From the
Rap1 proteins belong to the Ras superfamily of small molecular
weight GTP-binding proteins. Although Rap1 and Ras share approximately
50% overall amino acid sequence identity, the effector domains of the
two proteins are identical, suggesting either similar or antagonistic
signaling roles. Several pathways leading to Ras activation have been
defined, including those initiated by agonist binding to tyrosine
kinase or G
Guanine nucleotide-binding proteins, such as the heterotrimeric
G-proteins and members of the Ras superfamily, are GTPases involved in
various signal transduction processes
(1, 2) . Rap
proteins share 50% sequence identity and basic biochemical properties
with Ras
(3, 4) . Both of these proteins function as
molecular switches in which the GTP-bound conformation represents the
active form of the molecule while the GDP-bound conformation is the
inactive form. Two cellular factors that regulate this interconversion
of Rap have been identified: a Rap-GDP dissociation stimulator, Rap-GDS
(5, 6) , and a Rap-GTPase-activating protein, Rap-GAP
(7, 8, 9) .
Crystallographic studies of Ras
indicate that the difference between the GTP-active and GDP-inactive
structures derives mainly from two regions: switch I and switch II
(10) . Interestingly, switch I encompasses residues 32-40,
defined as the effector domain
(11, 12, 13) .
These amino acids are identical in Rap1 and Ras, raising the
possibility that the proteins share similar or antagonistic functions.
In fact, Rap1-GTP interacts in vitro with two proteins known
to bind the effector domain in Ras: p120-GAP
(14, 15) and p74 Raf kinase
(16) . Moreover, a role for Rap1
in antagonizing Ras function is strongly supported in several model
systems, including Xenopus oocytes
(17) ,
Drosophila(18) , and mammalian cells
(19, 20, 21) . However, the precise cellular
function of Rap1 remains unclear. Identification of a signaling pathway
that inputs signals into Rap proteins may assist in deciphering its
function. First insights into this signaling function came from studies
demonstrating that activation of the heterotrimeric G-protein, G
Phosphorylation of
Rap1b does not affect its basal GDP/GTP exchange reaction rate, basal
GTPase activity, or the GAP-stimulated GTPase activity
(23) .
However, experiments performed in vitro suggested that
phosphorylation by protein kinase A plays a role in Rap1 activation
(26) and consequently implicated Rap1 as one of the effectors of
cAMP action. Here we show that stimulation of cAMP signaling in
vivo results in activation of Rap1b. To our knowledge, this
represents the first demonstration of hormonal regulation of Rap
proteins.
We have developed a system that allows us to search for
agonists capable of activating Rap1b in vivo, as measured by
an increase in the ratio of bound GTP/GDP. Independent, stable cell
lines expressing Rap1b constructions were isolated making use of an
IPTG-inducible system
(27) , which avoided the potential
inhibitory activity of the expressed protein (Fig. 1, A and B). Exogenous Rap1b proteins were tagged at their
NH
These data demonstrate that Rap1b is activated
in vivo in response to cAMP, strongly implicating the
former's involvement in some aspect of cAMP signaling. The effect
of raising intracellular cAMP levels on the transformed phenotype is
well documented
(38, 39) . Several recent reports showed
that one component of cAMP's action is the inhibition of Map
kinase activation
(40, 41, 42) . Moreover, cAMP
interferes with the MAP kinase pathway through competitive inhibition
at a site downstream of p21 but upstream of Raf-1
(43) .
Interestingly, the effects of cAMP are very similar to those obtained
after stable expression of Rap Val-12. We
We thank S. Baim and M. Labow for a gift of the LAP267
system and Fernando Ribeiro-Neto, Michael Campa, and Eddie Wood for
critical reading of the manuscript and valuable suggestions during this
work.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-coupled receptors. Nothing is known about such
events for Rap1 proteins. The cAMP-mediated inhibition of Ras-dependent
MAP kinase activation is well documented and resembles that caused by
expression of GTPase-deficient Rap1. We have developed a system whereby
signals leading to Rap1b activation, i.e. an increase in
Rap1b-bound GTP/GDP ratio, can be measured. We report here that
treatment of cells with agents that elevate intracellular cAMP levels
result in Rap1b activation. These results demonstrate for the first
time agonist-dependent activation of Rap1 proteins.
by prostaglandins, led to cAMP-dependent phosphorylation of Rap1b
(22, 23, 24, 25) .
