Tumor Suppressor PTEN Inhibits Integrin- and Growth Factor–mediated Mitogen-activated Protein (MAP) Kinase Signaling Pathways

The tumor suppressor PTEN dephosphorylates focal adhesion kinase (FAK) and inhibits integrin-mediated cell spreading and cell migration. We demonstrate here that expression of PTEN selectively inhibits activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. PTEN expression in glioblastoma cells lacking the protein resulted in inhibition of integrin-mediated MAP kinase activation. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)- induced MAPK activation were also blocked. To determine the specific point of inhibition in the Ras/Raf/ MEK/ERK pathway, we examined these components after stimulation by fibronectin or growth factors. Shc phosphorylation and Ras activity were inhibited by expression of PTEN, whereas EGF receptor autophosphorylation was unaffected. The ability of cells to spread at normal rates was partially rescued by coexpression of constitutively activated MEK1, a downstream component of the pathway. In addition, focal contact formation was enhanced as indicated by paxillin staining. The phosphatase domain of PTEN was essential for all of these functions, because PTEN with an inactive phosphatase domain did not suppress MAP kinase or Ras activity. In contrast to its effects on ERK, PTEN expression did not affect c-Jun NH₂-terminal kinase (JNK) or PDGF-stimulated Akt. Our data suggest that a general function of PTEN is to down-regulate FAK and Shc phosphorylation, Ras activity, downstream MAP kinase activation, and associated focal contact formation and cell spreading.     

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.     

Suppression of integrin activation by activated Ras or Raf does not correlate with bulk activation of ERK MAP kinase

The rapid modulation of ligand-binding affinity ("activation") is a central property of the integrin family of cell adhesion receptors. The Ras family of small GTP-binding proteins and their downstream effectors are key players in regulating integrin activation. H-Ras can suppress integrin activation in fibroblasts via its downstream effector kinase, Raf-1. In contrast, to H-Ras, a closely related small GTP-binding protein R-Ras has the opposite activity, and promotes integrin activation. To gain insight into the regulation of integrin activation by Ras GTPases, we created a series of H-Ras/R-Ras chimeras. We found that a 35-amino acid stretch of H-Ras was required for full suppressive activity. Furthermore, the suppressive chimeras were weak activators of the ERK1/2 MAP kinase pathway, suggesting that the suppression of integrin activation may be independent of the activation of the bulk of ERK MAP kinase. Additional data demonstrating that the ability of H-Ras or Raf-1 to suppress integrin activation was unaffected by inhibition of bulk ERK1/2 MAP kinase activation supported this hypothesis. Thus, the suppression of integrin activation is a Raf kinase induced regulatory event that can be mediated independently of bulk activation of the ERK MAP-kinase pathway.     

Regulation of human immunodeficiency virus type 1 infectivity by the ERK mitogen-activated protein kinase signaling pathway

ERK1 and ERK2 mitogen-activated protein kinases (MAPK) play a critical role in regulation of cell proliferation and differentiation in response to mitogens and other extracellular stimuli. Mitogens and cytokines that activate MAPK in T cells have been shown to activate human immunodeficiency virus type 1 (HIV-1) replication. Little is known about the signal transduction pathways that activate HIV-1 replication in T cells upon activation by extracellular stimulation. Here, we report that activation of MAPK through the Ras/Raf/MEK signaling pathway enhances the infectivity of HIV-1 virions. Virus infectivity was enhanced by treatment of cells with MAPK stimulators, such as serum and phorbol myristate acetate, as well as by coexpression of constitutively activated Ras, Raf, or MEK (MAPK kinase) in the absence of extracellular stimulation. Treatment of cells with PD 098059, a specific inhibitor of MAPK activation, or with a MAPK antisense oligonucleotide reduced the infectivity of HIV-1 virions without significantly affecting virus production or the levels of virion-associated Gag and Env proteins. MAPK has been shown to regulate HIV-1 infectivity by phosphorylating Vif (X. Yang and D. Gabuzda, J. Biol. Chem. 273:29879-29887, 1998). However, MAPK activation enhanced virus infectivity in some cells lines that do not require Vif function. The HIV-1 Rev, Tat, p17(Gag), and Nef proteins were directly phosphorylated by MAPK in vitro, suggesting that other HIV-1 proteins are potential substrates for MAPK phosphorylation. These results suggest that activation of the ERK MAPK pathway plays a role in HIV-1 replication by enhancing the infectivity of HIV-1 virions through Vif-dependent as well as Vif-independent mechanisms. MAPK activation in producer cells may contribute to the activation of HIV-1 replication when T cells are activated by mitogens and other extracellular stimuli.     

