From the Ruttenberg Cancer Center, Mount Sinai School of Medicine,
New York, New York 10029 and the
Department of
Cellular and Molecular Pharmacology, and § Pharmaceutical
Chemistry, University of California,
San Francisco, California 94143
Received for publication, December 18, 2000, and in revised form, February 8, 2001
Modification of the ATP pocket on protein kinases
allows selective use of an ATP analogue that exhibits high affinity for the altered kinases. Using this approach, we altered the ATP-binding site on JNK and identified
N6-(2-phenythyl)-ATP, a modified form of ATP
that exhibits high specificity and affinity for the modified, but not
the wild type form, of JNK. Using modified JNK and its ATP analogue
enables the detection of novel JNK substrates. Among substrates
identified using this approach is heterogeneous nuclear
ribonucleoprotein K, which is involved in transcription and
post-transcriptional mRNA metabolism. The newly identified
substrate can be phosphorylated by JNK on amino acids 216 and 353, which contribute to heterogeneous nuclear ribonucleoprotein K mediated
transcriptional activities.
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INTRODUCTION |
The family of mitogen-activated protein kinases
(MAPK)1 consists of
evolutionarily conserved proteins that play a central role in
protecting cells from stress and DNA damage (1-5). Major components within the MAPK family are extracellular signal-regulated kinases (ERKs), stress-activated protein kinases (SAPK/JNK), and p38 (5, 6).
Whereas ERKs are preferentially activated by mitogens (7, 8), the JNK
and p38 pathways are triggered primarily by inflammatory cytokines and
by a diverse array of cellular stresses, including UV light and
hydrogen peroxide (9-12). Each stress kinase is activated by a defined
set of upstream protein kinases that are selectively triggered by
signals elicited from cell surface receptors or membrane-anchored proteins, or changes due to altered balance of reactive oxygen species
(13, 14). MAPKs phosphorylate proteins located at the plasma membrane,
cytoplasm, and nucleus (15). In non-stimulated cells, MAPKs are largely
cytoplasmic. Upon activation, a portion of the MAPKs translocates to
the nucleus (16-21). The duration of activation of MAPKs influences
the extent of their nuclear translocation and, thus, their access
to transcription factors (22-27).
Stress-activated protein kinases are encoded by three genes (JNK
1-3; reviewed in Ref. 28) that are alternatively spliced to
create more than 10 isoforms (29). Whereas JNK1 and
JNK2 are expressed in most human tissues, JNK3 is
seen primarily in brain, heart, and testis. Originally found as
microtubule-associated kinases (30), stress-activated kinases were
found to bind the amino-terminal domain of c-Jun (31) and to
phosphorylate c-Jun on Ser-63 and -73 (32). In normal growing cells,
JNK activity as a kinase is limited by the inhibitory effect of GSTpi
(33), and yet it efficiently targets ubiquitination and degradation of
its associated proteins, as demonstrated for c-Jun, ATF2, and p53
(34-37). In response to stress, JNK phosphorylation on both Thr-183
and Thr-185 residues by MKK4/7 (9, 27) leads to phosphorylation of JNK
substrates, which include c-Jun (38, 39), ATF-2 (40, 41), c-Myc (42),
Bcl2 (43), and p53 (36, 44, 45).
For a family of genes that are ubiquitously expressed in more than 10 isoforms, the number of substrates identified to date is surprisingly
limited. Attempts to identify JNK substrates by the two-hybrid screen
in most cases failed, probably because of the nature of JNK-targeted
ubiquitination of its bound substrates. To identify new putative
substrates for this kinase, we adopted the approach developed by Shokat
and colleagues (46-49), wherein the ATP pocket in a given kinase can
be altered so that it exhibits high affinity for selected forms of ATP.
This approach enables performing kinase reactions in the presence of
whole cellular proteins and yet selectively identifying specific
substrates, thus providing a "semi" in vivo setting for
identifying putative substrates. Here we describe the use of this
approach to modify JNK, identify the highest affinity ATP analogue, and
subsequently identify putative new substrates. We demonstrate the
ability of this technique to identify heterogeneous nuclear
ribonucleoprotein K (hnRNP-K, or K protein) as a new MAPK/JNK substrate.
hnRNP-K (K protein) belongs to a large family of nuclear RNA-binding
proteins that form complexes with RNA polymerase II transcripts (50,
51). The hnRNP-K protein has been implicated in diverse molecular and
cellular functions, including nuclear-cytoplasmic shuttling and RNA
transcription and translation (52). The K homology motifs,
originally found in hnRNP-K, are implicated in RNA binding (53). Import
and export of K protein is mediated via the KNS domain (aa 323-390),
which confers bidirectional transport across the nuclear envelope and
represents a novel shuttling pathway (54). The K protein has also been
shown to regulate translation in the cytoplasm. Together with another K
homology domain protein, hnRNP-E1, it binds to a CU-rich
"differentiation control element" in the 3'-untranslated region of
15-lipoxygenase mRNA and silences the translation of
this message in immature erythroid precursor cells (55).
The mammalian (53, 56) and the Drosophila melanogaster
homologues (57) of K protein have been implicated in transcription. Transactivation by the K protein involves an increase in RNA synthesis of various reporter genes (56). The K protein has also been found to
bind to a single-stranded DNA sequence of the human c-myc promoter and to affect transcription of c-myc as well as of
Sp1 and Sp3 (58). Interconversion of duplex and
single-stranded DNA (59) and association with the C/EBP
(60) are
among the mechanisms that may explain the effect of K protein on transcription.
