Rap1 is a small, Ras-like GTPase that was first identified as a protein that could suppress the oncogenic transformation of cells by Ras. Rap1 is activated by several extracellular stimuli and may be involved in cellular processes such as cell proliferation, cell differentiation, T-cell anergy and platelet activation. At least three different second messengers, namely diacylglycerol, calcium and cyclic AMP, are able to activate Rap1 by promoting its release of the guanine nucleotide GDP and its binding to GTP. Here we report that activation of Rap1 by forskolin and cAMP occurs independently of protein kinase A (also known as cAMP-activated protein kinase). We have cloned the gene encoding a guanine-nucleotide-exchange factor (GEF) which we have named Epac (exchange protein directly activated by cAMP). This protein contains a cAMP-binding site and a domain that is homologous to domains of known GEFs for Ras and Rap1. Epac binds cAMP in vitro and exhibits in vivo and in vitro GEF activity towards Rap1. cAMP strongly induces the GEF activity of Epac towards Rap1 both in vivo and in vitro. We conclude that Epac is a GEF for Rap1 that is regulated directly by cAMP and that Epac is a new target protein for cAMP.

SUMMARY Cell-to-cell communication is a major process that allows bacteria to sense and coordinately react to the fluctuating conditions of the surrounding environment. In several pathogens, this process triggers the production of virulence factors and/or a switch in bacterial lifestyle that is a major determining factor in the outcome and severity of the infection. Understanding how bacteria control these signaling systems is crucial to the development of novel antimicrobial agents capable of reducing virulence while allowing the immune system of the host to clear bacterial infection, an approach likely to reduce the selective pressures for development of resistance. We provide here an up-to-date overview of the molecular basis and physiological implications of cell-to-cell signaling systems in Gram-negative bacteria, focusing on the well-studied bacterium Pseudomonas aeruginosa . All of the known cell-to-cell signaling systems in this bacterium are described, from the most-studied systems, i.e., N -acyl homoserine lactones (AHLs), the 4-quinolones, the global activator of antibiotic and cyanide synthesis (GAC), the cyclic di-GMP (c-di-GMP) and cyclic AMP (cAMP) systems, and the alarmones guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), to less-well-studied signaling molecules, including diketopiperazines, fatty acids (diffusible signal factor [DSF]-like factors), pyoverdine, and pyocyanin. This overview clearly illustrates that bacterial communication is far more complex than initially thought and delivers a clear distinction between signals that are quorum sensing dependent and those relying on alternative factors for their production.

Epac1 (cAMP-GEFI) and Epac2 (cAMP-GEFII) are closely related guanine nucleotide exchange factors (GEFs) for the small GTPase Rap1, which are directly regulated by cAMP. Here we show that both GEFs efficiently activate Rap2 as well. A third member of the family, Repac (GFR), which lacks the cAMP dependent regulatory sequences, is a constitutive activator of both Rap1 and Rap2. In contrast to Epac1, Epac2 contains a second cAMP binding domain at the N terminus, as does the Epac homologue from Caenorhabditis elegans. Affinity measurements show that this distal cAMP binding domain (the A-site) binds cAMP with much lower affinity than the cAMP binding domain proximal to the catalytic domain (the B-site), which is present in both Epac1 and Epac2. Deletion mutant analysis shows that the high affinity cAMP binding domains are sufficient to regulate the GEFs in vitro. Interestingly, isolated fragments containing the B-sites of either Epac1 or Epac2, but not the A-site from Epac2, inhibit the catalytic domains in trans. This inhibition is relieved by the addition of cAMP. In addition to the cAMP binding domains, both Epac1 and Epac2 have a DEP domain. Deletion of this domain does not affect regulation of Epac1 activity but affects membrane localization. From these results, we conclude that all three members of the Epac family regulate both Rap1 and Rap2. Furthermore, we conclude that the catalytic activity of Epac1 is constrained by a direct interaction between GEF and high affinity cAMP binding domains in the absence of cAMP. Epac1 becomes activated by a release of this inhibition when cAMP is bound.

