Constitutive Activation Of Extracellular Signal-regulated Kinase Research Articles

Density-dependent ERK MAPK expression regulates MMP-9 and influences growth.

Previous work has shown that expression of the extracellular signal-regulated kinase (ERK) is decreased by high density in normal fibroblast cells, and this was correlated with increased expression of mitogen-activated protein kinase phosphatases. Because of these differences in ERK regulation upon contact inhibition, it is likely that other cellular responses may be influenced by the attainment of a contact-inhibited state. Expression of matrix metalloproteinase-9 and cadherin cleavage were both found to be decreased upon reaching high culture density. Inhibition of ERK activity with the MEK inhibitor PD98059 resulted in increased expression of cadherins, while constitutive activation of ERK through the use of expression of an ERK construct with a D319N sevenmaker mutation resulted in decreased expression of cadherins and enhanced colony formation of HT-1080 fibrosarcoma cells. Taken together, these results corroborate a role for the regulation of ERK upon the attainment of a contact-inhibited state with increased expression of cadherins.

NCS-Rapgef2, the Protein Product of the Neuronal Rapgef2 Gene, Is a Specific Activator of D1 Dopamine Receptor-Dependent ERK Phosphorylation in Mouse Brain.

The neuritogenic cAMP sensor (NCS), encoded by the Rapgef2 gene, links cAMP elevation to activation of extracellular signal-regulated kinase (ERK) in neurons and neuroendocrine cells. Transducing human embryonic kidney (HEK)293 cells, which do not express Rapgef2 protein or respond to cAMP with ERK phosphorylation, with a vector encoding a Rapgef2 cDNA reconstituted cAMP-dependent ERK activation. Mutation of a single residue in the cyclic nucleotide-binding domain (CNBD) conserved across cAMP-binding proteins abrogated cAMP-ERK coupling, while deletion of the CNBD altogether resulted in constitutive ERK activation. Two types of mRNA are transcribed from Rapgef2 in vivo. Rapgef2 protein expression was limited to tissues, i.e., neuronal and endocrine, expressing the second type of mRNA, initiated exclusively from an alternative first exon called here exon 1', and an alternative 5' protein sequence leader fused to a common remaining open reading frame, which is termed here NCS-Rapgef2. In the male mouse brain, NCS-Rapgef2 is prominently expressed in corticolimbic excitatory neurons, and striatal medium spiny neurons (MSNs). Rapgef2-dependent ERK activation by the dopamine D1 agonist SKF81297 occurred in neuroendocrine neuroscreen-1 (NS-1) cells expressing the human D1 receptor and was abolished by deletion of Rapgef2. Corticolimbic [e.g., dentate gyrus (DG), basolateral amygdala (BLA)] ERK phosphorylation induced by SKF81297 was significantly attenuated in CamK2α-Cre+/- ; Rapgef2cko/cko male mice. ERK phosphorylation in nucleus accumbens (NAc) MSNs induced by treatment with SKF81297, or the psychostimulants cocaine or amphetamine, was abolished in male Rapgef2cko/cko mice with NAc NCS-Rapgef2-depleting AAV-Synapsin-Cre injections. We conclude that D1-dependent ERK phosphorylation in mouse brain requires NCS-Rapgef2 expression.

Real-time genomic profiling of histiocytoses identifies early-kinase domain BRAF alterations while improving treatment outcomes.

Many patients with histiocytic disorders such as Langerhans cell histiocytosis (LCH) or Erdheim-Chester disease (ECD) have treatment-refractory disease or suffer recurrences. Recent findings of gene mutations in histiocytoses have generated options for targeted therapies. We sought to determine the utility of prospective sequencing of select genes to further characterize mutations and identify targeted therapies for patients with histiocytoses. Biopsies of 72 patients with a variety of histiocytoses underwent comprehensive genomic profiling with targeted DNA and RNA sequencing. Fifteen patients (21%) carried the known BRAF V600E mutation, and 11 patients (15%) carried various mutations in MAP2K1, which we confirm induce constitutive activation of extracellular signal-regulated kinase (ERK) and were sensitive to inhibitors of mitogen-activated protein kinase kinase (MEK, the product of MAP2K1). We also identified recurring ALK rearrangements, and 4 LCH patients with an uncommon in-frame deletion in BRAF (N486_P490del or N486_T491>K), resulting in constitutive activation of ERK with resistance to V600E-specific inhibitors. We subsequently describe clinical cases where patients with aggressive multisystem LCH experience dramatic and sustained responses to monotherapy with either dabrafenib or trametinib. These findings support our conclusion that comprehensive genomic profiling should be regularly applied to these disorders at diagnosis, and can positively impact clinical care.

