Rap1-mediated activation of extracellular signal-regulated kinases by cyclic AMP is dependent on the mode of Rap1 activation

Like other small G proteins of the Ras superfamily, Rap1 is activated by distinct guanine nucleotide exchange factors (GEFs) in response to different signals to elicit cellular responses. Activation of Rap1 by cyclic AMP (cAMP) can occur via cAMP-dependent protein kinase A (PKA)-independent and PKA-dependent mechanisms. PKA-independent activation of Rap1 by cAMP is mediated by direct binding of cAMP to Rap1-guanine nucleotide exchange factors (Rap1-GEFs) Epac1 (exchange protein directly activated by cAMP 1) and Epac2 (Epac1 and Epac2 are also called cAMP-GEFI and -GEFII). The availability of cAMP analogues that selectively activate Epacs, but not PKA, provides a specific tool to activate Rap1. It has been argued that the inability of these analogues to regulate extracellular signal-regulated kinases (ERKs) signaling despite activating Rap1 provides evidence that Rap1 is incapable of regulating ERKs. We confirm that the PKA-independent activation of Rap1 by Epac1 activates a perinuclear pool of Rap1 and that this does not result in ERK activation. However, we demonstrate that this inability to regulate ERKs is not a property of Rap1 but is rather a property of Epacs themselves. The addition of a membrane-targeting motif to Epac1 (Epac-CAAX) relocalizes Epac1 from its normal perinuclear locale to the plasma membrane. In this new locale it is capable of activating ERKs in a Rap1- and cAMP-dependent manner. Rap1 activation by Epac-CAAX, but not wild-type Epac, triggers its association with B-Raf. Therefore, we propose that its intracellular localization prevents Epac1 from activating ERKs. C3G (Crk SH3 domain Guanine nucleotide exchanger) is a Rap1 exchanger that is targeted to the plasma membrane upon activation. We show that C3G can be localized to the plasma membrane by cAMP/PKA, as can Rap1 when activated by cAMP/PKA. Using a small interfering RNA approach, we demonstrate that C3G is required for the activation of ERKs and Rap1 by cAMP/PKA. This activation requires the GTP-dependent association of Rap1 with B-Raf. These data demonstrate that B-Raf is a physiological target of Rap1, but its utilization as a Rap1 effector is GEF specific. We propose a model that specific GEFs activate distinct pools of Rap1 that are differentially coupled to downstream effectors.     

Divergent roles for Ras and Rap in the activation of p38 mitogen-activated protein kinase by interleukin-1

We have found that lethal toxin from Clostridium sordellii, which specifically inactivates the low molecular weight G proteins Ras, Rap, and Rac, inhibits the activation of p38 mitogen-activated protein kinase (MAPK) by interleukin-1 (IL-1) in EL4.NOB-1 cells and primary fibroblasts. The target protein involved appeared to be Ras, because transient transfections with dominant negative RasN17 inhibited p38 MAPK activation by IL-1. Furthermore, transfections of cells with constitutively active RasVHa-activated p38 MAPK. Further evidence for Ras involvement came from the observation that IL-1 caused a rapid activation of Ras in the cells and from the inhibitory effects of the Ras inhibitors manumycin A and damnacanthal. Toxin B from Clostridium difficile, which inactivates Rac, Cdc42, and Rho, was without effect. Dominant negative versions of Rac (RacN17) or Rap (Rap1AN17) did not inhibit the response. Intriguingly, transfection of cells with dominant negative Rap1AN17 activated p38 MAPK. Furthermore, constitutively active Rap1AV12 inhibited p38 MAPK activation by IL-1, consistent with Rap antagonizing Ras function. IL-1 also activated Rap in the cells, but with slower kinetics than Ras. Our studies therefore provide clear evidence using multiple approaches for Ras as a signaling component in the activation of p38 MAPK by IL-1, with Rap having an inhibitory effect.     

AF-6 controls integrin-mediated cell adhesion by regulating Rap1 activation through the specific recruitment of Rap1GTP and SPA-1

In the present study, we showed that SPA-1, a Rap1 GTPase-activating protein (GAP), was bound to a cytoskeleton-anchoring protein AF-6. SPA-1 and AF-6 were co-immunoprecipitated in the 293T cells transfected with both cDNAs as well as in normal thymocytes. In vitro binding studies using truncated fragments and their mutants suggested that SPA-1 was bound to the PDZ domain of AF-6 via probable internal PDZ ligand motif within the GAP-related domain. The motif was conserved among Rap1 GAPs, and it was shown that rapGAP I was bound to AF-6 comparably with SPA-1. RapV12 was also bound to AF-6 via the N-terminal domain, and SPA-1 and RapV12 were co-immunoprecipitated only in the presence of AF-6, indicating that they could be brought into close proximity via AF-6 in cells. Immunostaining analysis revealed that SPA-1 and RapV12 were co-localized with AF-6 at the cell attachment sites. In HeLa cells expressing SPA-1 in a tetracycline-regulatory manner, expression of AF-6 inhibited endogenous Rap1GTP and beta(1) integrin-mediated cell adhesion to fibronectin in SPA-1-induced conditions, whereas it affected neither of them in SPA-1-repressed conditions. These results suggested that AF-6 could control integrin-mediated cell adhesion by regulating Rap1 activation through the recruitment of both SPA-1 and Rap1GTP via distinct domains.     

