Multiple Grb2-Mediated Integrin-Stimulated Signaling Pathways to ERK2/Mitogen-Activated Protein Kinase: Summation of Both c-Src- and Focal Adhesion Kinase-Initiated Tyrosine Phosphorylation Events

Fibronectin receptor integrin-mediated cell adhesion triggers intracellular signaling events such as the activation of the Ras/mitogen-activated protein (MAP) kinase cascade. In this study, we show that the nonreceptor protein-tyrosine kinases (PTKs) c-Src and focal adhesion kinase (FAK) can be independently activated after fibronectin (FN) stimulation and that their combined activity promotes signaling to extracellular signal-regulated kinase 2 (ERK2)/MAP kinase through multiple pathways upstream of Ras. FN stimulation of NIH 3T3 fibroblasts promotes c-Src and FAK association in the Triton-insoluble cell fraction, and the time course of FN-stimulated ERK2 activation paralleled that of Grb2 binding to FAK at Tyr-925 and Grb2 binding to Shc. Cytochalasin D treatment of fibroblasts inhibited FN-induced FAK in vitro kinase activity and signaling to ERK2, but it only partially inhibited c-Src activation. Treatment of fibroblasts with protein kinase C inhibitors or with the PTK inhibitor herbimycin A or PP1 resulted in reduced Src PTK activity, no Grb2 binding to FAK, and lowered levels of ERK2 activation. FN-stimulated FAK PTK activity was not significantly affected by herbimycin A treatment and, under these conditions, FAK autophosphorylation promoted Shc binding to FAK. In vitro, FAK directly phosphorylated Shc Tyr-317 to promote Grb2 binding, and in vivo Grb2 binding to Shc was observed in herbimycin A-treated fibroblasts after FN stimulation. Interestingly, c-Src in vitro phosphorylation of Shc promoted Grb2 binding to both wild-type and Phe-317 Shc. In vivo, Phe-317 Shc was tyrosine phosphorylated after FN stimulation of human 293T cells and its expression did not inhibit signaling to ERK2. Surprisingly, expression of Phe-925 FAK with Phe-317 Shc also did not block signaling to ERK2, whereas FN-stimulated signaling to ERK2 was inhibited by coexpression of an SH3 domain-inactivated mutant of Grb2. Our studies show that FN receptor integrin signaling upstream of Ras and ERK2 does not follow a linear pathway but that, instead, multiple Grb2-mediated interactions with Shc, FAK, and perhaps other yet-to-be-determined phosphorylated targets represent parallel signaling pathways that cooperate to promote maximal ERK2 activation.     

Death effector domain protein PEA-15 potentiates Ras activation of extracellular signal receptor-activated kinase by an adhesion-independent mechanism

PEA-15 is a small, death effector-domain (DED)-containing protein that was recently demonstrated to inhibit tumor necrosis factor-alpha-induced apoptosis and to reverse the inhibition of integrin activation due to H-Ras. This led us to investigate the involvement of PEA-15 in Ras signaling. Surprisingly, PEA-15 activates the extracellular signal receptor-activated kinase (ERK) mitogen-activated protein kinase pathway in a Ras-dependent manner. PEA-15 expression in Chinese hamster ovary cells resulted in an increased mitogen-activated protein kinase kinase and ERK activity. Furthermore, PEA-15 expression leads to an increase in Ras guanosine 5'-triphosphate loading. PEA-15 bypasses the anchorage dependence of ERK activation. Finally, the effects of PEA-15 on integrin signaling are separate from those on ERK activation. Heretofore, all known DEDs functioned in the regulation of apoptosis. In contrast, the DED of PEA-15 is essential for its capacity to activate ERK. The ability of PEA-15 to simultaneously inhibit apoptosis and potentiate Ras-to-Erk signaling may be of importance for oncogenic processes.     

Differential activation of the Ras/extracellular-signal-regulated protein kinase pathway is responsible for the biological consequences induced by the Axl receptor tyrosine kinase.

