1: Oncogene. 2005 Apr 14;24(16):2654-66. 

A 5'-distal enhanceosome in the PDGF-A gene is activated in choriocarcinoma
cells via ligand-independent binding of vitamin D receptor and constitutive jun
kinase signaling.

Pedigo NG, Zhang H, Bruno ME, Kaetzel CS, Dugan AR, Shanehsaz P, Hennigan RF,
Xing Z, Koszewski NJ, Kaetzel DM.

Department of Molecular and Biomedical Pharmacology, University of Kentucky
College of Medicine, Lexington, KY 40536, USA.

Overexpression of platelet-derived growth factor A-chain (PDGF-A) is clearly
linked to autocrine and paracrine stimulation of malignant growth in many human
cancers. We have shown previously that PDGF-A overexpression in choriocarcinoma,
hepatoma and lung carcinoma cell lines is driven by the activity of a 66 bp
enhancer element (ACE66) located approximately 7 kb upstream of the PDGF-A
transcription start site. In this study, the ACE66 element is shown to be
activated in JEG-3 choriocarcinoma cells through synergistic interactions
between consensus DNA motifs for binding of vitamin D receptor, AP1 and ELK1.
Binding of the vitamin D/retinoid-X receptor (VDR/RXRalpha) heterodimer to the
ACE66 element was reconstituted in vitro with recombinant VDR/RXRalpha and with
JEG-3 nuclear extract, and was verified in living JEG-3 cells by chromatin
immunoprecipitation analysis. Transcriptional activity of the ACE66 element, as
well as occupancy of the element by VDR/RXRalpha, was shown to be independent of
stimulation with the hormonal VDR ligand, 1,25-dihydroxyvitamin D3. The jun
kinase pathway of mitogen-activated protein kinase (MAPK) signaling was shown to
activate the ACE66 enhancer, most likely through activation of factors binding
to the AP1 element. These results identify a novel mechanism of transcriptional
enhancement involving ligand-independent activity of the VDR/RXR heterodimer and
MAPK signaling pathways that appears to play an important role in the
overexpression of PDGF in many different settings of human malignancy.

PMID: 15829977 [PubMed - indexed for MEDLINE]


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2: Oncogene. 2005 Jun 16;24(26):4193-205. 

Mitogen regulated induction of FRA-1 proto-oncogene is controlled by the
transcription factors binding to both serum and TPA response elements.

Adiseshaiah P, Peddakama S, Zhang Q, Kalvakolanu DV, Reddy SP.

Department of Environmental Health Sciences, Bloomberg School of Public Health,
The Johns Hopkins University, Baltimore, MD 21205, USA.

FRA-1, a member of the FOS family of transcription factors, is overexpressed in
a variety of human tumors, and contributes to tumor progression. In addition to
mitogens, various toxicants and carcinogens persistently induce FRA-1 expression
in vitro and in vivo. Although the mitogen induced expression of c-FOS is
relatively well understood, it is poorly defined in the case of FRA-1. Our
recent analysis of the FRA-1 promoter has shown a critical role for a TRE
located at -318 in mediating the TPA-induced expression. The -379 to -283 bp
promoter segment containing a critical TRE (-318), however, is insufficient for
the induction of FRA-1 promoter. Here, we show that a 40-bp (-276/-237) segment,
comprising a TCF binding site and the CArG box (collectively known as serum
response element, SRE), and an ATF site, is also necessary for the FRA-1
induction by TPA and EGF. Interestingly, the -283 to +32 bp FRA-1 promoter
fragment containing an SRE and an ATF site alone was also insufficient to confer
TPA sensitivity to a reporter gene. However, in association with the -318 TRE,
the SRE and ATF sites imparted a strong TPA-inducibility to the reporter.
Similarly, EGF also required these motifs for the full induction of this gene.
Using ChIP assays we show that, in contrast to c-Jun, SRF, Elk1, ATF1 and CREB
proteins bind to SRE and ATF sites of the FRA-1 promoter, constitutively.
RNAi-mediated knockdown of endogenous SRF, ELK1 and c-JUN protein expression
significantly reduced TPA-stimulated FRA-1 promoter activity. Thus, a bipartite
enhancer formed by an upstream TRE and the downstream SRE and ATF sites and the
cognate factors is necessary and sufficient for the regulation of FRA-1 in
response to mitogens.

PMID: 15806162 [PubMed - indexed for MEDLINE]


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3: J Immunol. 2004 Aug 15;173(4):2552-61. 

Transcriptional regulation of the human TLR9 gene.

Takeshita F, Suzuki K, Sasaki S, Ishii N, Klinman DM, Ishii KJ.

Section of Retroviral Immunology, Center for Biologics Evaluation and Research,
Food and Drug Administration, Bethesda, MD 20892, USA.
takesita@yokohama-cu.ac.jp

To clarify the molecular basis of human TLR9 (hTLR9) gene expression, the
activity of the hTLR9 gene promoter was characterized using the human myeloma
cell line RPMI 8226. Reporter gene analysis and EMSA demonstrated that hTLR9
gene transcription was regulated via four cis-acting elements, cAMP response
element, 5'-PU box, 3'-PU box, and a C/EBP site, that interacted with the CREB1,
Ets2, Elf1, Elk1, and C/EBPalpha transcription factors. Other members of the
C/EBP family, such as C/EBPbeta, C/EBPdelta, and C/EBPepsilon, were also
important for TLR9 gene transcription. CpG DNA-mediated suppression of TLR9 gene
transcription led to decreased binding of the trans-acting factors to their
corresponding cis-acting elements. It appeared that suppression was mediated via
c-Jun and NF-kappaB p65 and that cooperation among CREB1, Ets2, Elf1, Elk1, and
C/EBPalpha culminated in maximal transcription of the TLR9 gene. These findings
will help to elucidate the mechanism of TLR9 gene regulation and to provide
insight into the process by which TLR9 evolved in the mammalian immune system.

PMID: 15294971 [PubMed - indexed for MEDLINE]


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4: J Mol Med. 2004 Aug;82(8):530-8. Epub 2004 May 6. 

The zinc finger transcription factor Egr-1 is upregulated in arsenite-treated
human keratinocytes.

Al-Sarraj A, Thiel G.

Department of Medical Biochemistry and Molecular Biology, Building 44,
University of Saarland Medical Center, 66421 Homburg, Germany.

