(Received for publication, May 9, 1995; and in revised form, July 10, 1995)
From the
Activation of p70 in cells stimulated with serum
correlates with the phosphorylation of seven sites. Pretreatment of
Swiss 3T3 cells with the immunosuppressant rapamycin blocks
phosphorylation of four of these sites (Thr
,
Thr
, Ser
, and Ser
), whereas
phosphorylation proceeds in the remaining three sites
(Ser
, Thr
, and Ser
). If
rapamycin is added post-serum stimulation, the pattern of
phosphorylation is qualitatively similar except that Ser
is still highly phosphorylated. The inhibitory effect of
rapamycin on serum-induced p70
activation and the
phosphorylation of Thr
, Thr
,
Ser
, and Ser
is rescued by FK506, providing
further evidence that the inhibitory effect is exerted through a
complex of rapamycin-FKBP12. Wortmannin treatment pre- or post-serum
stimulation inhibits phosphorylation of the same set of sites as
rapamycin, supporting the argument that both agents act on the same
pathway. Likewise, methylxanthine phosphodiesterase inhibitors block
p70
activation and phosphorylation of the same set of
sites as wortmannin and rapamycin. However, other agents that raise
intracellular cAMP levels have no inhibitory effect, leading to the
hypothesis that the inhibitory actions of methylxanthines on
p70
activity are not through activating protein kinase A
but through inhibition of an upstream kinase. Together the results
indicate that there are two kinase signaling pathways that must
converge to activate p70
and that only one of these
pathways is sensitive to rapamycin, wortmannin, and methylxanthine
inhibition.
p70 and p85
represent two isoforms
of the same kinase that are encoded by a common gene and are identical
except for a 23-amino acid extension at the amino terminus of
p85
(see Refs 1 and 2). Furthermore, both isoforms lie on
a p21
-p42
/p44
independent
signal transduction pathway(3, 4) , which bifurcates
at the level of the receptor(4) . Whereas p70
seems to be restricted to the cytoplasm(5) , the
amino-terminal extension of p85
harbors a nuclear
localization signal that constitutively targets it to the
nucleus(5) . The major substrate of the kinase in both
compartments of the cell appears to be the 40 S ribosomal protein S6
(see (6) ), whose multiple phosphorylation in the cytoplasm has
been implicated in the selective translational up-regulation of a
family of essential gene products(7, 8) . Consistent
with this finding, either microinjection of neutralizing antibodies
into cells (9) or treatment of cells with the immunosuppressant
rapamycin, which selectively blocks p70
/p85
phosphorylation and activation (10, 11, 12) , severely impedes cell cycle
progression.
Activation of p70/p85
is
associated with multiple phosphorylation of the enzyme, which can be
monitored as the slower migration of a family of bands on SDS-PAGE, (
)which collapse into a single band following treatment with
phosphatase or rapamycin(10, 13) . Initially,
phosphorylation at four major sites, Ser
,
Ser
, Thr
and Ser
, which are
clustered in a putative autoinhibitory domain, was found to parallel
p70
activation(14) . Substitution of these four
residues with acidic amino acids mimicked phosphorylation at these
sites(15) . Unexpectedly, the acidic form of the kinase was
still inactivated by rapamycin, and more surprisingly, inactivation was
shown to be associated with the dephosphorylation of a distinct set of
sites, with no effect observed on the phosphorylation of the four
identified sites(15) . Recently, these novel
rapamycin-sensitive sites have been identified as Thr
in
the catalytic domain and Thr
and Ser
located in a linker domain that couples the catalytic and
autoinhibitory domains. (
)Each of the sites was found to
reside in an atypical trypsin cleavage product, which largely explained
the difficulty in their identification. Earlier rapamycin studies (15) led to the hypothesis that at least two sets of
phosphorylation events regulate kinase activity, one of which was
controlled by mitogens and a second that appeared to be constitutively
activated. However, in re-examining this data, it was noted that in the
presence of serum, the parent and the acidic mutant exhibited a similar
mobility shift when analyzed by Western blots of SDS-PAGE(15) .
