Dephosphorylation Of Extracellular Signal-regulated Kinase Research Articles

Surfactant protein D prevents mucin overproduction in airway goblet cells via SIRPα

Mucin overproduction is a common feature of chronic airway diseases such as asthma and chronic obstructive pulmonary disease (COPD), and exacerbates their underlying respiratory condition. Surfactant protein D (SP-D) protects against airway diseases through modulation of immune reactions, but whether it also exerts direct effects on airway epithelial cells has remained unclear. Therefore, we sought to investigate the inhibitory role of SP-D on mucin production in airway epithelial cells. We prepared air–liquid interface (ALI) cultures of human primary bronchial epithelial cells (HBECs), which recapitulated a well-differentiated human airway epithelium. Benzo(a)pyrene (BaP), a key toxicant in cigarette smoke, induced mucin 5AC (MUC5AC) production in ALI-cultured HBECs, airway secretory cell lines, and airway epithelia of mice. Then, the protective effects of SP-D against the BaP-induced mucin overproduction were examined. BaP increased MUC5AC production in ALI cultures of HBECs, and this effect was attenuated by SP-D. SP-D also suppressed the BaP-induced phosphorylation of extracellular signal-regulated kinase (ERK) and MUC5AC expression in NCI-H292 goblet-like cells, but not in NCI-H441 club-like cells. Signal regulatory protein α (SIRPα) was found to be expressed in HBECs and NCI-H292 cells but absent in NCI-H441 cells. In NCI-H292 cells, SP-D activated SH2 domain-containing tyrosine phosphatase-1 (SHP-1), downstream of SIRPα, and knockdown of SIRPα abolished the suppressive effects of SP-D on BaP-induced ERK phosphorylation and MUC5AC production. Consistent with these in vitro findings, intratracheal instillation of SP-D prevented the BaP-induced phosphorylation of ERK and Muc5ac expression in airway epithelial cells in a mouse model. SP-D acts directly on airway epithelial cells to inhibit mucin secretion through ligation of SIRPα and SHP-1-mediated dephosphorylation of ERK. Targeting of SIRPα is therefore a potential new therapeutic approach to suppression of mucin hypersecretion in chronic airway diseases such as COPD and asthma.

Dusp6 deficiency attenuates neutrophil-mediated cardiac damage in the acute inflammatory phase of myocardial infarction

Dual-specificity phosphatase 6 (DUSP6) serves a specific and conserved function on the dephosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). We previously identified Dusp6 as a regenerative repressor during zebrafish heart regeneration, therefore we propose to investigate the role of this repressor in mammalian cardiac repair. Utilizing a rat strain harboring Dusp6 nonsense mutation, rat neutrophil-cardiomyocyte co-culture, bone marrow transplanted rats and neutrophil-specific Dusp6 knockout mice, we find that Dusp6 deficiency improves cardiac outcomes by predominantly attenuating neutrophil-mediated myocardial damage in acute inflammatory phase after myocardial infarction. Mechanistically, Dusp6 is transcriptionally activated by p38-C/EBPβ signaling and acts as an effector for maintaining p-p38 activity by down-regulating pERK and p38-targeting phosphatases DUSP1/DUSP16. Our findings provide robust animal models and novel insights for neutrophil-mediated cardiac damage and demonstrate the potential of DUSP6 as a therapeutic target for post-MI cardiac remodeling and other relevant inflammatory diseases.

Reevaluation of the role of LIP-1 as an ERK/MPK-1 dual specificity phosphatase in the C. elegans germline

