Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Between 30% and 50% of patients with advanced-stage anaplastic large-cell lymphoma (ALCL) harbor the balanced chromosomal rearrangement t(2;5)(p23;q35), which results in the generation of the fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). To further study survival signaling by NPMALK, we generated Ba/F3 cell lines with either inducible or constitutive expression of NPM-ALK and examined the regulation of the AKT target FOXO3a. We hypothesized that NPM-ALK signaling through phosphoinositol 3-kinase (PI 3-kinase) and AKT would regulate FOXO3a, a member of the forkhead family of transcription factors, thereby stimulating proliferation and blocking programmed cell death in NPM-ALK-transformed cells. In Ba/F3 cells with induced or constitutive expression of NPM-ALK, concomitant AKT activation and phosphorylation of its substrate, FOXO3a, was observed. In addition, transient expression of NPM-ALK in U-20S cells inhibited FOXO3a-mediated transactivation of reporter gene expression. Furthermore, NPM-ALK-induced FOXO3a phosphorylation in Ba/F3 cells resulted in nuclear exclusion of this transcriptional regulator, up-regulation of cyclin D2 expression, and down-regulation of p27(kip1) and Bim-1 expression. NPMALK reversal of proliferation arrest and of p27(kip1) induction was dependent on the phosphorylation of FOXO3a. Thus, FOXO3a is a barrier to hematopoietic transformation that is overcome by phosphorylation and cytoplasmic relocalization induced by the expression of NPM-ALK.
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PMID:NPM-ALK fusion kinase of anaplastic large-cell lymphoma regulates survival and proliferative signaling through modulation of FOXO3a. 1496 11

Constitutive overexpression of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is a key oncogenic event in anaplastic large-cell lymphomas with the characteristic chromosomal aberration t(2;5)(p23;q35). Proteins that interact with ALK tyrosine kinase play important roles in mediating downstream cellular signals, and are potential targets for novel therapies. Using a functional proteomic approach, we determined the identity of proteins that interact with the ALK tyrosine kinase by co-immunoprecipitation with anti-ALK antibody, followed by electrospray ionization and tandem mass spectrometry (MS/MS). A total of 46 proteins were identified as unique to the ALK immunocomplex using monoclonal and polyclonal antibodies, while 11 proteins were identified in the NPM immunocomplex. Previously reported proteins in the ALK signal pathway were identified including PI3-K, Jak2, Jak3, Stat3, Grb2, IRS, and PLCgamma1. More importantly, many proteins previously not recognized to be associated with NPM-ALK, but with potential NPM-ALK interacting protein domains, were identified. These include adaptor molecules (SOCS, Rho-GTPase activating protein, RAB35), kinases (MEK kinase 1 and 4, PKC, MLCK, cyclin G-associated kinase, EphA1, JNK kinase, MAP kinase 1), phosphatases (meprin, PTPK, protein phosphatase 2 subunit), and heat shock proteins (Hsp60 precursor). Proteins identified by MS were confirmed by Western blotting and reciprocal immunoprecipitation. This study demonstrates the utility of antibody immunoprecipitation and subsequent peptide identification by tandem mass spectrometry for the elucidation of ALK-binding proteins, and its potential signal transduction pathways.
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PMID:Identification of NPM-ALK interacting proteins by tandem mass spectrometry. 1496 12