Arsenite is a human carcinogen that may induce cancer in skin, liver, kidney,
bladder or lung. Arsenite executes its toxic effects by the induction of
signaling cascades. In particular, the activation of the stress-induced protein
kinase c-Jun N-terminal protein kinase and p38 and the phosphorylation and
activation of the transcription factor c-Jun have been linked to the biological
effects of arsenite. We analyzed whether arsenite has an impact on the
biosynthesis of the zinc finger transcription factor Egr-1. Egr-1 transcription
is upregulated following treatment of cells with hormones, cytokines or toxic
chemicals, and thus Egr-1 integrates many signaling cascades with changes in
gene expression patterns. Here, we show by Western blot experiments that
arsenite induces a transient synthesis of Egr-1 in human HaCaT keratinocytes.
Egr-1 biosynthesis was activated by arsenite concentrations insufficient for the
induction of c-Jun biosynthesis. This arsenite-triggered Egr-1 biosynthesis was
completely inhibited by the mitogen-activated protein kinase kinase inhibitor
PD98059 and by AG1487, an epidermal growth factor (EGF) receptor-specific
tyrosine kinase inhibitor. These results indicate that activation of the EGF
receptor as well as stimulation of the mitogen activated/extracellular
signal-regulated protein kinase is essential for arsenite-induced upregulation
of Egr-1. Moreover, we detected an elevated transcriptional activation potential
of the ternary complex factor Elk1, a key transcriptional regulator of serum
response element-driven gene transcription. The Egr-1 5'-flanking region
contains five serum response elements. Accordingly, we observed an increase in
Egr-1 promoter activity as a result of arsenite treatment. The fact that low
concentrations of arsenite are sufficient to induce Egr-1 biosynthesis suggests
that Egr-1 may be an integral part of arsenite-triggered signaling cascades
leading to tumor formation or cell death via alterations of the cellular genetic
program.

PMID: 15292961 [PubMed - indexed for MEDLINE]


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5: Mol Cell Biol. 2004 Jun;24(11):4613-26. 

Mitochondrial AKAP121 binds and targets protein tyrosine phosphatase D1, a novel
positive regulator of src signaling.

Cardone L, Carlucci A, Affaitati A, Livigni A, DeCristofaro T, Garbi C, Varrone
S, Ullrich A, Gottesman ME, Avvedimento EV, Feliciello A.

Dipartimento di Biologia e Patologia Molecolare e Cellulare, via S. Pansini, 5,
80131 Naples, Italy. feliciel@unina.it

A-kinase anchor protein 121 (AKAP121) and its spliced isoform AKAP84 anchor
protein kinase A (PKA) to the outer membrane of mitochondria, focusing and
enhancing cyclic AMP signal transduction to the organelle. We find that
AKAP121/84 also binds PTPD1, a src-associated protein tyrosine phosphatase. A
signaling complex containing AKAP121, PKA, PTPD1, and src is assembled in vivo.
PTPD1 activates src tyrosine kinase and increases the magnitude and duration of
epidermal growth factor (EGF) signaling. EGF receptor phosphorylation and
downstream activation of ERK 1/2 and Elk1-dependent gene transcription are
enhanced by PTPD1. Expression of a PTPD1 mutant lacking catalytic activity
inhibits src and downregulates ERK 1/2 but does not affect the activity of c-Jun
N-terminal kinase 1/2 and p38alpha mitogen-activated protein kinase. AKAP121
binds to and redistributes PTPD1 from the cytoplasm to mitochondria and inhibits
EGF signaling. Our findings indicate that PTPD1 is a novel positive regulator of
src signaling and a key component of the EGF transduction pathway. By binding
and/or targeting the phosphatase on mitochondria, AKAP121 modulates the
amplitude and persistence of src-dependent EGF transduction pathway. This
represents the first example of physical and functional interaction between
AKAPs and a protein tyrosine phosphatase.

PMID: 15143158 [PubMed - indexed for MEDLINE]


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6: J Cell Biochem. 2004 May 15;92(2):258-69. 

P53 down-regulates matrix metalloproteinase-1 by targeting the communications
between AP-1 and the basal transcription complex.

Sun Y, Zeng XR, Wenger L, Firestein GS, Cheung HS.

Department of Medicine, University of Miami School of Medicine, Miami, Florida
33101, USA. ysun@med.miami.edu

We have previously reported that human matrix metalloproteinase-1 (MMP1) is a
p53 target gene subject to down-regulation (Sun et al. [1999]: J Biol Chem
274:11535-11540]. In the present study, we demonstrate that the down-regulation
of the human -83MMP1 promoter fragment by p53 was abolished when the -72AP-1
site was eliminated and that a GAL4-cJun-mediated but not a GAL4-Elk1-mediated
induction of pFR-luci was effectively inhibited by p53 suggesting an AP-1
dependent but AP-1 binding independent mechanism. Results from gel mobility
shift assays were consistent with an AP-1 binding independent mechanism. We also
demonstrate that both p300 and TATA box binding proteins cooperated with the
transcription factor AP-1 to induce the promoter of MMP1; however, p53 only
inhibited the p300-mediated induction of the MMP1 promoter and the inhibition
was -72AP-1 dependent. Furthermore, the down-regulation of the MMP1 promoter and
mRNA by p53 could be reversed by p300 and by a p53 binding p300 fragment that
had no coactivator activity. Taken together, these results indicate that p53
down-regulates MMP1 mainly by disrupting the communications between the
transactivator AP-1 and the basal transcriptional complex, which are partially
mediated by p300. Finally, by using p53 truncated mutant constructs, we
demonstrate that both the N-terminal activation domain and the C-terminal
oligomerization domains of p53 were required for the down-regulation of MMP1
transcription. Copyright 2004 Wiley-Liss, Inc.

PMID: 15108353 [PubMed - indexed for MEDLINE]


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7: Eur J Neurosci. 2003 Aug;18(3):473-85. 

N-methyl-d-aspartate-triggered neuronal death in organotypic hippocampal
cultures is endocytic, autophagic and mediated by the c-Jun N-terminal kinase
pathway.

Borsello T, Croquelois K, Hornung JP, Clarke PG.

Institut de Biologie cellulaire et de Morphologie, Universite de Lausanne, Rue
du Bugnon 9, CH-1005, Switzerland.

Acute excitotoxic neuronal death was studied in rat organotypic hippocampal
slices exposed to 100 micro mN-methyl-d-aspartate. Fulgurant death of pyramidal
neurons occurred in the CA1 and CA3 regions and was already detectable within 2
h of the N-methyl-d-aspartate administration. Morphologically, the neuronal
death was neither apoptotic nor necrotic but had the hallmarks of autophagic
neuronal death, as shown by acid phosphatase histochemistry in both CA1 and CA3
and by electron microscopy in CA1. The dying neurons also manifested strong
endocytosis of horseradish peroxidase or microperoxidase, occurring probably by
a fluid phase mechanism, and followed, surprisingly, by nuclear entry. In
addition to these autophagic and endocytic characteristics, there were
indications that the c-Jun N-terminal kinase pathway was activated. Its target
c-Jun was selectively phosphorylated in CA1, CA3 and the dentate gyrus and
c-Fos, the transcription of which is under the positive control of c-Jun
N-terminal kinase target Elk1, was selectively up-regulated in CA1 and CA3. All
these effects, the neuronal death itself and the associated autophagy and
endocytosis, were totally prevented by a cell-permeable inhibitor of the
interaction between c-Jun N-terminal kinase and certain of its targets. These
results show that pyramidal neurons undergoing excitotoxic death in this
situation are autophagic and endocytic and that both the cell death and the
associated autophagy and endocytosis are under the control of the c-Jun
N-terminal kinase pathway.

