1. No category

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1994 84: 1780-1789
Activation of Ras and formation of GAP complex during TPA-induced
monocytic differentiation of HL-60 cells
K Katagiri, S Hattori, S Nakamura, T Yamamoto, T Yoshida and T Katagiri
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Activation of Ras and Formation of GAP Complex During TPA-Induced
Monocytic Differentiation of HL-60 Cells
By Koko Katagiri, Seisuke Hattori, S h u n Nakarnura, Tadashi Yarnarnoto, Takeshi Yoshida, and Takuya Katagiri
In this study, it was shown that theproportion of guanosine
triphosphate (GTP)-bound active Ras increased in TPA (12o-tetradecanoyl phorbol-13-acetate)-inducedmonocytic differentiation of HL-60 cells. The increase of active Ras was
observed at 24 hours after TPA stimulation and attained to
threefold (15%) over the proportion in nontreated HL-60
cells. Herbimycin A, an inhibitor of tyrosine kinase, prevented the activation ofRas,as
well as the induction of
monocytic differentiation. In parallel with the activation of
Ras, the proteins with molecular weights of 52, 56, 62, and
190 kD were tyrosine-phosphorylated and formed a complex
with GTPase-activating protein (GAP) for Ras. In addition to
the 116-kD GAP (type I GAP), the 100-kD GAP (type II GAP)
molecule was markedly induced at 24 hours after TPA stimu-
lation of HL-60 cells. These phenomena sustained for a further 24 hours during monocytic differentiation. However,
they were notobserved during retinoic acid-induced granulocytic differentiation of the cells. The HL-60 transfectants,
which expressed
a
dominant inhibitory H a m s Asnl7,
showed a low level of tyrosine-phosphorylated GAP-associated proteins and did not undergo full differentiation in response to TPA. Taken together, these data indicate that the
activation of Ras and GAP complex formation mutually correlate and function downstream of protein-tyrosine kinases
in thesignaling pathway for monocytic differentiation of HL60 cells.
0 1994 by The American Society of Hematology.
I
cells, Ras plays an important role in the process of neuronal
differentiation of PC12 cells that is triggered by the tyrosine
kinase receptor.’?“’ When PC12 cells respond to nerve
growth factor, rapid accumulation of the guanosine triphosphate (GTP)-bound form of Ras occurred.” The role of Ras
in the PC12 cell differentiation can be mimickedby the
action of mutationally activated Ras.I3 Nerve growth factorinduced neurite outgrowth was blocked by the microinjection
of an anti-Ras monoclonal antibody (MoAb)I4or a dominant
inhibitory ras mutant.”
The mammalian Ras proteins bind guanine nucleotides
and possess intrinsic GTPase activity. It alternates between
active GTP-bound and inactive guanosine diphosphate
(GDP)-bound
The relative activities of guanine
nucleotide-releasing factors (GRFs) and Ras GTPase-activating proteins (GAPs) acting on Ras at any particular time
may determine the activation state of Ras. A family of GRFs
directly activate Ras, and SedGrb2 mediates the interaction
Furof SOS GRF with activated tyrosine kinase receptors.’8-22
thermore, it was recently reported that p95’”’ (Vav), which
is specifically expressed in the hematopoietic cells, has the
GRF activity toward Ras and may be directly regulated by
tyrosine kina~e.~’,’~
The GAP stimulates the intrinsic GTP hydrolytic activity
of Ras and promotes the return of active GTP-bound R2s to
the inactive GDP-bound state.*’-?*Therefore, GAP might be
anticipated to function as a negative regulator of Ras. The
GAP contains two src homology 2 (SH2) domains responsible for interaction with tyrosine-phosphorylated protein^.'^.^'
It has been shown that GAP is coimmunoprecipitated with
two tyrosine-phosphorylated proteins with molecular
weights of 62 and 190 kD from lysates of cells expressing
activated tyrosine kinases or cells stimulated with growth
factor^.'^.^' The complex formation of GAP with these proteins inhibits GAP activity, which results in the activation
ofRas.”.’? Therefore, functional states of both GRFs and
GAPs may be controlled by tyrosine kinases in the signaling
pathway.