The fidelity of a signaling pathway depends on its tight regulation in space and time. Extracellular signal-regulated kinase (ERK) controls wide-ranging cellular processes to promote organismal development and tissue homeostasis. ERK activation depends on a reversible dual phosphorylation on the TEY motif in its active site by ERK kinase (MEK) and dephosphorylation by DUSPs (dual specificity phosphatases). LIP-1, a DUSP6/7 homolog, was proposed to function as an ERK (MPK-1) DUSP in the Caenorhabditis elegans germline primarily because of its phenotype, which morphologically mimics that of a RAS/let-60 gain-of-function mutant (i.e., small oocyte phenotype). Our investigations, however, reveal that loss of lip-1 does not lead to an increase in MPK-1 activity invivo. Instead, we show that loss of lip-1 leads to 1) a decrease in MPK-1 phosphorylation, 2) lower MPK-1 substrate phosphorylation, 3) phenocopy of mpk-1 reduction-of-function (rather than gain-of-function) allele, and 4) a failure to rescue mpk-1-dependent germline or fertility defects. Moreover, using diverse genetic mutants, we show that the small oocyte phenotype does not correlate with increased ectopic MPK-1 activity and that ectopic increase in MPK-1 phosphorylation does not necessarily result in a small oocyte phenotype. Together, these data demonstrate that LIP-1 does not function as an MPK-1 DUSP in the C. elegans germline. Our results caution against overinterpretation of the mechanistic underpinnings of orthologous phenotypes, since they may be a result of independent mechanisms, and provide a framework for characterizing the distinct molecular targets through which LIP-1 may mediate its several germline functions.

Ginsenoside Rk2 Protects against Ulcerative Colitis via Inactivating ERK/MEK Pathway by SIRT1.

Chinese traditional medicine is widely used in the treatment of ulcerative colitis (UC). Ginsenoside Rk2 is a newly discovered dammarane triterpenoid saponin isolated from ginseng. Our study aimed to investigate the effects of Ginsenoside Rk2 on UC. Human clones of colorectal adenocarcinoma Caco-2 cells and human intestinal epithelial THP-1 cells were co-cultured to establish a UC model in vitro. Cell viability and apoptosis were analyzed by cell counting kit 8 (CCK-8) and flow cytometry assay, respectively. Inflammatory cytokines' mRNA levels were measured by real-time quantitative polymerase chain reaction (RT-qPCR). Western blot was applied to examine the protein expression of apoptosis-associated proteins and the activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MEK) pathway. Furthermore, fisetin, an ERK kinase activator, was used to carry out rescue experiment. SRT1720, an activator of SIRT1, was applied to increase the SIRT1 protein levels while SIRT1 inhibitor nicotinamide (NAM) exerted the opposite effect. Ginsenoside Rk2 promoted cell viability, suppressed cell apoptosis, and reduced the release of pro-inflammatory cytokines including interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor-α (TNF-α) of HT-29 cells in UC model in a concentration-dependent manner. Meanwhile, the inhibitory effects of Ginsenoside Rk2 on the ERK/MEK pathway strengthened with the increase of concentration, and was verified by fisetin application. Furthermore, the upregulation of SIRT1 induced by Ginsenoside Rk2 prompted dephosphorylation of ERK and MEK to attenuate ERK/MEK pathway activation and reduced inflammatory progress, which was confirmed by SRT1720 as well as NAM. Ginsenoside Rk2 inactivated ERK/MEK pathway by regulating SIRT1 to restore the cellular function of human intestinal epithelial THP-1 cells. Therefore, Ginsenoside Rk2 may be effective in the treatment of UC.

Mitogen-activated protein kinases are involved in cucurbitacin D-induced antitumor effects on adult T-cell leukemia cells

Adult T cell leukemia (ATL) is an aggressive and malignant blood disease. We previously reported that steroid-structured cucurbitacin D (CuD) induces apoptosis in ATL cells. In this study, we investigated the effects of mitogen-activated protein kinase (MAPK) signaling inhibitors on CuD-induced cell death in peripheral blood lymphocytes (PBLs) isolated from ATL/acute lymphoblastic leukemia (ALL) patients and two human leukemia cell lines (MT-1 and MT-4). PBLs were isolated from an ATL/ALL patient as well as from a healthy donor. Cell surface markers were examined using flow cytometry. Serum cytokine levels were estimated using LEGENDplex or analyzed at the Center for Clinical and Translational Research of Kyushu University Hospital. Cell proliferation was assessed using the Cell Titer-Glo luminescent cell viability assay. Protein expression was determined by western blotting. PBLs from patients highly expressed CD4 and CD5. Serum from the patient contained high levels of interleukin (IL)-8, IL-10, IL-18, and interferon-γ compared to the healthy donor. CuD-induced cell death was enhanced by the mitogen-activated protein kinase kinase (MEK)1/2 inhibitor U0126. However, a c-Jun N-terminal kinase (JNK) inhibitor prevented CuD-induced cell death. Immunoblot analyses revealed that CuD reduced the phosphorylation of extracellular signal-regulated kinase (ERK), p38, and JNK, and co-treatment with CuD and U0126 did not affect the phosphorylation of ERK. MEK1/2 and p38 inhibitors enhanced CuD-induced cell death, and U0126 enhanced the CuD-induced de-phosphorylation of ERK in MT-1 and MT-4 cells. We conclude that CuD reduces ERK activation, resulting in enhanced antitumor effects on leukemic cells.

