From the Departments of Central Nervous System
Diseases, ¶ Vascular and Metabolic Diseases, and
Discovery
Chemistry of F. Hoffmann-LaRoche, Ltd., CH-4070 Basel, Switzerland
and the Departments of ** Epidemiology and Public Health and
Immunobiology, Yale University School of
Medicine, New Haven, Connecticut 06520-0834
Received for publication, July 3, 2000, and in revised form, October 13, 2000
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ABSTRACT |
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RSKB, a p90 ribosomal S6 protein kinase with two
catalytic domains, is activated by p38- and extracellular
signal-regulated kinase mitogen-activated protein kinase
pathways. The sequences between the two catalytic domains and of the
C-terminal extension contain elements that control RSKB activity. The
C-terminal extension of RSKB presents a putative bipartite
713KRX14KRRKQKLRS737
nuclear location signal. The distinct cytoplasmic and nuclear locations of various C-terminal truncation mutants supported the hypothesis that the nuclear location signal was essential to
direct RSKB to the nuclear compartment. The
725APLAKRRKQKLRS737 sequence also
was essential for the intermolecular association of RSKB with p38. The
activation of RSKB through p38 could be dissociated from p38 docking,
because RSKB truncated at Ser681 strongly responded
to p38 pathway activity. Interestingly, The p90 ribosomal S6 protein kinases
(RSKs)1 are a family of
Ser/Thr protein kinases composed of two catalytic domains, each with canonical ATP-binding site and activation loop sequences. In
addition, RSKs contain a regulatory linker sequence connecting the two
kinase domains and an extended C-terminal tail. RSKs comprise RSK1-RSK3, which are stimulated through the ERK pathway (1-6); the
more recently identified mitogen- and stress-activated protein kinase
type 1 and RSKB, which are activated by both the p38 and ERK pathways
(7-9); and RSK4 (10). RSKs are involved in many diverse functions,
such as regulation of glycogen metabolism by phosphorylating glycogen
synthase kinase-3 and the G-subunit of protein phosphatase 1, and cell
survival of cerebellar neurons through phosphorylation of BAD (reviewed
in Refs. 11-13). RSKs function in the control of M phase entry of
oocytes during meiosis and chromatin remodeling through histone H3
phosphorylation (14, 15). Furthermore, RSKs participate in the
regulation of transcription factors and coregulators, such as CREB
(3-5, 7, 8), CREB-binding protein and p300 (16), c-Fos (17), and
estrogen receptor (18). Deficient mutants of RSK2 in man are linked to
Coffin-Lowry syndrome, characterized by mental retardation and
malformations (19). Deletion of RSK4 is common in patients with
X-linked mental retardation (10). Interestingly, RSKB maps to the
BBS1 locus (20), which is associated with
Bardet-Biedl syndrome with manifestations reminiscent of Coffin-Lowry
syndrome (21), and may be a candidate BBS gene.
Many Ser/Thr-kinases present a resting state characterized by an
autoinhibitory conformation of the C-terminal extension. Phosphorylations, or interactions with other proteins, that relax autoinhibition, are required for the activation of these enzymes. For
example, the activation of MAPKAPK2, in addition to
phosphorylation of Thr205 in the catalytic site, correlated
with phosphorylation of a threonine in a
PXTP318 motif in the C-terminal tail,
which contains an autoinhibitory domain with homology to the
amphiphilic A-helix of other kinases (22-24). In CaMKII, a C-terminal
segment blocks the catalytic site in the absence of calmodulin; upon
calmodulin binding, a threonine within that segment is phosphorylated,
disrupting autoinhibition and leading to a calcium-independent active
state of the enzyme (25, 26). When compared with these single-domain
enzymes, RSKs with two kinase domains and regulatory sites in linker
and C-terminal tail present more complex regulation. Commonly, the N-terminal kinase of RSKs phosphorylates substrates, whereas the C-terminal kinase has a role in regulating RSKs activity. For example,
the stepwise activation of RSK1 involved phosphorylations of a
threonine in the C-terminal activation loop and of a serine in the
linker through ERK, and further phosphorylations of linker and
N-terminal activation loop sites through autophosphorylation (27). RSK2
is activated through integrating signals from two independent upstream
kinase pathways, ERK and 3-phosphoinositide-dependent protein kinase 1, targeting the C-terminal and N-terminal domains, respectively (28). Specific docking sites in the C-terminal tail of
RSKs, facilitating interaction with upstream MAPKs, in some instances
were found to be essential for activation (8, 29, 30). The profound
control exerted by C-terminal tail elements was further demonstrated by
the constitutive activity generated by truncation or mutation in the
conserved putative autoinhibitory C-terminal helix of RSK2 (31).
RSK1-RSK3 interact with ERK independent of activation state and locate
both to the cytoplasm and nucleus under resting conditions; upon
activation, the complex translocates to the nucleus (32). Sequence
comparison suggested that elements directing RSKs in general to the
nuclear compartment reside in the C-terminal tail (29). Thus, the
control of subcellular location, autoinhibition, and protein-protein
recognition in the association with upstream MAPKs all appear to be
functions of the C-terminal sequences of RSKs.
Here, we present a study of regulatory sites in the C-terminal tail of
RSKB. Sequential truncations of the C-terminal tail revealed elements
mediating nuclear location and p38 association. The C-terminal kinase
of RSKB has sequence similarities with CaMKs. The structure of rat
CaMKI (33) was of particular interest, because it presented an
autoinhibited conformation with a key AFN motif adjoining a helical
stretch of the C-terminal extension (26, 34); the phenylalanine of this
motif played a crucial role in the binding of the C-terminal extension
to the body of CaMKI and maintaining the resting state (33). This AFN
motif is conserved in RSKB. Sequence alignment and structure prediction suggested a study of the role of Phe709 within that motif
in the control of RSKB function.
Reagents--
Standard reagents were from various sources as
reported (8, 35, 36). Transfectam procedure was from Promega (Madison, WI). CREBtide (123KRREILSRRPSYRK136) was
purchased from Genosys Biotechnologies (Lake Front Circle, TX).
Antibodies to p38 (C-20) and epitope tag FLAG (antibody M2) were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Sigma,
respectively. SB202190
(4-(4-fluorophenyl-2-2-(4-hydroxyphenyl)-5-(4-pyridyl)-imidazole) was obtained from Dr. Wyss (Hoffmann-La Roche, Switzerland). Phorbol myristate acetate was from Sigma. PD98059 was purchased from Calbiochem.
