(Received for publication, June 30, 1995; and in revised form, August 29, 1995)
From the
Treatment of Swiss mouse 3T3 fibroblasts with certain cyclic
nucleotide phosphodiesterase inhibitors (theophylline, SQ 20,006, and
MY-5445) prevents the activation of the M 70,000
S6 kinase (p70
) induced by a variety of external stimuli.
Concentrations giving half-maximal inhibition were 800, 50, and 25
µM, respectively. Western blot analysis and immunocomplex
kinase assays showed that these compounds inhibit the phosphorylation
and activation of p70
without affecting the erk-encoded mitogen-activated protein (MAP) kinases or the rsk-encoded S6 kinase (p90
). A distinct
collection of cAMP and cGMP agonists and analogues did not suppress
p70
activation, indicating that 1) high intracellular
cyclic nucleotide concentrations do not antagonize the p70
pathway and 2) phosphodiesterase inhibitors block p70
activation by a mechanism that is independent of cAMP or cGMP
production. The effect of theophylline and SQ 20,006, but not MY-5445,
on p70
signaling may be due in part to the inhibition of
a phosphatidylinositol 3-kinase that acts upstream of
p70
. Finally, in contrast to many other cell types, cAMP
and cGMP were also found to have no inhibitory effect on the MAP
kinase/p90
signaling pathway in Swiss 3T3 fibroblasts.
Addition of mitogens to quiescent mammalian cells induces a
signaling cascade that leads to the multiple phosphorylation of 40 S
ribosomal protein S6(1) . S6 phosphorylation is thought to
increase the rate of synthesis of certain proteins which are required
for efficient G progression and whose mRNAs contain a
polypyrimidine tract at the 5` end(2) . Two families of
mitogen-stimulated S6 kinases have been identified: the M
70,000 S6 kinases (p70
) (
)(1, 3, 4) and the M
90,000 ribosomal S6 kinase
(p90
)(5) . Enzymes in both families are activated
by phosphorylation of serine/threonine
residues(6, 7, 8) . p90
is
activated by mitogen-activated protein (MAP) kinases (9) and
participates in a signaling network that includes ras, raf-1, and
Mek1(10) . By contrast, p70
lies on a distinct
pathway that does not appear to include MAP kinases (11) .
Injection of antibodies that neutralize p70 activity (12) and use of the immunosuppressant rapamycin, which blocks
the activation of the enzyme(13, 14) , has suggested
that p70
function during the G
phase of the
cell cycle is important for proliferation in some cell types. Three
serines and one threonine clustered at the carboxyl terminus of
p70
become phosphorylated in response to mitogen
treatment(15) . However, recent data have shown that deletion
of the carboxyl terminus (16) or mutation of the four
mitogen-induced phosphorylation sites to acidic residues (
)yields p70
molecules which can still be
activated by mitogens. Therefore, the contribution of these sites to
enzyme activation remains unclear. In addition to the four
mitogen-responsive phosphorylation sites, p70
contains
other phosphates that turn over very slowly and that appear to be
essential for enzyme activity. Rapamycin induces the dephosphorylation
of these unmapped sites and therefore prevents the activation of
p70
(17) .
Little is known about the signaling
components that function upstream of p70. One approach to
identify participants in the p70
pathway has been to
study the mechanism of action of inhibitors of the pathway. For
example, Kunz and co-workers (18) showed that rapamycin
suppresses the growth of Saccharomyces cerevisiae by
interacting with two gene products encoded by TOR1 and TOR2. Homologous proteins were subsequently found in higher
eucaryotes(19) . These proteins show significant homology to
the catalytic subunit of mammalian phosphatidylinositol (PtdIns)
3-kinase(20) , which plays an important role in mitogenesis and
other cellular responses(21, 22) . It was subsequently
shown that wortmannin and other specific inhibitors of mammalian PtdIns
3-kinase also prevent p70
activation induced by a variety
of agents(23, 24, 25) . Together, these
results suggested that PtdIns 3-kinase or a related enzyme might be
involved in the activation of p70
. This conclusion is
supported by the recent observation that expression of a constitutively
active PtdIns 3-kinase leads to activation of p70
and
phosphorylation of a novel site within the kinase catalytic
domain(26) .