ERK Dephosphorylation through MKP1 Deacetylation by SIRT1 Attenuates RAS-Driven Tumorigenesis.

The role of Situin 1 (SIRT1) in tumorigenesis is still controversial due to its wide range of substrates, including both oncoproteins and tumor suppressors. A recent study has demonstrated that SIRT1 interferes in the Kirsten rat sarcoma viral oncogene homolog (KRAS)-driven activation of the Raf-mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK pathway, thereby inhibiting tumorigenesis. However, the molecular mechanism of SIRT1 as a tumor suppressor in RAS-driven tumorigenesis has been less clearly determined. This study presents evidence that the ectopic expression of SIRT1 attenuates RAS- or MEK-driven ERK activation and reduces cellular proliferation and transformation in vitro. The attenuation of ERK activation by SIRT1 results from prompt dephosphorylation of ERK, while MEK activity remains unchanged. We identified that MKP1, a dual specific phosphatase for MAPK, was deacetylated by SIRT1. Deacetylation of MKP1 by direct interaction with SIRT1 increased the binding affinity to ERK which in turn facilitated inactivation of ERK. Taken together, these results suggest that SIRT1 would act as a tumor suppressor by modulating RAS-driven ERK activity through MKP1 deacetylation.

Oscillations and bistability in a model of ERK regulation.

This work concerns the question of how two important dynamical properties, oscillations and bistability, emerge in an important biological signaling network. Specifically, we consider a model for dual-site phosphorylation and dephosphorylation of extracellular signal-regulated kinase (ERK). We prove that oscillations persist even as the model is greatly simplified (reactions are made irreversible and intermediates are removed). Bistability, however, is much less robust-this property is lost when intermediates are removed or even when all reactions are made irreversible. Moreover, bistability is characterized by the presence of two reversible, catalytic reactions: as other reactions are made irreversible, bistability persists as long as one or both of the specified reactions is preserved. Finally, we investigate the maximum number of steady states, aided by a network's "mixed volume" (a concept from convex geometry). Taken together, our results shed light on the question of how oscillations and bistability emerge from a limiting network of the ERK network-namely, the fully processive dual-site network-which is known to be globally stable and therefore lack both oscillations and bistability. Our proofs are enabled by a Hopf bifurcation criterion due to Yang, analyses of Newton polytopes arising from Hurwitz determinants, and recent characterizations of multistationarity for networks having a steady-state parametrization.

Ligand-mediated dephosphorylation signaling for MAP kinase

Extracellular signal-regulated kinase (ERK), also known as classical mitogen-activated protein kinase, plays critical roles in cell regulation. ERK is activated through phosphorylation by a cascade of protein kinases including MEK. Various ligands activate the MEK/ERK pathway through receptor-dependent cell signaling. In cultured cells, many ligands such as growth factors, hormones, cytokines and vasoactive peptides elicit transient activation of MEK/ERK, often peaking at ~10 min after the cell treatment. Here, we describe a novel biological event, in which ligand-mediated cell signaling results in the dephosphorylation of MEK/ERK. Neuromedin N and neurotensin, peptides derived from the same precursor polypeptide, elicit cell signaling through the neurotensin receptors. In cultured human pulmonary artery smooth muscle cells (PASMCs), but not in human pulmonary artery endothelial cells (PAECs), we found that both neuromedin N and neurotensin promoted the dephosphorylation of ERK and MEK. Human PASMCs were found to express neurotensin receptor (NTR)-1, −2 and −3, while human PAECs only express NTR3. Neuromedin N-mediated dephosphorylation was suppressed by small chemical inhibitors of protein phosphatase 1/2A and peptidyl-prolyl isomerase. Transmission electron microscopy showed the formation of endocytic vesicles in response to neuromedin N treatment, and dephosphorylation did not occur when sorting nexin 9, a critical regulator of the endocytic vesicle formation, was knocked down. We conclude that neuromedin N and neurotensin elicit a unique dephosphorylation signaling in the MEK/ERK pathway that is regulated by endocytosis. Considering the pathophysiological importance of the MEK/ERK pathway, this discovery of the dephosphorylation mechanism should advance the field of cell signaling.