Expression Constructs, RSKB Truncations, and RSKB
Mutations--
Expression constructs for MAPKs and wt-RSKB were
generated as described (8). Mutated MAPK/ERK kinase was obtained from Stratagene (La Jolla, CA). RSKB point mutants were generated by site-directed mutagenesis using the Altered Sites in vitro
mutagenesis system (Promega) according to the recommendations of the
manufacturer. Expression plasmids of truncated RSKB were constructed by
replacing the NotI-SalI insert of
Flag-wt-RSKB/pALTER (8) by the NotI-SalI fragment
of the polymerase chain reaction product generated using the forward
primer 5'-GAAAATCATCGACTTCGGG and one of the following reverse primers,
(i) 5'-ACTAGGAGCTCGCGCTGCCGT ( Immunostaining and Immunoblotting--
One day after
transfection with the indicated plasmids, HEK293 cells were harvested,
and 20,000 cells/cm2 were seeded on
poly-D-lysine-coated coverslips and cultivated for 1 additional day in minimal essential medium containing 0.3% fetal calf
serum. The slides were washed in phosphate-buffered saline, fixed for 5 min in 4% formaldehyde in phosphate-buffered saline, and processed for
immunohistochemistry as previously reported (8). For p38 detection,
C-20 antibody (10 µg/ml) was used as primary reagent, with Texas
red-labeled goat anti-rabbit immunoglobulin from Jackson ImmunoResearch
(West Grove, PA) as secondary antibody. For FLAG-tagged truncated and
wt-RSKB detection, M2 antibody (10 µg/ml) was used as primary
reagent, with fluorescein isothiocyanate-labeled goat anti-mouse
antibody from Dako (Glostrup, Denmark) as secondary antibody. Cells
were analyzed on a Leica confocal fluorescence microscope. Subcellular
RSKB localization was scored for each mutant by counting the first 100 successive, positively transfected cells using a Leica DRMB
fluorescence microscope and assigning a nuclear, mixed (i.e.
strong nuclear combined with weak cytoplasmic), or homogeneous
cytoplasmic staining pattern. Immunoblots were incubated with specific
primary and secondary horseradish-conjugated antibodies and revealed by
chemiluminescence (ECL, Amersham Pharmacia Biotech) and Storm
PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA).
Cell Culture, Transfection, and Extract Preparation--
HEK293
(ATCC CRL 1573) cells were cultured in humidified air with 5%
CO2 at 37 °C. Cells were cultured in minimal essential medium supplemented with 10% fetal calf serum, 2 mM
L-glutamine, 100 units/ml penicillin, 10 µg/ml
streptomycin, pH 7.4. Transfections were done by the Transfectam
procedure as recommended by manufacturer. In parallel transfection
experiments, total amount of DNA was normalized with empty vector. In
studies of cells stimulated by the cotransfected mMKK6/p38 upstream
kinase pathway, a constitutively active mutant of MKK6 (mMKK6) was used
(8). Transfected cells were cultured for 2 days, including serum
starvation for the last 16 h in 0.3% fetal calf serum-containing
medium. The p38 kinase inhibitor SB202190 (10 µM) was
added together with the starvation medium; a second dose of SB202190
(10 µM) was added 1 h before harvesting the cells.
After stimulation, cells were washed with ice-cold phosphate-buffered
saline, and extracts were prepared with lysis buffer (50 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 500 µM
dithiothreitol, 1% Triton X-100, 5 mM NaPPi, 1 mM Na3VO4, 50 mM NaF,
CompleteTM protease inhibitor mixture (Roche Molecular Biochemicals)).
Cell lysates were cleared at 14,000 rpm for 10 min at 4 °C. Protein
concentration was determined using the BCA reagents (Pierce).
In Vitro Translation and Gel Shift Assay for p38
Binding--
wt-RSKB and truncated RSKB genes were in vitro
transcribed with T7 RNA polymerase and translated at 30 °C for 90 min in the presence of [35S]methionine using the TNT
coupled rabbit reticulocyte lysate system as specified by Promega.
Aliquots of reticulocyte lysate containing wt-RSKB or the various
truncated RSKBs were analyzed by SDS-PAGE and autoradiography to verify
expression of the in vitro translated proteins. 5-10 µl
of the reticulocyte lysate were used in the p38 binding assay performed
in 2 mM Tris, 30 mM HEPES, 10 mM
MgCl2, 1 mM dithiothreitol (pH 7.4) in a final volume of 25 µl. Active recombinant Flag-tagged human p38 Immunoprecipitation and Kinase Assays--
Cell extracts were
subjected to M2 immunoprecipitation followed by kinase assay using
CREBtide as a substrate. Cell extracts normalized to total protein
content (200 µg unless specified otherwise) were precleared twice
with 30 µl of protein G-Sepharose slurry for 20 min at 4 °C under
constant agitation. Antibody was added to precleared lysates and
incubated overnight at 4 °C. 30 µl of protein G-Sepharose slurry
were added. After 1 h of incubation, immune complexes were
pelleted and washed twice with {min}-kinase buffer (50 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 140 mM KCl) supplemented with 5 mM NaPPi. For
in vitro kinase assays, beads were resuspended in 25 µl of
{plus}-kinase buffer (33 µM CREBtide, 30 µM ATP, 10 mM MgCl2, 0.02 µCi/µl [ Alignment and Model Building--
All alignments were obtained
with the help of the in-house program
Xsae2 using a modified
version of CLUSTAL V (37). Sequences were accessed through the National
Center for Biotechnology Information and Swiss-Prot data bases under
the following accession numbers: AJ010119 (RSKB), L07597 (RSK1), P51812
(RSK2), Q15349 (RSK3), Q14012 (human CaMKI, kcc1_human), and Q63450 (rat CaMKI, kcc1_rat). All modeling calculations were made on a Silicon
Graphics Octane with a single R12000 processor using in-house modeling
package Moloc (38, 39). Model building was performed in a three-step
procedure. An initial C-
Refinement of the peptide model was performed using Moloc. Neither
water nor other molecules were added. In the first step, only amino
acid side chains were allowed to move while all backbone atoms were
kept in fixed positions. This step greatly removed repulsive
interactions between side chains, further improved RSKB Locates to the Nucleus--
Earlier immunostainings of
transfected HEK293 cells had shown that RSKB and its isolated
C-terminal domain located to the nucleus, whereas the N-terminal domain
distributed in the cytoplasm (8). The C-terminal tail sequence of RSKB
contains a putative bipartite
713KRX14KRRKQKLRS nuclear location
signal (NLS) (Fig. 1). To investigate its
role, mutants were generated with successive truncations of the
C-terminal tail, Elements of the RSKB C-terminal Tail Mediate Association with
p38--
RSKB was first identified as a partial cDNA encoding the
sequence downstream of Leu603 (RSKB Truncated RSKBs Distinctly Respond to Upstream MAPK
Activation--
To investigate whether association with p38 and
activation by p38 can be dissociated, M2 antibody precipitation
kinase assays were performed with lysates from HEK293 cells that had
been transfected with the various truncated RSKB mutants together with
mMKK6/p38 cotransfection and cultured in the presence of the p38
inhibitors SB202190, as indicated in Fig.
