cAMP inhibition of Akt is mediated by activated and phosphorylated Rap1b

Rap1b has been implicated in the transduction of the cAMP mitogenic signal. Rap1b is phosphorylated and activated by cAMP, and its expression in cells where cAMP is mitogenic leads to an increase in G(1)/S phase entry and tumor formation. The PCCL3 thyroid follicular cells represent a differentiated and physiologically relevant system that requires thyrotropin (TSH), acting via cAMP, for a full mitogenic response. In this model system, cAMP stimulation of DNA synthesis requires activation and phosphorylation of Rap1b by the cAMP-dependent protein kinase A (PKA). This scenario presents the challenge of identifying biochemical processes involved in the phosphorylation-dependent Rap1b mitogenic action. In thyroid cells, Akt has been implicated in the stimulation of cell proliferation by TSH and cAMP. However, the mechanism(s) by which cAMP regulates Akt activity remains unclear. In this study we show that in PCCL3 cells 1) TSH inhibits Akt activity via cAMP and PKA; 2) Rap1b is required for cAMP inhibition of Akt; and 3) transduction of the cAMP signal into Akt requires activation as well as phosphorylation of Rap1b by PKA.     

Epac, in synergy with cAMP-dependent protein kinase (PKA), is required for cAMP-mediated mitogenesis

cAMP stimulates proliferation in many cell types. For many years, cAMP-dependent protein kinase (PKA) represented the only known cAMP effector. PKA, however, does not fully mimic the action of cAMP, indicating the existence of a PKA-independent component. Since cAMP-mediated activation of the G-protein Rap1 and its phosphorylation by PKA are strictly required for the effects of cAMP on mitogenesis, we hypothesized that the Rap1 activator Epac might represent the PKA-independent factor. Here we report that Epac acts synergistically with PKA in cAMP-mediated mitogenesis. We have generated a new dominant negative Epac mutant that revealed that activation of Epac is required for thyroid-stimulating hormone or cAMP stimulation of DNA synthesis. We demonstrate that Epac's action on cAMP-mediated activation of Rap1 and cAMP-mediated mitogenesis depends on the subcellular localization of Epac via its DEP domain. Disruption of the DEP-dependent subcellular targeting of Epac abolished cAMP-Epac-mediated Rap1 activation and thyroid-stimulating hormone-mediated cell proliferation, indicating that an Epac-Rap-PKA signaling unit is critical for the mitogenic action of cAMP.     

Coordinated regulation of Rap1 and thyroid differentiation by cyclic AMP and protein kinase A

Originally identified as an antagonist of Ras action, Rap1 exhibits many Ras-independent effects, including a role in signaling pathways initiated by cyclic AMP (cAMP). Since cAMP is a critical mediator of the effects of thyrotropin (TSH) on cell proliferation and differentiation, we examined the regulation of Rap1 by TSH in a continuous line of rat thyroid-like cells. Both cAMP and protein kinase A (PKA) contribute to the regulation of Rap1 activity and signaling by TSH. TSH activates Rap1 through a cAMP-mediated and PKA-independent mechanism. TSH phosphorylates Rap1 in a PKA-dependent manner. Interference with PKA activity blocked phosphorylation but not the activation of Rap1. Rather, PKA inhibitors prolonged Rap1 activation, as did expression of a Rap1A mutant lacking a PKA phosphorylation site. These results indicate that PKA elicits negative feedback regulation on cAMP-stimulated Rap1 activity in some cells. The dual regulation of Rap1 by cAMP and PKA extends to downstream effectors. The ability of TSH to stimulate Akt phosphorylation was markedly enhanced by the expression of activated Rap1A and was repressed in cells expressing a putative dominant-negative Rap1A mutant. Although the expression of activated Rap1A was sufficient to stimulate wortmannin-sensitive Akt phosphorylation, TSH further increased Akt phosphorylation in a phosphatidylinositol 3-kinase- and PKA-dependent manner. The ability of TSH to phosphorylate Akt was impaired in cells expressing a Rap1A mutant that could be activated but not phosphorylated. These findings indicate that dual signals, Rap1 activation and phosphorylation, contribute to TSH-stimulated Akt phosphorylation. Rap1 plays an essential role in cAMP-regulated differentiation. TSH effects on thyroid-specific gene expression, but not its effects on proliferation, were markedly enhanced in cells expressing activated Rap1A and repressed in cells expressing a dominant-negative Rap1A mutant. These findings reveal complex regulation of Rap1 by cAMP including PKA-independent activation and PKA-dependent negative feedback regulation. Both signals appear to be required for TSH signaling to Akt.     

