Ras Stimulation Research Articles

Vav mediates Ras stimulation by direct activation of the GDP/GTP exchange factor Ras GRP1.

Here we describe a new signaling cross-talk between the Vav/Rac1 and Ras pathways that is established through the stimulation of RasGRP1, an exchange factor for Ras subfamily GTPases. This interaction is crucial for Ras activation in lymphoid cells, since this GTPase cannot become activated in the absence of Vav proteins. The activation of RasGRP1 requires both the generation of diacylglycerol via phospho lipase C-γ and the induction of actin polymerization, two responses induced by Vav and Rac1 that facilitate the translocation of RasGRP1 to juxtamembrane areas of the cell. Consistent with this, the cross-talk can be activated by tyrosine-phosphorylated wild-type Vav, oncogenic Vav and constitutively active Rac1. Conversely, Ras activation can be blocked in lymphocytes and ectopic systems using inhibitors affecting either phospholipase C-γ or F-actin polymerization. These results indicate that a relay mechanism exists in lymphoid and other cells helping in the generation of robust signaling responses by the Rac/Rho and Ras pathways upon receptor engagement.

Ras Protein Activation Is a Key Event in Activity-dependent Survival of Cerebellar Granule Neurons

Neuronal activity promotes the survival of cerebellar granule neurons (CGNs) during the postnatal development of cerebellum. CGNs that fail to receive excitatory inputs will die by apoptosis. This process could be mimicked in culture by exposing CGNs to either a physiological concentration of KCl (5 mm or K5) plus N-methyl-d-aspartate (NMDA) or to 25 mm KCl (K25). We have previously described that a 24-h exposure to NMDA (100 μm) or K25 at 2 days in vitro induced long term survival of CGNs in K5 conditions. Here we have studied the molecular mechanisms activated at 2 days in vitro in these conditions. First we showed that NMDA or K25 addition promoted a rapid stimulation of PI3K and a biphasic phosphorylation on Ser-473 of Akt, a PI3K substrate. Interestingly, we demonstrated that only the first wave of Akt phosphorylation is necessary for the NMDA- and K25-mediated survival. Additionally, we detected that both NMDA and K25 increased ERK activity with a similar time-course. Moreover, our results showed that NMDA-mediated activation of the small G-protein Ras is necessary for PI3K/Akt pathway activation, whereas Rap1 was involved in NMDA phosphorylation of ERK. On the other hand, Ras, but not Rap1, mediates K25 activation of PI3K/Akt and MEK/ERK pathways. Because neuroprotection by NMDA or K25 is mediated by Ras (and not by Rap1) activation, we propose that Ras stimulation is a crucial event in NMDA- and K25-mediated survival of CGNs through the activation of PI3K/Akt and MEK/ERK pathways.

Renal responses to short term non‐pressor AngII ± intrarenal RAS blockade in rats

Long term (14d) AngII infusion augments intrarenal AngII production and raises blood pressure; ACE inhibition (ACEi) blunts both responses. This study aimed to determine 1) the renal responses to a short term (3 d) non‐pressor dose of AngII and 2) whether the responses were blunted by ACEi treatment implicating local AngII production. Male Sprague‐Dawley rats were infused via osmotic minipumps with either vehicle (control) or AngII (200 ng/kg/min) ± the ACEi enalapril (30 mg/kg/day in drinking water). There were no significant differences in body weight gain, overnight urine volume, or urinary Na and K excretion among the groups. Abundance of renal transporters was determined by quantitative immunoblot, and normalized to control values, defined as 1.0. Control AngII AngII + enalapril Cortex NHE3 total 1.00 ± 0.05 1.51 ± 0.14* 1.37 ± 0.02* NHE3‐P 1.00 ± 0.07 1.37 ± 0.09 1.45 ± 0.13* NKCC total 1.00 ± 0.09 1.70 ± 0.23* 1.57 ± 0.11 NKCC‐P 1.00 ± 0.17 1.96 ± 0.20* 2.13 ± 0.17* NCC total 1.00 ± 0.05 1.33 ± 0.08* 1.12 ± 0.03 NCCpS71 1.00 ± 0.07 1.29 ± 0.09* 1.01 ± 0.04 SPAK total 1.00 ± 0.19 2.71 ± 0.55* 2.87 ± 0.40* Medulla NKCC total 1.00 ± 0.04 0.70 ± 0.05* 0.75 ± 0.07* NKCC‐P 1.00 ± 0.03 0.82 ± 0.11 1.22 ± 0.28 SPAK total 1.00 ± 0.02 0.99 ± 0.06 0.98 ± 0.03 p<0.05 vs. control. Conclusion1) 3 d AngII infusion increases cortical NHE3, NKCC, NCC and SPAK while decreasing medullary NKCC abundance. 2) Blocking intrarenal RAS with enalapril blunted the effects of AngII infusion on NCC total, NCCp, and NKCC indicating these are early targets of intrarenal RAS stimulation and, thus, intrarenal ACE inhibition. NIH R01 DK 083785.