A second possible class of inhibitors of the
p70 pathway was suggested by work of Thomas and
co-workers(27) , who showed that pretreatment of Swiss mouse
3T3 fibroblasts with theophylline or SQ 20,006 blocked the
serum-induced phosphorylation of S6. We demonstrate here that these two
compounds block S6 phosphorylation by selectively inhibiting the
activation of p70
. Theophylline and SQ 20,006 are best
known as nonspecific cyclic nucleotide phosphodiesterase
inhibitors(28, 29) that might raise the intracellular
concentration of cAMP and cGMP, leading to the activation of cAMP- and
cGMP-dependent protein kinases (PKA and PKG, respectively). It has
recently been shown that cAMP antagonizes p70
activation
in T cells (30) and the MAP kinase/p90
pathway in
a number of other cell
types(31, 32, 33, 34, 35) .
However, we show that inhibition of p70
activation in
Swiss 3T3 fibroblasts by theophylline and SQ 20,006 is independent of
increased cyclic nucleotide concentrations or PKA activation. Finally,
we find that cyclic nucleotides do not negatively regulate either the
p70
or the MAP kinase/p90
pathways in this
cell type.
Wild-type S49 mouse lymphoma cells (subclone 24.3.2) were grown as
described previously(38) . To make extracts, 1-2
10
cells were centrifuged at 500
g for 5
min, washed twice with cold phosphate-buffered saline (PBS; 137 mM NaCl, 3 mM KCl and 12 mM P
, pH 7.4),
and resuspended in 400 µl of extraction buffer. The cells were
homogenized with 10 strokes in a Potter-Elvehjem homogenizer, and the
homogenates were centrifuged at 4 °C. Supernatants were retained.
BAC-1 macrophages were grown to confluence in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and colony-stimulating factor-1. Cells were starved for colony-stimulating factor-1 for 27 h before use. Cells extract supernatants were prepared as described above for the fibroblasts.
For S6 kinase immunocomplex
assays, extract supernatants were diluted into immunoprecipitation
buffer (50 mM Tris, 1% Triton X-100, 50 mM NaCl, 20
mM NaF, 1 mM benzamidine, 5 mM EGTA, 10
mM PP, 30 mM pNPP, 200 µM vanadate, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 0.1% BSA,
0.1 mM DTT, and 0.1 mM PMSF, pH 7.2) and the
solutions were incubated with antibodies to p70
for 2 h
at 4 °C. Protein A-agarose beads (Sigma) which had been
preincubated with immunoprecipitation buffer were added, and the
samples were incubated for 1 h at 4 °C. The beads were washed twice
with immunoprecipitation buffer and twice with S6 kinase assay buffer
without DTT. S6 kinase assays were then performed as described above.
For MAP kinase immunocomplex assays, extract supernatants in
immunoprecipitation buffer were incubated with antibody 122 against
p42 as described above. After washing with
immunoprecipitation buffer, the protein A-agarose beads were washed
twice with MAP kinase assay buffer (30 mM Tris, pH 8, 20
mM MgCl
, 2 mM MnCl
, 0.1%
Triton X-100, and 0.1 mM DTT). MAP kinase assays were
initiated by adding 15 µl of MAP kinase assay buffer containing 10
µM ATP, 2 µM PKI, 10 µg of myelin basic
protein, and 0.33 µl of [
P]ATP. After
30 min at 37 °C the reactions were stopped by adding 10 µl of
SDS sample buffer and heating at 95 °C. The reactions were
subjected to electrophoresis on SDS-20% polyacrylamide gels and
autoradiography.
