A function for phosphoinositide 3-kinase beta lipid products in coupling beta gamma 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.     

Phosphoinositide 3-kinases in lysophosphatidic acid signaling: regulation and cross-talk with the Ras/mitogen-activated protein kinase pathway

Recent reports have shown that phosphoinositide 3-kinases (PI3Ks) mediate various biological activities of lysophosphatidic acid (LPA), including cell proliferation or survival. In addition, these enzymes have been proposed to be early intermediates of mitogen-activated protein kinase (MAPK) activation. Here we summarize our current knowledge of the mechanisms underlying these observations. p110gamma is an isoform of PI3K that can be activated in vitro by Gbetagamma subunits and was therefore considered as the logical candidate to mediate responses induced by G protein-coupled receptor (GPCR) agonists. In agreement with this, p110gamma has been involved in different biochemical models linking Gbetagamma to MAPK activation. Nevertheless, its apparent tissue-specific distribution has raised questions regarding the physiological relevance of these models. In addition, LPA can activate p110beta, a member of the phosphotyrosine-dependent PI3K subfamily that participates in the mitogenic effect of LPA. Its activation is thought to involve a synergistic effect of Gbetagamma and phosphotyrosine motifs provided by a transactivated EGF receptor/Gab1 pathway. We are currently studying a possible role of p110beta upstream from Ras, suggesting that this protein could provide a novel connection between betagamma and the MAPK pathway.     

Activation of c-fos promoter by Gbetagamma-mediated signaling: involvement of Rho and c-Jun N-terminal kinase

Several extracellular stimuli mediated by G protein-coupled receptors activate c-fos promoter. Recently, we and other groups have demonstrated that signals from G protein-coupled receptors stimulate mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. The activation of these three MAPKs is mediated in part by the G protein betagamma subunit (Gbetagamma). In this study, we characterized the signals from Gbetagamma to c-fos promoter using transient transfection of c-fos luciferase into human embryonal kidney 293 cells. Activation of m2 muscarinic acetylcholine receptor and overexpression of Gbetagamma, but not constitutively active Galphai2, stimulated c-fos promoter activity. The c-fos promoter activation by m2 receptor and Gbetagamma was inhibited by beta-adrenergic receptor kinase C-terminal peptide (betaARKct), which functions as a Gbetagamma antagonist. MEK1 inhibitor PD98059 and kinase-deficient mutant of JNK kinase, but not p38 MAPK inhibitor SB203580, attenuated the m2 receptor- and Gbetagamma-induced c-fos promoter activation. Activated mutants of Ras and Rho stimulated the c-fos promoter activity, and the dominant negative mutants of Ras and Rho inhibited the c-fos promoter activation by m2 receptor and Gbetagamma. Moreover, c-fos promoter activation by m2 receptor, Gbetagamma, and active Rho, but not active Ras, was inhibited by botulinum C3 toxin. These data indicated that both Ras- and Rho-dependent signaling pathways are essential for c-fos promoter activation mediated by Gbetagamma.     

Platelet-derived growth factor-dependent activation of phosphatidylinositol 3-kinase is regulated by receptor binding of SH2-domain-containing proteins which influence Ras activity.

Upon binding of platelet-derived growth factor (PDGF), the PDGF beta receptor (PDGFR) undergoes autophosphorylation on distinct tyrosine residues and binds several SH2-domain-containing signal relay enzymes, including phosphatidylinositol 3-kinase (PI3K), phospholipase C gamma (PLC gamma), the GTPase-activating protein of Ras (RasGAP), and the tyrosine phosphatase SHP-2. In this study, we have investigated whether PDGF-dependent PI3K activation is affected by the other proteins that associate with the PDGFR. We constructed and characterized a series of PDGFR mutants which contain binding sites for PI3K as well as one additional protein, either RasGAP, SHP-2, or PLC gamma. While all of the receptors had wild-type levels of PDGF-stimulated tyrosine kinase activity and associated with comparable amounts of PI3K activity, their abilities to trigger accumulation of PI3K products in vivo differed dramatically. The wild-type receptor, as well as receptors that recruited PI3K or PI3K and SHP-2, were all capable of fully activating PI3K. In contrast, receptors that associated with PI3K and RasGAP or PI3K and PLC gamma displayed a greatly reduced ability to stimulate production of PI3K products. When this series of receptors was tested for their ability to activate Ras, we observed a strong positive correlation between Ras activation and PI3K activation. Further investigation of the relationship between Ras and PI3K indicated that Ras was upstream of PI3K. Thus, activation of PI3K requires not only binding of PI3K to the tyrosine-phosphorylated PDGFR but accumulation of GTP-bound Ras as well. Furthermore, PLC gamma and RasGAP negatively modulate PDGF-dependent PI3K activation. Finally, PDGF-stimulated signal relay can be regulated by altering the ratio of SH2-domain-containing enzymes that are recruited to the PDGFR.     

