From the Department of Cell Biology, Harvard
Medical School, Boston, Massachusetts 02115 and the ¶ Freie
Universitat Berlin, Institut fur Biochemie, Thielallee 63, 14195 Berlin, Germany
Received for publication, August 2, 2000, and in revised form, November 1, 2000
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ABSTRACT |
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Ribosomal S6 kinase 2 (S6K2) is a recently identified serine/threonine protein kinase
that phosphorylates the 40 S ribosomal protein S6 in vitro.
S6K2 is highly homologous to S6K1 in the core kinase and linker
regulatory domains but differs from S6K1 in the N- and C-terminal
regions and is differently localized primarily to the nucleus because
of a C-terminal nuclear localization signal unique to S6K2. We
have recently demonstrated that S6K2 is regulated similarly to S6K1 by
the mammalian target of rapamycin pathway and by multiple PI3-K pathway
effectors in vivo. However, deletion of the C-terminal
domain of S6K2 enhances kinase activity, whereas analogous deletion of
S6K1 is inhibitory. Here, we characterize the S6K2 C-terminal motifs
that confer this differential regulation. We demonstrate that the
inhibitory effects of the S6K2 C-terminal domain are only partly
attributable to the nuclear localization signal but that three
C-terminal proline-directed potential mitogen-activated protein kinase
phosphorylation sites are critical mediators of this inhibitory effect.
Site-specific mutation of these sites to alanine completely
desensitizes S6K2 to activating inputs, whereas mutation to aspartic
acid to mimic phosphorylation results in an activated enzyme which is
hypersensitive to activating inputs. Pretreatment of cells with the
mitogen-activated protein-extracellular signal-regulated kinase kinase
(MEK) inhibitor U0126 inhibited S6K2 activation to a greater extent
than S6K1. Furthermore, S6K2 mutants with C-terminal deletion or acidic
phosphorylation site mutations displayed greatly reduced U0126
sensitivity. Thus, MEK-dependent inputs to C-terminal phosphorylation
sites appear to be essential for relief of S6K2 inhibition but less
critical for activation of S6K1. These data suggest a mechanism by
which weak PI3-K agonists can regulate S6 phosphorylation and selective
translation in the presence of mitogen-activated protein kinase signaling.
S6 phosphorylation is a conserved mitogenic response that
regulates translation of 5'-terminal oligopyrimidine tract-containing mRNAs encoding components of the protein synthetic machinery. This
critical translational response that regulates ribosome biosynthesis is
mediated by the ubiquitously expressed serine/threonine protein kinase
ribosomal S6 kinase 1 (S6K1)1
(1, 2). Drosophila and mice lacking S6K1 exhibit a small animal phenotype, implicating this kinase in regulation of cell size
(3, 4). However, S6 phosphorylation and 5'-terminal oligopyrimidine
tract-containing mRNA translation appear normal in cells derived
from mice lacking S6K1, suggesting a compensatory mechanism for these
functions. S6K2, a mitogen-responsive S6K1 homolog, has recently been
identified by our lab and others as a candidate for the compensatory S6
kinase (4-7).
Despite a high degree of sequence homology between S6K1 and S6K2
overall, it is likely that their physiological functions do not overlap
entirely. The small size phenotype persists in the S6K1-deleted mice
despite the presence of S6K2 (4). Both isoforms ( S6K2 shares a conserved four domain organization with S6K1. Both
contain a highly conserved core catalytic kinase domain and linker
regulatory region. Interaction between regulatory N-terminal acidic and
C-terminal basic domains is thought to maintain S6K1 in an inactive
conformation in unstimulated cells. In this inactive state, a
pseudosubstrate region in the C terminus may occlude the catalytic
site. Phosphorylation of C-terminal proline-directed motifs is thought
to disrupt the autoinhibitory interaction of the N- and C-terminal
domains, exposing other critical regulatory sites in the linker and
catalytic regions (9, 10). Three of the four C-terminal
proline-directed sites, as well as other mitogen-stimulated regulatory
phosphorylation sites identified in S6K1, are conserved in S6K2,
suggesting that these related kinases may share similar activation
mechanisms. Several studies have implicated MEK-dependent
signals in regulation of the S6K1 C-terminal proline-directed sites
(11-13), which conform to the consensus motif for ERK phosphorylation
sites (14).
