CalDAG-GEFIII activation of Ras, R-ras, and Rap1

We characterized a novel guanine nucleotide exchange factor (GEF) for Ras family G proteins that is highly homologous to CalDAG-GEFI, a GEF for Rap1 and R-Ras, and to RasGRP/CalDAG-GEFII, a GEF for Ras and R-Ras. This novel GEF, referred to as CalDAG-GEFIII, increased the GTP/GDP ratio of Ha-Ras, R-Ras, and Rap1 in 293T cells. CalDAG-GEFIII promoted the guanine nucleotide exchange of Ha-Ras, R-Ras, and Rap1 in vitro also, indicating that CalDAG-GEFIII exhibited the widest substrate specificity among the known GEFs for Ras family G proteins. Expression of CalDAG-GEFIII was detected in the glial cells of the brain and the glomerular mesangial cells of the kidney by in situ hybridization. CalDAG-GEFIII activated ERK/MAPK most efficiently, followed by CalDAG-GEFII and CalDAG-GEFI in 293T cells. JNK activation was most prominent in cells expressing CalDAG-GEFII, followed by CalDAG-GEFIII and CalDAG-GEFI. Expression of CalDAG-GEFIII induced neuronal differentiation of PC12 cells and anchorage-independent growth of Rat1A cells less efficiently than did CalDAG-GEFII. Thus, co-activation of Rap1 by CalDAG-GEFIII apparently attenuated Ras-MAPK-dependent neuronal differentiation and cellular transformation. Altogether, CalDAG-GEFIII activated a broad range of Ras family G proteins and exhibited a biological activity different from that of either CalDAG-GEFI or CalDAG-GEFII.     

RA-GEF, a novel Rap1A guanine nucleotide exchange factor containing a Ras/Rap1A-associating domain, is conserved between nematode and humans

A yeast two-hybrid screening for Ras-binding proteins in nematode Caenorhabditis elegans has identified a guanine nucleotide exchange factor (GEF) containing a Ras/Rap1A-associating (RA) domain, termed Ce-RA-GEF. Both Ce-RA-GEF and its human counterpart Hs-RA-GEF possessed a PSD-95/DlgA/ZO-1 (PDZ) domain and a Ras exchanger motif (REM) domain in addition to the RA and GEF domains. They also contained a region homologous to a cyclic nucleotide monophosphate-binding domain, which turned out to be incapable of binding cAMP or cGMP. Although the REM and GEF domains are conserved with other GEFs acting on Ras family small GTP-binding proteins, the RA and PDZ domains are unseen in any of them. Hs-RA-GEF exhibited not only a GTP-dependent binding activity to Rap1A at its RA domain but also an activity to stimulate GDP/GTP exchange of Rap1A both in vitro and in vivo at the segment containing its REM and GEF domains. However, it did not exhibit any binding or GEF activity toward Ras. On the other hand, Ce-RA-GEF associated with and stimulated GDP/GTP exchange of both Ras and Rap1A. These results indicate that Ce-RA-GEF and Hs-RA-GEF define a novel class of Rap1A GEF molecules, which are conserved through evolution.     

Identification of guanine nucleotide exchange factors (GEFs) for the Rap1 GTPase. Regulation of MR-GEF by M-Ras-GTP interaction