4. Role of Phe709 in Control of RSKB Activation--
A
sequence alignment revealed that the C-terminal domains of RSKB and
CaMKs have similarities. The structure of CaMKs is characterized by
autoinhibited conformations in the resting state (26, 33, 34). A search
through sequences of proteins deposited in the Protein Data Bank (40)
revealed a particularly interesting similarity between the C-terminal
extensions of rat CaMKI (33) and RSKB (Fig.
5A). The CaMKI structure
presented an inhibited state with a helical stretch of the C-terminal
extension pointing to the ATP and substrate binding sites. Sequence
alignment revealed an AFN motif that was conserved between the
C-terminal extensions of rat/human CaMKI and RSKB but not found in
RSK1-RSK3 (Fig. 5A). The central Phe of this
306AFN308 motif pointed into a hydrophobic
pocket, thus contributing to the binding of the C-terminal extension to
the body of CaMKI (Fig. 5B) (33). Further scrutiny showed
that Leu26, Phe31, Gly100,
Gly101, Glu102, and Leu148 in rat
CaMKI in the resting state are in direct contact with Phe307 (Fig. 5B); all of these contact residues,
homologous to Leu417, Phe422,
Gly488, Gly489, Glu490, and
Leu537 in RSKB, respectively, are conserved in RSKB and
very likely form a similar structure. In RSKB, Phe709 is
homologous to Phe307 in rat CaMKI and was predicted to play
a similarly crucial role in attaching the C-terminal extension to the
body of the C-terminal kinase domain and thereby blocking access of ATP
to its binding pocket.
To test this hypothesis, Phe709 was mutated to Ala to
generate F709A-RSKB. HEK293 cells were transfected with this mutant,
cultured in the absence of stimuli with and without SB202190, and
normalized precipitation kinase assays were performed. As shown in Fig.
6A, F709A-RSKB displayed
substantially elevated basal activity in nonstimulated cells.
Interestingly, the high basal F709A-RSKB activity was sensitive to
SB202190 treatment of the cells (Fig. 6A). Previous studies
had shown that wt-RSKB is not sensitive to SB202190 (36); the
structural configuration of the ATP binding site fold required for
sensitivity to pyridinyl imidazole inhibitors (44), as well as control
studies showing no direct effect of SB202190 on F709A-RSKB in
precipitation kinase assays practically excluded that the F709A
mutation had converted RSKB into a SB202190-sensitive conformation.
Thus, the inhibition documented in Fig. 6A most likely did
not result from a direct effect on F709A-RSKB, the presumed target of
SB202190, rather, was p38, which thus appeared to control basal
F709A-RSKB activity. F709A-RSKB in HEK293 cells was then stimulated by
cotransfection with mMKK6/p38 (Fig. 6B). F709A-RSKB
responded to p38 pathway activation to slightly higher levels than
wt-RSKB (Fig. 6B). SB202190 reduced activation through the
p38 pathway to background levels. These data were corroborated by
studies of HEK293 cells transfected with F709A-RSKB and stimulated by
treatment with arsenite (data not shown).
The F709A mutation was then introduced into truncated
Thr687 Is a Potential Control Site of RSKB
Activation--
The C-terminal RSKB sequence around Thr687
fits the minimal MAPK consensus phosphorylation site,
RSK family kinases are regulated through complex mechanisms
associated with phosphorylations in the two catalytic domain sequences, the linker sequence, and the C-terminal tail sequence. Studies of RSK1 (27, 46), RSK2 (29, 31), and RSKB and mitogen- and
stress-activated protein kinase type 1 (7, 8) demonstrated the
important control exerted by elements in the C-terminal sequences of
RSKs. For example, a mutation of a C-terminal tyrosine converted RSK2
to a constitutively active enzyme, suggesting that a putative autoinhibitory structure element controlled the basal activity of the
enzyme (31). Furthermore, a conserved element of the C termini of
RSK1-RSK3 corresponding to 722LAQRRVRKLPSTTL in RSK1
mediated the interaction with ERK, and complex formation between RSK2
and ERK was essential for the activation of RSK2 in vivo
(29). The present effects of truncations and point mutations of RSKB
indicated that the C-terminal extension contained several elements
controlling activation. The intermolecular association of RSKB with p38
depended on an element within the 725APLAKRRKQKLRS737-RSKB sequence that was also
essential for nuclear targeting, indicating an overlap of docking site
and NLS. In addition, RSKB, when truncated at Asn724, had
also lost responsiveness to activation through the p38 pathway, but
interestingly, the further truncation at Ser681 in
An element within the 725APLAKRRKQKLRS737
sequence, consistent with the nuclear location signal sequence
consensus, also was essential to target RSKB to the nucleus, suggesting
an overlap between p38 association and nuclear targeting sites. The
preferential nuclear location of RSKB was independent of stimulation by
mMKK6/p38 cotransfection, suggesting that RSKB locates constitutively
to the nucleus. A similar sequence element was responsible for the
nuclear location of MAPKAP2 under resting conditions; however, in
contrast to RSKB, MAPKAP2 was exported to the cytoplasm upon activation
through p38 (47, 48). The subcellular location of MAPKAP2 appeared tightly regulated by the removal of the C-terminal putative
autoinhibitory helix as a result of phosphorylation of
Thr317, which may unmask an adjacent nuclear export signal
(47, 48). In contrast, the present findings suggested that the
cytoplasmic location of the RSKB deletion mutants truncated at
Ser681 and Asn724 resulted from the absence of
the nuclear location signal rather than from
activation-dependent nuclear export.
The inhibited state of 725-772-RSKB
was nearly nonresponsive to p38. Sequence alignment with the
autoinhibitory C-terminal extension of
Ca+2/calmodulin-dependent protein kinase I
predicted a conserved regulatory 708AFN710
motif. Alanine mutation of the key Phe709 residue resulted
in strongly elevated basal level RSKB activity. A regulatory role also
was assigned to Thr687, which is located in a
mitogen-activated protein kinase phosphorylation consensus site. These
findings support that the RSKB C-terminal extension contains elements
that control activation threshold, subcellular location, and p38 docking.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
682-772-RSKB), (ii)
5'-ATTAGAGCTCATTCTCCACGCTCTT (
725-772-RSKB), and (iii)
5'-ATTAGGAGCTCCGCAGCTTCTGCTT (
738-772-RSKB).
was purified by affinity chromatography from Escherichia coli
expression. 1 µg of unlabeled p38
was used in the binding assay.