PI3K is required for insulin-stimulated but not EGF-stimulated ERK1/2 activation

The Ras/Raf/extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathway is known to cross-talk with other signaling pathways, including phosphatidylinositol 3-kinase (PI3K)/Akt pathway. However, the role of PI3K in ERK-1/2 activation induced by tyrosine kinase receptors was not fully understood. Here, we report that two structurally distinct PI3K inhibitors, wortmannin and LY294002, inhibited insulin-induced activation of ERK1/2 but had no effect on EGF-induced activation of ERK1/2 in hepatocellular carcinoma BEL-7402 and SMMC-7721 cells, breast cancer MCF-7 cells, and prostate cancer LNCaP cells. Although protein kinase C could act as a mediator between PI3K and ERK1/2, protein kinase C inhibitor chelerythrine chloride did not inhibit insulin-induced ERK1/2 activation. Both insulin- and EGF-induced ERK1/2 activation are strictly dependent on Ras activation, however, wortmannin only inhibited insulin-induced, but not EGF-induced Ras activation. These results indicate that PI3K plays different roles in the activation of Ras/ERK1/2 signaling by insulin and EGF, and that insulin-stimulated, but not EGF-stimulated, ERK1/2 and Akt signalings diverge at PI3K.     

Pathway crosstalk between Ras/Raf and PI3K in promotion of M-CSF-induced MEK/ERK-mediated osteoclast survival

While M-CSF-mediated MEK/ERK activation promotes osteoclast survival, the signaling pathway by which M-CSF activates MEK/ERK is unresolved. Functions for PI3K, Ras, and Raf have been implicated in support of osteoclast survival, although interaction between these signaling components has not been examined. Therefore, the interplay between PI3K, Ras and Raf in M-CSF-promoted MEK/ERK activation and osteoclast survival was investigated. M-CSF activates Ras to coordinate activation of PI3K and Raf/MEK/ERK, since Ras inhibition decreased PI3K activation and PI3K inhibition did not block M-CSF-mediated Ras activation. As further support for Ras-mediated signaling, constitutively active (ca) Ras promoted MEK/ERK activation and osteoclast survival, which was blocked by inhibition of PI3K or Raf. Moreover, PI3K-selective or Raf-selective caRas were only partially able to promote osteoclast survival when compared to parental caRas. We then examined whether PI3K and Raf function linearly or in parallel downstream of Ras. Expression of caPI3K increased MEK/ERK activation and promoted osteoclast survival downstream of M-CSF, supporting this hypothesis. Blocking Raf did not decrease osteoclast survival and MEK/ERK activation promoted by caPI3K. In addition, PI3K-selective Ras-mediated survival was not blocked by Raf inhibition. Taken together, our data support that Raf signaling is separate from Ras/PI3K signaling and PI3K signaling is separate from Ras/Raf signaling. These data therefore support a role for Ras in coordinate activation of PI3K and Raf acting in parallel to mediate MEK/ERK-promoted osteoclast survival induced by M-CSF.     

Crystal structure and functional analysis of Ras binding to its effector phosphoinositide 3-kinase gamma

Ras activation of phosphoinositide 3-kinase (PI3K) is important for survival of transformed cells. We find that PI3Kgamma is strongly and directly activated by H-Ras G12V in vivo or by GTPgammaS-loaded H-Ras in vitro. We have determined a crystal structure of a PI3Kgamma/Ras.GMPPNP complex. A critical loop in the Ras binding domain positions Ras so that it uses its switch I and switch II regions to bind PI3Kgamma. Mutagenesis shows that interactions with both regions are essential for binding PI3Kgamma. Ras also forms a direct contact with the PI3Kgamma catalytic domain. These unique Ras/PI3Kgamma interactions are likely to be shared by PI3Kalpha. The complex with Ras shows a change in the PI3K conformation that may represent an allosteric component of Ras activation.     

