(Received for publication, October 10, 1995; and in revised form, January 10, 1996)
From the
The mammalian Ras GTPase-activating protein
(p120) interacts with activated members of the Ras
superfamily of GTP-binding proteins to accelerate their deactivation by
sharply increasing their rates of GTP hydrolysis. Among the Ras-family
proteins interacting with p120
is Rap1A/Krev1, whose
activity is not affected by p120
but which competes
with Ras for p120
. A second protein that interacts
with p120
is p190
, which activates
the GTPase of guanine nucleotide-binding proteins of the Rho family
(including Rac1 and Rac2). Both these p120
-binding
proteins are of interest in connection with the regulation of the
respiratory burst oxidase, Rap1A/Krev1 because it copurifies with
cytochrome b
, and p190
because
it inhibits the Rac2-dependent activation of the respiratory burst
oxidase in a cell-free system. Using an 18-mer antisense
oligonucleotide, we were able to decrease the expression of
p120
in Epstein-Barr virus-transformed B lymphocytes.
Under conditions where p120
expression was
significantly depressed by antisense oligonucleotides, we observed a
40% increase in protein kinase C-dependent but not receptor-dependent
O
production. In contrast, sense and
scrambled oligonucleotides had no effect on either
p120
expression or O
production. Our results suggest a role for p120
as a negative regulator in the protein kinase C-mediated
activation of the respiratory burst oxidase.
The products of the mammalian N-, K-, and Ha-ras genes are 21-kDa guanine nucleotide-binding proteins
that possess a low intrinsic GTPase activity(1, 2) .
They play a significant role in both the control of normal cell growth (3) and its malignant subversion (4) and have been
implicated as key elements in signal transduction in lymphoid and
myeloid cell lines(5, 6, 7) . The activity of
Ras depends on the ratio of bound GTP to GDP, a value that is
determined by certain regulatory proteins: guanine nucleotide exchange
factors such as Sos (8) and Vav(9) , which activate Ras
by catalyzing the exchange of bound GDP for GTP, and GTPase-activating
proteins such as the NF-1 gene product (10) and
p120, which deactivate Ras by stimulating the
conversion of bound GTP to GDP(11) .
p120 is a monomeric 120-kDa cytosolic protein that greatly accelerates
the conversion of p21
GTP to the conformationally
inactive GDP-bound form(12, 13) . Accordingly,
p120
has been proposed as a negative regulator of Ras
in cellular processes. This proposal is consistent with the finding
that overexpression of p120
blocks oncogenic
transformation (14, 15) and inhibits Ras-dependent
cellular signaling(16) . In addition, since the site of
interaction of p21
GTP with p120
overlaps the effector region of Ras, it has been proposed that
p120
may also serve as an effector protein of active
Ras(17, 18) .
p120 consists of a
C-terminal domain that interacts with Ras and an N-terminal portion
containing two Src homology (SH2) domains and one intervening SH3
domain (reviewed in (19) ). The
p21
GTP-p120
complex may interact
with downstream effectors via these Src-homology domains. SH2 domains
bind phosphotyrosine residues and have been implicated in the
interactions of tyrosine kinases of the Src family with their effector
proteins(20, 21) . Several members of the Src family
of protein-tyrosine kinases (e.g. c-Src and Lck) have been
shown to associate with and phosphorylate
p120
(22, 23, 24) . In
fibroblasts transformed by cytoplasmic and receptor-like tyrosine
kinases(25) , p120
forms complexes with two
phosphotyrosine-containing proteins, p62 and p190
.
The complex with p62 appears to involve the SH2 domains of
p120
(26, 27) , and the formation of
the p120
-p190
complex is dependent
on phosphorylation(28) , suggesting that it too involves the
p120
SH2 domains. The complex itself has diminished
p120
activity in vitro(28) .
Tyrosine-phosphorylated p62 and p190
have been
detected in anti-p120
immunoprecipitates from various
activated or transformed B- and T-cell lines(30, 31) ,
and were proposed to participate in signal transduction in these cells.