Furthermore, in both cases, rapamycin treatment caused a similar
increase in electrophoretic mobility of the protein. These data
suggested that one or more of the rapamycin-sensitive phosphorylation
sites was involved in the p70
mobility
shift(15) . Since a similar mobility shift is observed during
the rapid activation of p70
by
mitogens(4, 10) , which is blocked by rapamycin (10) , this finding raises the question of whether the
rapamycin-sensitive phosphorylation sites are also regulated by
mitogens.
Since p70 activation has not yielded to in vitro reconstitution(3, 16) , a number of
indirect approaches have been exploited in an attempt to identify key
regulatory points in this signaling
pathway(4, 17, 18) . The establishment of
regulatory points has been extremely valuable in elucidating other
signal transduction pathways(19) . In the case of
p70
, this line of study has led to the finding that the
antibiotic wortmannin, which blocks phosphatidylinositol 3-OH kinase
activation, also blocks p70
activation(17, 18) . Though the role of
phosphatidylinositol 3-OH kinase in regulating this pathway has been
questioned(4, 20) , wortmannin clearly inhibits a key
step in the p70
pathway. In contrast to rapamycin,
wortmannin does not inhibit TPA activation of p70
,
leading to a model in which the wortmannin block has been placed
upstream of the site of rapamycin action(18) . Exploiting a
similar line, Monfar et al.(21) have recently
demonstrated that raising intracellular levels of cAMP in T cells
either blocks interleukin 2-induced p70
activation or
causes the immediate inactivation of the kinase in post-interleukin
2-stimulated cells. These findings have led to an expansion of the
model above in which activation of protein kinase A acts as a key
negative regulator of p70
as well as phosphatidylinositol
3-OH kinase, whose activation is also blocked by raising intracellular
cAMP levels(21) . To raise cAMP levels, Monfar et al.(21) co-treated cells with forskolin, an activator of
adenylate cyclase, and IBMX, a phosphodiesterase inhibitor. However,
methylxanthines, such as IBMX, have been shown to also act as protein
kinase inhibitors(22) . Indeed, previous studies have
demonstrated that treatment of cells with methylxanthines alone is
sufficient to block S6 phosphorylation, whereas raising intracellular
cAMP with prostglandin E
, a potent adenylate cyclase
agonist, had no inhibitory effect on this response(23) . These
latter findings raise the possibility that methylxanthines themselves
may block p70
activation and, if selective, could be
potential tools for identifying upstream kinases in this pathway.
The mapping of individual phosphorylation sites involved in
regulating p70 enables the assessment of these models and
allows for the determination of whether the newly identified sites of
phosphorylation are also implicated in p70
activation.
Here we have employed p70
mutants as well as
two-dimensional phosphopeptide analysis of endogenous p70
to establish whether any of the newly identified
rapamycin-sensitive sites of phosphorylation are also involved in
mitogenic activation of p70
. Next we have assessed
whether rapamycin and wortmannin inhibition of p70
activation is paralleled by the dephosphorylation of the same or
a distinct set of sites. Finally, we have examined whether
methylxanthines are capable of blocking p70
activation,
as opposed to other agents that raise intracellular cAMP levels, and
have examined the sites of p70
phosphorylation affected
by such treatment.
Figure 1:
Multiple phosphorylation of p70 regulates its activity. A, schematic representation of
p70
showing the location of the regulatory
phosphorylation sites. AD represents the autoinhibitory
domain. The model is drawn to scale(51) . B, the
activity of parent Myc-p70
(1) ,
Myc-p70
A
(2) , or
p70
D
E (3) expressed in human 293
cells was determined in an immunocomplex assay before (stippledbars) and after (solidbars)
stimulation with 10% FCS for 1 h. All activities were normalized for
p70
expression levels by quantitation of Western blots
probed with the 9E10 anti-Myc antibody, and decorated with rabbit
anti-mouse IgG followed by
I-conjugated protein A
(Amersham Corp.). Quantitation of bound
I-protein A was
done using a PhosphorImager and ImageQuant software (Molecular
Dynamics).