Negative regulation of the alpha interferon-induced antiviral response by the Ras/Raf/MEK pathway

Interferon (IFN) is one of the molecules released by virus-infected cells, resulting in the establishment of an antiviral state within infected and neighboring cells. IFN-induced antiviral response may be subject to modulation by the cellular signaling environment of host cells which impact the effectiveness of viral replication. Here, we show that cells with an activated Ras/Raf/MEK signaling cascade allow propagation of viruses in the presence of IFN. Ras-transformed (RasV12) and vector control NIH 3T3 cells were infected with vesicular stomatitis virus (VSV) or an IFN-sensitive vaccinia virus (delE3L) in the presence of alpha interferon. While IFN protected vector control cells from infection by both viruses, RasV12 cells were susceptible to viral infection regardless of the presence of IFN. IFN sensitivity was restored in RasV12 cells upon RNA interference (RNAi) knockdown of Ras. We further investigated which elements downstream of Ras are responsible for counteracting IFN-induced antiviral responses. A Ras effector domain mutant that can only stimulate the Raf kinase family of effectors was able to suppress the IFN response and allow VSV replication. IFN-induced antiviral mechanisms were also restored in RasV12 cells by treatment with a MEK inhibitor (U0126 or PD98059). Moreover, by using RNAi to MEK1 and MEK2, we determined that MEK2, rather than MEK1, is responsible for suppression of the IFN response. In conclusion, our results suggest that activation of the Ras/Raf/MEK pathway downregulates IFN-induced antiviral response.     

The Small GTP-binding Protein R-Ras Can Influence Integrin Activation by Antagonizing a Ras/Raf-initiated Integrin Suppression Pathway

The rapid modulation of ligand-binding affinity ("activation") is a central property of the integrin family of cell adhesion receptors. The small GTP-binding protein Ras and its downstream effector kinase Raf-1 suppress integrin activation. In this study we explored the relationship between Ras and the closely related small GTP-binding protein R-Ras in modulating the integrin affinity state. We found that R-Ras does not seem to be a direct activator of integrins in Chinese hamster ovary cells. However, we observed that GTP-bound R-Ras strongly antagonizes the Ras/Raf-initiated integrin suppression pathway. Furthermore, this reversal of the Ras/Raf suppressor pathway does not seem to be via a competition between Ras and R-Ras for common downstream effectors or via an inhibition of Ras/Raf-induced MAP kinase activation. Thus, R-Ras and Ras may act in concert to regulate integrin affinity via the activation of distinct downstream effectors.     

Stimulation of mitogen-activated protein kinase by oncogenic Ras p21 in Xenopus oocytes. Requirement for Ras p21-GTPase-activating protein interaction.

p21ras plays an important role in the control of cell proliferation. The molecular mechanisms implicated are unknown. We report that the injection of oncogenic Lys12 Ras into Xenopus laevis oocytes promoted the activation of mitogen-activated protein kinase (MAP kinase) after a lag of about 90 min. MAP kinase activity was 10-fold higher 4 h after injection of oncogenic Lys12 Ras than after injection of nononcogenic Gly12 Ras. The stimulated MAP kinase activity remained at a plateau for at least 18 h. Maximal stimulation was obtained with 5 ng of Lys12 Ras, which is similar to the amount that elicits germinal vesicle breakdown. DEAE-Sephacel chromatography of extracts from Lys12 Ras-injected oocytes showed one peak of MAP kinase. MAP kinase activation by Lys12 Ras was associated with tyrosine phosphorylation of MAP kinase (p42). As previously shown, the S6-kinase II (likely pp90rsk), which is activated in vitro by MAP kinase, was also activated by oncogenic Lys12 Ras. Lys12 Ras with an additional mutation (Glu38) in the effector region that binds GTPase-activating protein (GAP) did not promote MAP kinase or S6 kinase activations. Thus, GAP may be involved downstream to Ras in these activation processes. Our results indicate that the Ras-GAP complex promotes MAP kinase activation in oocytes. This supports the idea that Ras-GAP controls MAP kinase, a kinase implicated in the action of various stimuli.     

Involvement of Ras and Ral in chemotactic migration of skeletal myoblasts

In skeletal myoblasts, Ras has been considered to be a strong inhibitor of myogenesis. Here, we demonstrate that Ras is involved also in the chemotactic response of skeletal myoblasts. Expression of a dominant-negative mutant of Ras inhibited chemotaxis of C2C12 myoblasts in response to basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and insulin-like growth factor 1 (IGF-1), key regulators of limb muscle development and skeletal muscle regeneration. A dominant-negative Ral also decreased chemotactic migration by these growth factors, while inhibitors for phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase (MEK) showed no effect. Activation of the Ras-Ral pathway by expression of an activated mutant of either Ras, the guanine-nucleotide dissociation stimulator for Ral, or Ral resulted in increased motility of myoblasts. The ability of Ral to stimulate motility was reduced by introduction of a mutation which prevents binding to Ral-binding protein 1 or phospholipase D. These results suggest that the Ras-Ral pathway is essential for the migration of myoblasts. Furthermore, we found that Ras and Ral are activated in C2C12 cells by bFGF, HGF and IGF-1 and that the Ral activation is regulated by the Ras- and the intracellular Ca(2+)-mediated pathways. Taken together, our data indicate that Ras and Ral regulate the chemotactic migration of skeletal muscle progenitors.     

