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 (S6K1), through
phosphorylation of the 40 S ribosomal protein S6 and regulation of
5'-terminal oligopyrimidine tract mRNAs, is an important
regulator of cellular translational capacity. S6K1 has also been
implicated in regulation of cell size. We have recently identified
S6K2, a homolog of S6K1, which phosphorylates S6 in vitro
and is regulated by the phosphatidylinositide 3-kinase (PI3-K) and
mammalian target of rapamycin pathways in vivo. Here, we
characterize S6K2 regulation by PI3-K signaling intermediates and
compare its regulation to that of S6K1. We report that S6K2 is
activated similarly to S6K1 by the PI3-K effectors phosphoinositide-dependent kinase 1, Cdc42, Rac, and
protein kinase C The 70-kDa ribosomal S6 kinase 1 (S6K1)1 is a ubiquitously
expressed serine/threonine protein kinase that phosphorylates the 40 S
ribosomal protein S6 in response to mitogen stimulation (1). S6
phosphorylation up-regulates translation of mRNAs with 5'-terminal oligopyrimidine tracts, many of which encode ribosomal proteins and
translation elongation factors (2). S6K1 activation thus up-regulates
ribosome biosynthesis and enhances the translational capacity of the cell.
Deletion of S6K1 in Drosophila and mice has implicated S6K1
in regulation of cell size. The Drosophila knockout has a
high incidence of embryonic lethality, but surviving flies exhibit a
marked reduction in size that is cell autonomous (3). Mice lacking S6K1
through targeted disruption also exhibit a small animal phenotype (4).
Interestingly, S6 phosphorylation and 5'-terminal oligopyrimidine tract
mRNA translation were found to be normal in fibroblasts derived
from mice lacking S6K1, suggesting a compensatory mechanism for these
S6K1 functions. Our lab and others have recently identified S6K2, a
homolog of S6K1 that phosphorylates S6 in vitro (4-7).
Elevated S6K2 mRNA levels have been reported in the S6K1 knockout
mice (4). Drosophila are thought to express only S6K1, which
may account for the more severe S6K1 knockout phenotype in flies. S6K2
is a good candidate kinase that may supply some but not all of the
functions of S6K1 in the knockout mouse, because the small animal
phenotype persists despite the presence of S6K2, S6 phosphorylation,
and 5'-terminal oligopyrimidine tract mRNA translation.
There are two isoforms of both S6K1 and S6K2 derived from alternative
splicing at the N terminus. The p70S6K1 S6K1 and S6K2 are highly homologous overall, with the greatest sequence
homology in the kinase domain and adjacent regulatory linker domain. A
schematic diagram of S6K1 and S6K2 outlining regions of homology,
features unique to S6K2, and regulatory phosphorylation sites is
provided in Fig. 1. Seven of eight
mitogen-stimulated regulatory phosphorylation sites identified in S6K1
are conserved in human S6K2. There are interesting differences in S6K2
primary structure that may confer differential regulation and functions to this kinase. There are regions of sequence divergence between S6K1
and S6K2 in the N- and C-terminal domains. In addition to the nuclear
localization signal, the C terminus of S6K2 also contains a
proline-rich domain not found in S6K1.
but that S6K2 is more sensitive to basal activation
by myristoylated protein kinase C
than is S6K1. The
C-terminal sequence of S6K2 is divergent from that of S6K1. We
find that the S6K2 C terminus plays a greater role in S6K2 regulation
than does the S6K1 C terminus by functioning as a potent inhibitor of
activation by various agonists. Removal of the S6K2 C terminus results
in an enzyme that is hypersensitive to agonist-dependent
activation. These data suggest that S6K1 and S6K2 are similarly
activated by PI3-K effectors but that sequences unique to S6K2
contribute to stronger inhibition of its kinase activity. Understanding
the regulation of the two S6K homologs may provide insight into the
physiological roles of these kinases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
II isoform is cytosolic,
whereas p85 S6K1
I is nuclear (8). In contrast, both isoforms of S6K2
(p54 S6K2
II, p60 S6K2
I) (5, 6) are primarily nuclear, because of
the presence of a C-terminal putative nuclear localization signal
sequence (NLS) (7) not found in S6K1. Point mutation of the putative
NLS (K474M) results in cytosolic immunolocalization of S6K2
II (7).
