Differential role of beta(1C) and beta(1A) integrin cytoplasmic variants in modulating focal adhesion kinase, protein kinase B/AKT, and Ras/Mitogen-activated protein kinase pathways

The integrin cytoplasmic domain modulates cell proliferation, adhesion, migration, and intracellular signaling. The beta(1) integrin subunits, beta(1C) and beta(1A), that contain variant cytoplasmic domains differentially affect cell proliferation; beta(1C) inhibits proliferation, whereas beta(1A) promotes it. We investigated the ability of beta(1C) and beta(1A) to modulate integrin-mediated signaling events that affect cell proliferation and survival in Chinese hamster ovary stable cell lines expressing either human beta(1C) or human beta(1A). The different cytodomains of either beta(1C) or beta(1A) did not affect either association with the endogenous alpha(2), alpha(V), and alpha(5) subunits or cell adhesion to fibronectin or TS2/16, a mAb to human beta(1). Upon engagement of endogenous and exogenous integrins by fibronectin, cells expressing beta(1C) showed significantly inhibited extracellular signal-regulated kinase (ERK) 2 activation compared with beta(1A) stable cell lines. In contrast, focal adhesion kinase phosphorylation and Protein Kinase B/AKT activity were not affected. Selective engagement of the exogenously expressed beta(1C) by TS2/16 led to stimulation of Protein Kinase B/AKT phosphorylation but not of ERK2 activation; in contrast, beta(1A) engagement induced activation of both proteins. We show that Ras activation was strongly reduced in beta(1C) stable cell lines in response to fibronectin adhesion and that expression of constitutively active Ras, Ras 61 (L), rescued beta(1C)-mediated down-regulation of ERK2 activation. Inhibition of cell proliferation in beta(1C) stable cell lines was attributable to an inhibitory effect of beta(1C) on the Ras/MAP kinase pathway because expression of activated MAPK kinase rescued beta(1C) antiproliferative effect. These findings show that the beta(1C) variant, by means of a unique signaling mechanism, selectively inhibits the MAP kinase pathway by preventing Ras activation without affecting either survival signals stimulated by integrins or cellular interactions with the extracellular matrix. These findings highlight a role for beta(1)-specific cytodomain sequences in maintaining an intracellular balance of proliferation and survival signals.     

Contortrostatin activates ERK2 and tyrosine phosphorylation events via distinct pathways

We report that cells adhering to contortrostatin show transient increases in activation of Extracellular signal Regulated Kinase 2 (ERK2). The kinetics and degree of activation are similar to cells adhering to fibronectin or vitronectin. We have recently shown that contortrostatin induces tyrosine phosphorylation in tumor cells. Contortrostatin is shown here to stimulate activation of ERK2 in suspended cells, but this activation follows a different dose-response pattern than contortrostatin-induced tyrosine phosphorylation. Since contortrostatin induces tyrosine phosphorylation via alphavbeta3, we explored the effects of an alphavbeta3-blocking antibody, 7E3, on contortrostatin-stimulated ERK2 activation. While 7E3 completely blocks the effect of contortrostatin on tyrosine phosphorylation, this antibody had no effect on activation of ERK2. In cells lacking expression of alphavbeta3, tyrosine phosphorylation was unaffected by contortrostatin treatment, but ERK2 was activated. This is strong evidence that contortrostatin is regulating tyrosine phosphorylation events and ERK2 activation via separate pathways and through different integrin receptors.     

Co-application of NMDA and dopamine-induced rapid translation of RSK2 in the mature hippocampus