Toll-like receptor 4-mediated cAMP production up-regulates B-cell activating factor expression in Raw264.7 macrophages

B-cell activating factor (BAFF) plays a role in the generation and the maintenance of mature B cells. Lipopolysaccharide (LPS) increased BAFF expression through the activation of toll-like receptor 4 (TLR4)-dependent signal transduction. Here, we investigated the mechanism of action on mouse BAFF (mBAFF) expression by cAMP production in Raw264.7 mouse macrophages. mBAFF expression was increased by the treatment with a cAMP analogue, dibutyryl-cAMP which is the activator of protein kinase A (PKA), cAMP effector protein. PKA activation was measured by the phosphorylation of cAMP-response element binding protein (CREB) on serine 133 (S133). cAMP production and CREB (S133) phosphorylation were augmented by LPS-stimulation. While mBAFF promoter activity was enhanced by the co-transfection with pS6-RSV-CREB, it was reduced by siRNA-CREB. PKA inhibitor, H-89, reduced CREB (S133) phosphorylation and mBAFF expression in control and LPS-stimulated macrophages. Another principal cAMP effector protein is cAMP-responsive guanine nucleotide exchange factor (Epac), a Rap GDP exchange factor. Epac was activated by the treatment with 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (CPT), Epac activator, as judged by the measurement of Rap1 activation. Basal level of mBAFF expression was increased by CPT treatment. LPS-stimulated mBAFF expression was also slightly enhanced by co-treatment with CPT. In addition, dibutyryl-cAMP and CPT enhanced mBAFF expression in bone marrow-derived macrophages (BMDM). With these data, it suggests that the activation of PKA and cAMP/Epac1/Rap1 pathways could be required for basal mBAFF expression, as well as being up-regulated in the TLR4-induced mBAFF expression.     

Galpha and Gbeta gamma require distinct Src-dependent pathways to activate Rap1 and Ras

The Src tyrosine kinase is necessary for activation of extracellular signal-regulated kinases (ERKs) by the beta-adrenergic receptor agonist, isoproterenol. In this study, we examined the role of Src in the stimulation of two small G proteins, Ras and Rap1, that have been implicated in isoproterenol's signaling to ERKs. We demonstrate that the activation of isoproterenol of both Rap1 and Ras requires Src. In HEK293 cells, isoproterenol activates Rap1, stimulates Rap1 association with B-Raf, and activates ERKs, all via PKA. In contrast, the activation by isoproterenol of Ras requires Gbetagamma subunits, is independent of PKA, and results in the phosphoinositol 3-kinase-dependent activation of AKT. Interestingly, beta-adrenergic stimulation of both Rap1 and ERKs, but not Ras and AKT, can be blocked by a Src mutant (SrcS17A) that is incapable of being phosphorylated and activated by PKA. Furthermore, a Src mutant (SrcS17D), which mimics PKA phosphorylation at serine 17, stimulates Rap1 activation, Rap1/B-Raf association, and ERK activation but does not stimulate Ras or AKT. These data suggest that Rap1 activation, but not that of Ras, is mediated through the direct phosphorylation of Src by PKA. We propose that the beta(2)-adrenergic receptor activates Src via two independent mechanisms to mediate distinct signaling pathways, one through Galpha(s) to Rap1 and ERKs and the other through Gbetagamma to Ras and AKT.     

PKA phosphorylation of Src mediates cAMP's inhibition of cell growth via Rap1.

In fibroblast cells, cAMP antagonizes growth factor activation of ERKs and cell growth via PKA and the small G protein Rap1. We demonstrate here that PKA's activation of Rap1 was mediated by the Rap1 guanine nucleotide exchange factor C3G, the adaptor Crk-L, the scaffold protein Cbl, and the tyrosine kinase Src. Src was required for cAMP activation of Rap1 and the inhibition of ERKs and cell growth. PKA activated Src both in vitro and in vivo by phosphorylating Src on serine 17 within its amino terminus. This phosphorylation was required for cAMP's activation of Src and Rap1, as well as cAMP's inhibition of ERKs and cell proliferation. This study identifies an antiproliferative role for Src in the physiological regulation of cell growth by cAMP.     

CAMP activates Rap1 in differentiating mouse male germ cells: a new signaling pathway mediated by the cAMP-activated exchange factor Epac?