After incubation for 30 min at 30 °C, 5 µl of SDS-free loading
buffer was added to the reaction that was then electrophoresed on a
7.5% native polyacrylamide gel at 7 mA overnight using standard
SDS-PAGE running buffer
(acrylamide/N,N'-methylene-bisacrylamide weight ratio of 29:1). The gels were fixed for 15 min (10% acetic acid, 40% ethanol) and amplified for 30 min in NAM-P 100 amplifier solution (Amersham Pharmacia Biotech). Gels were dried and autoradiographed with Kodak Scientific X-OMAT AR film overnight.
-33P]ATP, in {min}-kinase buffer) and
incubated at 22 °C for 30 min under constant agitation. Similar to
wt-RSKB (36), SB202190 (10 µM) added directly to the
kinase reactions had no effect on the elevated basal activities of
F709A- and T687E-RSKB. Furthermore, 10 µM SB202190 had no
effect on stimulated F709A-RSKB activity when added to the in
vitro kinase reaction with precipitates from cells activated by
mMKK6/p38 cotransfection and cultured without SB202190. A minor
reduction of stimulated activity (<15%) of T687E-RSKB was seen when
10 µM SB202190 was added to kinase assays with
precipitates from cells activated by mMKK6/p38 cotransfection. A
possible effect of SB202190 on T687E-RSKB depends on relative
affinities and binding kinetics of ATP versus SB202190 for
the C-terminal ATP binding site; ATP concentrations in the in
vitro kinase assay and in vivo are 30 µM
and in the mM range, respectively. The assay may also be
influenced by coprecipitated p38, and spontaneous decay kinetics of
activity of distinct RSKB mutants. Thus, the assay format may explain
the weak reduction of stimulated T687E-RSKB activity by SB202190
independently of a hypothetical direct effect of the inhibitor.
Reactions were stopped by addition of 100 µl of 0.75% phosphoric
acid, and 100 µl of the mix reaction were filtered in a 96-well
phosphocellulose filter plate (Millipore, Bedford, MA), washed (five
times) with 100 µl of 0.75% phosphoric acid, washed once with
ethanol, and air-dried. Bound radioactivity was measured in a Packard
top counter, using 100 µl of microscintillation mixture (Packard).
model of the C-terminal domain of RSKB was
built by fitting the aligned sequence on the C-
template of rat
CaMK-I (33) (Protein Data Bank accession number 1A06 (40)).
This model was subsequently improved by searching a Moloc internal data
base of loops obtained from highly resolved protein structures. A loop
selection was made on the basis of minimal steric interactions with the
rest of the model. Subsequently newly introduced loops were optimized with the Moloc C-
force field. In a next step, a full atom model was
generated.
and
angles were obtained for aligned amino acids from the x-ray structure.
angles were also adopted from the
template where possible, and in cases of nonidentical amino acids generated by using the most probable value, applying the method
of Ponder and Richards (41). An energy calculation of the initial full
peptide structure revealed regions with bad van der Waals contacts of
amino acid side chains that were subsequently improved by manually
adjusting the relevant
angles. Newly inserted loop regions were
then optimized individually, with the rest of the protein kept
stationary. Repulsive van der Waals interactions were removed manually
where necessary.
angles of
nonconserved amino acids, and revealed regions with unfavorable
interactions. In further optimization, only C-
atoms were kept in a
fixed position, and all other atoms were allowed to move. In a third
round of optimization, no atoms were kept stationary, but positional
constraints were applied to C-
atoms. The quality of the model was
then checked with (i) Moloc internal programs, (ii) a program by Luthy
et al. (42), and (iii) PROCHECK (43).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
682-772-RSKB,
725-772-RSKB, and
738-772-RSKB (Fig.
1), and their subcellular location was determined in HEK293 cell
transfectants by immunostaining for the fused Flag epitope. wt-RSKB and
738-772-RSKB presented clearly dominant nuclear
staining, but some cells showed cytoplasmic and mixed
nuclear/cytoplasmic staining; to quantify, the first 100 successive
transfected cells were counted up in the fluorescence microscope with
each mutant and assigned to one of the three staining patterns as
defined under "Experimental Procedures" (Table
I and Fig. 2). In contrast to wt-RSKB
and
738-772-RSKB,
725-772-RSKB and
682-772-RSKB, both of which lack the NLS,
were predominantly found in the cytoplasm (Table I and Fig. 2). This
indicates that elements of the sequence between Ala725 and
Ser737 were essential to direct RSKB to the nuclear
compartment.
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Fig. 1.
Schema of RSKB sequence and C-terminal tail
mutations. The general structure with N- and C-terminal kinase and
linker phosphorylation sites (P) is indicated. The putative
bipartite NLS, 713KR ... K729RRKQK, is
boxed. The residues targeted for mutation,
Thr687 and Phe709, are indicated. The C termini
of the successive truncations in 725-772-RSKB,
738-772-RSKB, and
682-772-RSKB are
marked by arrows. A stretch of predicted helical structure
of the C-terminal tail 698VRSGLNATFMAFN710
sequence is indicated.
Percentages of transfected cells with nuclear, cytoplasmic, and mixed
nuclear/cytoplasmic location of 682-772-RSKB,
725-772-RSKB,
738-772-RSKB, and wt-RSKB
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Fig. 2.
RSKB nuclear location is
determined by the NLS element. HEK293 cells were
transfected with deletion mutants 682-772-RSKB,
725-772-RSKB, and
738-772-RSKB and
wt-RSKB, as indicated. All RSKB constructs were tagged with Flag
epitope. After transfection cells were cultured for 1 day, seeded on
poly-D-lysine-coated coverslips, and cultured for an
additional day. For immunodetection, the cells were
simultaneously stained with M2 and fluorescein
isothiocyanate-conjugated secondary antibodies and with C-20 anti-p38
and Texas red-labeled secondary antibodies. Confocal microscopy
showing two panels at higher and lower magnification are shown
for each mutant, as indicated.
N1 (8)) in
intracellular interaction screens using p38 as bait, indicating that
essential elements for p38 docking are located in the C-terminal tail
(8). To more closely define these elements, p38 coprecipitation studies
were performed with the C-terminal truncation mutants of RSKB, which
were introduced in HEK293 cells with and without
mMKK6/His6-p38 cotransfection. Normalized amounts of cell
lysates were precipitated with M2 antibody, and the precipitates were
probed by immunoblotting with an anti-p38 antibody. As shown in Fig.
