From the Department of Immunology, The Scripps Research Institute,
La Jolla, California 92037, the Department of
Biochemistry, Emory University, Atlanta, Georgia 30322, the
§ Institut für Medizinische Strahlenkunde und
Zellforschung, D-97078 Würzburg, Germany, and the
¶ Novartis Pharma AG, CH-4002 Basel, Switzerland
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ABSTRACT |
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A novel protein kinase whose activity can be
stimulated by mitogen in vivo was cloned and characterized.
The cDNA of this gene encodes an 802-amino acid protein (termed
RLPK) with the highest homology (37% identity) to the two protein
kinase families, p90RSK and p70RSK. Like
p90RSR, but not p70RSK, RLPK also contains two
complete nonidentical protein kinase domains. RLPK mRNA is widely
expressed in all human tissues examined and is enriched in the brain,
heart, and placenta. In HeLa cells, transiently expressed
epitope-tagged RLPK can be strongly induced by epidermal growth factor,
serum, and phorbol 12-myristate 13-acetate, but only moderately
up-regulated by tumor necrosis factor- Two families of ribosomal S6 protein kinase,
p90RSK and p70RSK, have been identified in
mammals (1-4). Members of p90RSK family contain two
nonidentical, but complete, kinase domains and have molecular masses
ranging from 85 to 92 kDa (5-9). The two p70RSK isoforms
have molecular masses of 70 and 85 kDa, but are believed to be
transcribed from a single gene by a differential splicing (10, 11).
Unlike p90RSK, p70RSK contains only one kinase
domain, which is about 51% identical to N-terminal kinase domain of
p90RSK. These two protein families were collectively called
RSK because of their ability to phosphorylate ribosomal S6 protein of
the 40 S ribosomal subunit (10, 12-17). However, the fact that
specific inhibition by rapamycin of the p70RSK but not
p90RSK inhibited growth factor-mediated phosphorylation of S6 protein
in vivo demonstrated that p70RSK, rather than
any isoforms of the p90RSK, was really responsible for
growth-associated phosphorylation of S6 protein (18-20).
p90RSK appears to have a broad range of substrates in
vitro that include glycogen synthase kinase 3, Nur 77, c-Fos (21,
22), serum response factor (23), 40 S ribosomal subunit, and histones H1, H3, and H2B (12, 24). In addition, estrogen receptor In this study, we reported cloning and characterization of RLPK, a
novel protein kinase with two kinase domains. The overall sequence
identity of this kinase to p90RSKs is about 37%, which is
much lower than that within the p90RSK family (79-82%),
suggesting that while it is homologous to p90RSKs, it is
not an isoform of p90RSK family. Despite the distinct amino
acid sequence of RLPK, we have found similarities in its substrate
specificity and activation profile to that of both p90RSK
and p70RSK. We demonstrated that the intrinsic activity of
recombinant RLPK is not dependent on phosphorylation and cannot be
regulated by MAP kinases in vitro. Therefore, RLPK may
represent another class of kinase with two kinase domains, and its
regulation mechanism and function may differ from that of
p90RSK and p70RSK.
Materials--
Pfu DNA polymerase and PHAS-I were
purchased from Stratagene (La Jolla CA). S6 peptide (RRRLSSLRA, amino
acids 231-239), activated and partially purified p70RSK
and p90RSK were purchased from Upstate Biotechnology (Lake
Placid, NY). Epidermal growth factor (EGF), tumor necrosis factor- cDNA Cloning of RLPK--
As reported previously (37), two
peptide sequences, LXTPCYTPYYAP and LXTPCTANFVAP
from MAPKAP-K2/3 and p90RSK2/3, respectively, were used to
search for new homologues of human MAP kinase-regulated protein kinase
in the GenBankTM data base of expressed sequence tags. Two
clones containing LXTP motif flanked by unknown sequences
were found (GenBankTM accession numbers N57096 and H09985).
One of the clones, N57096, after being completely sequenced, was found
to encode a part of a potentially new protein kinase showing homology
to p70RSK and p90RSK. Thus, we named this
putative protein as RLPK (for RSK-like protein kinase). Clone N57096
was used as a probe to screen a human placental library. 5 × 105 clones were screened, and two positive clones were
isolated. The longer one was completely sequenced and found to contain
the full-length coding region of the protein.