Construction of an IPTG
Control vector plasmid
(pL7-Hygro) was constructed in a triple ligation reaction by combining
a SalI/ NruI fragment from the vector pCEP4
(Invitrogen), containing the hygromycin gene with an
Ecl136II/ BamHI fragment from pL7-CAT
(27) ,
and a BamHI/ XhoI fragment from the vector pCGN
(28) containing the -inducible,
Epitope-tagged Rap1b Expression System
-globin intron and polyadenylation
signal. Different Rap1b clones were made by subcloning
Ecl136II/ BamHI fragments from pCGN-Rap1b
(29) into pL7-Hygro at the StuI/ BamHI site.
Stable Cell Line Production
The NIH3T3 fibroblast
derivative, LISN C4, which overexpresses the IGF-1 receptor was used
for all experiments
(30) . These cells were cotransfected using
Lipofectin reagent (Life Technologies, Inc.) with a 10-fold excess (10
µg) of the LAP 267 plasmid
(27) containing the mutant
lac repressor/VP16 fusion gene together with 1 µg of the
plasmid expressing wild type and mutant Rap1b. Rap1b expression was
under the control of a promoter containing several copies of the
lac operator. Colonies were selected for 2 weeks in
Dulbecco's modified Eagle's medium (DMEM) supplemented with
5% calf serum in the presence of 200 µg/ml hygromycin and clonal
lines were selected by dilution cloning in 96-well microtiter dishes in
DMEM with 5% calf serum in the presence of 4 µg/ml insulin. To
select lines with physiological levels of Rap1b expression, clonal
lines were then expanded and induced for 48 h in the presence of 5
mM IPTG. Cells were lysed and the transfected protein was
immunoprecipitated with the monoclonal antibody, which recognizes the
epitope tag, clone 12CA5 (BAbco). The level of expression of the Rap1b
constructs closely approximates that of the endogenous protein, as
estimated from Western blotting with the M90 antibody (data not shown).
All reagents were from Sigma unless otherwise specified.
Rap1b-bound GTP/GDP Measurements
Cells were grown
in the presence of 5 mM IPTG in 10-cm diameter dishes for 24
h. Subconfluent cells were then serum-starved for an additional
18-20 h in DMEM containing 0.7% bovine serum albumin. Cells were
incubated in phosphate-free DMEM for 45 min and then metabolically
labeled with P
(250 µCi/ml) for 3 h. The
medium was aspirated and replaced with medium containing 50
µM forskolin or 100 µM
8-(4-chlorophenylthio)-cAMP (CPT-cAMP) in combination with 100
µM IBMX or medium containing equivalent amounts of
dimethyl sulfoxide. Stimulations were stopped by washing the cells one
time in cold phosphate-buffered saline, followed by the addition of
lysis buffer (1% Nonidet P-40, 50 mM Tris (pH 7.5), 500
mM NaCl, 10 mM MgCl
) containing
approximately 100 nM recombinant Rap1b loaded with GTP
S.
Lysates were precleared at 4 °C with protein A-Sepharose (Pharmacia
Biotech Inc.) for 20 min. Exogenous Rap1b was immunoprecipitated from
lysates with the 12CA5 antibody, followed by incubation with protein
A-Sepharose containing 1 mM cold GTP and ATP. Precipitates
were washed extensively in lysis buffer containing 0.25% sodium
deoxycholate. The phosphorylation state of Rap1b was analyzed by
removing 20% of the sample for analysis on 10-20% gradient
polyacrylamide gels. The remaining sample was incubated at 70 °C
for 20 min and loaded onto polyethyleneimine cellulose plates. The
nucleotides were resolved in 0.75 M
KH
P0
, pH 3.4. Rap1b bound GDP and GTP were
quantitated using a Molecular Dynamics PhosphorImager. The percentage
of GTP was calculated using the equation, GTP/(1.5 GDP + GTP).
terminus with the HA epitope. The epitope does not
modify the subcellular localization of Rap1b or its ability to be
phosphorylated by protein kinase A in vivo(29) . The
addition of the epitope served a dual function. It allowed us to
analyze different Rap1b constructions independently of the endogenous
protein for which specific immunoprecipitating antibodies do not exist.
It also allowed us to include GTP
S-loaded recombinant Rap1b in the
cell lysis buffer to inhibit postlysis GAP activity, a function
fulfilled by the Y13-259 antibody in the Ras nucleotide assay
(31) . This methodological consideration allowed us to reliably
measure activation of Rap1 as reflected by an increase in the
GTP/GDP-bound ratio.