RasGRP1 Is Essential for Ras Activation by the Tumor Promoter 12-O-Tetradecanoylphorbol-13-acetate in Epidermal Keratinocytes

RasGRP1 is a guanine nucleotide exchange factor for Ras that binds with high affinity to diacylglycerol analogs like the phorbol esters. Recently, we demonstrated a role for RasGRP1 in skin carcinogenesis and suggested its participation in the action of tumor-promoting phorbol esters like 12-O-tetradecanoylphorbol-13-acetate (TPA) on Ras pathways in epidermal cells. Given the importance of Ras in carcinogenesis, we sought to discern whether RasGRP1 was a critical pathway in Ras activation, using a RasGRP1 knockout (KO) mouse model to examine the response of keratinocytes to TPA. In contrast to the effect seen in wild type keratinocytes, Ras(GTP) levels were barely detected in RasGRP1 KO cells even after 60 min of exposure to phorbol esters. The lack of response was rescued by enforced expression of RasGRP1. Furthermore, small hairpin RNA-induced silencing of RasGRP1 abrogated the effect of TPA on Ras. Analysis of Ras isoforms showed that both H-Ras and N-Ras depended on RasGRP1 for activation by TPA, whereas activation of K-Ras could not be detected. Although RasGRP1 was dispensable for ERK activation in response to TPA, JNK activation was reduced in the KO keratinocytes. Notably, TPA-induced phosphorylation of JNK2, but not JNK1, was reduced by RasGRP1 depletion. These data identify RasGRP1 as a critical molecule in the activation of Ras by TPA in primary mouse keratinocytes and suggest JNK2 as one of the relevant downstream targets. Given the role of TPA as a skin tumor promoter, our findings provide additional support for a role for RasGRP1 in skin carcinogenesis.

Proteolytic Degradation of SCOP in the Hippocampus Contributes to Activation of MAP Kinase and Memory

Because activation of ERK1/2 MAP kinase (MAPK) is critical for hippocampus-dependent memory, there is considerable interest in mechanisms for regulation of MAPK during memory formation. Here we report that MAPK and CREB-mediated transcription are negatively regulated by SCOP (suprachiasmatic nucleus [SCN] circadian oscillatory protein) and that SCOP is proteolyzed by calpain when hippocampal neurons are stimulated by brain-derived neurotrophic factor (BDNF), KCl depolarization, or NMDA. Moreover, training for novel object memory decreases SCOP in the hippocampus. To determine if hippocampus-dependent memory is influenced by SCOP in vivo, we generated a transgenic mouse strain for the inducible overexpression of SCOP in the forebrain. Overexpression of SCOP completely blocked memory for novel objects. We conclude that degradation of SCOP by calpain contributes to activation of MAPK during memory formation.