The GAP-associated tyrosine phosphoproteins p62and
p190 were recently cloned from fib rob last^."^^^ The p62 has
a significant homology to a putative heterogeneous nuclear
N HEMATOPOIESIS, progenitor cells proliferate and differentiate along multiple cell lineages. This process is
highly controlled through successive stages of differentiation
and growth arrest. The induction of specific differentiation
in in vitro culture enables us to dissect and analyze myelopoiesis. The human myeloblastic cell line HL-60 iswell
known to be led both to the monocytic differentiation and
to the granulocytic one.’ The former is induced by TPA (120-tetradecanoylphorbol- 13-acetate) and the latter by retinoic
acid (RA).’ These in vitro systems are available for elucidating the molecules involved in the myeloid differentiation.
Protein tyrosine kinases (PTKs) are important regulatory
proteins that control cellular growth and differentiation.’ Recently, we reported that both fyn and lyn tyrosine kinases
were expressed abundantly during TPA-induced monocytic
differentiation.3The inhibitors of PTK activity, such as herbimycin A and genistein, markedly inhibited monocytic differentiation, suggesting that PTK is involved in the differentiation process? The tyrosine-phosphorylated proteins in
TPA-treated cells include cytoskeletal proteins, tubulins,
which are associated with Fyn and Lyn PTKs.‘
The signal from tyrosine kinase has been suggested to be
transmitted to Ras through specific protein-protein interactions.‘”’ In addition to the growth of various origins of the
From the Tokyo Institute for Immunopharmacology, Inc, Takada,
Toshima-ku;the Division of Biochemistry and Cellular Biology, National Institute of Neuroscience, National Center of Neurology and
Psychiatry, Ogawahigashi, Kodaira;Department of Oncology, Institute of Medical Science, University of Tokyo, Shirokanedai, Minatoku; and the Department of Immunology, Center for Basic Research,
The Kitasato Institute, Shirokane, Minato-ku, Tokyo, Japan.
Submitted December 13, 1993; accepted May 6, 1994.
Address reprint requests to Takuya Katagiri, PhD, Department
of Immunology, Center for Basic Research, The Kitasato Institute,
5-9-1 Shirokane, Minato-ku, Tokyo, 108, Japan.
The publication costsof this article were defrayedin part by page
chargepayment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-4971/94/8406-0004$3.00/0
1780
Blood, Vol 84, No 6 (September 15). 1994: pp 1780-1789
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1781
RAsIGAP IN MONOCYTIC DIFFERENTIATION
ribonucleoprotein protein and has the ability to bind to single-stranded DNA and to RNA.33 The p62 is assumed to
play a role in some aspect of mRNA processing or utilization. Because a large portion of p190 is identical to a nuclear
transcription factor,34p190 may transmit a signal to the nucleus for modulation of specific cellular genes. p190 also
has GAP activity towards Rhomac family,35which regulates
the reorganization of actin cytoskeleton in cell growth and
differentiati~n.~~
Therefore, the formation of molecular complexes consisting of GAP, p62, and p190 is supposed to
function as the effectors for Ras. Alternatively, it is possible
that a p62- and pl90-mediated signaling pathway operates
independently of Ras followed by tyrosine kinase activation.
In this study, we have reported that, during TPA-induced
monocytic differentiation of HL-60 cells, the GTP-bound
active form of Ras increased in a PTK-dependent manner,
and that tyrosine-phosphorylated proteins of 52, 56, 62, and
190 kD associated withGAP. These signaling molecules
were indicated to function in the induction of monocytic
differentiation.
MATERIALS AND METHODS
Cells. HL-60 cells were suspended with RPM1 1640 medium
(GIBCO, Grand Island, NY) containing 10% fetal calf serum (Sigma
Chemical CO,St Louis, MO). For differentiation experiments, growing cells were subcultured at a density of 2 X IOs celldmL and
treated either with TPA (10 ng/mL; Sigma) or RA (1 pmoVL; Sigma)
in the presence or absence of herbimycin A (0.4 pg/mL) for the
indicated times? Herbimycin A was kindly donated by Yoshimasa
Uehara (National Institute of Health, Tokyo, Japan).
