©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cyclic AMP-dependent Activation of Rap1b (*)

Daniel L. Altschuler (2)(§)(¶), Scott N. Peterson(¶) (1), Michael C. Ostrowski (2)(§), Eduardo G. Lapetina (1)(**)

From the (1) Division of Cell Biology, Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709 and the (2) Department of Microbiology, Duke University Medical Center, Durham, North Carolina 27710-0001

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-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.


INTRODUCTION

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 by prostaglandins, led to cAMP-dependent phosphorylation of Rap1b (22, 23, 24, 25) .

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.


EXPERIMENTAL PROCEDURES

Construction of an IPTG-inducible, Epitope-tagged Rap1b Expression System

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 -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 GTPS. 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 KHP0, 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).


RESULTS AND DISCUSSION

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 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 GTPS-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, ATPS, 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.

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() 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by NCI Grant CA53271.

Both authors contributed equally to this work.

**
To whom correspondence should be addressed: Division of Cell Biology, Burroughs Wellcome Co., 3030 Cornwallis Rd., Research Triangle Park, NC 27709. Tel.: 919-315-4435; Fax: 919-315-0286.

The abbreviations used are: IPTG, isopropyl-1-thio--D-galactopyranoside; DMEM, Dulbecco's modified Eagle's medium; CPT-cAMP, 8-(4-chlorophenylthio)-cAMP; IBMX, isobutylmethylxanthine; GTPS, guanosine 5`-3- O-(thio)triphosphate; ATPS, adenosine 5`-3- O-(thio)triphosphate.

D. L. Altschuler, S. N. Peterson, M. C. Ostrowski, and E. G. Lapetina, unpublished results.


ACKNOWLEDGEMENTS

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.


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