Tumor suppressor p53 restricts Ras stimulation of RhoA and cancer cell motility

Many features of the cancer cell phenotype emerge as a result of cooperation between multiple oncogenic mutations. Here we show that activated Ras(V12) and loss of p53 function can cooperate to promote cell motility, a feature closely associated with cancer progression to malignancy. Our analysis indicates that Ras(V12) and loss of p53 synergistically induce RhoA activity, revealing a previously unknown role for p53 in tumor suppression. p53 prevents activation of RhoA and thus induction of cell motility by Ras(V12) through a simple signaling circuit, which integrates multiple inputs that converge on RhoA. Our data suggest that p53 suppresses cancer progression to malignancy by modulating the quality of Ras signaling.

Ras Stimulation of E2F Activity and a Consequent E2F Regulation of Integrin α6β4 Promote the Invasion of Breast Carcinoma Cells

Active Ras proteins contribute to breast carcinogenesis and progression. Here, we provide evidence that active H-Ras regulates the expression and activity of the E2F family of transcription factors in SUM-159 breast carcinoma cells. In addition, we show by using a DNA-binding mutant of E2F, as well as expression of specific E2Fs that are transcriptionally active, that the active E2Fs1-3 can mediate the H-Ras-dependent invasion of SUM-159 cells. The inhibitory E2Fs4-5, in contrast, do not influence invasion. One mechanism by which the active E2Fs promote H-Ras-dependent invasion seems to be their ability to increase expression of the beta4 integrin subunit, a component of the alpha6beta4 integrin that is known to enhance carcinoma invasion. Specifically, expression of E2Fs1-3 increased beta4 mRNA, protein, and cell surface expression. The active E2Fs were unable to stimulate invasion in cells that expressed a beta4 short hairpin RNA. This effect of the active E2Fs on beta4 expression does not seem to result from E2F-mediated beta4 transcription because the beta4 promoter lacks known E2F binding motifs. In summary, the data reported here indicate a novel mechanism by which H-Ras can promote the invasion of breast carcinoma cells. This mechanism links active H-Ras, transcriptionally active E2F, and the alpha6beta4 integrin in a common pathway that culminates in enhanced alpha6beta4-dependent invasion.

Ras Signaling and Transcriptional Synergy at a Flexible Ets-1/Pit-1 Composite DNA Element Is Defined by the Assembly of Selective Activation Domains

Pit-1 and Ets-1 binding to a composite element synergistically activates and targets Ras-mitogen-activated protein kinase signaling to the rat prolactin promoter. These transcriptional responses appear to depend on three molecular features: organization of the Ets-1/Pit-1 composite element, physical interaction of these two factors via the Pit-1 homeodomain (amino acids 199-291) and the Ets-1 regulatory III domain (amino acids 190-257), and assembly of their transcriptional activation domains (TADs). Here we show that the organization of the Ets-1/Pit-1 composite element tolerates significant flexibility with regard to Ras stimulation and synergy. Specifically, the putative monomeric Pit-1 binding site can be substituted with bona fide binding sites for either a Pit-1 monomer or dimer, and these sites tolerated a separation of 28 bp. Additionally, we show that the physical interaction of Ets-1 and Pit-1 is not required for Ras responsiveness or synergy because block mutations of the Pit-1 interaction surface in Ets-1, which reduced Ets-1/Pit-1 binding in vitro, did not significantly affect Ets-1 stimulation of Ras responsiveness or synergy. We also show differential use of distinct TAD subtypes and Pit-1 TAD subregions to mediate either synergy or Ras responsiveness. Specifically, TADs from Gal4, VP16, or Ets-2 regulatory III domain linked to Ets-1 DNA binding domain constructs restored synergy to these TAD/Ets-1 DNA binding domain fusions. Conversely, deletion of the defined Pit-1 TAD (amino acids 2-80) retained synergy, but not Ras responsiveness. Consequently, we further defined the Pit-1 amino-terminal TAD into region 1 (R1, amino acids 2-45) and region 2 (R2, amino acids 46-80). R1 appears to regulate basal and synergistic responses, whereas the Ras response was mapped to R2. In summary, Ras responsiveness and Pit-1/Ets-1 synergy are mediated through the assembly of distinct TADs at a flexible composite element, indicating that different mechanisms underlie these two transcriptional responses and that the Pit-1 R2 subregion represents a novel, tissue-specific Ras-responsive TAD.

