(Received for publication, December 18, 1995; and in revised form, February 5, 1996)
From the
In order to study the regulation of the ribosomal protein S6
kinase, p70, by G protein-coupled receptors, Rat-1
fibroblasts were stably transfected with two versions of the
adrenergic receptor. Stimulation of clone 1C cells,
which express 3.5 pmol/mg of protein of the human
receptor, with the
agonist UK 14304 led to a
transient increase in p70
activity. UK 14304 also
activated p70
in a clone expressing the porcine
receptor (400 fmol/mg of protein). Lysophosphatidic
acid (LPA), acting through endogenous G protein-coupled receptors, also
activated p70
in
receptor-transfected
and in nontransfected cells. Activation of p70
by both UK
14304 and LPA was accompanied by increased phosphorylation of the
protein. Rapamycin completely blocked the activation of p70
by both agents. Activation of p70
by UK 14304 and
by LPA, but not by platelet-derived growth factor (PDGF), was blocked
by preincubation of cells with pertussis toxin. Wortmannin, a selective
inhibitor of phosphoinositide (PI) 3-OH kinase, prevented activation of
p70
by UK 14304, LPA, and PDGF. These data indicate that
p70
is regulatable by G
-coupled receptor
agonists in a pertussis toxin-sensitive fashion in Rat-1 fibroblasts
and that activation of p70
by such agents appears to
involve an isoform of PI 3-kinase.
Mitogenic stimulation of cells results in increased
phosphorylation of the ribosomal protein S6. Increased S6
phosphorylation correlates with elevated rates of translation and may
be partly responsible for the increase in protein synthesis triggered
by mitogens(5) . Phosphorylation of S6 occurs on five closely
positioned sites and is catalyzed by a protein kinase known as
p70(6, 7, 8) . Prevention of
the activation of p70
represses cell growth and in some
cell types blocks entry into S
phase(9, 10, 11, 12) .
The
mechanism of activation of p70 has not been fully
characterized but appears to involve its phosphorylation on multiple
sites by more than one protein kinase(13, 14) .
Various lines of evidence indicate that p70
activation is
independent of the p21
/MAP (
)kinase
pathway(15, 16) . For example, p21
and p74
mutants block activation of
MAP kinases but not p70
and certain PDGF receptor
mutants, which activate p21
normally, fail to
activate p70
(16) . The immunosuppressive
macrolide rapamycin completely blocks the activation of p70
but has no effect on MAP kinase activation (17, 18) and as such has become a useful tool for
delineating p70
-specific signaling events. Other studies
have shown that the PI 3-kinase inhibitor wortmannin prevents
activation of p70
by a range of
agonists(19, 20, 21, 22, 23) ,
suggesting that PI 3-kinase lies upstream of p70
.
Mitogen receptors can be subdivided into those which either are or
couple to a tyrosine kinase (the tyrosine kinase class) and those which
couple to heterotrimeric G proteins (the GPCRs). Although it was
considered that these two classes signalled via distinct and mutually
exclusive mechanisms, recent evidence has challenged this view. For
example, the p21/MAP
kinase pathway,
originally thought only to be activated by tyrosine kinase receptors,
is now known to be activated by several
GPCRs(24, 25, 26, 27, 28, 29) .
These include the
adrenergic, m
muscarinic, and LPA receptors which couple to the pertussis
toxin-sensitive G
subfamily. The mechanisms linking
G
-linked receptors with the MAP kinase and other mitogenic
signaling pathways are still unclear but accumulating evidence from
several laboratories indicates an involvement of G protein
subunit complexes(30, 31, 32) .
As a means
to study the mitogenic signaling of GPCRs, we have previously
transfected Rat-1 fibroblasts with the human -C10
adrenergic receptor and shown that the receptor interacts directly with
two pertussis toxin-sensitive G proteins, G
and
G
, and thus induces both adenylyl cyclase inhibition and
phospholipase D activation(33, 34) . Activation of the
receptor also results in a mitogenic response characterized by a
pertussis toxin-sensitive activation of the
p21
/MAP kinase pathway and DNA
synthesis(26, 29) . Given the role of p70
in mitogenic signaling, we reasoned that this kinase should also
be activated following stimulation of
receptors in
these cells. Our results show that this does occur and thus provide the
first demonstration of p70
activation by a receptor
acting solely through G
. We further show that wortmannin
attenuates activation of p70
suggesting that an isoform
of PI 3-kinase is involved in the activation mechanism.
To examine possible coupling of GPCRs to p70,
we used Rat-1 fibroblasts stably transfected with either the human
adrenergic receptor (clone 1C) or with an
equivalent HA-tagged version of the porcine receptor (clone TAG WT3).
