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N G Ahn

Publications and source records attributed to N G Ahn.

At least 37 records · Page 2Linked to original sources
PubMed1999

Enhancement of beta-globin locus control region-mediated transactivation by mitogen-activated protein kinases through stochastic and graded mechanisms.

Activation of the mitogen-activated protein kinase (MAPK) pathway enhances long-range transactivation by the beta-globin locus control region (LCR) (W. K. Versaw, V. Blank, N. M. Andrews, and E. H. Bresnick, Proc. Natl. Acad. Sci. USA 95:8756-8760, 1998). The enhancement requires tandem recognition sites for the hematopoietic transcription factor NF-E2 within the hypersensitive site 2 (HS2) subregion of the LCR. To distinguish between mechanisms of induction involving the activation of silent promoters or the increased efficacy of active promoters, we analyzed basal and MAPK-stimulated HS2 enhancer activity in single, living cells. K562 erythroleukemia cells stably transfected with constructs containing the human Agamma-globin promoter linked to an enhanced green fluorescent protein (EGFP) reporter, with or without HS2, were analyzed for EGFP expression by flow cytometry. When most cells in a population expressed EGFP, MAPK augmented the activity of active promoters. However, under conditions of silencing, in which cells reverted to a state with no measurable EGFP expression, MAPK activated silent promoters. Furthermore, studies of populations of EGFP-expressing and non-EGFP-expressing cells isolated by flow cytometry showed that MAPK activation converted nonexpressing cells into expressing cells and increased expression in expressing cells. These results support a model in which MAPK elicits both graded and stochastic responses to increase HS2-mediated transactivation from single chromatin templates.

5' Untranslated Regions↗
PubMed1999

Inhibition of mitogen-activated kinase signaling sensitizes HeLa cells to Fas receptor-mediated apoptosis.

The Fas receptor (FasR) is an important physiological mediator of apoptosis in various tissues and cells. However, there are also many FasR-expressing cell types that are normally resistant to apoptotic signaling through this receptor. The mitogen-activated protein kinase (MAPK) signaling cascade has, apart from being a growth-stimulating factor, lately received attention as an inhibitory factor in apoptosis. In this study, we examined whether MAPK signaling could be involved in protecting FasR-insensitive cells. To this end, we used different approaches to inhibit MAPK signaling in HeLa cells, including treatment with the MAPK kinase inhibitor PD 98059, serum withdrawal, and expression of dominant-interfering MAPK kinase mutant protein. All of these treatments were effective in sensitizing the cells to FasR-induced apoptosis, demonstrating that MAPK indeed is involved in the control of FasR responses. The MAPK-mediated control seemed to occur at or upstream of caspase 8, the initiator caspase in apoptotic FasR responses. Transfection with the constitutively active MAPK kinase abrogated FasR-induced apoptosis also in the presence of cycloheximide, indicating that the MAPK-generated suppression of FasR-mediated apoptotic signaling is protein synthesis independent. In cells insensitive to FasR-induced apoptosis, stimulation of the FasR with an agonistic antibody resulted in significant MAPK activation, which was inhibited by PD 98059. When different cell types were compared, the FasR-mediated MAPK activation seemed proportional to the degree of FasR insensitivity. These results suggest that the FasR insensitivity is likely to be a consequence of FasR-induced MAPK activation, which in turn interferes with caspase activation.

Apoptosis↗
PubMed1998

Activation of the MKK/ERK pathway during somatic cell mitosis: direct interactions of active ERK with kinetochores and regulation of the mitotic 3F3/2 phosphoantigen.