We have recently determined that S6K1 and S6K2 are regulated similarly
by effectors of the PI3-K pathway, including Cdc42, Rac, protein kinase
C Plasmids and Mutagenesis--
Eukaryotic expression vectors
encoding rat p70 S6K1 Cell Culture and Transfection--
HEK293E cells were cultured
in Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum, 0.2 units/ml penicillin, and 200 ng/ml streptomycin.
Cells were seeded at 2 × 106/60-mm dish 3-4 h prior
to calcium phosphate transfection with 6-10 µg of total DNA. Cells
were washed with phosphate-buffered saline after 18-20 h and starved
in serum-free Dulbecco's modified Eagle's medium for 24 h prior
to stimulation and lysis. U2OS cells were cultured in media as
above and transfected with 2 µg of total DNA using LipofectAMINE
reagent (Life Technologies, Inc.) according to the manufacturer's
protocol. U2OS cells were starved and lysed as described for HEK293 cells.
Cell Lysis and Immunoblotting--
Cells were pretreated for 30 min with U0126 or Me2SO vehicle, then stimulated with 100 nM insulin or 50 ng/ml EGF for 30 min. Cells were washed
with phosphate-buffered saline and scraped in lysis buffer (10 mM KPO4, 1 mM EDTA, 10 mM MgCl2, 50 mM
Immune Complex Kinase Assay--
One-third of total lysate was
immunoprecipitated using The PI3-K and mammalian target of rapamycin pathways contribute to
the mitogen-dependent activation of S6K1 and S6K2. These kinases are highly homologous overall but diverge in the N- and C-terminal domains. Because deletion of the C-terminal domain dramatically potentiates S6K2 activation (15), whereas truncation of
S6K1 is inhibitory (9, 10), we sought to determine which S6K2
C-terminal motifs may mediate the inhibitory effect on the kinase.
Disruption of the Putative Nuclear Localization Signal Modestly
Enhances S6K2 Activation by PI3-K Effectors--
Because the
C-terminal domain of S6K2 contains several features distinct from S6K1,
we isolated the effects of these differences by mutagenesis. Two unique
features of the S6K2 C terminus are the presence of a polyproline-rich
domain and a nuclear localization sequence. Previous analysis of the
polyproline-rich domain suggests that its deletion does not affect S6K2
regulation (5). However, mutation of a single amino acid in the
putative nuclear localization signal within the context of full-length
HA-S6K2 (K474M) results in cytoplasmic localization of the kinase in
transfected HEK293 cells by immunolocalization (7). We assayed the
ability of cotransfected Myc-PDK1 to activate the putative nuclear
localization sequence point mutant HA-S6K2-K474M in HEK293 cells. This
point mutant was activated to a greater extent than wild type HA-S6K2 by Myc-PDK1 (Fig. 1). However, this
activity was intermediate between that of wild type and HA-S6K2- MEK-dependent Activation of S6K2--
A common feature
of the S6K1 and S6K2 C termini is the presence of several
proline-directed phosphorylation sites. Mitogen-induced phosphorylation
of four proline-directed motifs in the C terminus of S6K1
(Ser411, Ser418, Thr421, and
Ser424) is thought to be an early step in S6K1 activation
(18). Three of these sites (Ser410, Ser417, and
Ser423) are conserved in S6K2. To determine the
contribution of these putative phosphorylation sites to regulation of
S6K2, we mutated these sites to aspartic acid (HA-S6K2-D3) to mimic
phosphorylation or to alanine (HA-S6K2-A3) to prevent phosphorylation.
