Endogenous expression and protein kinase A-dependent phosphorylation of the guanine nucleotide exchange factor Ras-GRF1 in human embryonic kidney 293 cells

We have previously reported the Ras-dependent activation of the mitogen-activated protein kinases p44 and p42, also termed extracellular signal-regulated kinases (ERK)1 and 2 (ERK1/2), mediated through Gs-coupled serotonin receptors transiently expressed in human embryonic kidney (HEK) 293 cells. Whereas Gi- and Gq-coupled receptors have been shown to activate Ras through the guanine nucleotide exchange factor (GEF) called Ras-GRF1 (CDC25Mm) by binding of Ca2+/calmodulin to its N-terminal IQ domain, the mechanism of Ras activation through Gs-coupled receptors is not fully understood. We report the endogenous expression of Ras-GRF1 in HEK293 cells. Serotonin stimulation of HEK293 cells transiently expressing Gs-coupled 5-HT7 receptors induced protein kinase A-dependent phosphorylation of the endogenous human Ras-GRF1 on Ser927 and of transfected mouse Ras-GRF1 on Ser916. Ras-GRF1 overexpression increased basal and serotonin-stimulated ERK1/2 phosphorylation. Mutations of Ser916 inhibiting (Ser916Ala) or mimicking (Ser916Asp/Glu) phosphorylation did not alter these effects. However, the deletion of amino acids 1-225, including the Ca2+/calmodulin-binding IQ domain, from Ras-GRF1 reduced both basal and serotonin-stimulated ERK1/2 phosphorylation. Furthermore, serotonin treatment of HEK293 cells stably expressing 5-HT7 receptors increased [Ca2+]i, and the serotonin-induced ERK1/2 phosphorylation was Ca2+-dependent. Therefore, both cAMP and Ca2+ may contribute to the Ras-dependent ERK1/2 activation after 5-HT7 receptor stimulation, through activation of a guanine nucleotide exchange factor with activity towards Ras.     

Phosphorylation of the Ras-GRF1 exchange factor at Ser916/898 reveals activation of Ras signaling in the cerebral cortex

The Ras-GRF1 exchange factor, which is regulated by increases in intracellular calcium and the release of G beta gamma subunits from heterotrimeric G proteins, plays a critical role in the activation of neuronal Ras. Activation of G protein-coupled receptors stimulates an increase in the phosphorylation of Ras-GRF1 at certain serine residues. The first of these sites to be identified, Ser(916) in the mouse sequence (equivalent to Ser(898) in the rat sequence), is required for full activation of the Ras exchange factor activity of Ras-GRF1 by muscarinic receptors. We demonstrate here that Ras-GRF1 is highly expressed in rat brain compared with the Sos exchange factor and that there is an increase in incorporation of (32)P into Ser(898) of brain Ras-GRF1 following activation of protein kinase A. Phosphorylation of Ras-GRF1 at Ser(916) is also required for maximal induction of Ras-dependent neurite outgrowth in PC12 cells. A novel antibody (termed 2152) that selectively recognizes Ras-GRF1 when it is phosphorylated at Ser(916/898) confirmed the regulated phosphorylation of Ras-GRF1 by Western blotting in both model systems of transfected COS-7 and PC12 cells and also of the endogenous protein in rat forebrain slices. Indirect confocal immunofluorescence of transfected PC12 cells using antibody 2152 demonstrated reactivity only under conditions in which Ras-GRF1 was phosphorylated at Ser(916/898). Confocal immunofluorescence of cortical slices of rat brain revealed widespread and selective phosphorylation of Ras-GRF1 at Ser(898). In the prefrontal cortex, there was striking phosphorylation of Ras-GRF1 in the dendritic tree, supporting a role for Ras activation and signal transduction in neurotransmission in this area.     