Although the Ras subfamily of GTPases consists of approximately 20 members, only a limited number of guanine nucleotide exchange factors (GEFs) that couple extracellular stimuli to Ras protein activation have been identified. Furthermore, no novel downstream effectors have been identified for the M-Ras/R-Ras3 GTPase. Here we report the identification and characterization of three Ras family GEFs that are most abundantly expressed in brain. Two of these GEFs, MR-GEF (M-Ras-regulated GEF, KIAA0277) and PDZ-GEF (KIAA0313) bound specifically to nucleotide-free Rap1 and Rap1/Rap2, respectively. Both proteins functioned as Rap1 GEFs in vivo. A third GEF, GRP3 (KIAA0846), activated both Ras and Rap1 and shared significant sequence homology with the calcium- and diacylglycerol-activated GEFs, GRP1 and GRP2. Similarly to previously identified Rap GEFs, C3G and Smg GDS, each of the newly identified exchange factors promoted the activation of Elk-1 in the LNCaP prostate tumor cell line where B-Raf can couple Rap1 to the extracellular receptor-activated kinase cascade. MR-GEF and PDZ-GEF both contain a region immediately N-terminal to their catalytic domains that share sequence homology with Ras-associating or RalGDS/AF6 homology (RA) domains. By searching for in vitro interaction with Ras-GTP proteins, PDZ-GEF specifically bound to Rap1A- and Rap2B-GTP, whereas MR-GEF bound to M-Ras-GTP. C-terminally truncated MR-GEF, lacking the GEF catalytic domain, retained its ability to bind M-Ras-GTP, suggesting that the RA domain is important for this interaction. Co-immunoprecipitation studies confirmed the interaction of M-Ras-GTP with MR-GEF in vivo. In addition, a constitutively active M-Ras(71L) mutant inhibited the ability of MR-GEF to promote Rap1A activation in a dose-dependent manner. These data suggest that M-Ras may inhibit Rap1 in order to elicit its biological effects.     

Neuronal calcium activates a Rap1 and B-Raf signaling pathway via the cyclic adenosine monophosphate-dependent protein kinase

Activity-dependent regulation of neuronal events such as cell survival and synaptic plasticity is controlled by increases in neuronal calcium levels. These actions often involve stimulation of intracellular kinase signaling pathways. For example, the mitogen-activated protein kinase, or extracellular signal-regulated kinase (ERK), signaling cascade has increasingly been shown to be important for the induction of gene expression and long term potentiation. However, the mechanisms leading to ERK activation by neuronal calcium are still unclear. In the present study, we describe a protein kinase A (PKA)-dependent signaling pathway that may link neuronal calcium influx to ERKs via the small G-protein, Rap1, and the neuronal Raf isoform, B-Raf. Thus, in PC12 cells, depolarization-mediated calcium influx led to the activation of B-Raf, but not Raf-1, via PKA. Furthermore, depolarization also induced the PKA-dependent stimulation of Rap1 and led to the formation of a Rap1/B-Raf signaling complex. In contrast, depolarization did not lead to the association of Ras with B-Raf. The major action of PKA-dependent Rap1/B-Raf signaling in neuronal cells is the activation of ERKs. Thus, we further show that, in both PC12 cells and hippocampal neurons, depolarization-induced calcium influx stimulates ERK activity in a PKA-dependent manner. Given the fact that both Rap1 and B-Raf are highly expressed in the central nervous system, we suggest that this signaling pathway may regulate a number of activity-dependent neuronal functions.     

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.     

Identification of the mechanisms regulating the differential activation of the mapk cascade by epidermal growth factor and nerve growth factor in PC12 cells

In PC12 cells, epidermal growth factor (EGF) transiently stimulates the mitogen-activated protein (MAP) kinases, ERK1 and ERK2, and provokes cellular proliferation. In contrast, nerve growth factor (NGF) stimulation leads to the sustained activation of the MAPKs and subsequently to neuronal differentiation. It has been shown that both the magnitude and longevity of MAPK activation governs the nature of the cellular response. The activations of MAPKs are dependent upon two distinct small G-proteins, Ras and Rap1, that link the growth factor receptors to the MAPK cascade by activating c-Raf and B-Raf, respectively. We found that Ras was transiently stimulated upon both EGF and NGF treatment of PC12 cells. However, EGF transiently activated Rap1, whereas NGF stimulated prolonged Rap1 activation. The activation of the ERKs was due almost exclusively (>90%) to the action of B-Raf. The transient activation of the MAPKs by EGF was a consequence of the formation of a short lived complex assembling on the EGF receptor itself, composed of Crk, C3G, Rap1, and B-Raf. In contrast, NGF stimulation of the cells resulted in the phosphorylation of FRS2. FRS2 scaffolded the assembly of a stable complex of Crk, C3G, Rap1, and B-Raf resulting in the prolonged activation of the MAPKs. Together, these data provide a signaling link between growth factor receptors and MAPK activation and a mechanistic explanation of the differential MAPK kinetics exhibited by these growth factors.     