Rap1, a Ras-like G-protein, is implicated in the signaling of various cellular processes as morphogenesis, differentiation, cell adhesion and spreading, and maintenance of T cell anergy and B cell activation. The effectors that mediate Rap1 signaling have not yet been definitely identified, with the exception of B-Raf which, however, is restricted to neuronal tissues and a small subset of other cell types, including in particular male germ cells. We previously showed that in mouse spermatids Rap1 could interact with B-Raf giving rise to a signaling complex. Here we investigated about the possible molecules which "switch on" Rap1 finding that cAMP could in vivo activate endogenous Rap1. Spermatid-enriched cell cultures stimulated with 8-(4-chlorophenylthio)-cyclic AMP yielded higher levels of GTP-bound Rap1 than unstimulated cells. Since cAMP-induced Rap1 activation is actually retained to occur through Epac, we checked whether this recently discovered Rap1 exchange factor is expressed in male germ cells. Our findings indicate that Epac is present in spermatogenic cells and exhibits a preferential subplasmalemmal localization, although it shows also an intracellular location, more or less pronounced depending on the type of spermatogenic cell examined. Taken together, our data show that cAMP activates Rap1 in differentiating male germ cells which express the cAMP sensor Epac, thus suggesting that this activation might occur directly through Epac.     

Cyclic AMP inhibits extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/Akt pathways by inhibiting Rap1

Cyclic AMP inhibited both ERK and Akt activities in rat C6 glioma cells. A constitutively active form of phosphatidylinositol 3-kinase (PI3K) prevented cAMP from inhibiting Akt, suggesting that the inactivation of Akt by cAMP is a consequence of PI3K inhibition. Neither protein kinase A nor Epac (Exchange protein directly activated by cAMP), two known direct effectors of cAMP, mediated the cAMP-induced inhibition of ERK and Akt phosphorylation. Cyclic AMP inhibited Rap1 activation in C6 cells. Moreover, inhibition of Rap1 by a Rap1 GTPase-activating protein-1 also resulted in a decrease in ERK and Akt phosphorylation, which was not further decreased by cAMP, suggesting that cAMP inhibits ERK and Akt by inhibiting Rap1. The role of Rap1 in ERK and Akt activity was further demonstrated by our observation that an active form of Epac, which activated Rap1 in the absence of cAMP, increased ERK and Akt phosphorylation. Inhibition of ERK and/or PI3K pathways mediated the inhibitory effects of cAMP on insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 gene expression. Moreover, cAMP, as well as ERK and PI3K inhibitors produced equivalent stimulation and inhibition, respectively, of p27(Kip1) and cyclin D2 protein levels, potentially explaining the observation that cAMP prevented C6 cells from entering S phase.     

Cyclic adenosine 5'-monophosphate-stimulated neurotensin secretion is mediated through Rap1 downstream of both Epac and protein kinase A signaling pathways

Neurotensin (NT), a gut peptide, plays important roles in gastrointestinal secretion, inflammation, and growth of normal and neoplastic tissues. cAMP regulates the secretion of hormones via its effector proteins protein kinase A (PKA) or Epac (exchange protein directly activated by cAMP). The small GTPase Rap1 can be activated by both PKA and Epac; however, the role of Rap1 in hormone secretion is unknown. Here, using the BON human endocrine cell line, we found that forskolin (FSK)-stimulated NT secretion was reduced by inhibition of Rap1 expression and activity. FSK-stimulated NT secretion was enhanced by overexpression of either wild-type or constitutively active Rap1. Epac activators and wild-type Epac enhanced NT release and Rap1 activity. In contrast, overexpression of a cAMP binding mutant, EpacR279E, decreased NT release and Rap1 activity. PKA activation increased NT release and Rap1 activity. FSK-stimulated NT release was reduced by PKA inhibition and the dominant negative Rap1N17. NT secretion, stimulated by Epac activation, was reduced by PKA inhibition; NT release, stimulated by PKA activation, was enhanced by wild-type Epac but reduced by the mutant EpacR279E. Finally, prostaglandin E2 (PGE2), a physiological agent that increases cAMP, stimulated NT secretion via cAMP/PKA/Rap1. Importantly, we demonstrate that PKA and Epac mediate the cAMP-induced NT secretion synergistically by converging at the common downstream target protein Rap1. Moreover, PGE2, a potent mediator of inflammation and associated with colorectal carcinogenesis, stimulates NT release suggesting a possible link between PGE2 and NT on intestinal inflammatory disorders and colorectal cancers.     