The p120
-associated p190
stimulates
the intrinsic GTPase activity of Rac1 and Rac2, which are Rho family
guanine nucleotide-binding proteins(32) . In human phagocytes,
Rac2 plays a significant role in the activation of the respiratory
burst oxidase(33, 34, 35) . The human oxidase
consists of two membrane-bound components (gp91
and p22
) and at least three
cytosolic components (p47
,
p67
, and Rac2) that after cellular activation
assemble to form the active enzyme (reviewed in (36) ).
p190
has been shown to inhibit the Rac2-dependent
activation of the respiratory burst oxidase in a cell-free
system(37) , pointing to a role for this
p120
-associated protein in the regulation of the
oxidase. The same oxidase has also been found in normal human B-cells
and EBV-transformed (
)B
lymphocytes(38, 39) , and Rac2 plays a significant
role in the activation of the lymphocyte oxidase(33) . Since we
were able to detect p190
in p120
immunoprecipitates from cultured EBV-transformed B lymphocytes
and since there have been several reports of the use of antisense
oligonucleotides in these cells (33, 40, 41) , we used antisense
oligonucleotides to study the effects of p120
(and
possibly associated proteins) on O
production. In this report we describe the results of this study.
Low passage cells ()were washed 3 times in
Opti-MEM and then incubated with 0.1 mM oligonucleotides for
24 h in serum-free Opti-MEM at a starting cell density of 4
10
/ml. The cells were then washed once in HBSS and used
either for immunoblotting or for the measurement of
O
production.
Using a monoclonal antibody against human
p120, we were able to immunoprecipitate
p120
, together with the associated
tyrosine-phosphorylated proteins p62 and p190
, from
resting and stimulated EBV-transformed B-cells (data not shown).
Blotting of cell lysates with antibodies against p120
and the oxidase components p47
and p67
showed that p120
could be easily detected in
lysates of even small amounts of cells (<5
10
).
The t
for the turnover of p120
was 8-12 h, as measured in pulse-chase experiments with
S-labeled cysteine/methionine (Fig. 1). In view of
this observation and reports that oligonucleotides are readily taken up
by EBV-transformed B lymphocytes (33, 40, 41) we decided to use antisense
oligonucleotides to study the effect of p120
on the
respiratory burst oxidase activity in this cell type.
Figure 1:
Turnover of
p120 in EBV-transformed B lymphocytes. The t
for p120
was determined as
described under ``Results.'' Top, a representative
immunoblot showing p120
precipitated from aliquots of
cultured cells at various times. Bottom,
S in the
immunoprecipitated p120
. The results show the amount
of
S present at each time point, expressed as a fraction
of the amount of
S present at 0 time (mean ± 1
S.E.; n = 3). Radioactivity at zero time was 42,700
± 1300 counts/24 h (S.D.).
Subconfluent
B-cell cultures were cultured without serum for 24 h in the presence of
p120-specific antisense, sense, or scrambled
oligonucleotides. The cells were then assayed for 1) expression of
p120
, and 2) O
production. In preliminary experiments with oligonucleotides
end-labeled with
P, we showed that the added
oligonucleotides were taken up by the cells in a time-dependent
fashion, with maximum uptake at 4 h as previously reported (40) (data not shown). In addition, we found by HPLC that the
oligonucleotide was not significantly degraded after culture for 24 h
in medium alone and that even when cultured in the presence of the
cells
20% of the oligonucleotide initially added was present after
24 h (Fig. 2).
Figure 2:
Stability of oligonucleotides in culture.
Experiments were conducted as described in the text. Left,
representative HPLC tracings of antisense oligonucleotide incubated
with cells for 0 (above) and 8 (below) h. In each trace, the broad asymmetric peak on the left represents phenol, and the sharp peak on the right is the oligonucleotide. Right, oligonucleotide levels as
a function of time. Three incubations were conducted in the absence of
cells, and two were conducted in the presence of cells. The results are
presented as the mean ± S.E. of the values at zero time. A at zero time was 0.31 ± 0.13 and 0.68
± 0.23 for incubations carried out in the absence and presence
of cells, respectively.