RA-GEF-1, a guanine nucleotide exchange factor for Rap1, is activated by translocation induced by association with Rap1*GTP and enhances Rap1-dependent B-Raf activation

We previously identified RA-GEF-1, a novel guanine nucleotide exchange factor (GEF) for Rap1 with the ability to associate with Rap1.GTP at its Ras/Rap1-associating (RA) domain. Because it possesses a PSD-95/DlgA/ZO-1 (PDZ) domain, it was also named PDZ-GEF. In this report, we have examined the role of the RA domain of this protein in Rap1-mediated cellular responses. A mutant of RA-GEF-1 (RA-GEF-1DeltaRA) carrying a 21-residue deletion at its RA domain fully retains the in vitro GEF activity toward Rap1 but completely loses the Rap1 binding activity. In contrast, RA-GEF-1DeltaRA, expressed in COS-7 cells, exhibits a 3-fold reduction in its in vivo GEF activity toward Rap1 compared with wild-type RA-GEF-1 as examined by the Rap1 pull-down assay. Correspondingly, when coexpressed with wild-type Rap1, RA-GEF-1DeltaRA is unable to further activate B-Raf, whereas RA-GEF-1 stimulates B-Raf as efficiently as activated Rap1. Consistent with these observations, coexpression of activated Rap1 induces translocation of RA-GEF-1, which is otherwise located in the cytoplasm, to the perinuclear compartment, where Rap1 is also predominantly localized. This localization almost coincides with that of the Golgi apparatus, which was detected by anti-trans-Golgi-network 38 antibody. RA-GEF-1DeltaRA fails to show the translocation. These results indicate that RA-GEF-1 defines a novel category of GEF that is translocated to a particular subcellular compartment by association with the GTP-bound form of a small GTPase and catalyzes activation of the GDP-bound form present in the compartment, thereby causing an amplification of cellular responses induced by the small GTPase.     

Highly conserved toxicity of Saccharomyces cerevisiae Rap1p

Budding yeast ( Saccharomyces cerevisiae ) Rap1p has been expressed in fission yeast ( Schizosaccharo-myces pombe ) under the control of the regulatable fructose bisphosphatase ( fbp ) promoter. When the fbp promoter was derepressed, cells containing the complete RAP1 gene failed to show any significant growth, suggesting that Rap1p is toxic. A derivative of Rap1p that has a temperature-sensitive mutation in the DNA-binding domain was not toxic in cells grown at 37°C, a temperature at which DNA binding by rap1p ^ts is severely inhibited. Removal of a short region downstream of the DNA-binding domain, including a region previously shown to be essential for Rap1p toxicity in budding yeast, also abolished the toxic effect. The toxic effect of Rap1p has therefore been conserved between two distantly related yeasts. In budding yeast, overexpression of Rap1p also caused changes to the lengths of the telomeric repeats. No effects on telomeres were detected in fission yeast.     

Negative regulation of Rap1 activation by the Cbl E3 ubiquitin ligase

Cbl functions as an adaptor protein by interacting with other signalling molecules to form multimolecular complexes. Previous studies have proposed that Cbl is also a positive regulator of CrkL–C3G signalling, which leads to Rap1 activation. However, there is a lack of genetic evidence for a physiological function of Cbl in regulating this pathway. Here, we show that Cbl deficiency results in enhanced activation of Rap1. Cbl was shown to promote the ubiquitylation of CrkL without any apparent effect on its stability. Remarkably, the membrane translocation of C3G, its association with CrkL, and the guanine-nucleotide exchange activity of C3G were all increased in Cbl^−/− thymocytes. Consistent with a function of Rap1 in integrin activation, enhanced integrin-mediated cell adhesion was also seen in Cbl^−/− thymocytes. Thus, Cbl negatively regulates Rap1 activation, probably through a proteolysis-independent E3-ubiquitin-ligase activity of Cbl that modulates protein–protein interactions.     

Glucagon transiently stimulates mTORC1 by activation of an EPAC/Rap1 signaling axis

Activation of the protein kinase mechanistic target of rapamycin (mTOR) in both complexes 1 and 2 (mTORC1/2) in the liver is repressed during fasting and rapidly stimulated in response to a meal. The effect of feeding on hepatic mTORC1/2 is attributed to an increase in plasma levels of nutrients, such as amino acids, and insulin. By contrast, fasting is associated with elevated plasma levels of glucagon, which is conventionally viewed as having a counter-regulatory role to insulin. More recently an expanded role for glucagon action in post-prandial metabolism has been demonstrated. Herein we investigated the impact of insulin and glucagon on mTORC1/2 activation. In H4IIE and HepG2 cultures, insulin enhanced phosphorylation of the mTORC1 substrates S6K1 and 4E-BP1. Surprisingly, the effect of glucagon on mTORC1 was biphasic, wherein there was an acute increase in phosphorylation of S6K1 and 4E-BP1 over the first hour of exposure, followed by latent suppression. The transient stimulatory effect of glucagon on mTORC1 was not additive with insulin, suggesting convergent signaling. Glucagon enhanced cAMP levels and mTORC1 stimulation required activation of the glucagon receptor, PI3K/Akt, and exchange protein activated by cAMP (EPAC). EPAC acts as the guanine nucleotide exchange factor for the small GTPase Rap1. Rap1 expression enhanced S6K1 phosphorylation and glucagon addition to culture medium promoted Rap1-GTP loading. Signaling through mTORC1 acts to regulate protein synthesis and we found that glucagon promoted an EPAC-dependent increase in protein synthesis. Overall, the findings support that glucagon elicits acute activation of mTORC1/2 by an EPAC-dependent increase in Rap1-GTP.