The NPM-ALK fusion protein is found in up to 75% of pediatric anaplastic large cell lymphomas (ALCL). The ALK kinase becomes constitutively activated and triggers malignant transformation. Molecular targeting of the tumor-specific NPM-ALK fusion by gene-silencing methods seems to be a promising approach both for the treatment of ALCL and to decipher signaling pathways used by NPM-ALK. We designed and evaluated three chemically synthesized small interfering RNAs (siRNAs) for downregulation of the NPM-ALK fusion mRNA. Compared to HeLa cells transfected with the NPM-ALK expression plasmid only and to an siRNA containing two point mutations, the most potent anti-NPM-ALK siRNA reduced NPM-ALK protein expression in HeLa cells to almost undetectable levels, and the number of cells stained positively for NPM-ALK decreased by 80%. With respect to signaling, expressing of NPM-ALK increased the activity of AKT and ERK in HeLa cells, and this effect could be blocked by the specific siRNA targeting NPM-ALK. Expression of endogenous NPM-ALK mRNA in SR786 ALCL cells decreased by 50%-60% in cells transfected with the NPM-ALK siRNA. However, the amount of NPM-ALK protein was not influenced by a single transfection of the siRNAs against NPM-ALK. Repeated transfections over 8 days led to a significant reduction in NPM-ALK protein but without induction of apoptosis. We believe that the long protein half-life of NPM-ALK, at least 48 hours, limits the application of transiently transfected siRNAs. Nevertheless, RNA interference (RNAi) offers a suitable technique to dissect signaling pathways employed by NPM-ALK and may potentially be used to develop siRNA-based gene therapeutic approaches against NPM-ALK-positive lymphomas.
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PMID:Design and evaluation of chemically synthesized siRNA targeting the NPM-ALK fusion site in anaplastic large cell lymphoma (ALCL). 1500 Aug 27

NPM-ALK characterizes anaplastic large cell lymphoma (ALCL), as does the high expression of CD30, a feature shared with H-RS cells of classic Hodgkin's lymphoma. In H-RS cells, ligand-independent signaling by overexpressed CD30 drives constitutive NF-kappaB activation, which is absent in ALCL cells. Here we show that NPM-ALK impedes CD30 signaling and NF-kappaB activation, dependent on both ALK kinase activity and the N-terminal NPM domain. NPM-ALK transduction into H-RS cell lines abrogates recruitment and aggregation of TRAF proteins, inducing an ALCL-like morphology and phenotype. TRAF2 associates with NPM-ALK at a consensus binding motif located in the kinase domain. Thus, NPM-ALK abrogates CD30-driven NF-kappaB activation and can also induce an ALCL phenotype, distinguishing ALCL cells from H-RS cells of T cell origin.
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PMID:The NPM-ALK oncoprotein abrogates CD30 signaling and constitutive NF-kappaB activation in anaplastic large cell lymphoma. 1509 42

Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is a constitutively active fusion tyrosine kinase involved in lymphomagenesis of human anaplastic large cell lymphomas (ALCL), the maturation and activity of which depend on the association with the heat shock protein (hsp) 90 protein chaperone. Targeting hsp90 by the ansamycins geldanamycin and 17-allyl-amino-demethoxygeldanamycin (17-AAG) promotes degradation of several proteins through the ubiquitin-proteasome pathway, including oncogenic Raf, v-Src, erbB2, and BCR-ABL. We have previously shown that 17-AAG prevents hsp90/NPM-ALK complex formation and fosters NPM-ALK turnover, perhaps through its association with the hsp70 chaperone. Here, we show that inhibition of the proteasome activity by the potent and specific compound pyrazylcarbonyl-Phe-Leu-boronate (PS-341) blocks 17-AAG-induced down-regulation of NPM-ALK, which becomes detergent-insoluble and relocates into ubiquitin-rich perinuclear vesicles that represent aggregated polyubiquitinated forms of the protein. Kinase activity was not mandatory for proteasomal degradation of NPM-ALK, because kinase-defective NPM-ALK was even more rapidly degraded upon 17-AAG treatment. Prolonged exposure to the proteasome inhibitor was shown to trigger caspase-3-mediated apoptosis in proliferating ALCL cells at nanomolar concentrations. However, we verified that the accumulation of detergent-insoluble NPM-ALK in ALCL cells was not a spurious consequence of PS341-committed apoptosis, because caspase inhibitors prevented poly(ADP-ribose) polymerase cleavage whereas they did not affect partitioning of aggregated NPM-ALK. In line with these observations, the carboxyl hsp70-interacting ubiquitin ligase (CHIP), was shown to increase basal ubiquitination and turnover of NPM-ALK kinase, supporting a mechanism whereby NPM-ALK proceeds rapidly toward hsp70-assisted ubiquitin-dependent proteasomal degradation, when chaperoning activity of hsp90 is prohibited by 17-AAG.
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PMID:Ubiquitination and proteasomal degradation of nucleophosmin-anaplastic lymphoma kinase induced by 17-allylamino-demethoxygeldanamycin: role of the co-chaperone carboxyl heat shock protein 70-interacting protein. 1512 67