ERK-regulated αB-crystallin induction by matrix detachment inhibits anoikis and promotes lung metastasis in vivo.

Evasion of extracellular matrix detachment-induced apoptosis (“anoikis”) is a defining characteristic of metastatic tumor cells. The ability of metastatic carcinoma cells to survive matrix detachment and escape anoikis enables them to disseminate as viable circulating tumor cells and seed distant organs. Here we report that αB-crystallin, an antiapoptotic molecular chaperone implicated in the pathogenesis of diverse poor-prognosis solid tumors, is induced by matrix detachment and confers anoikis-resistance. Specifically, we demonstrate that matrix detachment downregulates extracellular-signal regulated kinase (ERK) activity and increases αB-crystallin protein and mRNA levels. Moreover, we show that ERK inhibition in adherent cancer cells mimics matrix detachment by increasing αB-crystallin protein and mRNA levels, while constitutive ERK activation suppresses αB-crystallin induction during matrix detachment. These findings indicate that ERK inhibition is both necessary and sufficient for αB-crystallin induction by matrix detachment. To examine the functional consequences of αB-crystallin induction in anoikis, we stably silenced αB-crystallin in two different metastatic carcinoma cell lines. Strikingly, silencing αB-crystallin increased matrix detachment-induced caspase activation and apoptosis but did not affect cell viability of adherent cancer cells. In addition, silencing αB-crystallin in metastatic carcinoma cells reduced the number of viable circulating tumor cells and inhibited lung metastasis in two orthotopic models, but had little or no effect on primary tumor growth. Taken together, our findings point to αB-crystallin as a novel regulator of anoikis-resistance that is induced by matrix detachment-mediated suppression of ERK signaling and promotes lung metastasis. Our results also suggest that αB-crystallin represents a promising molecular target for antimetastatic therapies.

The Rac GTPase effector p21-activated kinase is essential for hematopoietic stem/progenitor cell migration and engraftment

AbstractThe p21-activated kinases (Paks) are serine/threonine kinases that are major effectors of the Rho guanosine 5′\\x{2011}triphosphatase, Rac, and Cdc42. Rac and Cdc42 are known regulators of hematopoietic stem and progenitor cell (HSPC) function, however, a direct role for Paks in HSPCs has yet to be elucidated. Lin-Sca1+c-kit+ (LSK) cells from wild-type mice were transduced with retrovirus expressing Pak inhibitory domain (PID), a well-characterized inhibitor of Pak activation. Defects in marrow homing and in vitro cell migration, assembly of the actin cytoskeleton, proliferation, and survival were associated with engraftment failure of PID-LSK. The PID-LSK demonstrated decreased phosphorylation of extracellular signal-regulated kinase (ERK), whereas constitutive activation of ERK in these cells led to rescue of hematopoietic progenitor cell proliferation in vitro and partial rescue of Pak-deficient HSPC homing and engraftment in vivo. Using conditional knock-out mice, we demonstrate that among group A Paks, Pak2−/− HSPC show reduced homing to the bone marrow and altered cell shape similar to PID-LSK cells in vitro and are completely defective in HSPC engraftment. These data demonstrate that Pak proteins are key components of multiple engraftment-associated HSPC functions and play a direct role in activation of ERK in HSPCs, and that Pak2 is specifically essential for HSPC engraftment.

SP-1 regulation of MMP-9 expression requires Ser586 in the PEST domain.