The mechanistic basis of regulation of K protein activities is not well
understood. The carboxyl terminus of the K protein consists of the
SH3-binding cluster, which is required for binding of K protein with
Vav, an association implicated in the cytoplasmic localization of K
protein (61, 62). K protein has been shown to associate with c-Src (63,
64), through which it has been implicated in regulating processing,
trafficking, or translation of mRNA. Src as well as protein kinase
C
and an interleukin 1-responsive K protein kinase have been shown
to phosphorylate hnRNP K, although the significance of this
phosphorylation is not known (65, 66). Here we characterize the
phosphorylation of K protein by JNK and demonstrate that K protein
phosphorylation is required for its contribution to
AP1-dependent transcriptional activities.
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MATERIALS AND METHODS |
Cells--
Human embryo kidney cells (293T) were maintained in
Dulbecco's modified Eagle's medium supplemented with calf serum
(10%) and antibiotics.
Plasmids--
HnRNP K cDNA was polymerase chain reaction
amplified using an HA-tagged primer and cloned into pCDNA3 or
pGEX-4T-2. Mutations on serine phosphorylation sites (S116A, S216A,
S284A, and S353A) of hnRNP K and in the ATP pocket of JNK2 (M108G and
L168A) were performed using the Quick Change Site-directed Mutagenesis
Kit (Stratagene) and confirmed by DNA sequencing. Mutated hnRNP K cDNA was also subcloned into the pGEX-4T-2 plasmid. GST-Jun was expressed and purified as described previously (28). pGEX-4T-2-hnRNP K
fusion protein was expressed in Escherichia coli BL21 and
purified using immobilized glutathione beads (Pierce) by standard methods.
32P Labeling of Phenythyl-ADP--
E.
coli nucleoside diphosphate kinase (0.1 mg) was mixed with 100 µCi of [
-32P]ATP (6000 Ci/mmol, Amersham Pharmacia
Biotech) in HBS (150 mM NaCl, 20 mM HEPES, pH
7.4) containing 5 mM MgCl2 and equilibrated at
room temperature for 5 min followed by separation on a Bio-spin column
(p-6). Phenythyl-ADP (5 µl of 0.1 mM) was added to the phosphorylated nucleoside diphosphate kinase for 10 min at room temperature before the reaction mixture was heated (80 °C for 2 min)
and subsequently spun down to pellet the denatured nucleoside diphosphate kinase (24).
Protein Kinase Assays--
Protein kinase assays were carried
out using a fusion protein (GST-Jun) or whole cell extract that was
dialyzed against kinase buffer (20 mM HEPES, pH 7.4, 0.5 mM EGTA, 1 mM dithiothreitol, 2 mM
MgCl2, 2 mM MnCl2, 5 mM
NaF, 0.1 mM NaVO3, 5 mM
-glycerolphosphate, and 75 mM NaCl) as described
previously (33). Briefly, 2 µg of GST-Jun or 100 µg of lysate were
incubated with immunopurified HA-JNK or HA-JNK-as3 in the presence of
kinase buffer containing 3 µCi of [
-32P]ATP or
32P-labeled N6-(2-phenythyl)-ATP and
25 µM cold ATP for 30 min at 30 °C. Phosphorylated GST-Jun was separated on SDS-PAGE, and phosphorylated lysate was separated by two-dimensional PAGE followed by staining and autoradiography.
Two-dimensional Gel Electrophoresis--
Isoelectric focusing
(IEF) gels (consist of 8 M urea, 4%
acrylamide/bisacrylamide, 2% Nonidet P-40, 1.6% Pharmalyte pH 5-8, and 0.4% Pharmalyte pH3-10) were prepared in gel tube (inner
diameter: 2 mm × 18 cm; BioRad). Following polymerization (3 h),
IEF gels were pre-run for 1 h at 200 V with 10 mM
H3P04 as anode butter and 20 mM
NaOH as cathode buffer. Samples were loaded (40 µl) and run at 400 V
for 14-16 h followed by 800 V for 1 h. IEF gels were removed from
tube and equilibrated in SDS sample buffer for 15 min at room
temperature before they were placed on 12% SDS-polyacrylamide gel and
run at 40 mA until the dye front reached to the bottom of the gel.
Microsequencing--
Tandem nanoflow electrospray mass
spectrometry employing a PE Sciex QSTAR instrument was used to
determine the sequence of peptides obtained by tryptic digestion of 3 silver-stained spots. The peptide sequences obtained for the
corresponding spots were: GGRGGSRAR, NTDEMVELR, and NLPPPPPPR, each of
which exhibited 100% identity with the hnRNP-K protein.
Purification of JNK for Kinase Assay--
293T cells were
transfected with pcDNA3, wt-HA-JNK2, or HA-JNK2-as3 by standard
calcium phosphate precipitation methods. Thirty hours later, cells were
exposed to UV-C (60 J/m2) and harvested after 45 min.
Protein samples were prepared from cells as previously described
(31).
Orthophosphate in Vivo Labeling--
Cells were cultured in
phosphate-free medium for 1 h before addition of
[32P]orthophosphate (1 mCi/plate) for 1 h. HA
antibody immunoprecipitated HA-K was separated on SDS-PAGE and
transferred onto nitrocellulose membrane following autoradiography and
Western blot.
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RESULTS |
Identification of K Protein as a JNK Substrate Using a Mutant Form
of JNK and a Corresponding Form of Modified ATP--
To enable
selective screening for putative JNK substrates, we modified the
ATP-binding site on JNK (JNK analogue specific 3; JNK-as3) so that it
utilizes an ATP analogue. Based on the success of engineering both
tyrosine and serine/threonine kinases from a wide variety of protein
kinase families including, Src, Fyn, CDK2, Cla4 (yeast Pak
kinase), CaMKII, and fus3 (yeast MAPK), we have
identified two key residues that must be modified in wild-type kinase
to allow recognition of ATP analogues modified at the N (6) position
(45-48, 67). These two residues in JNK are M108G and L168A,
respectively, and are analogous to the mutations required to create
Cla4-as3 (Fig. 1; Ref. 68). The mutations
M108G and L168A maintained JNK inducibility by UV as well as its
substrate recognition (Fig.