Northern Blot Analysis--
A multiple human tissue mRNA
blot containing 2 mg of poly(A)+ RNA from eight different
tissues was purchased from CLONTECH. The blot was
hybridized to a probe prepared by random priming part of the cDNA
of RLPK in clone N57096 with [ cDNA Constructs and Recombinant Proteins--
The bacteria
expression plasmids for His6-tagged proteins or their
mutants were constructed by inserting the full coding region of the
cDNAs, generated by polymerase chain reaction using Pfu polymerase, into the pET-11 vector (Novagen, Madison, WI). The proteins
were expressed and purified as described previously (38). The mammalian
expression plasmid for RLPK was constructed by subcloning hemagglutinin-epitope tagged full-length RLPK cDNA into pcDNA3 vector (Invitrogen, San Diego, CA). All point mutations and truncations including human CREB N-terminal 2-158, RLPK(581A), RLPK(581D), RLPK(700A), and RLPK(700D) were created by a polymerase chain reaction-based method using Pfu DNA polymerase (39). The
sequence of the oligonucleotides used for creating those constructs is available upon request. Each mutation was subjected to DNA sequencing to confirm the correct amino acid replacement. Expression and purification of MKK6(E), MKK7(D), MEK1(E), HSP27, eIF4-e, wild type
PRAK, p38 In Vitro Protein Kinase Assay--
In vitro kinase
assays were performed using standard experimental conditions as
described (38). Briefly, 2.5 µg of bacterially expressed and Ni-NTA
column-purified wild type or mutant proteins of RLPK were used in a
total reaction of 40 µl containing 10 µg of substrate protein, 100 µM cold ATP, and 10 µCi of [ Cell Transfection and Extracellular Stimulation--
HeLa cells
were plated in six-well plates at 60% confluence in Dulbecco's
modified Eagle's medium supplemented with 10% FBS. After 12 h,
the cells were transfected with expression vectors containing HA-tagged
RLPK or empty pcDNA3 vector using LipofectAMINE (Life Technologies,
Inc.). Thirty-six hours after transfection the cells were incubated in
the presence or absence of different stimuli. For the stimulation by
EGF, insulin-like growth factor, and serum, the transfected cells were
washed twice by phosphate-saline buffer and kept in nonserum
Dulbecco's modified Eagle's medium for 5 h before the treatment.
Various inhibitors, as indicated in Fig. 3B, were added 30 min before EGF stimulation to the cells. HA-tagged RLPK was
immunoprecipitated using anti-HA monoclonal antibody-conjugated agarose
beads and quantitated by Western blot with monoclonal anti-HA antibody.
HA-RLPK from each treatment was used in in vitro kinase
assay using histone 2B as substrate. HSP27 was used as substrate for
PRAK. The relative RLPK kinase activity by various stimuli and
inhibitors was normalized to the amount of each HA-tagged protein
detected on Western blot and then compared with the control activity.