The tissue inhibitor of metalloproteinase-1 (TIMP1) is expressed in a subset of malignant lymphomas and can inhibit tumor spread and promote cell survival. Recent data suggest that TIMP1 expression may be regulated by signal transducer and activator of transcription (STAT)-3. Thus, we tested the hypothesis that TIMP1 expression is related to STAT3 activation in lymphomas, with a focus on anaplastic large cell lymphomas (ALCLs), which are known to express high levels of phosphorylated/active STAT3 (pSTAT3). Specific inhibition of STAT3 with a dominant-negative construct led to concentration-dependent down-regulation of TIMP1 expression in two anaplastic lymphoma kinase (ALK)(+) ALCL cell lines, Karpas 299 and SU-DHL-1. Using cDNA microarrays, ALK(+) ALCL cell lines consistently expressed the highest TIMP1 level among 29 lymphoma cell lines of various subtypes. The association between TIMP1 expression and high level of STAT3 activation was validated by Western blots and immunostaining using antibodies specific for pSTAT3 and TIMP1. We further evaluated the relationship between TIMP1 expression and STAT3 activation in 43 ALCL tumors (19 ALK(+) and 24 ALK(-)) using immunohistochemistry and a tissue microarray. The TIMP1(+) group had a mean of 64% pSTAT3(+) cells as compared to 23% pSTAT3(+) cells in the TIMP1(-) group (P = 0.002). As expected, TIMP1 positivity was higher in the ALK(+) group (15 of 19, 79%) compared with the ALK(-) group (5 of 24, 21%; P = 0.0002) because NPM-ALK restricted to ALK(+) tumors was previously shown to activate STAT3. In conclusion, STAT3 directly contributes to the high level of TIMP1 expression in ALK(+) ALCL, and TIMP1 expression correlates with high level of STAT3 activation in ALCL. TIMP1, as a downstream target of STAT3, may mediate the anti-apoptotic effects of STAT3.
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PMID:Signal transducer and activator of transcription-3 activation contributes to high tissue inhibitor of metalloproteinase-1 expression in anaplastic lymphoma kinase-positive anaplastic large cell lymphoma. 1616 Oct 18

Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is an aberrant fusion gene product expressed in a subset of cases of anaplastic large cell lymphoma (ALCL). It has been shown that NPM-ALK binds to and activates signal transducer and activator of transcription 3 (STAT3) in vitro, and that STAT3 is constitutively active in ALK(+) ALCL cell lines and tumors. In view of the oncogenic potential of STAT3, we further examined its biological significance in ALCL using two ALK(+) ALCL cell lines (Karpas 299 and SU-DHL-1) and an adenoviral vector that carries dominant-negative STAT3 (AdSTAT3DN). Infection by AdSTAT3DN led to the expression of STAT3DN in both ALK(+) ALCL cell lines at a similar efficiency. Subcellular fractionation studies showed that a significant proportion of the expressed STAT3DN protein translocated to the nucleus, despite the fact that STAT3DN has a mutation at residue 705(tyrosine --> phenylalanine), a site that is believed to be crucial for STAT3 activation and nuclear translocation. Introduction of STAT3DN induced apoptosis and G(1) cell cycle arrest. Western blot studies showed that expression of STAT3DN resulted in caspase-3 cleavage, downregulation of Bcl-2, Bcl-xL, cyclin D3, survivin, Mcl-1, c-Myc and suppressor of cytokine signaling 3. These results support the concept that STAT3 activation is pathogenetically important in ALCL cells by deregulating the expression of multiple target proteins that are involved in the control of apoptosis and cell cycle progression.
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PMID:Selective inhibition of STAT3 induces apoptosis and G(1) cell cycle arrest in ALK-positive anaplastic large cell lymphoma. 1518 87

