Activation of JNK by EPAC is independent of its activity as a Rap Guanine Nucleotide Exchanger

Guanine Nucleotide Exchange Factors (GEFs) and their associated GTP binding proteins (G-proteins) are key regulatory elements in the signal transduction machinery that relays information from the extracellular environment into specific intracellular responses. Among them, the MAPK cascades represent ubiquitous downstream effector pathways. We have previously described that, analogous to the Ras-dependent activation of the Erk-1/2 pathway, members of the Rho family of small G-proteins activate the JNK cascade when GTP is loaded by their corresponding GEFs. Searching for novel regulators of JNK activity we have identified EPAC as a strong activator of JNK-1. EPAC (Exchange Protein Activated by cAMP) is a member of a growing family of GEFs that specifically display exchange activity on the Rap subfamily, of Ras small G-proteins. We report here that while EPAC activates the JNK several fold, a constitutively active (G12V) mutant of Rap1b does not, suggesting that Rap-GTP is not sufficient to transduce EPAC-dependent JNK activation. Moreover, EPAC signaling to the JNKs was not blocked by inactivation of endogenous Rap suggesting that Rap activation is not necessary for this response. Consistent with these observations, domain deletion mutant analysis shows that the catalytic GEF domain is dispensable for EPAC-mediated activation of JNK. These studies identified a region overlapping the REM domain as critical for JNK activation. Consistent with this, an isolated REM domain from EPAC is sufficient to activate JNK. We conclude that EPAC signals to the JNK cascade through a new mechanism that does not involve its canonical catalytic action, i.e. Rap-specific GDP/GTP exchange. This represents not only a novel way to activate the JNKs but also a yet undescribed mechanism of downstream signaling by EPAC. supernatants incubated 60min Beads four After Laemmli were fractionated in a and transferred a HA-Rap1-GTP was visualized by western blotting with an anti-HA antibody


INTRODUCTION
Epac (exchange protein activated by cAMP) is a newly discovered guanine nucleotide exchange factor (GEF) 1 that selectively activates members of the Rap family of G-proteins (1)(2)(3). Rap proteins, like all G-proteins, are biochemical transducers (4,5) which function as allosteric regulatory elements, switching between an inactive GDPbound and an active GTP-bound conformation (4). The switch mechanism consists of activation by exchange of bound GDP for GTP, and inactivation by hydrolysis of GTP into GDP, catalyzed by GEFs and GTPase-activating proteins (GAPs), respectively. Similar to all GEFs, Epac acts catalytically on the rate-limiting step for G protein activation, i.e. dissociation of bound GDP (4).
The involvement of Rap1 in signaling mechanisms is demonstrated by the variety of second messengers mediating its activation. Recently, a number of exchange factors were identified as mediators of these specific activities (6). Among them, Epac was characterized as a molecule responsible for cAMP-dependent Rap1 activation (1,2).
Epac is a multidomain protein comprised of a C-terminal catalytic and an N-terminal regulatory module. The C-terminal module encompasses the Rap-specific GEF catalytic core and the Ras exchange motif (REM) (1) . According to the structure solved for SOS proliferative conditions depending on the cellular setting (9,12). The discovery of additional MAPKs as ERK-5 and others add diversity to the MAPK scenario (15).
The signaling cascades that end in MAPKs and regulate their activity include several sequential events of phosphorylation in the cytoplasm that define phosphorylation cascades. MAPKs are activated by dual phosphorylation at two residues, a tyrosine and a threonine by kinases known as MAPKKs (16). Particularly JNK is activated by the prototypical JNKKs, MKK4 (SEK1) and MKK7 (SEK2), which are in turn activated by upstream phosphorylation events. A large group of JNKKKs has been reported (13). However, the mechanisms involved in the activation of these JNKKKs, and the link between a specific stress signal and its mediators are still ill defined. The module SOS/Ras/Raf/MEK/Erk1-2 constitutes a typical example of a MAPK signaling pathway (10) . In recent years we have contributed to the development of the dominant concept that probably all of the MAPKs are activated in an analogous fashion when we discovered that Dbl, a GEF specific for the Rho family of small Gproteins can activate the JNK pathway (17) through the Rho family members Rac and Cdc42 and a series of kinases that include MLK3, MEKK and SEK (17)(18)(19)(20).
It is well now established that the Rho-like small G-proteins Rac and Cdc42 represent a link between environmental stimuli and JNK activation (21). These Gproteins as well as their corresponding GEFs are also regulators of the actin cytoskeleton dynamics (22). Expression of the small G-protein Rap1b in Swiss3T3 fibroblasts induces an anchorage-dependent transformed phenotype (23), accompanied by changes in focal contacts and actin cytoskeleton (unpubl. observ.). These results