Figure 1:
IPTG-inducible
expression of Rap1b in LISN C4 fibroblasts (30). A, cells were
cotransfected with the LAP 267 plasmid (27) containing a mutant lac repressor fused to the VP16 transcriptional activation domain
together with a plasmid expressing wild type and mutant Rap1b under the
control of a minimal promoter containing several lactose operator
sequences. B, expression of exogenous, epitope
( epit)-tagged Rap1b protein. Clonal lines selected after
dilution cloning were grown in DMEM with 5% calf serum for 48 h in the
presence or absence of 5 mM IPTG. Cells were lysed and
immunoprecipitated with the monoclonal antibody clone 12CA5 (BAbco).
Lanes 1 and 2, vector alone; lanes3 and 4, wild-type Rap1b, pKSS; lanes5 and 6, S179A/S180A double mutant, pKAA; lanes7 and 8, Rap Val-12, pVal; lanes9 and 10, Rap1b Gly-181, pCys.
Cells expressing wild-type Rap1b were
stimulated as indicated, lysed, and the epitope-tagged Rap1b was
immunoprecipitated with the 12CA5 antibody. The phosphorylation state
and the Rap1b-bound nucleotides were then determined. Preliminary
agonist screening assays clearly showed that cAMP-elevating agents
increased the level of phosphorylation and the bound GTP/GDP ratio.
These included both receptor-mediated agents such as prostaglandin
E and non-receptor-mediated agents such as forskolin and
CPT-cAMP. The time course of the forskolin/IBMX-dependent activation of
Rap1b is shown in Fig. 2. Forskolin/IBMX treatment resulted in a
rapid increase in Rap1b phosphorylation (Fig. 2 A) and in
general was accompanied by a 3-5-fold increase in the bound
GTP/GDP ratio compared with the basal ratio (Fig. 2 B).
These values are comparable with those observed for Ras activation with
tyrosine kinase receptor agonists
(32) . The forskolin-dependent
activation of Rap1b clearly showed a biphasic profile with a rapid
first phase (0.5-1 min) and a second sustained phase (5-30
min). This response was consistently observed. Attempts to inhibit the
decrease in Rap-GTP at the 2.5-min time point were made with several
phosphatase inhibitors: NaF, vanadate, okadaic acid, ATP
S, and
-glycerophosphate. All failed to change the biphasic profile
observed. The activation probably represents a balance of in vivo reactions, exchange activation, and GAP inhibition, although
general NDP kinases and nucleotide triphosphatases cannot be ruled out.
Figure 2:
Analysis of Rap1b phosphorylation and
bound nucleotide in cells expressing wild-type protein after
forskolin/IBMX treatment. A, time course of phosphorylation of
Rap1b after stimulating cells with forskolin ( FK)/IBMX
followed by immunoprecipitation of Rap1b with the antibody 12CA5.
B, Rap1b-bound guanine nucleotide resolved by
polyethyleneimine cellulose thin layer chromatography. Ab,
antibody; ORI, origin.
We tested the possibility that an agonist-dependent increase in GTP
hydrolysis over exchange activation might explain the decrease in the
bound GTP/GDP ratio observed at 2.5 min. We generated a cell line
expressing a Val-12 mutation using the same inducible system. As
reported before, Rap Val-12 is impaired in its GTPase activity and
resistant to the effects of Rap-GAP
(33) and so represents an
invaluable tool for the examination of GAP activity versus exchange mechanisms. Rap Val-12 showed a 5-fold higher basal
GTP/GDP ratio compared with the wild-type protein, confirming that the
GTPase was inhibited. However, the kinetic profile after forskolin or
CPT-cAMP stimulation showed the same biphasic behavior (Fig. 3).
This result suggests that the involvement of GAP inhibition in the
overall cAMP-dependent activation reaction is not significant. As
reported before, Rap-GAP is a substrate for protein kinase A
(34) , and in agreement with our data, in vitro phosphorylation did not affect its specific activity
(35) .
Treatment of cells expressing Rap Val-12 with prostaglandin E (10 µM) resulted in a slightly greater than 2-fold
increase (23% basal to 51%) in bound GTP within 1 min, and this then
dropped to 37% by 20 min poststimulation (data not shown).
Figure 3:
Analysis of Rap1b bound nucleotide in
cells expressing Rap Val-12. Cells were treated with either forskolin
( FK)/IBMX or CPT-cAMP/IBMX ( CPT), a permeable analog
of cAMP. A comparison of the percent GTP bound to Rap Val-12 after
forskolin () or CPT-cAMP (
) is shown. Results shown here
are representative of at least three independent experiments. The
absolute levels of activation varied from experiment to experiment, but
the profile shown did not.