The mitogen-activated protein (MAP) kinase pathway, which includes extracellular signal-regulated protein kinases 1 and 2 (ERK1, ERK2) and MAP kinase kinases 1 and 2 (MKK1, MKK2), is well-known to be required for cell cycle progression from G1 to S phase, but its role in somatic cell mitosis has not been clearly established. We have examined the regulation of ERK and MKK in mammalian cells during mitosis using antibodies selective for active phosphorylated forms of these enzymes. In NIH 3T3 cells, both ERK and MKK are activated within the nucleus during early prophase; they localize to spindle poles between prophase and anaphase, and to the midbody during cytokinesis. During metaphase, active ERK is localized in the chromosome periphery, in contrast to active MKK, which shows clear chromosome exclusion. Prophase activation and spindle pole localization of active ERK and MKK are also observed in PtK1 cells. Discrete localization of active ERK at kinetochores is apparent by early prophase and during prometaphase with decreased staining on chromosomes aligned at the metaphase plate. The kinetochores of chromosomes displaced from the metaphase plate, or in microtubule-disrupted cells, still react strongly with the active ERK antibody. This pattern resembles that reported for the 3F3/2 monoclonal antibody, which recognizes a phosphoepitope that disappears with kinetochore attachment to the spindles, and has been implicated in the mitotic checkpoint for anaphase onset (Gorbsky and Ricketts, 1993. J. Cell Biol. 122:1311-1321). The 3F3/2 reactivity of kinetochores on isolated chromosomes decreases after dephosphorylation with protein phosphatase, and then increases after subsequent phosphorylation by purified active ERK or active MKK. These results suggest that the MAP kinase pathway has multiple functions during mitosis, helping to promote mitotic entry as well as targeting proteins that mediate mitotic progression in response to kinetochore attachment.

3T3 Cells↗
PubMed1998

Mitogen-activated protein kinases (Erk1,2) phosphorylate Lys-Ser-Pro (KSP) repeats in neurofilament proteins NF-H and NF-M.

Mammalian neurofilament proteins, particularly midsized (NF-M) and heavy (NF-H) molecular weight neurofilament proteins, are highly phosphorylated in axons. Neurofilament function depends on the state of phosphorylation of the numerous serine/threonine residues in these proteins. Most phosphorylation occurs in the lys-ser-pro (KSP) repeats in the C-terminal tail domains of NF-H and NF-M. In our previous study, cyclin-dependent kinase 5 (cdk5) was shown to phosphorylate specifically the KSPXK repeats in rat NF-H. Because 80% of the repeats are of the KSPXXXK type, it was of interest to determine which kinase phosphorylates these motifs. Using a synthetic KSPXXXK peptide to screen for a specific kinase, we fractionated rat brain extracts by column chromatography and identified extracellular signal-regulated kinase (Erk2) activated by an upstream activator, the mitogen-activated protein kinase kinase MAPKK (MEK), by Western blot analysis, sequence identification, and inhibition by a specific MEK inhibitor (PD 98059). The fraction containing Erk2, as well as bacterially expressed Erk1 and Erk2, phosphorylated all types of KSP motifs in peptides (KSPXK, KSPXXK, KSPXXXK, and KSPXXXXK) derived from NF-M and NF-H. They also phosphorylated an expressed 24 KSPXXXK repeat NF-H polypeptide, an expressed NF-H as well as dephosphorylated native rat NF-H, and NF-M proteins with accompanying decreases in their respective electrophoretic mobilities. A comparative kinetic study of Erk2 and cdk5 phosphorylation of KSPXK and KSPXXXK peptides revealed that, in contrast to cdk5, which phosphorylated only the KSPXK peptide, Erk2 could phosphorylate both. The preferred substrate for Erk2 was KSPXXXK peptide. The MEK inhibitor PD 98059 also inhibited phosphorylation of NF-H, NF-M, and microtubule-associated protein (MAP) in primary rat hippocampal cells and caused a decrease in neurite outgrowth, suggesting that Erk1,2 may play an important role in neurite growth and branching. These data suggest that neuronal Erk1 and Erk2 are capable of phosphorylating serine residues in diverse KSP repeat motifs in NF-M and NF-H.

Adenosine Triphosphate↗
PubMed1998

Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor.

Anthrax lethal toxin, produced by the bacterium Bacillus anthracis, is the major cause of death in animals infected with anthrax. One component of this toxin, lethal factor (LF), is suspected to be a metalloprotease, but no physiological substrates have been identified. Here it is shown that LF is a protease that cleaves the amino terminus of mitogen-activated protein kinase kinases 1 and 2 (MAPKK1 and MAPKK2) and that this cleavage inactivates MAPKK1 and inhibits the MAPK signal transduction pathway. The identification of a cleavage site for LF may facilitate the development of LF inhibitors.