Antibodies. '2'I-labeled monoclonal antiphosphotyrosine PY-20
(ICN Radiochemicals, Irvine, CA), monoclonal anti-GAP antibody
(Santa Cruz Biotechnology, Inc, Santa Cruz, CA), polyclonal antiGAP antibody (Upstate Biotechnology, Inc, Lake Placid, NY), polyclonal anti-Vav antibody (Santa Cruz Biotechnology), '2'I-labeled
monoclonal antimouse Ig (ICN), 1251-labeledmonoclonal antirabbit
Ig (ICN), monoclonal anti-Ras antibody (NC-RASW), and peroxidase-linked antimouse Ig F(ab)2fragments (Amersham CO,Arlington
Height, IL) were used in the immunoprecipitation and immunoblotting as described below.
Immunoprecipitation. TPA- or RA-treated HL-60 cells (5 X IO6)
were collected by centrifugation and suspended at 0°C for 30 minutes
with 1 mL of ristocetin-induced platelet aggregation (RIPA) lysis
buffer (1% Triton X-100, 10 mmoVL Tris-HC1, 150 mmoVL NaCI,
2 mmoVL EDTA, 10 mg/mL aprotinin, 10 mmom NaF, 1 mmoVL
Na3VO4;pH 7.6).3.4 The supernatant was precleared by incubation
with excess amount of protein G-Sepharose 4B (Pharmacia Fine
Chemicals, Piscataway, NJ). The cleared lysate was incubated with
various kinds of antibodies and protein G-Sepharose 4B. The immunoprecipitates were washed with the lysis buffer extensively.
Zmmunoblotting. The 10 mL of the cell lysates or the immunoprecipitated proteins were subjected to electrophoresis on 8.5% or
12% polyacrylamiddsodium dodecyl sulfate gel. The transfer of the
proteins to polyvinylidene difluoride membrane and blotting with
'251-labeledPY-20 or monoclonal anti-GAP antibody plus 'Z'I-labeled antimouse Ig antibody were performed as described.' Monoclonal anti-Ras antibody, peroxidase-linked antimouse Ig F(ab)2fragments and enhanced chemiluminescence (ECL) system (Amersham)
were also used for Western blotting analysis.
Analysis of Ras-bound GDP/GTP. Cells were washed twice with
Tris-buffered saline (20 mmol/L Tris-HC1 {pH 7.51, 150 mmoVL
NaCI) and were labeled for 6 hours in 1 mL of phosphate-free
Dulbecco's modified Eagle's medium supplemented with 0.05 mCi/
mL of 32P,. Nucleotides bound to Ras were analyzed as described."
Cytochemical demonstration of nonspecz3c esterase. HL-60
cells that had been stimulated with TPA in the absence or presence
of herbimycin A were washed twice with phosphate-buffered saline.
Cells were collected by centrifugation onto glass slides, fixed at 4°C
for 30 seconds by a buffered formalin aceton mixture (pH 6.6; 20
mg Na2HP04, 100 mg KH2P04, 30 mL H,O, 45 mL aceton, and 25
mL formalin). Cells were incubated for 30 minutes at 37°C in the
reaction medium (10 mg Fast Garnet GBC per 9.5 mL of 0.067 moll
L phosphate buffer, pH 6.3 and 10 mg alpha-naphtyl butyrate per
0.5 mL ethylene glycol monomethyl ether; Muto Pure Chemicals
CO,LTD, Tokyo, Japan). The cells were washed with water and
counterstained for 10 minutes with Carrazi's Hematoxylin (Wako
Pure Chemical Industries, Ltd, Tokyo, Japan). The cells were then
washed with water, dried, and mounted with Gewount (Biomeda
Corp, Foster City, CA).
Transfection of HL-60 cells with a dominant inhibitory Ha-ras
expression plasmid. The plasmid pMMTVrasH(Asn-17) was constructed as described previously.'5 The Ha-ras Asn-17 gene is expressed from a mouse mammary tumor virus (MMTV) long terminal
repeat. Transfection of 4 X lo6of HL-60 cells with 10 mg of plasmid
DNAs was performed by electroporation. The transfected cells were
diluted, and 2 X 10"of the cells per well were plated onto 96well microplates. Two days after transfecton, G418 (GIBCO, Grand
Island, NY) was added at 1 mg/mL of final concentration, and subclones were isolated after 4 to 6 weeks of G418 treatment and were
propagated in the presence of G4 18.