Regorafenib induces extrinsic and intrinsic apoptosis through inhibition of ERK/NF-κB activation in hepatocellular carcinoma cells.

The aim of the present study was to investigate the role of NF-κB inactivation in regorafenib-induced apoptosis in human hepatocellular carcinoma SK-HEP-1 cells. SK-HEP-1 cells were treated with different concentrations of the NF-κB inhibitor 4-N-[2-(4-phenoxyphenyl)ethyl]quinazoline-4,6-diamine (QNZ) or regorafenib for different periods. The effects of QNZ and regorafenib on cell viability, expression of NF-κB-modulated anti-apoptotic proteins and apoptotic pathways were analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, western blotting, DNA gel electrophoresis, flow cytometry and NF-κB reporter gene assay. Inhibitors of various kinases including AKT, c-Jun N-terminal kinase (JNK), P38 and extracellular signal-regulated kinase (ERK) were used to evaluate the mechanism of regorafenib-induced NF-κB inactivation. The results demonstrated that both QNZ and regorafenib significantly inhibited the expression of anti-apoptotic proteins and triggered extrinsic and intrinsic apoptosis. We also demonstrated that regorafenib inhibited NF-κB activation through ERK dephosphorylation. Taken all together, our findings indicate that regorafenib triggers extrinsic and intrinsic apoptosis through suppression of ERK/NF-κB activation in SK-HEP-1 cells.

Cell Density-Dependent Increase in Tyrosine-Monophosphorylated ERK2 in MDCK Cells Expressing Active Ras or Raf.

The extracellular signal-regulated kinase (ERK) is one of the principal hub proteins that transmit growth signals from upstream oncogene products including Ras and BRaf to downstream effector proteins. However, there are both reports supporting and refuting the increase in ERK activity in cancer tissues expressing the active Ras and BRaf proteins. We considered that the cell density might account for this discrepancy. To examine this possibility, we prepared Madin-Darby canine kidney (MDCK) cells that expressed an active HRas, NRas, KRas, or BRaf and an ERK biosensor based on the principle of Förster resonance energy transfer (FRET). As we anticipated, expression of the active Ras or BRaf increased ERK activity at low cell densities. However, the ERK activity was markedly suppressed at high cell densities irrespective of the expression of the active Ras or BRaf. Western blotting analysis with Phos-tag gel revealed the decrease of tyrosine and threonine-diphosphorylated active ERK and the increase of tyrosine-monophosphorylated inactive ERK at high cell density. In addition, we found that calyculin A, an inhibitor for PPP-subfamily protein serine/threonine phosphatases, decreased the tyrosine-monophosphorylated ERK. Our study suggests that PPP-subfamily phosphatases may be responsible for cell density-dependent ERK dephosphorylation in cancer cells expressing active Ras or BRaf protein.

The Role of Phospholipase D and MAPK Signaling Cascades in the Adaption of Lichen Microalgae to Desiccation: Changes in Membrane Lipids and Phosphoproteome.