We find that these sites play a major role in activation of S6K2,
because HA-S6K2-D3 exhibits elevated basal and EGF- or
insulin-stimulated activity similar to that of HA-S6K2-
The Ser-Pro motifs in S6K2 conform to a consensus motif for ERK
substrates (14), and S6K1 has been shown to be modestly inhibited by
pharmacological inhibition of MEK activity (11, 12). To determine
whether S6K2 activation requires this pathway, HEK293 cells transfected
with HA-S6K2 or HA-S6K1 were treated with varying doses of the MEK
inhibitor U0126 prior to stimulation with EGF. EGF-stimulated HA-S6K1
activity was modestly inhibited (0-30% inhibition, n = 4) by maximal doses (5-10 µM) of U0126 in multiple
experiments (Fig. 4A; see also
Fig. 7). By contrast, EGF-stimulation of HA-S6K2 was potently inhibited
by U0126 in a dose-dependent manner, with 50-90%
inhibition by maximal doses of U0126 in multiple experiments (Fig. 4;
see also Figs. 6 and 7). HA-S6K2 is also potently inhibited by U0126 in
U2OS cells (Fig. 4B). The greater sensitivity of S6K2
relative to S6K1 was also noted in assays of the endogenous kinases in
HEK293 (93% inhibition of S6K2, 45% inhibition of S6K1; Fig.
5A) and U2OS cells (Fig.
5B), suggesting that MEK-regulated kinases may be more
important for activation of S6K2 than S6K1.
To identify potential targets of the MEK pathway in S6K2, we assessed
the effects of U0126 on the HA-S6K2 C-terminal mutants. EGF-stimulated
activity of the D3 or
We have employed EGF in these studies because it is an agonist for both
S6K and ERK activation. However, at physiological levels of insulin
receptor, insulin is a potent activator of S6K1 and S6K2 but a poor
agonist for ERKs (11). In contrast to EGF, we find that
insulin-stimulated HA-S6K2 activity is insensitive to U0126 (Fig.
8), suggesting that MEK-independent
signals relieve S6K2 autoinhibition in response to insulin. Thus, EGF
and insulin activation of S6K2 may employ distinct signal transduction
mechanisms.
Studies in mice lacking S6K1 reveal that S6K1 and S6K2 may mediate
both common and nonredundant functions in vivo. S6K2
possesses unique regions of primary structure, as well as a primarily
nuclear subcellular localization distinct from S6K1. Understanding the regulation of these related kinases may provide insight into the physiological functions unique to each kinase. We have recently demonstrated similarities and notable differences in the regulation of
S6K1 and S6K2 by PI3-K pathway effectors. Of particular interest is the
observation that the C-terminal domain of S6K2 mediates a potent
inhibitory influence on kinase activity; deletion of the C terminus of
S6K2 activates the kinase, whereas analogous deletion of S6K1 is
inhibitory (15). In the present study, we dissect the roles of the
distinct features of the S6K2 C terminus and conclude that the
proline-directed phosphorylation sites are MEK-regulated and contribute
the major regulatory influence of this domain on S6K2 kinase activity.
A conspicuous feature unique to the C terminus of S6K2 is the nuclear
localization signal. Disruption of this basic sequence by a single
point mutation (K474M) confers predominantly cytosolic expression to
HA-S6K2-K474M as assayed by immunofluorescence (7). We demonstrate here
that this mutant is more sensitive to activation by insulin or
cotransfected PI3-K effectors. However, cytosolic localization alone
does not account for the dramatic potentiation of the S6K2- The S6K2 proline-directed phosphorylation sites appear to play an
essential role in relieving S6K2 autoinhibition. In fact, acidic
substitution of these sites (S6K2-D3) results in a mutant that is
regulated very similarly to S6K2- Our data suggest that different kinases may phosphorylate the
C-terminal proline-directed sites in response to different upstream signals and that MEK-dependent kinases play a more critical
role in regulation of S6K2 than S6K1. We demonstrate that S6K2 is more sensitive than S6K1 to MEK inhibition by U0126. Further, it is likely
that the C-terminal Ser-Pro motifs may be targets of
MEK-dependent kinases, because S6K2-D3 and S6K2- The kinases that regulate the C-terminal sites in S6K1 and S6K2
in vivo are not known, but ERK1/2 or cdc2 (M-phase promoting factor) can phosphorylate the S6K1 C terminus in
vitro (21). Previous studies have indicated that Ras and Raf are
neither necessary nor sufficient for S6K1 activation by mitogens (22).