RESULTS
We reported previously that the expressions of different
src family PTKs were markedly induced during TPA-induced monocytic and RA-induced granulocytic differentiation of HL-60 cells.3The immunoblotting using antiphosphotyrosine MoAb PY-20 showed that both TPA and RA
induced a tyrosine phosphorylation of various kinds of proteins (refer to Fig 3A): Because the connection of tyrosine
kinases to Ras in the signaling pathway was generally bel i e ~ e d , ~we
' investigated whether or not activation of Ras
occurs in the TPA- or RA-treated HL-60 cells and analyzed
Ras-bound nucleotides in these cells. As shown in Fig lA,
the proportion of GTP-bound Ras increased up to 15% of
the total amount of Ras at 48 hours after TPA stimulation
of HL-60 cells, but RA stimulation did not cause an accumulation of Ras-GTP. The level of GTP-bound Ras innontreated HL-60 cells and in RA-treated HL-60 cells was approximately 6%of total Ras. The increase in the relative
amount of GTP-bound Ras was not observed within short
periods (3 minutes and 3 hours; data not shown), but became
remarkable at 24 hours after TPA stimulation of HL-60 cells
and was sustained for another 24 hours during monocytic
differentiation (Fig IB).
We reported that herbimycin A, an inhibitor of PTK, prevented HL-60 cells from acquiring macrophage-like features
such as plastic and cellular adherence after stimulation (Fig
2A).4 In addition, herbimycin A completely inhibited TPAinduced activity of nonspecific esterase, a marker enzyme
for monocytes (Fig 2A).38 Asshown in Fig 2B, the increase
of GTP-bound Ras in TPA-treated HL-60 cells was abro-
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KATAGlRl ET AL
1782
A
NC
None RA
TPA
-GDP
-GTP
-ori
0
10
20
30
40
5 0
Hours after TPA stimulation
Fig 1. (A) Accumulation of Ras-GTP in monocytic differentiation,
but not in granulocytic differentiation of HL-60 cellsis shown. HL-60
cells that weretreated with or without TPA or RA for48 hours were
labeledwith -PI. The cell lysateswere subjected to immunoprecipitation with or without (NCI monoclonal anti-Ras antibody V13-259. The
nucleotides in the immunoprecipitates were analyzed by thin-layer
chromatography.The origin (ori) and the positions of GDP and GTP
are indicated. (B1 Time courseof Ras activation during monocytic
differentiation is shown. HL-60 cells were treated for the indicated
times with TPA, and guanine nucleotides that were bound to Ras
were analyzed. The relative amount of GTP as a percentage of Rasassociated guanine nucleotides (mean f SEM; n + 3) is shown.
gated by herbimycin A. These data suggest that Ras plays
roles downstream of tyrosine kinase(s) for the induction of
monocytic differentiation.
Recently, the GAP-associated tyrosine phosphoproteins,
p62 and p190, were supposed to function as the effectors
downstream of signals emitted by R~S."..'~
Figure 3A showed
that in nontreated HL-60 cells, phosphotyrosine-containing
proteins were not detected, and TPA and RA induced a
tyrosine phosphorylation of various kinds of proteins. The
tyrosine phosphorylation of proteins with molecular weights
of 62 and 190 kD were observed in TPA-treated HL-60 cells
(Fig 3A). To examine whether these phosphorylated proteins
associate with GAP during the monocytic differentiation, the
lysates from TPA-treated HL-60 cells were immunoprecipitated with several kinds of monoclonal and polyclonal antiGAP antibodies, and the precipitates were analyzed by immunoblotting using '251-labeledantiphosphotyrosine MoAb
(PY-20). As shown in Fig 3B, anti-GAP immunoprecipitates
from TPA-treated HL-60 cells contained several kinds of
tyrosine-phosphorylated proteins (p190. p62, p56, and p52).
In contrast, tyrosine-phosphorylated proteins were not present in anti-GAP immunoprecipitate from RA-treated cells
(Fig 3B). These tyrosine-phosphorylated molecules in antiGAP immunoprecipitates were clearly visible at 24 hours
after the TPA stimulation and increased thereafter until the
completion of monocytic differentiation (Fig 3C). By using
monoclonal anti-p62 antibody (data not shown), the tyrosinephosphorylated 62 kD protein in this study was proven to
be GAP-associated protein ~ 6 2p56
. ~and
~ p52 proteins were
neither tubulin nor Shc proteins, which have close molecular
weights (data not shown) and have yet to be identified.