When the cells were incubated for 24 h in the
presence of the antisense oligonucleotide, expression of
p120 was inhibited (Fig. 3). Immunoblots of
cells treated with the antisense oligonucleotide showed a substantial
reduction in the level of p120
as compared with
untreated cells or cells treated with sense or scrambled
oligonucleotides. By contrast, the levels of p47
and
p67
were not significantly altered by incubation with
antisense oligonucleotide, as revealed by parallel immunostaining of
blots with specific antibodies against these two proteins. These
results indicated that the suppression of p120
expression by the antisense oligonucleotide was specific and did
not affect the expression of two irrelevant proteins related to the
respiratory burst oxidase in this cell type.
Figure 3:
Effect of p120 antisense
oligonucleotide on p120
expression in EBV-transformed
B lymphocytes. EBV-transformed B lymphocytes were incubated for 24 h
with 0.1 mM antisense (A), sense (S), or
scrambled (SC) oligonucleotide. Cell proteins were then
precipitated with acetone and analyzed by SDS-PAGE and immunoblotting
as described in the text, probing with anti-p120
(1:2000), anti-p47
(1:5000), and
anti-p67
(1:1000) as indicated. The immunoblot
shown here is representative of blots from nine separate
experiments.
O production by EBV-transformed
B-cells treated with antisense, sense, or scrambled oligonucleotides
was detected by luminol-enhanced chemiluminescence of cells stimulated
with phorbol, Pansorbin, or ionomycin. When antisense-treated cells
were stimulated with phorbol we observed a significant enhancement of
O
release in comparison with cells
preincubated with sense or scrambled sequences (Fig. 4, top). This enhancement always correlated with a reduced
expression of p120
and was not observed in cells
incubated in the presence of the oligonucleotide for 3 days, conditions
under which the expression of p120
had returned to
levels seen after incubation with sense or scrambled oligos (data not
shown). At 10 min, burst enhancement averaged 147 ± 10% (S.E.)
in cells treated with 0.1 mM antisense oligonucleotide. When
O
formation was triggered by Pansorbin,
however, which acts through the cell's surface immunoglobulin
receptors, the burst-enhancing effect of antisense oligonucleotides was
not seen (Fig. 4, middle).
Figure 4:
Effect of p120 antisense
oligonucleotide on O
production by
EBV-transformed B lymphocytes. Cells pretreated for 24 h with
oligonucleotides as indicated were stimulated with phorbol (top), Pansorbin (middle), or ionomycin (bottom). O
production was
determined as luminol-enhanced chemiluminescence as described under
``Materials and Methods.'' Each point represents the
mean ± S.E. from five (phorbol, Pansorbin) or four (ionomycin)
separate experiments. Controls for the phorbol-, Pansorbin-, and
ionomycin-stimulated cells were the 10, 10, and 8 min scrambled
oligonucleotide values, respectively. Absolute values for control
relative luminescence units at these time points, expressed as
Luminoskan units, were 2.05 ± 0.05 for phorbol-stimulated cells,
0.49 ± 0.15 for Pansorbin-stimulated cells, and 0.64 ±
0.10 for ionomycin-treated cells (mean ± S.E.), respectively. No
light was emitted by unstimulated cells. By analysis of variance,
differences between antisense versus scrambled and antisense versus sense results were significant for phorbol-treated and
ionomycin-treated cells (p < 0.01 for all four comparisons)
but were not significant for Pansorbin-treated cells.
,
antisense;
, sense;
, scrambled.
O production by Pansorbin-treated
cells was much lower than that of phorbol-treated cells. To determine
whether the difference in the effect of antisense oligonucleotides on
phorbol-stimulated versus Pansorbin-stimulated cells was due
to the weakness of the latter stimulus, antisense experiments were
carried out on cells treated with ionomycin, another weak stimulus.