PMID: 12911744 [PubMed - indexed for MEDLINE]


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8: Mol Cancer Res. 2003 May;1(7):541-50. 

Protein tyrosine phosphatase epsilon inhibits signaling by mitogen-activated
protein kinases.

Toledano-Katchalski H, Kraut J, Sines T, Granot-Attas S, Shohat G, Gil-Henn H,
Yung Y, Elson A.

Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot
76100, Israel.

Mitogen-activated protein kinases (MAPKs) mediate signaling from the cell
membrane to the nucleus following their phosphorylation at conserved threonine
and tyrosine residues within their activation loops. We show that protein
tyrosine phosphatase epsilon (PTP epsilon) inhibits ERK1 and ERK2 kinase
activity and reduces their phosphorylation; in agreement, ERK phosphorylation is
increased in fibroblasts and in mammary tumor cells from mice genetically
lacking PTP epsilon. PTP epsilon inhibits events downstream of ERKs, such as
transcriptional activation mediated by Elk1 or by the serum response element.
PTP epsilon also inhibits transcriptional activation mediated by c-Jun and C/EBP
binding protein (CHOP) but not that mediated by the unrelated NFkB, attesting
that it is broadly active within the MAPK family but otherwise specific. The
effect of PTP epsilon on ERKs is at least in part indirect because
phosphorylation of the threonine residue in the ERK activation loop is reduced
in the presence of PTP epsilon. Nonetheless, PTP epsilon is present in a
molecular complex with ERK, providing PTP epsilon with opportunity to act on ERK
proteins also directly. We conclude that PTP epsilon is a physiological
inhibitor of ERK signaling. Slow induction of PTP epsilon and its lack of
nuclear translocation following mitogenic stimulation suggest that PTP epsilon
functions to prevent inappropriate activation and to terminate prolonged, rather
than acute, activation of ERK in the cytosol.

PMID: 12754301 [PubMed - indexed for MEDLINE]


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9: Am J Physiol Cell Physiol. 2003 Oct;285(4):C922-34. Epub 2003 May 7. 

G alpha 13-mediated transformation and apoptosis are permissively dependent on
basal ERK activity.

Adarichev VA, Vaiskunaite R, Niu J, Balyasnikova IV, Voyno-Yasenetskaya TA.

Department of Pharmacology, University of Illinois, Chicago, IL 60612, USA.

We previously reported that the alpha-subunit of heterotrimeric G13 protein
induces either mitogenesis and neoplastic transformation or apoptosis in a
cell-dependent manner. Here, we analyzed which signaling pathways are required
for G alpha 13-induced mitogenesis or apoptosis using a novel mutant of G alpha
13. We have identified that in human cell line LoVo, the mutation encoding
substitution of Arg260 to stop codon in mRNA of G alpha 13 subunit produced a
mutant protein (G alpha 13-T) that lacks a COOH terminus and is endogenously
expressed in LoVo cells as a polypeptide of 30 kDa. We found that G alpha 13-T
lost its ability to promote proliferation and transformation but retained its
ability to induce apoptosis. We found that full-length G alpha 13 could
stimulate Elk1 transcription factor, whereas truncated G alpha 13 lost this
ability. G alpha 13-dependent stimulation of Elk1 was inhibited by
dominant-negative extracellular signal-regulated kinase (MEK) but not by
dominant-negative MEKK1. Similarly, MEK inhibitor PD-98059 blocked G alpha
13-induced Elk1 stimulation, whereas JNK inhibitor SB-203580 was ineffective. In
Rat-1 fibroblasts, G alpha 13-induced cell proliferation and foci formation were
also inhibited by dominant-negative MEK and PD-98059 but not by
dominant-negative MEKK1 and SB-203580. Whereas G alpha 13-T alone did not induce
transformation, coexpression with constitutively active MEK partially restored
its ability to transform Rat-1 cells. Importantly, full-length but not G alpha
13-T could stimulate Src kinase activity. Moreover, G alpha 13-dependent
stimulation of Elk1, cell proliferation, and foci formation were inhibited by
tyrosine kinase inhibitor, genistein, or by dominant-negative Src kinase,
suggesting the involvement of a Src-dependent pathway in the G alpha 13-mediated
cell proliferation and transformation. Importantly, truncated G alpha 13
retained its ability to stimulate apoptosis signal-regulated kinase ASK1 and
c-Jun terminal kinase, JNK. Interestingly, the apoptosis induced by G alpha 13-T
was inhibited by dominant-negative ASK1 or by SB-203580.

PMID: 12736137 [PubMed - indexed for MEDLINE]


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10: Anticancer Res. 2002 Mar-Apr;22(2A):799-804. 

The retinoid-inducible gene I: effect on apoptosis and mitogen-activated kinase
signal pathways.

Huang SL, Shyu RY, Yeh MY, Jiang SY.

Graduate Institute of Life Sciences, National Defense Medical Center, Taipei,
Taiwan.

BACKGROUND: The retinoid-inducible gene I (RIG1), belonging to the family of
type II tumor suppressor genes, was isolated from human gastric cancer cells
treated with all-trans retinoic acid. The activity of the RIG1 gene was
investigated in this study. MATERIALS AND METHODS: HtTA cervical and TSGH9201
gastric cancer cells were transiently transfected with expression vectors that
synthesized RIG1-myc or RIG1-EGFP fusion protein. Cell growth was analyzed by
measuring the incorporation of bromodeoxyuridine. Apoptosis was evaluated by the
formation of in situ DNA breakage. The activities of mitogen-activated kinase
signal pathways were analyzed using signal pathway trans-reporting systems.
RESULTS: Expression of the RIG1-myc fusion protein resulted in decreased cell
growth. Both RIG1-EGFP and RIG1-myc fusion proteins induced cellular apoptosis
that was characterized by the presence of apoptotic bodies and in situ DNA
breakage. The transactivation activities of Elk1, c-Jun and CHOP proteins were
suppressed by 80, 50 and 88%, respectively, in HtTA cells expressing the
RIG1-myc fusion protein for two days. Similarly, the transactivation activities
of the CHOP protein was suppressed in TSGH9201 and HtTA cells transiently
expressing RIG1-myc and RIG1-EGFP, respectively. CONCLUSION: The RIG1 fusion
proteins exhibited growth suppressive and apoptosis-inducing activity. The
protein negatively-regulated signal pathways of extracellular signal-regulated
kinase, c-Jun N-terminal kinase and p38 mitogen-activated kinase.