Phosphatase Treatment of Recombinant RLPK--
About 20 µg of
recombinant RLPK bound to the Ni-NTA beads was washed three times with
the binding buffer (40 mM Tris of pH 6.5, 0.3 M
NaCl, and 10 mM imidazole) and split equally into two parts
(namely 1 and 2). Part 2 was further treated with 5 units of potato
acid phosphatase in phosphatase buffer (40 mM Tris, pH 6.5, 5 mM dithiothreitol, 0.1 M NaCl) at 30 °C
for 15 min with shaking. Part 1 was incubated under the same
conditions, but without potato acid phosphatase. After three washes
with ice-cold binding buffer at pH of 7.9, RLPK from both parts was
eluted out from Ni-NTA beads and used in kinase assay. The same
procedure was used for PRAK except that kinase was pre-activated in the
beads by incubating with 5 µg of GST-p38 Cloning of a Novel Human Protein Kinase with Two Kinase
Domains--
We reported previously cloning of a novel MAP
kinase-regulated protein kinase by searching a conserved MAP kinase
regulatory site in the GenBankTM data base (37). Another
candidate for an unknown protein kinase containing the sequence LKTP
located in the T-loop was found. It was encoded in part by two EST
clones (GenBankTM accession numbers N57096 and H09985
containing overlapping cDNA sequence). The full-length sequence of
this gene was cloned by using the insert of the N57096 EST clone as a
probe to screen a human placental library. The longest cDNA
isolated from the screening had a length of 2840-base pair with an
in-frame stop codon preceding the first ATG predicted as the starting
codon for the gene. The open reading frame contained 2406 base pairs, which encoded 802 amino acids. Alignment of the deduced protein to the
GenBankTM data base indicated that it was most similar to
p70RSK and p90RSK, representing two different
groups of S6 protein kinases. Thus, we named this putative protein
kinase RLPK, which stands for RSK-like protein kinase. Protein domain
analysis (using the Expasy profile domain search program) revealed
that, like p90RSKs, RLPK contained two nonidentical, but
complete, protein kinase domains. The first kinase domain was located
in amino acids 40-340, and the second one was within amino acids
420-720. Protein sequence analysis indicated that RLPK was 36.7, 36.8, 36.7 and 36.7% identical to human p70RSK,
p90RSK1, p90RSK2, and p90RSK3,
respectively, and had less than 28% identity to any other known human
protein kinases. Like p90RSK, the N-terminal kinase domain
of RLPK had homology to p70RSK, while the C-terminal kinase
domain was similar to MAP kinase-regulated protein kinases such as
MAPKAPK2, MNK1, and PRAK. Although RLPK and p90RSK shared
the common structural feature of two kinase domains, the percentages of
identity among three p90RSK isoforms were much higher
(79-82%) than that between p90RSKs and RLPK. As was shown
in a computer-generated phylogenetic tree (Fig.
1B), RLPK is rather dissimilar
to p90RSK group as p70RSK. This suggested that
RLPK was not a new isoform of p90RSK, but rather
represented a new class of two-kinase domain kinase and that RLPK may
have diverged earlier in its structure and physiological function from
a two-kinase domain ancestral protein kinase than did isoforms of the
RSK family under evolutionary selection pressure.
Northern blot analysis showed a single band of 4.4-kilobase transcript
of RLPK in all eight tissues examined; however, the mRNA of this
gene seemed much more abundant in heart, brain, and placenta than in
lung, kidney, and liver (Fig. 1C).
Kinase Activity of RLPK in Vitro--
Wild type RLPK was expressed
as His6-tagged protein in bacteria and purified for
biochemical analysis. The recombinant protein was tested in
vitro on a panel of proteins, which were known substrates for
various kinases, including MAP kinase, MAPK-activated protein kinases,
and RSKs (Fig. 2A). We found
that histone 2B was the most preferred substrate in this panel. Myelin
basic protein, histone 1, and CREB were also phosphorylated by RLPK,
but to a lesser extent and the rest of others were poor substrates.
Since it was known that S6 protein, one of the components in small
ribosomal 40 S, was a preferred substrate for both p90RSK
and p70RSK, we sought to compare the activity of RLPK
toward S6 protein with that of p90RSK and
p70RSK. Because p90RSK and p70RSK
used in our experiment were partially purified, the absolute quantities
of the two enzymes were not determined. Therefore, we first normalized
RLPK activity to the p90RSK and p70RSK using
histone 2B as substrate. A peptide from S6 protein
(231RRRLSSLRA239), widely used as standard
in vitro substrate for both types of RSKs or related protein
kinases, was used in the in vitro kinase assay. As shown in
Fig. 2C, RLPK possessed an activity toward the S6 peptide
similar to that of p90RSK and p70RSK.
RLPK Is Activated by Growth-related Stimuli in Culture
Cells--
To determine which extracellular stimuli can activate RLPK
in vivo, a variety of agonists were tested for their ability
to activate transient expressed HA-tagged RLPK in HeLa cells. The activity of RLPK was measured in an immunokinase assay with histone 2B
as the substrate. As shown in Fig.