2a). To identify the modified
form of ATP that exhibits the highest affinity to JNK-as3 we compared
four forms of ATP, of which N6-(2-phenythyl)-ATP
exhibited the strongest ability to inhibit phosphorylation of c-Jun by
modified but not wt forms of JNK when added in excess as the cold form
of ATP (Fig. 2b). 32P-Labeled
N6-(2-phenythyl)-ATP also exhibited the highest
affinity for the mutant but not for the wt form of JNK in its ability
to phosphorylate c-Jun (Fig. 2c). Given the low
cross-reactivity with the wt form of JNK and the high affinity for the
modified JNK, these observations suggest that
N6-(2-phenythyl)-ATP could be used to enable
detection of specific JNK substrates in the presence of endogenous form
of JNK.

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Fig. 1.
Rationale for mutating specific residues
within ATP pocket of JNK. Former modifications of both tyrosine
and serine/threonine kinases from a variety of protein kinase families
including, Src, Fyn, CDK2, Cla4 (yeast Pak kinase),
CaMKII, and fus3 (yeast mapk), led to the
identification of two key residues that must be modified in wild-type
kinase to allow recognition of ATP analogs modified at the N-6
position. These two residues in JNK are M108G and L168A, respectively,
and are analogous to the mutations required to create Cla4-as3.
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Fig. 2.
Generation of ATP pocket mutant JNK and
identification of ATP analogue. a, in vitro
kinase assay using GST-Jun as substrate and wt- or JNK-as3 (analog
specific 3, mutated on aa M108G,L168A) immunopurified from UV-treated
(60 J/m2) 293T cells was carried out as previously
described (33), demonstrating that mutations of JNK did not alter its
activation by UV and phosphorylation of c-Jun (upper panel).
Lower panel reveals that equal amounts of immunopurified
kinase and substrate were used. b, in vitro
kinase assay using modified ATP (N6-(benzyl)-ATP
(Benz-ATP), N6-(1-methylbutyl)-ATP
(meth-ATP), N6-(cyclopentyl)-ATP
(Cycl-ATP), and N6-(2-phenethyl)-ATP)
as competitor. Kinase reactions were carried out as above in the
presence of 200 µM modified ATP (upper panel).
Lower panel is Coomassie Brilliant Blue
(CBB)-stained GST-Jun substrate. c,
in vitro kinase assay using 32P-labeled
Phen-ATP. Kinase reaction was carried out in the kinase buffer
containing GST-Jun substrate, wt-JNK, or JNK-as3 and
32P-Phen-ATP (2 µCi) for 30 min at 30 °C. Lower
panel is Coomassie Brilliant Blue-stained substrate.
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To identify proteins specifically phosphorylated by modified JNK,
cellular extracts were incubated with modified JNK that had been
immunopurified from 293T overexpressing cells in the presence of
32P-labeled N6-(2-phenythyl)-ATP.
Phosphorylated proteins were separated on two-dimensional gels followed
by silver staining (Fig. 3b)
and subsequent autoradiography (Fig. 3a). Following
alignment of the phosphorylated proteins to those seen in the
silver-stained gels, we isolated the corresponding spots. Tandem
nanoflow electrospray mass spectrometry of silver-stained spots, which
corresponded to the phosphorylated proteins (Fig. 3, a and
b), identified 3 peptide sequences, each of which exhibited
100% homology with hnRNP-K protein (Fig. 3c).

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Fig. 3.
Identification of hnRNP-K as JNK
substrate. a, separation of JNK-as3-specific
substrates. An in vitro kinase assay was carried out using
protein extracts (100 µg) as substrates in the presence of
32P-Phen-ATP (2 µCi) and JNK-as3 or wt JNK as indicated
in the figure followed by two-dimensional PAGE separation using IEF in
the first dimension and SDS-PAGE in the second. The figure depicts
autoradiograph of the actual kinase reaction. Arrows
indicate the position of the spots subjected to microsequencing.
b, reaction performed as indicated in panel a,
shown is the silver-stained gel with the corresponding spots that were
dissected for microsequencing. c, outlined are
the three peptides identified in microsequencing reaction revealed full
homology with the hnRNP-K amino acid sequence.
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Phosphorylation of K Protein by JNK and ERK in Vitro--
The
full-length cDNA of the K protein was cloned into bacterial and
mammalian expression vectors. Bacterially expressed and purified
GST-tagged K protein was efficiently phosphorylated in vitro
by the immunopurified active form of JNK but not by immunopurified p38
(Fig. 4a). Further analysis
carried out using proteins prepared from cells before and after UV
treatment revealed efficient phosphorylation of the K protein as early
as 30 min after UV treatment and to lesser degrees by proteins prepared
2 h after UV irradiation (Fig. 4b). Forced expression
of the constitutively active form of ERK and to a lesser degree of JNK
was also efficient in leading to the phosphorylation of K protein (Fig.
4b). These observations confirm that the K protein can serve
as a substrate for JNK and ERK phosphorylation.