The S6K2
I and
II isoforms may reside in distinct nuclear
compartments based on subcellular fractionation studies (6).
View larger version (20K):
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Fig. 1.
Primary structure of S6K1 and S6K2.
Major domain boundaries and mitogen-stimulated phosphorylation sites
are indicated. The solid black bars indicate regions of high
sequence homology. The gray box indicates the homologous
acidic domain of S6K1 and S6K2. The diagonally striped box
indicates the unique proline-rich region of S6K2. The vertically
striped bar indicates the sequence boundaries of the S6K2- CT
mutant.
In both S6K1 and S6K2, the conserved core catalytic and linker domains are flanked by regulatory N- and C-terminal domains. For S6K1, it is thought that interaction between N-terminal acidic residues and C-terminal basic residues inhibits the kinase by allowing a C-terminal pseudosubstrate region to occlude the kinase domain. Mitogen-stimulated phosphorylation of the C-terminal Ser/Thr-Pro motifs is believed to disrupt these interactions, relieving autoinhibition of S6K1 and exposing other regulatory sites, including the major rapamycin-sensitive site, T389, and the catalytic activation loop site, Thr229 (9, 10).
The PI3 kinase (PI3-K) and mTOR signaling pathways mediate multiple mitogen-stimulated phosphorylation events that lead to S6K1 activation. Consistent with the important roles of these pathways in S6K1 regulation, S6K1 activation is inhibited by pharmacological inhibitors of these pathways (11, 12). The immunosuppressant rapamycin, an inhibitor of mTOR, leads to rapid and complete dephosphorylation and inactivation of S6K1 (11). The role of mTOR in S6K1 activation may be suppression of an S6K1 phosphatase (13). There is also evidence suggesting that mTOR may directly phosphorylate S6K1 (14). S6K2 activation is also sensitive to rapamycin, and the analogous rapamycin-sensitive mitogen-stimulated S6K1 phosphorylation sites are conserved in S6K2 (Thr388, Ser410, Ser417, and Ser423) (4-7).
Multiple PI3-K effectors provide distinct inputs to S6K1 activation.
Phosphoinositide-dependent kinase 1 (PDK1) is a
constitutively active kinase whose access to many substrates is
regulated by PI3-K-derived phosphatidylinositol 3,4-bisphosphate
and phosphatidylinositol 3,4,5-trisphosphate (15).
Phosphorylation of Thr229 in the S6K1 catalytic domain
activation loop by PDK1 is a critical activating input (15, 16). PDK1
may also phosphorylate Thr389 (17). The PI3-K- and
PDK1-regulated atypical PKC isoforms and
have also been
implicated in S6K1 regulation (18, 19). These atypical PKCs interact
with S6K1, but it is not yet known whether they directly phosphorylate
S6K1. We have reported growth factor-independent coimmunoprecipitation
of PDK1, PKC
, and S6K1, suggesting the existence of preassembled
complexes of PI3-K-regulated signaling molecules (18). Our lab has also
demonstrated association of the PI3-K-regulated Rho family G proteins
Cdc42 and Rac with S6K1 and has shown that these G proteins contribute
to S6K1 activation (20). Interestingly, Cdc42 and PKC
or PKC
have
recently been shown to associate (21). The mechanism of Cdc42/Rac
activation of S6K1 is not yet known but requires isoprenylation of the
G protein, suggesting that membrane targeting is important (20).
Preliminary studies using pharmacological inhibitors suggest that, like
S6K1, S6K2 is regulated by the PI3-K and mTOR signaling pathways
(4-7). We have shown that a constitutively active PI3-K p110 subunit
activates S6K2 and that S6K2 is activated by PDK1 (6). Others have
found that S6K2, like S6K1, can be regulated by the PI3-K effector
Akt/PKB (7). However, further characterization of the signaling
intermediates that regulate S6K2 has not yet been addressed. Given the
differences in subcellular localization and primary sequence and the
lack of complete functional redundancy between these homologs in the
S6K1 knockout mice (4), we aimed to examine in detail the regulation of
S6K2. Here, we report that although S6K2 is regulated similarly to S6K1
by PI3-K effectors, the C terminus of S6K2 exerts a more potent
inhibitory effect on the kinase.