Figure 1:
Effect of cyclic nucleotide reagents on
the EGF-induced activation of S6 kinase. Fibroblasts were pretreated as
described below and then at t = 0 min 5 nM EGF
was added for the indicated times. Extract supernatants were prepared
and assayed for S6 kinase activity as described under
``Experimental Procedures.'' Results are means of at least
two independent experiments. A, fibroblasts were pretreated
with 5 mM theophylline (), 1 mM SQ 20,006
(
) or without drug (
) for 15 min. B, fibroblasts
were untreated (
) or pretreated with 300 µM PGE
for 15 min (
) or with 500 µM 8-Br-cAMP (
) or 500 µMS
-8-Br-cAMPS (
) for 30 min. C,
fibroblasts were untreated (
), or pretreated with 500 µM 8-Br-cGMP (
) for 30 min or with 300 µM MY-5445
(
) or 300 µM SNAP (
) for 15
min.
Figure 2:
Dose
response for inhibition of S6 kinase activation. Fibroblasts were
pretreated for 15 min with increasing concentrations of theophylline
(), SQ 20,006 (
) or MY-5445 (
) and then stimulated
for 20 min with 5 nM EGF. S6 kinase activity was measured in
cell extract supernatants. Results show averages of at least two
determinations.
Figure 3:
Selective inhibition of p70 activation and phosphorylation. A, separation of
p70
and p90
by anion exchange
chromatography. Fibroblasts (4
15 cm plates) were treated with
PBS (
), 5 nM EGF for 2.5 min (
), 1 mM SQ
20,006 for 15 min (
), or 1 mM SQ 20,006 for 15 min
followed by 5 nM EGF for 2.5 min (
). Cell extract
supernatants were subjected to chromatography on a Mono Q column and
fractions were assayed for S6 kinase activity (see ``Experimental
Procedures''). B, cells were pretreated as described
below and then treated without (lane 1) or with (lanes
2-9) 5 nM EGF for 20 min. p70
was
then assayed in immunoprecipitates (upper panel) or subjected
to Western blot analysis (lower panel) as described in
``Experimental Procedures.'' The upper panel shows
an autoradiograph of
P
incorporated into S6
during the kinase assay. Pretreatments were: lanes 1 and 2, PBS, 15 min; lane 3, 5 mM theophylline,
15 min; lane 4, 1 mM SQ 20,006, 15 min; lane
5, 500 µM 8-Br-cAMP, 30 min; lane 6, 500
µMS
-8-Br-cAMPS, 30 min; lane
7, 300 µM PGE
, 15 min; lane 8,
500 µM 8-Br-cGMP, 30 min; and lane 9, 300
µM MY-5445, 15 min.
Figure 6:
Effect of cyclic nucleotide reagents on
MAP kinases and p90. A, fibroblasts were treated
with 5 nM EGF for the indicated times and p42
and p44
were detected on Western blots. B, cells were pretreated as described below and then treated
without (lane 1) or with 5 nM EGF (lanes
2-9), 1 nM insulin (lanes 10 and 11) or 384 pM PDGF (lanes 12 and 13) for 5 min. p42
in cell extract supernatants
was then assayed in immunoprecipitates (upper panel) and
p42
and p44
were subjected to Western
blot analysis (lower panel) as described under
``Experimental Procedures.'' The upper panel shows
an autoradiograph of
P
incorporated into
myelin basic protein during the kinase assay. Pretreatments were: lanes 1, 2, 10, and 12, PBS, 15
min; lane 3, 5 mM theophylline, 15 min; lane
4, 1 mM SQ 20,006, 15 min; lanes 5, 11,
and 13, 500 µM 8-Br-cAMP, 30 min; lane
6, 500 µMS
-8-Br-cAMPS, 30 min; lane 7, 300 µM PGE
, 15 min; lane
8, 500 µM 8-Br-cGMP, 30 min; and lane 9, 300
µM MY-5445, 15 min. C, Western blot analysis of
p90
. Cell treatments were the same as described above for B.
All evidence obtained
so far indicates that p70 is activated by
phosphorylation(7, 15, 17, 26) .