Animals↗
PubMed1998

Feedback regulation of Raf-1 and mitogen-activated protein kinase (MAP) kinase kinases 1 and 2 by MAP kinase phosphatase-1 (MKP-1).

Inactivation of growth factor-regulated mitogen-activated protein (MAP) kinases (ERK1 and ERK2) has been proposed to occur in part through dephosphorylation by the dual specificity MAP kinase phosphatase-1 (MKP-1), an immediate early gene that is induced by mitogenic signaling. In this study, we examined the effect of MKP-1 on signaling components upstream of ERK1 and ERK2. Coexpression of MKK1 or MKK2 with MKP-1 resulted in 7-10-fold activation of mitogen-activated protein kinase kinase (MKK), which required the presence of regulatory serine phosphorylation sites. Endogenous MKK1 and MKK2 were also activated upon MKP-1 expression. Raf-1, a direct regulator of MKK1 and MKK2, was activated under these conditions, and a synergistic activation of MKK was observed upon coexpression of Raf-1 and MKP-1. This effect did not appear to involve synthesis of autocrine growth factors or the inhibition of basal extracellular signal-regulated kinase (ERK) activity but was inhibited by a dominant negative Ras mutant, indicating that MKP-1 enhances Ras-dependent activation of Raf-1 in a cell autonomous manner. This study demonstrates positive feedback regulation of Raf-1 and MKK by the MKP-1 immediate early gene and a potential mechanism for activating Raf-1/MKK signaling pathways alternative to those involving ERK.

3T3 Cells↗
PubMed1998

Deuterium exchange mass spectrometry as a probe of protein kinase activation. Analysis of wild-type and constitutively active mutants of MAP kinase kinase-1.

Wild-type and constitutively active mutants of human MAP kinase kinase-1 (MKK1) were analyzed by deuterium exchange mass spectrometry using a protocol that minimized loss of deuterium during analysis due to back exchange. The observed peptides accounted for 335 out of 393 residues. Not counting overlap peptides, three peptides showed decreased exchange in constitutively active compared to wild-type MKK1 and nine showed increased exchange. Backbone amides in which exchange rates decreased upon kinase activation were observed near the regulatory phosphorylation sites Ser218 and Ser222 and the adjacent beta9 strand. These decreases are consistent with electrostriction or reduced solvent access due to domain closure or formation of new hydrogen or salt bonds around the catalytic cleft and within the activation lip. Increased exchange upon activation was observed within six peptides derived from helix C and the five-stranded beta sheet from the N-proximal lobe of the conserved kinase domain and in one peptide located at the interface between the N- and C-proximal lobes. Two amides that underwent increased exchange were specifically localized between residues 68 and 69 in beta1 and 140 and 142 in beta5. These residues probably form contacts with each other on opposite sites of the beta sheet as well as with helix C. These increases appeared to represent localized fluctuations, rather than rigid body rearrangements, suggesting that MKK1 activation requires enhanced flexibility within the N-proximal lobe, perhaps to accommodate ATP binding, phosphotransfer, or ADP release.

Adenosine Triphosphate↗
PubMed1998

Activation of the mitogen-activated protein kinase pathway in U937 leukemic cells induces phosphorylation of the amino terminus of the TATA-binding protein.