However, other data suggest a role for MEK-dependent
signals in S6K1 activation. Inducible Raf, although less potent than
serum stimulation, was reported to activate S6K1 in an ERK-independent
manner (13). Scott and Lawrence (11) have shown that ERK1/2 are not
involved in insulin-stimulated phosphorylation of S6K1 or of the
rapamycin-sensitive translational regulator 4E-BP1/PHAS-I
(eIF-4E binding protein 1/phosphorylated heat- and
acid-stable protein regulated by
insulin) but that the MEK inhibitor PD098059 blunts insulin
stimulation of these events. A recent study also implicates a role for
basal MEK activity, independent of ERK1/2, for phosphorylation of S6K1, 4E-BP1/PHAS-I and stimulation of protein synthesis in response to
insulin or phorbol ester (12). Consistent with these reports, our data
demonstrate a modest inhibition of S6K1 when MEK is inhibited.
It is likely that two distinct mechanisms, both requiring basal MEK
activity, influence C-terminal regulation of S6K1 and S6K2. Our data
demonstrate that MEK activity is essential for EGF stimulation of S6K2
but only partially contributes to S6K1 activation. By contrast, S6K2
activation by insulin is minimally affected by U0126, suggesting that
different growth factors utilize distinct pathways to regulate S6K2. In
the HEK293 cells utilized in this study, insulin is a potent agonist
for PI3-K but a weak agonist for ERK1/2, whereas EGF is a less potent
PI3-K agonist but a potent ERK activator. Based on our data, we
hypothesize that in the presence of a strong PI3-K signal,
MEK-independent kinases may be able to phosphorylate the S6K2 C
terminus, whereas growth factors that are weaker PI3-K agonists may
require MEK-dependent signals to stimulate C-terminal
phosphorylation. Alternatively, insulin and EGF may activate other
distinct pathways that influence the S6K2 C terminus.
Deletion of the S6K1 gene by homologous recombination suggests that
S6K1 and S6K2 may serve both distinct and common functions. Because S6
phosphorylation and 5'-terminal oligopyrimidine tract-containing mRNA translation are preserved in MEFs from S6K1-deficient mice (4), it is likely that both S6K1 and S6K2 may mediate these functions.
The small size phenotype of these mice, however, suggests that S6K1 may
have functions for which S6K2 cannot entirely compensate (4). The
primarily nuclear localization of both S6K2 isoforms also suggests that
S6K2 may have targets discrete from those of the cytosolic p70 S6K1
isoform. We have demonstrated similarities in the pathways that
regulate S6K1 and S6K2, as well as notable differences, particularly in
regulation of the C-terminal domain. Differential regulation by MEK
signaling, along with discrete subcellular localization, may confer
specificity toward potentially unique substrates of these S6 kinases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
I and
II) of
S6K2 appear to be localized primarily to the nucleus because of a
C-terminal nuclear localization sequence (6, 7). In contrast, the
70-kDa
II S6K1 isoform is predominantly cytosolic, but the 85-kDa
I isoform, which contains an N-terminal nuclear localization
sequence, is nuclear (8). Finally, there are regions in the N- and
C-terminal domains of S6K2 that lack homology to S6K1 (5).
, and PDK1 (15). Both kinases are also activated by overexpression
of Akt/protein kinase B (7). Inhibition of S6K1 and S6K2
activity by rapamycin treatment of cells implicates them as effectors
of the nutrient-sensitive mammalian target of rapamycin pathway (6).
Despite these similarities, we recently reported a major difference in
the role of the C-terminal domain in S6K2 regulation. Deletion of the
C-terminal domain of S6K2 enhances basal kinase activity and renders
S6K2 hypersensitive to activation by growth factors and PI3-K-regulated
effectors (15). In contrast, deletion of the analogous region of S6K1 inhibits kinase activity and does not result in the dramatic
potentiation of activation seen with S6K2. Here, we explore the roles
of the unique features of the C-terminal region of S6K2 to address the mechanism of S6K2 regulation. We find that nuclear localization contributes to the lesser activity of S6K2 but is not the primary inhibitory influence on its activity. We report that the
proline-directed phosphorylation sites are essential for activation of
full-length S6K2 but not S6K1 and that these sites confer greater
sensitivity of S6K2 to MEK-inhibition than S6K1.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
II (HA-S6K1/pRK7) or human p54 S6K2
II
(HA-S6K2/pcDNA3) under the control of the cytomegalovirus promoter
have been described (6). Alignments of human S6K1 and S6K2 isoforms
identifying primary sequence homology, domain junctions, and
phosphorylation sites have been published (5, 15). HA-S6K plasmids were
mutagenized using the Quik-Change polymerase chain reaction-based
method (Stratagene). To generate HA-S6K2-
CT, a stop codon was
introduced at amino acid 399. HA-S6K1-
CT and HA-S6K1-D3E mutants
have been described (9). Plasmids encoding GST-Cdc42 mutants (16) and
Myc-PDK1/pcDNA3 (17) have been described elsewhere.