Rac1, a small GTPase, regulates macrophage MMP (matrix metalloproteinase)-9 in an ERK (extracellular-signal-regulated kinase)- and SP (specificity protein)-1-dependent manner. SP-1 contains a PEST (Pro-Glu-Ser-Thr) domain that may modulate protein stability. We hypothesize that Thr578, Ser586 and/or Ser587 in the PEST domain are required for SP-1 stability and MMP-9 expression secondary to activation of ERK, a serine/threonine kinase. We determined the effects of Rac1 and ERK on MMP-9 expression driven by SP-1WT (wild-type) and the SP-1 mutants T578A, S586A and S587A. Expression of WT and mutant SP-1 increased MMP9 promoter activity in alveolar macrophages. However, constitutively active Rac1 suppressed MMP9 promoter activity in cells expressing SP-1WT, SP-1T578A and SP-1S587A, but not SP-1S586A. Furthermore, constitutive ERK activation, which was inhibited by Rac1, significantly increased MMP9 transcription in cells expressing SP-1WT, but not SP-1S586A. As Rac1 activation and ERK inactivation increased degradation of SP-1WT and not SP-1S586A, the results of the present study suggest that SP-1 stability mediated at Ser586 regulates MMP9 transcription. Ex vivo, alveolar macrophages obtained from patients with asbestosis had less MMP-9 expression that was associated with decreased SP-1 expression and ERK activation. These observations demonstrate that Ser586 in the PEST domain of SP-1 is important for MMP9 gene expression in alveolar macrophages and highlight the importance of these proteins in pulmonary fibrosis.

Opposing Actions of Extracellular Signal-regulated Kinase (ERK) and Signal Transducer and Activator of Transcription 3 (STAT3) in Regulating Microtubule Stabilization during Cardiac Hypertrophy

Excessive proliferation and stabilization of the microtubule (MT) array in cardiac myocytes can accompany pathological cardiac hypertrophy, but the molecular control of these changes remains poorly characterized. In this study, we examined MT stabilization in two independent murine models of heart failure and revealed increases in the levels of post-translationally modified stable MTs, which were closely associated with STAT3 activation. To explore the molecular signaling events contributing to control of the cardiac MT network, we stimulated cardiac myocytes with an α-adrenergic agonist phenylephrine (PE), and observed increased tubulin content without changes in detyrosinated (glu-tubulin) stable MTs. In contrast, the hypertrophic interleukin-6 (IL6) family cytokines increased both the glu-tubulin content and glu-MT density. When we examined a role for ERK in regulating cardiac MTs, we showed that the MEK/ERK-inhibitor U0126 increased glu-MT density in either control cardiac myocytes or following exposure to hypertrophic agents. Conversely, expression of an activated MEK1 mutant reduced glu-tubulin levels. Thus, ERK signaling antagonizes stabilization of the cardiac MT array. In contrast, inhibiting either JAK2 with AG490, or STAT3 signaling with Stattic or siRNA knockdown, blocked cytokine-stimulated increases in glu-MT density. Furthermore, the expression of a constitutively active STAT3 mutant triggered increased glu-MT density in the absence of hypertrophic stimulation. Thus, STAT3 activation contributes substantially to cytokine-stimulated glu-MT changes. Taken together, our results highlight the opposing actions of STAT3 and ERK pathways in the regulation of MT changes associated with cardiac myocyte hypertrophy.

Computational modelling of cancerous mutations in the EGFR/ERK signalling pathway

BackgroundThe Epidermal Growth Factor Receptor (EGFR) activated Extracellular-signal Regulated Kinase (ERK) pathway is a critical cell signalling pathway that relays the signal for a cell to proliferate from the plasma membrane to the nucleus. Deregulation of the EGFR/ERK pathway due to alterations affecting the expression or function of a number of pathway components has long been associated with numerous forms of cancer. Under normal conditions, Epidermal Growth Factor (EGF) stimulates a rapid but transient activation of ERK as the signal is rapidly shutdown. Whereas, under cancerous mutation conditions the ERK signal cannot be shutdown and is sustained resulting in the constitutive activation of ERK and continual cell proliferation. In this study, we have used computational modelling techniques to investigate what effects various cancerous alterations have on the signalling flow through the ERK pathway.ResultsWe have generated a new model of the EGFR activated ERK pathway, which was verified by our own experimental data. We then altered our model to represent various cancerous situations such as Ras, B-Raf and EGFR mutations, as well as EGFR overexpression. Analysis of the models showed that different cancerous situations resulted in different signalling patterns through the ERK pathway, especially when compared to the normal EGF signal pattern. Our model predicts that cancerous EGFR mutation and overexpression signals almost exclusively via the Rap1 pathway, predicting that this pathway is the best target for drugs. Furthermore, our model also highlights the importance of receptor degradation in normal and cancerous EGFR signalling, and suggests that receptor degradation is a key difference between the signalling from the EGF and Nerve Growth Factor (NGF) receptors.ConclusionOur results suggest that different routes to ERK activation are being utilised in different cancerous situations which therefore has interesting implications for drug selection strategies. We also conducted a comparison of the critical differences between signalling from different growth factor receptors (namely EGFR, mutated EGFR, NGF, and Insulin) with our results suggesting the difference between the systems are large scale and can be attributed to the presence/absence of entire pathways rather than subtle difference in individual rate constants between the systems.