Ribosomal S6 kinase2 (RSK2) is known to take part in several signal transduction cascades including Mitogen Activated Protein Kinase/Extracellular Regulated Kinase (MAPK/ERK). Following our recent observation that ERK can serve as a coincidence detector for fast and slow neurotransmission in the hippocampus, we analyzed the status of RSK2 phosphorylation subsequent to application of NMDA, dopamine, or both to preparations of mature hippocampal slices in Sprague-Dawley rats. RSK2 was indeed phosphorylated; however, in addition, the amount of RSK2 protein (60%) was induced within 10 min following stimulation. Moreover, the induced expression of RSK2 could be detected in both the cell body layer and the dendrites of hippocampal CA1 cells. Pharmacological analysis showed that RSK2 induction was MAPK ERK Kinase (MEK)-ERK independent, but mammalian Target of Rapamycin (mTOR) and translation dependent. We suggest that the fast kinetics of RSK2 translation that follows physiological stimulations, together with recent observations that its over-expression is vital for the attenuation of major signal transduction cascades, indicate an expanded physiological function of RSK2 in neurons, and sheds new light on the role of RSK2 in the Coffin-Lowry syndrome.     

Platelet ERK2 activation by thrombin is dependent on calcium and conventional protein kinases C but not Raf-1 or B-Raf

Extracellular signal-regulated kinase (ERK) activation pathways have been well characterized in a number of cell types but very few data are available for platelets. The thrombin-induced signaling pathway leading to ERK2 activation in platelets is largely uncharacterized. In this study, we investigated the kinases involved in thrombin-induced ERK2 activation in conditions of maximal ERK2 activation. We found that thrombin-induced mitogen-activated protein kinase/ERK kinase (MEK)1/2 activation was necessary for ERK2 phosphorylation. We obtained strong evidence that conventional protein kinase Cs (PKCs) and calcium are involved in thrombin-induced ERK2 activation. First, ERK2 and MEK1/2 phosphorylation was totally inhibited by low concentrations (1 microM) of RO318425, a specific inhibitor of conventional PKCs. Second, Ca(2+), from either intracellular pools or the extracellular medium, was necessary for ERK2 activation and conventional PKC activation, excluding the involvement of a new class of calcium-insensitive PKCs. Third, LY294002 and wortmannin had no significant effect on ERK2 activation, even at concentrations that inhibit phosphatidylinositol (PI)3-kinase (5 microM to 25 microM and 50 nM, respectively). This suggests that PI3-kinase was not necessary for ERK2 activation and therefore, that PI3-kinase-dependent atypical PKCs were not involved. Surprisingly, in contrast to proliferative cells, we found that the serine/threonine kinases Raf-1 and B-Raf were not an intermediate kinase between conventional PKCs and MEK1/2. After immunoprecipitation of Raf-1 and B-Raf, the basal glutathione S-transferase-MEK1 phosphorylation observed in resting platelets was not upregulated by thrombin and was still observed in the absence of anti-Raf-1 or anti-B-Raf antibodies. In these conditions, the in vitro cascade kinase assay did not detect any MEK activity. Thus in platelets, thrombin-induced ERK2 activation is activated by conventional PKCs independently of Raf-1 and B-Raf activation.     

Dual role of cAMP and involvement of both G-proteins and ras in regulation of ERK2 in Dictyostelium discoideum.

Dictyostelium discoideum expresses two Extracellular signal Regulated Kinases, ERK1 and ERK2, which are involved in growth, multicellular development and regulation of adenylyl cyclase. Binding of extracellular cAMP to cAMP receptor 1, a G-protein coupled cell surface receptor, transiently stimulates phosphorylation, activation and nuclear translocation of ERK2. Activation of ERK2 by cAMP is dependent on heterotrimeric G-proteins, since activation of ERK2 is absent in cells lacking the Galpha4 subunit. The small G-protein rasD also activates ERK2. In cells overexpressing a mutated, constitutively active rasD, ERK2 activity is elevated prior to cAMP stimulation. Intracellular cAMP and cAMP-dependent protein kinase (PKA) are essential for adaptation of the ERK2 response. This report shows that multiple signalling pathways are involved in regulation of ERK2 activity in D.discoideum.     