Phorbol ester treatment of U937 leukemic cells results in the activation of numerous protein kinase pathways, followed by cell cycle arrest and differentiation into macrophage-like cells. Because major changes in gene transcription are associated with this process, the role of general transcription factors in the cell response to phorbol esters was examined. Experiments demonstrate that phorbol ester treatment of U937 cells stimulates the phosphorylation of the TATA-binding protein (TBP); this phosphorylation occurs within 30 min and is still apparent, although greatly reduced, at 4 h. The following results demonstrate that TBP phosphorylation occurs as a result of activation of an extracellular signal-regulated kinase (ERK) protein kinase: (a) overexpression of mitogen-activated protein kinase phosphatase-1 blocks phorbol 12-myristate 13-acetate (PMA)-induced phosphorylation of TBP both in vitro and in vivo; (b) pretreatment with the ERK kinase kinase inhibitor PD098059 also blocks PMA-induced phosphorylation of TBP both in vitro and in vivo; and (c) phosphorylation of TBP is observed when serum-starved NIH 3T3 cells are stimulated with fresh serum, another activator of the ERK pathway. TBP can be phosphorylated in vitro by extracts of U937 cells or by bacterially expressed activated ERK2; the phosphorylation sites were mapped to ERK kinase consensus sites in the TBP amino-terminal domain. Using glutathione S-transferase-TBP fusion proteins, cellular proteins that bind specifically to the TBP amino terminus have been identified. These observations suggest that ERK-mediated phosphorylation of TBP occurs during the PMA-induced differentiation of U937 cells and the stimulation of the G0-G1 transition in fibroblasts and could play a role in the regulation of TBP protein interactions and thus regulate gene transcription during these two processes.

3T3 Cells↗
PubMed1997

Phosphorylation within the transactivation domain of adenovirus E1A protein by mitogen-activated protein kinase regulates expression of early region 4.

A critical role of the 289-residue (289R) E1A protein of human adenovirus type 5 during productive infection is to transactivate expression of all early viral transcription. Sequences within and proximal to conserved region 3 (CR3) promote expression of these viral genes through interactions with a variety of transcription factors requiring the zinc binding motif in CR3 and in some cases a region at the carboxy-terminal end of CR3, including residues 183 to 188. It is known that 3',5' cyclic AMP (cAMP) reduces the level of phosphorylation of the 289R E1A protein through the activation of protein phosphatase 2A by the E4orf4 protein. This study was designed to identify the E1A phosphorylation sites affected by E4orf4 expression and to determine their importance in regulation of E1A activity. We report here that two previously unidentified sites at Ser-185 and Ser-188 are the targets for decreased phosphorylation in response to cAMP. At least one of these sites, presumably Ser-185, is phosphorylated in vitro by purified mitogen-activated protein kinase (MAPK), and both are hyperphosphorylated in cells which express a constitutively active form of MAPK kinase. Analysis of E1A-mediated transactivation activity indicated that elevated phosphorylation at these sites increased expression of the E4 promoter but not that of E3. We have recently shown that one or more E4 products induce cell death due to p53-independent apoptosis, and thus it seems likely that one role of the E4orf4 protein is to limit production of toxic E4 products by limiting expression of the E4 promoter.

Adenovirus E1A Proteins↗
PubMed1997

Megakaryocytic differentiation induced by constitutive activation of mitogen-activated protein kinase kinase.

The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic stem cells. Phorbol esters, which initiate megakaryocyte differentiation in this cell line, caused a rapid increase in extracellular-signal-regulated kinase (ERK), which remained elevated for 2 h and returned to near-basal levels by 24 h. In the absence of extracellular stimuli, ERK could be activated by expression of constitutively active mutants of mitogen-activated protein (MAP) kinase kinase (MKK), resulting in cell adhesion and spreading, increased cell size, inhibition of cell growth, and induction of the platelet-specific integrin alphaIIb beta3, all hallmarks of megakaryocytic differentiation. In contrast, expression of wild-type MKK had little effect. In addition, constitutively active MKK suppressed the expression of an erythroid marker, alpha-globin, indicating the ability to suppress cellular responses necessary for alternative cell lineages. The MKK inhibitor PD98059 blocked MKK/ERK activation and cellular responses to phorbol ester, demonstrating that activation of MKK is necessary and sufficient to induce a differentiation program along the megakaryocyte lineage. Thus, the MAP kinase cascade, which promotes cell growth and proliferation in many cell types, instead inhibits cell proliferation and initiates lineage-specific differentiation in K562 cells, establishing a model system to investigate the mechanisms by which this signal transduction pathway specifies cell fate and developmental processes.

Base Sequence↗
PubMed1996

Interdependent domains controlling the enzymatic activity of mitogen-activated protein kinase kinase 1.