The immunoblotting using monoclonal anti-GAP antibody
showed that TPA treatment significantly increased the level
of type I p1 16 GAP (Fig 4). In the same cells, expression
of the type I1 p100 GAP molecule was induced. In contrast,
RA treatment increased the level of p116 GAP but not of
p100 GAP (Fig 4). By using the other two kinds of antiGAP antibodies, the induction of p100 GAP proteinwas
also clearly shown in the TPA-treated HL-60 cells (data not
shown).
Because it has been reported that Vav, which is expressed
in hematopoietic cells, is tyrosine-phosphorylated and shows
GRF activity toward Ras, we examined tyrosine phosphorylation of Vav in TPA-treated HL-60 cells. As shown in Fig
5, the fourfold to fivefold increase in tyrosine phosphorylation of Vav was observed at 24 hours after TPA stimulation.
A dominant inhibitory mutation of Ha-rus that changes
Ser-17 to Asn-17 in the gene product p21 [p21(Asn-I7)Harus]'' was used to investigate the role of Ras in the monocytic differentiation. The cells transfected with pMMTVrusH(Asn-17) construct yielded (3418-resistant transformants.
We screened such transformants to select the clones that
were unable to undergo monocytic differentiation by TPA
even in the absence of the inducer, dexamethasone, because
dexamethasone itself had aninhibitory effect on the differentiation. Among such clones, T-13 and T-65 were chosen
for further analyses, which showed the quite lowlevel of
adherence and morphologic changes as compared with the
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RAS/GAP IN MONOCYTIC DIFFERENTIATION
1783
b
"
B
20
I
JS
Herbimycin A
Fig 2. (A) Inhibition of cellular and plastic adhesion and nonspecific esterase activity in TPA-treated HL-60 cells by herbimycin A is shown.
HL-60 cells (2 x lo5 calls/mL) were plated on dishes (35 mm diameter; Falcon Labware, Oxnard, CA). TPA (10 ng/mL) or TPA plus herbimycin
A (0.4 mg/mL) were then
added. (Lane al After 2 days, pictures were takenusing a microscope (Olympus CK2; Olympus, Tokyo, Japan) (original
.
b) After 2 days, cells were incubated in the reaction medium containing alpha-naphtyl butyrate and Fast Garnet
magnification ~ 1 0 0 ) (Lane
GBC and were counterstained with Hematoxylin as described in Materials and Methods (original magnification ~ 6 0 0 )(B)
. Inhibition of Ras
activation by herbimycin A is shown. HL-60 cells (5 x 10') that had been treated with TPA or TPA plus herbimycin A (0.4 mg/mL) were labeled
with 32Pi.The amounts of Ras-GTP in the cells were measured as in Fig 1.
parental clone (Fig 6A). In contrast, T-42 and T-54 showed
similar extent of adherence as that observed in the original
HL-60 cells. T-42 and T-54 were selected as controls (Fig
6A). Figure 6B showed that the basal expressions of Ras in
T-13 and T-65 were threefold more thanthose in the parental
HL-60 and increased even more (1.5-fold to twofold) after
stimulation with dexamethasone (data not shown). In contrast, T-42 and T-54 did not show the increased levels of
Ras protein (Fig 6B), even if exposed to dexamethasone
(data not shown). Therefore, the increased levels of Ras in
T-13 and T-65 clones should represent the expression of
mutant Ha-rus Asn-17 protein, whereas T-42 and T-54 expressed only the neomycin-resistant gene. It is also reported
that basal expression of p21 (Asn-l 7) in PC12 cells in the
absence of the inducer blocked the morphologic change by
NGF.
The amount of tyrosine-phosphorylated GAP-associated
proteins was significantly (30% to 50%) attenuated in TPAtreated T-13 and T-65 as compared withthe TPA-treated
parental HL-60 cells, T-42, and T-54 (Fig 6C). The basal
expressions of GAP proteins were not different among these
clones (data not shown). These data suggest that the mutant
Ha-rus gene product inhibits both the increase in the amount
of tyrosine-phosphorylated GAP-associated proteins and the
TPA-induced monocytic differentiation of HL-60 cells,
The various kinds of tyrosine kinases such as Fyn, Lvn,"
H c ~ , 'Brk,""
~
Fes.4' and focal adhesion PTK4' were expressed
during TPA-induced monocytic differentiation of HL-60
cells (Katagiri et al' and unpublished data). Surprisingly,
however, none of these tyrosine kinases were detected in
anti-GAP immunoprecipitate (data not shown). Furthermore,
the anti-macrophage colony-stimulating factor, anti-granulocyte-macrophage colony-stimulating factor, and anti-fms
antibodies did not inhibit the tyrosine phosphorylation and
monocytic differentiation of TPA-treated HL-60 cells (data
not shown). Therefore, it is notlikelythat
these receptor
tyrosine kinases were involved in the differentiation, but we
can not exclude the possibility that TPA directly activates
the receptor tyrosine kinases.