3A, RLPK activity was
significantly stimulated by 12-myristate 13-acetate, EGF, and serum,
while the proinflammatory cytokine tumor necrosis factor-
To assess which signal pathways were involved in regulating RLPK
activity in vivo, a panel of inhibitors with either specific or broad inhibitory effects on PKC, tyrosine kinase,
p70RSK, MEK1, and p38 RLPK Can Be Phosphorylated, but Not Activated, by MAP Kinases in
Vitro--
As shown in Fig. 1A, RLPK contained a putative
MAP kinase phosphorylation site, LXTP, in the T-loop of
C-terminal kinase domain. Therefore, RLPK could be a MAP
kinase-regulated protein kinase. To test this hypothesis, we performed
a coupled kinase assay in which different MAP kinases activated by
their corresponding MKKs were included in the kinase reaction to
measure kinase activity of RLPK using histone 2B as substrate. As shown
in Fig. 4A, RLPK was
apparently phosphorylated by activated p38 We have identified and cloned a novel RSK-like protein kinase,
RLPK. This protein shows 37% overall identity to p70RSK
and p90RSK and has been demonstrated to share similar
in vitro substrates to these two families of kinases.
Structurally like p90RSKs, RLPK contains two kinase
domains. The N-terminal kinase domain of RLPK is homologous to
p70RSK and the C-terminal kinase domain shows homology to a
group of known MAP kinase-regulated protein kinases. In addition to
this two-kinase domain structure, the activation profile of RLPK is also more similar to p90RSK than to p70RSK in
that RLPK is insensitive to rapamycin inhibition. However, by contrast,
several structural features of RLPK distinguish it from the p90RSK
family. First, the overall similarity of RLPK to the p90RSK
group as a whole is much lower than that within the p90RSK
family (37% versus 79-82%). Second, RLPK contains an
additional 49-amino acid C-terminal serine and threonine-rich
tail, which is missing in all isoforms of p90RSKs. Third,
although the Thr-581 residue of RLPK falls in the conserved LXTP motif in the T-loop of the C-terminal kinase domain,
the residue in the X position is a positively charged
lysine. This is unique to all of known MAP kinase-regulated protein
kinase, including all members of p90RSKs that have a
neutral (methionine) residue instead (Fig. 1A). These
features indicate that RLPK represents a new class of protein kinase
rather than being an isoform of p90RSK.
RLPK activity was found to be tightly controlled in vivo
(Fig. 3A). However, kinase activity associated with
recombinant RLPK was not influenced by phosphorylation with MAP kinase
or dephosphorylation with phosphatase. These data argue against our
original prediction that RLPK is a MAP kinase-regulated protein kinase.
However, if the following assumptions are true, RLPK may still be
considered a MAP kinase-regulated protein kinase: 1) pre-existing
post-translational modifications such as phosphorylation or protease
cleavage is required for RLPK to be regulated by MAP kinases; 2) other
co-factors are involved in RLPK regulation by MAP kinases. It has been
proposed that the two kinase domains in p90RSK comprise
different substrate specificities and that the N-terminal domain was
believed to be responsible for the phosphorylation of c-Fos and S6
protein (9). We have found that the sequence identity between
N-terminal kinase domain of RLPK and p90RSKs is much higher
(~51%) than the overall sequence identity (~37%). The sequence
identity between N-terminal kinase domain of RLPK and that of
p70RSK is also 51%. Such high similarity may account for
the similar substrate specificity observed among these different groups
of kinases. When the C-terminal kinase domain sequence of RLPK and that
of the p90RSKs are compared, only 39% sequence identity is
found, suggesting this domain in RLPK may differ more from its
p90RSK counterpart in its function or regulation mechanism
than the N-terminal kinase domain. As we mentioned in the results
section, the C-terminal kinase domain of RLPK is similar to MAP
kinase-regulated protein kinases. Although we showed that
phosphorylation of RLPK by ERK or p38 in vitro had no
influence on its activity toward histone 2B, we cannot exclude the
possibility that phosphorylation did enhance the kinase activity of the
C-terminal kinase domain, and the inability to detect such activation
in our kinase assay would be the result of the inability of the
C-terminal kinase domain to use histone 2B as substrate. Nevertheless,
the role of phosphorylation of RLPK by MAP kinases still needs further investigation.