EXPERIMENTAL PROCEDURES
Cell Lines and Transfections: HEK 293T cells, were maintained in Dulbecco's modified Eagle's medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum. Transfections were performed by the calcium phosphate precipitation technique, adjusting the total amount of plasmid DNA to 3-6mg/plate with empty vector.
Additional transfections were performed using FuGene trasfection reagent (Roche-Boehringer), adjusting the total amount of DNA plasmid to 0.5-1mg/plate as directed by the manufacturer.
Autoradiography was performed with the aid of an intensifying screen. Parallel immunoprecipitates were processed for western-blot analysis using the same antiserum as described (17). To assay p38 activity, cells were transfected with a plasmid that expresses HA-p38 which was immunoprecipitated from the cell lysates with a specific antibody against the HA epitope (MMS-101R, Covance) and processed as described  (25). The EPAC REM and DEP domains were isolated by PCR amplification using oligonucleotides 5'acGGATCCacagtgatgtctggcacc3' and 5'tgGAATTCtcactgctcgctgccacccgc3' for R E M a n d 5 ' a c G G A T C C a t g a c c c g a g a c c g g a a g t a c c 3 ' a n d to a nitrocellulose membrane. HA-Rap1-GTP was visualized by western blotting with an anti-HA antibody as previous described (17).

EPAC is a JNK activator:
Expression of the Rap1-specific GEF Epac into HEK293T cells triggered a strong JNK response, about a six-fold increase in its kinase activity. This Epacmediated effect was similar in magnitude to the one produced by canonical JNK activators as Onc-Dbl and the protein synthesis inhibitor anisomycin ( fig.1, upper panel and bars). The lower panel of fig. 1 shows that co-expression of the GEFs did not alter the JNK protein levels which is indicative of changes in specific JNK activity rather than changes in the amount of enzyme present. As reported previously for MAPK activation (27), parallel experiments showed that a GFP-JNK fusion protein translocates to the nucleus upon contransfection with plasmids that express Epac or Onc-Dbl (data not shown).

Opposite to Epac, a constitutively active mutant of Rap1b fails to stimulate JNK activity
We have previously found that expression of constitutively active forms of small G-proteins of the Rho subfamily, were highly effective in triggering JNK activation to levels comparable as its corresponding GEFs, Ost and Dbl (17). Epac is a Rap-specific GEF that turns out to be a significant JNK activator ( fig 1). If its effects on JNK activity are conveyed through Rap activation, we reasoned that expression of a constitutively active Rap form should fully mimic Epac's action. We found that while Rac1QL and Epac display a five-fold induction in JNK activation, (fig 2A), expression of a GTPase deficient form of Rap1b (RapG12V) did not produce any changes in JNK activity, despite high levels of expression as measured in western blots ( fig.2A and 2B).
The HA-RapG12V construct is expressed on its active form when transfected into HEK293T cells as shown by its ability to bind to the Rap binding domain of RalGDS, which binds Rap1 on its active GTP-bound conformation (not shown). These results demonstrate that in contrast to Epac, Rap1b-G12V does not activate JNK, suggesting that the enzymatic activity described for Epac (GTP loading unto Rap) is not sufficient to transduce Epac's effects on JNK activity.

Rap activity is not necessary for EPAC-mediated JNK activation.
Results from the two preceding sections suggest two potential alternatives for Epac signaling. One of them would be mediated by Rap activation, as has been previously demonstrated, while the other one would be Rap-independent. The first scenario should be mimicked by expression of RapG12V; meanwhile the second one should not be altered by the presence of Rap inhibitory molecules. We observed no JNK activation by RapG12V; therefore in order to assess the second possibility,  3A). This seemingly contradictory result could be explained assuming that RapN17 is actually an inhibitor of the upstream GEF regulator Epac, and not of the small Gprotein itself, acting presumably through titration of "free" Epac molecules, thereby preventing Epac actions including JNK activation.
Since RapG12V does not mimic Epac's action on JNK, and expression of a downstream negative regulator of Rap1 (RapGAP), has no effect on Epacstimulated JNK activity, we conclude that Rap1 activity should not be necessary for Epac-mediated JNK activation.