DISCUSSION
In our preceding reports, we have proposed an idea that
PTKs play a crucial role in TPA-induced monocytic differentiation of HL-60 cell^."^.^ In the current study, it was found
that both the activation of Ras and theincrease in the amount
of tyrosine-phosphorylated GAP-associated proteins occurred in a PTK-dependent manner during monocytic differentiation. Ras and GAP were suggested to function downstream of the PTK-signaling pathway during the monocytic
differentiation.
The dominant negative mutant Ha-rus gene product inhibited monocytic differentiation (Fig 5). indicating that the
activation of Ras was an essential event for the monocytic
differentiation of HL-60 cells induced by TPA. The role
of Ras in cell differentiation was shown in the neuronal
differentiation of PC- 12 cells induced by NGF.'.'' Our findings in this study again strengthened the importance of Ras
in cell differentiation as well as in cell proliferation.
The activation of Ras during TPA-induced monocytic differentiation of HL-60 cells was dependent on tyrosine-phos-
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KATAGlRl ET AL
1784
TPA
RA
TPA
0
PQ
(3 +Q
(3.
48 hr
24
RA
Q
P
L\.(3
dp
0 24 48hr
200-
200-
97.4-
97.4 -
69-
6946-
'..
8
46-
30-
A
Anti-P.Tyr Blot
B
Anti-P.Tyr Blot
a-GAP Immunoprecipitates
200
-
97.4
-
0
9
24
48 h r
"
"
69-
46-
30-
C
Anti-P.TyrBlot
Fig 3. (A) Tyrosine phosphorylation patterns of TPA- or RA-treated HL-60 cells are
shown. The RlPA lysates from TPA- or RA-treated HL-60 cells (5 x lo' cells) for 0,24,
and 48 hours were immunoblotted with
'251-lableled PY-20. Theexposure time was 24
hours at -80°C with an intensifier screen. (B) Formation ofcomplex of GAP with tyroin monocytic differentiation, but notin granulosine-phosphorylated proteins is shown
cytic differentiation, of HL-60 cells. The RlPA lysates from TPA- or RA-treated HL-60
cells (2.5 x 106cells) for 48 hours were immunoprecipitated with polyclonal anti-GAP
antibody or control rabbit IgG and protein G-Sepharose. Anti-GAP-immunoprecipitated proteins weresubjected t o electrophoresis, transferred t o a polyvinylidene difluoridefilter, and immunoblotted
with '251-labeled PY-20.
The exposuretime was12 hours
at -80°C with an intensifierscreen. IC) Time course of GAP complex formation during
monocytic differentiation isshown. Cell lysates from 2.5 x 10' cells of HL-60 cells that
had been incubated with TPA for the indicated times were immunoprecipitated with
monoclonal anti-GAP and analyzed by immunoblotting with 'z51-labeledPY-20. The
exposure time was 12 hours at -80°C with an intensifier screen.
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R A ~ G A PIN MONOCYTIC DIFFERENTIATION
TPA
0 24 48 hr
RA
0 24
48 hr
200GAP1
116-
I
97.4-
1 GAPll
69
46
already identified as GAP-associated proteins,''.'4but also
the unknown p56 and p52.
Type I1 GAP was reported to be generated by alternative
splicing of type I GAP and to lack the hydrophobic amino
terminus characteristic to type I GAP.'s.JhSo far. the expression of type I1 GAP has been observed only in theplacenta.'"''
In this study, type 11 GAP in additiontotype
I GAP was induced in the monocytic differentiation. The
expression of type I1 GAP might be related to the association
of p56 andp52with GAP, because both appear to occur
characteristically in TPA-treated HL-60 cells. Therefore, our
system provides a goodmodel for the analysis of type 11
GAP function.