Experiments with the protein kinase C inhibitor GFX showed that
ionomycin, like phorbol but unlike Pansorbin, activated
O
production by EBV-transformed B
lymphocytes through a protein kinase C-dependent mechanism (Fig. 5). Although levels of O
production in response to ionomycin were similar to those seen
with Pansorbin, enhanced O
production
in response to ionomycin was increased to 133 ± 8% (S.E.) of
control by the antisense oligonucleotide.
Figure 5:
Effect of the protein kinase C inhibitor
GFX on phorbol-, ionomycin-, and Pansorbin-triggered
O formation by EBV-transformed B
lymphocytes. Cells were preincubated with 0.5 µM GFX for 5
min at 37 °C and then stimulated with phorbol, ionomycin, or
Pansorbin as described under ``Materials and Methods.''
O
production was determined as
luminol-enhanced chemiluminescence as described under ``Materials
and Methods.'' The results represent the mean ± S.E. of
four separate experiments, expressed as relative Luminoskan units.
, control; &cjs2113;, GFX.
p120 has
been connected to tyrosine kinase-mediated signaling pathways, where it
is thought to function downstream of activated Src-like protein
tyrosine kinases or growth factor receptor tyrosine
kinases(49, 56) . In T-cells, p120
is bound to Lck and becomes specifically phosphorylated by this
Src-like tyrosine kinase(23) . Since Lck has been reported to
play a significant role in EBV-induced growth of transformed B
lymphocytes(40) , and since antisense oligonucleotides against
Lck inhibited the growth of EBV-transformed B-cells, we examined the
effects of the p120
antisense oligonucleotide on this
parameter. We found that when p120
expression was
significantly reduced, the cell count after 24 h was low (Fig. 6). Despite this decrease in cell proliferation,
O
production was increased in cells
incubated with the antisense oligonucleotide.
Figure 6:
Effect of p120 antisense
oligonucleotide on cell counts in cultures of EBV-transformed B
lymphocytes. Cells were cultured in the presence of various
oligonucleotides as described under ``Materials and
Methods.'' After 24 h in culture, the cells were isolated by
centrifugation (2000
g for 3 min at room temperature),
resuspended in Hanks' balanced salts solution, evaluated for
viability, and counted in a hemocytometer. The results represent the
mean ± S.E. of five separate experiments. For evaluating the
significance of differences between experimental and control cultures
(the controls were the cultures containing scrambled oligonucleotide), p values as determined by a paired t test are shown
in the figure.
Antisense strategy is a useful approach for elucidating the
role of different proteins in signal transduction(46) . Using
an 18-mer antisense oligonucleotide spanning the start codon of the
p120 message, we examined the function of
p120
in the control of respiratory burst oxidase
activity in EBV-transformed B lymphocytes. In agreement with Cheung and
Dosch(40) , we observed that these cells took up
oligonucleotides in a time-dependent fashion and that the antisense
oligonucleotide, but not the sense or scrambled oligonucleotide, was
able to reduce the expression of p120
. When
p120
expression was reduced,
O
production by phorbol-and
ionomycin-stimulated lymphocytes increased, although
O
production by protein A
(Pansorbin)-stimulated cells remained unchanged. Both phorbol- and
ionomycin-stimulated O
production, but
not the Pansorbin-triggered burst, appear to be dependent on the
activation of protein kinase C, as indicated by results obtained with
GFX. The antisense results, therefore, point to a specific role for
p120
in the protein kinase C-dependent activation of
the respiratory burst oxidase in EBV-transformed B-cells.