Classically, lichen phycobionts are described as poikilohydric organisms able to undergo desiccation due to the constitutive presence of molecular protection mechanisms. However, little is known about the induction of cellular responses in lichen phycobionts during drying. The analysis of the lipid composition of the desiccated lichen microalga Asterochloris erici revealed the unusual accumulation of highly polar lipids (oligogalactolipids and phosphatidylinositol), which prevents the fusion of membranes during stress, but also the active degradation of cone-shaped lipids (monogalactosyldiacylglycerol and phosphatidylethanolamine) to stabilize membranes in desiccated cells. The level of phosphatidic acid increased 7-fold during desiccation, implicating a possible role for phospholipase D (PLD) in the response to osmotic stress. Inhibition of PLD with 1-butanol markedly impaired the recovery of photosynthesis activity in A. erici upon desiccation and salt stress (2 M NaCl). These two hyperosmotic stresses caused the phosphorylation of c-Jun N-terminal kinase (JNK) and p38-like mitogen-activated protein kinase (MAPK) and the dephosphorylation of extracellular signal-regulated kinase (ERK). The incubation with 1-butanol reduced the phosphorylation of JNK-like proteins and increased the dephosphorylation of ERK-like proteins, which indicates an upstream control of MAPK cascades by PLD. The phosphoproteome showed that desiccation caused the phosphorylation of several proteins in A. erici, most of them involved in protein turnover. The results demonstrate that lichen phycobionts possess both constitutive and inducible protective mechanisms to acquire desiccation tolerance. Among others, these responses are controlled by the PLD pathway through the activation of MAPK cascades.

Targeted inhibition of MEK1 by cobimetinib leads to differentiation and apoptosis in neuroblastoma cells

BackgroundNeuroblastoma (NB) is one of the most common childhood malignancies. Currently, high risk NB carries a poor outcome and significant treatment related toxicities and, thus has been a focus for new therapeutics research in pediatric oncology. In this study, we evaluated the effects of the MEK inhibitor cobimetinib, as a single agent and in combinations, on the growth, survival and differentiation properties against a molecularly representative panel of NB cell lines.MethodsIn vitro anti-proliferative activity of cobimetinib alone or in combination was investigated by cell viability assays and its target modulatory activity was evaluated using phospho-kinases antibody arrays and western blot analysis. To determine the effect of combination with cis-RA on differentiation and resulting enhanced cellular cytotoxicity, the expression of glial fibrillary acidic protein (GFAP) and microtubule-associated protein 2 (MAP2) expression levels were examined by immuno-fluorescence.ResultsOur findings show that cobimetinib alone induced a concentration-dependent loss of cell viability in all NB cell lines. In addition, cobimetinib showed feedback activation of MEK1/2, and the dephosphorylation of extracellular signal-regulated kinases (ERK1/2) and c-RAF, providing information on the biological correlates of MEK inhibition in NB. Combined treatment with cis-RA, led to differentiation and enhanced sensitization of NB cells lines to cobimetinib.ConclusionCollectively, our results provide evidence that cobimetinib, in combination with cis-RA, represents a feasible option to develop novel treatment strategies for refractory NB.

P53 Protein-mediated Up-regulation of MAP Kinase Phosphatase 3 (MKP-3) Contributes to the Establishment of the Cellular Senescent Phenotype through Dephosphorylation of Extracellular Signal-regulated Kinase 1/2 (ERK1/2)

Growth arrest is one of the essential features of cellular senescence. At present, the precise mechanisms responsible for the establishment of the senescence-associated arrested phenotype are still incompletely understood. Given that ERK1/2 is one of the major kinases controlling cell growth and proliferation, we examined the possible implication of ERK1/2. Exposure of normal rat epithelial cells to etoposide caused cellular senescence, as manifested by enlarged cell size, a flattened cell body, reduced cell proliferation, enhanced β-galactosidase activity, and elevated p53 and p21. Senescent cells displayed a blunted response to growth factor-induced cell proliferation, which was preceded by impaired ERK1/2 activation. Further analysis revealed that senescent cells expressed a significantly higher level of mitogen-activated protein phosphatase 3 (MKP-3, a cytosolic ERK1/2-targeted phosphatase), which was suppressed by blocking the transcriptional activity of the tumor suppressor p53 with pifithrin-α. Inhibition of MKP-3 activity with a specific inhibitor or siRNA enhanced basal ERK1/2 phosphorylation and promoted cell proliferation. Apart from its role in growth arrest, impairment of ERK1/2 also contributed to the resistance of senescent cells to oxidant-elicited cell injury. These results therefore indicate that p53-mediated up-regulation of MKP-3 contributes to the establishment of the senescent cellular phenotype through dephosphorylating ERK1/2. Impairment of ERK1/2 activation could be an important mechanism by which p53 controls cellular senescence.