PMID: 12014653 [PubMed - indexed for MEDLINE]


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11: Blood. 2002 Feb 15;99(4):1388-97. 

Regulation of the Erk2-Elk1 signaling pathway and megakaryocytic differentiation
of Bcr-Abl(+) K562 leukemic cells by Gab2.

Dorsey JF, Cunnick JM, Mane SM, Wu J.

Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute,
Tampa, FL 33612, USA.

In the blast crisis phase of chronic myelogenous leukemia (CML), Bcr-Abl(+)
myeloblasts fail to undergo terminal maturation. The extracellular
signal-regulated kinase (Erk) mitogen-activated protein (MAP) kinase has been
shown to mediate terminal differentiation of myeloid cells. Interestingly,
Bcr-Abl(+) CML cell lines established from blast crisis were found to have low
Erk MAP kinase activity. In this study, we analyzed the role of the Gab2 docking
protein in regulation of the Erk MAP kinase in Bcr-Abl(+) K562 human CML cells.
Overexpression of Gab2 in K562 cells resulted in transcriptional activation of
the c-fos serum response element (SRE) promoter, whereas overexpression of SHP2,
Grb2, and CrkL had no effect. Activation of the c-fos SRE transcriptional
activity by Gab2 required tyrosine 604, which is a SHP2 docking site on Gab2,
and the SHP2 tyrosine phosphatase activity. Elk1, c-Jun, and CHOP
trans-reporting assays indicated that overexpression of Gab2 selectively
activated the Erk2-Elk1 signaling pathway. To determine cellular consequences of
elevating the Gab2 level in K562 cells, stable cell lines for
doxycycline-inducible expression of the wild-type Gab2 (Gab2WT) and an
SHP2-binding defective Gab2 (Gab2Tyr604Phe) were established. Analysis of these
cell lines indicated that induction of Gab2WT expression, but not Gab2Tyr604Phe
expression, led to Erk activation, growth arrest, cell spreading, and
enlargement; expression of megakaryocyte/platelet lineage-specific integrins
alphaIIb/beta3 (CD41/CD61); and upregulation of RNA for megakaryocyte/platelet
proteins. All of these changes are characteristics of megakaryocytic
differentiation. Together, these results reveal Gab2 as a limiting signaling
component for Erk MAP kinase activation and terminal differentiation of K562 CML
cells.

PMID: 11830491 [PubMed - indexed for MEDLINE]


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12: J Neurochem. 2002 Jan;80(1):119-25. 

MEK and ERK protect hypoxic cortical neurons via phosphorylation of Bad.

Jin K, Mao XO, Zhu Y, Greenberg DA.

Buck Institute for Age Research, Novato, California 94945, USA.
kjin@buckinstitute.org

We investigated the role of mitogen-activated protein kinase (MAPK) pathways in
hypoxic neuronal injury using primary cultures from murine cerebral cortex.
Hypoxia caused the death of approximately 50% of neurons at 16 h and
approximately 65% of neurons at 24 h. This was associated with
phospho-activation of the MAPK/extracellular signal-regulated kinase (ERK)
kinase MEK1/2 and its downstream target ERK1/2, but not p38 MAPK or c-Jun
N-terminal kinase (JNK), as detected by western blotting. The MEK1/2 inhibitor,
PD98059, increased neuronal death in hypoxic cultures, suggesting that MEK1/2
promotes neuronal survival, whereas the p38 inhibitors, SB202190 and SB203580,
had no effect. To identify downstream effects of ERK1/2 that might regulate
hypoxic neuronal death, we measured hypoxia-induced phosphorylation of three
ERK1/2 targets: the 90-kDa ribosomal protein S6 kinase (RSK), the transcription
factor ELK1, and the pro-apoptotic Bcl-2 family protein Bad. We observed
increased abundance of inactivated (phospho-)Bad, but no change in phospho-RSK
or phospho-ELK1. Moreover, the MEK inhibitor PD98059 reduced
phospho-inactivation of Bad in hypoxic cultures. These findings suggest that a
cell-survival program involving phospho-activation of MEK1/2 and ERK1/2 and
inactivation of Bad is mobilized in hypoxic neurons, and may help to regulate
neuronal fate following hypoxic-ischemic injury.

PMID: 11796750 [PubMed - indexed for MEDLINE]


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13: J Biol Chem. 2002 Mar 8;277(10):8741-8. Epub 2001 Dec 26. 

Selective in vivo inhibition of mitogen-activated protein kinase activation
using cell-permeable peptides.

Kelemen BR, Hsiao K, Goueli SA.

Genencor International, Palo Alto, California 94304, USA.

The extracellular signal-regulated kinase (ERK), a member of the
mitogen-activated protein kinases (MAPKs), is essential for cellular
proliferation and differentiation, and thus there exists great interest to
develop specific and selective inhibitors of this enzyme. Whereas small molecule
inhibitors PD098095 and U0126 have been used to study MAPK/ERK kinase (MEK),
their target selectivity has been questioned recently. The cross-reactivity of
ATP-directed inhibitors with other protein kinases prompted us to develop
structure-based selective peptide inhibitors of ERK activation. Based on a
MEK1-derived peptide, we developed inhibitors of ERK activation in vitro and in
vivo. The inclusion of either an alkyl moiety or a membrane-translocating
peptide sequence facilitated the cellular uptake of the peptide inhibitor and
prevented ERK activation in 4-phorbol 12-myristate 13-acetate-stimulated NIH 3T3
cells or nerve growth factor-treated PC12 cells in a concentration-dependent
manner. In addition, cell-permeable peptides inhibited ERK-mediated activation
of the transcriptional activity of ELK1. The peptides did not have an inhibitory
effect on the activity of two other closely related classes of MAPKs, c-Jun
amino-terminal kinase or p38 protein kinase. Thus, these peptides may serve as
valuable tools for investigating ERK activation and for selective investigation
of ERK-mediated responses. With the knowledge of other kinase interacting
domains, it would be possible to design cell-permeable inhibitors for
investigating diverse cellular signaling mechanisms and for possible therapeutic
applications.

PMID: 11756441 [PubMed - indexed for MEDLINE]


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14: Eur J Cell Biol. 2000 Dec;79(12):924-35. 

Nerve growth factor- and epidermal growth factor-regulated gene transcription in
PC12 pheochromocytoma and INS-1 insulinoma cells.

Groot M, Boxer LM, Thiel G.

Medical Biochemistry and Molecular Biology, University of Saarland Medical
School, Homburg, Germany.

