BCR/ABL activates Rap1 and B-Raf to stimulate the MEK/Erk signaling pathway in hematopoietic cells

The BCR/ABL fusion tyrosine kinase activates various intracellular signaling pathways, thus causing chronic myeloid leukemia (CML). Here we demonstrate that the inducible expression of BCR/ABL in a murine hematopoietic cell line, TonB210, leads to the activation of the Ras family small GTPase Rap1, which is inhibited by the ABL kinase inhibitor imatinib. The Rap1 activity in a CML cell line, K562, was also inhibited by imatinib. Inhibition of Rap1 activation by a dominant negative mutant of Rap1, Rap1-N17, or SPA-1 inhibited the BCR/ABL-induced activation of Elk-1. BCR/ABL also activated in a kinase activity-dependent manner the B-Raf kinase, which is an effector molecule of Rap1 and a potent activator of the MEK/Erk/Elk-1 signaling pathway. Together, these data suggest that, in addition to the well-established Ras/Raf-1 pathway, BCR/ABL activates the alternative signaling pathway involving Rap1 and B-Raf to activate Erk, which may play important roles in leukemogenesis.     

Erythropoietin-stimulated Raf-1 tyrosine phosphorylation is associated with the tyrosine kinase Lyn in J2E erythroleukemic cells.

The serine/threonine kinase Raf-1 is crucial for transducing intracellular signals emanating from numerous growth factors. Here we used the J2E erythroid cell line transformed by the nu-raf/nu-myc oncogenes to examine the effects of erythropoietin on endogenous Raf-1 activity. Despite the presence of constitutively active v-raf in these cells, Raf-1 exokinase activity increased after erythropoietin stimulation. This increase in enzymatic activity coincided with tyrosine phosphorylation of Raf-1 on residue Y341. Significantly, the tyrosine kinase Lyn coimmunoprecipitated with Raf-1, and Raf-1 was not tyrosine-phosphorylated in a J2E subclone lacking Lyn. Therefore, it was concluded that Lyn may be the kinase responsible for tyrosine phosphorylating Raf-1 and increasing its exokinase activity in response to erythropoietin.     

Calcium/calmodulin-dependent protein kinase II binds to Raf-1 and modulates integrin-stimulated ERK activation

Integrin activation generates different signalings in a cell type-dependent manner and stimulates cell proliferation through the Ras/Raf-1/Mek/Erk pathway. In this study, we demonstrate that integrin stimulation by fibronectin (FN), besides activating the Ras/Erk pathway, generates an auxiliary calcium signal that activates calmodulin and the Ca2+/calmodulin-dependent protein kinase II (CaMKII). This signal regulates Raf-1 activation by Ras and modulates the FN-stimulated extracellular signal-regulated kinase (Erk-1/2). The binding of soluble FN to integrins induced increase of intracellular calcium concentration associated with phosphorylation and activation of CaMKII. In two different cell lines, inhibition of CaMKII activity by specific inhibitors inhibited Erk-1/2 phosphorylation. Whereas CaMK inhibition affected neither integrin-stimulated Akt phosphorylation nor p21Ras or Mek-1 activity, it was necessary for Raf-1 activity. FN-induced Raf-1 activity was abrogated by the CaMKII specific inhibitory peptide ant-CaNtide. Integrin activation by FN induced the formation of a Raf-1/CaMKII complex, abrogated by inhibition of CaMKII. Active CaMKII phosphorylated Raf-1 in vitro. This is the first demonstration that CaMKII interplays with Raf-1 and regulates Erk activation induced by Ras-stimulated Raf-1. These findings also provide evidence supporting the possible existence of cross-talk between other intracellular pathways involving CaMKII and Raf-1.     