The virulence of the opportunistic human pathogen Pseudomonas aeruginosa PAO1 is controlled by an N-acyl-homoserine lactone (AHL)-dependent quorum-sensing system. During functional analysis of putative acylase genes in the P. aeruginosa PAO1 genome, the PA2385 gene was found to encode an acylase that removes the fatty acid side chain from the homoserine lactone (HSL) nucleus of AHL-dependent quorum-sensing signal molecules. Analysis showed that the posttranslational processing of the acylase and the hydrolysis reaction type are similar to those of the beta-lactam acylases, strongly suggesting that the PA2385 protein is a member of the N-terminal nucleophile hydrolase superfamily. In a bioassay, the purified acylase was shown to degrade AHLs with side chains ranging in length from 11 to 14 carbons at physiologically relevant low concentrations. The substituent at the 3 position of the side chain did not affect activity, indicating broad-range AHL quorum-quenching activity. Of the two main AHL signal molecules of P. aeruginosa PAO1, N-butanoyl-Lhomoserine lactone (C 4 -HSL) and N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C 12 -HSL), only 3-oxo-C 12 -HSL is degraded by the enzyme. Addition of the purified protein to P. aeruginosa PAO1 cultures completely inhibited accumulation of 3-oxo-C 12 -HSL and production of the signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone and reduced production of the virulence factors elastase and pyocyanin. Similar results were obtained when the PA2385 gene was overexpressed in P. aeruginosa. These results demonstrate that the protein has in situ quorum-quenching activity. The quorum-quenching AHL acylase may enable P. aeruginosa PAO1 to modulate its own quorum-sensing-dependent pathogenic potential and, moreover, offers possibilities for novel antipseudomonal therapies.Pseudomonas aeruginosa PAO1 is an opportunistic pathogen which causes disease mainly in individuals who are immunocompromised, have cystic fibrosis, or suffer from serious burn wounds. It utilizes two N-acyl-homoserine lactone (AHL)-dependent quorum-sensing systems, termed las and rhl, which together regulate an extensive set of cell population density and growth-phase-dependent virulence factors (7,22). The las and the rhl quorum-sensing systems employ N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C 12 -HSL) and N-butanoyl-L-homoserine lactone (C 4 -HSL), which function by activating the response regulator proteins LasR and RhlR, respectively. In diverse gram-negative bacteria, many different AHL signal molecules have been characterized. These all consist of a homoserine lactone (HSL) ring or nucleus which is connected via an amide bond to a fatty acid side chain of 4 to 14 carbon atoms in length that may contain an oxo or hydroxyl group at the 3Ј position and unsaturated bonds (Fig. 1).In P. aeruginosa PAO1, swarming motility, biofilm maturation, and the expression of virulence factors such as exoproteases, hemolysins, exotoxin A, exoenzyme S, pyocyanin, cyanide, and the cytotoxic lectins PA-IL and PA-IIL, as w...

Guanine nucleotide exchange factors (GEFs) activate Ras proteins by stimulating the exchange of GTP for GDP in a multistep mechanism which involves binary and ternary complexes between Ras, guanine nucleotide, and GEF. We present fluorescence measurements to define the kinetic constants that characterize the interactions between Ras, GEF, and nucleotides, similar to the characterization of the action of RCC1 on Ran [Klebe et al. (1995) Biochemistry 34, 12543-12552]. The dissociation constant for the binary complex between nucleotide-free Ras and the catalytic domain of mouse Cdc25, Cdc25(Mm285), was 4.6 nM, i.e., a 500-fold lower affinity than the Ras.GDP interaction. The affinities defining the ternary complex Ras. nucleotide.Cdc25(Mm285) are several orders of magnitude lower. The maximum acceleration by Cdc25(Mm285) of the GDP dissociation from Ras was more than 10(5)-fold. Kinetic measurements of the association of nucleotide to nucleotide-free Ras and to the binary complex Ras. Cdc25(Mm285) show that these reactions are practically identical: a fast binding step is followed by a reaction of the first order which becomes rate limiting at high nucleotide concentrations. The second reaction is thought to be a conformational change from a low- to a high-affinity nucleotide binding conformation in Ras. Taking into consideration all experimental data, the reverse isomerization reaction from a high- to a low-affinity binding conformation in the ternary complex Ras. GDP.Cdc25(Mm285) is postulated to be the rate-limiting step of the GEF-catalyzed exchange. Furthermore, we demonstrate that the disruption of the Mg2+-binding site is not the only factor in the mechanism of GEF-catalyzed nucleotide exchange on Ras.