Identification of annexin 1 as a PU.1 target gene in leukemia cells

To identify PU.1 downstream target genes, we first established PU.1-knockdown K562 (K562PU.1KD) cells expressing reduced levels of PU.1 by stably transfected PU.1 siRNAs. From microarray analysis, we found that several genes including annexin 1 were markedly induced in K562PU.1KD cells. Annexin 1 is a calcium- and phospholipid-binding protein and increased expression leads to the constitutive activation of extracellular signal-regulated kinase (ERK). Consistent with this, we observed constitutive activation of ERK in K562PU.1KD cells. Furthermore, we revealed the mRNA expression of annexin 1 was negatively correlated with PU.1 mRNA expression in 43 primary AML specimens (R=−0.31, p<0.042).

ERK Inhibition by SU1498 Alters Expression of Bcl‐2 Family Proteins in Breast Cancer Cells

The extracellular signal‐regulated kinase (ERK) pathway is vital to the cell survival. We have demonstrated the ability of SU1498, an inhibitor of ERK kinase activity, to sensitize breast cancer cells with constitutively active ERK to apoptosis. To identify the mechanisms involve, the expression of the antiapoptotic Bcl‐2 and Bcl‐xL and the proapoptotic Bad, Bax, Bik and Bak were examined under apoptotic conditions. One breast cell line (MCF‐10A) and three breast cancer cell lines (MCF 7, MDA‐MB‐231 and MDA‐MB‐468) were used to examine the effects of SU1498 and serum starvation on the expression of the Bcl‐2 family proteins. The MCF‐10A and MCF7 cells exhibit normal ERK regulation while the MDA‐MB‐231 and MDA‐MB‐468 cells lines have a constitutively activated ERK. Across all the cell lines, the combined treatments resulted in either decreased expression, or no changes in the expression, of the antiapoptotic Bcl2 proteins. The expression of the proapoptotic proteins Bad or Bak were dramatically upregulated only in those cells with constitutive ERK activation, while they tended to demonstrate reduced expression in the cells lines with normal ERK regulation. These cell lines with upregulate Bad and/or Bak expression were also those most sensitive to SU1498 induced cell death. The ability of SU1498 to sensitize breast cancer cells to apoptosis by inhibition of ERK may have therapeutic uses.Supported by NSERC

Heat shock protein Hsp72 controls oncogene-induced senescence pathways in cancer cells.

The heat shock protein Hsp72 is expressed at the elevated levels in various human tumors, and its levels often correlate with poor prognosis. Previously we reported that knockdown of Hsp72 in certain cancer cells, but not in untransformed breast epithelial cells, triggers senescence via p53-dependent and p53-independent mechanisms. Here we demonstrate that the p53-dependent pathway controlled by Hsp72 depends on the oncogenic form of phosphatidylinositol 3-kinase (PI3K). Indeed, upon expression of the oncogenic PI3K, epithelial cells began responding to Hsp72 depletion by activating the p53 pathway. Moreover, in cancer cell lines, activation of the p53 pathway caused by depletion of Hsp72 was dependent on oncogenes that activate the PI3K pathway. On the other hand, the p53-independent senescence pathway controlled by Hsp72 was associated with the Ras oncogene. In this pathway, extracellular signal-regulated kinases (ERKs) were critical for senescence, and Hsp72 controlled the ERK-activating kinase cascade at the level of Raf-1. Importantly, upon Ras expression, untransformed cells started responding to knockdown of Hsp72 by constitutive activation of ERKs, culminating in senescence. Therefore, Hsp72 is intimately involved in suppression of at least two separate senescence signaling pathways that are regulated by distinct oncogenes in transformed cells, which explains why cancer cells become "addicted" to this heat shock protein.

