Activation of mitogen-activated protein kinases (Erk1 and Erk2) cascade results in phosphorylation of NF-M tail domains in transfected NIH 3T3 cells.

Neurofilaments (NFs) are neuron-specific intermediate filaments, and are the major cytoskeletal component in large myelinated axons. Lysine-serine-proline (KSP) repeats in the tail domains of high molecular weight NF proteins (NF-M and NF-H) are extensively phosphorylated in vivo in the axon. This phosphorylation in the tail domain has been postulated to play an important role in mediating neuron-specific properties, including axonal caliber and conduction velocity. Recent studies have shown that the mitogen-activated protein kinases (extracellular signal-regulated kinases, Erk1 and Erk2) phosphorylate KSP motifs in peptide substrates derived from the NF-M and NF-H tail domains in vitro. However, it is not clear whether activation of the mitogen activated protein (MAP) kinase pathway is able to phosphorylate these domains in vivo. To answer this question, a constitutively active form of mitogen-activated Erk activating kinase (MEK1) was cotransfected with an NF-M expression construct into NIH 3T3 cells. The activated mutant, but not the dominant negative mutant, induced phosphorylation of NF-M. In addition, it was shown that epidermal growth factor, which induces the MAP kinase cascade in NIH 3T3 cells, also activated endogenous Erk1 and Erk2 and NF-M tail domain phosphorylation in the transfected cells. These results present direct evidence that in-vivo activation of Erk1 and Erk 2 is sufficient for NF-M tail domain phosphorylation in transfected cells.     

Phosphorylation of the head domain of neurofilament protein (NF-M): a factor regulating topographic phosphorylation of NF-M tail domain KSP sites in neurons

In neurons the phosphorylation of neurofilament (NF) proteins NF-M and NF-H is topographically regulated. Although kinases and NF subunits are synthesized in cell bodies, extensive phosphorylation of the KSP repeats in tail domains of NF-M and NF-H occurs primarily in axons. The nature of this regulation, however, is not understood. As obligate heteropolymers, NF assembly requires interactions between the core NF-L with NF-M or NF-H subunits, a process inhibited by NF head domain phosphorylation. Phosphorylation of head domains at protein kinase A (PKA)-specific sites seems to occur transiently in cell bodies after NF subunit synthesis. We have proposed that transient phosphorylation of head domains prevents NF assembly in the soma and inhibits tail domain phosphorylation; i.e. assembly and KSP phosphorylation in axons depends on prior dephosphorylation of head domain sites. Deregulation of this process leads to pathological accumulations of phosphorylated NFs in the soma as seen in some neurodegenerative disorders. To test this hypothesis, we studied the effect of PKA phosphorylation of the NF-M head domain on phosphorylation of tail domain KSP sites. In rat cortical neurons we showed that head domain phosphorylation of endogenous NF-M by forskolin-activated PKA inhibits NF-M tail domain phosphorylation. To demonstrate the site specificity of PKA phosphorylation and its effect on tail domain phosphorylation, we transfected NIH3T3 cells with NF-M mutated at PKA-specific head domain serine residues. Epidermal growth factor stimulation of cells with mutant NF-M in the presence of forskolin exhibited no inhibition of NF-tail domain phosphorylation compared with the wild type NF-M-transfected cells. This is consistent with our hypothesis that transient phosphorylation of NF-M head domains inhibits tail domain phosphorylation and suggests this as one of several mechanisms underlying topographic regulation.     

Acute activation of Erk1/Erk2 and protein kinase B/akt proceed by independent pathways in multiple cell types