Stimulation of JNK by EPAC is independent of its cAMP-dependent GEF activity.
The general organization of Epac is schematized in Fig.4A

Epac-mediated JNK activation is dissociated from Epac's GEF activity
Although the results described above strongly suggest that Epac's nucleotide exchange on Rap1 is not involved in Epac-mediated JNK activation, we directly tested this premise experimentally. We asked whether the presence of the GEF domain was  5A). These results demonstrate unequivocally that Epac activation of JNK is independent of its guanine nucleotide exchange function.  4A).

Finding a new efector domain in EPAC: A role for the REM domain in JNK
In order to assess this possibility we decided to isolate the REM domain and challenge its ability to activate JNK, another Epac region that contains the DEP domain was also isolated as a control and both were expressed as GST fusion proteins. Fig 6 shows that while being expressed to the same levels ( fig. 6A), GST-DEP did not induce changes in JNK activity but GST-REM exerted an effect comparable to N-Epac ( fig. 6B). We understand that Epac's REM domain is responsible for this JNK activation, as GST alone did not alter either JNK activity or expression levels ( fig. 6B bars, lower and middle panels). Thus, we have identified REM as the minimal Epac domain endowed with capability to fully stimulate JNK activity.

Signaling specificity: EPAC mediated JNK activation is not reproduced by other SAPKs as p38 MAPK.
To assay for JNK signaling specificity and to discard any effect on cell stress due to the over expression of Epac mutants we decided to test p38 kinase activity, a SAPK clearly distinct but closely related to JNK. Figure 7 shows that cotransfection with increasing amounts of a plasmid expressing N-Epac fails to augment p38 kinase activity (bars and upper panel) while it strongly activates the phosphorylating activity of

DISCUSSION
Signal transduction mediated by proteins that bind and hydrolyze GTP and its associated GEFs has been receiving growing interest.  (1). It is generally accepted that Epac exhibits a strict dependence on cAMP in regards to both its activation and its nucleotide exchange on Rap (1). To our knowledge no effect of Epac unrelated to cAMP and/or Rap G-proteins has been described to date other than JNK stimulation, as described here. This is a provocative conclusion with respect to Epac, although a precedent example of a cAMP-unrelated action pertinent to a protein that is typically activated by binding of cAMP exists-LPS induced PKA-mediated phosphorylation of p65 NFkB is independent of cAMP (32).
As is the case with Epac, binding of cAMP to PKAr relieves inhibitory constrains imposed by the CBD of PKAr, resulting in allosteric activation of PKAc (33). In this context, it is noteworthy to mention some similarities noted between the cAMP-independent actions of Epac and PKA. As with Epac, the cAMP-independent There is a previous report (29) that while analyzing JNK activation by other GEFs shows no JNK activation by Epac. We understand this difference might be due to a divergence in the pattern of proteins expressed by the cell lines being maintained in different labs. Our results provide strong evidence that EPAC signals to the JNK cascade. Moreover we provide evidences of the existence of a novel mechanism for EPAC signaling. Through a variety of complementary approaches, we prove that EPAC is acting, in this case, in a Rap-independent fashion. This is to our knowledge the first report of a GEF-independent signal transduction activity for EPAC and ends up adding a new ingredient in the increasingly complex scene of signaling by GEFs.
One important implication of this work is the intriguing possibility that Epac-REM/JNK stimulatory function is triggered upon stimulation with a yet unidentified stress signal. Although the upstream signaling agents feeding on Epac are not known from the present work, bona fide activators of JNK such as nutrients, growth factors, osmolarity, temperature, pH, radiation, etc.; may use Epac-dependent signal transduction processes independently of cAMP and Rap G-proteins. Our results strongly suggest that a cAMP-independent mechanism is involved in the transduction of environmental signals that are discriminated by Epac into activation of JNK. These predictions are currently being investigated.  were assayed for Rap expression using an antibody directed against the HA epitope.