the previous demonstrations that the functions
of the GM-CSF and IL-3 receptors that support proliferation of murine factor-dependent cell lines can be replaced
by known tyrosine kinase receptors such as the EGF and
M-CSF receptor^."^^^.^^ Taken together, these data suggest
From www.bloodjournal.org by guest on June 12, 2017. For personal use only.
1934
OKUDA ET AL
that the initial events of signal transduction do not require
receptor internalization.
Receptor internalization has been observed to occur
after the binding of several other hematopoietic growth
factors to their receptors. The best studied example is the
M-CSF receptor, c-fms. M-CSFlc-fms complexes are internalized within minutes, enter endosomes, and are degraded. After downmodulation, there is a refractory period
of several hours during which new receptors are synthesized and reaccumulate at the cell surface. PMA also
induces receptor downmodulation, but through a different
mechanism than ligand, because PMA does not induce
receptor autophosphorylation on tyrosine, and under conditions in which protein kinase C is itself downmodulated by
chronic PMA exposure, M-CSF is still able to induce
receptor internali~ation.~'.~
Interestingly, v-fms is relatively
refractory to downmodulation by either M-CSF or PMA.49
In the presence of M-CSF, the intrinsic tyrosine kinase of
c-fis can be activated at 4"C, and the substrates that are
phosphorylated 09 tyrosine at 4°C are similar to those
phosphorylated at 37°C.4.50Neither the receptor nor the
ligand are internalized at P C , indicating that signal transduction is normally initiated before, and does not require,
internalization.
The results presented here suggest that the functional
consequences of GM-CSF receptor internalization may be
similar in many ways to c-fms internalization. In both cases,
receptor internalization is not required for activation of
tyrosine kinase activity, can be induced by either ligand or
PMA, and probably serves primarily to limit receptor
activation by directing the receptorlligand complex to
endosomes for degradation. Further studies are warranted
to correlate the structure of the GM-CSF receptor complexes with both signal transduction and in'ternalization.
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1991 78: 1928-1935
Internalization of the granulocyte-macrophage colony-stimulating
factor receptor is not required for induction of protein tyrosine
phosphorylation in human myeloid cells
K Okuda, B Druker, Y Kanakura, M Koenigsmann and JD Griffin
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