PC12 and INS-1 cells both express the nerve growth factor (NGF) receptors trkA
and p75NTR and the epidermal growth factor receptor (EGF). In PC12 cells, NGF
treatment initiates a signaling cascade that ultimately leads to a change of the
genetic program of the cell. We have investigated the role of NGF in regulating
gene transcription in PC12 and INS-1 cells, in order to define if there are
NGF-regulated genes per se. Furthermore, to distinguish between growth factor
stimulation via receptor tyrosine kinases in general and NGF-specific changes in
gene transcription, we analyzed the effects of EGF on gene transcription. First,
we tested the biological activities of fusion proteins consisting of the
DNA-binding domain of the yeast transcription factor GAL4 and the
phosphorylation-dependent activation domains of the transcription factors Elk1,
CREB, ATF2 and c-jun in NGF- or EGF-treated PC12 cells. We found a striking
increase in the transcriptional activity of the GAL4-Elk1 fusion protein that is
a major substrate for the extracellular signal-regulated protein kinase (ERK).
This effect was observed in NGF- as well as in EGF-treated PC12 cells. In INS-1
cells, however, the activity of the GAL4-Elk1 fusion protein was induced by NGF,
but not by EGF. The effects of NGF and EGF on gene transcription were
subsequently studied with plasmids containing reporter genes under control of
the Egr-1, c-jun, HES-1 or Bc12 regulatory sequences. NGF stimulated Egr-1
promoter activities in PC12 and INS-1 cells, although the effect was much more
pronounced in PC12 cells than in INS-1 cells. EGF also stimulated Egr-1 promoter
activity in both PC12 and INS-1 cells. Stimulation of c-jun promoter activity by
NGF was observed only in PC12 cells. Deletion mutagenesis demonstrated the
importance of the 12-O-tetradecanoylphorbol-13-acetate response elements within
the c-jun promoter for basal and NGF-mediated transcriptional induction.
Likewise, NGF activated HES1 and Bcl2 P1 promoter activities in PC12 cells but
not in INS-1 cells and EGF did not show any effects on these promoters. We
conclude that in PC12 and INS-1 cells, NGF signaling leads to an activation of
the ERK subtype of mitogen-activated protein kinases in the nucleus and a
subsequent activation of Egr-1 gene transcription. The NGF-induced transcription
of the c-jun, HES1 and Bc12 genes is, in contrast, cell type-specific,
indicating that NGF can trigger different gene expression programs dependent on
the signaling pathways present in a particular cell type. EGF is clearly able to
activate gene transcription, suggesting that the differences in the biological
activities of EGF and NGF cannot be explained by the inability of EGF to
stimulate gene transcription.

PMID: 11152283 [PubMed - indexed for MEDLINE]


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15: Thyroid. 2000 Sep;10(9):747-52. 

The thyrotropin receptor is not involved in the activation of p42/p44
mitogen-activated protein kinases by thyrotropin preparations in Chinese hamster
ovary cells expressing the human thyrotropin receptor.

Correze C, Blondeau JP, Pomerance M.

Unite de Recherche, Transduction Hormonale et Regulation Cellulaire, U 486
INSERM, Faculte de Pharmacie, Chatenay-Malabry, France.

We studied whether bovine pituitary thyrotropin (bTSH) or human recombinant
thyrotropin (rhTSH) stimulated p42/p44 mitogen-activated protein kinases (MAPKs)
in Chinese hamster ovary cells expressing human thyrotropin receptor (CHO-hTSHR
cells). We show that p42/p44 MAPK phosphorylation was induced by both TSH
preparations at similar levels in CHO-hTSHR cells and in wild-type CHO cells. In
contrast, cyclic adenosine monophosphate (cAMP) production was stimulated by TSH
only in CHO-hTSHR cells, demonstrating that p42/p44 MAPK stimulation was
independent of the TSH receptor. Moreover, similar results were obtained with
two other cell lines: the FRTL-5 thyroid cell line and the CCL39 fibroblast cell
line. Maximal stimulation of p42/p44 MAPK phosphorylation was observed after a
5- to 10-minute incubation with bTSH and rhTSH preparations. At this time, the
phosphorylation of GST-Elk1 was also increased in a time- and
concentration-dependent manner by bTSH preparations. The phosphorylation of
p42/p44 MAPKs was abolished by PD 98059 and GF 109203X, indicating the
involvement of MAPK kinases (MEK 1/2) and protein kinase C. In contrast, the
activation of p42/p44 MAPKs was insensitive to H89, to cholera toxin and to
pertussis toxin. These data suggest that the protein kinase A pathway was not
implicated in p42/p44 MAPK activation by TSH preparations. Moreover, Gs or Gi/Go
proteins do not appear to participate in p42/p44 MAPK activation. We also showed
that these TSH preparations failed to induce activation of c-Jun NH2 terminal
kinase. We therefore conclude that the commercial TSH preparations used in this
study contained factor(s) responsible for the specific activation of p42/p44
MAPKs by a TSH receptor-independent mechanism.

PMID: 11041451 [PubMed - indexed for MEDLINE]


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16: Biochemistry. 2000 Aug 1;39(30):8917-28. 

Ets domain transcription factor PE1 suppresses human interstitial collagenase
promoter activity by antagonizing protein-DNA interactions at a critical AP1
element.

Bidder M, Loewy AP, Latifi T, Newberry EP, Ferguson G, Willis DM, Towler DA.

Department of Molecular Biology and Pharmacology, Washington University School
of Medicine, St. Louis, Missouri 63110, USA.