Therefore, theophylline and SQ 20,006 might reduce p70
activity either by affecting its phosphorylation or by
stimulating the degradation of the enzyme. To distinguish between these
two possibilities, we examined the protein levels and phosphorylation
state of p70
on immunoblots. p70
migrates
differently in SDS-polyacrylamide gels depending on its phosphorylation
state, with the highly phosphorylated and active form migrating more
slowly than the dephosphorylated, inactive enzyme(7) . In
resting cells the kinase was present mainly as hypophosphorylated
species and kinase activity in immunoprecipitates was low (Fig. 3B, lane 1). After EGF treatment
p70
activity increased and only the most highly
phosphorylated forms were detected on the immunoblot (Fig. 3B, lane 2). When EGF was added to cells
pretreated with theophylline or SQ 20,006 most of the p70
molecules remained hypophosphorylated and the kinase activity did
not increase (Fig. 3B, lanes 3 and 4). These data demonstrate that theophylline and SQ 20,006
either prevent the EGF-induced phosphorylation of p70
or
stimulate its dephosphorylation. In addition, no change in p70
protein levels was seen after treatment with theophylline or SQ
20,006 (Fig. 3B, lower panel), indicating that
these agents do not induce the degradation of the enzyme.
In addition to EGF, p70 is activated
by a wide variety of agonists that act by different mechanisms. These
include peptide growth factors that function through receptor-tyrosine
kinases (insulin and PDGF) or receptors coupled to guanosine
nucleotide-binding proteins (bombesin), the Ca
ionophore A23187, the protein kinase C activator PMA and
cycloheximide. To determine whether 8-Br-cAMP inhibits p70
activation induced by any of these other compounds, cells were
pretreated with or without 8-Br-cAMP and then exposed to one of these
p70
agonists. Cell extracts were prepared at those times
when p70
was maximally stimulated. Kinase activity was
increased to distinct levels and pretreatment with 8-Br-cAMP had little
or no effect on the activation of p70
induced by any of
the compounds tested (Table 2). By contrast, theophylline
strongly inhibited the activation of p70
by every agonist
tested (Table 2). Three conclusions can be made from these
results. First, cAMP and PKA do not antagonize the p70
activation pathway in fibroblasts. Second, theophylline and SQ 20,006
block p70
signaling by a mechanism that is independent of
cAMP or PKA. And third, the target of theophylline seems to be a common
regulatory element in p70
signaling pathways induced by
different agonists.
Figure 4:
Effect of 8-Br-cAMP on p70 activity in different cell types. Cycling S49 lymphoma cells or
Swiss fibroblasts were treated for 30 min with PBS (black
bars) or 500 µM 8-Br-cAMP (gray bars). BAC-1
macrophages were co-treated for 15 min with 24,000 units/ml of
colony-stimulating factor-1 in the presence (gray bars) or
absence (black bars) of 500 µM 8-Br-cAMP. Extract
supernatants were prepared and assayed for S6 kinase activity (see
``Experimental Procedures'').
Figure 5:
Dose-response for inhibition of PtdIns
3-kinase activity. Partially purified p110p85
heterodimers were incubated with increasing amounts of theophylline
(
), SQ 20,006 (
) or MY-5445 (
) and assayed for
lipid kinase activity (see ``Experimental
Procedures'').
To directly
examine the effect of cyclic nucleotide reagents on MAP kinase
activity, the erk-2-encoded p42 was
precipitated with a specific polyclonal antibody and assayed in
immunocomplexes. These assays showed that p42
was
strongly activated by EGF (Fig. 6B, upper
panel, lanes 1 and 2). This response was still
fully intact in cells pretreated with cAMP analogues or PGE
(Fig. 6B, upper panel, lanes
5-7). p42
was also activated normally in
cells pretreated with 8-Br-cGMP (Fig. 6B, upper
panel, lane 8). In contrast to the results obtained with
p70
( Fig. 1and Fig. 3B),
pretreatment of cells with theophylline, SQ 20,006, or MY-5445 did not
block the activation of p42
by EGF (Fig. 6B, upper panel, lanes 3, 4, and 9). Thus, these compounds do not disrupt all
EGF receptor-mediated responses. We also tested whether MAP kinase
activation induced by insulin or PDGF is affected by cAMP. Although
these two growth factors were relatively poor activators of
p42
at these concentrations, it was evident that
addition of 8-Br-cAMP had no inhibitory effect (Fig. 6B, upper panel, lanes
10-13). Examination of MAP kinases in these cell extracts on
Western blots indicated that the activation of p44
, like
p42
, was also resistant to high cyclic nucleotide levels
and phosphodiesterase inhibitors (Fig. 6B, lower
panel). In addition, the growth factor-induced mobility shift of
p90
was not affected by these reagents (Fig. 6C); these results confirm and extend the finding
in Fig. 3A. Thus, in Swiss 3T3 fibroblasts activation
of the erk-encoded MAP kinases and p90
is not
antagonized by cAMP or cGMP.