-glycerophosphate, 5 mM EGTA, 0.5% Nonidet P-40, 0.1%
Brij 35, 0.1% sodium deoxycholate, 1 mM sodium
orthovanadate, 40 mg/ml phenylmethylsulfonyl fluoride, 10 µg/ml
leupeptin, 5 µg/ml pepstatin, pH 7.28) and centrifuged at 15,000 × g for 10 min. Lysates (10% total) were subjected to 7.5% SDS-polyacrylamide gel electrophoresis, transferred to
nitrocellulose membrane, immunoblotted using
-HA,
-GST (Santa
Cruz),
-Myc (17),
-phospho-ERK1/2 (Sigma),
-S6K2 (6),
-S6K1
(16), or
-Rsk, and horseradish peroxidase-conjugated secondary
antibody, and detected with enhanced chemiluminescence reagents.
-HA antibody for transfected S6 kinases or
-S6K2 (6) or
-S6K1 (16) antibody for endogenous kinases, and
protein A-Sepharose. Immunoprecipitates were washed with 1 ml each of
buffer A (10 mM Tris, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 100 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, 2 mM
dithiothreitol, 10 µg/ml leupeptin, and 5 µg/ml pepstatin, pH
7.2), buffer B (buffer A except with 0.1% Nonidet P-40 and 1 M NaCl), and ST buffer (50 mM Tris-HCl, 5 mM Tris-base, 150 mM NaCl, pH 7.2). Kinase
activity toward a recombinant GST-S6 peptide (32 final amino acids of
ribosomal S6) in washed immunoprecipitates was assayed in a reaction
containing 20 mM HEPES, 10 mM
MgCl2, 50 µM ATP unlabeled, 5 µCi of
[
-32P]ATP (PerkinElmer Life Sciences), 3 ng/µl
protein kinase A inhibitor, pH 7.2, for 12 min at 30 °C.
Reactions were subjected to 12% SDS-polyacrylamide gel
electrophoresis, and the amount of 32P incorporated into
GST-S6 was assessed by autoradiography and quantitated by
phosphorimaging (Bio-Rad). Where indicated, the gel was immunoblotted
with
-HA antibody to verify equal immunoprecipitation of
HA-S6Ks.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
CT,
suggesting that although cytosolic localization of S6K2 facilitates its
activation by PI3-K effectors, other features of the S6K2 C-terminal
domain inhibit activation of the kinase.
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Fig. 1.
PDK1 regulation of HA-S6K2 wild type, K474M,
and CT. HEK293 cells were transfected
with 1.0 µg of HA-S6K2 wild type (wt) or K474M or 2.0 µg
of
CT in the pcDNA3 vector and 0.5 or 1.0 µg
Myc-PDK1/pcDNA3 as indicated. Transfected cells were quiesced in
serum-free medium for 24 h prior to 30 min of stimulation with 100 nM insulin. Cells were lysed as described, and protein
expression levels were assayed by immunoblotting with anti-HA or -Myc
antibodies. After Western blotting, lysates were normalized for HA-S6K2
expression levels prior to immune complex kinase assay. Activity of
HA-S6K2 constructs is indicated in the top panel. An anti-HA
Western blot of the immunoprecipitated kinase assay samples and an
anti-Myc Western blot of whole cell lysate are shown in the
bottom panel. Data are representative of three
experiments.