In MC3T3E1 calvarial osteoblasts, fibroblast growth factor receptor (FGFR)
signaling elicits multiple transcriptional responses, including upregulation of
the interstitial collagenase/matrix metalloproteinase 1 (MMP1) promoter. FGF
responsiveness maps to a bipartite Ets/AP1 element at base pairs -123 to -61 in
the human MMP1 promoter. Under basal conditions, the MMP1 promoter is repressed
in part via protein-DNA interactions at the Ets cognate, and minimally two
mechanisms convey MMP1 promoter upregulation by FGF2: (a) transcriptional
activation via Fra1/c-Jun containing DNA-protein interactions at the AP1 cognate
and (b) derepression of promoter activity regulated by the Ets cognate. To
identify osteoblast Ets repressors that potentially participate in gene
expression in the osteoblast, we performed reverse transcription-polymerase
chain reaction (RT-PCR) analysis of mRNA isolated from MC3T3E1 cells, using
degenerative amplimers to the conserved Ets DNA binding domain to survey the Ets
genes expressed by these cells. Six distinct Ets mRNAs were identified: Ets2,
Fli1, GABPalpha, SAP1, Elk1, and PE1. Of these, only PE1 has extensive homology
to the known Ras-regulated Ets transcriptional repressor, ERF. Therefore, we
cloned and characterized PE1 cDNA from a mouse brain library and performed
functional analysis of this particular Ets family member. A 2 kb transcript was
isolated from brain that encodes a approximately 57 kDa protein; the predicted
protein contains the known N-terminal Ets domain of PE1 and a novel C-terminal
domain with signficant homology to murine ERF. The murine PE1 open reading frame
(ORF) is much larger than the previously reported human PE1 ORF. Consistent with
this, affinity-purified rabbit anti-mouse PE1 antibody specifically recognizes
an approximately 66 kDa protein present only in the nuclear fraction of MC3T3E1
osteoblasts. Recombinant PE1 binds authentic AGGAWG Ets DNA cognates, and
transient transfection studies demonstrate that PE1 represses MMP1 promoter
activity. Surprisingly, although deletion of the MMP1 Ets cognate at nucleotides
-88 to -83 abrogates FGF2 induction, it does not prevent suppression of the
AP1-dependent MMP1 promoter by PE1. PE1 regulation maps to the MMP1 promoter
region -75 to -61, suggesting that PE1 suppresses transcription via
protein-protein interactions with AP1. Consistent with this, recombinant GST-PE1
specifically inhibits the formation of protein-DNA interactions on the MMP1 AP1
site (-72 to -66) when present in an admixture with MC3T3E1 crude nuclear
extract. In toto, these data indicate that PE1 participates in the
transcriptional regulation of the MMP1 promoter in osteoblasts. As observed with
other transcriptional repressors of MMP1 gene expression, transcriptional
suppression by PE1 occurs via inhibition of AP1-dependent promoter activity.

PMID: 10913304 [PubMed - indexed for MEDLINE]


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17: J Biol Chem. 2000 Jun 2;275(22):16466-72. 

IB1 reduces cytokine-induced apoptosis of insulin-secreting cells.

Bonny C, Oberson A, Steinmann M, Schorderet DF, Nicod P, Waeber G.

Division of Medical Genetics and the Department of Internal Medicine, CHUV
University Hospital, 1011 Lausanne Switzerland. christophe.bonny@chuv.hospvd.ch

IB1/JIP-1 is a scaffold protein that interacts with upstream components of the
c-Jun N-terminal kinase (JNK) signaling pathway. IB1 is expressed at high levels
in pancreatic beta cells and may therefore exert a tight control on signaling
events mediated by JNK in these cells. Activation of JNK by interleukin 1
(IL-1beta) or by the upstream JNK constitutive activator DeltaMEKK1 promoted
apoptosis in two pancreatic beta cell lines and decreased IB1 content by 50-60%.
To study the functional consequences of the reduced IB1 content in beta cell
lines, we used an insulin-secreting cell line expressing an inducible IB1
antisense RNA that lead to a 38% IB1 decrease. Reducing IB1 levels in these
cells increased phosphorylation of c-Jun and increased the apoptotic rate in
presence of IL-1beta. Nitric oxide production was not stimulated by expression
of the IB1 antisense RNA. Complementary experiments indicated that
overexpression of IB1 in insulin-producing cells prevented JNK-mediated
activation of the transcription factors c-Jun, ATF2, and Elk1 and decreased
IL-1beta- and DeltaMEKK1-induced apoptosis. These data indicate that IB1 plays
an anti-apoptotic function in insulin-producing cells probably by controlling
the activity of the JNK signaling pathway.

PMID: 10748095 [PubMed - indexed for MEDLINE]


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18: Endocr J. 1999 Aug;46(4):545-53. 

Transforming growth factor-beta stimulates articular chondrocyte cell growth
through p44/42 MAP kinase (ERK) activation.

Yonekura A, Osaki M, Hirota Y, Tsukazaki T, Miyazaki Y, Matsumoto T, Ohtsuru A,
Namba H, Shindo H, Yamashita S.

Department of Orthopedic Surgery, Nagasaki University School of Medicine, Japan.

Transforming growth factor-beta1 (TGF-beta1) stimulates articular chondrocyte
cell proliferation and extracellular matrix formation. We reported previously
that immediate and transient expression of c-fos mRNA through protein kinase C
activation is required for the mitogenic effect of TGF-beta1 on cultured rat
articular chondrocytes (CRAC). In gel kinase assays using myelin basic protein
(MBP) showed that total cell lysates from cells treated with TGF-beta1 caused
rapid phosphorylation of MBP, which suggests the involvement of
mitogen-activated protein kinase (MAPK) activation. To identify specific MAPK
pathways activated by TGF-beta1, we performed in vitro kinase assays using
specific substrates. TGF-beta1 induced a rapid activation of extracellular
signal regulated kinase (ERK) with a peak at 5 min, which decreased to basal
levels within 240 min after TGF-beta1 stimulation. In contrast, the c-jun
N-terminal kinase activity increased only about 2.5-fold after 240 min of
stimulation and p38 MAPK activity did not change significantly. ERK activation
by TGF-beta1 was also confirmed by in vivo phosphorylation assays of Elk1.
However, a specific MEK1 inhibitor, PD98059, significantly decreased TGF-beta1
induced Elk1 phosphorylation in a dose-dependent manner. Furthermore, PD98059
reduced the TGF-beta1-induced cell growth by 40%. These results indicate that
TGF-beta1 specifically activates MEK1 and subsequent ERK pathways in CRAC, and
that the activation of this MAPK pathway plays a role in the mitogenic response
to TGF-beta1.

PMID: 10580747 [PubMed - indexed for MEDLINE]


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19: Mol Cell Biol. 1999 Oct;19(10):7076-87. 

The net repressor is regulated by nuclear export in response to anisomycin, UV,
and heat shock.

Ducret C, Maira SM, Dierich A, Wasylyk B.

Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ULP,
67404 Illkirch Cedex, France.

The ternary complex factors (TCFs) are targets for Ras/mitogen-activated protein
kinase signalling pathways. They integrate the transcriptional response at the
level of serum response elements in early-response genes, such as the c-fos
proto-oncogene. An important aim is to understand the individual roles played by
the three TCFs, Net, Elk1, and Sap1a. Net, in contrast to Elk1 and Sap1a, is a
strong repressor of transcription. We now show that Net is regulated by
nuclear-cytoplasmic shuttling in response to specific signalling pathways. Net
is mainly nuclear under both normal and basal serum conditions. Net contains two
nuclear localization signals (NLSs); one is located in the Ets domain, and the
other corresponds to the D box. Net also has a nuclear export signal (NES) in
the conserved Ets DNA binding domain. Net is apparently unique among Ets
proteins in that a particular leucine in helix 1, a structural element,
generates a NES. Anisomycin, UV, and heat shock induce active nuclear exclusion
of Net through a pathway that involves c-Jun N-terminal kinase kinase and is
inhibited by leptomycin B. Nuclear exclusion relieves transcriptional repression
by Net. The specific induction of nuclear exclusion of Net by particular
signalling pathways shows that nuclear-cytoplasmic transport of transcription
factors can add to the specificity of the response to signalling cascades.