Majority of anaplastic large-cell lymphomas (ALCLs) are associated with the t(2;5)(p23;q35) translocation, fusing the NPM (nucleophosmin) and ALK (anaplastic lymphoma kinase) genes (NPM-ALK). Recent studies demonstrated that ALK may also be involved in variant translocations, namely, t(1;2)(q25;p23), t(2;3)(p23;q21), t(2;17)(p23;q23) and inv(2)(p23q35), which create the TPM3-ALK, TFG-ALK5, CLTC-ALK, and ATIC-ALK fusion genes, respectively. Although overexpression of NPM-ALK has previously been shown to transform fibroblasts, the transforming potential of variant X-ALK proteins has not been precisely investigated. We stably transfected the cDNAs coding for NPM-ALK, TPM3-ALK, TFG-ALK, CLTC-ALK or ATIC-ALK into nonmalignant NIH3T3 cells. All X-ALK variants are tyrosine phosphorylated and their subcellular distribution was in agreement with that observed in tumors. Moreover, our results show that the in vitro transforming capacity of NIH3T3-transfected cells are in relation to the level of X-ALK fusion proteins excepted for TPM3-ALK for which there is an inverse correlation. The differences between the five X-ALK variants with regard to proliferation rate, colony formation in soft agar, invasion, migration through the endothelial barrier and tumorigenicity seem to be due to differential activation of various signaling pathways such as PI3-kinase/AKT. These findings may have clinical implications in the pathogenesis and prognosis of ALK-positive ALCLs.
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PMID:Differential effects of X-ALK fusion proteins on proliferation, transformation, and invasion properties of NIH3T3 cells. 1520 56

Previous studies have shown that activation of the Raf/MEK/ERK pathway is necessary for G2/M transition. However, as for the activation state of MEK in mitosis the conclusion is not consistent. Here we show that MEK is inhibited in mitosis. In addition, we identify a multifunctional protein named B23 that strongly cross-reacts with a phospho-MEK antibody in mitotic cells. Sequence homology between the N-terminus surrounding Ser 4 of B23 and the Raf phosphorylation site on MEK suggests a mechanism for cross-reaction of the antibody. Thus, mutation of Ser 4 to alanine abolishes cross-reactivity between B23 and the phospho-MEK antibody. Our findings may explain the discrepancy of results obtained with the use of phospho-MEK antibody regarding the activation state of MEK in mitosis.
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PMID:MEK inhibition and phosphorylation of serine 4 on B23 are two coincident events in mitosis. 1535 59

Anaplastic large cell lymphoma (ALCL) is a highly proliferative neoplasm that frequently carries the t(2;5)(p23;q35) and aberrantly expresses nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). Previously, NPM-ALK had been shown to activate the phosphatidylinositol 3 kinase (PI3K)/Akt pathway. As the cyclin-dependent kinase (CDK) inhibitor p27(Kip1) (p27) is usually not expressed in ALCL, we hypothesized that activated Akt (pAkt) phosphorylates p27 resulting in increased p27 proteolysis and cell cycle progression. Here we demonstrate that inhibition of pAkt activity in ALCL decreases p27 phosphorylation and degradation, resulting in increased p27 levels and cell cycle arrest. Using immunohistochemistry, pAkt was detected in 24 (57%) of 42 ALCL tumors, including 8 (44%) of 18 ALK-positive tumors and 16 (67%) of 24 ALK-negative tumors, and was inversely correlated with p27 levels. The mean percentage of p27-positive tumor cells was 5% in the pAkt-positive group compared with 26% in the pAkt-negative group (P = .0076). These findings implicate that Akt activation promotes cell cycle progression through inactivation of p27 in ALCL.
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PMID:Inhibition of Akt increases p27Kip1 levels and induces cell cycle arrest in anaplastic large cell lymphoma. 1537 80


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