Biochemical characterisation of TCTP questions its function as a guanine nucleotide exchange factor for Rheb.

Translationally controlled tumour protein (TCTP) is involved in malignant transformation and regulation of apoptosis. It has been postulated to serve as a guanine nucleotide exchange factor for the small G-protein Rheb. Rheb functions in the PI3 kinase/mTOR pathway. The study presented here was initiated to characterise the interaction between TCTP and Rheb biochemically. Since (i) no exchange activity of TCTP towards Rheb could be detected in vitro, (ii) no interaction between TCTP and Rheb could be detected by NMR spectroscopy, and (iii) no effect of TCTP depletion in cells on the direct downstream targets of Rheb could be observed in vivo, this study shows that TCTP is unlikely to be a guanine nucleotide exchange factor for Rheb.

The role of Rap1 in integrin-mediated cell adhesion.

Rap1 is a member of the Ras-like small GTPases. Originally the protein was identified in a genome-wide screen for suppressors of Ras transformation, but the mechanism of this reversion remained elusive. We have investigated the signalling function of Rap1. We observed that Rap1 is activated by a large variety of stimuli, including growth factors, neurotransmitters and cytokines. Common second messengers like cAMP, diacylglycerol and calcium are mediators of this activation. These messengers activate guanine nucleotide exchange factors (GEFs), the most notable of which is Epac (exchange protein directly activated by cAMP). However, the downstream effectors of Rap1 are less clear. Although direct connections of Rap1 with the serine/threonine kinases Raf1 and B-raf have been reported, we were unable to find functional evidence for an interaction of endogenous Rap1 signalling with the Raf/extracellular-signal-regulated kinase (ERK) pathway. Instead we observe a clear connection of Rap1 with inside-out signalling to integrins. Indeed, introduction of a constitutively active Rap1 as well as Epac induces integrin-mediated cell adhesion, whereas inhibition of Rap1 signalling by the introduction of Rap1GAP (GTPase-activating protein) inhibits inside-out activation of integrins. More importantly, activation of a G(s)-protein-coupled receptor results in integrin-mediated cell adhesion, by a pathway involving Epac and Rap1. From these results, we conclude that one of the functions of receptor-induced Rap1 activation is inside-out regulation of integrins.

A new phospholipase-C-calcium signalling pathway mediated by cyclic AMP and a Rap GTPase.

Stimulation of phosphoinositide-hydrolysing phospholipase C (PLC) generating inositol-1,4,5-trisphosphate is a major calcium signalling pathway used by a wide variety of membrane receptors, activating distinct PLC-beta or PLC-gamma isoforms. Here we report a new PLC and calcium signalling pathway that is triggered by cyclic AMP (cAMP) and mediated by a small GTPase of the Rap family. Activation of the adenylyl cyclase-coupled beta2-adrenoceptor expressed in HEK-293 cells or the endogenous receptor for prostaglandin E1 in N1E-115 neuroblastoma cells induced calcium mobilization and PLC stimulation, seemingly caused by cAMP formation, but was independent of protein kinase A (PKA). We provide evidence that these receptor responses are mediated by a Rap GTPase, specifically Rap2B, activated by a guanine-nucleotide-exchange factor (Epac) regulated by cAMP, and involve the recently identified PLC-epsilon isoform.

Dynamic interaction of cAMP with the Rap guanine-nucleotide exchange factor Epac1.

Epac1 is a Rap-specific guanine-nucleotide exchange factor (GEF) which is activated by the binding of cAMP to a cyclic nucleotide monophosphate (cNMP)-binding domain. We investigated the equilibrium and dynamics of the interaction of cAMP and Epac1 using a newly designed fluorescence analogue of cAMP, 8-MABA-cAMP. We observed that the interaction of cAMP, measured by competition with 8-MABA-cAMP, with an isolated cNMP binding domain of Epac1 has an overall equilibrium constant (Kd) of 4 microM and that the kinetics of the interaction are highly dynamic. The binding properties of cAMP are apparently not affected when the catalytic domain is present, despite the fact that binding of cAMP results in activation of Epac1. This indicates that for the activation process, no appreciable binding energy is required. However, when bound to Rap1b, the apparent Kd of Epac to cAMP was about fivefold lower, suggesting that substrate interaction stabilizes cAMP binding. Since the fluorescent analogues used here were either less able or unable to induce activation of Epac1, we concluded that the binding of nucleotide to Epac and the activation of GEF activity are uncoupled processes and that thus appropriate cAMP analogues can be used as inhibitors of the Epac1-mediated signal transduction pathway of Rap.

RalGEF2, a pleckstrin homology domain containing guanine nucleotide exchange factor for Ral.

Ral is a ubiquitously expressed Ras-like small GTPase. Several guanine nucleotide exchange factors for Ral have been identified, including members of the RalGDS family, which exhibit a Ras binding domain and are regulated by binding to RasGTP. Here we describe a novel type of RalGEF, RalGEF2. This guanine nucleotide exchange factor has a characteristic Cdc25-like catalytic domain at the N terminus and a pleckstrin homology (PH) domain at the C terminus. RalGEF2 is able to activate Ral both in vivo and in vitro. Deletion of the PH domain results in an increased cytoplasmic localization of the protein and a corresponding reduction in activity in vivo, suggesting that the PH domain functions as a membrane anchor necessary for optimal activity in vivo.

Mechanism of regulation of the Epac family of cAMP-dependent RapGEFs.

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.