Vav mediates Ras stimulation by direct activation of the GDP/GTP exchange factor Ras GRP1.

Here we describe a new signaling cross-talk between the Vav/Rac1 and Ras pathways that is established through the stimulation of RasGRP1, an exchange factor for Ras subfamily GTPases. This interaction is crucial for Ras activation in lymphoid cells, since this GTPase cannot become activated in the absence of Vav proteins. The activation of RasGRP1 requires both the generation of diacylglycerol via phospho lipase C-gamma and the induction of actin polymerization, two responses induced by Vav and Rac1 that facilitate the translocation of RasGRP1 to juxtamembrane areas of the cell. Consistent with this, the cross-talk can be activated by tyrosine-phosphorylated wild-type Vav, oncogenic Vav and constitutively active Rac1. Conversely, Ras activation can be blocked in lymphocytes and ectopic systems using inhibitors affecting either phospholipase C-gamma or F-actin polymerization. These results indicate that a relay mechanism exists in lymphoid and other cells helping in the generation of robust signaling responses by the Rac/Rho and Ras pathways upon receptor engagement.

Elk-1 Pushes SRF Around

Murai and Treisman demonstrate an intriguing mechanism whereby physical interactions between transcription factors can shift the balance between Rho and Ras stimulation of serum response factor (SRF)-regulated gene transcription. SRF, a transcription factor that regulates many immediate-early genes, is itself stimulated by changes in actin dynamics mediated by Rho-family GTPases. However, the expression of some SRF-regulated genes, such as c-fos, is not stimulated by the Rho-actin pathway but is stimulated by Ras signaling through the mitogen-activated protein kinase (MAPK) pathway, which activates the ternary complex factors (TCFs), a family of transcription factors that bind cooperatively with SRF to DNA. Murai and Treisman investigated the role of interactions between SRF and the TCF protein Elk-1 in limiting c-fos sensitivity to RhoA-actin. Using c-fos promoter mutants transfected into NIH 3T3 cells, the authors demonstrated that deleting a TCF-binding site adjacent to the SRF binding site (ΔTCF mutant) rendered transcriptional activation after serum stimulation sensitive to latrunculin, a specific inhibitor of the RhoA-actin pathway. Serum stimulated expression of the ΔTCF construct, but this stimulation was blocked by latrunculin. In the presence of an Elk-1 mutant that interacted with the SRF and bound to the ΔTCF mutant, as shown by gel shift assay, expression from the ΔTCF construct was not inhibited by latrunculin. Using an Elk-1 mutant that bound DNA independently of the SRF, the authors determined that when the B box, a 20-amino acid region that mediates Elk-1 interaction with SRF, was mutated, then latrunculin still inhibited expression from the ΔTCF construct. Although physical interaction of SRF with the Elk-1 B box inhibited Rho-actin stimulation of the SRF, this interaction potentiated transcriptional activation by Elk-1. This suggests that TCF proteins, through direct interaction with the SRF, can modulate the relative sensitivity of target genes to Rho and Ras regulation.K. Murai, R. Treisman, Interaction of serum response factor (SRF) with the Elk-1 B box inhibits RhoA-actin signaling to SRF and potentiates transcriptional activation by Elk-1. Mol. Cell. Biol. 22, 7083-7092 (2002). [Abstract] [Full Text]