The activation of human mitogen-activated protein kinase kinase 1 (MKK1) is achieved by phosphorylation at Ser218 and Ser222 within a regulatory loop. Partial activation was achieved by replacing these residues with aspartic/glutamic acid. Higher activity was obtained by introducing four acidic residue substitutions in the regulatory loop, indicating that acidic residues in the loop stabilize an active configuration by the introduction of negative charge. Activation of MKK1 is also achieved by deleting residues 44-51, N-terminal to the consensus catalytic core. Although substitution of residues within this segment by alanine does not affect activity, introduction of proline residues elevates kinase activity, indicating that activation results from perturbation of secondary structure within residues 44-51. Pseudosubstrate inhibition, a commonly observed mechanism of kinase regulation, is not operative in this process. Both the acidic substitutions and the N-terminal deletion increase Vmax, V/K(m),ERK2, and V/K(m),ATP, as is also observed following phosphorylation of wild-type MKK1. A synergistic enhancement of these steady-state rate parameters occurs upon combining the mutations, suggesting that conformational changes induced by mutagenesis together mimic those seen upon phosphorylation.

Amino Acid Sequence↗
PubMed1996

Mass spectrometric analysis of 40 S ribosomal proteins from Rat-1 fibroblasts.

Although sequences of most mammalian ribosomal proteins are available, little is known about the post-translational processing of ribosomal proteins. To examine their post-translational modifications, 40 S subunit proteins purified from Rat-1 fibroblasts and their peptides were analyzed by liquid chromatography coupled with electrospray mass spectrometry. Of 41 proteins observed, 36 corresponded to the 32 rat 40 S ribosomal proteins with known sequences (S3, S5, S7, and S24 presented in two forms). The observed masses of S4, S6-S8, S13, S15a, S16, S17, S19, S27a, S29, and S30 matched those predicted. Sa, S3a, S5, S11, S15, S18, S20, S21, S24, S26-S28, and an S7 variant showed changes in mass that were consistent with N-terminal demethionylation and/or acetylation (S5 and S27 also appeared to be internally formylated and acetylated, respectively). S23 appeared to be internally hydroxylated or methylated. S2, S3, S9, S10, S12, S14, and S25 showed changes in mass inconsistent with known covalent modifications (+220, -75, +86, +56, -100, -117, and -103 Da, respectively), possibly representing novel post-translational modifications or allelic sequence variation. Five unidentified proteins (12,084, 13,706, 13,741, 13,884, and 34, 987 Da) were observed; for one, a sequence tag (PPGPPP), absent in any known ribosomal proteins, was determined, suggesting that it is a previously undescribed ribosome-associated protein. This study establishes a powerful method to rapidly analyze protein components of large biological complexes and their covalent modifications.

Amino Acid Sequence↗
PubMed1996

Positive feedback between MAP kinase and Mos during Xenopus oocyte maturation.

Mos is a serine-threonine protein kinase and a key regulator of meiosis. One function of Xenopus Mos is to activate mitogen-activated protein kinase (MAPK) through direct phosphorylation and activation of MAPK kinase (MAPKK). All three members of this signal cascade can individually induce hormone-independent reentry of oocytes into meiosis I. However, their inducing efficiency is reduced in the absence of protein synthesis. Here we show that de novo Mos synthesis is required for induction of meiosis I by active MAPKK or Mos-MAPK coinjection. In addition, MAPK efficiently phosphorylates Mos at Ser-3 in vitro. These results suggest that a positive feedback loop exists between MAPK and Mos during oocyte maturation. De novo synthesis of Mos, and other proteins, is required for progression from meiosis I to the metaphase arrest at meiosis II; therefore, one function of MAPK during normal Xenopus oocyte maturation might be to stimulate the synthesis or accumulation of Mos that is required for the completion of meiosis.

Animals↗
PubMed1996

Galpha12 and Galpha13 regulate extracellular signal-regulated kinase and c-Jun kinase pathways by different mechanisms in COS-7 cells.