9
Anti- GAP Blot
Fig 4. One hundred kilodaltons of GAP protein was induced in
monocytic differentiation, but not in granulocytic differentiation, of
HL-60 cells. The proteins in the lysates from 5 x 10' of HL-60 cells
that had been treated with TPA or RA for 24 and 48 hours were
separated on 9% polyacrylamide gel, transferred t o a polyvinylidene
difluoride filter, and analyzed by immunoblotting with monoclonal
anti-GAP antibody and '251-labeledantimouse lg. The exposure time
was 8 hours at -80°C with an intensifier screen.
200-
97.4-
69
phorylation (Fig 2). ~95'""is suggested to function as a GRF
for Ras, and its activity was directly stimulated by tyrosine
kinase^.^^." The tyrosine phosphorylation of p95'"' was
found to be greatly (fivefold) enhanced after TPA stimulation
(Fig 5). RA also induced the tyrosine-phosphorylation of
Vavwithout activation of Ras (Katagiri et al, manuscript
submitted). Both TPA and RA induced GAP expression:
however, formation of GAP complex, which is enzymatically less active, was observed only in TPA-treated cells.
Therefore, the activation of Vav might be cancelled by the
increase in the level of GAP activity in RA-treated cells.
HL-60 cells harbor a point mutation in the N-rus gene:'
whose product may preferentially bind GTP. However, contribution of N-Ras to the total Ras level seems to be rather
low, because the basal Ras-GTP level is not as high as is
observed in other cells expressing activated forms of R m Z X
Downwardet al" reported that treatment of T cells by
TPA results in the activation of Ras within several minutes.
We could not detect activation of Ras in such short periods
of time after TPA addition. Therefore, the mechanisms that
lead to the activation of Ras may be quite different between
the two systems.
The several kinds of tyrosine-phosphorylated proteins
were shown to associate with GAP during the monocytic
differentiation of HL-60 cells. These GAP-associated phosphoproteins contained not only p62 and p190, which were
-
46-
Anti-P.Tyr Blot
Anti-Vav Blot
Fig 5. Tyrosine phosphorylation of p95"' in TPA-treated HL-60
cells is shown. The RlPA lysates from TPA-treated HL-60 cells (2.5
x lo6 cells) for 0, 24, and 48 hours were immunoprecipitated with
polyclonal anti-Vav and protein
G-Sepharose. Anti-Vav-immunoprecipitated proteins weresubjected t o electrophoresis, transferred t o
a polyvinylidene difluoridefilter, and immunoblotted with'251-labeled
PY-20. The exposure time was 12 hours at -80°C with an intensifier
screen.
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KATAGlRl ET AL
T-42
T-13
T-54
B
...
" ~ "
_.""
./___
97.4
-
69
-
30
-
M r(l0
3021.5-
*p21 ras
14.3 Anti-P.Tyr Blot of
anti-GAP
immunoprecipitates
Fig 6. (A) Adhesion of a dominant inhibitory Ha-ras Asn-17 gene-transfected HL-60 cells. Ha-ras Asn-17 transfectants were prepared as
described in Materials andMethods. The cells were seeded in &well dishes at a lo6cells per well and treated with
10 nglmL of TPA. Clones,
T-13 and T-65 showed a diminished cellular and plastic adhesion. In contrast, T-42 and T-54 showed a strong adhesion similar to that of
parental HL-60 cells. Two days after TPA stimulation, pictures were taken using a microscope (Olympus C U I . (Original magnification x50.)
(B) Expression of Ha-Ras Asn-17 in HL-60 subclones is shown. Cell lysates from 5 x 10' cells of T-13, T-65, T-42, T-54, and HL-60 cells were
subjected t o sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 12% polyacrylamide gels, were transferred t o a polyvinylidene
difluoride filter, and were immunostained with monoclonal anti-Ras (NC-004155peroxidase-linked antimouse lg F(ablr fragments and then
treated withAmersham ECL reagents. (C) Decreased GAPcomplex formation inT-13 and T-65 cells is shown. Cell lysates from lo6 cells of T13, T-65, T-42, T-54, and parentalHL-60 cells that hadbeen incubated with TPA for 48 hours were immunoprecipitated with monoclonal antiGAP and analyzed by immunoblotting with '251-labeled PY-20.The exposure time was 12 hours at -80°C with an intensifier screen.