Elk-1 Pushes SRF Around

Murai and Treisman demonstrate an intriguing mechanism whereby physical interactions between transcription factors can shift the balance between Rho and Ras stimulation of serum response factor (SRF)-regulated gene transcription. SRF, a transcription factor that regulates many immediate-early genes, is itself stimulated by changes in actin dynamics mediated by Rho-family GTPases. However, the expression of some SRF-regulated genes, such as c- fos , is not stimulated by the Rho-actin pathway but is stimulated by Ras signaling through the mitogen-activated protein kinase (MAPK) pathway, which activates the ternary complex factors (TCFs), a family of transcription factors that bind cooperatively with SRF to DNA. Murai and Treisman investigated the role of interactions between SRF and the TCF protein Elk-1 in limiting c- fos sensitivity to RhoA-actin. Using c- fos promoter mutants transfected into NIH 3T3 cells, the authors demonstrated that deleting a TCF-binding site adjacent to the SRF binding site (ΔTCF mutant) rendered transcriptional activation after serum stimulation sensitive to latrunculin, a specific inhibitor of the RhoA-actin pathway. Serum stimulated expression of the ΔTCF construct, but this stimulation was blocked by latrunculin. In the presence of an Elk-1 mutant that interacted with the SRF and bound to the ΔTCF mutant, as shown by gel shift assay, expression from the ΔTCF construct was not inhibited by latrunculin. Using an Elk-1 mutant that bound DNA independently of the SRF, the authors determined that when the B box, a 20-amino acid region that mediates Elk-1 interaction with SRF, was mutated, then latrunculin still inhibited expression from the ΔTCF construct. Although physical interaction of SRF with the Elk-1 B box inhibited Rho-actin stimulation of the SRF, this interaction potentiated transcriptional activation by Elk-1. This suggests that TCF proteins, through direct interaction with the SRF, can modulate the relative sensitivity of target genes to Rho and Ras regulation. K. Murai, R. Treisman, Interaction of serum response factor (SRF) with the Elk-1 B box inhibits RhoA-actin signaling to SRF and potentiates transcriptional activation by Elk-1. Mol. Cell. Biol. 22 , 7083-7092 (2002). [Abstract] [Full Text]

A Function for Phosphoinositide 3-Kinase β Lipid Products in Coupling βγ to Ras Activation in Response to Lysophosphatidic Acid

Although Gbetagamma is thought to mediate mitogen-activated protein kinase (MAPK) activation in response to G protein-coupled receptor stimulation, the mechanisms involved in this pathway have not been clearly defined. Phosphoinositide 3-kinase (PI3K) has been proposed as an early intermediate in this process, but its role has remained elusive. We have observed that dominant negative mutants of p110beta, but not of p110gamma, inhibited MAPK stimulation in response to lysophosphatidic acid (LPA). The role of p110beta was located upstream from Ras. To determine which of the lipid or protein kinase activities of p110beta were important for Ras activation, we produced a mutant p110beta lacking the lipid but not the protein kinase activity. This protein displayed a dominant negative activity similar to a kinase-dead mutant, indicating that p110beta lipid kinase activity was essentially involved in Ras activation. In agreement, overexpression of the lipid phosphatase PTEN was found to specifically inhibit Ras stimulation induced by LPA. In addition, we have observed that the PH domain-containing adapter protein Gab1, which is involved in p110beta activation during LPA stimulation, is also implicated in this pathway downstream of p110beta. Indeed, both membrane redistribution and phosphorylation of Gab1 were reduced in the presence of PI3K inhibitors or dominant negative p110beta. Downstream of Gab1, the tyrosine phosphatase SHP2 was found to mediate Ras activation in response to LPA and to be recruited through PI3K and Gab1, because transfection of Gab1 mutant deficient for SHP2 binding inhibited Ras activation without interfering with PI3K activation. We conclude that LPA-induced Ras activation is mediated by a p110beta/Gab1/SHP2 pathway. Moreover, we present data indicating that p110beta is effectively the target of betagamma in this pathway, suggesting that the p110beta/Gab1/SHP2 pathway provides a novel link between betagamma and Ras by integrating two early events of LPA signaling, i.e. Gbetagamma release and tyrosine kinase receptor transactivation.