Doxorubicin attenuates serotonin-induced long-term synaptic facilitation by phosphorylation of p38 mitogen-activated protein kinase.

Doxorubicin (DOX) is an anthracycline used widely for cancer chemotherapy. Its primary mode of action appears to be topoisomerase II inhibition, DNA cleavage, and free radical generation. However, in non-neuronal cells, DOX also inhibits the expression of dual-specificity phosphatases (also referred to as MAPK phosphatases) and thereby inhibits the dephosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK), two MAPK isoforms important for long-term memory (LTM) formation. Activation of these kinases by DOX in neurons, if present, could have secondary effects on cognitive functions, such as learning and memory. The present study used cultures of rat cortical neurons and sensory neurons (SNs) of Aplysia to examine the effects of DOX on levels of phosphorylated ERK (pERK) and phosphorylated p38 (p-p38) MAPK. In addition, Aplysia neurons were used to examine the effects of DOX on long-term enhanced excitability, long-term synaptic facilitation (LTF), and long-term synaptic depression (LTD). DOX treatment led to elevated levels of pERK and p-p38 MAPK in SNs and cortical neurons. In addition, it increased phosphorylation of the downstream transcriptional repressor cAMP response element-binding protein 2 in SNs. DOX treatment blocked serotonin-induced LTF and enhanced LTD induced by the neuropeptide Phe-Met-Arg-Phe-NH2. The block of LTF appeared to be attributable to overriding inhibitory effects of p-p38 MAPK, because LTF was rescued in the presence of an inhibitor (SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole]) of p38 MAPK. These results suggest that acute application of DOX might impair the formation of LTM via the p38 MAPK pathway.

Src inhibitors in suppression of papillary thyroid carcinoma growth

Papillary thyroid carcinoma is the most common thyroid malignancy. Most papillary thyroid carcinomas contain BRAF mutations or RET/PTC rearrangements, thus providing targets for biologic therapy. Our previous studies had suggested papillary thyroid carcinomas (PTCs) with a BRAF mutation and the RET/PTC1 rearrangement have different sensitivities to MEK1/2 inhibitors, suggesting different signaling transduction pathways were involved. Src signaling transduction pathway in PTC cells was examined using Src inhibitors (PP2, SU6656, or dasatinib) and si-Src RNA in vitro by Western blot analysis and proliferation analysis. An orthotopic xenograft mouse model was used for the in vivo studies using dasatinib. In PTC cells, Src inhibitors suppressed p-Src and p-FAK and inhibited cell growth. In addition, significant suppression and extension of the p-ERK1/2 dephosphorylation were detected in RET/PTC1-rearranged cells in combination with an MEK inhibitor (CI-1040). The Src family kinase/ABL inhibitor, dasatinib, significantly decreased tumor volume in mice inoculated with PTC cells carrying the RET/PTC1 rearrangement. In BRAF-mutated PTC cells, Src inhibitors effectively suppressed p-Src expression and dasatinib significantly decreased tumor volume with twice daily treatment. Src inhibitors effectively inhibited the Src signaling transduction pathway in PTC cells in vitro and dasatinib suppressed tumor growth in vivo. These results suggested that Src signaling transduction pathway plays an important role in regulating growth in PTC cells. Combination of Src and MEK1/2 inhibitors extended the dephosphorylation of extracellular signal-regulated kinase (ERK)1/2 in PTCs carrying the RET/PTC1 rearrangement suggesting that combination therapy with complementary inhibitors of other signaling transduction pathways may be needed to effectively suppress growth and induce apoptosis in these cells.