Role of Phosphoinositide 3-Kinase in Activation of Ras and Mitogen-Activated Protein Kinase by Epidermal Growth Factor

The paradigm for activation of Ras and extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase by extracellular stimuli via tyrosine kinases, Shc, Grb2, and Sos does not encompass an obvious role for phosphoinositide (PI) 3-kinase, and yet inhibitors of this lipid kinase family have been shown to block the ERK/MAP kinase signalling pathway under certain circumstances. Here we show that in COS cells activation of both endogenous ERK2 and Ras by low, but not high, concentrations of epidermal growth factor (EGF) is suppressed by PI 3-kinase inhibitors; since Ras activation is less susceptible than ERK2 activation, PI 3-kinase-sensitive events may occur both upstream of Ras and between Ras and ERK2. However, strong elevation of PI 3-kinase lipid product levels by expression of membrane-targeted p110alpha is by itself never sufficient to activate Ras or ERK2. PI 3-kinase inhibition does not affect EGF-induced receptor autophosphorylation or adapter protein phosphorylation or complex formation. The concentrations of EGF for which PI 3-kinase inhibitors block Ras activation induce formation of Shc-Grb2 complexes but not detectable EGF receptor phosphorylation and do not activate PI 3-kinase. The activation of Ras by low, but mitogenic, concentrations of EGF is therefore dependent on basal, rather than stimulated, PI 3-kinase activity; the inhibitory effects of LY294002 and wortmannin are due to their ability to reduce the activity of PI 3-kinase to below the level in a quiescent cell and reflect a permissive rather than an upstream regulatory role for PI 3-kinase in Ras activation in this system.     

PI3K/Akt-sensitive MEK-independent compensatory circuit of ERK activation in ER-positive PI3K-mutant T47D breast cancer cells

We explored the crosstalk between cell survival (phosphatidylinositol 3-kinase (PI3K)/Akt) and mitogenic (Ras/Raf/MEK/extracellular signal-regulated kinase (ERK)) signaling pathways activated by an epidermal growth factor (EGF) and analyzed their sensitivity to small molecule inhibitors in the PI3K-mutant estrogen receptor (ER)-positive MCF7 and T47D breast cancer cells. In contrast to MCF7 cells, ERK phosphorylation in T47D cells displayed resistance to MEK inhibition by several structurally different compounds, such as U0126, PD 098059 and PD 198306, MEK suppression by small interfering RNA (siRNA) and was also less sensitive to PI3K inhibition by wortmannin. Similar effect was observed in PI3K-wild type ER-positive BT-474 cells, albeit to a much lesser extent.MEK-independent ERK activation was induced only by ErbB receptor ligands and was resistant to inhibition of several kinases and phosphatases that are known to participate in the regulation of Ras/mitogen-activated protein kinase (MAPK) cascade. Although single agents against PDK1 or Akt did not affect EGF-induced ERK phosphorylation, a combination of PI3K/Akt and MEK inhibitors synergistically suppressed ERK activation and cellular growth. siRNA-mediated silencing of class I PI3K or Akt1/2 genes also significantly decreased U0126-resistant ERK phosphorylation.Our data suggest that in T47D cells ErbB family ligands induce a dynamic, PI3K/Akt-sensitive and MEK-independent compensatory ERK activation circuit that is absent in MCF7 cells. We discuss candidate proteins that can be involved in this activation circuitry and suggest that PDZ-Binding Kinase/T-LAK Cell-Originated Protein Kinase (PBK/TOPK) may play a role in mediating MEK-independent ERK activation.     

Gbetagamma subunits stimulate p21-activated kinase 1 (PAK1) through activation of PI3-kinase and Akt but act independently of Rac1/Cdc42

The p21-activated kinase (PAK) family is homologous to the yeast sterile 20 (Ste20) and regulates a wide variety of cellular responses, including cell morphology, proliferation, and survival. In this study we examined the activation of PAK1 by Gbetagamma subunits. Co-transfection of COS7 cells with Gbeta1gamma2 or Gbeta1gamma5 was sufficient to induce agonist-independent activation of PAK1. Expression of dominant/negative Rac, Cdc42, or Ras did not inhibit this Gbetagamma-dependent activation. Wortmannin, which inhibits phosphoinositide 3-kinase (PI3-kinase) activity, and expression of a dominant/negative form of Akt were sufficient to abrogate the activation of PAK1 that was induced by Gbetagamma. These results reveal that stimulation of PAK1 by Gbetagamma can occur via a PI3-kinase and Akt pathway that does not require Rac1 or Cdc42.     