It was reported earlier that treatment of Swiss mouse 3T3
fibroblasts with the nonselective phosphodiesterase inhibitors
theophylline and SQ 20,006 blocks the mitogen-induced phosphorylation
of ribosomal protein S6(27) . We show here that these two
compounds mediate this effect by inhibiting the activation of
p70 (Fig. 3). Theophylline and SQ 20,006 did not
act as general kinase inhibitors or disrupt all receptor-mediated
responses, as shown by the fact that activation of MAP kinases and
p90
was not inhibited (Fig. 3A and 6).
Thus, like rapamycin (13, 14) and
wortmannin(23, 24, 25) , theophylline and SQ
20,006 are selective inhibitors of the p70
pathway as
opposed to the MAP kinase/p90
pathway. Also like
rapamycin and wortmannin, theophylline and SQ 20,006 did not inhibit
p70
directly
, but rather caused a reduction
in the phosphate content of the protein (Fig. 3B). This
could be due to activation of a negative regulator of the p70
pathway or inhibition of a positive regulator. Inhibition by
theophylline was rapidly reversed by washing out the inhibitor even in
the presence of cycloheximide,
indicating that all of the
components required for p70
activation are still present
in theophylline-treated cells.
Theophylline and SQ 20,006 inhibit
cyclic nucleotide phosphodiesterases in
vitro(28, 29) . Therefore, one hypothesis to
explain the mechanism of inhibition of p70 would be that
the compounds increase intracellular cAMP levels, and as a result PKA
is activated and phosphorylates a regulatory protein in the p70
pathway. However, several lines of evidence indicate that
cAMP/PKA do not negatively regulate p70
in these cells.
First, theophylline and SQ 20,006 were not found to be effective cAMP
agonists under conditions in which p70
was inhibited (Table 1). This result is consistent with other reports that
theophylline has little or no effect on basal cAMP levels in mouse
fibroblasts(47) . Second, increasing the intracellular cAMP
concentration by addition of cell-permeant cAMP analogues or PGE
did not block the stimulation of p70
induced by EGF (Fig. 1B) or other agonists (Table 2), even
though PKA was strongly activated (Table 1). These results are
consistent with the observation made earlier that PGE
does
not inhibit serum-induced S6 phosphorylation in
vivo(27) . Finally, work by others has shown that cAMP/PKA
promotes the proliferation of Swiss mouse 3T3 fibroblasts(48) ;
therefore, this pathway would not be expected to interfere with the
activation of p70
, which is also required for efficient
cell cycle progression in this cell type(12, 13) .
In contrast to our results, Monfar et al.(30) recently reported that cAMP inhibits the
interleukin-2-mediated activation of p70 in CTLL-20 T
cells, in part by antagonizing the activity of PtdIns 3-kinase. This
discrepancy could be due to cell type or agonist specificity. Unlike
Swiss fibroblasts, T cells arrest in the G
phase of the
cell cycle in response to high cAMP concentrations (45) or
rapamycin treatment(49) . Since rapamycin inhibits
interleukin-2-mediated p70
activation(14) , it
seems plausible that the p70
pathway could also be the
target for a cAMP-dependent cell cycle block in T cells. On the other
hand, Monfar et al. (30) used forskolin/IBMX as a cAMP
agonist, raising the possibility that p70
activation in
CTLL-20 T cells might be sensitive to forskolin/IBMX and not to cAMP per se. Our finding that 8-Br-cAMP has no effect on
p70
activity in a T cell lymphoma line (Fig. 4)
also suggests that the sensitivity of T cell p70
to
IBMX/forskolin might be independent of cAMP. Macrophages also arrest in
the G
phase of the cell cycle in response to high cAMP
levels or exposure to rapamycin (46) . We found that 8-Br-cAMP
enhances rather than inhibits the activation of p70
by
colony-stimulating factor-1 in these cells (Fig. 4).