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Fig. 4.
hnRNP-K is phosphorylated by JNK or ERK
in vitro. a, immunokinase assay using
bacterially expressed and purified GST-hnRNP K (GST-K)
fusion protein as substrate and activated forms of HA-JNK or HA-p38 as
kinase. 293T cells (upper panel) transfected with either
HA-JNK or HA-p38 followed by UV irradiation and immunopurification
using HA antibodies. Middle panel depicts Coomassie
Blue-stained substrate. Lower panel depicts immunoblot of
HA-JNK or HA-p38 that were used for the kinase assay. b,
in vitro kinase assay using purified GST-K as substrate and
endogenous JNK or ERK as kinase that immunopurified from 293T cells
using monoclonal anti-JNK1 antibody (PharMingen) or polyclonal
anti-ERK2 antibody (Santa Cruz Biotech), respectively (upper
panel). Lower panel represents equal amount of
substrate stained with Coomassie Blue.
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To identify the JNK/ERK phosphoacceptor sites on the K protein, we
mutated the proline-driven serines at aa 116, 216, 284, and 353. Of the
four mutants, S216A and S353A exhibited the lowest degree of JNK
phosphorylation when compared with the wt protein (Fig.
5a). When subjected to
phosphorylation by ERK, the most efficient decrease in ERK
phosphorylation was observed with the S353A mutant. A noticeable
decrease was also seen in phosphorylation of the S284A mutant, whereas
the S216A mutant revealed a marginal decrease in phosphorylation when
compared with the wt form of the K protein (Fig. 5b). Of
interest is the slight shift in the Mr of the
S116A mutant, although the mutation did not affect the degree of
phosphorylation by either ERK or JNK (Fig. 5, a and b). These findings demonstrate that different residues on
the K protein may serve as the primary phosphoacceptor sites for JNK versus ERK.

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Fig. 5.
Identification of JNK phosphoacceptor sites
on the K protein. a, in vitro
phosphorylation of phosphomutant GST-K by JNK. GST-K mutated on
residues S116A (GST-K116), S216A (GST-K216),
S284A (GST-K284), or S353A (GST-K353) was
produced in bacteria and purified on glutathione beads prior to
phosphorylation by immunopurified JNK from UV-treated cells. The
upper panel depicts the autoradiograph, whereas the
lower panel shows the Coomassie Blue staining of the
substrate used in the reaction. The degree of phosphorylation was
quantified on the basis of densitometry scanning. Numbers
reflects change in % of phosphorylation. b, in
vitro phosphorylation of single phosphomutant GST-K by
immunopurified ERK. The analysis is similar to that described in
panel a, with the exception that the kinase used in the
reaction was ERK immunopurified from MEK-EL expressing cells.
c, in vitro phosphorylation of double mutant
GST-K by JNK. GST-K (mutation on S216A and S284A
(GST-K216/284), S216A and S353A (GST-K216/353),
and S284A and S353A (GST-K284/353)) were produced in
bacteria, purified on glutathione beads and subjected to
phosphorylation by JNK. The data shown are representative of at least
three experiments. d, in vitro phosphorylation of
double mutant GST-K by ERK. GST-K (wt or mutants as described in
panel c) were produced in bacteria, purified on glutathione
beads, and subjected to phosphorylation by ERK.
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Since no single mutant revealed complete loss of phosphorylation of K
protein by ERK, we mutated the K protein on multiple phosphoacceptor
sites. Among the K proteins that contain different combinations of
mutations on different phosphoacceptor sites, a double mutant (S216 and
S353) exhibited the most efficient decrease in JNK phosphorylation
(Fig. 5c), whereas a double mutant (S284A,S353A) exhibited
the most efficient decrease in ERK phosphorylation (Fig. 5d). A triple mutant (S216A,S284A,S353A) exhibited the same
degree of phosphorylation as that shown by the double mutant,
suggesting that the primary site for JNK phosphorylation consists of
serines 216 and 353 on the K protein. Analysis of secondary structure prediction of wt versus phosphomutant forms of the K protein
via the predict-protein software did not reveal change in the
conformation/structure of the K protein due to these mutations (data
not shown).
Phosphorylation of K Protein by JNK and ERK in Vivo--
To
confirm phosphorylation of the K protein by JNK or ERK in
vivo, we performed orthophosphate labeling of cells that had been
transfected with the K protein and the respective upstream kinases for
ERK, p38, or JNK. Immunoprecipitation of the K protein followed by
SDS-PAGE and autoradiography revealed efficient phosphorylation upon
expression of MEK-EL, a constitutively active form of MEK that
drives ERK phosphorylation. UV treatment and JNKK2(CAA) were also
capable of mediating phosphorylation of the K protein (Fig. 6).

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Fig. 6.
In vivo phosphorylation of
HA-hnRNP K by JNK and ERK. HA-hnRNP K was co-transfected to 293T
cells with or without the upstream kinase of p38, ERK, or JNK (MKK6,
MEK-EL, or JNKK2(CAA), respectively) or subjected to UV treatment as
indicated in the figure. Orthophosphate labeling was carried out for
2 h prior to protein extraction. 32P-Labeled HA-hnRNP
K was immunoprecipitated, washed extensively before separation on
SDS-PAGE, transferred to a nitrocellulose membrane, and subsequently
analyzed by means of autoradiography (upper panel) and
Western using antibodies to HA (lower panel) to reveal equal
loading of the HA-hnRNP-K protein. The arrows point to the
positions of the K protein, IgG, and a nonspecific HA-cross-reacting
band (ns).