Several
laboratories have presented evidence showing a link between
p120 and protein kinase C-dependent signaling
pathways. In T-cells, an increase in the amount of active p21
that was induced by phorbol but not by receptor-dependent stimuli
has been related to a protein kinase C-mediated inactivation of
p120
(7) . In fibroblasts, overexpression of
p120
inhibited the activation of mitogen-activated
protein kinase by phorbol but not by receptor-dependent
stimuli(16) , presumably by causing a selective blockade of
signals from activated protein kinase C due to the inactivation of
p21
. Thus, for both cell types, p120
has been proposed as a negative regulator in a protein kinase
C-dependent signaling pathway. Our results point to a similar role for
p120
as a down-regulator of a protein kinase
C-dependent signaling pathway leading to an activated NADPH-oxidase.
Among the factors that regulate the activity of small guanine
nucleotide-binding proteins is p190, a 190-kDa
protein that accelerates the deactivation of guanine nucleotide-binding
proteins of the Rho class(32) . p190
is of
interest in regard to oxidase activation because Rac2, one of its
targets, is known to participate in the activation of the respiratory
burst oxidase both in the cell-free system (47, 48) and in whole cells(33) . In accord
with this idea, p190
was shown to inhibit
Rac2-dependent oxidase activation in the cell-free system (37, 49) . p190
is known to
associate with p120
in many cell types (25, 26, 30, 49) and using
anti-phosphotyrosine antibodies, we found a phosphorylated 190-kDa
protein, which could be identified as p190
, in
immunoprecipitates of p120
from EBV-transformed B
lymphocytes (data not shown). The possibility therefore exists that the
deficiency of p120
induced by the antisense
oligonucleotide resulted in a deficiency of p190
,
leading to persistent oxidase activity due to a delay in the
deactivation of Rac2. Unfortunately, we were unable to detect
p190
on Western blots of extracts from
oligonucleotide-treated cells, so we have no direct evidence as to the
effect of the antisense oligonucleotide on p190
concentrations in the transformed lymphocytes. However, our
finding that phorbol-dependent but not receptor- (i.e. Pansorbin-) dependent oxidase activation is affected by the
antisense oligonucleotide is difficult to explain on the basis of an
indirect effect of p120
mediated through
p190
, because Rac2 is involved in both phorbol- and
receptor-mediated activation of the respiratory burst oxidase (33) so that an effect occurring via p190
should have involved both phorbol-activated and
receptor-activated O
production. The
results therefore suggest a direct effect of p120
on
the oxidase activation cascade.
Rap1A is a member of the Ras
superfamily of guanine nucleotide-binding proteins. Its participation
in the regulation of oxidase activity is suggested by its
copurification with cytochrome b(50) and by the finding that overexpression of Rap1A
mutants fixed in either the active or inactive conformation decrease
oxidase activity in transfected B lymphocytes(51) . Since
p120
has been shown to interact with the putative
effector region of rap1A in vitro(52) ,
p120
could participate in the physiological
regulation of Rap1A by competitively interfering with its deactivation
by Rap1-GAP, an 85-95-kDa Rap1-specific GTPase-activating protein (53) . Accelerated deactivation of Rap1A in the
p120
-depleted cell could therefore account for the
effect of the antisense oligonucleotide on oxidase activity. Whether a
mechanism based on Rap1A deactivation could account for the difference
between phorbol-activated and receptor-activated cells remains to be
determined.
A third alternative is presented by the interactions
between the N-terminal SH2 domain of p120 and various
proteins containing phosphorylated
tyrosines(19, 25, 54, 55) . The
inhibition in the growth of EBV-transformed B-cells by the
p120
antisense oligonucleotide could result from
interactions between p120
and proteins other than
p190
or p62, the two proteins that are commonly found
in p120
immunoprecipitates. The Src-like tyrosine
kinase Lck is a candidate of particular interest, since Lck forms a
complex in vitro with the phosphorylated N-terminal SH2 domain
of p120
(23) and is thought to play a
significant role in the transformation of EBV-transformed B
lymphocytes(40) . The inhibition of cell growth caused by
p120
antisense oligonucleotides might somehow be
connected to the interaction between p120
and Lck.