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.     

Role of phosphoinositide 3-kinase and endocytosis in nerve growth factor-induced extracellular signal-regulated kinase activation via Ras and Rap1

Neurotrophins promote multiple actions on neuronal cells including cell survival and differentiation. The best-studied neurotrophin, nerve growth factor (NGF), is a major survival factor in sympathetic and sensory neurons and promotes differentiation in a well-studied model system, PC12 cells. To mediate these actions, NGF binds to the TrkA receptor to trigger intracellular signaling cascades. Two kinases whose activities mediate these processes include the mitogen-activated protein (MAP) kinase (or extracellular signal-regulated kinase [ERK]) and phosphoinositide 3-kinase (PI3-K). To examine potential interactions between the ERK and PI3-K pathways, we studied the requirement of PI3-K for NGF activation of the ERK signaling cascade in dorsal root ganglion cells and PC12 cells. We show that PI3-K is required for TrkA internalization and participates in NGF signaling to ERKs via distinct actions on the small G proteins Ras and Rap1. In PC12 cells, NGF activates Ras and Rap1 to elicit the rapid and sustained activation of ERKs respectively. We show here that Rap1 activation requires both TrkA internalization and PI3-K, whereas Ras activation requires neither TrkA internalization nor PI3-K. Both inhibitors of PI3-K and inhibitors of endocytosis prevent GTP loading of Rap1 and block sustained ERK activation by NGF. PI3-K and endocytosis may also regulate ERK signaling at a second site downstream of Ras, since both rapid ERK activation and the Ras-dependent activation of the MAP kinase kinase kinase B-Raf are blocked by inhibition of either PI3-K or endocytosis. The results of this study suggest that PI3-K may be required for the signals initiated by TrkA internalization and demonstrate that specific endocytic events may distinguish ERK signaling via Rap1 and Ras.     

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.     

Mechanism of the spatio-temporal regulation of Ras and Rap1

Epidermal growth factor (EGF) activates Ras and Rap1 at distinct intracellular regions. Here, we explored the mechanism underlying this phenomenon. We originally noticed that in cells expressing Epac, a cAMP-dependent Rap1 GEF (guanine nucleotide exchange factor), cAMP activated Rap1 at the perinuclear region, as did EGF. However, in cells expressing e-GRF, a recombinant cAMP-responsive Ras GEF, cAMP activated Ras at the peripheral plasma membrane. Based on the uniform cytoplasmic expression of Epac and e-GRF, GEF did not appear to account for the non-uniform increase in the activities of Ras and Rap1. In contrast, when we used probes with reduced sensitivity to GTPase-activating proteins (GAPs), both Ras and Rap1 appeared to be activated uniformly in the EGF-stimulated cells. Furthermore, we calculated the local rate constants of GEFs and GAPs from the video images of Ras activation and found that GAP activity was higher at the central plasma membrane than the periphery. Thus we propose that GAP primarily dictates the spatial regulation of Ras family G proteins, whereas GEF primarily determines the timing of Ras activation.     