Dynamic Regulation of Extracellular Signal-Regulated Kinase (ERK) by Protein Phosphatase 2A Regulatory Subunit B56γ1 in Nuclei Induces Cell Migration

Extracellular signal-regulated kinase (ERK) signalling plays a central role in various biological processes, including cell migration, but it remains unknown what factors directly regulate the strength and duration of ERK activation. We found that, among the B56 family of protein phosphatase 2A (PP2A) regulatory subunits, B56γ1 suppressed EGF-induced cell migration on collagen, bound to phosphorylated-ERK, and dephosphorylated ERK, whereas B56α1 and B56β1 did not. B56γ1 was immunolocalized in nuclei. The IER3 protein was immediately highly expressed in response to costimulation of cells with EGF and collagen. Knockdown of IER3 inhibited cell migration and enhanced dephosphorylation of ERK. Analysis of the time course of PP2A-B56γ1 activity following the costimulation showed an immediate loss of phosphatase activity, followed by a rapid increase in activity, and this activity then remained at a stable level that was lower than the original level. Our results indicate that the strength and duration of the nuclear ERK activation signal that is initially induced by ERK kinase (MEK) are determined at least in part by modulation of the phosphatase activity of PP2A-B56γ1 through two independent pathways.

Short and long access to cocaine self-administration activates tyrosine phosphatase STEP and attenuates GluN expression but differentially regulates GluA expression in the prefrontal cortex

Dephosphorylation of extracellular signal-regulated kinase (ERK) and cyclic AMP response element binding protein (CREB) in the dorsomedial prefrontal cortex (dmPFC) at the end of short access (ShA) cocaine self-administration is implicated in cocaine seeking. However, what receptors and phosphatases mediate this effect and whether ERK/CREB and related phospho-proteins in the dmPFC react similarly during early withdrawal from long access (LgA) cocaine self-administration are unknown. The effects of ShA vs. LgA cocaine self-administration on the phosphorylation of protein phosphatase 2A (PP2A) and striatal-enriched protein tyrosine phosphatase (STEP), as well as GluN and GluA receptor subtype expression in the dmPFC during early withdrawal, were compared. Rats self-administered cocaine or received saline during 2- or 6-h daily sessions for 10-11days. Two hours after the final session, the dmPFC was dissected out and processed for immunoblotting. Similar to previous findings after ShA cocaine, phospho-ERK and phospho-CREB in the dmPFC were decreased after LgA cocaine. Cocaine elevated phospho-PP2A (deactivation) and decreased phospho-STEP (activation) in both ShA and LgA cocaine rats. GluN1, GluN2B, and phospho-GluN2B Tyr1472 in the dmPFC were decreased after ShA and LgA cocaine. Further, a significant reduction of GluA2, GluA1, and phospho-GluA1 Ser845 was found only in LgA rats. Activation of phospho-STEP may underlie ERK and CREB dephosphorylation in the dmPFC as well as internalization and degradation of GluN complexes during early withdrawal from both ShA and LgA cocaine self-administration, whereas differential alteration of AMPA receptor subunits after ShA and LgA cocaine self-administration depends on cocaine intake.

Induction of Vascular Endothelial Growth Factor Secretion by Childhood Acute Lymphoblastic Leukemia Cells via the FLT-3 Signaling Pathway

Human leukemia cells secrete VEGF, which can act in a paracrine manner within the bone marrow microenvironment to promote leukemia cell survival and proliferation. The FLT-3 receptor tyrosine kinase plays an essential role in regulating normal hematopoiesis, but its constitutive activation via mutation in acute leukemias is generally associated with poor outcome. The aim of this study was to investigate interactions between the FLT-3 and VEGF signaling pathways in acute leukemia using cell lines and ex vivo cultures of pediatric acute lymphoblastic leukemia cells following expansion of direct patient explants in immunodeficient mice. Different xenograft lines exhibited variable cell surface FLT-3 expression, as well as basal and FLT-3 ligand-induced VEGF secretion, whereas the MV4;11 cell line, which expresses constitutively active FLT-3, secreted high levels of VEGF. The FLT-3 inhibitor, SU11657, significantly reduced VEGF secretion in three of six xenograft lines and MV4;11 cells, in conjunction with inhibition of FLT-3 tyrosine phosphorylation. Moreover, exposure of xenograft cells to the FLT-3-blocking antibody, D43, also reduced VEGF secretion to basal levels and decreased FLT-3 tyrosine phosphorylation. In terms of downstream signaling, SU11657 and D43 both caused dephosphorylation of extracellular signal-regulated kinase 1/2, with no changes in AKT or STAT5 phosphorylation. Finally, partial knockdown of FLT-3 expression by short interfering RNA also resulted in inhibition of VEGF secretion. These results indicate that FLT-3 signaling plays a central role in the regulation of VEGF secretion and that inhibition of the FLT-3/VEGF pathway may disrupt paracrine signaling between leukemia cells and the bone marrow microenvironment.