CT in HEK293
cells (Fig. 2), whereas substitution of
two of these sites to alanine (S417A, S423A results in an inactive
HA-S6K2-A2 mutant (Fig. 2A). Similarly, when all three sites
are mutated to alanine, the HA-S6K2-A3 cannot be activated by EGF (see
Fig. 7) or insulin stimulation (data not shown). These findings
indicate that these C-terminal phosphorylation sites are essential for
activation of the full-length kinase. This is surprising because
mutation of the corresponding sites in S6K1 to alanine (A4) reduces
kinase activity 5-fold, but the kinase retains mitogen-responsive
activity (see Fig. 7) (19). Like HA-S6K2-
CT (15), HA-S6K2-D3 is
hypersensitive to activation by insulin, PDK1, or Cdc42V12 (Fig.
3). Notably, the analogous acidic mutant
of S6K1, HA-S6K1-D3E (S411D, S418D, T421E, and S424D), demonstrates
elevated basal and insulin-stimulated activity but is not
hypersensitive to Cdc42V12 or PDK1 (data not shown). In multiple
experiments, HA-S6K2-
CT and -D3 mutants exhibit comparable activities, suggesting that these three sites may largely account for
the inhibitory effect conferred by the intact C-terminal domain.
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Fig. 2.
Acidic substitution of the C-terminal Ser-Pro
motifs enhances growth factor activation of HA-S6K2. A,
mutations of the C-terminal Ser-Pro motifs stimulate (D3) or
inhibit (A2) EGF-stimulated S6K2 activity. HEK293 cells were
transfected with 1.0 µg of HA-S6K2 wild type (wt),
HA-S6K2- CT (
CT), HA-S6K2-D3
(D3), or HA-S6K2-A2 (A2) mutants in the
pcDNA3 vector. Cells were quiesced in serum-free medium for 24 h prior to 30 min of stimulation with 50 ng/ml EGF. Cells were lysed
and subjected to immunoblotting and immune complex kinase assay. Fold
activation of HA-S6K2 mutants, with basal activity of HA-S6K2 wild type
equal to 1.0, is indicated in the top panel. The anti-HA
Western blot is shown in the bottom panel. Data are
representative of four experiments. B, mutations of the
C-terminal Ser-Pro motifs (D3) stimulate insulin-stimulated
S6K2 activity. Cells were transfected and starved as above but were
stimulated for 30 min with 100 nM insulin prior to lysis,
immunoblotting, and immune complex kinase assay. HA-S6K2 constructs
were expressed at similar levels. Data represent the average fold
activation relative to wild type S6K2 basal activity and S.E. of four
experiments.
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Fig. 3.
Acidic substitution of the C-terminal Ser-Pro
motifs enhances of HA-S6K2 activation by PI3-K pathway effectors.
HEK293 cells were transfected with 1.0 µg of HA-S6K2 wild type
(wt) or HA-S6K2-D3 (D3) mutant in the pcDNA3
vector and 2 µg of GST-Cdc42V12/pEBG, 2 µg of Myc-PDK1/pcDNA3
or pEBG. Cells were quiesced as above prior to 30 min of stimulation
with 100 nM insulin and lysis. Activity of HA-S6K2
constructs is indicated in the top panel. Anti-HA, -Myc, or
-GST Western blots are shown in the bottom panel. This
experiment is representative of two experiments.
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Fig. 4.
HA-S6K2 is more sensitive than HA-S6K1 to
inhibition by U0126. A, dose-dependent
inhibition of HA-S6K2, but not HA-S6K1, by U0126 in HEK293 cells.
HEK293 cells were transfected with 1.0 µg of HA-S6K2/pcDNA3 or
0.5 µg of HA-S6K1/pRK7. Cells were quiesced in serum-free medium for
24 h and then pretreated for 30 min with the indicated dose of
U0126 or Me2SO vehicle. Cells were then stimulated with 50 ng/ml EGF for 30 min and lysed as in Fig. 2. The top panel
indicates the percentage of maximal EGF-stimulated HA-S6K activity in
the absence (100%) or presence of the indicated doses of U0126.