To understand the mechanism of Axl signaling, we have initiated studies to delineate downstream components in interleukin-3-dependent 32D cells by using a chimeric receptor containing the recombinant epidermal growth factor (EGF) receptor extracellular and transmembrane domains and the Axl kinase domain (EAK [for EGF receptor-Axl kinase]). We have previously shown that upon exogenous EGF stimulation, 32D-EAK cells are capable of proliferation in the absence of interleukin-3. With this system, we determined that EAK-induced cell survival and mitogenesis are dependent upon the Ras/extracellular-signal-regulated protein kinase (ERK) cascade. Although the phosphatidylinositol-3 kinase pathway is activated upon EAK signaling, it appears to be dispensable for the biological actions of the Axl kinase. Furthermore, we demonstrated that different threshold levels of Ras/ERK activation are needed to induce a block to apoptosis or proliferation in 32D cells. Recently, we have identified an Axl ligand, GAS6. Surprisingly, GAS6-stimulated 32D-Axl cells exhibited no blockage to apoptosis or mitogenic response which is correlated with the absence of Ras/ERK activation. Taken together, these data suggest that different extracellular domains dramatically alter the intracellular response of the Axl kinase. Furthermore, our data suggest that the GAS6-Axl interaction does not induce mitogenesis and that its exact role remains to be determined.     

The Ras/Raf/MEK/extracellular signal-regulated kinase pathway induces autocrine-paracrine growth inhibition via the leukemia inhibitory factor/JAK/STAT pathway

Sustained activation of the Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway can lead to cell cycle arrest in many cell types. We have found, with human medullary thyroid cancer (MTC) cells, that activated Ras or c-Raf-1 can induce growth arrest by producing and secreting an autocrine-paracrine factor. This protein was purified from cell culture medium conditioned by Raf-activated MTC cells and was identified by mass spectrometry as leukemia inhibitory factor (LIF). LIF expression upon Raf activation and subsequent activation of JAK-STAT3 was also observed in small cell lung carcinoma cells, suggesting that this autocrine-paracrine signaling may be a common response to Ras/Raf activation. LIF was sufficient to induce growth arrest and differentiation of MTC cells. This effect was mediated through the gp130/JAK/STAT3 pathway, since anti-gp130 blocking antibody or dominant-negative STAT3 blocked the effects of LIF. Thus, LIF expression provides a novel mechanism allowing Ras/Raf signaling to activate the JAK-STAT3 pathway. In addition to this cell-extrinsic growth inhibitory pathway, we find that the Ras/Raf/MEK/ERK pathway induces an intracellular growth inhibitory signal, independent of the LIF/JAK/STAT3 pathway. Therefore, activation of the Ras/Raf/MEK/ERK pathway can lead to growth arrest and differentiation via at least two different signaling pathways. This use of multiple pathways may be important for "fail-safe" induction and maintenance of cell cycle arrest.     

Ras-mutant Cancer Cells Display B-Raf Binding to Ras That Activates Extracellular Signal-regulated Kinase and Is Inhibited by Protein Kinase A Phosphorylation

The small G protein Ras regulates proliferation through activation of the mitogen-activated protein (MAP) kinase (ERK) cascade. The first step of Ras-dependent activation of ERK signaling is Ras binding to members of the Raf family of MAP kinase kinase kinases, C-Raf and B-Raf. Recently, it has been reported that in melanoma cells harboring oncogenic Ras mutations, B-Raf does not bind to Ras and does not contribute to basal ERK activation. For other types of Ras-mutant tumors, the relative contributions of C-Raf and B-Raf are not known. We examined non-melanoma cancer cell lines containing oncogenic Ras mutations and express both C-Raf and B-Raf isoforms, including the lung cancer cell line H1299 cells. Both B-Raf and C-Raf were constitutively bound to oncogenic Ras and contributed to Ras-dependent ERK activation. Ras binding to B-Raf and C-Raf were both subject to inhibition by the cAMP-dependent protein kinase PKA. cAMP inhibited the growth of H1299 cells and Ras-dependent ERK activation via PKA. PKA inhibited the binding of Ras to both C-Raf and B-Raf through phosphorylations of C-Raf at Ser-259 and B-Raf at Ser-365, respectively. These studies demonstrate that in non-melanocytic Ras-mutant cancer cells, Ras signaling to B-Raf is a significant contributor to ERK activation and that the B-Raf pathway, like that of C-Raf, is a target for inhibition by PKA. We suggest that cAMP and hormones coupled to cAMP may prove useful in dampening the effects of oncogenic Ras in non-melanocytic cancer cells through PKA-dependent actions on B-Raf as well as C-Raf.     