Figure 2:
Serum-induced activation of p70 in Swiss 3T3 cells. A, p70
activity in
extracts from quiescent cells (lane1) or cells
stimulated with 10% FCS for 15 min (lane2) was
assayed after immunoprecipitation with p70
M5 antibody as
described under ``Experimental Procedures.'' Extracts (20
µg of total protein) from quiescent cells (lane3) or cells serum-stimulated for 15 min (lane4) were subjected to Western blot analysis. Derivatives i-iv represent increasingly phosphorylated forms of
p70
. Cells labeled with
P
were
incubated in the absence (B) or presence (C) of 10%
FCS for 15 min. Immunoprecipitated p70
was subjected to
two-dimensional tryptic phosphopeptide mapping following performic acid
oxidation as described under ``Experimental Procedures.''
Identified phosphorylation sites of p70
are numbered
according to the p70
sequence(51) , and the
origin is indicated with an arrow.
Figure 3:
Effect of rapamycin pretreatment or
posttreatment on activation and phosphorylation of p70 in
response to serum. Swiss 3T3 cells were labeled with
P
and incubated in the absence (A and B, lane1) or the presence of 10% FCS (A and B, lane2) for 15 min or were
preincubated for 30 min with 5 nM rapamycin and then
stimulated with 10% FCS for 15 min (A and B, lane3, and C) or first stimulated with 10% FCS for
15 min and then treated with 5 nM rapamycin for 30 min (A and B, lane4, and D). A, total p70
activity was measured in an in
vitro immunocomplex kinase assay as in Fig. 2. One unit of
kinase incorporates 1 pmol of P
/min into S6. B,
the amount of
P
incorporated into p70
was quantitated by Cerenkov counting following
immunoprecipitation of the kinase as described under
``Experimental Procedures.'' C and D,
two-dimensional tryptic phosphopeptide maps of
P
-labeled p70
from cells
pretreated (C) or posttreated (D) with rapamycin was
carried out as in Fig. 2.
Figure 4:
Reversal of rapamycin-induced
dephosphorylation of p70 by FK506. A, Swiss 3T3
cells were stimulated with 10% FCS for 30 min in the absence (A, lanes1 and 4, and D)
or presence of 5 nM rapamycin (A, lanes2 and 3, B and C). Cultures
were then washed and incubated for an additional 3 h in DMEM containing
10% FCS and either 5 µM FK506 (A, lanes3 and 4, C and D) or the
carrier, 0.02% ethanol alone (A, lanes1 and 2, B). Whole cell lysates were subjected to S6 kinase
assays as described previously under ``Experimental
Procedures.'' B-D, two-dimensional tryptic
phosphopeptide maps of p70
were analyzed from cells
treated as in A, lanes2, 3, and 4, respectively, which had been prelabeled with
P
. Two-dimensional tryptic phosphopeptide
analysis was carried out as in Fig. 2.
Figure 5:
Effect of wortmannin on p70. A (inset), Swiss 3T3 cells stimulated with 10% FCS
for 15 min were then incubated with increasing concentrations of
wortmannin for an additional 30 min. Cell extracts were prepared and
assayed as described under ``Experimental Procedures.'' A and B, cells were preincubated for 30 min with 200 nM wortmannin and then stimulated with serum for 15 min (lane1) or first stimulated with 10% FCS for 15 min (lane2) and then treated with 200 nM wortmannin for
15 min (lane3) or 30 min (lane4).
Cell extracts were prepared and either assayed for (A) S6
kinase activity or (B) the mobility of p70
on
Western blots as described under ``Experimental Procedures.'' C, two-dimensional tryptic phosphopeptide maps of p70
from cells pretreated with wortmannin for 30 min prior to
stimulation with 10% FCS for 15 min; D, maps of p70
from cells first stimulated with 10% FCS for 15 min followed by
the addition of wortmannin for 30 min. Cells were prelabeled with
P
, and extracts were prepared as in A and B. Two-dimensional phosphopeptide analysis was
carried out as described in Fig. 2.
Figure 6:
Effect of cAMP or phosphodiesterase
inhibitors on p70 activity. Swiss 3T3 cells were
preincubated for 30 min without (lane1) or with 500
µM 8-bromo-cAMP (lane2), 50 µM forskolin (lane3), 500 µM IBMX (lane4), or 1.2 mM SQ20006 (lane5) and then stimulated with 10% FCS for 15 min. Whole
cell lysates were assayed for kinase activity as described under
``Experimental Procedures.''