Although RLPK is activated by growth factors, as are p90RSK
and p70RSK, the studies using chemical inhibitors indicated
that the upstream signal pathways of RLPK differ from both
p90RSK and p70RSK. However, the possibility
that the different kinase domains phosphorylate specific substrates
suggested that the data present in Fig. 3 may only partially represent
the activation and regulation of RLPK, since stress stimuli may have
increased RLPK activity to substrates other than histone 2B. The
partial inhibition of RLPK activation by PD98059 and SB203580 may have
also resulted from the use of unsuitable substrate for the kinase
assay. A key question to be investigated is the existence and
identification of specific substrates for each kinase domain.
Crystal structure studies have revealed that some kinases such as ERK2
functioned as dimers and that phosphorylation is required for the dimer
formation, which is essential for the kinase normal ligand-dependent relocalization (42). The two kinase domain structure of RLPK and p90RSK may be functionally equivalent
to a dimerized pair of kinase molecules. If this is true, a study of
truncated RLPK derivatives containing two kinase domains independently
would provide valuable insight into their separate regulation and
substrate specificity. Although the independent activities of each
kinase domain is important, a cooperative effect between the two kinase
domains is also potentially of great interest. Furthermore, as
discussed above, the role of post-translational modification of RLPK in
kinase relocalization and interaction with other proteins needs to be defined.
and other stress-related
stimuli. The activity of RLPK stimulated by epidermal growth factor was
not inhibited by several known protein kinase C inhibitors nor by
rapamycin, a known specific inhibitor for p70RSK, but could
be inhibited by herbimycin A, a tyrosine kinase inhibitor, and
partially inhibited by PD98059 or SB203580, inhibitors for the
mitogen-activated protein kinase pathways. Recombinant RLPK possesses
high phosphorylation activity toward histone 2B and the S6 peptide,
RRRLSSLRA. Although purified recombinant RLPK can be phosphorylated by
ERK2 and p38
in vitro, its activity is not affected by
this phosphorylation. Moreover, the treatment of RLPK with acid
phosphatase did not reduce its in vitro kinase activity.
These data suggest that RLPK is structurally similar to previously
isolated RSKs, but its regulatory mechanism may be distinct from either
p70RSK or p90RSKs.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
(25),
actin-binding protein 280 (26), and
CREB1 (27) were reported to
be regulated by p90RSK in vivo. It has also been
shown that p90RSK2 forms complexes with MAP kinase ERK1
(28, 29) and with a nuclear regulatory protein CBP (30). The scope of
these interactions indicate the complexity of the regulation and
function of p90RSKs. Although both p70RSK and
p90RSK respond to growth factor-related stimuli, quite
different mechanisms have been implicated in the regulation of these
kinases. The p70RSK is rapamycin-sensitive and relies on
multisite phosphorylation to be activated (31-33). The interactions
with Rho family members such as RAC1 and Cdc2 may be crucial for
p70RSK activation (34). As for p90RSKs, ERK1
and ERK2 were believed to be responsible, at least in part, for their
activation (4, 35, 36). Despite the progress in this field, regulation
mechanisms and biological functions of these two RSK protein kinase
families are still largely unknown.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
(TNF-
), insulin-like growth factor-1, and phorbol 12-myristate
13-acetate were purchased from Genzyme (Cambridge, MA). Calcium
ionophore A23187, histone H1, rapamycin, herbimycin A, genistein,
GF109203, H7, HA1004, and erbstatin analog were purchased from Sigma.
Ro318220, PD98059 and SB203580 were purchased from Calbiochem. Potato
acid phosphatase, myelin basic protein, anisomycin, and arsenite were purchased from Sigma. Histone 2B and anti-HA epitope antibody for
Western blot were purchased from Boehringer Mannheim. eIF-4e cDNA
was obtained from Dr. C. G. Proud (University of Kent at Canterbury). CREB cDNA was obtained from Dr. R. A. Maurer
(Oregon Health Sciences University, Portland, OR). The cDNA clones
of N57096 and H09985 were purchased from Research Genetics (Huntsville,
AL). Ni-NTA beads for His-tagged protein purification were purchased
from Qiagen and glutathione-Sepharose 4B beads for GST fusion protein
purification was purchased from Amersham Pharmacia Biotech. HA-affinity
matrix beads was purchased from Berkley Antibody Co.