The dominant negative Ras mutant, N17Ras, can inhibit signaling independently of blocking Ras activation

Ras plays an important role in a variety of cellular functions, including growth, differentiation, and oncogenic transformation. For instance, Ras participates in the activation of Raf, which phosphorylates and activates mitogen-activated protein kinase kinase (MEK), which then phosphorylates and activates extracellular signal-regulated kinase (ERK), a mitogen-activated protein (MAP) kinase. Activation of MAP kinase appears to be essential for propagating a wide variety of extracellular signals from the plasma membrane to the nucleus. N17Ras, a GDP-bound dominant negative mutant, is used widely as an interfering mutant to assess Ras function in vivo. Surprisingly, we observed that expression of N17Ras inhibited the activity and phosphorylation of Elk-1, a physiological substrate of MAP kinases, in response to phorbol myristate acetate. The activity and phosphorylation of the MAP kinase hemagglutinin epitope (HA)-ERK1 were not affected by N17Ras in response to the same stimulus. Additionally, expression of N17Ras, but not L61S186Ras, a GTP-bound interfering mutant, inhibited MEK-induced Elk-1 phosphorylation, suggesting that inhibition of Elk-1 may be unique to GDP-bound Ras mutants. Finally, we observed that V12Ras-induced focus formation in NIH3T3 cells is inhibited by coexpression of GDP-bound Ras mutants, such as N17, A15, and N17N69. Therefore, N17Ras and V12 Ras may be codominant with respect to Elk-1 activation and cellular transformation. These results indicate that N17Ras appears to have at least two distinguishable functions: interference with endogenous Ras activation and inhibition of Elk-1 and transfomation. Furthermore, our data imply the possibility that GDP-bound Ras, like N17Ras, may have a direct role in signal transduction.     

ERK phosphorylation potentiates Elk-1-mediated ternary complex formation and transactivation.

Induction of the human c-fos proto-oncogene by mitogens depends on the formation of a ternary complex by p62TCF with the serum response factor (SRF) and the serum response element (SRE). We demonstrate that Elk-1, a protein closely related to p62TCF in function, is a nuclear target of two members of the MAP kinase family, ERK1 and ERK2. Phosphorylation of Elk-1 increases the yield of ternary complex in vitro. At least five residues in the C-terminal domain of Elk-1 are phosphorylated upon growth factor stimulation of NIH3T3 cells. These residues are also phosphorylated by purified ERK1 in vitro, as determined by a combination of phosphopeptide sequencing and 2-D peptide mapping. Conversion of two of these phospho-acceptor sites to alanine impairs the formation of ternary complexes by the resulting Elk-1 proteins. Removal of these serine residues also drastically diminishes activation of the c-fos promoter in epidermal growth factor-treated cells. Analogous mutations at other sites impair activation to a lesser extent without affecting ternary complex formation in vitro. Our results indicate that phosphorylation regulates ternary complex formation by Elk-1, which is a prerequisite for the manifestation of its transactivation potential at the c-fos SRE.     

Protein kinase C regulates integrin-induced activation of the extracellular regulated kinase pathway upstream of Shc

Adhesion of fibroblasts to extracellular matrices via integrin receptors is accompanied by extensive cytoskeletal rearrangements and intracellular signaling events. The protein kinase C (PKC) family of serine/threonine kinases has been implicated in several integrin-mediated events including focal adhesion formation, cell spreading, cell migration, and cytoskeletal rearrangements. However, the mechanism by which PKC regulates integrin function is not known. To characterize the role of PKC family kinases in mediating integrin-induced signaling, we monitored the effects of PKC inhibition on fibronectin-induced signaling events in Cos7 cells using pharmacological and genetic approaches. We found that inhibition of classical and novel isoforms of PKC by down-regulation with 12-0-tetradeconoyl-phorbol-13-acetate or overexpression of dominant-negative mutants of PKC significantly reduced extracellular regulated kinase 2 (Erk2) activation by fibronectin receptors in Cos7 cells. Furthermore, overexpression of constitutively active PKCalpha, PKCdelta, or PKCepsilon was sufficient to rescue 12-0-tetradeconoyl-phorbol-13-acetate-mediated down-regulation of Erk2 activation, and all three of these PKC isoforms were activated following adhesion. PKC was required for maximal activation of mitogen-activated kinase kinase 1, Raf-1, and Ras, tyrosine phosphorylation of Shc, and Shc association with Grb2. PKC inhibition does not appear to have a generalized effect on integrin signaling, because it does not block integrin-induced focal adhesion kinase or paxillin tyrosine phosphorylation. These results indicate that PKC activity enhances Erk2 activation in response to fibronectin by stimulating the Erk/mitogen-activated protein kinase pathway at an early step upstream of Shc.     