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.     

Alpha-thrombin-mediated phosphatidylinositol 3-kinase activation through release of Gbetagamma dimers from Galphaq and Galphai2

Chinese hamster embryonic fibroblasts (IIC9 cells) express the Galpha subunits Galphas, Galphai2, Galphai3, Galphao, Galpha(q/11), and Galpha13. Consistent with reports in other cell types, alpha-thrombin stimulates a subset of the expressed G proteins in IIC9 cells, namely Gi2, G13, and Gq as measured by an in vitro membrane [35S]guanosine 5'-O-(3-thio)triphosphate binding assay. Using specific Galpha peptides, which block coupling of G-protein receptors to selective G proteins, as well as dominant negative xanthine nucleotide-binding Galpha mutants, we show that activation of the phosphatidylinositol 3-kinase/Akt pathway is dependent on Gq and Gi2. To examine the role of the two G proteins, we examined the events upstream of PI 3-kinase. The activation of the PI 3-kinase/Akt pathway by alpha-thrombin in IIC9 cells is blocked by the expression of dominant negative Ras and beta-arrestin1 (Phillips-Mason, P. J., Raben, D. M., and Baldassare, J. J. (2000) J. Biol. Chem. 275, 18046-18053, and Goel, R., Phillips-Mason, P. J., Raben, D. M., and Baldassare, J. J. (2002) J. Biol. Chem. 277, 18640-18648), indicating a role for Ras and beta-arrestin1. Interestingly, inhibition of Gi2 and Gq activation blocks Ras activation and beta-arrestin1 membrane translocation, respectively. Furthermore, expression of the Gbetagamma sequestrant, alpha-transducin, inhibits both Ras activation and membrane translocation of beta-arrestin1, suggesting that Gbetagamma dimers from Galphai2 and Galphaq activate different effectors to coordinately regulate the PI 3-kinase/Akt pathway.     

Galectin-1 augments Ras activation and diverts Ras signals to Raf-1 at the expense of phosphoinositide 3-kinase

Ras proteins activate diverse effector molecules. Depending on the cellular context, Ras activation may have different biological consequences: induction of cell proliferation, senescence, survival, or death. Augmentation and selective activation of particular effector molecules may underlie various Ras actions. In fact, Ras effector-loop mutants interacting with distinctive effectors provide evidence for such selectivity. Interactions of active Ras with escort proteins, such as galectin-1, could also direct Ras selectivity. Here we show that in comparison with Ras transfectants, H-Ras/galectin-1 or K-Ras4B/galectin-1 co-transfectants exhibit enhanced and prolonged epidermal growth factor (EGF)-stimulated increases in Ras-GTP, Raf-1 activity, and active extracellular signal-regulated kinase. Galectin-1 antisense RNA inhibited these EGF responses. Conversely, Ras and galectin-1 co-transfection inhibited the EGF-stimulated increase in phosphoinositide 3-kinase (PI3K) activity. Galectin-1 transfection also inhibited Ras(G12V)-induced PI3K but not Raf-1 activity. Galectin-1 co-immunoprecipitated with Ras(G12V) or with Ras(G12V/T35S) that activate Raf-1 but not with Ras(G12V/Y40C) that activates PI3K. Thus, galectin-1 binds active Ras and diverts its signal to Raf-1 at the expense of PI3K. This demonstrates a novel mechanism controlling the duration and selectivity of the Ras signal. Ras gains selectivity when it is associated with galectin-1, mimicking the selectivity of Ras(T35S), which activates Raf-1 but not PI3K.     