-32P]dCTP as described
before (38). After exposure to an x-ray film, the blot was stripped in
1% SDS solution by boiling for 1 min and re-hybridized to a
[
-32P]dCTP-labeled human
-actin cDNA probe.
, ERK2, JNK2, GST fusion protein of ATF-2(1-109) and
GST-p38
were performed as described previously (37, 40).
-32P]ATP.
In the coupling kinase assay, the reactions included 1 µg of purified
p38, JNK2, or ERK2 and 0.2 µg of the corresponding activator MKK6(E),
MKK7(D), or MEK1(E), respectively. The reactions were stopped by adding
an equal amount of 2 × SDS sample buffer. The proteins were then
separated on SDS-PAGE. The intensities of 32P-labeled
proteins were determined by phosphoimage analysis. For the S6 peptide
phosphorylation assay, 50 ng of S6 peptide and an appropriate amount of
protein kinase, which had been determined by normalizing equal activity
of each kinase toward histone 2B, were incubated in a kinase reaction
as described above. The same kinase reaction without S6 peptide was
used as the control. The reactions were stopped by adding 8 volumes of
75 mM phosphoric acid and loaded onto phosphocellulose spin
filters (Pierce). After three washes with 75 mM phosphoric
acid, the incorporated 32P in the peptide substrates
absorbed on the filter was determined by liquid scintillation counting.
and 1 µg of MKK6(E) in
the presence of 300 µM ATP at 30 °C in kinase assay
buffer with shaking for 15 min. GST-p38 and MKK6 were removed by
washing the beads with the phosphatase buffer.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
A, amino acid sequence comparison of
human RLPK (the second line), p70RSK (designated as S6K70),
and three p90RSK isoforms (designated as RSK1, RSK2, and
RSK3, respectively) by Lasergene Megaline program (DNAStar, Madison,
WI). Residues conserved between two or more proteins are boxed. The conserved threonine
residues (Thr-581 in RLPK) in the LXTP motif are marked by a
triangle underneath the sequences. The cDNA sequence of
RLPK has been deposited in the DDBJ/EMBL/GenBankTM data
base under the accession number of AF08000. B, ancestral
relationships among RLPK, p70RSK, and p90RSKs
are presented as a phylogenetic tree created by the Lasergene Megaline
program. The scale under the tree indicates the distance between
sequences in substitution event units. C, distribution of
RLPK transcripts in different tissues. A Northern blot containing 2 µg of poly(A) mRNA isolated from various human tissues was
hybridized to a RLPK probe (upper panel). The same blot was
stripped and re-hybridized to a human -actin probe to provide a
reference for equal loading of the samples in each lane (lower
panel). The arrowhead indicates the positions of
transcripts of
-actin.
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Fig. 2.
In vitro kinase activity of RLPK.
A, approximately 2 µg of recombinant RLPK purified from
bacteria was used in each kinase reaction with 10 µg of
His6-HSP27, His6-CREB(2-158),
His6-eIF-4e, Phas-1, myelin basic protein, histone 2B,
histone 1, or GST-ATF2(1-109) as substrates. The kinase reactions were
stopped by adding SDS sample buffer, and the reaction products were
analyzed by SDS-PAGE. Substrate phosphorylation was detected by
autoradiography as shown. Comparable results were obtained in two
independent experiments. B, confirmation of an equal amount
of each protein used in A by a parallel loading in a
separate SDS-PAGE stained by the Coomassie Blue. C,
comparison of the relative activities of RLPK, p90RSK, and
p70RSK to phosphorylate the S6 peptide (RRRLSSLRA). The
amounts of protein kinases used in this assay were normalized to
histone 2B phosphorylation activity (data not shown). The
phosphorylation activity for each kinase was defined as the total
counts of counts/min from a kinase reaction containing both kinase and
the S6 peptide subtracting the counts from the autophosphorylation of
the corresponding kinase. The results shown here for S6 peptide
phosphorylation represent three independent experiments.
and cell
stress stimuli calcium ionophore A23187, anisomycin, and arsenite also increased RLPK activity, but to a lesser extent. A Western blot demonstrated the amount of RLPK that was used in each reaction (lower panel of Fig. 3A).
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Fig. 3.