Figure 7:
Effect of SQ20006 on p70
activity and phosphorylation state. A, whole cell lysates from
Swiss 3T3 cells, first stimulated with 10% FCS for 15 min and then
treated with 1.2 mM SQ20006 for the indicated times were
subjected to S6 kinase assays as described under ``Experimental
Procedures.'' A (inset), Total MAP kinase
activity from cell extracts of time point 0 and 30 min was done as
described under ``Experimental Procedures.'' A,
equal amounts of protein (16 µg) from cell extracts at indicated
times were analyzed by Western blotting as described under
``Experimental Procedures.'' C and D,
two-dimensional tryptic phosphopeptide maps of p70
from
cells pretreated with 1.2 mM SQ20006 for 30 min prior to
stimulation with 10% FCS for 15 min (C) or from cells first
stimulated with 10% FCS for 15 min followed by the addition of 1.2
mM SQ20006 for 30 min (D).
P
-labeled p70
was subjected to
the two-dimensional phosphopeptide analysis as described in Fig. 2.
As wortmannin does not block TPA activation of
p70, whereas rapamycin does, each agent has been argued
to attack a unique target in the p70
signaling pathway.
If both agents operate on the same pathway, the wortmannin target would
be situated more proximal to the cell surface receptor, with the
rapamycin target situated downstream. To determine where SQ20006 acts
in this pathway, cells were stimulated with TPA in the presence or
absence of all three agents. The results show that wortmannin has no
effect on TPA activation of p70
, as shown by
others(17) , whereas rapamycin and SQ20006 block kinase
activity (Fig. 8). In contrast to p70
activation,
all three agents have no effect on p42
activation (Fig. 8). These results suggest that SQ20006 operates very
similarly to rapamycin, possibly inhibiting a common target.
Figure 8:
Effect of rapamycin, wortmannin, and
SQ20006 on TPA activation of p70. Swiss 3T3 cells were
incubated in the absence or presence of 0.5 µM TPA for 30
min following pretreatment for 30 min with either 1.2 mM SQ20006 (SQ), 200 nM wortmannin (WM),
or 5 nM rapamycin (RAP). p70
and MAP
kinase activity were measured following immunoprecipitation of equal
amounts of protein from each cell extracts as described under
``Experimental Procedures.''
From the data presented here, it is evident that the
phosphorylation of Thr, Thr
, and
Ser
are largely responsible for the mobility shifts
observed on Western blots following mitogenic stimulation. The most
likely reason these sites were not detected in the initial analysis (14) was that all three reside in atypically cleaved tryptic
peptides.
This problem is further compounded by the fact
that the cleavage efficiency of these peptides is poor and varies
between batches of trypsin. The ability of the marcrolide to induce an
equivalent increase in mobility of the wild type p70
and
the p70
D
E mutant on SDS-PAGE (15) is
consistent with the conclusion that the mobility shifts in p70
are due to the rapamycin-sensitive sites. Furthermore, the
phosphorylation of the rapamycin-insensitive sites, like the
rapamycin-sensitive sites, appears to have a large impact on kinase
activity (Fig. 1). Thus, as has been described for other kinases (36) , only a subset of sites cause changes in electrophoretic
mobility, and therefore, interpretations concerning the extent of
p70
phosphorylation and activation by mobility shift
should be treated cautiously.
Recently Chung et
al.(17) , employing specific point mutants of the PDGF
receptor, provided evidence for two separate signaling pathways leading
to p70 activation. One pathway was regulated through
tyrosines 740 and 751 and hypothesized to be mediated through
activation of phosphatidylinositol 3-OH kinase, whereas the second
pathway was regulated by phosphorylation of tyrosines 1009 and 1021,
apparently signaling through protein lipase C
. Interestingly, the
phosphatidylinositol 3-OH kinase inhibitor wortmannin only blocked
signaling from tyrosines 740 and 751 and not tyrosines 1009 and 1021,
while rapamycin blocked p70
activation through both
pathways(17) . These results led to the hypothesis that, in the
pathway mediated by tyrosines 740 and 751, the rapamycin block lies
downstream of the wortmannin block(18) . The hypothesis that
both agents inhibit p70
activation through different
components, which are located on the same signaling pathway, is
consistent with their ability to block the same set of phosphorylation
sites in the kinase (Figs. 3-5). Indeed, this same set of
phosphorylation sites is also sensitive to SQ20006 treatment (Fig. 7).