ERK2- and p90Rsk2-dependent Pathways Regulate the CCAAT/Enhancer-binding Protein-β Interaction with Serum Response Factor

The serum response element (SRE) of the c-fos promoter is a convergence point for mitogenic signaling pathways. Several transcription factors regulate SRE, including serum response factor (SRF), ternary complex factors, and CCAAT/enhancer-binding protein-beta (C/EBPbeta). C/EBPbeta can interact with both SRF and the ternary complex factor family member Elk-1, but only in response to activated Ras. Transactivation of the SRE by C/EBPbeta is also greatly stimulated by Ras. The Ras effectors that signal to C/EBPbeta are unknown. In this report, we demonstrate that a consensus MAPK site in C/EBPbeta is necessary for Ras stimulation of both C/EBPbeta-SRF interaction and transactivation of the SRE by C/EBPbeta. To dissect signaling pathways activated downstream of Ras, different Ras effector constructs were analyzed. We show that activated forms of Raf and phosphatidylinositol 3-kinase stimulate C/EBPbeta-SRF interaction. We also show a novel selectivity for the MAPK family member ERK2, where dominant-negative ERK2, but not dominant-negative ERK1, blocks Ras stimulation of C/EBPbeta-SRF interaction. In addition, recombinant C/EBPbeta protein is phosphorylated by ERK2, but not by ERK1, in vitro. Finally, we demonstrate a requirement for p90(Rsk2) in regulation of C/EBPbeta-SRF interaction. These data show that multiple Ras effectors are required to regulate C/EBPbeta and SRF association.

A Critical Role for Phosphoinositide 3-Kinase Upstream of Gab1 and SHP2 in the Activation of Ras and Mitogen-activated Protein Kinases by Epidermal Growth Factor

Although the mechanisms involved in the activation of mitogen-activated protein kinases (MAPK) by receptor tyrosine kinases do not display an obvious role for phosphoinositide 3-kinases (PI3Ks), we have observed in the nontransformed cell line Vero stimulated with epidermal growth factor (EGF) that wortmannin and LY294002 nearly abolished MAPK activation. The effect was observed under strong stimulation and was independent of EGF concentration. In addition, three mutants of class Ia PI3Ks were found to inhibit MAPK activation to an extent similar to their effect on Akt/protein kinase B activation. To determine the importance of PI3K lipid kinase activity in MAPK activation, we have used the phosphatase PTEN and the pleckstrin homology domain of Tec kinase. Overexpression of these proteins, but not control mutants, was found to inhibit MAPK activation, suggesting that the lipid products of class Ia PI3K are necessary for MAPK signaling. We next investigated the location of PI3K in the MAPK cascade. Pharmacological inhibitors and dominant negative forms of PI3K were found to block the activation of Ras induced by EGF. Upstream from Ras, although association of Grb2 with its conventional effectors was independent of PI3K, we have observed that the recruitment of the tyrosine phosphatase SHP2 required PI3K. Because SHP2 was also essential for Ras activation, this suggested the existence of a PI3K/SHP2 pathway leading to the activation of Ras. In addition, we have observed that the docking protein Gab1, which is involved in PI3K activation during EGF stimulation, is also implicated in this pathway downstream of PI3K. Indeed, the association of Gab1 with SHP2 was blocked by PI3K inhibitors, and expression of Gab1 mutant deficient for binding to SHP2 was found to inhibit Ras stimulation without interfering with PI3K activation. These results show that, in addition to Shc and Grb2, a PI3K-dependent pathway involving Gab1 and SHP2 is essential for Ras activation under EGF stimulation.

The p38 Pathway Provides Negative Feedback for Ras Proliferative Signaling

Ras activates three mitogen-activated protein kinases (MAPKs) including ERK, JNK, and p38. Whereas the essential roles of ERK and JNK in Ras signaling has been established, the contribution of p38 remains unclear. Here we demonstrate that the p38 pathway functions as a negative regulator of Ras proliferative signaling via a feedback mechanism. Oncogenic Ras activated p38 and two p38-activated protein kinases, MAPK-activated protein kinase 2 (MK2) and p38-related/activated protein kinase (PRAK). MK2 and PRAK in turn suppressed Ras-induced gene expression and cell proliferation, whereas two mutant PRAKs, unresponsive to Ras, had little effect. Moreover, the constitutive p38 activator MKK6 also suppressed Ras activity in a p38-dependent manner whereas arsenite, a potent chemical inducer of p38, inhibited proliferation only in a tumor cell line that required Ras activity. MEK was required for Ras stimulation of the p38 pathway. The p38 pathway inhibited Ras activity by blocking activation of JNK, without effect upon ERK, as evidenced by the fact that PRAK-mediated suppression of Ras-induced cell proliferation was reversed by coexpression of JNKK2 or JNK1. These studies thus establish a negative feedback mechanism by which Ras proliferative activity is regulated via signaling integrations of MAPK pathways.