Central Role of Protein Kinase Cε in Constitutive Activation of ERK1/2 and Rac1 in the Malignant Cells of Hairy Cell Leukemia

We have previously identified the presence of Ras/Raf-independent constitutive activation of extracellular signal-regulated kinase (ERK) in the hairy cells (HCs) of hairy cell leukemia. The aim of the present study was to characterize the signaling components involved in this activation and their relationship to the reported activation of Rac1. We found that both Rac1 and ERK activation in HCs are downstream of active Src and protein kinase C (PKC). Inhibition with toxin B showed that Rac1 plays no role in ERK activation in HCs. However, toxin B inhibited p60src and the Rac1-GEF Vav, demonstrating a positive feedback/activation of p60src by Rac1. Treatment with specific small interfering RNA for various PKC isoforms, or with PKC isoform-specific inhibitors, demonstrated a central role for PKCepsilon in the constitutive activation of Rac1 and ERK in HCs. PKCepsilon and active ERK were mutually associated and co-localized with mitochondria in HCs. Furthermore, active PKCepsilon was nitrated on tyrosine, pointing to a reactive oxygen species-dependent mechanism of activation. By being involved in activation of ERK and Rac1, PKCepsilon plays roles in both the survival of HCs and in the cytoskeletal dynamics responsible for the distinctive morphology and tissue homing of these cells. Our study therefore describes novel aspects of signaling important for the pathogenesis of hairy cell leukemia.

Constitutive Activation of Extracellular Signal-Regulated Kinase Predisposes Diffuse Large B-Cell Lymphoma Cell Lines to CD40-Mediated Cell Death

CD40 promotes survival, proliferation, and differentiation of normal B cells but can cause activation-induced cell death in malignant B lymphocytes. CD40 ligand and anti-CD40 antibodies have been used successfully to induce apoptosis in lymphoma lines both in vitro and in xenograft tumor models. Although this makes CD40 an attractive target for antitumor therapies, the response of malignant B cells to CD40 signaling is variable, and CD40 stimulation can enhance proliferation and can increase chemoresistance in some cell lines. It would therefore be useful to identify markers that predict whether a specific cell line or tumor will undergo apoptosis when stimulated with CD40 and to identify targets downstream of CD40 that affect only the apoptotic arm of CD40 signaling. We have analyzed gene expression patterns in CD40-sensitive and CD40-resistant diffuse large B-cell lymphoma (DLBCL) cell lines to identify signaling pathways that are involved in CD40-mediated apoptosis. CD40-resistant lines expressed pre-B-cell markers, including RAG and VPREB, whereas CD40-sensitive cells resembled mature B cells and expressed higher levels of transcripts encoding several members of the CD40 signaling pathway, including LCK and VAV. In addition, CD40-sensitive DLBCL cell lines also displayed constitutive activation of extracellular signal-regulated kinase (ERK) and failed to undergo apoptosis when ERK phosphorylation was inhibited. In contrast, CD40-resistant lines showed no constitutive activation of ERK and no increase in ERK activity in response to CD40 stimulation. Our results suggest that constitutive activation of ERK may be required for death signaling by CD40.

Activation of the extracellular signal-regulated kinase by complement C5b-9

Extracellular signals may be transmitted to nuclear or cytoplasmic effectors via the mitogen-activated protein kinases. In the passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury, proteinuria, and activation of phospholipases and protein kinases. This study addresses the complement-mediated activation of the extracellular signal-regulated kinase (ERK). C5b-9 induced ERK threonine202/tyrosine204 phosphorylation (which correlates with activation) in GEC in culture and PHN in vivo. Expression of a dominant-inhibitory mutant of Ras reduced complement-mediated activation of ERK, but activation was not affected significantly by downregulation of protein kinase C. Complement-induced ERK activation resulted in phosphorylation of cytosolic phospholipase A2 and was, in part, responsible for phosphorylation of mitogen-activated protein kinase-associated protein kinase-2, but did not induce phosphorylation of the transcription factor, Elk-1. Activation of ERK was attenuated by drugs that disassemble the actin cytoskeleton (cytochalasin D, latrunculin B), and these compounds interfered with the activation of ERK by mitogen-activated protein kinase kinase (MEK). Overexpression of a constitutively active RhoA as well as inhibition of Rho-associated kinase blocked complement-mediated ERK activation. Complement cytotoxicity was enhanced after disassembly of the actin cytoskeleton but was unaffected after inhibition of complement-induced ERK activation. However, complement cytotoxicity was enhanced in GEC that stably express constitutively active MEK. Thus complement-induced ERK activation depends on cytoskeletal remodelling and affects the regulation of distinct downstream substrates, while chronic, constitutive ERK activation exacerbates complement-mediated GEC injury.