3A, wt-RSKB and
738-772-RSKB coprecipitated p38 independent of
activation. In contrast,
725-772-RSKB and
682-772-RSKB were unable to coprecipitate p38, even
though all wt and truncated RSKB were expressed at similar levels (Fig.
3B). The essential role of the
725APLAKRRKQKLRS737 sequence for the physical
association between RSKB and p38 was confirmed in gel band shift assays
(Fig. 3C). wt-RSKB and truncated RSKB were translated
in vitro, incubated with and without recombinant active p38
purified from E. coli expression, and electrophoresed through nondenaturing PAGE. The retarded electrophoretic migration of
RSKB in the presence of p38 was readily apparent with wt-RSKB and
738-772-RSKB and with the isolated C-terminal domain
but absent with
725-772- and
682-772-RSKB, as well as with the isolated N-terminal
domain (Fig. 3C). These findings suggested that the sequence
between Ala725 and Ser737 contained elements
essential for association with p38, as well as for nuclear
targeting.
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Fig. 3.
Intermolecular association of p38
with RSKB depends on the 725APLAKRRKQKLRS737
sequence independent of activation. A, immunoblots
revealing coprecipitated endogenous and transfected
His6-tagged p38 in various RSKB precipitates. HEK293 cells
were transfected with 682-772-RSKB,
725-772-RSKB,
738-772-RSKB, and wt-RSKB
Flag fusion expression constructs (
682-772,
725-772,
738-772, and wt,
respectively). Cells were either stimulated by cotransfection of
mMKK6/p38 or left nonstimulated, as indicated, and cultured for 48 h. Cell lysates (50 µg of total protein) were precipitated with M2
antibody, and immunoblots were performed using a pan-anti-p38 antibody
as revealing reagent. Compared with endogenous p38, transfected p38 in
the experiment with cells stimulated by mMKK6/p38 cotransfection is
electrophoretically retarded by the His6-tag (left
panel); revealed by PhosphorImager. B, expression of
the various mutated and of wt-RSKB. HEK293 cells were transfected with
the various Flag-RSKB expression constructs (15 µg of plasmid DNA) in
parallel. The lysates normalized to protein content were subjected
to 7% (F709A-, S687A-, S687E-, and wt-RSKB) and 12%
(all truncated RSBK mutants) SDS-PAGE and analyzed by immunoblotting.
The figure is a composite of independent gels; the stained bands are
aligned side-by-side, but in the gels, they ran to apparent
molecular masses consistent with full-length and truncated RSKB,
respectively. Membranes were probed by M2 antibody and revealed by
autoradiography. C, retarded electrophoretic migration of
in vitro translated RSKB in the presence of recombinant
human p38
. [35S]Methionine-labeled wt-RSKB (wt,
lanes 2-4), isolated C-terminal catalytic domain (C-term,
lanes 5 and 6), isolated N-terminal catalytic domain
(N-term, lanes 7 and 8), and C-terminally
truncated RSKB mutants
682-772-RSKB
(
682-772, lanes 9 and 10),
725-772-RSKB (
725-772, lanes
11 and 12), and
738-772-RSKB
(
738-772, lanes 13 and 14) were
incubated with or without 1 µg of p38
or 1 µg of bovine serum
albumin (BSA), as indicated. The samples were subjected
to 7.5% PAGE under native conditions to reveal retardation due
to RSKB/p38 association. 14C-Methylated protein molecular
mass markers are shown in lane 1, in kDa; shown is an
autoradiogram.
738-772-RSKB was
activated by the p38 pathway to about half the extent of wt-RSKB. In
contrast, the activation of
725-772-RSKB was
substantially reduced to nearly the background level. Interestingly,
the further truncation in
682-772-RSKB resulted in a
renewed response to mMKK6/p38 activation (Fig. 4), suggesting that the
lack of response of
725-772-RSKB to p38 activation did
not result from the absence of those sequence elements found essential
for p38 association. These data suggested that RSKB activation was
under the control of C-terminal sequence elements downstream of
Ser682 that were independent of the elements mediating the
strong association with p38 reflected by coprecipitation, since
activation and coprecipitation could be dissected.
View larger version (17K):
[in a new window]
Fig. 4.
C-terminal truncations reveal an inhibitory
effect of the 725APLAKRRKQKLRS737
sequence. HEK293 cells were transfected with
682-772-RSKB,
725-772-RSKB,
738-772-RSKB, and wt-RSKB Flag fusion expression
constructs (
682-772,
725-772,
738-772, and wt, respectively). Cells
were stimulated by cotransfection of mMKK6/p38 (MKK6) or
left unstimulated (US) and cultured with the p38 inhibitor
SB202190, as indicated. Cell lysates (normalized to total protein
content) were immunoprecipitated with M2 antibody, and kinase assays
were performed using CREBtide as substrate. A representative (of
n = 3) experiments is shown. Incorporated radioactivity
was determined in a Packard top counter (mean cpm ± S.D.).
View larger version (74K):
[in a new window]
Fig. 5.
Sequence alignment and structure comparison
point to a crucial C-terminal AFN motif. A, alignment
of C-terminal sequences of human RSK1, RSK2, RSK3, RSKB and rat/human
CaMK1. The respective accession numbers in the National Center for
Biotechnology Information and Swiss-Prot data bases are L07597, P51812,
Q15349, AJ010119, Q63450, and Q14012. B, model of RSKB in
the region of contact between the AFN motif with the body of the
C-terminal catalytic domain, as predicted by fitting the aligned
sequence to the CaMKI structure template (33) and refinement. C-
representation of RSKB (red), showing side chains of the AFN
sequence (cyan), with the crucial Phe709
pointing in a pocket lined by residues Leu417,
Phe422, Gly488, Gly489,
Glu490, and Leu537 (yellow) that are
highly conserved between RSKB and rat/human CaMKI.
View larger version (19K):
[in a new window]
Fig. 6.
Elevated basal activity and enhanced
responses to p38 pathway stimulation of F709A-RSKB. A,
HEK293 cells were transfected with F709A-RSKB and wt-RSKB Flag fusion
expression constructs and cultured with the p38 inhibitor SB202190, as
indicated. Kinase assays were performed with M2 antibody precipitates
from normalized lysates of unstimulated cells. A representative (of
n = 4) series of parallel experiments is shown (mean
and S.D. of F709A-RSKB basal activity, 7.6 ± 3.2-fold).