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JNK Phosphorylation of HnRNP K Increases Its
Transcriptional Activity--
The cellular functions attributed to the
K protein include inhibition of RNA translation (55) and
transcriptional activation (53, 56-60). Since phosphorylation of the K
protein by JNK did not affect its ability to elicit inhibition of RNA
translation (49), we have further elucidated the role of JNK in K
protein ability to contribute to transcriptional activities. Among
promoters that were affected by K protein are those that consist of the AP1 site (53). Given the role of JNK in the activation of c-Jun and
ATF2, primary factors that mediate transcription from AP1 sites, we
have elucidated the possible contribution of JNK to K protein effect on
transcription from AP1 sites.
To this end we have co-transfected Jun-LUC construct with either wt or
phosphomutant (216/353) forms of the K protein into 293T cells. To
modulate the degree of JNK, activity cells were also transfected with
either the constitutive active JNK upstream, kinase (JNKK2(CAA)), or
with a dominant negative JNK construct (JNK(APF)) in conjunction with
UV treatment as indicated. Whereas forced expression of JNKK2(CAA)
alone is sufficient to increase Jun-LUC activity (67%),
co-transfection of K and JNKK2(CAA) further induce (>3-fold) Jun-LUC
activity (Fig. 7a). The latter
increase is dependent on K protein phosphorylation by JNK since
co-expression of the phosphomutant K protein (K216/353) with JNKK2(CAA)
did not increase Jun-LUC activities. The effect of JNK on K proteins ability to facilitate transcription from AP1 site is specific for JNK
since the mutant form of JNK (on its phosphoacceptor sites, which
render it as a dead kinase and as a dominant negative for endogenous
JNK) failed to mediate increase in AP1 activities. Further support for
the role of JNK in K protein contribution to AP1-mediated transcription
comes from the analysis of Jun-LUC activities in UV-treated cells.
Whereas UV irradiation led to a noticeable increase (>3-fold which was
comparable to the effect of JNKK2CAA) this increase was completely
attenuated upon JNK(APF) expression. This observation suggests that the
ability to increase AP1-dependent transcriptional
activities via K protein is dependent on JNK activities. A smaller
(60%) increase in AP1-mediated transcription was also seen in
UV-treated cells that were co-transfected with the phosphomutant form
of K, probably due to the effect of UV on the endogenously expressed K
protein, which could not be out-competed by exogenous expression of the
phosphomutant K form (49). Indeed, co-expression of phosphomutant K and
JNK(APF) attenuated the UV-mediated increase in Jun-LUC activities.

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Fig. 7.
JNK phosphorylation of K protein contributes
to its transcriptional activities from AP1-bearing promoters.
a, Jun-LUC construct (0.1 µg) and wt-k (0.5 µg) or mut-K
(k216/353, 0.5 µg) were co-transfected with constitutive active JNK
upstream kinase (JNKK2(CAA), 0.5 µg) with dominant negative JNK
construct (JNK(APF), 0.5 µg), and with -galactosidase construct.
24 h after transfection (Gene Jammer into 293 cells) cells were
treated with mock or with UV (50 J/m2). Proteins were
prepared 8 h after UV treatment and used for measurement of
-galactosidase activities as well as LUC activities (luciferase
measurement kit; Promega). Values shown were normalized per
transfection efficiency and represent mean of three experiments.
b, experiment was performed as described in panel
a, with the exception that MEKK1 (0.1 µg) was transfected as
indicated.
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Further studies on K protein contribution to transcription from
promoters bearing the AP1 site was carried out using the upstream kinase MEKK1 in its constitutively active form (
MEKK1). This powerful construct efficiently activates most stress kinase cascades, including JNK, p38, MAPK, and IKK. Forced expression of
MEKK1 efficiently activated transcription of Jun-LUC (8-fold), which was
further increased (up to 11-fold) upon K protein expression (Fig.
7b). Increase observed upon K protein expression was
attenuated by JNK-APF. Along these lines, phosphomutant K was not able
to augment the transcriptional activities that were mediated by
MEKK1. Expression of JNK(APF) in combination with the phosphomutant
form of the K protein reduced some of the increase (25%) mediated by
MEKK1, probably due to the limited effects of these constructs on
the endogenous form of K protein. Together, these findings point to the
role of JNK in acquiring K protein ability to contribute to
transcriptional activation, as shown here for Jun-LUC bearing promoter sequences.
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DISCUSSION |
Among the stress-activated protein kinases, JNK has been well
characterized as central to the cells decision for life or death in
response to most cellular stress-inducing agents. JNK elicits its
potent regulatory function through tight regulation of its substrates,
which in most cases are bound to JNK and concomitantly targeted by JNK
for ubiquitination and degradation under non-stressed conditions. After
exposure to any of the diverse stimuli that can activate it, the kinase
efficiently phosphorylates the substrate, gaining in stability and
activity. Despite the large amount of data accumulated so far with
regard to JNKs ability to regulate transcription factors and other
stress-related proteins, the number of stress kinases remains small,
considering that JNK comprises a family of 3 genes that appear in over
10 different isoforms. It is imperative to identify new JNK substrates
that may form part of the stress response and therefore dictate the
fate of the stressed cell. Here we demonstrate a new approach to the
identification of potential new JNK substrates. Using the technique
originally developed by Kevan and colleagues (45-48) for the
Src gene, we demonstrate the ability to modify the
ATP pocket on JNK and consequently to utilize a modified form of ATP
that exhibit high affinity toward the modified substrate. This new
match enables selective phosphorylation of JNK substrates when the
modified kinase and ATP are supplied. Importantly, the modification
does not hamper JNK activation by stress, nor does it affect the
recognition of JNK substrates, as shown here for c-Jun. Using this
approach, the current studies demonstrate the identification of hnRNP-K
as a JNK and ERK substrate. The phosphoacceptor sites for JNK and ERK
on the K protein are different, and indeed, ERK phosphorylation results
in biological consequences different from those of phosphorylation by
JNK (49). Whereas ERK phosphorylation on aa 284 and 353 contributes to
K protein nuclear export and concomitant inhibition of RNA translation (49), phosphorylation by K protein on aa 216 and 353 increases the
transcriptional effects of the K protein. This finding illustrates the
diverse forms of regulation of K protein by varying protein kinases,
each of which contributes to different K protein functions.