ERK Pathway Positively Regulates the Expression of Sprouty Genes

Sprouty was originally identified as an inhibitor of Drosophila development-associated receptor tyrosine kinase (RTK) signaling. Although RTK signaling has been shown to induce Sprouty gene expression, the precise induction pathway downstream of RTK remains unclear. As RTK signaling pathway includes activation of extracellular signal-regulated kinases (ERKs), we have examined a correlation between activation of ERKs and induction of Sprouty gene expression. All reagents which induce the activation of ERKs induce Sprouty gene expression; these agents include not only growth factors which bind to RTK but also phorbol 12-myristate-13-acetate and active Raf-1 kinase. Furthermore, the Sprouty gene expression induced by all those agents is totally suppressed when the cells are pretreated with specific inhibitors of ERK kinase (MEK). Human tumor cells which exhibit constitutive activation of ERKs show elevated expression of Sprouty genes, which is abolished by treatment of these cells with MEK inhibitors. All these findings clearly indicate that Sprouty gene expression is positively regulated by the ERK pathway downstream of RTK.

Dual Potential of Extracellular Signal-Regulated Kinase for the Control of Cell Survival

Extracellular signal-regulated kinase (ERK) controls cell proliferation positively or negatively depending on the cellular contexts in which ERK is activated. In this report, we address differential roles of transient and constitutive activation of ERK in cell survival. Under a basal culture condition, mesangial cells showed modest, sustained ERK activity. The activity of ERK was transiently upregulated (15–60 min) in response to hydrogen peroxide (H2O2). The cells exposed to H2O2 exhibited shrinkage of the cytoplasm, condensation/fragmentation of nuclei and DNA ladder typical of apoptosis. Inhibition of the transient ERK activation by MEK inhibitor PD098059 attenuated the H2O2-induced apoptosis. In contrast, inactivation of the constitutive ERK by PD098059 induced morphologic and biochemical features of apoptosis under the unstimulated, basal culture condition. Consistently, transfection with dominant-negative mutants of ERK1 and ERK2 inhibited H2O2-induced apoptosis, whereas it facilitated apoptosis of unstimulated cells. These results suggested the novel, dual potential of ERK for the regulation of cell survival. Transient upregulation of ERK participates in the induction of apoptosis, whereas basal, constitutive activity of ERK is required for the maintenance of cell survival.

Constitutive Activation of Extracellular Signal-Regulated Kinase in Human Acute Leukemias: Combined Role of Activation of MEK, Hyperexpression of Extracellular Signal-Regulated Kinase, and Downregulation of a Phosphatase, PAC1

Extracellular signal-regulated kinase (ERK) is an important intermediate in signal transduction pathways that are initiated by many types of cell surface receptors. It is thought to play a pivotal role in integrating and transmitting transmembrane signals required for growth and differentiation. Constitutive activation of ERK in fibroblasts elicits oncogenic transformation, and recently, constitutive activation of ERK has been observed in some human malignancies, including acute leukemia. However, mechanisms underlying constitutive activation of ERK have not been well characterized. In this study, we examined the activation of ERK in 79 human acute leukemia samples and attempted to find factors contributing to constitutive ERK activation. First, we showed that ERK and MEK were constitutively activated in acute leukemias by in vitro kinase assay and immunoblot analysis. However, in only one half of the studied samples, the pattern of ERK activation was similar to that of MEK activation. Next, by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analysis, we showed hyperexpression of ERK in a majority of acute leukemias. In 17 of 26 cases (65.4%) analyzed by immunoblot, the pattern of ERK expression was similar to that of ERK activation. The fact of constitutive activation of ERK in acute leukemias suggested to us the possibility of an abnormal downregulation mechanism of ERK. Therefore, we examined PAC1, a specific ERK phosphatase predominantly expressed in hematopoietic tissue and known to be upregulated at the transcription level in response to ERK activation. Interestingly, in our study, PAC1 gene expression in acute leukemias showing constitutive ERK activation was significantly lower than that in unstimulated, normal bone marrow (BM) samples showing minimal or no ERK activation (P =.002). Also, a significant correlation was observed between PAC1 downregulation and phosphorylation of ERK in acute leukemias (P =.002). Finally, by further analysis of 26 cases, we showed that a complementary role of MEK activation, ERK hyperexpression, and PAC1 downregulation could contribute to determining the constitutive activation of ERK in acute leukemia. Our results suggest that ERK is constitutively activated in a majority of acute leukemias, and in addition to the activation of MEK, the hyperexpression of ERK and downregulation of PAC1 also contribute to constitutive ERK activation in acute leukemias.