Activation and inhibition of RLPK in cultured
cells. A, the activity of various extracellular stimuli
were tested for stimulating RLPK in HeLa cells under the conditions as
described under "Experimental Procedures." Samples of approximately
106 HeLa cells, transiently transfected with HA-tagged
RLPK, were treated with tumor necrosis factor- (100 ng/ml),
12-myristate 13-acetate (100 nM), Ca2+
ionophore A13287 (5 µM), arsenite (200 µM),
anisomycin (50 ng/ml), EGF (1 ng/ml), insulin-like growth factor
(2 ng/ml), or 20% serum at 37 °C for 20 min. RLPK was
immunoprecipitated from the cell lysates using HA-affinity metrics
beads. Kinase activity in each of immunoprecipitates was assayed using
histone 2B as substrate (upper panel), and the amount of
HA-RLPK used in each kinase reaction was determined by Western blotting
(lower panel). The relative -fold activation of RLPK by
different stimuli was calculated using the intensity of phosphorylated
H2B that has been normalized to the amount of HA-RLPK used in each
reaction to compare with that of the control. B, various
inhibitors were tested for their ability to inhibit RLPK activity
in vivo. HeLa cells that transiently expressed HA-RLPK were
serum-starved for 12 h and then pretreated with GF-10923X (10 µm), H7 (20 µm), HA1004 (20 µm), Ro318220 (5 µm), herbimycin A
(10 µg/ml), genistein (20 µg/ml), rapamycin (5 ng/ml), erbstatin
analog (1.5 µg/ml), PD98059 (20 µM), or SB203580 (20 µM) at 37 °C for 20 min before EGF (1 ng/ml)
stimulation. After 30-min EGF stimulation, HA-tagged RLPK was
immunoprecipitated from cell lysate. Kinase activity in each of the
immunoprecipitates was assayed (upper panel) and HA-RLPK was
quantitated (lower panel) as described for A. The
relative inhibition of EGF-induced RLPK activation by each drug was
defined by following formula: (1
A/C) × 100%, in which A represents intensity of phosphorylated H2B
from a given reaction after normalization to the amount of HA-RLPK used
in that reaction, and C is the intensity of phosphorylated
H2B from EGF-induced sample without any drug treatment.
/
were used. HeLa cells that
transiently expressed HA-tagged RLPK were pretreated with individual
inhibitors before EGF stimulation. As shown in Fig. 3B,
tyrosine kinase inhibitor herbimycin A had the most profoundly
inhibitory effect with more than 60% inhibition being observed.
Inhibitions by two other tyrosine inhibitors, genistein and erbstatin
analog, were also observed, suggesting that EGF stimulation of RLPK was
mediated through a tyrosine kinase. There was a partial inhibition of
RLPK activity when the MEK1 inhibitor, PD98059, or the p38 inhibitor,
SB203580, was used, suggesting a potential direct or indirect
involvement of both ERK and p38 MAP kinases in RLPK signal pathway. In
contrast, rapamycin, a p70RSK specific inhibitor in
vivo, did not show any inhibitory effect on RLPK activation nor
did the PKC inhibitors, GF109230X, H7, or Ro318220. When Ro318220 was
used in an in vitro kinase assay, the IC50 for
RLPK was determined to be ~500 nM (data not shown) that
was much higher than that of p90RSK (5 nM) and
p70RSK (100 nM) (41). Therefore, it appeared
that the regulation of RLPK was apparently different from that of
p70RSK and may also be distinct from that of
p90RSK. Furthermore, it suggested that EGF-mediated
activation of RLPK did not necessarily go through PKC.