B, HEK293 cells transfected with F709A-RSKB
(F709A) and wt-RSKB (wt) Flag fusion expression
constructs were activated by mMKK6/p38 cotransfection (MKK6)
or left unstimulated (US) and cultured with SB202190, and
kinase assays were performed with M2 antibody precipitates from
normalized cell lysates using CREBtide as substrate, as indicated.
Incorporated radioactivity was determined in a Packard top counter. A
representative (of n = 3) experiments is shown (mean
cpm ± S.D.).
725-772-RSKB to generate
725-772[F709A]-RSKB. As shown in Fig.
7, the combination of this truncation with the F709A mutation sufficed to partially overcome the lack of
response of
725-772-RSKB in nonstimulated and
stimulated cells. These combined data suggested that sequence elements
between Ser682 and Asn724 had a function in
inhibitory RSKB control, which was unmasked both through the truncation
at Asn724 and through the F709A mutation. To further probe
the role of Phe709 in RSKB control, a molecular model of
the C-terminal domain of RSKB was built by fitting the aligned sequence
to the template of the rat CaMKI structure (33). This model was
improved and refined according to standard procedures (see under
"Experimental Procedures"); it presented the C-terminal extension
in contact with the body of the C-terminal catalytic domain in a
structural arrangement that was consistent both with the proposed
homology between Phe307 and Phe709 in CaMKI and
RSKB and with a functional role of Phe709, as suggested by
the effect of the F709A mutation on RSKB basal level activity.
View larger version (16K):
[in a new window]
Fig. 7.
The F709A mutation mitigates the lack of
response of 725-772-RSKB to
p38 pathway stimulation. HEK293 cells were transfected with
725-772[F709A]-RSKB
(
725-772[F709A]),
725-772-RSKB
(
725-772), and wt-RSKB (wt) Flag
fusion expression constructs, stimulated by mMKK6/p38 cotransfection
(MKK6) or left unstimulated (US), and cultured
with SB202190, and kinase assays were performed with M2 antibody
precipitates from normalized cell lysates, using CREBtide as substrate,
as indicated. Incorporated radioactivity determined in a Packard top
counter. A representative (of n = 4) series of
experiments is shown (mean cpm ± S.D.).
X(S/T)P, (where
represents Pro or aliphatic)
(45) and is adjacent to the 698VRSGLNATFMAFN710
sequence containing elements of the predicted inhibitory control function. This is an intriguing parallel to MAPKAP2, in which phosphorylation of Thr317 contributed to activation (22);
Thr317 is located N-terminal to the autoinhibitory A-helix
of MAPKAPK2, and its phosphorylation may destabilize the helix or its
interaction with the hydrophobic catalytic cleft (22).
Thr687 therefore was mutated to Glu (T687E-RSKB), to mimic
the effect of phosphorylation, and to Ala (T687A-RSKB). T687E-RSKB and
T687A-RSKB expression constructs were introduced in HEK293 cells,
lysates were prepared, and normalized precipitation kinase assays were performed as described above. As shown in Fig.
8A, T687E-RSKB had
substantially higher basal activity when compared with wt-RSKB and
T687A-RSKB. Similarly to F709A-RSKB, the high basal activity of
T687E-RSKB was fully sensitive to SB202190 treatment of cells (Fig.
8B). From control studies, the inhibitor had no effect on basal T687E-RSKB activity but weakly (<15%) inhibited the in
vivo stimulated activity. This weak inhibition of stimulated
activity may result from the distinct in vivo and in
vitro assay conditions (see under "Experimental Procedures")
rather than from a direct effect of the inhibitor. Furthermore, it is
very unlikely that the T687E mutation converted the C-terminal ATP
binding site fold of RSKB into a SB202190-sensitive conformation (44).
Thus, the presumed target of SB202190 in Fig. 8B is p38,
suggesting that the high basal activity of T687E-RSKB depended on p38
activity. Furthermore, F709A-, T687A-, and T687E-RSKB were introduced
in HEK293 cells that were activated by cotransfection with mMKK6/p38, and precipitation kinase assays were performed, as indicated in Fig.
8C. It was found that all mutants responded to both p38 and ERK pathway activation; F709A-RSKB reached highest activation levels.
T687E-RSKB reacted similar to wt-RSKB, and interestingly, the T687A
mutation was permissive. These data are supported by findings in
precipitation kinase assays performed with lysates of HEK293 cells
transfected with either F709A-, T687A-, or T687E-RSKB alone and
activated by treatment with arsenite and phorbol myristate acetate,
as indicated in Fig. 8D.
View larger version (33K):
[in a new window]
Fig. 8.
Responses of T687A-RSKB and T687E-RSKB point
to a regulatory role of Thr687. HEK293 cells were
transfected with T687A-, T687E-, F709A-, and wt-RSKB Flag fusion
expression constructs, stimulated through mMKK6/p38 cotransfection or
phorbol myristate acetate and arsenite treatment, and cultured for
48 h with SB202190, as indicated. M2 antibody precipitation kinase
assays from normalized cell lysates using CREBtide as substrate.
Incorporated radioactivity was determined in a Packard top counter.
Representative (of n 3) series of experiments are
shown (mean cpm ± S.D.). A, kinase activities of
T687E-, T687A-, and wt-RSKB in unstimulated cells determined in
parallel assays, as indicated. B, parallel kinase assays of
T687E- and wt-RSKB in unstimulated cells cultured with SB202190, as
indicated. C, parallel precipitation kinase assays of
T687E-, T687A-, F709A-, and wt-RSKB in unstimulated cells
(US) and cells stimulated through mMKK6/p38 cotransfection
(MKK6), as indicated. D, parallel kinase assays
of T687E-, T687A-, F709A-, and wt-RSKB in unstimulated cells and cells
stimulated by treatment with arsenite (0.5 mM) and phorbol
myristate acetate (100 ng/ml) for 30 min, as indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
682-772-RSKB largely restored responsiveness. This
activation of
682-772-RSKB depended on
stimulation through the cotransfected MEKK6/p38 pathway and was fully
sensitive to SB202190, suggesting that p38 phosphorylated and activated
682-772-RSKB, even though activation through a distinct
unknown kinase pathway that would have to be under the control of p38
or sensitive to SB202190 cannot be excluded entirely. When extrapolated
to wt-RSKB, these findings suggested that in contrast to RSK2, where the formation of a complex between the upstream MAPK and the C-terminal tail was essential for activation (29), the strong physical association
of RSKB with p38 and its activation through the p38 pathway are
separate functions. The nonresponsiveness of
725-772-RSKB to p38 activation thus likely was due to
the presence of inhibitory elements between Ser682 and
Asn724 rather than to the absence of a p38 docking site;
given the nearly complete block of
725-772-RSKB, these
inhibitory sequences in wt-RSKB presumably are counterbalanced by
elements farther downstream of Asn724.