Pattern formation by receptor tyrosine kinases: analysis of the Gurken gradient in Drosophila oogenesis

Spatial patterns of cell differentiation in developing tissues can be controlled by receptor tyrosine kinase (RTK) signaling gradients, which may form when locally secreted ligands activate uniformly expressed receptors. Graded activation of RTKs can span multiple cell diameters, giving rise to spatiotemporal patterns of signaling through the Extracellular Signal Regulated/Mitogen Activated Protein Kinase (ERK/MAPK), which connects receptor activation to multiple aspects of tissue morphogenesis. This general mechanism has been identified in numerous developmental contexts, from body axis specification in insects to patterning of the mammalian neocortex. We review recent quantitative studies of this mechanism in Drosophila oogenesis, an established genetic model of signaling through the Epidermal Growth Factor Receptor (EGFR), a highly conserved RTK.     

Extracellular ATP-induced proliferation of adventitial fibroblasts requires phosphoinositide 3-kinase, Akt, mammalian target of rapamycin, and p70 S6 kinase signaling pathways

Extracellular nucleotides are increasingly recognized as important regulators of growth in a variety of cell types. Recent studies have demonstrated that extracellular ATP is a potent inducer of fibroblast growth acting, at least in part, through an ERK1/2-dependent signaling pathway. However, the contributions of additional signaling pathways to extracellular ATP-mediated cell proliferation have not been defined. By using both pharmacologic and genetic approaches, we found that in addition to ERK1/2, phosphatidylinositol 3-kinase (PI3K), Akt, mammalian target of rapamycin (mTOR), and p70 S6K-dependent signaling pathways are required for ATP-induced proliferation of adventitial fibroblasts. We found that extracellular ATP acting in part through G(i) proteins increased PI3K activity in a time-dependent manner and transient phosphorylation of Akt. This PI3K pathway is not involved in ATP-induced activation of ERK1/2, implying activation of independent parallel signaling pathways by ATP. Extracellular ATP induced dramatic increases in mTOR and p70 S6K phosphorylation. This activation of the mTOR/p70 S6 kinase (p70 S6K) pathway in response to ATP is because of independent contributions of PI3K/Akt and ERK1/2 pathways, which converge on the level of p70 S6K. ATP-dependent activation of mTOR and p70 S6K also requires additional signaling inputs perhaps from pathways operating through Galpha or Gbetagamma subunits. Collectively, our data demonstrate that ATP-induced adventitial fibroblast proliferation requires activation and interaction of multiple signaling pathways such as PI3K, Akt, mTOR, p70 S6K, and ERK1/2 and provide evidence for purinergic regulation of the protein translational pathways related to cell proliferation.     

MEKK2 regulates focal adhesion stability and motility in invasive breast cancer cells

MEK Kinase 2 (MEKK2) is a serine/threonine kinase that functions as a MAPK kinase kinase (MAP3K) to regulate activation of Mitogen-activated Protein Kinases (MAPKs). We recently have demonstrated that ablation of MEKK2 expression in invasive breast tumor cells dramatically inhibits xenograft metastasis, but the mechanism by which MEKK2 influences metastasis-related tumor cell function is unknown. In this study, we investigate MEKK2 function and demonstrate that silencing MEKK2 expression in breast tumor cell significantly enhances cell spread area and focal adhesion stability while reducing cell migration. We show that cell attachment to the matrix proteins fibronectin or Matrigel induces MEKK2 activation and localization to focal adhesions. Further, we reveal that MEKK2 ablation enhances focal adhesion size and frequency, thereby linking MEKK2 function to focal adhesion stability. Finally, we show that MEKK2 knockdown inhibits fibronectin-induced Extracellular Signal-Regulated Kinase 5 (ERK5) signaling and Focal Adhesion Kinase (FAK) autophosphorylation. Taken together, our results strongly support a role for MEKK2 as a regulator of signaling that modulates breast tumor cell spread area and migration through control of focal adhesion stability.     

Activation of extracellular signal-regulated kinases 1 and 2 by insulin-like growth factor I in human osteosarcoma MG-63 cells.

The activation of extracellular signal-regulated kinases 1 and 2 by insulin-like growth factor I in human osteosarcoma MG-63 cells was examined by Mono Q ion exchange chromatography of cell extracts and measurement of myelin basic protein kinase activity, and by immunoblotting of cell extracts with a phospho-specific extracellular signal-regulated kinase antibody. Extracellular signal-regulated kinase 1 appeared to be activated in resting cells and addition of insulin-like growth factor I resulted in the activation primarily of extracellular signal-regulated kinase 2. Extracellular signal-regulated kinase 2 was found in the nucleus after addition of insulin-like growth factor I.     