We used two inhibitors of the signaling enzyme phosphatidylinositol 3-kinase (PtdIns3K), wortmannin and LY294002, to evaluate the potential involvement of PtdIns3K in the activation of the MAP kinases (MAPK), Erk1 and Erk2. In dose-response studies carried out on six different cell lines and a primary cell culture, we analyzed the ability of the inhibitors to block phosphorylation of protein kinase B/akt (PKB/akt) at Ser473 as a measure of PtdIns3K activity, or the phosphorylation of Erk1/2 at activating Thr/Tyr sites as a measure of the extent of activation of MAPK/Erk kinase (MEK/Erk). In three different hemopoietic cell lines stimulated with cytokines, and in HEK293 cells, stimulated with serum, either wortmannin or LY294002, but never both, could partially block phosphorylation of Erks. The same observations were made in a B-cell line and in primary fibroblasts. In only one cell type, the A20 B cells, was there a closer correlation between the PtdIns3K inhibition by both inhibitors, and their corresponding effects on Erk phosphorylation. However, this stands out as an exception that gives clues to the mechanism by which cross-talk might occur. In all other cells, acute activation of the pathway leading to Erk phosphorylation could proceed independently of PtdIns3K activation. In a biological assay comparing these two pathways, the ability of LY294002 and the MEK inhibitor, U0126, to induce apoptosis were tested. Whereas LY294002 caused death of cytokine-dependent hemopoietic cells, U0126 had little effect, but both inhibitors together had a synergistic effect. The data show that these two pathways are regulating very different downstream events involved in cell survival.     

MKP3 negatively modulates PDGF-induced Akt and Erk5 phosphorylation as well as chemotaxis

MAP kinase phosphatase-3 (MKP3), also known as DUSP6 or Pyst1, is a dual specificity phosphatase considered to selectively dephosphorylate extracellular-signal-regulated kinase 1/2 (Erk1/2). Here, we report that in NIH3T3 cells, MKP3 is induced in response to platelet-derived growth factor (PDGF)-BB treatment in an Erk1/2- and phosphatidylinositol 3-kinase (PI3K)-dependent manner, but independently of Erk5 expression. Silencing of MKP3 expression did not affect PDGF-BB-induced Erk1/2 or p38 phosphorylation; however, their basal level of phosphorylation was elevated. Furthermore, we found that PDGF-BB-mediated activation of Erk5 and Akt was enhanced when the MKP3 expression was reduced. Interfering with Mek1/2 or PI3K using the inhibitors CI-1040 and LY-294002, respectively, inhibited PDGF-BB-induced MKP3 expression. Functionally, we found that MKP3 silencing did not affect cell proliferation, but enhanced the chemotactic response toward PDGF-BB. Although both Akt and Erk5 have been linked to increased cell survival, downregulation of MKP3 did not alter the ability of PDGF-BB to protect NIH3T3 cells from starvation-induced apoptosis. However, we observed an increased apoptosis in untreated cells with reduced MKP3 expression. In summary, our data indicate that there is negative cross-talk between Erk1/2 and Erk5 that involves regulation of MKP3 expression, and that PI3K in addition to promoting Akt phosphorylation also negatively modulates Akt, through MKP3 expression.     

Phosphatidylinositol 3-kinase contributes to Erk1/Erk2 MAP kinase activation associated with hepatocyte growth factor-induced cell scattering

MAP kinase cascade-dependent responses were investigated during scattering of HepG2 human hepatoma cells stimulated by HGF or phorbol ester. Inhibition of phosphatidylinositol 3-kinase with LY294002 prevented completely the dissociation of cells. Inhibition of MAP kinase kinase (MEK) with PD98059 prevented the development of characteristic morphological changes associated with cell migration. EGF, which failed to induce cell scattering, caused a short-term increase in the phosphorylation of Erk1/Erk2 MAP kinases. On the contrary, HGF or phorbol ester stimulated the phosphorylation of MAP kinases for a long time. Experiments performed with LY294002 indicated that phosphatidylinositol 3-kinase contributed to the HGF-stimulated phosphorylation of Erk1/Erk2. This finding was confirmed by the demonstration that the MAP kinase cascade-dependent expression of a high-Mr (>300 kDa) protein pair appearing in the course of cell scattering was inhibited by LY294002 in HGF-induced cells but was not inhibited in phorbol ester-treated cells.     