Stimulation of 1C cells (which express around 3.5 pmol of receptor/mg
of membrane protein(33) ) with the
agonist UK
14304 led to a transient increase in p70
activity
measured by an immunocomplex kinase assay (Table 1). Maximal
activation (approximately 3-fold) was observed around 10-20 min
after stimulation and although activity declined thereafter it remained
above basal levels for at least 3 h (data not shown). In TAG WT3 cells
(which express around 400 fmol of receptor/mg of membrane protein), UK
14304 activated p70
to a lesser extent than in 1C cells (Table 1). In parental Rat-1 cells, which do not express
detectable levels of
receptor(33) , UK 14304
did not significantly increase p70
activity. In addition
to the effects of UK 14304, the mitogenic glycerophospholipid LPA,
acting through endogenously expressed GPCRs, activated p70
in all three cell types (Table 1). Thus, activation of
p70
by G protein-coupled agonists may be a generalized
phenomenon in these cells which does not require heterologous high
level expression of the appropriate receptor.
As expected, the
tyrosine kinase receptor ligand PDGF also strongly coupled to
p70 activation in both
receptor-transfected and nontransfected cells (Table 1).
The relative activation of p70
induced by PDGF, and
indeed by LPA, was substantially higher in the untransfected cells, but
this appears to reflect elevated basal p70
activities in
both the TAG WT3 and 1C cells (Table 1). Attempts to
down-regulate basal p70
activity in these cells by
manipulating the conditions used for serum deprivation were
unsuccessful (data not shown). Similarly, elevated MAP kinase basal
activity in these cells has previously been reported(29) .
Activation of p70 correlates strongly with its
phosphorylation on multiple sites(13, 14) . Therefore,
we next examined whether the
receptor-mediated
activation of p70
was accompanied by enhanced
phosphorylation of the protein. Phosphorylation of p70
results in a reduction in the relative mobility of the protein on
SDS-polyacrylamide gel electrophoresis, and this is often used as an
indicator of p70
activation status(17) . Fig. 1shows an immunoblot of p70
following
treatment of cells with different agonists. In parental Rat-1 cells,
PDGF and, to a lesser extent, LPA induced the appearance of slower
migrating forms of p70
, whereas UK 14304 had no such
effect. Immunoblotting of clone 1C cells revealed that most of the
p70
protein was detected as the slower migrating forms
even in unstimulated cells, consistent with the elevated basal
p70
activities in these cells (Table 1).
Phosphatase treatment of p70
immunoprecipitated from LPA-
or UK 14304-treated clone 1C cells resulted in the disappearance of the
most slowly migrating forms of p70
and the coincident
appearance of a single band with faster mobility (Fig. 1B). This effect was also evident in samples
prepared from unstimulated clone 1C cells indicating that p70
exhibits increased basal phosphorylation as well as elevated
basal activity in these cells.
Figure 1:
G protein-coupled receptor activation
results in increased phosphorylation of p70. A,
Rat-1 cells (left panel) or clone 1C cells (right
panel) were left untreated (C) or treated for 10 min with
10 ng/ml PDGF (P), 10 µM LPA (L), or 1
µM UK 14304 (U). Lysates were prepared, and 25
µg of lysate protein from each sample was immunoblotted with
anti-p70
. The fastest and slowest of the four
immunoreactive bands representing various phosphorylation states of the
enzyme are indicated by the arrowheads. B, p70
immunoprecipitated from control, LPA, or UK 14304-stimulated
clone 1C cells were treated or not with potato acid phosphatase and
then subjected to immunoblotting with anti-p70
antibodies
as described under ``Experimental Procedures.'' C,
clone 1C cells were metabolically labeled with
P
and then left untreated (control) or treated with LPA or UK
14304. p70
was immunoprecipitated from the cells as
described under ``Experimental Procedures'' and
electrophoresed, and the dried gel was exposed to x-ray film for 24 h.
The p70
bands were excised from the gel and counted for
radioactivity. Duplicate immunoprecipitations from a single experiment,
performed on one other occasion with similar results, are
shown.
To confirm that activation of
p70 by GPCRs results in increased phosphorylation of the
protein, cells were metabolically labeled with
P
, and the levels of phosphate incorporated
into p70
were measured. Fig. 1C shows
that both UK 14304 and LPA induce an increase in the phosphorylation of
p70
. Counting of the
P incorporated into the
p70
bands revealed that LPA induced a 43.0 ± 4.0% (n = 4) increase and UK 14304 induced a 65.9 ±
4.3% (n = 4) increase in p70
phosphorylation. For comparison, PDGF, the most potent activator
of p70
used in this study, increased the phosphorylation
of p70
by around 2-fold in Rat-1 cells (data not shown).
Taken together, these results show that activation of p70
by GPCRs correlates with enhanced phosphorylation of the protein.
Whether G
-mediated signals result in phosphorylation of the
same sites on p70
induced by other effectors remains to
be determined.
The immunosuppressive drug rapamycin blocks the
activation of p70 by preventing or reversing its
phosphorylation at key sites necessary for
activity(11, 12, 13, 14, 17, 18) .