Stimulation of the ERK pathway by GTP-loaded Rap1 requires the concomitant activation of Ras,protein kinase C,and protein kinase A in neuronal cells

The small GTPases Ras or Rap1 were suggested to mediate the stimulatory effect of some G protein-coupled receptors on ERK activity in neuronal cells. Accordingly, we reported here that pituitary adenylate cyclase-activating polypeptide (PACAP), whose G protein-coupled receptor triggers neuronal differentiation of the PC12 cell line via ERK1/2 activation, transiently activated Ras and induced the sustained GTP loading of Rap1. Ras mediated peak stimulation of ERK by PACAP, whereas Rap1 was necessary for the sustained activation phase. However, PACAP-induced GTP-loading of Rap1 was not sufficient to account for ERK activation by PACAP because 1) PACAP-elicited Rap1 GTP-loading depended only on phospholipase C, whereas maximal stimulation of ERK by PACAP also required the activity of protein kinase A (PKA), protein kinase C (PKC), and calcium-dependent signaling; and 2) constitutively active mutants of Rap1, Rap1A-V12, and Rap1B-V12 only minimally stimulated the ERK pathway compared with Ras-V12. The effect of Rap1A-V12 was dramatically potentiated by the concurrent activation of PKC, the cAMP pathway, and Ras, and this potentiation was blocked by dominant-negative mutants of Ras and Raf. Thus, this set of data indicated that GPCR-elicited GTP loading of Rap1 was not sufficient to stimulate efficiently ERK in PC12 cells and required the permissive co-stimulation of PKA, PKC, or Ras.     

Characterisation of PDZ-GEFs,a family of guanine nucleotide exchange factors specific for Rap1 and Rap2

PDZ-GEF1 (RA-GEF/nRapGEP/CNrasGEF) is a guanine nucleotide exchange factor (GEF) characterised by the presence of a PSD-95/DlgA/ZO-1 (PDZ) domain, a Ras-association (RA) domain and a region related to a cyclic nucleotide binding domain (RCBD). These domains are in addition to a Ras exchange motif (REM) and GEF domain characteristic for GEFs for Ras-like small GTPases. PDZ-GEF1 efficiently exchanges nucleotides of both Rap1 and Rap2, but has also been implicated in mediating cAMP-induced Ras activation through binding of cAMP to the RCBD. Here we describe a new family member, PDZ-GEF2, of which we isolated two splice variants (PDZ-GEF2A and 2B). PDZ-GEF2 contains, in addition to the domains characteristic for PDZ-GEF1, a second, less conserved RCBD at the N-terminus. PDZ-GEF2 is also specific for both Rap1 and Rap2. We further investigated the possibility that PDZ-GEF2, like PDZ-GEF1, is a cAMP-responsive GEF for Ras. However, in contrast to previous results, we did not find any effect of either PDZ-GEF1 or PDZ-GEF2 on Ras in the absence or presence of cAMP. Moreover, affinity measurements by isothermic calorimetry showed that the RCBD of PDZ-GEF1 does not bind cAMP with a physiologically relevant affinity. We conclude that both PDZ-GEF1 and 2 are specific for Rap1 and Rap2 and unresponsive to cAMP and various other nucleotides.     

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.     

Extracellular signal-regulated kinase-dependent proliferation is mediated through the protein kinase A/B-Raf pathway in human uveal melanoma cells