Role of guanine nucleotide exchange factor P-Rex-2b in sphingosine 1-phosphate-induced Rac1 activation and cell migration in endothelial cells

Endothelial cell (EC) migration has an important role in angiogenesis. Sphingosine-1 phosphate (S1P) stimulates EC migration via activation of Gi proteins. In this study, we characterized a mouse guanine nucleotide exchange factor (GEF) P-Rex2b for its regulation by Gbetagamma and PI3K and its role in S1P-induced Rac1 activation and cell migration in ECs. We found that co-expression of Gbetagamma or an active form of PI3K (PI3K(AC)) with P-Rex2b increased the SRE.Luciferase (SRE.L) reporter gene activity that can be stimulated by the Rho family of small GTPases including Rac1. Co-expression with P-Rex2b of Gbetagamma and PI3K(AC) or wild type PI3Kgamma that can be activated by Gbetagamma led to further increases in the reporter gene activity. Together with the finding that co-expression of Gbetagamma and/or PI3K(AC) increased the levels of active Rac1, we conclude that P-Rex2b is a Rac GEF that can be regulated by Gbetagamma and PI3K. Additionally, we demonstrated that Gbetagamma interacted with P-Rex2b, probably through P-Rex2b sequences at the PH domain and that the DEP and PDZ domains of P-Rex2b exerted an inhibitory effect on P-Rex2b's activity because their deletion increased the SER.L reporter gene activity. Furthermore, we found that P-Rex2b is involved in S1P-induced Rac1 activation and cell migration in ECs because siRNA-mediated suppression of P-Rex2b expression in ECs-diminished Rac1 activation and cell migration in response to S1P. Therefore, P-Rex2b is a physiologically significant Rac1 GEF that has an important role in the regulation of EC migration.     

A novel signaling pathway for beta-adrenergic receptor-mediated activation of phosphoinositide 3-kinase in H9c2 cardiomyocytes

Stimulation of cardiac beta-adrenergic receptors (beta-AR) activates both the G(s)- and G(i)-coupled signaling cascades, including the phosphoinositide 3 kinase (PI3K) pathway, that have important physiological implications. Multiple isoforms of PI3K exist in the heart. The goals of this study were to examine the intracellular signaling pathways linking beta-AR to PI3K and to identify the PI3K isoform mediating this transactivation in a cardiac context. Acute beta-AR stimulation with isoproterenol resulted in increased tyrosine kinase-associated PI3K activity and phosphorylation of Akt and p70S6K in H9c2 cardiomyocytes. Cotreatment with ICI-118,551, but not CGP-20712, abolished the increase in PI3K activity, suggesting a beta(2)-AR-mediated event. PI3K activation was also abrogated by cotreatment with pertussis toxin, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2, a selective Src-family tyrosine kinases inhibitor), or AG-1296 [selective platelet-derived growth factor receptor (PDGFR) inhibitor] but not with an inhibitor for protein kinase A, protein kinase C, Ras, adenylyl cyclase, epidermal growth factor receptor, or insulin-like growth factor-1 receptor. beta-AR stimulation induced an increase in tyrosine phosphorylation of PDGFR, which was abolished by inhibition of Src either by PP2 or small interfering RNA. Moreover, H9c2 cardiomyocytes stably transfected with a vector expressing a Gbetagamma sequestrant peptide derived from the COOH-terminus of beta-AR kinase-1 failed to activate PI3K after beta-AR stimulation, suggesting Gbetagamma is required for the transactivation. Furthermore, acute beta-AR stimulation in vivo resulted in increases in PDGFR-associated PI3K and PI3Kalpha isoform activities but not the activities of other isoforms (PI3Kbeta, -delta, -gamma) in adult mouse heart. Taken together, these data provide in vitro and in vivo evidence for a novel mechanism of beta-AR-mediated transactivation of cardiac PI3Kalpha via sequential involvement of Galpha(i)/Gbetagamma, Src, and PDGFR.     