The mechanism underlying this effect has not been characterized
completely, but recent reports have identified the protein kinase
FRAP/RAFT (38, 39, 40) as a specific target
for rapamycin in mammalian cells. The activation of p70
by UK 14304, LPA, and PDGF in the present study was completely
blocked by pretreatment of cells with rapamycin (Fig. 2)
indicating that there are commonalities in the mechanisms employed by
both G protein-coupled agonists and other agents to elicit activation
of p70
. In contrast, only the activation of p70
by UK 14304 and LPA was blocked by pretreatment of cells with
pertussis toxin (Fig. 3), consistent with their effects being
mediated via the G
family of heterotrimeric G proteins.
Although pertussis toxin pretreatment also led to a significant
reduction in the p70
activity precipitated from
PDGF-stimulated cells (Fig. 4), this is entirely accounted for
by pertussis toxin-mediated inhibition of basal p70
activity. Thus, the relative increases in p70
activity induced by PDGF in control and pertussis toxin-treated
cells were 42 ± 4% and 45 ± 3% (n = 3),
respectively.
Figure 2:
Rapamycin prevents activation of
p70. Clone 1C cells were pretreated (open bars)
or not (filled bars) with 100 nM rapamycin for 10 min
prior to the addition of PDGF (10 ng/ml), LPA (10 µM), or
UK 14304 (1 µM) for 10 min. Lysates were prepared, and
p70
activity was measured on the immune complex as
described under ``Experimental Procedures.'' *, significant
inhibitory effect of rapamycin (p < 0.05, Student's t test)
Figure 3:
Pertussis toxin prevents activation of
p70 by G
-linked agonists. Clone 1C cells were
pretreated (open bars) or not (filled bars) with 25
ng/ml pertussis toxin for 16 h prior to the addition of PDGF (10
ng/ml), LPA (10 µM), or UK 14304 (1 µM) for
an additional 10 min. Lysates were prepared, and p70
activity was measured on the immune complex as described under
``Experimental Procedures.'' *, significant effect of
pertussis toxin treatment (p < 0.05, Student's t test).
Figure 4:
Wortmannin blocks activation of
p70 by UK14304. Clone 1C cells were pretreated for 5 min
with the indicated concentrations of wortmannin prior to the addition
of 1 µM UK 14304 for 10 min. Lysates were prepared, and
p70
activity was measured on the immune complex as
described under ``Experimental Procedures.'' Data are
expressed as a percentage of the maximal stimulatory effect of UK 14304
in the absence of wortmannin and are taken from a single experiment
done on three other occasions with similar
results.
Although the precise mechanisms of activation of
p70 are unknown, a number of studies using the selective
inhibitor wortmannin indicate that PI 3-kinase lies upstream of
p70
in a signaling cascade induced by a number of
tyrosine kinase receptors (19, 20, 21, 22, 23) . More
recent work suggests that p70
contains a set of
wortmannin-sensitive phosphorylation
sites(13, 14, 41) . We therefore sought
evidence for PI 3-kinase involvement in the activation of p70
by GPCRs. Pretreatment of clone 1C cells with wortmannin led to a
dose-dependent attenuation of p70
activation by UK 14304 (Fig. 4). Approximately 50% inhibition occurred with
concentrations of wortmannin around 30 nM with complete
inhibition evident at 50-100 nM. These concentrations
are similar to those reported to inhibit p70
activation
by other growth
factors(19, 20, 21, 22, 23) .
Wortmannin also prevented the full activation of p70
by
LPA (Table 2). We also tested the effects of LY 294002
(2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), another PI 3-kinase
inhibitor, structurally unrelated to wortmannin(20) .
Preincubation of clone 1C cells with 50 µM LY 294002
completely blocked the activation of p70
induced by UK
14304 as well as by LPA and PDGF (data not shown). Together, these data
suggest that PI 3-kinase is required for the activation of p70
by G
-linked agonists in Rat-1 cells. Whether this is
the same PI 3-kinase isoform believed to regulate p70
in
response to tyrosine kinase receptor ligands is not known. It is worth
noting that specific G protein
-activated forms of PI
3-kinase have been identified (1, 2, 3) and,
very recently, a novel G protein-activated PI 3-kinase was isolated and
cloned(4) . This protein, termed PI 3-kinase-
, is
regulated directly by both
and
G protein subunits and
is also inhibited by wortmannin. It will be of interest to determine
whether this isoform of PI 3-kinase is stimulated by UK 14304 and other
G protein-coupled ligands and whether it is required for the activation
of p70
.
In conclusion, we have shown that stimulation
of both endogenous and heterologously expressed G-coupled
receptors results in a pertussis toxin-sensitive activation and
coincident hyperphosphorylation of the S6 kinase p70
. The
activation is sensitive to both rapamycin and wortmannin and as such
may be similar mechanistically to that stimulated by ligands signaling
via tyrosine kinase receptors. Future work will be directed toward
understanding the precise mechanisms underlying the effects of
G
-coupled agonists on p70
, particularly the
potential role of
signaling complexes.