Intestinal epithelial cancer cell anoikis resistance: EGFR‐mediated sustained activation of Src overrides Fak‐dependent signaling to MEK/Erk and/or PI3‐K/Akt‐1

Herein, we investigated the survival roles of Fak, Src, MEK/Erk, and PI3‐K/Akt‐1 in intestinal epithelial cancer cells (HCT116, HT29, and T84), in comparison to undifferentiated and differentiated intestinal epithelial cells (IECs). We report that: (1) cancer cells display striking anoikis resistance, as opposed to undifferentiated/differentiated IECs; (2) under anoikis conditions and consequent Fak down‐activation, cancer cells nevertheless exhibit sustained Fak–Src interactions and Src/MEK/Erk activation, unlike undifferentiated/differentiated IECs; however, HCT116 and HT29 cells exhibit a PI3‐K/Akt‐1 down‐activation, as undifferentiated/differentiated IECs, whereas T84 cells do not; (3) cancer cells require MEK/Erk for survival, as differentiated (but not undifferentiated) IECs; however, T84 cells do not require Fak and HCT116 cells do not require PI3‐K/Akt‐1, in contrast to the other cells studied; (4) Src acts as a cornerstone in Fak‐mediated signaling to MEK/Erk and PI3‐K/Akt‐1 in T84 cells, as in undifferentiated IECs, whereas PI3‐K/Akt‐1 is Src‐independent in HCT116, HT29 cells, as in differentiated IECs; and (5) EGFR activity inhibition abrogates anoikis resistance in cancer cells through a loss of Fak–Src interactions and down‐activation of Src/MEK/Erk (T84, HCT116, HT29 cells) and PI3‐K/Akt‐1 (T84 cells). Hence, despite distinctions in signaling behavior not necessarily related to undifferentiated or differentiated IECs, intestinal epithelial cancer cells commonly display an EGFR‐mediated sustained activation of Src under anoikis conditions. Furthermore, such sustained Src activation confers anoikis resistance at least in part through a consequent sustenance of Fak–Src interactions and MEK/Erk activation, thus not only overriding Fak‐mediated signaling to MEK/Erk and/or PI3‐K/Akt‐1, but also the requirement of Fak and/or PI3‐K/Akt‐1 for survival. J. Cell. Biochem. 107: 639–654, 2009. © 2009 Wiley‐Liss, Inc.     

Altered gene expression profiles of NIH3T3 cells regulated by human lung cancer associated gene CT120

CT120, a novel membrane-associated gene implicated in lung carcinogenesis, was previously identified from chromosome 17pl 3.3 locus, a hot mutation spot involved in human malignancies. In the present study, we further determined that CT120 ectopic expression could promote cell proliferation activity of NIH3T3 cells using MTS assay, and monitored the downstream effects of CT120 in NIH3T3 cells with Atlas mouse cDNA expression arrays. Among 588 known genes, 133 genes were found to be upregulated or downregulated by CT120. Two major signaling pathways involved in cell proliferation, cell survival and anti-apoptosis were overexpressed and activated in response to CT120:One is the Raf/MEK/Erk signal cascades and the other is the PI3K/Akt signal cascades, suggesting that CT120 might contribute, at least in part, to the constitutively activation of Erk and Akt in human lung caner cells. In addition, some tumor metastasis associated genes cathepsin B, cathepsin D, cathepsin L, MMP-2/TIMP-2 were also upregulated by CT120, upon which CT120 might be involved in tumor invasiveness and metastasis. In addition, CT120 might play an important role in tumor progression through modulating the expression of some candidate “Lung Tumor Progression”genes including B-Raf, Rab-2, BAX, BAG-1, YB-1, and Cdc42.     

Requirement of Erk, but not JNK, for arsenite-induced cell transformation.

Trivalent arsenic (arsenite, As3+) is a human carcinogen, which is associated with cancers of skin, lung, liver, and bladder. However, the mechanism by which arsenite causes cancer is not well understood. In this study, we found that exposure of Cl 41 cells, a well characterized mouse epidermal cell model for tumor promotion, to a low concentration of arsenite (<25 microM) induces cell transformation. Interestingly, arsenite induces Erk phosphorylation and increased Erk activity at doses ranging from 0.8 to 200 microM, while higher doses (more than 50 microM) are required for activation of JNK. Arsenite-induced Erk activation was markedly inhibited by introduction of dominant negative Erk2 into cells, while expression of dominant negative Erk2 did not show inhibition of JNK and MEK1/2. Furthermore, arsenite-induced cell transformation was blocked in cells expressing the dominant negative Erk2. In contrast, overexpression of dominant negative JNK1 was shown to increase cell transformation even though it inhibits arsenite-induced JNK activation. Our results not only show that arsenite induces Erk activation, but also for the first time demonstrates that activation of Erk, but not JNK, by arsenite is required for its effects on cell transformation.     