and ERK2 (lanes 2 and 6), but not by activated JNK2 (lane
4). However, the activity of RLPK toward histone 2B was not
influenced by the phosphorylation as compared with the control
(lane 1). These results suggested that either RLPK was not
regulated by MAP kinase phosphorylation or that the recombinant RLPK
had been already phosphorylated at regulatory sites during its
biosynthesis or during the process of purification. To examine the
second possibility, we took two approaches. First we employed
dephosphorylation, in which the bacterially expressed wild type RLPK
was treated with potato acid phosphatase before being used in a kinase
assay. As shown in Fig. 4B, phosphatase treatment did not
substantially reduce the RLPK activity toward histone 2B. In contrast,
the activity of PRAK, a characterized p38-regulated protein kinase (37)
as the control, was almost completely abolished by the same phosphatase
treatment. Thus, phosphorylation may not be required for this RLPK
activity. In the second approach, mutations on the putative MAP kinase
phosphorylation sites were created. Since previous studies have
suggested that p90RSKs were regulated by ERK in
vivo (36) and the Thr residue in LXTP motif of
p90RSK2 is the regulatory phosphorylation site (4), we
reasoned that if the conserved threonine residue in the LXTP
sequence of RLPK was an operational regulatory site, mutation of this
site to a nonphosphorylatable alanine residue would prevent activation
of RLPK, otherwise, mutation to a negative charged aspartic acid would
mimic a phosphate group and result in constitutively activated RLPK. We
constructed two sets of mutants by converting Thr-581 and Thr-700 to
alanines or aspartic acids. Thr-700 was selected as another site to be
mutated because the surrounding sequence, PLMTPD (located immediately
downstream of the XI kinase subdomain in the C-terminal kinase domain
of RLPK), was very similar to that of the conserved regulatory motif.
As shown in Fig. 4C, none of the mutants of RLPK exhibited
substantial change in their kinase activity toward histone 2B as
compared with that of the wild type RLPK, implying that neither Thr-581
nor Thr-700 functioned as a regulatory site in RLPK. Collectively, our
data suggested that histone 2B phosphorylation activity by bacterially
expressed RLPK was not subjected to direct regulation by ERK, JNK, and
p38 MAP kinases nor necessarily dependent on phosphorylation.
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Fig. 4.
Effects of phosphorylation and mutation on
RLPK activity. A, the effects of three different MAP
kinases, p38 , JNK2, and ERK2 (1 µg of each), activated by their
corresponding upstream activators, MKK6(E), MKK7(D), and MEK1(E) (0.2 µg of each), respectively, (not shown in the figure), on the activity
of RLPK (3 µg) were assessed by in vitro coupling kinase
assays using histone 2B (10 µg) as substrate. The plus
signs indicate the presence of the proteins in the reaction. The
phosphorylation of the proteins was determined by phosphoimaging. The
position of each protein on the SDS-PAGE are as marked. B,
effect of dephosphorylation on recombinant RLPK activity. His-tagged
wild type RLPK bound to the Ni-NTA beads was incubated with or without
potato acid phosphatase under the conditions as described under
"Experimental Procedures," then subjected to kinase assay using
histone 2B as substrate. As a control, His-tagged PRAK was
pre-activated by GST-p38 and MKK6 on the Ni-NTA beads and then treated
with phosphatase under the same conditions as RLPK. The substrate used
in PRAK kinase assay was HSP27 (10 µg). C, kinase activity
of mutant RLPK. An equal amount (3 µg) of recombinant RLPK,
RLPK(T581A), RLPK(T581D), RLPK(T700A), or RLPK(700D) was used in
protein kinase assay with histone 2B (10 µg) as substrate.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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ACKNOWLEDGEMENTS |
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We thank Dr. Christopher G. Proud for eIF-4E expression plasmid, Dr. Richard A. Maurer for CREB expression plasmid, and Janet V. Kuhns for secretarial assistance.
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Addendum |
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During the preparation of this manuscript, Deak et al. (43) reported the sequence of a human kinase termed MSK1 that is identical to RLPK.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM51417 and AI41637 and American Heart Association Grant-in-aid 950076090. This is publication number 11890-IMM from the Department of Immunology at The Scripps Research Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF08000.
Established investigator of the American Heart Association. To
whom correspondence should be addressed: Dept. of Immunology, The
Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA
92037. Tel.: 619-784-8704; Fax: 619-784-8227; E-mail: jhan{at}scripps.edu.
The abbreviations used are: CREB, cAMP response element-binding protein; MAP, mitogen-activated protein; EGF, epidermal growth factor; HA, hemagglutinin; GST, glutathione S-transferase; Ni-NTA, nickel-nitrilotriacetic acid; PAGE, polyacrylamide gel electrophoresis; CBP, CREb-binding protein; EST, expressed sequence tags.
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