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.     

Involvement of the MAP kinase ERK2 in MUC1 mucin signaling

MUC1 mucin is a receptor-like glycoprotein expressed abundantly in various cancer cell lines as well as in glandular secretory epithelial cells, including airway surface epithelial cells. The role of this cell surface mucin in the airway is not known. In an attempt to understand the signaling mechanism of MUC1 mucin, we established a stable cell line from COS-7 cells expressing a chimeric receptor consisting of the extracellular and transmembrane domains of CD8 and the cytoplasmic (CT) domain of MUC1 mucin (CD8/MUC1 cells). We previously observed that treatment of these cells with anti-CD8 antibody resulted in tyrosine phosphorylation of the CT domain of the chimera. Here we report that treatment of CD8/MUC1 cells with anti-CD8 resulted in activation of extracellular signal-regulated kinase (ERK) 2 as assessed by immunoblotting, kinase assay, and immunocytochemistry. The activation of ERK2 was completely blocked either by a dominant negative Ras mutant or in the presence of a mitogen-activated protein kinase kinase (MEK) inhibitor. We conclude that tyrosine phosphorylation of the CT domain of MUC1 mucin leads to activation of a mitogen-activated protein kinase pathway through the Ras-MEK-ERK2 pathway. Combined with the existing data by others, it is suggested that one of the roles of MUC1 mucin may be regulation of cell growth and differentiation via a common signaling pathway, namely the Grb2-Sos-Ras-MEK-ERK2 pathway.     

Erk 5 is necessary for sustained PDGF-induced Akt phosphorylation and inhibition of apoptosis

Extracellular regulated kinase (Erk) 5 is a member of the mitogen activated protein (MAP) kinase family that has been implicated in both cell proliferation and survival. In the present study, we found that stimulation with platelet-derived growth factor (PDGF)-BB leads to a transient activation of Erk5, which was shown to be dependent on recruitment of both Src kinases and the tyrosine phosphatase Shp2 to the activated PDGF receptor β (PDGFRβ). We could also demonstrate that Shp2 docking to the receptor is critical for Src kinase activation, suggesting that Shp2 may contribute to Erk5 activation through its involvement in Src kinase activation. Under control conditions, PDGF-BB promoted a sustained Akt phosphorylation. However, reduction of the expression of Erk5 by siRNA resulted in only a transient Akt phosphorylation, and an inability of PDGF-BB to suppress caspase 3 activation and inhibit apoptotic nuclear morphological changes such as condensed or fragmented chromatin under serum-free conditions.     

Hydrogen peroxide-mediated phosphorylation of ERK1/2, Akt/PKB and JNK in cortical neurones: dependence on Ca2+ and PI3-kinase

Primary cortical neurones exposed to an oxidative insult in the form of hydrogen peroxide (H(2)O(2)) for 30 min showed a concentration-dependent increase in oxidative stress followed by a delayed NMDA receptor-dependent cell death measured 24 h later. Extracellular signal-regulated protein kinase (ERK1/2), c-jun N-terminal kinase (JNK) and the kinase Akt/PKB may regulate neuronal viability in response to oxidative insults. Using phospho-specific antibodies, a 15-min stimulation of neurones with H(2)O(2) (100 microm - 1 mm) produced a concentration-dependent phosphorylation of ERK1/2 and Akt/PKB that was partly dependent on extracellular Ca(2+) and phosphatidylinositol 3-kinase (PI3-K). Higher concentrations of H(2)O(2) (1 mm) also stimulated a phosphorylation of JNK which was totally dependent on extracellular Ca(2+) but not PI3-K. H(2)O(2)-induced phosphorylation of ERK1/2, Akt/PKB or JNK were unaffected by the NMDA channel blocker MK801. Blocking ERK1/2 activation with the upstream inhibitor U0126 (10 microm) enhanced H(2)O(2)-induced (100-300 microm range) neurotoxicity and inhibited H(2)O(2)-mediated phosphorylation of the cyclic AMP regulatory binding protein (CREB), suggesting that ERK1/2 signals to survival under these conditions. At higher concentrations (mm), H(2)O(2)-stimulated a phosphorylation of c-jun. It is likely, therefore, that subjecting neurones to moderate oxidative-stress recruits pro-survival signals to CREB but during severe oxidative stress pro-death signals through JNK and c-jun are dominant.     