Many growth factors and agonists for G protein-coupled receptors activate mitogen-activated protein (MAP) kinase pathways, including the extracellular signal-regulated kinase (ERK) pathway and the c-Jun kinase (JNK) pathway. Transient transfection of dominant negative and constitutively active pathway components in COS-7 cells shows that two G protein subunits, Galpha12 and Galpha13, inhibit the ERK pathway and stimulate the JNK pathway. Constitutively active (GTPase-deficient) Galpha12 and Galpha13 both inhibit ERK pathway activation by epidermal growth factor. A Galpha13/alphaz chimera, which responds to stimulation by Gi-coupled receptors, mediates inhibition of ERK via such a receptor, the dopamine-2 receptor. In addition, expression of a dominant negative mutant of the GTPase, Cdc42, blocks activation of the JNK pathway by Galpha12 and Galpha13 but does not alter inhibition of ERK activation by the same Galpha proteins; conversely, mutationally activated Cdc42 stimulates the JNK pathway but has no effect on the ERK pathway. Our results show that different mechanisms mediate two effects of Galpha12 and Galpha13: the ERK pathway inhibition is mediated at the level of MAP kinase kinase in a Ras- and Raf-independent fashion, whereas the JNK pathway stimulation is mediated by Cdc42.

Animals↗
PubMed1996

Induction of mitogen-activated protein kinase phosphatase 1 by the stress-activated protein kinase signaling pathway but not by extracellular signal-regulated kinase in fibroblasts.

The intracellular mechanisms involved in the activation of extracellular signal-regulated kinase (ERK) are relatively well understood. However, the intracellular signaling pathways which regulate the termination of ERK activity remain to be elucidated. Mitogen-activated protein kinase phosphatase 1 (MKP-1) has been shown to dephosphorylate and inactivate ERK in vitro and in vivo. In the present study, we show in NIH3T3 fibroblasts that activation of the stress-activated protein kinase (SAPK) pathway by either specific extracellular stress stimuli or via induction of MEKK, an upstream kinase of SAPK, results in MKP-1 gene expression. In contrast, selective stimulation of the ERK pathway by 12-O-tetradecanoylphorbol-13-acetate or following expression of constitutively active MEK, the upstream dual specificity kinase of ERK did not induce the transcription of MKP-1. Hence, these findings demonstrate the existence of cross-talk between the ERK and SAPK signaling cascades since activation of SAPK induced the expression of MKP-1 that can inactivate ERK. This mechanism may modulate the cellular response to stimuli which employ the SAPK signal transduction pathway.

3T3 Cells↗
PubMed1996

Constitutively active mitogen-activated protein kinase kinase 1 (MAPKK1) and MAPKK2 mediate similar transcriptional and morphological responses.

Both mitogen-activated protein kinase kinase 1 (MAPKK1) and MAPKK2 function downstream of the proto-oncogene product Raf in signaling pathways that affect cell proliferation and differentiation. The isoforms were previously shown to be differentially regulated in two significant ways: (a) MAPKK1, but not MAPKK2, was phosphorylated and inactivated by the cyclin-dependent kinase p34cdc2; and (b) p21 Ras formed a ternary complex with Raf/MAPKK1 but not with Raf/MAPKK2. To further characterize the regulation and function of the two isoforms, we compared their mode of activation by v-Mos and examined the transcriptional and morphological responses that they mediate in cultured mammalian cells. v-Mos enhanced the enzymatic activity of both isoforms to the same extent, by about 600-fold. Constitutively active MAPKK2 mutants were generated by introducing the same deletion and amino acid substitutions that have been shown to activate MAPKK1, suggesting that the conformational changes that lead to their activation are analogous. These mutants potentiated transcription from a promoter containing AP1-responsive elements and induced morphological transformation when expressed in mammalian cells, matching outcomes observed with constitutively active MAPKK1. The specific activity of p42 MAPK in the transformed cells was 3-fold higher than in cells expressing wild-type MAPKK, thereby implicating p42 MAPK as a common effector in vivo, and suggesting that sustained activation of p42 MAPK may represent a critical factor that contributes to the development of the transformed state. Altogether, the results demonstrate that the two isoforms elicit similar responses in vivo despite differences in their regulation.

3T3 Cells↗