Western blots for anti-HA or anti-phospho-ERK1/2 (both quiescent and
EGF-stimulated) are shown in the bottom panel. Data are
representative of four experiments. B,
dose-dependent inhibition of HA-S6K2 by U0126 in U2OS
cells. U2OS cells were transfected with 2.0 µg of HA-S6K2/pcDNA3
using LipofectAMINE. After overnight recovery in medium with
10% fetal bovine serum, cells were quiesced for 24 h in
serum-free media prior to treatment with the indicated doses of U0126
and 50 ng/ml EGF as above. Lysates were normalized for total protein
content following Bradford Assay (Bio-Rad reagent), and equal HA-S6K2
expression levels were confirmed by immunoblotting. Equal amounts of
protein were subjected to immune complex kinase assay. The percentage
of maximal EGF-stimulated HA-S6K2 activity in the absence (100%) or
presence of the indicated doses of U0126 is presented. Data are
representative of two experiments.
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Fig. 5.
Endogenous S6K2 is more sensitive than S6K1
to inhibition by U0126. A, endogenous S6K2 is more
sensitive to U0126 than endogenous S6K1 in HEK293 cells. HEK293 cells
were quiesced in serum-free medium for 24 h before pretreatment
with the indicated doses of U0126 or Me2SO vehicle and then
stimulated with 50 ng/ml EGF for 30 min. Cells were lysed, and immune
complex kinase assays using anti-S6K2 or anti-S6K1 antibodies were
performed. The top panel indicates the percentage of maximal
EGF-stimulated S6K activity in the absence (100%) or presence of the
indicated doses of U0126. Western blots for anti-S6K2, anti-S6K1, and
anti-phospho-ERK1/2 are shown in the bottom panel and
include lanes for quiescent and unstimulated samples. Data are
representative of two experiments. B, endogenous S6K2 is
more sensitive to U0126 than endogenous S6K1 in U2OS cells. U2OS cells
were treated and subjected to immunoblotting with anti-S6K2, anti-S6K1,
anti-phospho-ERK1/2, and anti-Rsk (included as a control ERK-regulated
substrate) and immune complex kinase assay as above. Data are presented
as the percentages of maximal EGF-stimulated S6K activity in the
absence (100%) or presence of the indicated doses of U0126. Data are
representative of three experiments.
CT HA-S6K2 mutants was partially sensitive to
U0126 but to a lesser extent than wild type (Fig. 6). HA-S6K2-A3 exhibits only low basal
activity that is unresponsive to mitogen stimulation. Thus, no further
inhibition of this mutant was observed with U0126 (Fig.
7). Notably, the EGF-stimulated activity
of HA-S6K1-A4 was insensitive to U0126 (Fig. 7). The C-terminal
proline-directed motifs appear to be MEK-regulated in both S6K1 and
S6K2. However, we demonstrate that the HA-S6K2-D3 and
CT mutants are
partially sensitive to MEK inhibition, whereas HA-S6K1-A4 is resistant
to U0126. These data suggest that in S6K2, other sites outside the C
terminus may integrate additional MEK-dependent inputs. In
contrast, the C-terminal sites appear to be the major determinants of
MEK-sensitivity in S6K1.
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Fig. 6.
Activating C-terminal HA-S6K2 mutations
reduce sensitivity to U0126. HEK293 cells were transfected with
1.0 µg of the indicated HA-S6K2 plasmid. Cells were serum-starved for
24 h and then pretreated with Me2SO or 5 µM U0126 for 30 min prior to 30 min of stimulation with
50 ng/ml EGF. Cells were lysed and subjected to immune complex kinase
assay. Equivalent amounts of protein were expressed as assessed by
anti-HA Western blotting. The maximal EGF-stimulated activity in the
absence of U0126 for each construct was normalized to 100%. Data
averaged from four experiments are reported as the percentages of
maximal activity and S.E. of EGF-stimulated activity after U0126
treatment.
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Fig. 7.
Alanine mutation of the C-terminal
proline-directed motifs results in greater inhibition of HA-S6K2 than
HA-S6K1. HEK293 cells were transfected with 1.0 µg of HA-S6K2
wild type (wt) or 1.25 µg of A3 constructs in pcDNA3
or 0.5 µg of HA-S6K1 wild type or A4 mutant in pRK7. Cells were
starved and stimulated with 50 ng/ml EGF as above and lysed for
analysis by Western blotting and kinase assay. Activity of anti-HA
immunoprecipitates is shown in the top panel. The anti-HA
and anti-phospho-ERK1/2 Western blots are shown in the bottom
panel. Data are representative of at least two experiments.