RGS protein specificity towards Gq- and Gi/o-mediated ERK 1/2 and Akt activation, in vitro

Extracellular Regulated Kinases (ERK) and Protein Kinase B (Akt) are intermediaries in relaying extracellular growth signals to intracellular targets. Each pathway can become activated upon stimulation of G protein-coupled receptors mediated by G(q) and G(i/o) proteins subjected to regulation by RGS proteins. The goal of the study was to delineate the specificity in which cardiac RGS proteins modulate G(q)and G(i/o)-induced ERK and Akt phosphorylation. To isolate G(q)- and G(i/o)-mediated effects, we exclusively expressed muscarinic M(2) or M(3) receptors in COS-7 cells. Western blot analyses demonstrated increase of phosphorylation of ERK 1.7-/3.3-fold and Akt 2.4-/6-fold in M(2)-/M(3)- expressing cells through carbachol stimulation. In co-expressions, M(3)/G(q)-induced activation of Akt was exclusively blunted through RGS3s/RGS3, whereas activation of ERK was inhibited additionally through RGS2/RGS5. M(2)/G(i/o) induced Akt activation was inhibited by all RGS proteins tested. RGS2 had no effect on M(2)/G(i/o)-induced ERK activation. The high degree of specificity in RGS proteins-depending modulation of G(q)- and G(i/o)-mediated ERK and Akt activation in the muscarinic network cannot merely be attributed exclusively to RGS protein selectivity towards G(q) or G(i/o) proteins. Counter-regulatory mechanisms and inter-signaling cross-talk may alter the sensitivity of GPCR-induced ERK and Akt activation to RGS protein regulation.     

The N-Terminal Domain of ERK1 Accounts for the Functional Differences with ERK2

The Extracellular Regulated Kinase 1 and 2 transduce a variety of extracellular stimuli regulating processes as diverse as proliferation, differentiation and synaptic plasticity. Once activated in the cytoplasm, ERK1 and ERK2 translocate into the nucleus and interact with nuclear substrates to induce specific programs of gene expression. ERK1/2 share 85% of aminoacid identity and all known functional domains and thence they have been considered functionally equivalent until recent studies found that the ablation of either ERK1 or ERK2 causes dramatically different phenotypes. To search a molecular justification of this dichotomy we investigated whether the different functions of ERK1 and 2 might depend on the properties of their cytoplasmic-nuclear trafficking. Since in the nucleus ERK1/2 is predominantly inactivated, the maintenance of a constant level of nuclear activity requires continuous shuttling of activated protein from the cytoplasm. For this reason, different nuclear-cytoplasmic trafficking of ERK1 and 2 would cause a differential signalling capability. We have characterised the trafficking of fluorescently tagged ERK1 and ERK2 by means of time-lapse imaging in living cells. Surprisingly, we found that ERK1 shuttles between the nucleus and cytoplasm at a much slower rate than ERK2. This difference is caused by a domain of ERK1 located at its N-terminus since the progressive deletion of these residues converted the shuttling features of ERK1 into those of ERK2. Conversely, the fusion of this ERK1 sequence at the N-terminus of ERK2 slowed down its shuttling to a similar value found for ERK1. Finally, computational, biochemical and cellular studies indicated that the reduced nuclear shuttling of ERK1 causes a strong reduction of its nuclear phosphorylation compared to ERK2, leading to a reduced capability of ERK1 to carry proliferative signals to the nucleus. This mechanism significantly contributes to the differential ability of ERK1 and 2 to generate an overall signalling output.