Mutated B-Raf-mediated constitutive activation of ERK1/2 is involved in about 66% of cutaneous melanoma. By contrast, activating mutations in B-RAF are rare in ocular melanoma. This study aimed to determine the role of wild-type B-Raf ((WT)B-Raf) in uveal melanoma cell growth. We used cell lines derived from primary tumors of uveal melanoma to assess the role of (WT)B-Raf in cell proliferation and to characterize its upstream regulators and downstream effectors. Melanoma cell lines expressing (WT)B-Raf and (WT)Ras grew with similar proliferation rates, showed constitutive activation of ERK1/2, and had similar levels of B-Raf expression and B-Raf kinase activity as melanoma cell lines expressing the activating V600E mutation ((V600E)B-Raf). They were equally as sensitive to pharmacological inhibition of MEK1/2 for cell proliferation and transformation as (V600E)B-Raf cells. siRNA-mediated depletion of Raf-1 did not affect either ERK1/2 activation, whereas siRNA-mediated depletion of B-Raf reduced cell proliferation by up to 65% through the inhibition of ERK1/2 activation, irrespective of the mutational status of B-Raf. Pharmacological inhibition of cAMP-dependent protein kinase (PKA) and siRNA-mediated depletion of PKA greatly reduced B-Raf activity, ERK1/2 activation, and cell proliferation in (WT)B-Raf cells, whereas it did not affect (V600E)B-Raf cells, demonstrating a key role of PKA in mediating (WT)B-Raf/ERK signaling for uveal melanoma cell growth. Moreover, inactivation or depletion of PKA did not affect Rap-1 activity, and Rap-1 depletion did not affect either B-Raf activity or ERK1/2 activation. This ruled out a role for Rap1 in the PKA-mediated B-Raf/ERK activation in (WT)B-Raf cells. Finally, we demonstrated the importance of cyclin D1 in mediating PKA/(WT)B-Raf signaling for cell proliferation. Altogether, our results suggest that the PKA/B-Raf pathway is a potential target for therapeutic strategies against (WT)B-Raf-expressing uveal melanoma.     

Ectopic B-Raf expression enhances extracellular signal-regulated kinase (ERK) signaling in T cells and prevents antigen-presenting cell-induced anergy

T cells that receive stimulation through the T cell receptor (TCR) in the absence of costimulation become anergic and are refractory to subsequent costimulation. This unresponsiveness is associated with the constitutive activation of the small G protein, Rap1, and the lack of Ras-dependent activation of ERK. Recent studies suggest that Rap1 can activate the MAP kinase kinase kinase B-Raf that is either endogenously or ectopically expressed. Peripheral T cells generally do not express B-Raf; therefore, to test the hypothesis that ectopic expression of B-Raf could permit Rap1 to activate ERK signaling, we generated transgenic mice expressing B-Raf within peripheral T cells. This converted Rap1 into an activator of ERK, to enhance ERK activation and proliferation following TCR engagement in the absence of costimulation. When T cells were incubated with engineered APCs presenting antigen on I-Ek and expressing low levels of B7, they became anergic, displayed constitutive activation of Rap1, and were deficient in Ras and ERK activation. However, when incubated with the same APCs, T cells expressing the B-Raf transgene proliferated upon restimulation and displayed elevated ERK activation. Thus B-Raf expression and enhanced ERK activation is sufficient to prevent anergy in a model of APC-induced T cell anergy. However, studies using anti-TCR antibody-induced anergy showed that the ability of ERKs to reverse T cell anergy is dependent on the anergic model utilized.     

Stimulation of phospholipase C-epsilon by the M3 muscarinic acetylcholine receptor mediated by cyclic AMP and the GTPase Rap2B

Stimulation of phospholipase C (PLC) by G(q)-coupled receptors such as the M(3) muscarinic acetylcholine receptor (mAChR) is caused by direct activation of PLC-beta enzymes by Galpha(q) proteins. We have recently shown that G(s)-coupled receptors can stimulate PLC-epsilon, apparently via formation of cyclic AMP and activation of the Ras-related GTPase Rap2B. Here we report that PLC stimulation by the M(3) mAChR expressed in HEK-293 cells also involves, in part, similar mechanisms. M(3) mAChR-mediated PLC stimulation and [Ca(2+)](i) increase were reduced by 2',5'-dideoxyadenosine (dd-Ado), a direct adenylyl cyclase inhibitor. On the other hand, overexpression of Galpha(s) or Epac1, a cyclic AMP-regulated guanine nucleotide exchange factor for Rap GTPases, enhanced M(3) mAChR-mediated PLC stimulation. Inactivation of Ras-related GTPases with clostridial toxins suppressed the M(3) mAChR responses. The inhibitory toxin effects were mimicked by expression of inactive Rap2B, but not of other inactive GTPases (Rac1, Ras, RalA, Rap1A, and Rap2A). Activation of the M(3) mAChR induced GTP loading of Rap2B, an effect strongly enhanced by overexpression of Galpha(s) and inhibited by dd-Ado. Overexpression of PLC-epsilon and PLC-beta1, but not PLC-gamma1 or PLC-delta1, enhanced M(3) mAChR-mediated PLC stimulation and [Ca(2+)](i) increase. In contrast, expression of a catalytically inactive PLC-epsilon mutant reduced PLC stimulation by the M(3) mAChR and abrogated the potentiating effect of Galpha(s). In conclusion, our findings suggest that PLC stimulation by the M(3) mAChR is a composite action of PLC-beta1 stimulation by Galpha(q) and stimulation of PLC-epsilon apparently mediated by G(s)-dependent cyclic AMP formation and subsequent activation of Rap2B.     