Polar opposites: Erk direction of CD4 T cell subsets

Effective immune responses depend upon appropriate T cell differentiation in accord with the nature of an infectious agent, and the contingency of differentiation depends minimally on TCR, coreceptor, and cytokine signals. In this reverse genetic study, we show that the MAPK Erk2 is not essential for T cell proliferation in the presence of optimum costimulation. Instead, it has opposite effects on T-bet and Gata3 expression and, hence, on Th1 and Th2 differentiation. Alternatively, in the presence of TGF-β, the Erk pathway suppresses a large program of gene expression, effectively limiting the differentiation of Foxp3(+) regulatory T cells. In the latter case, the mechanisms involved include suppression of Gata3 and Foxp3, induction of Tbx21, phosphorylation of Smad2,3, and possibly suppression of Socs2, a positive inducer of Stat5 signaling. Consequently, loss of Erk2 severely impeded Th1 differentiation while enhancing the development of Foxp3(+)-induced T regulatory cells. Selected profiles of gene expression under multiple conditions of T cell activation illustrate the opposing consequences of Erk pathway signaling.     

Involvement of ligand occupancy in Insulin-like growth factor-I (IGF-I) induced cell growth in osteoblast like MC3T3-E1 cells

Growth factors and matrix proteins regulate the proliferation and differentiation of osteoblasts. The insulin-like growth factor (IGF) system comprises IGF-I, IGF-II, and six high-affinity IGF-binding proteins (IGFBPs). IGFs stimulate cell growth in many types of tissue; IGF-binding proteins regulate cellular actions and can affect cell growth. IGF-I is involved in differentiation, proliferation, and matrix formation in osteoblasts; IGFBP-5 is associated with the extracellular matrix (ECM) and can potentiate the actions of IGF-I. We investigated the effect of ECM proteins on the responses of MC3T3-E1 osteoblast cells to IGF-I and IGFBP-5. In addition, because extracellular signal-regulated kinases 1 and 2 (Erk 1/2) affect cell growth, we evaluated the effects of IGFBP-5 on Erk 1/2 phosphorylation in MC3T3-E1 cells. IGF-I caused an increase in IGFBP-5 expression in cultured MC3T3-E1 cells, and IGF-I plus IGFBP-5 significantly increased cell growth. Likewise, the addition of IGF-I and IGFBP-5 to cultured MC3T3-E1 cells increased the synthesis of the ECM proteins osteopontin (OPN) and thrombospondin-1 (TSP-1), which can bind to alphaVbeta3 integrin receptors on the cell surface. By contrast, the addition of an antibody against ECM proteins inhibited the effects of OPN and TSP-1 on IGFBP-5 expression. The stimulatory effect of IGFBP-5 was mediated via Erk 1/2 activation. These data suggest that IGFBP-5 regulates Erk 1/2 phosphorylation in cultured MC3T3-E1 cells via ECM proteins that may ultimately stimulate the growth of osteoblasts. We determined whether occupation of the alphaVbeta3 integrin receptor affects IGF-I receptor (IGF-IR)-mediated signaling and function in MC3T3-E1 osteoblast cells. Occupation of the alphaVbeta3 integrin receptor with ECM proteins induced IGF-I-stimulated IGF-IR phosphorylation. Conversely, in the presence of the alphaVbeta3-specific disintegrin echistatin, IGF-I-stimulated IGF-IR activation was inhibited. IGF-I-stimulated IGF-IR phosphorylation was accompanied by IRS-1 phosphorylation and MAPK activation. However, these effects were attenuated by echistatin. Thus, occupancy of the alphaVbeta3 disintegrin receptor modulates IGF-I-induced IGF-IR activation and IGF-IR-mediated function in MC 3T3-E1 osteoblasts.     