Since
theophylline and SQ 20,006 also inhibit cGMP-specific
phosphodiesterases in vitro(28, 29) , we
explored the possibility that cGMP and PKG might be involved in the
inhibition of p70 activation. Although MY-5445, a
selective inhibitor of cGMP-specific phosphodiesterases, strongly
blocked the activation of p70
, 8-Br-cGMP and SNAP, a
guanylyl cyclase activator, had no effect (Fig. 1C and Table 2). These results strongly suggest that inhibition of
p70
by MY-5445 is not a consequence of increased cGMP
production or PKG activity.
Having ruled out the possibility that
cAMP/PKA or cGMP play a role in theophylline-induced p70 inhibition, we searched for alternative mechanisms that might
mediate this response. PtdIns 3-kinase, which has been proposed to act
as a positive upstream regulator of
p70
(23, 24, 25, 26) ,
seemed to be a likely target. Indeed, we show here that theophylline
and SQ 20,006, but not MY-5445, also inhibit PtdIns 3-kinase activity in vitro (Fig. 5). However, inhibition of PtdIns
3-kinase cannot be the only reason why theophylline blocks the
p70
pathway. We showed earlier that wortmannin, a
specific inhibitor of PtdIns 3-kinase, is a poor inhibitor of bombesin-
and PMA-induced p70
activation(25) . We concluded
from those studies that some pathways leading to p70
activation are independent of PtdIns 3-kinase(25) . By
contrast, theophylline strongly inhibited the p70
response to all of the agonists listed in Table 2,
including bombesin and PMA. These data suggest that theophylline might
act on another target in addition to PtdIns 3-kinase. This target
appears to be a regulatory element that functions either independently
of all activation pathways (such as a p70
phosphatase) or
in every pathway (such as a p70
kinase).
Theophylline
has several other known cellular effects that could play a role in its
ability to inhibit the activation of p70. Theophylline
mobilizes intracellular Ca
by opening ryanodine
receptor Ca
channels (50) and is a potent
antagonist of adenosine receptors(29, 51) . The
possible role of Ca
or adenosine receptors in the
activation of p70
remains to be tested. More importantly,
many purine analogues and derivatives such as theophylline are
competitive inhibitors of kinases due to a structural similarity to ATP (52) . This property may account for the ability of
theophylline to inhibit PtdIns 3-kinase activity in vitro (Fig. 5). Therefore, theophylline may inhibit the
p70
pathway by targeting an upstream kinase that is
required for a response to all agonists tested so far. Since no such
upstream component has been identified yet, this hypothesis cannot be
tested directly. SQ 20,006 and MY-5445 bear some structural resemblance
to theophylline and may act in a similar manner. Identification or
design of specific and potent inhibitors of p70
activation, based on the compounds we identified here, could lead
to the isolation of upstream activators that function in this pathway.
We show here that the activation of p42/p44
and p90
in Swiss fibroblasts is also resistant to
high cAMP and cGMP levels (Fig. 6). MAP kinase activation in
PC12 cells is similarly resistant to high cAMP levels(31) . By
contrast, negative regulation of the MAP kinase/p90
cascade by cAMP has been observed in Xenopus oocytes,
Rat1 cells, smooth muscle cells, CHO cells, and adipocytes stimulated
with a variety of
agonists(32, 33, 34, 35) . In some
of these cell types high cAMP levels also antagonize cell proliferation (33, 34) or meiotic cell division(35) .
Several different mechanisms may contribute to inhibition of MAP kinase
activation, including a reduced activation of Raf-1 due to
phosphorylation by PKA(34) .
Our results, together with
those from other laboratories, indicate that cyclic
nucleotide-dependent signaling may interact in a variety of ways with
the p70 and MAP kinase/p90
pathways. These
interactions may be important for generating cell type-specific
responses to identical physiological conditions. Identification of
regulatory components in the p70
pathway will allow us to
further elucidate the mechanisms of cell type-specific control.