Requirement of p38 MAPK for a cell-death pathway triggered by vorinostat in MDA-MB-231 human breast cancer cells

Vorinostat is a histone deacetylase inhibitor that effectively suppresses cancer-cell proliferation by inducing cell-cycle arrest and/or apoptosis. We now show the involvement of p38 mitogen-activated protein kinase (MAPK) in the regulation of vorinostat-induced apoptosis in MDA-MB-231 human breast cancer cells. Vorinostat induced the hyperacetylation of histone H3, which correlated to apoptosis induction. Vorinostat-induced apoptosis occurred in parallel with the phosphorylation of p38 MAPK and the dephosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). Knockdown of p38 MAPK prominently abrogated apoptosis induction and was accompanied by decreased caspase-3 cleavage. These findings support the notion that the activation of the p38 MAPK pathway followed by caspase-3 cleavage is responsible for vorinostat-induced apoptosis in MDA-MB-231 cells.

Phosphodiesterase 3A (PDE3A) Deletion Suppresses Proliferation of Cultured Murine Vascular Smooth Muscle Cells (VSMCs) via Inhibition of Mitogen-activated Protein Kinase (MAPK) Signaling and Alterations in Critical Cell Cycle Regulatory Proteins

Cyclic nucleotide phosphodiesterase 3 (PDE3) is an important regulator of cyclic adenosine monophosphate (cAMP) signaling within the cardiovascular system. In this study, we examined the role of PDE3A and PDE3B isoforms in regulation of growth of cultured vascular smooth muscle cells (VSMCs) and the mechanisms by which they may affect signaling pathways that mediate mitogen-induced VSMC proliferation. Serum- and PDGF-induced DNA synthesis in VSMCs grown from aortas of PDE3A-deficient (3A-KO) mice was markedly less than that in VSMCs from PDE3A wild type (3A-WT) and PDE3B-deficient (3B-KO) mice. The reduced growth response was accompanied by significantly less phosphorylation of extracellular signal-regulated kinase (ERK) in 3A-KO VSMCs, most likely due to a combination of greater site-specific inhibitory phosphorylation of Raf-1(Ser-²⁵⁹) by protein kinase A (PKA) and enhanced dephosphorylation of ERKs due to elevated mitogen-activated protein kinase phosphatase 1 (MKP-1). Furthermore, 3A-KO VSMCs, compared with 3A-WT, exhibited higher basal PKA activity and cAMP response element-binding protein (CREB) phosphorylation, higher levels of p53 and p53 phosphorylation, and elevated p21 protein together with lower levels of Cyclin-D1 and retinoblastoma (Rb) protein and Rb phosphorylation. Adenoviral overexpression of inactive CREB partially restored growth effects of serum in 3A-KO VSMCs. In contrast, exposure of 3A-WT VSMCs to VP16 CREB (active CREB) was associated with inhibition of serum-induced DNA synthesis similar to that in untreated 3A-KO VSMCs. Transfection of 3A-KO VSMCs with p53 siRNA reduced p21 and MKP-1 levels and completely restored growth without affecting amounts of Cyclin-D1 and Rb phosphorylation. We conclude that PDE3A regulates VSMC growth via two complementary pathways, i.e. PKA-catalyzed inhibitory phosphorylation of Raf-1 with resulting inhibition of MAPK signaling and PKA/CREB-mediated induction of p21, leading to G₀/G₁ cell cycle arrest, as well as by increased accumulation of p53, which induces MKP-1, p21, and WIP1, leading to inhibition of G₁ to S cell cycle progression.