RGS19 inhibits Ras signaling through Nm23H1/2-mediated phosphorylation of the kinase suppressor of Ras

Besides serving as signal terminators for G protein pathways, several regulators of G protein signaling (RGS) can also modulate cell proliferation. RGS19 has previously been shown to enhance Akt signaling despite impaired Ras signaling. The present study examines the mechanism by which RGS19 inhibits Ras signaling. In HEK293 cells stably expressing RGS19, serum-induced Ras activation and phosphorylations of Raf/MEK/ERK were significantly inhibited, while cells expressing RGS2, 4, 7, 8, 10, or 20 did not exhibit this inhibitory phenotype. Conversely, siRNA-mediated knockdown of RGS19 enabled partial recovery of serum-induced ERK phosphorylation. Interestingly, two isoforms of the tumor metastasis suppressor Nm23 (H1 and H2) were upregulated in 293/RGS19 cells. As a nucleoside diphosphate kinase, Nm23H1 can phosphorylate the kinase suppressor of Ras (KSR). Elevated levels of phosphorylated KSR were indeed detected in the nuclear fractions of 293/RGS19 cells. Co-immunoprecipitation assays revealed that Nm23H1/2 can form complexes with RGS19, Ras, or KSR. siRNA-mediated knockdown of Nm23H1/2 allowed 293/RGS19 cells to partially recover their ERK responses to serum treatment, while overexpression of Nm23H1/2 in HEK293 cells suppressed the serum-induced ERK response. This study demonstrates that expression of RGS19 can suppress Ras-mediated signaling via upregulation of Nm23.     

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.     

Kinase Suppressor of Ras 2 (KSR2) Regulates Tumor Cell Transformation via AMPK

Kinase suppressor of Ras 1 (KSR1) and KSR2 are scaffolds that promote extracellular signal-regulated kinase (ERK) signaling but have dramatically different physiological functions. KSR2(-/-) mice show marked deficits in energy expenditure that cause obesity. In contrast, KSR1 disruption has inconsequential effects on development but dramatically suppresses tumor formation by activated Ras. We examined the role of KSR2 in the generation and maintenance of the transformed phenotype in KSR1(-/-) mouse embryo fibroblasts (MEFs) expressing activated Ras(V12) and in tumor cell lines MIN6 and NG108-15. KSR2 rescued ERK activation and accelerated proliferation in KSR1(-/-) MEFs. KSR2 expression alone induced anchorage-independent growth and synergized with the transforming effects of Ras(V12). Similarly, RNA interference (RNAi) of KSR2 in MIN6 and NG108-15 cells inhibited proliferation and colony formation, with concomitant defects in AMP-activated protein kinase (AMPK) signaling, nutrient metabolism, and metabolic capacity. While constitutive activation of AMPK was sufficient to complement the loss of KSR2 in metabolic signaling and anchorage-independent growth, KSR2 RNAi, MEK inhibition, and expression of a KSR2 mutant unable to interact with ERK demonstrated that mitogen-activated protein (MAP) kinase signaling is dispensable for the transformed phenotype of these cells. These data show that KSR2 is essential to tumor cell energy homeostasis and critical to the integration of mitogenic and metabolic signaling pathways.     