View larger version (27K):
[in a new window]
Fig. 8.
Insulin-stimulated HA-S6K2 activity is
insensitive to U0126. HEK293 cells were transfected with 1 µg of
HA-S6K2/pcDNA3. Cells were quiesced and pretreated with 5 µM U0126 as above, prior to 30 min of stimulation with
100 nM insulin. Cells were lysed, and equal protein
expression levels were verified by anti-HA Western blot prior to immune
complex kinase assay. Data are presented as fold activation of HA-S6K2,
mean and S.D., n = 2.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
CT
mutant, because HA-S6K2-
CT is significantly more active than
HA-S6K2-K474M. Activation of wild type S6K2 by cytosolic proteins such
as Cdc42 and PDK1 suggests that the kinase may exit the nucleus during
the course of its activation. It is likely that cytosolic localization
of the K474M mutant facilitates interaction with cytosolic S6K2
activators. However, as the nuclear localization sequence is rich in
basic residues, it is also possible that disruption of the basic
sequence itself may lessen the inhibitory potential of the C terminus.
CT, suggesting that phosphorylation
of these sites is sufficient to disrupt C-terminal mediated inhibitory
intermolecular or intramolecular interactions. The current model of
S6K1 activation based on structure function analyses postulates that an
interaction between basic residues in the C-terminal pseudosubstrate
domain and an acidic region in the N terminus contributes to kinase
autoinhibition, which is disrupted by mitogen-stimulated
phosphorylation of the C-terminal proline-directed sites. The acidic
residues are conserved between the S6K1 and S6K2 N termini, suggesting
that this mechanism may also apply to S6K2. However, the kinases
diverge completely between the end of the acidic region and beginning
of the conserved catalytic domain. It is possible that the divergent N-
and C-terminal regions of S6K2 may form a stronger autoinhibitory
intramolecular interaction than occurs in S6K1, which may account for
the diminished specific activity of S6K2 relative to S6K1. It is
interesting to note that only three of the four mitogen-stimulated S6K1
proline-directed sites are conserved in human S6K2. It is attractive to
speculate that the lesser mitogen-stimulated net negative charge
because of three sites, in the context of a more basic C terminus
(because of the basic nuclear localization signal) may render S6K2 more sensitive to autoinhibition via N- and C-terminal
charge-dependent interaction. The enhanced sensitivity of
S6K2-D3 to activation by PDK1 or Cdc42V12 relative to the corresponding
S6K1-D3E mutant further supports a stronger influence of these
C-terminal proline-directed sites in regulation of S6K2 than S6K1.
CT are
less sensitive to U0126 inhibition than wild type S6K2. Residual MEK
dependence could potentially be mediated by the proline-directed S370
site, because the analogous S6K1 S371 site, which is phosphorylated by
an unknown kinase, is essential for S6K1 activity (20). Interestingly, the residual EGF-stimulated activity of the HA-S6K1-A4 mutant is
insensitive to U0126, suggesting that the C-terminal proline-directed motifs may be the major targets of the MEK pathway in S6K1 but that
additional MEK-dependent inputs may regulate S6K2.
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ACKNOWLEDGEMENTS |
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We thank members of the Blenis laboratory and John Hwa for critical reading of this manuscript.
![]() |
FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM51405 (to J. B.).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.
§ Recipient of a Postdoctoral Fellowship from the American Cancer Society. Present address: Div. of Vascular Surgery, Dartmouth Medical School, Dartmouth-Hitchcock Medical Center, Lebanon, NH 03756.
Recipient of the Charles A. King Trust Fellowship Award from
the Medical Foundation.
** To whom correspondence should be addressed: Dept. of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. E-mail: jblenis@hms.harvard.edu.
Published, JBC Papers in Press, December 6, 2000, DOI 10.1074/jbc.M009972200
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ABBREVIATIONS |
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The abbreviations used are: S6K, ribosomal S6 kinase; PI3-K, phosphoinositide 3-kinase; ERK, extracellular signal-regulated kinase; PDK1, phosphoinositide-dependent kinase 1; MEK, mitogen-activated protein-ERK kinase; EGF, epidermal growth factor; HA, hemagglutinin; GST, glutathione S-transferase.
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