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
1787
RAsIGAP IN MONOCYTIC DIFFERENTIATION
It was reported that GAP-associated proteinsp62 and p190
bound tighly to GAP in vitro. This binding is dependent on
the tyrosine-phosphorylation of GAP-associated proteins and
occurs through the N-terminal SH2 region of GAP that recongnizes p h o s p h o t y r ~ s i n e .The
~ ~ *N-terminal
~ ~ ~ ~ SH2 domain
of GAP conjugated with agarose beads also bound tyrosinephosphorylated GAP-associated proteins, as above, in the lysate of TPA-treated HL-60 cells (datanot shown). Therefore,
tyrosine-phosphorylation of GAP-associated proteins seems
to be a necessary event for a formation of GAP complex
through its SH2 domain in PA-treated HL-60 cells.
The active form of Ras was assumed to affect the tertiary
structure of GAP and enables its SH2 domain to interact
efficientlywithphosphoproteins:’
The dominant inhibitory
mutant Ha-rus Asn-17 decreasedthe amount of tyrosine-phosphorylatedGAP-associatedproteins in the TPA-stimulated
HL-60 cells. This result suggests that active Ras might promote the formation of a complex betweenGAP and tyrosinephosphorylated GAP-associated proteins
in TPA-treated cells.
GAP was shown to serve as an effector of Ras in the K+
channel linked to a muscarinic receptor in atrial membranes!8 Recently, the N-terminal domain of GAP was reported to show the same effe~t.4~
The N-terminal domain of
GAP directly interacts with p190, and exerts effector functions for regulating the cytoskeletal organization and the cell
adhe~ion.4~
In TPA-treated HL-60 cells, GAP also serves
as an effector of Ras and influences the functions of its
downstream target molecules, which might be GAP-associated proteins. Accordingly, the GAP complex might play
crucial roles for coupling Ras with the downstream signaling
molecules in the monocytic differentiation.
We summarized our results in Fig 7, where the Ras and
GAP fit into the model for differentiation-inducing signal
triggered by activation of protein kinase CO (PKCP) by
TPA. The activation of PKCP results in the expression of
early response genes:’ the products of which might lead to
the induction of various kinds of PTKs at several hours after
TPA tim mu la ti on.^ The PTKs phosphorylate Vav and GAPassociated proteins and the activation of Ras is then induced.
GTP-bound active Ras may cause conformational change of
GAP to promote GAP complex formation. GAP complex
may transduce signals from Ras to the nucleus and affect the
organization of the cytoskelton. Mitogen-activated protein
kinase functions at the distal end of a rus-dependent signaling pathway, transducing signals to the n u ~ l e u s . ~ ’These
”~
signals altogether might elicit the activation of a cascade of
genes that code for the complete expression of macrophagespecific phenotypes.
Our findings strongly suggest that Ras and GAP work in
the FTK-mediated differentiation of the cells. Future studies
aimed at identifying the GAP-associated proteins p56 and
p52 and the tyrosine kinases involved in tyrosine phosphorylation of GAP-associated proteins will aid to elucidate the
signaling pathway that activates a genetic program for monocytic differentiation.
ACKNOWLEDGMENT
We wish to thank Dr Susumu Nishimura (Merck-Banyu Pharmaceutical CO) for his kind gift of anti-Ras antibody, and Dr Geoffrey
TPA
I/
PKCp
I
Fig 7. A model for involvement of Ras-GAP in TPA-induced signal
transduction pathways of HL-60 cells is shown. Based on our observations, we suggest the following outline for a signal transduction
pathway thet results in terminal cell differentiation. The interaction
of TPA with PKCp causes its activation and translocation. The activated PKC#J induces expression of early response genes (iun/fos),
that mayconduct the induction of various kinds of PTKs. PTKs phosphorylate Vav and GAP-associated proteins at tyrosine residues. By
tyrosine phosphorylation of these proteins, GRF activity of Vav is
stimulated, and GAP activity is suppressed, resulting in theactivation
of Ras. Active Rar may allow the SH2 region of GAP to form GAP
complex. The signal triggered by Ras-GTP is also transmitted by Raf,
mitogen-activated protein kinase kinase (MAPKK), and MAP kinase
(MAPK) to thenucleus.
M. Cooper (Harvard Medical School, Boston, MA) for providing
Ha-ras expression plasmids.
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