725-772-RSKB, in view of the
renewed response to activation after the further truncation in
682-772-RSKB, pointed to the existence of an
autoinhibitory sequence element(s) between residues Ser682
and Asn724. A sequence alignment focused interest on an AFN
motif that was conserved between CaMKI and RSKB but absent in
RSK1-RSK3. The structure of CaMKI (33) revealed a crucial role of the
306AFN308 motif of the CaMKI C-terminal
extension; the central Phe307 pointed into a hydrophobic
pocket and contributed to the binding of the C-terminal extension to
the body of the enzyme. This conformation of the C-terminal extension
with helical stretches pointing to the ATP and substrate
binding sites and blocking access of ATP to its site, related
to the autoinhibited state of CaMKI. The elevated basal activity of
F709A-RSKB when compared with wt-RSKB supported the view that
Phe709 played a similar regulatory role in raising the
activation threshold as the homologous Phe307 in rat CaMKI;
this view, furthermore, is consistent with the predictions of a
molecular model obtained by fitting the aligned RSKB sequence to the
rat CaMKI structure template (33). Interestingly, the introduction of
the F709A mutation into
725-772-RSKB produced less
pronounced effects on basal and stimulated activation levels than might
have been expected, suggesting that the perturbation of the putative
autoinhibitory element by the F709A mutation in
725-772-RSKB did not compensate for the absence of the
activation-promoting influence of sequence elements downstream of
Asn724 in wt-RSKB. Furthermore, the elevated basal activity
of T687E-RSKB suggested that Thr687 also is a regulatory
site, consistent with its location in a MAPK phosphorylation consensus
site. Similarly to F709A-RSKB, the difference between T687E-RSKB and
wt-RSKB was more readily apparent with regards to basal activity than
in altered responsiveness to upstream pathway activation. Intriguingly,
this elevated basal activity of both F709A- and T687E-RSKB was
sensitive to SB202190 treatment of the cells. wt-RSKB is not sensitive
to SB202190, and in view of the structural requirements in the ATP
binding site for sensitivity to pyridinyl imidazole class inhibitors
(44) and of control studies, it was practically excluded that either of
the RSKB mutants had become SB202190-sensitive. Although an unknown
kinase pathway between mMKK6/p38 and RSKB, which would have to be under
p38 control or sensitive to SB202190, cannot be excluded entirely, the
most likely target of the inhibitor in the present studies was p38.
This then suggested that similar to the persistent activation of
wt-RSKB in tumor necrosis factor
-stimulated cells (36), the
elevated activities of both F709A- and T687E-RSKB in nonstimulated
cells depended on basal p38 activity, suggesting that the two mutations
lowered an activation threshold. Interestingly,
682-772-RSKB, lacking the putative inhibitory element
between Ser682 and Asn724, but also lacking the
p38 docking site, had no elevated basal activity in nonstimulated
cells. Additional studies are required to elucidate whether this
results from a distinct role of p38 docking in basal and burst
activation of RSKB or from a location to nuclear compartment(s) with
specific activation conditions.
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ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge the excellent technical support of C. Sanner-Bubbendorf and M. Dellenbach and the interest and support of Dr. N. H. Ruddle (Yale University, New Haven, CT).
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FOOTNOTES |
---|
* 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.
§ These authors contributed equally to this work.
§§ To whom correspondence should be addressed: Dept. of Epidemiology and Public Health, Yale University School of Medicine, P. O. Box 208034, New Haven, CT 06520-8034. Tel.: 1-203-785-7440; Fax: 1-203-785-6130; E-mail: werner.lesslauer@yale.edu.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M005822200
2 C. Broger, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: RSKs, family of 90 kDa ribosomal S6 protein kinases; MAPK, mitogen-activated protein kinase; p38, stress-activated 38kDa MAPK; ERK, extracellular signal-regulated kinase; RSKB, ribosomal S6 protein kinase B; CaMK, Ca+2/calmodulin-dependent protein kinase; MKK6, p38 MAPK kinase; mMKK6, constitutively active mutant of MKK6; MAPKAPK2, MAPK-activated protein kinase type 2; NLS, nuclear location signal; CREB, cAMP response element-binding protein; wt, wild-type; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Blenis, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5889-5892[Abstract] |
2. | Bohm, M., Moellmann, G., Cheng, E., Alvarez-Franco, M., Wagner, S., Sassone-Corsi, P., and Halaban, R. (1995) Cell Growth Differ. 6, 291-302[Abstract] |
3. | Xing, J., Ginty, D. D., and Greenberg, M. E. (1996) Science 273, 959-963[Abstract] |
4. |
Pende, M.,
Fisher, T. L.,
Simpson, P. B.,
Russell, J. T.,
Blenis, J.,
and Gallo, V.
(1997)
J. Neurosci.
17,
1291-1301 |
5. |
De Cesare, D.,
Jacquot, S.,
Hanauer, A.,
and Sassone-Corsi, P.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
12202-12207 |
6. |
Xing, J.,
Kornhauser, J. M.,
Xia, Z.,
Thiele, E. A.,
and Greenberg, M. E.
(1998)
Mol. Cell. Biol.
18,
1946-1955 |
7. |
Deak, M.,
Clifton, A. D.,
Lucocq, L. M.,
and Alessi, D. R.
(1998)
EMBO J.
17,
4426-4441 |
8. |
Pierrat, B.,
da Silva Correia, J.,
Mary, J. L.,
Tomás-Zuber, M.,
and Lesslauer, W.
(1998)
J. Biol. Chem.
273,
29661-29671 |
9. |
New, L.,
Zhao, M.,
Li, Y.,
Basset, W. W.,
Feng, Y.,
Ludwig, S.,
Padova, F. D.,
Gram, H.,
and Han, J.
(1999)
J. Biol. Chem.
274,
1026-1032 |
10. | Yntema, H. G., van den Helm, B., Kissing, J., van Duijnhoven, G., Poppelaars, F., Chelly, J., Moraine, C., Fryns, J. P., Hamel, B. C., Heilbronner, H., Pander, H. J., Brunner, H. G., Ropers, H. H., Cremers, F. P., and van Bokhoven, H. (1999) Genomics 62, 332-343[CrossRef][Medline] [Order article via Infotrieve] |
11. | Frodin, M., and Gammeltoft, S. (1999) Mol. Cell. Endocrinol. 151, 65-77[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Nebreda, A. R.,
and Gavin, A. C.