Upregulation of Ras/Raf/ERK1/2 signaling and ERK5 in the brain of autistic subjects

Autism is a neurodevelopmental disorder characterized by impairments in social interaction, verbal communication and repetitive behaviors. A number of studies have shown that the Ras/Raf/ERK1/2 (extracellular signal-regulated kinase) signaling pathway plays important roles in the genesis of neural progenitors, learning and memory. Ras/Raf/ERK1/2 and ERK5 have also been shown to have death-promoting apoptotic roles in neural cells. Recent studies have shown a possible association between neural cell death and autism. In addition, two recent studies reported that a deletion of a locus on chromosome 16, which included the mitogen-activated protein kinase 3 (MAPK3) gene that encodes ERK1, is associated with autism. Most recently, our laboratory detected that Ras/Raf/ERK1/2 signaling activities were significantly enhanced in the brain of BTBR mice that model autism, as they exhibit many autism-like behaviors. We thus hypothesized that Ras/Raf/ERK1/2 signaling and ERK5 could be abnormally regulated in the brain of autistic subjects. In this study, we show that the expression of Ras protein was significantly elevated in the frontal cortex of autistic subjects. C-Raf phosphorylation was increased in the frontal cortex, while both C-Raf and A-Raf activities were enhanced in the cerebellum of autistic subjects. We also detected that both the protein expression and activities of ERK1/2 were significantly upregulated in the frontal cortex of autistic subjects, but not in the cerebellum. Furthermore, we showed that ERK5 protein expression is upregulated in both frontal cortex and cerebellum of autistic subjects. These results suggest that the upregulation of Ras/Raf/ERK1/2 signaling and ERK5 activities mainly found in the frontal cortex of autistic subjects may be critically involved in the pathogenesis of autism.     

Convergence of signaling pathways on the activation of ERK in B cells

The B cell receptor (BCR) initiates three major signaling pathways: the Ras pathway, which leads to extracellular signal-regulated kinase (ERK) activation; the phospholipase C-gamma pathway, which causes calcium mobilization; and the phosphoinositide 3-kinase (PI 3-kinase) pathway. These combine to induce different biological responses depending on the context of the BCR signal. Both the Ras and PI 3-kinase pathways are important for B cell development and activation. Several model systems show evidence of cross-regulation between these pathways. Here we demonstrate through the use of PI 3-kinase inhibitors and a dominant-negative PI 3-kinase construct that the BCR-induced phosphorylation and activation of ERK is dependent on PI 3-kinase. PI 3-kinase feeds into the Ras signaling cascade at multiple points, both upstream and downstream of Ras. We also show that ERK activation is dependent on phospholipase C-gamma, in keeping with its dependence on calcium mobilization. Last, the activation of PI 3-kinase itself is completely dependent on Ras. We conclude that the PI 3-kinase and Ras signaling cascades are intimately connected in B cells and that the activation of ERK is a signal integration point, since it requires simultaneous input from all three major signaling pathways.     

Involvement of the ERK signaling cascade in protein kinase C-mediated cell cycle arrest in intestinal epithelial cells