Rap1 couples cAMP signaling to a distinct pool of p42/44MAPK regulating excitability,synaptic plasticity,learning, and memory

Learning-induced synaptic plasticity commonly involves the interaction between cAMP and p42/44MAPK. To investigate the role of Rap1 as a potential signaling molecule coupling cAMP and p42/44MAPK, we expressed an interfering Rap1 mutant (iRap1) in the mouse forebrain. This expression selectively decreased basal phosphorylation of a membrane-associated pool of p42/44MAPK, impaired cAMP-dependent LTP in the hippocampal Schaffer collateral pathway induced by either forskolin or theta frequency stimulation, decreased complex spike firing, and reduced the p42/44MAPK-mediated phosphorylation of the A-type potassium channel Kv4.2. These changes correlated with impaired spatial memory and context discrimination. These results indicate that Rap1 couples cAMP signaling to a selective membrane-associated pool of p42/44MAPK to control excitability of pyramidal cells, the early and late phases of LTP, and the storage of spatial memory.     

A novel Epac-Rap-PP2A signaling module controls cAMP-dependent Akt regulation

Rap1b has been implicated in the transduction of the cAMP mitogenic signal. It is phosphorylated and activated by cAMP, and its expression in models where cAMP is mitogenic leads to proliferation and tumorigenesis. Akt is a likely downstream effector of cAMP-Rap1 action. cAMP elevation induced a rapid and transient Akt inhibition that required activated and phosphorylated Rap1b. However, the mechanism(s) by which cAMP-Rap regulates Akt remains unclear. Here we show that (i) upstream regulators, PIK and PDK1, are not the target(s) of the cAMP inhibitory action; (ii) constitutively active Akt and calyculin A-stimulated Akt are resistant to cAMP inhibition, suggesting the action of a phosphatase; (iii) cAMP increases the rate of Akt dephosphorylation, directly implicating an Akt-phosphatase; (iv) Epac- and protein kinase A (PKA)-specific analogs synergistically inhibit Akt, indicating the involvement of both cAMP-dependent effector pathways; (v) H89 and dominant negative Epac 279E block cAMP-inhibitory action; (vi) Epac associates in a complex with Akt and PP2A, and the associated-phosphatase activity is positively modulated by cAMP in a PKA- and Rap1-dependent manner; (vii) like its action on Akt inhibition, PKA- and Epac-specific analogs synergistically activate Epac-associated PP2A; and (viii) dominant negative PP2A blocks cAMP-inhibitory action. Thus, we uncovered a novel cAMP-Epac/PKA-Rap1b-PP2A signaling module involved in Akt regulation that may represent a physiological event in the process of cAMP stimulation of thyroid mitogenesis.     

Use of an activation-specific probe to show that Rap1A and Rap1B display different sensitivities to activation by forskolin in rat1 cells

Rap1A and Rap1B are small GTPases of the Ras superfamily whose activation can be measured using a probe that interacts specifically with the GTP-bound forms of Rap1A and Rap1B. Using this procedure we demonstrate that the cyclic AMP-elevating agent forskolin activates both Rap1A and Rap1B in Rat1 cells. Whilst the protein kinase A inhibitor H89 ablated the ability of forskolin to cause cAMP response element binding protein (CREB) phosphorylation in Rat1 cells, it did not affect the ability of forskolin to activate either Rap1A and Rap1B. Forskolin differentially activated Rap1A and Rap1B isoforms in a time- and dose-dependent manner. The cAMP-specific type 4 family phosphodiesterase inhibitor rolipram potentiated the rate of activation of both Rap1A and Rap1B by forskolin challenge of Rat1 cells. Challenge of Rat1 cells with rolipram alone was able to elicit the phosphorylation of CREB but not activation of either Rap1A or Rap1B.     

Reactive oxygen species (ROS) play a critical role in the cAMP-induced activation of Ras and the phosphorylation of ERK1/2 in Leydig cells