PMID: 10490644 [PubMed - indexed for MEDLINE]


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20: J Biol Chem. 1999 May 21;274(21):15173-80. 

Stimulation of Elk1 transcriptional activity by mitogen-activated protein
kinases is negatively regulated by protein phosphatase 2B (calcineurin).

Tian J, Karin M.

Laboratory of Gene Regulation and Signal Transduction, Department of
Pharmacology, University of California, San Diego, La Jolla, California
92093-0636, USA.

Cellular calcium (Ca2+) and the Ca2+-binding protein calmodulin (CaM) regulate
the activities of Ca2+/CaM-dependent protein kinases and protein phosphatase 2B
(calcineurin). Functional interactions between CaM kinases and mitogen-activated
protein (MAP) kinases were described. In this report, we describe cross-talk
between calcineurin and mitogen-activated protein kinase signaling. Calcineurin
was found to specifically down-regulate the transcriptional activity of
transcription factor Elk1, following stimulation of this activity by the ERK,
Jun N-terminal kinase, or p38 MAP kinase pathways. Expression of constitutively
activated calcineurin or activation of endogenous calcineurin by Ca2+ ionophore
decreased the phosphorylation of Elk1 at sites that positively regulate its
transcriptional activity. Calcineurin specifically dephosphorylates Elk1 at
phosphoserine 383, a site whose phosphorylation by MAP kinases makes a critical
contribution to the enhanced transcriptional activity of Elk1. The cross-talk
between calcineurin and MAP kinases is of physiological significance as low
doses of Ca2+ ionophore which by themselves are insufficient for c-fos induction
can actually inhibit induction of c-fos expression by activators of MAP kinases.
Thus through the effect of calcineurin on Elk1 phosphorylation, Ca2+ can have a
negative effect on expression of Elk1 target genes. This mechanism explains why
different levels of intracellular Ca2+ can result in very different effects on
gene expression.

PMID: 10329725 [PubMed - indexed for MEDLINE]


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21: Biol Chem. 1998 Aug-Sep;379(8-9):1101-11. 

Enzyme-linked immunosorbent assay for measurement of JNK, ERK, and p38 kinase
activities.

Forrer P, Tamaskovic R, Jaussi R.

Institute of Medical Radiobiology of the Paul Scherrer Institute and the
University of Zurich, Villigen PSI, Switzerland.

A rapid enzyme-linked immunosorbent assay for the enzyme activity measurement of
three well-known mitogen-activated protein (MAP) kinases, JNK2, ERK2, and p38 is
described. The assay involves immobilization of the respective kinase substrates
c-Jun, Elk1, or ATF2 on microtiter plates, addition of the kinase reaction
mixture, and measurement of substrate phosphorylation using
phospho-epitope-specific antibodies. This novel procedure represents a marked
improvement to conventional radioactive MAP kinase assays in terms of
quantification, precision, performance at physiological ATP concentration, high
throughput, time consumption and amenability to automation. In addition to the
standard solid phase assay using plastic-bound protein substrates, we developed
an alternative solution phase protocol using soluble protein substrates. By
comparing the results of the two assays, we found that MAP kinases retained much
of their substrate specificity in the phosphorylation of immobilized protein
substrates. Interestingly, we observed a strong preference of JNK2 and p38 for
the phosphorylation of dimeric over monomeric substrates. We further
characterized the kinase inhibitory activity of olomoucine, staurosporine, and
SB 203580 for JNK2, ERK2, and p38. Taken together, this assay could assist in
the biochemical characterization of MAP kinases and in identifying potent and
specific inhibitors of these enzymes.

PMID: 9792443 [PubMed - indexed for MEDLINE]


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22: Biochem J. 1998 Oct 1;335 ( Pt 1):19-26. 

Insulin-stimulated expression of c-fos, fra1 and c-jun accompanies the
activation of the activator protein-1 (AP-1) transcriptional complex.

Griffiths MR, Black EJ, Culbert AA, Dickens M, Shaw PE, Gillespie DA, Tavare JM.

Department of Biochemistry, School of Medical Sciences, University of Bristol,
Bristol BS8 1TD, UK.

The activator protein-1 (AP-1) transcriptional complex is made up of members of
the Fos (c-Fos, FosB, Fra1, Fra2) and Jun (c-Jun, JunB, JunD) families and is
stimulated by insulin in several cell types. The mechanism by which insulin
activates this complex is not well understood but it is dependent on the
activation of the Erk1 and Erk2 isoforms of mitogen-activated protein kinases.
In the current study we show that the AP-1 complex isolated from
insulin-stimulated cells contained c-Fos, Fra1, c-Jun and JunB. The activation
of the AP-1 complex by insulin was accompanied by (i) a transient increase in
c-fos expression, and the transactivation of the ternary complex factors Elk1
and Sap1a, in an Erk1/Erk2-dependent fashion; (ii) a substantial increase in the
expression of Fra1 protein and mRNA, which was preceded by a transient decrease
in its electrophoretic mobility upon SDS/PAGE, indicative of phosphorylation;
and (iii) a sustained increase in c-jun expression without increasing c-Jun
phosphorylation on serines 63 and 73 or activation of the stress-activated
kinase JNK/SAPK. In conclusion, insulin appears to stimulate the activity of the
AP-1 complex primarily through a change in the abundance of the components of
this complex, although there may be an additional role for Fra1 phosphorylation.

PMID: 9742208 [PubMed - indexed for MEDLINE]


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23: J Biol Chem. 1997 Dec 19;272(51):32163-8. 

c-Jun NH2-terminal kinases target the ubiquitination of their associated
transcription factors.

Fuchs SY, Xie B, Adler V, Fried VA, Davis RJ, Ronai Z.

Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, New York
10029, USA.

Regulatory proteins are often ubiquitinated, depending on their phosphorylation
status as well as on their association with ancillary proteins that serve as
adapters of the ubiquitination machinery. We previously demonstrated that c-Jun
is targeted for ubiquitination by its association with inactive c-Jun
NH2-terminal kinase (JNK). Phosphorylation by activated JNK protects c-Jun from
ubiquitination, thus by prolonging its half-life. In the study reported here, we
determined the ability of JNK to target ubiquitination of its other substrates
(Elk1 and activating transcription factor 2 (ATF2)) and associated proteins
(ATF2 and JunB). We demonstrate that phosphorylation by JNK protects ATF2, but
not Elk1, from JNK-targeted ubiquitination. We also show that association of
inactive JNK with JunB or ATF2 is necessary to target them for ubiquitination.
Unlike its targeting of c-Jun, JNK requires additional cellular components, yet
to be identified, to target the ubiquitination of ATF2. Elk1 is phosphorylated
by JNK, but JNK neither associates with nor targets Elk1 for ubiquitination. The
implications for the dual role of JNK in the regulation of ubiquitination and
stability of c-Jun, ATF2, and JunB in normally growing versus stressed cells are
discussed.