Ras stimulates DNA topoisomerase II alpha through MEK: a link between oncogenic signaling and a therapeutic target.

Topoisomerase II alpha (topo II alpha) is a major target of antitumor treatments. In an effort to determine why this protein might be a better target in tumor cells than in normal cells, we attempted to determine if the altered proliferative signaling in a tumor cell might effect the levels of expression of the topo II alpha gene. In support of this idea, it was found that topo II alpha was elevated following microinjection of oncogenic Ras protein. Oncogenic ras was further shown to stimulate the topo II alpha promoter. Stimulation by ras was independent of the normal cell cycle regulation of this promoter. Transactivation of topo II alpha by ras required both the MEK/ERK pathway, and the stress-associated protein kinase (SAPK) signaling pathway. As a direct confirmation that both ERK and SAPK were involved in topo II alpha regulation, a constitutively active MEKK that stimulates these two kinases simultaneously was shown to strongly induce topo II alpha promoter activity. Activation of either pathway alone, on the other hand, only slightly stimulated the topo II alpha promoter. Deletion analyses showed that elements near both the 5' and 3' ends of the promoter were responsible for the ras stimulation. Site-directed mutagenesis further demonstrated that an Ets-like binding site near the 5' end (-480 to -475) was one of the responsive elements. Taken together, these studies demonstrate the direct role of Ras signaling in stimulation of topo II alpha expression, and thereby establish a link between the action of a common tumor mutation and the target of multiple anti-tumor reagents.

EGF induced SOS phosphorylation in PC12 cells involves P90 RSK-2.

SOS, the guanine nucleotide exchange factor for Ras, becomes phosphorylated on serine and threonine residues following stimulation of cells with growth factors. These phosphorylations may play a role in negative feedback of Ras stimulation and have been shown to be mediated in part by the MAP kinases Erk-1 and Erk-2. Here we show that in addition to MAP kinase, a major mitogen activated kinase for SOS is p90 Rsk-2, a downstream target of MAP kinase. p90 Rsk-2 phosphorylates SOS in an in gel assay and also in solution in vitro. The ability of p90 Rsk-2 to phosphorylate SOS increases greatly following EGF treatment of PC12 cells and is blocked by expression of N17 Ras or treatment with the MEK inhibitor PD98059. Phosphopeptide mapping revealed that the sites phosphorylated by p90 Rsk-2 in vitro were also phosphorylated in intact cells in response to EGF treatment. Several major sites of in vivo phosphorylation correlated with p90 Rsk-2 phosphorylation sites rather than MAP kinase sites. It is therefore likely that p90 Rsk-2 plays an important role in the down regulation of the Ras activation pathway through SOS.

Selective facilitative effect of post-trial reticular stimulation in discriminative learning in the rat

Ninety-eight Sprague-Dawley rats, implanted with electrodes in the mesencephalic tegmentum (reticular activating system, RAS) served as subjects in two experiments. In the first experiment (n = 42) we investigated the effects of a RAS stimulation (5 μ A, 300 Hz, 90 sec in duration) on the acquisition of a positively reinforced light–dark discrimination in a T-maze. In the second experiment (n = 56) the reinforcement and the treatment were dissociated by comparing the effects of the RAS stimulation administered after correct or incorrect choices, during the same discrimination task. In the two experiments, despite large differences in learning conditions, the results show a considerable learning facilitation by administering the RAS stimulation immediately after each trial. This facilitation does not seem to be due to an interaction between reinforcement and stimulation, since the results of experiment 2 show the maximum facilitation in animals stimulated after each (non-reinforced) error, compared to subjects stimulated after each (reinforced) correct choice. These results are discussed both in terms of consolidation processes and in terms of comparison of the cue values of S + and S - in a discriminative learning situation.