Stimulation of Ras guanine nucleotide exchange activity of Ras-GRF1/CDC25(Mm) upon tyrosine phosphorylation by the Cdc42-regulated kinase ACK1

Ras-GRF1 is a brain-specific guanine nucleotide exchange factor (GEF) for Ras, whose activity is regulated in response to Ca(2+) influx and G protein-coupled receptor signals. In addition, Ras-GRF1 acts as a GEF for Rac when tyrosine-phosphorylated following G protein-coupled receptor stimulation. However, the mechanisms underlying the regulation of Ras-GRF1 functions remain incompletely understood. We show here that activated ACK1, a nonreceptor tyrosine kinase that belongs to the focal adhesion kinase family, causes tyrosine phosphorylation of Ras-GRF1. On the other hand, kinase-deficient ACK1 exerted no effect. GEF activity of Ras-GRF1 toward Ha-Ras, as defined by in vitro GDP binding and release assays, was augmented after tyrosine phosphorylation by ACK1. In contrast, GEF activity toward Rac1 remained latent, implying that ACK1 does not represent a tyrosine kinase that acts downstream of G protein-coupled receptors. Consistent with enhanced Ras-GEF activity, accumulation of the GTP-bound form of Ras within the cell was shown through the use of Ras-binding domain pull-down assays. Furthermore, Ras-dependent activation of ERK2 by Ras-GRF1 was enhanced following co-expression of activated ACK1. These results implicate ACK1 as an upstream modulator of Ras-GRF1 and suggest a signaling cascade consisting of Cdc42, ACK1, Ras-GRF1, and Ras in neuronal cells.     

Basis for signaling specificity difference between Sos and Ras-GRF guanine nucleotide exchange factors

Sos and Ras-GRF are two families of guanine nucleotide exchange factors that activate Ras proteins in cells. Sos proteins are ubiquitously expressed and are activated in response to cell-surface tyrosine kinase stimulation. In contrast, Ras-GRF proteins are expressed primarily in central nervous system neurons and are activated by calcium/calmodulin binding and by phosphorylation. Although both Sos1 and Ras-GRF1 activate the Ras proteins Ha-Ras, N-Ras, and Ki-Ras, only Ras-GRF1 also activates the functionally distinct R-Ras GTPase. In this study, we determined which amino acid sequences in these exchange factors and their target GTPases are responsible for this signaling specificity difference. Analysis of chimeras and individual amino acid exchanges between Sos1 and Ras-GRF1 revealed that the critical amino acids reside within an 11-amino acid segment of their catalytic domains between the second and third structurally conserved regions (amino acids (aa) 828-838 in Sos1 and 1057-1067 in Ras-GRF1) of Ras guanine nucleotide exchange factors. In Sos1, this segment is in helix B, which is known to interact with the switch 2 region of Ha-Ras. Interestingly, a similar analysis of Ha-Ras and R-Ras chimeras did not identify the switch 2 region of Ha-Ras as encoding specificity. Instead, we found a more distal protein segment, helix 3 (aa 91-103 in Ha-Ras and 117-129 in R-Ras), which interacts instead primarily with helix K (aa 1002-1016) of Sos1. These findings suggest that specificity derives from the fact that R-Ras-specific amino acids in the region analogous to Ha-Ras helix 3 prevent a functional interaction with Sos1 indirectly, possibly by preventing an appropriate association of its switch 2 region with helix B of Sos1. Although previous studies have shown that helix B of Sos1 and helix 3 of Ha-Ras are involved in promoting nucleotide exchange on Ras proteins, this study highlights the importance of these regions in establishing signaling specificity.     

Maintenance of CDC42 GDP-bound state by Rho-GDI inhibits MAP kinase activation by the exchange factor Ras-GRF. evidence for Ras-GRF function being inhibited by Cdc42-GDP but unaffected by CDC42-GTP

The function of the Ras guanine nucleotide exchange factor Ras-GRF/cdc25(Mn) is subject to tight regulatory processes. We have recently shown that the activation of the Ras/MAPK pathway by Ras-GRF is controlled by the Rho family GTPase Cdc42 through still unknown mechanisms. Here, we report that retaining Cdc42 in its GDP-bound state by overexpressing Rho-GDI inhibits Ras-GRF-mediated MAPK activation. Conversely, Ras-GRF basal and LPA- or ionomycin-stimulated activities were unaffected by a constitutively active GTP-bound Cdc42. Moreover, the Cdc42 downstream effectors MLK3, ACK1, PAK1, and WASP had no detectable influence on Ras-GRF-mediated MAPK activation. In contrast, promoting GDP release from Cdc42 with the Rho family GEF Dbl or with ionomycin suppressed the restraint exerted by Cdc42 on Ras-GRF activity. We conclude that Cdc42-GDP inhibits Ras-GRF-induced MAPK activation, but neither Cdc42-GTP nor the Cdc42 downstream effectors affect Ras-GRF performance. Interestingly, the loss of the GDP-bound state by Cdc42 abolishes its inhibitory effects on Ras-GRF function. These results suggest that the Cdc42 mechanism of action may not be solely restricted to activation of downstream signaling cascades when GTP-loaded. Furthermore, the GDP-bound form may be acting as an inhibitory molecule down-modulating parallel signaling routes such as the Ras/MAPK pathway.     

