cAMP controls many cellular processes mainly through the activation of protein kinase A (PKA). However, more recently PKA-independent pathways have been established through the exchange protein directly activated by cAMP (Epac), a guanine nucleotide exchange factor for the small GTPases Rap1 and Rap2. In this report, we show that cAMP can induce integrin-mediated cell adhesion through Epac and Rap1. Indeed, when Ovcar3 cells were treated with cAMP, cells adhered more rapidly to fibronectin. This cAMP effect was insensitive to the PKA inhibitor H-89. A similar increase was observed when the cells were transfected with Epac. Both the cAMP effect and the Epac effect on cell adhesion were abolished by the expression of Rap1–GTPase-activating protein, indicating the involvement of Rap1 in the signaling pathway. Importantly, a recently characterized cAMP analogue, 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate, which specifically activates Epac but not PKA, induced Rap-dependent cell adhesion. Finally, we demonstrate that external stimuli of cAMP signaling, i.e., isoproterenol, which activates the Gαs-coupled β2-adrenergic receptor can induce integrin-mediated cell adhesion through the Epac-Rap1 pathway. From these results we conclude that cAMP mediates receptor-induced integrin-mediated cell adhesion to fibronectin through the Epac-Rap1 signaling pathway.

DNA synthesis in Escherichia coli is inhibited transiently after UV irradiation. Induced replisome reactivation or ''replication restart'' occurs shortly thereafter, allowing cells to complete replication of damaged genomes. At the present time, the molecular mechanism underlying replication restart is not understood. DNA polymerase II (pol II), encoded by the dinA (polB) gene, is induced as part of the global SOS response to DNA damage. Here we show that pol II plays a pivotal role in resuming DNA replication in cells exposed to UV irradiation. There is a 50-min delay in replication restart in mutant cells lacking pol II. Although replication restart appears normal in ⌬umuDC strains containing pol II, the restart process is delayed for >90 min in cells lacking both pol II and UmuD 2 C. Because of the presence of pol II, a transient replication-restart burst is observed in a ''quick-stop'' temperature-sensitive pol III mutant (dnaE486) at nonpermissive temperature. However, complete recovery of DNA synthesis requires the concerted action of both pol II and pol III. Our data demonstrate that pol II and UmuD 2 C act in independent pathways of replication restart, thereby providing a phenotype for pol II in the repair of UV-damaged DNA.DNA polymerase II (pol II), encoded by the damage-inducible dinA (polB) gene of Escherichia coli, is regulated at the transcriptional level by the LexA repressor (1-4) and is induced Ϸ7-fold from Ϸ50 to 350 molecules per damaged cell (4). Although pol II was discovered in 1970 (5) and was characterized biochemically shortly thereafter (6, 7), it has remained an enigma, in contrast to polymerases I and III, which carry out clearly defined roles in DNA repair and replication, respectively (8).Genetic studies reveal that pol II may be involved in repairing DNA damaged by UV irradiation (9) or oxidation (10), in bypassing abasic lesions in vivo in the absence of heat shock induction (11) and in the repair of interstrand cross-links (12). It has been shown that pol II catalyzes episomal DNA synthesis in vivo (13,14) and synthesizes chromosomal DNA in a pol III antimutator (dnaE915) background (14). Pol II is able to catalyze processive synthesis in vitro in the presence of ␤-sliding clamp and clamp-loading ␥-complex, suggesting a role for pol II in replication and repair (15)(16)(17).After UV irradiation, a replication fork may be become stalled when encountering a DNA template lesion (18). In this paper, we report that pol II plays a pivotal role in replication restart (19), i.e., induced replisome reactivation (20-22), a process whereby ''reinitiation'' of DNA synthesis on UVdamaged DNA allows lesion bypass to occur in an error-free repair pathway. Our data provide a well defined role for pol II in repairing UV-induced chromosomal DNA damage. MATERIALS AND METHODSBacterial Strains and Growth Conditions. The E. coli K-12 strains used in this study are listed in Table 1. To avoid complications arising from the use of nonisogenic strains, we moved the previously generated polB (10)...

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 integrinmediated 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.

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