Activity-dependent NMDA receptor-mediated activation of protein kinase B/Akt in cortical neuronal cultures

The serine/threonine protein kinase B (PKB)/Akt is a phosphoinositide 3-kinase (PI3K) effector that is thought to play an important roll in a wide variety of cellular events. The present study examined whether PKB activation in cortical neuronal cultures is coupled with synaptic activity. A 1-h incubation of neuronal cultures with tetrodotoxin (TTX), the PI3K inhibitor wortmannin, the NMDA receptor antagonist MK-801 or removal of extracellular calcium significantly reduced basal levels of phospho(Ser473)-PKB, indicating that activity-dependent glutamate release maintains PKB activation through an NMDA receptor-PI3K pathway. A 5-min exposure to NMDA (50 micro m) in the presence of TTX increased phospho-PKB back to levels observed in the absence of TTX. NMDA stimulation of phospho-PKB was blocked by wortmannin, the CaMKII inhibitor KN-93, MK-801, and removal of extracellular calcium. We have previously shown that NMDA receptors can bi-directionally regulate activation of extracellular-signal regulated kinase (ERK), and NMDA receptor stimulation of PKB in the present study appeared to mirror activation of ERK. These results suggest that in cultured cortical neurons, PKB activity is dynamically regulated by synaptic activity and is coupled to NMDA receptor activation. In addition, NMDA receptor activation of ERK and PKB may occur through overlapping signaling pathways that bifurcate at the level of Ras.     

H-Ras isoform modulates extracellular matrix synthesis, proliferation, and migration in fibroblasts

Ras GTPases are ubiquitous plasma membrane transducers of extracellular stimuli. In addition to their role as oncogenes, Ras GTPases are key regulators of cell function. Each of the Ras isoforms exhibits specific modulatory activity on different cellular pathways. This has prompted researchers to determine the pathophysiological roles of each isoform. There is a proven relationship between the signaling pathways of transforming growth factor-β1 (TGF-β1) and Ras GTPases. To assess the individual role of H-Ras oncogene in basal and TGF-β1-mediated extracellular matrix (ECM) synthesis, proliferation, and migration in fibroblasts, we analyzed these processes in embryonic fibroblasts obtained from H-Ras knockout mice (H-ras(-/-)). We found that H-ras(-/-) fibroblasts exhibited a higher basal phosphatidylinositol-3-kinase (PI3K)/Akt activation than wild-type (WT) fibroblasts, whereas MEK/ERK 1/2 activation was similar in both types of cells. Fibronectin and collagen synthesis were higher in H-ras(-/-) fibroblasts and proliferation was lower in H-ras(-/-) than in WT fibroblasts. Moreover, H-Ras appeared indispensable to maintain normal fibroblast motility, which was highly restricted in H-ras(-/-) cells. These results suggest that H-Ras (through downregulation of PI3K/Akt activation) could modulate fibroblast activity by reducing ECM synthesis and upregulating both proliferation and migration. TGF-β1 strongly increased ERK and Akt activation in WT but not in H-ras(-/-) fibroblasts, suggesting that H-Ras is necessary to increase ERK 1/2 activation and to maintain PI3K downregulation in TGF-β1-stimulated fibroblasts. TGF-β1 stimulated ECM synthesis and proliferation, although ECM synthesis was higher and proliferation lower in H-ras(-/-) than in WT fibroblasts. Hence, H-Ras activation seems to play a key role in the regulation of these effects.     

Activation of PI3K and R‐ras signaling promotes the extension of sensory axons on inhibitory chondroitin sulfate proteoglycans

Chondroitin sulfate proteoglycans (CSPGs) are extracellular inhibitors of axon extension and plasticity, and cause growth cones to exhibit dystrophic behaviors. Phosphoinositide 3‐kinase (PI3K) is a lipid kinase activated by axon growth promoting signals. In this study, we used embryonic chicken dorsal root ganglion neurons to determine if CSPGs impair signaling through PI3K. We report that CSPGs inhibit PI3K signaling in axons and growth cones, as evidenced by decreased levels of phosphorylated downstream kinases (Akt and S6). Direct activation of PI3K signaling, using a cell permeable phosphopeptide (PI3Kpep), countered the effects of CSPGs on growth cones and axon extension. Both overnight and acute treatment with PI3Kpep promoted axon extension on CSPG‐coated substrates. The R‐Ras GTPase is an upstream positive regulator of PI3K signaling. Expression of constitutively active R‐Ras promoted axon extension and growth cone elaboration on CSPGs and permissive substrata. In contrast, an N‐terminus‐deleted constitutively active R‐Ras, deficient in PI3K activation, promoted axon extension but not growth cone elaboration on CSPGs and permissive substrata. These data indicate that activation of R‐Ras‐PI3K signaling may be a viable approach for manipulating axon extension on CSPGs. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 918–933, 2014