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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.

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.

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.

Regulation of phosphoinositide 3-kinase (PI 3-kinase) can occur by binding of the regulatory p85 subunit to tyrosine-phosphorylated proteins and by binding of the p110 catalytic subunit to activated Ras. However, the way in which these regulatory mechanisms act to regulate PI 3-kinase in vivo is unclear. Here we show that several growth factors (basic fibroblast growth factor [bFGF], platelet-derived growth factor [PDGF], and epidermal growth factor [EGF; to activate an EGF receptor-Ret chimeric receptor]) all activate PI 3-kinase in vivo in the neuroectoderm-derived cell line SKF5. However, these growth factors differ in their ability to activate PI 3-kinase-dependent signaling. PDGF and EGF(Ret) treatment induced PI 3-kinase-dependent lamellipodium formation and protein kinase B (PKB) activation. In contrast, bFGF did not induce lamellipodium formation but activated PKB, albeit to a small extent. PDGF and EGF(Ret) stimulation resulted in binding of p85 to tyrosine-phosphorylated proteins and strong Ras activation. bFGF, however, induced only strong activation of Ras. In addition, while RasAsn17 abolished bFGF activation of PKB, PDGF- and EGF(Ret)-induced PKB activation was only partially inhibited and lamellipodium formation was unaffected. Interestingly, in contrast to activation of only endogenous Ras (bFGF), ectopic expression of activated Ras did result in lamellipodium formation. From this we conclude that, in vivo, p85 and Ras synergize to activate PI 3-kinase and that strong activation of only endogenous Ras exerts a small effect on PI 3-kinase activity, sufficient for PKB activation but not lamellipodium formation. This differential sensitivity to PI 3-kinase activation could be explained by our finding that PKB activation and lamellipodium formation are independent PI 3-kinase-induced events.

Ras is a small GTPase that cycles between an inactive GDP-bound and an active GTP-bound form. A large variety of ligands that stimulate cell surface receptors induce the activation of Ras. Thus far, this activation could only be measured by the increase of GTP bound to Ras, which was precipitated from radio-labelled cell extract. We have used the minimal Ras-binding domain (RBD) of Raf1 (aa 51-131) to identify in vivo activated Ras. This novel method is based on the observation that RBD binds RasGTP in vitro with a Kd of 20 nM whereas the affinity between RBD and RasGDP is three orders of magnitude lower. Here we show that the Gst-RBD fusion protein precipitates transfected RasL61 (RasGTP) but not RasN17 (RasGDP) from cell lysates. In addition, we demonstrate for two different cell lines that the increase in RasGTP is reflected by an increase in Ras bound to Gst-RBD. From these results we conclude that the minimal Ras-binding domain of Raf1 is an excellent activation specific-probe for Ras.

Nucleotide excision repair, which is defective in xeroderma pigmentosum (XP), involves incision of a DNA strand on each side of a lesion. We isolated a human gene homologous to yeast Rad1 and found that it corrects the repair defects of XP group F as well as rodent groups 4 and 11. Causative mutations and strongly reduced levels of encoded protein were identified in XP-F patients. The XPF protein was purified from mammalian cells in a tight complex with ERCC1. This complex is a structure-specific endonuclease responsible for the 5' incision during repair. These results demonstrate that the XPF, ERCC4, and ERCC11 genes are equivalent, complete the isolation of the XP genes that form the core nucleotide excision repair system, and solve the catalytic function of the XPF-containing complex.

The temperature dependence of the circular dichroism (CD) spectra of a series of deoxyadenylates (dA)n, n = 2, 3, 6, 9, 12, infinity, in aqueous solution was studied. The data were interpreted on the basis of a new conformational model for the stacked state suggested by our previous proton NMR studies on (dA)2 and (dA)3 [C. S. M. Olsthoorn, L. J. Bostelaar, J. H. van Boom & C. Altona (1980) Eur. J. biochem. 112, 95-110]. In this model the stacked regions of the single-stranded oligomers consist of residues taking up a geometry resembling that of the B-DNA genus of structures (all sugars S or C2'-endo) except those residues at the 3' end that do not 'feel' a following stacking interaction. The deoxyribose rings in the latter residues retain (or regain when melting out removes a stacking interaction somewhere along the chain) the conformational freedom (S in equilibrium N, N = C3'-endo) that these rings possess in the monomers 2'-deoxyadenosine 5'-methylphosphate or in 2'-deoxyadenosine 3',5'-bis(methylphosphate), as the case may be. It is shown that this model allows (a) construction of the CD spectra of (dA)n, n = 3, 6, 9, 12, from those of the dimer and the polymer; (b) the separation of the weak CD displayed by the regular S-S stacking mode and the far stronger CD exhibited by the 3'-end S-N stacking (the latter CD resembles that of the A-DNA genus of structures); (c) delineation of the thermodynamics of stacking. The melting temperature remains constant and independent of chain length (about 50 degrees C) whereas delta H degrees and delta S degrees show a slight increase in absolute values on increasing n from 2 to infinity owing to small cooperativity effects. Near 0 degrees C the dimer occurs for about 90% in the stacked form, the oligomers attain even higher conformational purities. It is suggested that premelting phenomena observed in the CD spectra of double-helical DNAs may also involve local transitions from the normal B-like ----S-S-s---- stacking mode to an A-like ----S-S-N---- stacking geometry.

Two examples of neighbouring group participation during the removal of protecting groups from phosphotriesters of partially or fully protected intermediates of nucleic acids are presented. The first example shows that ammonolysis of aryl groups from phosphotriesters of partially protected - 5'- hydroxy free - nucleic acids (e.g., 4b approximately to; Ar=2C1C 6H4) gives rise to the formation of unnatural nucleic acids (e.g., 7 approximately to and 8 approximately to). The second one illustrates that fluoride ion promoted hydrolysis of 2,2,2-trichloroethyl groups from phosphotriesters of fully protected nucleic acids (e.g., 18a approximately to), having t-butyldimethylsilyl groups at the 2'-positions, leads to the formation of a considerable amount of side-products (e.g., 20 approximately to and 21 approximately to).