The method developed and employed here for the identification of novel
JNK substrates could be employed for identification of JNK substrates
in various tissues, in response to different stimuli, at various stages
of development, and in promotion and progression of human tumors. Each
of the scenarios requires that the set of proteins be used against the
corresponding controls to assure that novel JNK substrates are
selected. Overall, the altered JNK and its corresponding ATP as
described in the current study open new horizons for elucidating novel
JNK substrates.
Implications of K protein phosphorylation by JNK are illustrated for K
proteins contribution to transcriptional activities, in this case via
AP1 sequences. Among the mechanisms underlying K protein ability to
confer increased transcriptional output are interconversion of duplex
and single-stranded DNA (59) and association with the C/EBP
(60),
each of which could be better affected by the phosphorylated form of
the K protein, which may increase affinity to associated proteins or
DNA. The increased effect of the K protein on transcription is expected
to have a wide effect on transcriptional output due to the general
nature of K protein effect on transcriptional regulation.
We thank David Morgan and Justin Blethrow for
advice, Michael Karin for JNKK2(CAA) and Stuart Aaronson for MEK-EL
constructs. We thank Shiraz Mujtaba and Ming-ming Zao for
computer-based analysis of K protein conformation. We also thank
members of the Ronai lab for advice and helpful comments during the
preparation of this manuscript.
The abbreviations used are:
MAPK, mitogen-activated protein kinase;
ERK, extracellular signal-regulated
kinase;
JNK, c-Jun NH2-terminal kinase;
hnRNP-K, human
nuclear ribonucleoprotein K;
aa, amino acid(s);
SH3, Src homology
domain 3;
HA, hemagglutinin;
GST, glutathione S-transferase;
PAGE, polyacrylamide gel electrophoresis;
wt, wild-type.
1.
|
Errede, B.,
and Levin, B. E.
(1993)
Curr. Opin. Cell Biol.
5,
254-260[Medline]
[Order article via Infotrieve]
|
2.
|
Davis, R. J.
(1994)
Trends Biochem. Sci.
19,
470-473[CrossRef][Medline]
[Order article via Infotrieve]
|
3.
|
Marshall, C. J.
(1994)
Curr. Opin. Genet. Dev.
4,
82-89[Medline]
[Order article via Infotrieve]
|
4.
|
Kyriakis, J. M.,
and Avruch, J.
(1996)
J. Biol. Chem.
271,
24313-24316[Free Full Text]
|
5.
|
Schaeffer, H. J.,
and Weber, M. J.
(1999)
Mol. Cell. Biol.
19,
2435-2444[Free Full Text]
|
6.
|
Widmann, C.,
Gibson, S.,
Jarpe, M. B.,
and Johnson, G. L.
(1999)
Physiol. Rev.
79,
143-180[Abstract/Free Full Text]
|
7.
|
Lopez-Ilasaca, M.
(1998)
Biochem. Pharmacol.
56,
269-277[CrossRef][Medline]
[Order article via Infotrieve]
|
8.
|
Kyriakis, J. M.,
and Avruch, J.
(1996)
Bioessays
18,
567-577[Medline]
[Order article via Infotrieve]
|
9.
|
Dérijard, B.,
Hibi, M.,
Wu, I.-H.,
Barrett, T.,
Su, B.,
Deng, T.,
Karin, M.,
and Davis, R. J.
(1994)
Cell
76,
1025-1037[Medline]
[Order article via Infotrieve]
|
10.
|
Adler, V.,
Schaffer, A.,
Kim, J.,
Dolan, L. R.,
and Ronai, Z.
(1995)
J. Biol. Chem.
270,
26071-26077[Abstract/Free Full Text]
|
11.
|
Karin, M.
(1995)
J. Biol. Chem.
270,
16483-16486[Free Full Text]
|
12.
|
Minden, A.,
and Karin, M.
(1997)
Biochim. Biophys. Acta
1333,
F85-104[CrossRef][Medline]
[Order article via Infotrieve]
|
13.
|
Adler, V.,
Yin, Z.,
Tew, K. D.,
and Ronai, Z.
(1999)
Oncogene
18,
6104-6111[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Kamata, H.,
and Hirata, H.
(1999)
Cell Signal.
11,
1-14[CrossRef][Medline]
[Order article via Infotrieve]
|
15.
|
Moriguchi, T.,
Gotoh, Y.,
and Nishida, E.
(1996)
Adv. Pharmacol.
36,
121-137[Medline]
[Order article via Infotrieve]
|
16.
|
Chen, R.-H.,
Sarnecki, C.,
and Blenis, J.
(1992)
Mol. Cell. Biol.
12,
915-927[Abstract]
|
17.
|
Chen, Y. R.,
Wang, X.,
Templeton, D.,
Davis, R. J.,
and Tan, T. H.
(1996)
J. Biol. Chem.
271,
31929-31936[Abstract/Free Full Text]
|
18.
|
Gonzalez, F. A.,
Seth, A.,
Raden, D. L.,
Bowman, D. S.,
Fay, F. S.,
and Davis, R. J.
(1993)
J. Cell Biol.
122,
1089-1101[Abstract]
|
19.
|
Lenormand, P.,
Sardet, C.,
Pages, G.,
L'Allemain, G.,
Brunet, A.,
and Pouysségur, J.