Calcium and calmodulin are essential for Ras-GRF1-mediated activation of the Ras pathway by lysophosphatidic acid

The exchange factor Ras-GRF1, also called CDC25Mm, couples calcium signaling and G-protein-coupled receptors to Ras and downstream effectors. Here we show that when expressed in different cell lines Ras-GRF1 strongly enhances the level of active Ras (Ras-GTP) and the activity of mitogen-activated protein kinases (MAPK). Moreover, in NIH 3T3 fibroblasts it potentiates the effect of lysophosphatidic acid (LPA) on Ras protein and MAPK activity. Calmodulin and cytosolic free calcium are essential for Ras and MAPK activation induced by LPA and mediated by Ras-GRF1, as shown by the finding that BAPTA-AM, an intracellular calcium chelator, and calmodulin inhibitors completely abolished this effect. This report demonstrates the relevance of calmodulin in addition to calcium for the response of Ras-GRF1 to LPA.     

The Rho family GTPase Cdc42 regulates the activation of Ras/MAP kinase by the exchange factor Ras-GRF

The Ras guanine-nucleotide exchange factor Ras-GRF/Cdc25(Mn) harbors a complex array of structural motifs that include a Dbl-homology (DH) domain, usually found in proteins that interact functionally with the Rho family GTPases, and the role of which is not yet fully understood. Here, we present evidence that Ras-GRF requires its DH domain to translocate to the membrane, to stimulate exchange on Ras, and to activate mitogen-activated protein kinase (MAPK). In an unprecedented fashion, we have found that these processes are regulated by the Rho family GTPase Cdc42. We show that GDP- but not GTP-bound Cdc42 prevents Ras-GRF recruitment to the membrane and activation of Ras/MAPK, although no direct association of Ras-GRF with Cdc42 was detected. We also demonstrate that catalyzing GDP/GTP exchange on Cdc42 facilitates Ras-GRF-induced MAPK activation. Moreover, we show that the potentiating effect of ionomycin on Ras-GRF-mediated MAPK stimulation is also regulated by Cdc42. These results provide the first evidence for the involvement of a Rho family G protein in the control of the activity of a Ras exchange factor.     

The N-terminal pleckstrin, coiled-coil, and IQ domains of the exchange factor Ras-GRF act cooperatively to facilitate activation by calcium.

We have recently shown that the neuronal exchange factor p140 Ras-GRF becomes activated in vivo in response to elevated calcium levels [C. L. Farnsworth, N. W. Freshney, L. B. Rosen, A. Ghosh, M. E. Greenberg, and L. A. Feig, Nature (London) 376:524-527, 1995]. Activation is mediated by calcium-induced calmodulin binding to an IQ domain near the N terminus of Ras-GRF. Here we show that the adjacent N-terminal pleckstrin homology (PH), coiled-coil, and IQ domains function cooperatively to allow Ras-GRF activation. Deletion of the N-terminal PH domain redistributes a large percentage of Ras-GRF from the particulate to the cytosolic fraction of cells and renders the protein insensitive to calcium stimulation. A similar cellular distribution and biological activity are observed when only the core catalytic domain is expressed. Although the PH domain is necessary for particulate association of Ras-GRF, it is not sufficient for targeting the core catalytic domain to this cellular location. This requires the PH domain and the adjacent coiled-coil and IQ sequences. Remarkably, this form of Ras-GRF is constitutively activated. The PH and coiled-coil domains must also perform an additional function, since targeting to the particulate fraction of cells is not sufficient to allow Ras-GRF activation by calcium. A Ras-GRF mutant containing the PH domain from Ras-GTPase-activating protein in place of its own N-terminal PH domain localizes to the particulate fraction of cells but does not respond to calcium. Similar phenotypes are seen with mutant Ras-GRFs containing point mutations in either the PH or coiled-coil domain. These findings argue that the N-terminal PH, coiled-coil, and IQ domains of Ras-GRF function together to connect Ras-GRF to multiple components in the particulate fractions of cells that are required for responsiveness of the protein to calcium signaling.     