Activation of the LH receptor (LHR) in Leydig cells results in the phosphorylation of ERK1/2 by cAMP-dependent and cAMP-independent pathways. Here we examine the mechanisms by which cAMP stimulates ERK1/2 phosphorylation. We show that the stimulation of steroidogenesis is not necessary or sufficient to stimulate the phosphorylation of ERK1/2 but that other cAMP-dependent mitochondrial functions are involved. Using MA-10 cells as a model, we showed that cAMP analogs increase reactive oxygen species (ROS) formation and that an uncoupler of oxidative phosphorylation and a ROS scavenger prevent this increase. These two compounds also inhibit the increase in ERK1/2 phosphorylation provoked by cAMP analogs, thus suggesting that the cAMP-induced phosphorylation of ERK1/2 is mediated by mitochondrial ROS. In agreement with this hypothesis we also show that a reduction in glutathione levels, which alters the redox state of MA-10 cells, potentiates the effect of cAMP on ERK1/2 phosphorylation. Measurements of the dephosphorylation of ERK and the activation of Ras showed that the ROS scavenger prevents the cAMP-provoked activation of Ras and that cAMP, with or without a ROS scavenger, has little or no effect on the dephosphorylation of ERK. Lastly, we show that the uncoupler of oxidative phosphorylation and the ROS scavenger also prevent the ability of cAMP analogs to increase ERK1/2 phosphorylation in primary cultures of mouse Leydig cells. We conclude that, in Leydig cells, cAMP enhances the phosphorylation of ERK1/2 via a mitochondria-derived, ROS-dependent activation of Ras.     

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.     

Rap1 activation plays a regulatory role in pancreatic amylase secretion

Rap1 is a member of the Ras superfamily of small GTP-binding proteins and is localized on pancreatic zymogen granules. The current study was designed to determine whether GTP-Rap1 is involved in the regulation of amylase secretion. Rap1A/B and the two Rap1 guanine nucleotide exchange factors, Epac1 and CalDAG-GEF III, were identified in mouse pancreatic acini. A fraction of both Rap1 and Epac1 colocalized with amylase in zymogen granules, but only Rap1 was integral to the zymogen granule membranes. Stimulation with cholecystokinin (CCK), carbachol, and vasoactive intestinal peptide all induced Rap1 activation, as did calcium ionophore A23187, phorbol ester, forskolin, 8-bromo-cyclic AMP, and the Epac-specific cAMP analog 8-pCPT-2'-O-Me-cAMP. The phospholipase C inhibitor U-73122 abolished carbachol- but not forskolin-induced Rap1 activation. Co-stimulation with carbachol and 8-pCPT-2'-O-Me-cAMP led to an additive effect on Rap1 activation, whereas a synergistic effect was seen on amylase release. Although the protein kinase A inhibitor H-89 abolished forskolin-stimulated CREB phosphorylation, it did not modify forskolin-induced GTP-Rap1 levels, excluding PKA participation. Overexpression of Rap1 GTPase-activating protein, which blocked Rap1 activation, reduced the effect of 8-bromo-cyclic AMP, 8-pCPT-2'-O-Me-cAMP, and vasoactive intestinal peptide on amylase release by 60% and reduced CCK- as well as carbachol-stimulated pancreatic amylase release by 40%. These findings indicate that GTP-Rap1 is required for pancreatic amylase release. Rap1 activation not only mediates the cAMP-evoked response via Epac1 but is also involved in CCK- and carbachol-induced amylase release, with their action most likely mediated by CalDAG-GEF III.     

Rap1A is a substrate for cyclic AMP-dependent protein kinase in human neutrophils

The Ras-related protein, Rap1B, has previously been shown to serve as a PKA substrate in vitro and to be phosphorylated by cAMP elevating agents in human platelets. We have purified a Rap1 protein that serves as a PKA substrate from human neutrophils, and we now identify this protein as Rap1A. A 23-kDa protein that co-migrated with recombinant Rap1A was phosphorylated in electroporated human neutrophils upon stimulation by cAMP in the presence of [gamma-32P]ATP. This protein could be immunoprecipitated by the Rap1A/B-specific antibody, R61. The 23-kDa phosphoprotein was monitored during the purification of Rap1 from neutrophil membrane extracts and was shown to copurify with Rap1 during the DEAE Sephacel, heptylamine Sepharose, and MonoQ chromatography steps utilized. The purified protein was phosphorylated to an extent of 1 mol phosphate/mol GTP gamma S bound. This protein was identified as Rap1A by: 1) amino acid sequence analysis; and 2) immunoblotting with a Rap1A-specific antibody. The amino acid phosphorylated on Rap1A by PKA was a serine residue. The site of phosphorylation was indicated by carboxypeptidase digestion and confirmed using a mutant recombinant Rap1A lacking the relevant serine (serine-180). Rap1A, not Rap1B, appears to be the major 23-kDa PKA substrate in human neutrophils. It is possible that Rap1A plays a role in human neutrophils in mediating the inhibitory effects of cAMP-elevating agents upon chemoattractant-stimulated cell activation.