Zap-70-independent Ca(2+) mobilization and Erk activation in Jurkat T cells in response to T-cell antigen receptor ligation

The tyrosine kinase ZAP-70 has been implicated as a critical intermediary between T-cell antigen receptor (TCR) stimulation and Erk activation on the basis of the ability of dominant negative ZAP-70 to inhibit TCR-stimulated Erk activation, and the reported inability of anti-CD3 antibodies to activate Erk in ZAP-70-negative Jurkat cells. However, Erk is activated in T cells receiving a partial agonist signal, despite failing to activate ZAP-70. This discrepancy led us to reanalyze the ZAP-70-negative Jurkat T-cell line P116 for its ability to support Erk activation in response to TCR/CD3 stimulation. Erk was activated by CD3 cross-linking in P116 cells. However, this response required a higher concentration of anti-CD3 antibody and was delayed and transient compared to that in Jurkat T cells. Activation of Raf-1 and MEK-1 was coincident with Erk activation. Remarkably, the time course of Ras activation was comparable in the two cell lines, despite proceeding in the absence of LAT tyrosine phosphorylation in the P116 cells. CD3 stimulation of P116 cells also induced tyrosine phosphorylation of phospholipase C-gamma1 (PLCgamma1) and increased the intracellular Ca(2+) concentration. Protein kinase C (PKC) inhibitors blocked CD3-stimulated Erk activation in P116 cells, while parental Jurkat cells were refractory to PKC inhibition. The physiologic relevance of these signaling events is further supported by the finding of PLCgamma1 tyrosine phosphorylation, Erk activation, and CD69 upregulation in P116 cells on stimulation with superantigen and antigen-presenting cells. These results demonstrate the existence of two pathways leading to TCR-stimulated Erk activation in Jurkat T cells: a ZAP-70-independent pathway requiring PKC and a ZAP-70-dependent pathway that is PKC independent.     

CrkL plays a role in SDF-1-induced activation of the Raf-1/MEK/Erk pathway through Ras and Rac to mediate chemotactic signaling in hematopoietic cells

Intracellular signaling mechanisms regulating SDF-1-induced chemotaxis of hematopoietic cells have remained elusive. Here we demonstrate that overexpression of the adaptor molecule CrkL enhances SDF-1-induced chemotaxis of hematopoietic BaF3 and 32Dcl3 cells. Overexpression of CrkL also enhanced SDF-1-induced activation of the Raf-1/MEK/Erk signaling pathway as well as that of the small GTPases Ras, Rap1, and Rac, while a dominant negative mutant of Ras or Rac suppressed CrkL-enhanced Erk activation. SDF-1 stimulation induced tyrosine phosphorylation of CrkL, which was inhibited by the Src family kinase inhibitor PP1 or by dominant negative mutants of Lyn, thus indicating that Lyn mediated SDF-1-induced phosphorylation of CrkL. However, inhibition of the Lyn kinase activity failed to affect SDF-1-induced activation of the small GTPases and Erk. On the other hand, SDF-1-induced activation of the Erk signaling pathway as well as chemotaxis was inhibited by overexpression of a CrkL mutant lacking the N-terminal SH3 domain, which mediates interaction with various signaling molecules including guanine nucleotide exchange factors for the Ras and Rho family GTPases. SDF-1-induced chemotaxis was also inhibited by the dominant negative Ras or Rac mutant as well as by the MEK inhibitor PD98059. These results indicate that CrkL mediates SDF-1-induced activation of the Raf-1/MEK/Erk signaling pathway through Ras as well as Rac in hematopoietic cells and, thereby, plays important roles in the induction of chemotactic response.     

Protein kinase C decreases the hepatocyte growth factor-induced activation of Erk1/Erk2 MAP kinases

HGF and phorbol ester induce the scattering of HepG2 cells. Recently, we have reported that the motility and morphological responses that accompany this process require the activation of Erk1/Erk2 MAP kinases, and phosphatidylinositol 3-kinase contributes to the activation of Erk1/Erk2 in HGF-induced cells. The cell scattering-associated appearance of a high-M(r) (>300 kDa) protein pair has also been observed, and has been proven to be a sensitive marker of the intensity of Erk1/Erk2 activation. Our present study demonstrates that in HGF-induced cells protein kinase C and phosphatidylinositol 3-kinase regulate oppositely the expression of these cell scattering-associated proteins. While in phorbol ester-treated cells the sustained activation of protein kinase C is essential for this expression, in HGF-induced cells the inhibition of protein kinase C with bisindolylmaleimide I stimulates the expression. Protein kinase C reduces the HGF-induced phosphorylation of Erk1/Erk2, and in this way it can limit the intensity of Erk1/Erk2-dependent gene-expression     