Inhibition of the Raf-1 kinase by cyclic AMP agonists causes apoptosis of v-abl-transformed cells.

Here we investigate the role of the Raf-1 kinase in transformation by the v-abl oncogene. Raf-1 can activate a transforming signalling cascade comprising the consecutive activation of Mek and extracellular-signal-regulated kinases (Erks). In v-abl-transformed cells the endogenous Raf-1 protein was phosphorylated on tyrosine and displayed high constitutive kinase activity. The activities of the Erks were constitutively elevated in both v-raf- and v-abl-transformed cells. In both cell types the activities of Raf-1 and v-raf were almost completely suppressed after activation of the cyclic AMP-dependent kinase (protein kinase A [PKA]), whereas the v-abl kinase was not affected. Raf inhibition substantially diminished the activities of Erks in v-raf-transformed cells but not in v-abl-transformed cells, indicating that v-abl can activate Erks by a Raf-1-independent pathway. PKA activation induced apoptosis in v-abl-transformed cells while reverting v-raf transformation without severe cytopathic effects. Overexpression of Raf-1 in v-abl-transformed cells partially protected the cells from apoptosis induced by PKA activation. In contrast to PKA activators, a Mek inhibitor did not induce apoptosis. The diverse biological responses correlated with the status of c-myc gene expression. v-abl-transformed cells featured high constitutive levels of expression of c-myc, which were not reduced following PKA activation. Myc activation has been previously shown to be essential for transformation by oncogenic Abl proteins. Using estrogen-regulated c-myc and temperature-sensitive Raf-1 mutants, we found that Raf-1 activation could protect cells from c-myc-induced apoptosis. In conclusion, these results suggest (i) that Raf-1 participates in v-abl transformation via an Erk-independent pathway by providing a survival signal which complements c-myc in transformation, and (ii) that cAMP agonists might become useful for the treatment of malignancies where abl oncogenes are involved, such as chronic myeloid leukemias.     

Transient activation of RAF-1, MEK, and ERK2 coincides kinetically with ternary complex factor phosphorylation and immediate-early gene promoter activity in vivo.

We have investigated the early in vivo signaling events triggered by serum that lead to activation of the c-fos proto-oncogene in HeLa cells. Both RAF-1 and MEK kinase activities are fully induced within 3 min of serum treatment and quickly decrease thereafter, slightly preceding the activation and inactivation of p42MAPK/ERK2. ERK2 activity correlates tightly with a transient phosphatase-sensitive modification of ternary complex factor (TCF), manifested by the slower electrophoretic mobility of TCF-containing protein-DNA complexes. These induced complexes in turn correlate with the activity of the c-fos, egr-1, and junB promoters. Phorbol ester treatment induces the same events but with slower and prolonged kinetics. Inhibition of serine/threonine phosphatase activities by okadaic acid treatment reverses the repression of the c-fos promoter either after induction or without induction. This corresponds to the presence of the induced complexes and of ERK2 activity, as well as to the activation of a number of other kinases. Inhibition of tyrosine phosphatase activities by sodium vanadate treatment delays but does not block ERK2 inactivation, TCF dephosphorylation, and c-fos repression. The tight linkage in vivo between the activity of MAP kinase, TCF phosphorylation, and immediate-early gene promoter activity is consistent with the notion that a stable ternary complex over the serum response element is a direct target for the MAP kinase signaling cascade. Furthermore, serine/threonine phosphatases are implicated in regulating the kinase cascade, as well as the state of TCF modification and c-fos promoter activity, in vivo.     