Thyrotropin-induced mitogenesis is Ras dependent but appears to bypass the Raf-dependent cytoplasmic kinase cascade.

Cellular growth control requires the coordination and integration of multiple signaling pathways which are likely to be activated concomitantly. Mitogenic signaling initiated by thyrotropin (TSH) in thyroid cells seems to require two distinct signaling pathways, a cyclic AMP (cAMP)-dependent signaling pathway and a Ras-dependent pathway. This is a paradox, since activated cAMP-dependent protein kinase disrupts Ras-dependent signaling induced by growth factors such as epidermal growth factor and platelet-derived growth factor. This inhibition may occur by preventing Raf-1 protein kinase from binding to Ras, an event thought to be necessary for the activation of Raf-1 and the subsequent activation of the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinases (MEKs) and MAP kinase (MAPK)/ERKs. Here we report that serum-stimulated hyperphosphorylation of Raf-1 was inhibited by TSH treatment of Wistar rat thyroid cells, indicating that in this cell line, as in other cell types, increases in intracellular cAMP levels inhibit activation of downstream kinases targeted by Ras. Ras-stimulated expression of genes containing AP-1 promoter elements was similarly inhibited by TSH. On the other hand, stimulation of thyroid cells with TSH resulted in stimulation of DNA synthesis which was Ras dependent but both Raf-1 and MEK independent. We also show that Ras-stimulated DNA synthesis required the use of this kinase cascade in untreated quiescent cells but not in TSH-treated cells. These data suggest that in TSH-treated thyroid cells, Ras might be able to signal through effectors other than the well-studied cytoplasmic kinase cascade.     

Rac1 GTPase is activated by hepatitis B virus replication--involvement of HBX

Hepatitis B virus (HBV) is a causative agent for liver diseases including hepatocellular carcinoma. Understanding its interactions with cellular proteins is critical in the elucidation of the mechanisms of disease progression. Using a cell-based HBV replication system, we showed that HBV replication in HepG2 cells resulted in a cellular morphological changes displaying membrane rufflings and lamellipodia like structures reminiscent of cells expressing constitutively activated Rac1. We also showed that activated Rac1 resulted in increased viral replication. HBV replication specifically activated wild type Rac1, but not Cdc42. The Rac1 activation by HBV replication also resulted in the phosphorylation of ERK1/2 and AKT, the downstream targets of Rac1 signaling cascade. The smallest HBV viral protein, HBX, was able to activate the endogenous Rac1 and induce membrane ruffling when transfected into cells. Significantly, HBX was found to directly interact with a Rac1 nucleotide exchange factor (betaPIX) through a SH3 binding motif. Taken together, we have shown the interaction of HBV with the Rho GTPase, affecting cell morphology through the Rac1 activation pathway. HBV may possibly make use of an activated Rac1 signaling pathway for increased replication and resultant metastatic effects.     

Aberrant Ras regulation and reduced p190 tyrosine phosphorylation in cells lacking p120-Gap.

The Ras guanine nucleotide-binding protein functions as a molecular switch in signalling downstream of protein-tyrosine kinases. Ras is activated by exchange of GDP for GTP and is turned off by hydrolysis of bound GTP to GDP. Ras itself has a low intrinsic GTPase activity that can be stimulated by GTPase-activating proteins (GAPs), including p120-Gap and neurofibromin. These GAPs possess a common catalytic domain but contain distinct regulatory elements that may couple different external signals to control of the Ras pathway. p120-Gap, for example, has two N-terminal SH2 domains that directly recognize phosphotyrosine motifs on activated growth factor receptors and cytoplasmic phosphoproteins. To analyze the role of p120-Gap in Ras regulation in vivo, we have used fibroblasts derived from mouse embryos with a null mutation in the gene for p120-Gap (Gap). Platelet-derived growth factor stimulation of Gap-/- cells led to an abnormally large increase in the level of Ras-GTP and in the duration of mitogen-activated protein (MAP) kinase activation compared with wild-type cells, suggesting that p120-Gap is specifically activated following growth factor stimulation. Induction of DNA synthesis in response to platelet-derived growth factor and morphological transformation by the v-src and EJ-ras oncogenes were not significantly affected by the absence of p120-Gap. However, we found that normal tyrosine phosphorylation of p190-rhoGap, a cytoplasmic protein that associates with the p120-Gap SH2 domains, was dependent on the presence of p120-Gap. Our results suggest that p120-Gap has specific functions in downregulating the Ras/MAP kinase pathway following growth factor stimulation, and in modulating the phosphorylation of p190-rhoGap, but is not required for mitogenic signalling.     