Earlier studies had shown that phosphodiesterase
inhibitors can inhibit or ablate serum-induced S6
phosphorylation(23, 37) . Recent results from Monfar et al.(21) employing the cAMP elevating agents
forskolin and IBMX, demonstrated that the two agents together prevent
interleukin 2-induced p70 activation in a T cell line,
leading them to conclude that this inhibitory effect was exerted
through raising intracellular levels of cAMP. The results presented
here demonstrate that, in Swiss 3T3 cells, raising cAMP levels either
by use of the nonhydrolyzable analogue 8-bromo-cAMP or an adenylate
cyclase agonist has no effect on serum-induced p70
activation (Fig. 6), consistent with earlier studies on S6
phosphorylation(23) . However, IBMX alone had a 25% inhibitory
effect on kinase activation, whereas the more potent phosphodiesterase
inhibitor SQ20006 (35) had a more pronounced effect. Recent
studies have shown that the structurally related 2-aminopurine
analogue, olomoucine, selectively inhibits a number of cell
cycle-regulated kinases both in vitro and in
vivo(38) . Olomoucine however, had no effect on
serum-induced p70
activation while blocking
p42
/p44
activation (data not shown),
suggesting that SQ20006 or similar structural analogues may be useful
tools in specifically analyzing the p70
signal
transduction pathway.
SQ20006 has a very similar p70 inhibitory profile to that of rapamycin. However, earlier studies
showed that SQ20006, at concentrations that completely inhibit
p70
activation, ablate serum-induced up-regulation of
protein synthesis(23) . In contrast, rapamycin only has a
marginal effect on global protein synthesis(7) . Although
having only a small effect on general protein synthesis, rapamycin
selectively suppresses the translational up-regulation of a family of
mRNA that are characterized by having a polypyrimidine tract at their
5` transcriptional start site(7) . Taken together this suggests
that SQ20006 is inhibiting the function of at least one other cell
component that is involved in the up-regulation of translation. Since
the inhibitory effect of SQ20006 appears to be exerted at
initiation(23) , this component may be one of the specific
factors involved in initiation of translation(39) .
It is
clear from the data presented here that the signaling events leading to
p70 activation are not all converging through a
rapamycin/wortmannin/SQ20006-sensitive pathway. Instead, there appear
to be at least two independent pathways required for p70
activation, only one of which is blocked by the inhibitors
employed here. The sites targeted by this pathway have been recently
identified as Thr
, Thr
, and
Ser
The complexity of p70 activation by
phosphorylation is further emphasized by the observation that
phosphorylation events mediated by these pathways may be
interdependent. The results presented here raise the possibility that
the phosphorylation of Thr
and Ser
may be
dependent on phosphorylation of Thr
and/or Ser
(Figs. 3-6). Such an interpretation may also hold true for
phosphorylation of Thr
. This site is only found
phosphorylated in the tryptic peptide Thr
-Lys
when Ser
is also phosphorylated(14) . In
contrast, the singly phosphorylated form of this peptide is only
phosphorylated on Ser
, implying that phosphorylation at
the threonine residue is dependent on prior phosphorylation at the
serine. Point mutations of individual sites, combined with
phosphopeptide maps will help to resolve the importance of each site in
hierarchal phosphorylation. Knowledge of the phosphorylation sites and
their mutual interdependence will be an important tool in identifying
the upstream kinases that regulate p70
activation.
Recent findings have indicated that p70/p85
plays a critical role in cell cycle
progression(9, 10, 47) . Furthermore, it
appears to mediate this effect through the phosphorylation of 40 S
ribosomal protein S6 and the subsequent translational up-regulation of
a family of mRNA transcripts that encode for components of the protein
synthetic machinery(7, 8) . Up-regulation of specific
translational components (48, 49) or obstruction of
gene products that regulate their function (50) can transform
cells or increase their susceptibility to transformation. These
observations may explain why p70
is among the most highly
conserved mammalian enzymes, having the identical sequence in man,
mouse, rat, and rabbit. A highly regulated mechanism of p70
control would be consistent with the loss of this control leading
to a constitutive growth state. The use of specific inhibitors should
provide invaluable tools in probing regulatory pathways that govern
p70
/p85
activation and mechanisms that link
the kinase to translational control.