(1993)
J. Cell Biol.
122,
1079-1088[Abstract]
|
20.
|
Reszka, A. A.,
Seger, R.,
Diltz, C. D.,
Krebs, E. G.,
and Fischer, E. H.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
8881-8885[Abstract]
|
21.
|
Fukuda, M.,
Gotoh, Y.,
and Nishida, E.
(1997)
EMBO J.
16,
1901-1908[Abstract/Free Full Text]
|
22.
|
Chou, S.-Y.,
Baichwal, V.,
and Ferrell, J. E., Jr.
(1992)
Mol. Biol. Cell
3,
1117-1130[Abstract]
|
23.
|
Gille, H.,
Sharrocks, A. D.,
and Shaw, P. E.
(1992)
Nature
358,
414-416[CrossRef][Medline]
[Order article via Infotrieve]
|
24.
|
Hunter, T.,
and Karin, M.
(1992)
Cell
70,
375-387[Medline]
[Order article via Infotrieve]
|
25.
|
Seth, A.,
Gonzalez, F. A.,
Gupta, S.,
Raden, D. L.,
and Davis, R. J.
(1992)
J. Biol. Chem.
267,
24796-24804[Abstract/Free Full Text]
|
26.
|
Cheng, J.-T.,
Cobb, M. H.,
and Baer, R.
(1993)
Mol. Cell. Biol.
13,
801-808[Abstract]
|
27.
|
Hibi, M.,
Lin, A.,
Smeal, T.,
Minden, A.,
and Karin, M.
(1993)
Genes Dev.
7,
2135-2148[Abstract]
|
28.
|
Davis, R.-J.
(2000)
Cell
103,
239-252[Medline]
[Order article via Infotrieve]
|
29.
|
Gupta, S.,
Barrett, T.,
Whitmarsh, A. J.,
Cavanagh, J.,
Sluss, H. K.,
Derijard, B.,
and Davis, R. J.
(1996)
EMBO J.
15,
2760-2770[Abstract]
|
30.
|
Kyriakis, J. M.,
and Avruch, J.
(1990)
J. Biol. Chem.
265,
17355-17363[Abstract/Free Full Text]
|
31.
|
Adler, V.,
Polotskaya, A.,
Wagner, F.,
and Kraft, A. S.
(1992)
J. Biol. Chem.
267,
17001-17005[Abstract/Free Full Text]
|
32.
|
Pulverer, B. J.,
Kyriakis, J. M.,
Avruch, J.,
Nikolakaki, E.,
and Woodgett, J. R.
(1991)
Nature
353,
670-674[CrossRef][Medline]
[Order article via Infotrieve]
|
33.
|
Adler, V.,
Yin, Z.,
Fuchs, S. Y.,
Benezra, M.,
Rosario, L.,
Tew, K. D.,
Pincus, M. R.,
Sardana, M.,
Henderson, C. J.,
Wolf, C. R.,
Davis, R.,
and Ronai, Z.
(1999)
EMBO J.
18,
1321-1334[Abstract/Free Full Text]
|
34.
|
Fuchs, S. Y.,
Dolan, L. R.,
Davis, R.,
and Ronai, Z.
(1996)
Oncogene
13,
1529-1533
|
35.
|
Fuchs, S. Y.,
Xie, B.,
Adler, V. A.,
Fried, V. A.,
Davis, R. J.,
and Ronai, Z.
(1997)
J. Biol. Chem.
272,
32163-32168[Abstract/Free Full Text]
|
36.
|
Fuchs, S. Y.,
Adler, V.,
Buschmann, T.,
Wu, X.,
and Ronai, Z.
(1998)
Genes Dev.
12,
2543-2547
|
37.
|
Trier, M.,
Staszewski, L. M.,
and Bohmann, D.
(1994)
Cell
78,
787-798[Medline]
[Order article via Infotrieve]
|
38.
|
Xia, Z.,
Dickens, M.,
Raingeaud, J.,
Davis, R. J.,
and Greenberg, M. E.
(1995)
Science
270,
1326-1331[Abstract]
|
39.
|
Verheij, M.,
Bose, R.,
Lin, X. H.,
Yao, B.,
Jarvis, W. D.,
Grant, S.,
Birrer, M. J.,
Szabo, E.,
Zon, L. I.,
Kyriakis, J. M.,
Haimovitz-Friedman, A.,
Fuks, Z.,
and Kolesnick, R. N.
(1996)
Nature
380,
75-79[CrossRef][Medline]
[Order article via Infotrieve]
|
40.
|
Westwick, J. K.,
Bielawska, A. E.,
Dbaibo, G.,
Hannun, Y. A.,
and Brenner, D. A.
(1995)
J. Biol. Chem.
270,
22689-22692[Abstract/Free Full Text]
|
41.
|
Noguchi, K.,
Kitanaka, C.,
Yamana, H.,
Kokubum, A.,
Mochizuki, T.,
and Kuchino, Y.
(1999)
J. Biol. Chem.
274,
32580-32587[Abstract/Free Full Text]
|
42.
|
Yamamoto, K.,
Ichijo, H.,
and Korsmeyer, S. J.
(1999)
Mol. Cell. Biol.
19,
8469-8478[Abstract/Free Full Text]
|
43.
|
Milne, D. M.,
Campbell, L. E.,
Campbell, D. G.,
and Meek, D. W.
(1995)
J. Biol. Chem.
270,
5511-5518[Abstract/Free Full Text]
|
44.
|
Fuchs, S. Y.,
Fried, V. A.,
and Ronai, Z.