We have reported previously that protein kinase C (PKC) signaling can mediate a program of cell cycle withdrawal in IEC-18 nontransformed intestinal crypt cells, involving rapid disappearance of cyclin D1, increased expression of Cip/Kip cyclin-dependent kinase inhibitors, and activation of the growth suppressor function of pocket proteins. In the current study, we present evidence to support a requisite role for PKC alpha in mediating these effects. Furthermore, analysis of the signaling events linking PKC/PKC alpha activation to changes in the cell cycle regulatory machinery implicate the Ras/Raf/MEK/ERK cascade. PKC/PKC alpha activity promoted GTP loading of Ras, activation of Raf-1, and phosphorylation/activation of ERK. ERK activation was found to be required for critical downstream effects of PKC/PKC alpha activation, including cyclin D1 down-regulation, p21(Waf1/Cip1) induction, and cell cycle arrest. PKC-induced ERK activation was strong and sustained relative to that produced by proliferative signals, and the growth inhibitory effects of PKC agonists were dominant over proliferative events when these opposing stimuli were administered simultaneously. PKC signaling promoted cytoplasmic and nuclear accumulation of ERK activity, whereas growth factor-induced phospho-ERK was localized only in the cytoplasm. Comparison of the effects of PKC agonists that differ in their ability to sustain PKC alpha activation and growth arrest in IEC-18 cells, together with the use of selective kinase inhibitors, indicated that the length of PKC-mediated cell cycle exit is dictated by the magnitude/duration of input signal (i.e. PKC alpha activity) and of activation of the ERK cascade. The extent/duration of phospho-ERK nuclear localization may also be important determinants of the duration of PKC agonist-induced growth arrest in this system. Taken together, the data point to PKC alpha and the Ras/Raf/MEK/ERK cascade as key regulators of cell cycle withdrawal in intestinal epithelial cells.     

Zn2+-induced ERK activation mediated by reactive oxygen species causes cell death in differentiated PC12 cells

Recent studies have provided evidence that Zn2+ plays a crucial role in ischemia- and seizure-induced neuronal death. However, the intracellular signaling pathways involved in Zn2+-induced cell death are largely unknown. In the present study, we investigated the roles of mitogen-activated protein kinases (MAPKs), such as c-Jun N-terminal kinase (JNK), p38 MAPK and extracellular signal-regulated kinase (ERK), and of reactive oxygen species (ROS) in Zn2+-induced cell death using differentiated PC12 cells. Intracellular accumulation of Zn2+ induced by the combined application of pyrithione (5 microM), a Zn2+ ionophore, and Zn2+ (10 microM) caused cell death and activated JNK and ERK, but not p38 MAPK. Preventing JNK activation by the expression of dominant negative SEK1 (SEKAL) did not attenuate Zn2+-induced cell death, whereas the inhibition of ERK with PD98059 and the expression of dominant negative Ras mutant (RasN17) significantly prevented cell death. Inhibition of protein kinase C (PKC) and phosphatidylinositol-3 kinase had little effect on Zn2+-induced ERK activation. Intracellular Zn2+ accumulation resulted in the generation of ROS, and antioxidants prevented both the ERK activation and the cell death induced by Zn2+. Therefore, we conclude that although Zn2+ activates JNK and ERK, only ERK contributes to Zn2+-induced cell death, and that ERK activation is mediated by ROS via the Ras/Raf/MEK/ERK signaling pathway.     

Expression of c-Myc in response to colony-stimulating factor-1 requires mitogen-activated protein kinase kinase-1

The mitogen-inducible gene c-myc is a key regulator of cell proliferation and transformation. Yet, the signaling pathway(s) that regulate its expression have remained largely unresolved. Using the mitogen-activated protein kinase kinase (MEK1/2) inhibitor PD98059 and dominant negative forms of Ras (N17) and ERK1 (K71R), we found that activation of Ras and extracellular signal-regulated kinase (ERK) is necessary for colony-stimulating factor-1 (CSF-1)-mediated c-Myc expression and DNA synthetic (S) phase entry. Quiescent NIH-3T3 cells expressing a partially defective CSF-1 receptor, CSF-1R (Y809F), exhibited impaired ERK1 activation and c-Myc expression and failed to enter the S phase of the cell division cycle in response to CSF-1 stimulation. Ectopic expression of a constitutively active form of MEK1 in cells expressing CSF-1R (Y809F) rescued c-Myc expression and S phase entry, but only in the presence of CSF-1-induced cooperating signals. Therefore, MEK1 participates in an obligate signaling pathway linking CSF-1R to c-Myc expression, but other signals from CSF-1R must cooperate with the MEK/ERK pathway to induce c-Myc expression and S phase entry in response to CSF-1 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.