SDF-1 synergistically enhances IL-3-induced activation of the Raf-1/MEK/Erk signaling pathway through activation of Rac and its effector Pak kinases to promote hematopoiesis and chemotaxis

Stromal cell-derived factor 1 (SDF-1) cooperates with cytokines to promote hematopoiesis. Here we demonstrate that SDF-1 activates Erk synergistically with interleukin-3 (IL-3) in hematopoietic cells. Small GTPases Ras and Rac were prominently activated by IL-3 and SDF-1, respectively. In accordance with this, Raf-1 was significantly activated by IL-3 but not by SDF-1. SDF-1 strongly induced phosphorylation of Raf-1 on S338, the target site for the Rac effector Paks, and enhanced the IL-3-induced activation of Raf-1 and MEK. Furthermore, the synergistic activation of Erk was inhibited by expression of a dominant-negative mutant of Pak1 or that of Rac and was enhanced by an activated mutant of Pak1. SDF-1 and IL-3 also showed synergistic effects on expansion of hematopoietic cells and on induction of chemotaxis, which were both inhibited by the MEK inhibitor PD98059. These results suggest that SDF-1 synergistically enhances IL-3-induced Erk activation by up-regulating Raf-1 activity through the Rac effector Pak kinases to promote hematopoiesis.     

B-Raf Inhibits Programmed Cell Death Downstream of Cytochrome c Release from Mitochondria by Activating the MEK/Erk Pathway

Growth factor-dependent kinases, such as phosphatidylinositol 3-kinase (PI 3-kinase) and Raf kinases, have been implicated in the suppression of apoptosis. We have recently established Rat-1 fibroblast cell lines overexpressing B-Raf, leading to activation of the MEK/Erk mitogen-activated protein kinase pathway. Overexpression of B-Raf confers resistance to apoptosis induced by growth factor withdrawal or PI 3-kinase inhibition. This is accompanied by constitutive activation of Erk without effects on the PI 3-kinase/Akt pathway. The activity of MEK is essential for cell survival mediated by B-Raf overexpression, since either treatment with the specific MEK inhibitor PD98059 or expression of a dominant inhibitory MEK mutant blocks the antiapoptotic activity of B-Raf. Activation of MEK is not only necessary but also sufficient for cell survival because overexpression of constitutively activated MEK, Ras, or Raf-1, like B-Raf, prevents apoptosis after growth factor deprivation. Overexpression of B-Raf did not interfere with the release of cytochrome c from mitochondria after growth factor deprivation. However, the addition of cytochrome c to cytosols of cells overexpressing B-Raf failed to induce caspase activation. It thus appears that the B-Raf/MEK/Erk pathway confers protection against apoptosis at the level of cytosolic caspase activation, downstream of the release of cytochrome c from mitochondria.     

The Mos/MAP kinase pathway stabilizes c-Fos by phosphorylation and augments its transforming activity in NIH 3T3 cells.

The c-mos proto-oncogene product, Mos, is a serine/threonine kinase that can activate ERK1 and 2 mitogen-activated protein (MAP) kinases by direct phosphorylation of MAPK/ERK kinase (MEK). ERK activation is essential for oncogenic transformation of NIH 3T3 cells by Mos. In this study, we examined how mitogenic and oncogenic signalling from the Mos/MEK/ERK pathway reaches the nucleus to activate downstream target genes. We show that c-Fos (the c-fos protooncogene product), which is an intrinsically unstable nuclear protein, is metabolically highly stabilized, and greatly enhances the transforming efficiency of NIH 3T3 cells, by Mos. This stabilization of c-Fos required Mos-induced phosphorylation of its C-terminal region on Ser362 and Ser374, and double replacements of these serines with acidic (Asp) residues markedly increased the stability and transforming efficiency of c-Fos even in the absence of Mos. Moreover, activation of the ERK pathway was necessary and sufficient for the c-Fos phosphorylation and stabilization by Mos. These results indicate that c-Fos undergoes stabilization, and mediates at least partly the oncogenic signalling, by the Mos/MEK/ERK pathway. The present findings also suggest that, in general, the ERK pathway may regulate the cell fate and function by affecting the metabolic stability of c-Fos.