Another
potential factor involved in the activation of Rap1b is Rap1-GDS. As
discussed above, GDS action is enhanced in a cell-free system after
Rap1b phosphorylation
(26) . Previous experiments in cell-free
systems led to the hypothesis that Rap1b phosphorylation is involved in
priming its own GDS-dependent activation
(26) . To test the
effect of Rap1b phosphorylation on forskolin-dependent activation, we
generated a cell line expressing a double mutant S179A/S180A (pKAA),
which is not phosphorylated by protein kinase A
(29) . Our
results indicate that phosphorylation is required for activation in the
first phase but is not strictly required in the second phase.
Stimulation of this cell line with forskolin/IBMX showed a smaller but
consistent activation over the time course analyzed (Fig. 4). The
differences in the kinetic profiles may indicate that full activation
of Rap1 by cAMP is complex, involving several factors, or that the
unphosphorylated Rap1 is a poorer substrate for activation and so
exhibits slower kinetics. It is possible that there is a functional
distinction between the two phases of activation in that they could be
responsible for triggering different signals. The phosphorylation data
for wild-type and mutant Rap1b do not show a biphasic profile. It is
possible that by gaining a greater understanding of the biphasic nature
of Rap1 activation we will understand more fully the role of Rap1
phosphorylation.
Figure 4:
A comparison of the percentage GTP bound
to wild-type and mutant Rap1b. Wild type pKSS (), pKAA (
),
and pCys Rap1b (
) are shown.
COOH-terminal post-translational modifications
include a series of events that are important not only for membrane
localization but also for appropriate targeting and interaction with
other proteins. Specifically, Rap1b is geranyl-geranylated at Cys-181
(36, 37) . In COS cells, Rap1b Gly-181 (pCys) mutants
failed to incorporate [C]mevalonolactone and
were shown by indirect immunofluorescence to be localized to the
cytosol. A stable cell line expressing IPTG-inducible Rap1b Gly-181 was
generated. The Rap1b Gly-181 protein was soluble and almost exclusively
localized in the S100 fraction (data not shown). In general, Ras
mutants at this position have a null phenotype. We tested whether Rap1b
Gly-181 mutants could be activated in response to forskolin/IBMX. We
were unable to detect any activation over the time course analyzed
(Fig. 4), even though the phosphorylation of Rap1b was more rapid
when compared with the wild-type protein (data not shown). These data
indicate that the presence of the isoprenoid moiety is required for the
activation machinery or that membrane localization is necessary for the
activation step. It is interesting to note that almost all of the
mammalian exchange factors isolated so far are soluble and able to
utilize the non-processed protein as a substrate. An alternative
explanation might be that even though the exchange factor is present in
a soluble form, a membrane-bound limiting cofactor is required for the
activation of the membrane-bound Rap protein. Demonstrating the
existence of this cofactor will require further analysis and
experimentation.
(
)
and
others have demonstrated that Rap Val-12 can inhibit MAP kinase
activation
(33) . Rap1 can also block signals to the Fos
promoter from c-K-Ras but not c-Raf1 in transient expression assays
(44) . In a recent report, phosphorylation of an
NH
-terminal Raf fragment by protein kinase A reduced the
affinity of Raf-1-(1-149) for Ras in vitro(45) .
This information provides a molecular basis for cAMP inhibition of the
Ras pathway but leaves open the possibility that other cellular factors
may contribute to the action of cAMP. It is tempting to speculate,
based upon several previously published results
(33, 38, 39, 40, 41, 42, 43, 44) ,
that Rap1b plays a role in the cAMP-dependent inhibition of the MAP
kinase pathway. There are several possible mechanisms by which the
perinuclear Rap1b might antagonize Ras, localized in the plasma
membrane. To date, we have been unable to obtain data to support direct
interaction of Rap1b and Raf-1 in mammalian cells. An intriguing
alternative is that upon activation, Rap may activate specific
phosphatases that impinge upon the Ras pathway at the Raf-1/MEK/Map
kinase activation step. Alternatively, Rap1 activation might trigger an
independent function, which in combination with Raf inhibition is
responsible for the full inhibitory action of cAMP. Expression of a
dominant negative Rap mutant protein may allow us to resolve which, if
either, of these possibilities is correct.
-D-galactopyranoside; DMEM,
Dulbecco's modified Eagle's medium; CPT-cAMP,
8-(4-chlorophenylthio)-cAMP; IBMX, isobutylmethylxanthine; GTP
S,
guanosine 5`-3- O-(thio)triphosphate; ATP
S, adenosine
5`-3- O-(thio)triphosphate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.