Phosphorylation of serine 916 of Ras-GRF1 contributes to the activation of exchange factor activity by muscarinic receptors

The Ras-GRF1 exchange factor is strongly implicated in the control of neuronal Ras. The activity of Ras-GRF1 is regulated by increases in intracellular calcium and the release of Gbetagamma subunits from heterotrimeric G-proteins. Increases in Ras-GRF1 activity toward Ras that are stimulated by receptors coupled to G-proteins are associated with enhanced phosphorylation of Ras-GRF1 on one or more serine residues. Co-expression of Ras-GRF1 with subtype 1 human muscarinic receptors in COS-7 cells allowed mapping of a carbachol-stimulated phosphorylation site to a region composed of residues 916-976. Site-directed mutagenesis replaced each of the serine residues within this region with alanine and demonstrated that serine 916 is a major site of in vivo phosphorylation of Ras-GRF1 in both COS-7 cells and NIH-3T3 fibroblasts. Serine 916 was a substrate for protein kinase A both in vivo and in vitro, suggesting a novel link between the cAMP and Ras signaling systems. Carbachol-dependent phosphorylation of serine 916 occurred through a protein kinase A-independent pathway, however. Full-length Ras-GRF1 that contains an alanine 916 mutation was only partially activated by carbachol, suggesting that phosphorylation at residue 916 is necessary for full activation. Phosphorylation of serine 916 in response to forskolin treatment did not, however, increase the activity of Ras-GRF1, indicating that it is not sufficient for activation.     

Ras/ERK signalling in cannabinoid tolerance: from behaviour to cellular aspects

We investigated the role of the Ras/extracellular-regulated kinase (ERK) pathway in the development of tolerance to Delta(9)-tetrahydrocannabinol (THC)-induced reduction in spontaneous locomotor activity by a genetic (Ras-specific guanine nucleotide exchange factor (Ras-GRF1) knock-out mice) and pharmacological approach. Pre-treatment of wild-type mice with SL327 (50 mg/kg i.p.), a specific inhibitor of mitogen-activated protein kinase kinase (MEK), the upstream kinase of ERK, fully prevented the development of tolerance to THC-induced hypolocomotion. We investigated the impact of the inhibition of ERK activation on the biological processes involved in cannabinoid tolerance (receptor down-regulation and desensitization), by autoradiographic cannabinoid CB1 receptor and cannabinoid-stimulated [(35)S]GTPgammaS binding studies in subchronically treated mice (THC, 10 mg/kg s.c., twice a day for 5 days). In the caudate putamen and cerebellum of Ras-GRF1 knock-out mice and SL327 pre-treated wild-type mice, CB1 receptor down-regulation and desensitization did not occur, suggesting that ERK activation might account for CB1 receptor plasticity involved in the development of tolerance to THC hypolocomotor effect. In contrast, the hippocampus and prefrontal cortex showed CB1 receptor adaptations regardless of the genetic or pharmacological inhibition of the ERK pathway, suggesting regional variability in the cellular events underlying the altered CB1 receptor function. These findings suggest that at least in the caudate putamen and cerebellum, the Ras/ERK pathway is essential for triggering the alteration in CB1 receptor function responsible for tolerance to THC-induced hypomotility.     

The Ras-GRF1 exchange factor coordinates activation of H-Ras and Rac1 to control neuronal morphology

The Ras-GRF1 exchange factor has regulated guanine nucleotide exchange factor (GEF) activity for H-Ras and Rac1 through separate domains. Both H-Ras and Rac1 activation have been linked to synaptic plasticity and thus could contribute to the function of Ras-GRF1 in neuronal signal transduction pathways that underlie learning and memory. We defined the effects of Ras-GRF1 and truncation mutants that include only one of its GEF activities on the morphology of PC12 phaeochromocytoma cells. Ras-GRF1 required coexpression of H-Ras to induce morphological effects. Ras-GRF1 plus H-Ras induced a novel, expanded morphology in PC12 cells, which was characterized by a 10-fold increase in soma size and by neurite extension. A truncation mutant of Ras-GRF1 that included the Ras GEF domain, GRFdeltaN, plus H-Ras produced neurite extensions, but did not expand the soma. This neurite extension was blocked by inhibition of MAP kinase activation, but was independent of dominant-negative Rac1 or RhoA. A truncation mutant of Ras-GRF1 that included the Rac GEF domains, GRFdeltaC, produced the expanded phenotype in cotransfections with H-Ras. Cell expansion was inhibited by wortmannin or dominant-negative forms of Rac1 or Akt. GRFdeltaC binds H-Ras.GTP in both pulldown assays from bacterial lysates and by coimmunoprecipitation from HEK293 cells. These results suggest that coordinated activation of H-Ras and Rac1 by Ras-GRF1 may be a significant controller of neuronal cell size.     