(1999)
Science
286,
1309-1310 |
13. | Cobb, M. H. (1999) Progr. Biophys. Mol. Biol. 71, 479-500[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Palmer, A.,
Gavin, A.,
and Nebreda, A.
(1998)
EMBO
17,
5037-5047 |
15. |
Sassone-Corsi, P.,
Mizzen, C. A.,
Cheung, P.,
Crosio, C.,
Monaco, L.,
Jacquot, S.,
Hanauer, A.,
and Allis, C. D.
(1999)
Science
285,
886-891 |
16. | Nakajima, T., Fukamizu, A., Takahashi, J., Gage, F. H., Fisher, T., Blenis, J., and Montminy, M. R. (1996) Cell 86, 465-474[Medline] [Order article via Infotrieve] |
17. | Fisher, T. L., and Blenis, J. (1996) Mol. Cell. Biol. 16, 1212-1219[Abstract] |
18. |
Joel, P. B.,
Smith, J.,
Sturgill, T. W.,
Fisher, T. L.,
Blenis, J.,
and Lannigan, D. A.
(1998)
Mol. Cell. Biol.
18,
1978-1984 |
19. | Merienne, K., Jacquot, S., Pannetier, S., Zeniou, M., Bankier, A., Gecz, J., Mandel, J. L., Mulley, J., Sassone-Corsi, P., and Hanauer, A. (1999) Nat. Genet. 22, 13-14[CrossRef][Medline] [Order article via Infotrieve] |
20. | Zhu, S., and Gerhard, D. S. (1998) Hum. Genet. 103, 674-680[CrossRef][Medline] [Order article via Infotrieve] |
21. | Bruford, E. A., Riise, R., Teague, P. W., Porter, K., Thomson, K. L., Moore, A. T., Jay, M., Warburg, M., Schinzel, A., Tommerup, N., Tornqvist, K., Rosenberg, T., Patton, M., Mansfield, D. C., and Wright, A. F. (1997) Genomics 41, 93-99[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Engel, K.,
Schultz, H.,
Martin, F.,
Kotlyarov, A.,
Plath, K.,
Hahn, M.,
Heinemann, U.,
and Gaestel, M.
(1995)
J. Biol. Chem.
270,
27213-21 |
23. | Stokoe, D., Campbell, D. G., Nakielny, S., Hidaka, H., Leevers, S. J., Marshall, C., and Cohen, P. (1992) EMBO J. 11, 3985-3994[Abstract] |
24. | Veron, M., Radzio-Andzelm, E., Tsigelny, I., Ten Eyck, L. F., and Taylor, S. S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10618-10622[Abstract] |
25. |
Yang, E.,
and Schulman, H.
(1999)
J. Biol. Chem.
274,
26199-26208 |
26. | Miller, S. G., Patton, B. L., and Kennedy, M. B. (1988) Neuron 1, 593-604[Medline] [Order article via Infotrieve] |
27. |
Dalby, N. K.,
Morrice, N.,
Caudwell, F. B.,
Avruch, J.,
and Cohen, P.
(1998)
J. Biol. Chem.
273,
1496-1505 |
28. |
Jensen, C. J.,
Buch, M. B.,
Krag, T. O.,
Hemmings, B. A.,
Gammeltoft, S.,
and Frodin, M.
(1999)
J. Biol. Chem.
274,
27168-27176 |
29. |
Smith, J. A.,
Poteet-Smith, C. E.,
Malarkey, K.,
and Sturgill, T. W.
(1999)
J. Biol. Chem.
274,
2893-2898 |
30. | Gavin, A., and Nebreda, A. (1999) Curr. Biol. 9, 281-284[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Poteet-Smith, C. E.,
Smith, J. A.,
Lannigan, D. A.,
Freed, T. A.,
and Sturgill, T. W.
(1999)
J. Biol. Chem.
274,
22135-22138 |
32. |
Zhao, Y.,
Bjorbaek, C.,
and Moller, E. M.
(1996)
J. Biol. Chem.
271,
29773-29779 |
33. | Goldberg, J., Nairn, A. C., and Kuriyan, J. (1996) Cell 84, 875-887[Medline] [Order article via Infotrieve] |
34. |
Rich, R. C.,
and Schulman, H.
(1998)
J. Biol. Chem.
273,
28424-28429 |
35. |
Da Silva, J.,
Pierrat, B.,
Mary, J. L.,
and Lesslauer, W.
(1997)
J. Biol. Chem.
272,
28373-28380 |
36. |
Tomás-Zuber, M.,
Mary, J. L.,
and Lesslauer, W.
(2000)
J. Biol. Chem.
275,
23549-23558 |
37. | Higgins, D. G., Bleasby, A. J., and Fuchs, R. (1992) Comput. Appl. Biosci. 8, 189-191[Abstract] |
38. | Gerber, P. R., and Mueller, K. (1995) J. Comp. Aided Mol. Design 9, 251-268 |
39. | Gerber, P. R. (1998) J. Comp. Aided Mol. Design 12, 37-51[CrossRef] |
40. |
Berman, H. M.,
Westbrook, J.,
Feng, Z.,
Gilliland, G.,
Bhat, T. N.,
Weissig, H.,
Shindyalov, I. N.,
and Bourne, P. E.
(2000)
Nucleic Acids Res.
28,
235-242 |
41. | Ponder, J. W., and Richards, F. M. (1987) J. Mol. Biol. 193, 775-791[Medline] [Order article via Infotrieve] |
42. | Luthy, R., Bowie, J. U., and Eisenberg, D. (1992) Nature 356, 83-85[CrossRef][Medline] [Order article via Infotrieve] |
43. | Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. Crystallogr. 26, 283-291[CrossRef] |
44. | Eyers, P. A., Craxton, M., Morrice, N., Cohen, P., and Goedert, M. (1998) Chem. Biol. 5, 321-328[Medline] [Order article via Infotrieve] |
45. |
Waskiewicz, A. J.,
Flynn, A.,
Proud, C. G.,
and Cooper, J. A.
(1997)
EMBO J.
16,
1909-1920 |
46. |
Gavin, A. C.,
Ainle, A. N.,
Chierici, E.,
Jones, M.,
and Nebreda, A. R.
(1999)
Mol. Biol. Cell
10,
2971-2986 |
47. |
Engel, K.,
Kotlyarov, A.,
and Gaestel, M.
(1998)
EMBO
17,
3363-3371 |
48. | Ben-Levy, R., Hooper, S., Wilson, R., Paterson, H., and Marshall, C. (1998) Curr. Biol. 8, 1047-1057 |