Identification and characterization of RA-GEF-2, a Rap guanine nucleotide exchange factor that serves as a downstream target of M-Ras

The Ras family small GTPase Rap is regulated by an array of specific guanine nucleotide exchange factors (GEFs) in response to upstream stimuli. RA-GEF-1 was identified as a novel Rap GEF, which possesses a Ras/Rap1-associating (RA) domain. Here we report a protein closely related to RA-GEF-1, named RA-GEF-2. Like RA-GEF-1, a putative cyclic nucleotide monophosphate-binding domain, a Ras exchanger motif, a PSD-95/DlgA/ZO-1 domain, and an RA domain in addition to the GEF catalytic domain are found in RA-GEF-2. However, RA-GEF-2 displays a different tissue distribution profile from that of RA-GEF-1. RA-GEF-2 stimulates guanine nucleotide exchange of both Rap1 and Rap2, but not Ha-Ras. The RA domain of RA-GEF-2 binds to M-Ras in a GTP-dependent manner, but not to other Ras family GTPases tested, including Ha-Ras, N-Ras, Rap1A, Rap2A, R-Ras, RalA, Rin, Rit, and Rheb, in contrast to the RA domain of RA-GEF-1, which specifically binds to Rap1. In accordance with this, RA-GEF-2 colocalizes with activated M-Ras in the plasma membrane in COS-7 cells, suggesting a role of RA-GEF-2 in the regulation of Rap1 and Rap2, particularly in the plasma membrane. In fact, an increase in the level of the GTP-bound form of plasma membrane-located Rap1 was observed when coexpressed with RA-GEF-2 and activated M-Ras. Thus, RA-GEF-2 acts as a GEF for Rap1 and Rap2 downstream of M-Ras in the plasma membrane, whereas RA-GEF-1 exerts Rap GEF function in perinuclear compartments including the Golgi apparatus.     

BCR/ABL activates Rap1 and B-Raf to stimulate the MEK/Erk signaling pathway in hematopoietic cells

The BCR/ABL fusion tyrosine kinase activates various intracellular signaling pathways, thus causing chronic myeloid leukemia (CML). Here we demonstrate that the inducible expression of BCR/ABL in a murine hematopoietic cell line, TonB210, leads to the activation of the Ras family small GTPase Rap1, which is inhibited by the ABL kinase inhibitor imatinib. The Rap1 activity in a CML cell line, K562, was also inhibited by imatinib. Inhibition of Rap1 activation by a dominant negative mutant of Rap1, Rap1-N17, or SPA-1 inhibited the BCR/ABL-induced activation of Elk-1. BCR/ABL also activated in a kinase activity-dependent manner the B-Raf kinase, which is an effector molecule of Rap1 and a potent activator of the MEK/Erk/Elk-1 signaling pathway. Together, these data suggest that, in addition to the well-established Ras/Raf-1 pathway, BCR/ABL activates the alternative signaling pathway involving Rap1 and B-Raf to activate Erk, which may play important roles in leukemogenesis.