Ras-dependent and -independent pathways target the mitogen-activated protein kinase network in macrophages.

Mitogen-activated protein kinases (MAPKs) are activated upon a variety of extracellular stimuli in different cells. In macrophages, colony-stimulating factor 1 (CSF-1) stimulates proliferation, while bacterial lipopolysaccharide (LPS) inhibits cell growth and causes differentiation and activation. Both CSF-1 and LPS rapidly activate the MAPK network and induce the phosphorylation of two distinct ternary complex factors (TCFs), TCF/Elk and TCF/SAP. CSF-1, but not LPS, stimulated the formation of p21ras. GTP complexes. Expression of a dominant negative ras mutant reduced, but did not abolish, CSF-1-mediated stimulation of MEK and MAPK. In contrast, activation of the MEK kinase Raf-1 was Ras independent. Treatment with the phosphatidylcholine-specific phospholipase C inhibitor D609 suppressed LPS-mediated, but not CSF-1-mediated, activation of Raf-1, MEK, and MAPK. Similarly, down-regulation or inhibition of protein kinase C blocked MEK and MAPK induction by LPS but not that by CSF-1. Phorbol 12-myristate 13-acetate pretreatment led to the sustained activation of the Raf-1 kinase but not that of MEK and MAPK. Thus, activated Raf-1 alone does not support MEK/MAPK activation in macrophages. Phosphorylation of TCF/Elk but not that of TCF/SAP was blocked by all treatments that interfered with MAPK activation, implying that TCF/SAP was targeted by a MAPK-independent pathway. Therefore, CSF-1 and LPS target the MAPK network by two alternative pathways, both of which induce Raf-1 activation. The mitogenic pathway depends on Ras activity, while the differentiation signal relies on protein kinase C and phosphatidylcholine-specific phospholipase C activation.     

Sprouty2 inhibits the Ras/MAP kinase pathway by inhibiting the activation of Raf.

Several genetic studies in Drosophila have shown that the dSprouty (dSpry) protein inhibits the Ras/mitogen-activated protein (MAP) kinase pathway induced by various activated receptor tyrosine kinase receptors, most notably those of the epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR). Currently, the mode of action of dSpry is unknown, and the point of inhibition remains controversial. There are at least four mammalian Spry isoforms that have been shown to co-express preferentially with FGFRs as compared with EGFRs. In this study, we investigated the effects of the various mammalian Spry isoforms on the Ras/MAP kinase pathway in cells overexpressing constitutively active FGFR1. hSpry2 was significantly more potent than mSpry1 or mSpry4 in inhibiting the Ras/MAP kinase pathway. Additional experiments indicated that full-length hSpry2 was required for its full potency. hSpry2 had no inhibitory effect on either the JNK or the p38 pathway and displayed no inhibition of FRS2 phosphorylation, Akt activation, and Ras activation. Constitutively active mutants of Ras, Raf, and Mek were employed to locate the prospective point of inhibition of hSpry2 downstream of activated Ras. Results from this study indicated that hSpry2 exerted its inhibitory effect at the level of Raf, which was verified in a Raf activation assay in an FGF signaling context.     

Inhibition of hepatitis B virus replication by MyD88 is mediated by nuclear factor-kappaB activation

In our previous paper, we reported that myeloid differential primary response protein (MyD88), a key adaptor in the signaling cascade of the innate immune response, inhibits hepatitis B virus (HBV) replication. The MyD88 activated nuclear factor-kappaB (NF-kappaB) signaling pathway and the intracellular upregulation of NF-kappaB signaling can induce an antiviral effect. Therefore, the association between the inhibition of HBV replication by MyD88 and NF-kappaB activation was investigated further. The results show that NF-kappaB activation was moderately increased after MyD88 expression. The strong activation of NF-kappaB by the IkappaB kinase complex IKKalpha/IKKbeta dramatically suppressed HBV replication; the MyD88 dominant negative mutant that abrogated NF-kappaB activity did not inhibit HBV replication. Furthermore, the IkappaBalpha dominant negative mutant restored the inhibition of HBV replication by MyD88. These results support a role for NF-kappaB activation in the inhibition of HBV replication and suggest a novel mechanism for the inhibition of HBV replication by MyD88 protein.