Activation of the Ras-GRF/CDC25Mm exchange factor by lysophosphatidic acid.

The Ras-GRF exchange factor can activate Ras-dependent responses following the activation of heterotrimeric G-protein and calcium signalling. In stable lines of NIH-3T3 fibroblasts that express Ras-GRF, the agonist lysophosphatidic acid (LPA) increases the phosphorylation state and activity of Ras-GRF. The stimulation of Ras-GRF can be demonstrated in vitro, in an assay using recombinant Ras substrate, and in situ, by a selective increase in the ability of LPA to stimulate mitogen-activated protein (MAP) kinase. The increase in Ras-GRF phosphorylation state, which occurs on serine residues, and the increase in exchange factor activity are blocked by pretreatment with pertussis toxin. Activation of Ras-GRF by LPA can also be inhibited by chelation of intracellular calcium and treatment of the Ras-GRF with protein phosphatase 1 (PP1), supporting a model in which Ras-GRF serves to integrate signals from multiple transduction pathways.     

Long-Term Potentiation in the CA1 Hippocampus Induced by NR2A Subunit-Containing NMDA Glutamate Receptors Is Mediated by Ras-GRF2/Erk Map Kinase Signaling

Background

NMDA-type glutamate receptors (NMDARs) are major contributors to long-term potentiation (LTP), a form of synaptic plasticity implicated in the process of learning and memory. These receptors consist of calcium-permeating NR1 and multiple regulatory NR2 subunits. A majority of studies show that both NR2A and NR2B-containing NMDARs can contribute to LTP, but their unique contributions to this form of synaptic plasticity remain poorly understood.

Methodology/Principal Findings

In this study, we show that NR2A and NR2B-containing receptors promote LTP differently in the CA1 hippocampus of 1-month old mice, with the NR2A receptors functioning through Ras-GRF2 and its downstream effector, Erk Map kinase, and NR2B receptors functioning independently of these signaling molecules.

Conclusions/Significance

This study demonstrates that NR2A-, but not NR2B, containing NMDA receptors induce LTP in pyramidal neurons of the CA1 hippocamus from 1 month old mice through Ras-GRF2 and Erk. This difference add new significance to the observation that the relative levels of these NMDAR subtypes is regulated in neurons, such that NR2A-containing receptors become more prominent late in postnatal development, after sensory experience and synaptic activity.     

Prenylation of target GTPases contributes to signaling specificity of Ras-guanine nucleotide exchange factors

Ras-GRF1 and Ras-GRF2 constitute a family of calmodulin-regulated guanine-nucleotide exchange factors (GEFs) that activate Ras proteins. Here we show that whereas Ras-GRF1 activated both Ha-Ras and R-Ras in cells, Ras-GRF2 activated only Ha-Ras. The inability of Ras-GRF2 to activate R-Ras was the consequence of the GTPase being post-translationally modified, since Ras-GRF2 activated unprocessed R-Ras as effectively as unprocessed Ha-Ras when assays were performed either in vivo or in vitro. Moreover, Ras-GRF2 failed to activate fully processed R-Ras in vitro. The particular C-terminal lipid attached to the GTPases played an important role in determining signaling specificity, since R-Ras became more responsive to Ras-GRF2 when it was farnesylated instead of geranylgeranylated. Similarly, Ha-Ras became less responsive to Ras-GRF2 when it was geranylgeranylated instead of farnesylated. Analysis of chimeras between Ras-GRF1 and Ras-GRF2 demonstrated that a 30-amino acid segment embedded with their catalytic domains was responsible for recognizing the presence of different lipids on Ras proteins. These results indicate that the specific lipid moiety attached to GTPases can contribute to signaling specificity of Ras-GEFs.