PMID: 9405416 [PubMed - indexed for MEDLINE]


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24: Circ Res. 1996 Aug;79(2):162-73. 

Stimulation of the stress-activated mitogen-activated protein kinase subfamilies
in perfused heart. p38/RK mitogen-activated protein kinases and c-Jun N-terminal
kinases are activated by ischemia/reperfusion.

Bogoyevitch MA, Gillespie-Brown J, Ketterman AJ, Fuller SJ, Ben-Levy R, Ashworth
A, Marshall CJ, Sugden PH.

National Heart and Lung Institute (Cardiac Medicine), Imperial College of
Science, University of London, UK.

It has recently been recognized that cellular stresses activate certain members
of the mitogen-activated protein kinase (MAPK) superfamily. One role of these
"stress-activated" MAPKs is to increase the transactivating activity of the
transcription factors c-Jun, Elk1, and ATF2. These findings may be particularly
relevant to hearts that have been exposed to pathological stresses. Using the
isolated perfused rat heart, we show that global ischemia does not activate the
42- and 44-kD extracellular signal-regulated (protein) kinase (ERK) subfamily of
MAPKs but rather stimulates a 38-kD activator of MAPK-activated protein kinase-2
(MAPKAPK2). This activation is maintained during reperfusion. The molecular
characteristics of this protein kinase suggest that it is a member of the
p38/reactivating kinase (RK) group of stress-activated MAPKs. In contrast,
stress-activated MAPKs of the c-Jun N-terminal kinase (JNK/SAPKs) subfamily are
not activated by ischemia alone but are activated by reperfusion following
ischemia. Furthermore, transfection of ventricular myocytes with activated
protein kinases (MEKK1 and SEK1) that may be involved in the upstream activation
of JNK/ SAPKs induces increases in myocyte size and transcriptional changes
typical of the hypertrophic response. We speculate that activation of multiple
parallel MAPK pathways may be important in the responses of hearts to cellular
stresses.

Publication Types:
    Review
    Review, Tutorial

PMID: 8755992 [PubMed - indexed for MEDLINE]


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25: J Cell Sci. 1995 Nov;108 ( Pt 11):3599-609. 

Neither ERK nor JNK/SAPK MAP kinase subtypes are essential for histone H3/HMG-14
phosphorylation or c-fos and c-jun induction.

Cano E, Hazzalin CA, Kardalinou E, Buckle RS, Mahadevan LC.

Nuclear Signalling Laboratory, Randall Institute, King's College London, UK.

The effects of EGF, TPA, UV radiation, okadaic acid and anisomycin on ERK and
JNK/SAPK MAP kinase cascades have been compared with their ability to elicit
histone H3/HMG-14 phosphorylation and induce c-fos and c-jun in C3H 10T1/2
cells. EGF and UV radiation activate both ERKs and JNK/SAPKs but to markedly
different extents; EGF activates ERKs more strongly than JNK/SAPKs, whereas UV
radiation activates JNK/SAPKs much more strongly than ERKs. Anisomycin and
okadaic acid activate JNK/SAPKs but not ERKs, and conversely, TPA activates ERKs
but not JNK/SAPKs. Nevertheless, all these agents elicit phosphorylation of
ribosomal and pre-ribosomal S6, histone H3 and HMG-14, and the induction of
c-fos and c-jun, showing that neither cascade is absolutely essential for these
responses. We then analysed the relationship between ERKs, JNK/SAPKs and the
transcription factors Elk-1 and c-Jun, implicated in controlling c-fos and
c-jun, respectively. JNK/SAPKs bind to GST-cJun1-79, and ERKs, particularly
ERK-2, to GST-Elk1(307-428); there is no cross-specificity of binding. Further,
GST-Elk1(307-428) binds preferentially to active rather than inactive ERK-2. In
vitro, JNK/SAPKs phosphorylate both GST-cJun1-79 and GST-Elk1(307-428), whereas
ERKs phosphorylate GST-Elk1(307-428) but not GST-cJun1-79. Thus, neither ERKs
nor JNK/SAPKs are absolutely essential for nuclear signalling and c-fos and
c-jun induction. The data suggest either that activation of a single MAP kinase
subtype is sufficient to elicit a complete nuclear response, or that other
uncharacterised routes exist.

PMID: 8586671 [PubMed - indexed for MEDLINE]


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26: Cell Growth Differ. 1995 Nov;6(11):1437-46. 

jun-NH2-terminal kinase activation mediated by UV-induced DNA lesions in
melanoma and fibroblast cells.

Adler V, Fuchs SY, Kim J, Kraft A, King MP, Pelling J, Ronai Z.

Molecular Carcinogenesis Program, American Health Foundation, Valhalla, New York
10595, USA.

jun-NH2-terminal kinase (JNK) belongs to a family of protein kinases that
phosphorylates c-Jun, ATF2, and Elk1 in response to various forms of stress
including UV irradiation and heat shock. Although in previous studies we have
demonstrated the importance of membrane components for JNK activation by UV
irradiation, here we have elucidated the role of DNA damage in this response. We
show that in vitro-irradiated or sonicated DNA that is added to proteins
prepared from UV-treated cells can further induce JNK activation in a
dose-dependent manner. When compared with UV-B (300 nm), UV-C (254 nm), which is
better absorbed by the DNA, is significantly more potent in activating JNK.
Furthermore, when wavelengths lower than 300 nm were filtered out, UV-B was no
longer able to activate JNK. With the aid of melanoma and fibroblast cells,
which exhibit different resistances to irradiation and require different UV
doses to generate the same number of DNA lesions, we demonstrate that above a
threshold level of 0.45 lesions and up to 0.75 lesions per 1875 bp, the degree
of JNK activation correlates with the amount of lesions induced by UV-C
irradiation. Finally, to explore the role of nuclear and mitochondrial DNA
(mtDNA) in mediating JNK activation after UV irradiation, we have used cells
that lacks mtDNA. Although the lack of mtDNA did not impair the ability of UV to
activate JNK, when enucleated, these cells had lost the ability to activate JNK
in response to UV irradiation. Overall, our results suggest that DNA damage in
the nuclear compartment is an essential component that acts in concert with
membrane-anchored proteins to mediate c-Jun phosphorylation by JNK.

PMID: 8562482 [PubMed - indexed for MEDLINE]


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