(1998)
Oncogene
17,
1483-1490[CrossRef][Medline]
[Order article via Infotrieve]
|
45.
|
Shah, K.,
Liu, Y.,
Deirmengian, C.,
and Shokat, K. M.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
3565-3570[Abstract/Free Full Text]
|
46.
|
Liu, Y.,
Shah, K.,
Yang, F.,
Witucki, L.,
and Shokat, K. M.
(1998)
Chem. Biol.
5,
91-102[Medline]
[Order article via Infotrieve]
|
47.
|
Gillespie, P. G.,
Gillespie, S. K.,
Mercer, J. A.,
Shah, K.,
and Shokat, K. M.
(1999)
J. Biol. Chem.
29,
31373-31381[CrossRef]
|
48.
|
Bishop, A. C.,
Ubersax, J. A.,
Petsch, D. T.,
Matheos, D. P.,
Gray, N. S.,
Blethrow, J.,
Shimizu, E.,
Tsien, J. Z.,
Schultz, P. G.,
Rose, M. D.,
Wood, J. L.,
Morgan, D. O.,
and Shokat, K. M.
(2000)
Nature
407,
395-401[CrossRef][Medline]
[Order article via Infotrieve]
|
49.
|
Habelhah, H.,
Shah, K.,
Huang, L.,
Ostareck-Lederer, A.,
Burlingame, A. L.,
Shokat, K. M.,
Hentze, M. W.,
and Ronai, Z.
(2001)
Nat. Cell Biol.
3,
325-330[CrossRef][Medline]
[Order article via Infotrieve]
|
50.
|
Krecic, A.,
and Swanson, M.
(1999)
Curr. Opin. Cell Biol.
11,
363-371[CrossRef][Medline]
[Order article via Infotrieve]
|
51.
|
Nakielny, S.,
and Dreyfuss, G.
(1999)
Cell
99,
677-690[Medline]
[Order article via Infotrieve]
|
52.
|
Bomsztyk, K.,
Seuningen, I.,
Suzuki, H.,
Denisenko, O.,
and Ostrowski, J.
(1997)
FEBS Lett.
403,
113-115[CrossRef][Medline]
[Order article via Infotrieve]
|
53.
|
Lee, M.-H.,
Mori, S.,
and Raychaudhuri, P.
(1996)
J. Biol. Chem.
271,
3420-3427[Abstract/Free Full Text]
|
54.
|
Michael, W.,
Eder, P.,
and Dreyfuss, G.
(1997)
EMBO J.
16,
3587-3598[Abstract/Free Full Text]
|
55.
|
Ostareck, D.,
Ostareck-Lederer, A.,
Wilm, M.,
Thiele, B.,
Mann, M.,
and Hentze, M.
(1997)
Cell
89,
597-606[Medline]
[Order article via Infotrieve]
|
56.
|
Tomonaga, T.,
and Levens, D.
(1995)
J. Biol. Chem.
270,
4875-4881[Abstract/Free Full Text]
|
57.
|
Hovemann, B.,
Reim, I.,
Werner, S.,
Katz, S.,
and Saumweber, H.
(2000)
Gene (Amst.)
245,
127-137[CrossRef][Medline]
[Order article via Infotrieve]
|
58.
|
Du, Q.,
Melnikova, I.,
and Gardner, P.
(1998)
J. Biol. Chem.
273,
19877-19888[Abstract/Free Full Text]
|
59.
|
Tomonaga, T.,
and Levens, D.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
5830-5835[Abstract/Free Full Text]
|
60.
|
Miau, L.-H.,
Chang, C.-J.,
Shen, B.-J.,
Tsai, W.-H.,
and Lee, S.-C.
(1998)
J. Biol. Chem.
273,
10784-10791[Abstract/Free Full Text]
|
61.
|
Hobert, O.,
Jallal, B.,
Schlessinger, J.,
and Ullrich, A.
(1994)
J. Biol. Chem.
269,
20225-20228[Abstract/Free Full Text]
|
62.
|
Bstelo, X.,
Suen, K.-L.,
Michael, W.,
Dreyfuss, G.,
and Barbacid, M.
(1995)
Mol. Cell Biol.
15,
1324-1332[Abstract]
|
63.
|
Taylor, S.,
and Shalloway, D.
(1994)
Nature
368,
867-874[CrossRef][Medline]
[Order article via Infotrieve]
|
64.
|
Weng, Z.,
Thomas, S.,
Rickles, R.,
Taylor, J.,
Brauer, A.,
Seidel-Dugan, C.,
Michael, W.,
Dreyfuss, G.,
and Brugge, J.
(1994)
Mol. Cell Biol.
14,
4509-4521[Abstract]
|
65.
|
Seuningen, I.,
Ostrowski, J.,
Bustelo, X.,
Sleath, P.,
and Bomsztyk, K.
(1995)
J. Biol. Chem.
270,
26976-26985[Abstract/Free Full Text]
|
66.
|
Schullery, D.,
Ostrowski, J.,
Denisenko, O.,
Stempka, L.,
Shnyreva, M.,
Suzuki, H.,
Gschwendt, M.,
and Bomsztyk, K.
(1999)
J. Biol. Chem.
274,
15101-15109[Abstract/Free Full Text]
|
67.
|
Liu, Y.,
Shah, K.,
Yang, F.,
Witucki, L.,
and Shokat, K. M.
(1998)
Bioorg. Med. Chem.
6,
1219-1226[CrossRef][Medline]
[Order article via Infotrieve]
|
68.
|
Weiss, E. L.,
Bishop, A. C.,
Shokat, K. M.,
and Drubin, D. G.
(2000)
Nat. Cell Biol.
10,
677-685[CrossRef]
|