Cyclic AMP and mitogen-activated protein kinases are required for glutamate-dependent cyclic AMP response element binding protein and Elk-1 phosphorylation in the dorsal striatum in vivo

Dopaminergic and glutamatergic signalling cascades are integrated in striatal medium spiny neurones by cyclic AMP response-element binding protein and Elk-1 phosphorylation. Phosphorylated cyclic AMP response-element binding protein and phosphorylated Elk-1 contribute to c-fos expression by binding to the calcium and cyclic AMP response-element and the serum response element, respectively, in the c-fos promoter. The role of cyclic AMP and mitogen-activated protein kinase signalling cascades in glutamate-induced cyclic AMP response-element binding protein and Elk-1 phosphorylation and Fos expression was investigated using semiquantitative immunocytochemistry in vivo. Intracerebroventricular infusion of the sodium channel blocker, tetrodotoxin, decreased the glutamate-induced increase in phosphorylated cyclic AMP response-element binding protein, phosphorylated Elk-1, and Fos immunoreactivity. Intracerebroventricular infusion of the mitogen-activated and extracellular signal-regulated kinase inhibitor, PD98059, the p38 mitogen-activated protein kinase inhibitor, SB203580, or the cyclic AMP inhibitor, Rp-8-Br-cAMPS, decreased glutamate-induced phosphorylated cyclic AMP response-element binding protein, phosphorylated Elk-1, and Fos immunoreactivity. Simultaneous infusion of glutamate and Sp-8-Br-cAMPS, a cyclic AMP analogue, augmented induction of Fos immunoreactivity but not phosphorylated cyclic AMP response-element binding protein or phosphorylated Elk-1 immunoreactivity. These data indicate that cyclic AMP and mitogen-activated protein kinase signalling cascades are necessary for glutamate to induce cyclic AMP response-element binding protein and Elk-1 phosphorylation and Fos expression in the striatum. Furthermore, neuronal activity plays an important role in glutamate-induced signalling cascades in vivo.     

Mechanisms of inhibition of the Ras-MAP kinase signaling pathway in 30.7b Ras 12 cells by tea polyphenols (-)-epigallocatechin-3-gallate and theaflavin-3,3'-digallate.

Our previous study showed that tea polyphenols inhibited MAP kinase and AP-1 activities in mouse epidermal JB6 cells and the corresponding H-ras-transformed cell line 30.7b Ras 12. The present study investigated the mechanisms of this inhibition. The cells were incubated with (-)-epigallocatechin-3-gallate (EGCG) or theaflavin-3,3'-digallate (TFdiG) (20 mM) for different times, and the cell lysate was analyzed by immunoblotting. EGCG treatment decreased the levels of phospho-Erk1/2 and -MEK1/2 time-dependently (by 60% at 60 min). TFdiG lowered their levels by 38%-50% at 15 min. TFdiG effectively decreased total Raf-1 protein levels, most likely through lysosomal degradation. EGCG did not affect protein levels or the activity of Raf-1 significantly but decreased its association with MEK1 as determined by co-immunoprecipitation. In addition, EGCG and TFdiG (10 mM) inhibited the phosphorylation of Elk-1 by isolated phospho-Erk1/2 in vitro. This inhibition of Erk1/2 activity is Elk-1 concentration-dependent and ATP concentration-independent, which suggests that EGCG and TFdiG interfere with the binding of the protein substrate to the kinase. The presently demonstrated specific mechanisms of inhibition of MAP kinases by EGCG and TFdiG may help us to understand the effects of tea consumption on cancer, inflammatory diseases, and cardiovascular diseases.