Activation of Mitogen-activated Protein Kinases by Formyl Peptide Receptors Is Regulated by the Cytoplasmic Tail

Wild type formyl peptide receptors (FPRwt) and receptors deleted of the carboxyl-terminal 45 amino acids (FPRdel) were stably expressed in undifferentiated HL-60 promyelocytes. Expression of FPRwt reconstituted N-formylmethionyl-leucyl-phenylalanine (FMLP)-stimulated extracellular signal-regulated kinase (ERK) and p38 kinase activity. Expression of FPRdel resulted in a 2-5-fold increase in basal ERK and p38 kinase activity, whereas FMLP failed to stimulate either mitogen-activated protein kinase (MAPK). Pertussis toxin abolished FMLP stimulation of both MAPKs in FPRwt cells but had no effect on either basal or FMLP-stimulated MAPK activity in FPRdel cells. FMLP stimulated a concentration-dependent increase in guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding in membranes from FPRwt but not FPRdel cells. GTPgammaS inhibited FMLP binding to FPRwt but not FPRdel membranes. Photoaffinity labeling with azidoanilide-[gamma-32P]GTP in the presence or absence of FMLP showed increased labeling only in FPRwt membranes. Immunoprecipitation of alphai2 and alphaq/11 from solubilized, photolabeled membranes showed that FPRwt were coupled to alphai2 but not to alphaq/11. FPRwt cells demonstrated calcium mobilization following stimulation with FMLP, whereas FPRdel cells showed no increase in intracellular calcium. We conclude that the carboxyl-terminal tail of FPRs is necessary for ligand-mediated activation of Gi proteins and MAPK cascades. Deletion of the carboxyl-terminal tail results in constitutive activation of ERK and p38 kinase through a Gi2-independent pathway.

Differential activation of T cell cytokine production by the extracellular signal-regulated kinase (ERK) signaling pathway.

Stimulation of T cells via the T cell receptor (TCR) activates a number of signaling pathways that are potentially involved in the elicitation of physiological responses, such as the production of cytokines. The extracellular signal-regulated kinases (ERK) are a group of molecules activated in response to TCR ligation, whose role in T cell cytokine production is controversial. In this study, we have asked whether ERK activation is coupled to the production of a number of T cell-derived cytokines, and whether particular cytokines are differentially affected by ERK activation. To address these questions, we have utilized a constitutively active version of the immediate upstream activator of both ERK1 and ERK2, mitogen-activated/extracellular signal-regulated kinase 1 (MEK1), to activate ERK signaling selectively in the absence of other TCR-activated signaling pathways. The effect of constitutive MEK/ERK activation on T cell cytokine production was measured by transiently co-transfecting newly activated mouse T cells with DNA encoding constitutively active MEK1 (CA-MEK1) and the human interleukin-2 (IL-2) receptor alpha chain (hCD25), purifying hCD25+ transfectants by flow-cytometric cell sorting, and measuring the production of IL-3, IL-4, interferon (IFN)-gamma and granulocyte/macrophage-colony-stimulating factor (GM-CSF) either in the presence or absence of ionomycin stimulation. Newly activated T cells were used in these experiments as they more closely resemble T cells activated in vivo than do transformed T cells or long-term established T cell clones. CA-MEK1 expression led to constitutive ERK activation, which acted synergystically with ionomycin treatment to stimulate cytokine production. Furthermore, these experiments revealed a hierarchy of cytokine responsiveness to MEK/ERK activation, such that the production of IL-3 was most affected, followed by GM-CSF, IFN-gamma, and IL-4.