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EXPERIMENTAL PROCEDURES |
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Plasmids and Mutagenesis--
Eukaryotic expression vectors
encoding rat p70 S6K1 II (HA-S6K1/pRK7) or human p54 S6K2
II
(HA-S6K2/pcDNA3) under the control of the cytomegalovirus promoter
have been described (6). A schematic alignment of rat S6K1
II and
human S6K2
II isoforms identifying domain junctions and
phosphorylation sites is indicated in Fig. 1. A detailed primary
sequence alignment has been published (5). 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 (Fig. 1). HA-S6K1-
CT has been described
(9). Plasmids encoding GST-Cdc42 or GST-Rac1 and mutants (20),
Myc-PDK1/pcDNA3 (22), and FLAG-PKC
and mutants (18) have been
described elsewhere .
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 1 × 106/60-mm dish 1 day prior to transfection. Cells were transfected with 6-10 µg of total DNA by the calcium phosphate method for 6 h, washed with phosphate-buffered saline, and allowed to recover in Dulbecco's modified Eagle's medium with 10% fetal bovine serum overnight. Cells were then starved in serum-free Dulbecco's modified Eagle's medium for 24 h prior to stimulation and lysis.
Cell Lysis and Immunoblotting--
Cells were pretreated for 30 min with wortmannin or vehicle then stimulated with 100 nM
insulin or 50 ng/ml epidermal growth factor 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
-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),
-PKC
(22), or
-Myc (22), and horseradish peroxidase-conjugated secondary antibody, and detected with enhanced chemiluminescence reagents.
Immune Complex Kinase Assay--
One-third of total lysate was
immunoprecipitated using -HA antibody and protein A-Sepharose.
Alternately, volumes of lysate assayed were normalized to reflect S6K
expression levels determined by Western blotting. 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 PKI,
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).
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RESULTS |
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S6K2, like S6K1, is regulated by the PI3-K pathway. S6K2 is activated by constitutively active p110 PI3-K and inhibited when cells are treated with wortmannin (6). Wortmannin-sensitive phosphorylation sites found in S6K1 are conserved in S6K2 and S6K1. Multiple PI3-K pathway effectors have been implicated in S6K1 activation. Given the differences in primary structure and subcellular localization between S6K2 and S6K1, we sought to determine whether S6K2 is regulated by the same downstream PI3-K effectors known to regulate S6K1 and to investigate the roles of divergent S6K2 C-terminal sequences.
Rho Family G Proteins Regulate S6K2--
Evidence suggests that
PI3-K regulates the Rho family G proteins Cdc42 and Rac through
activation of their guanine nucleotide exchange factors, such as Dbl
and Vav (23). We have previously shown that Cdc42 and Rac regulate
S6K1; cotransfection of GTPase-deficient constitutively active point
mutants of Cdc42 and Rac (G12V) activates HA-S6K1, and dominant
negative point mutants (T17N) with high GDP affinity (which sequester
guanine nucleotide exchange factors) antagonize growth factor
activation of S6K1 (20). Similarly, transient cotransfection of HEK293
cells with HA-S6K2 and GST-Cdc42V12 or GST-RacV12 enhanced basal
(2-5-fold) and insulin-stimulated (2-3-fold) activity as measured in
immune complex kinase assays (Fig.
2A). Consistent with a role
for Rho family G proteins in regulation of S6K2, cotransfection of
HA-S6K2 with the dominant negative GST-Cdc42N17 mutant inhibits insulin
stimulation of HA-S6K2, as well as HA-S6K1, activity (Fig.
2B). PDK1 activates S6K1 by phosphorylating
Thr229 in the catalytic activation loop (15, 16). It is
likely that Cdc42 contributes an S6K-activating function distinct from
that of PDK1, because cotransfection of submaximally activating levels of Myc-PDK1 and GST-Cdc42V12 cooperatively activate S6K2. We
demonstrate here for the first time that this is also the case with
S6K1 (Fig. 3).
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The mechanism by which Cdc42 activates S6K1 is not yet known, but
Cdc42V12 and S6K1 coimmunoprecipitate and a mutation that prevents Cdc42 isoprenylation (C189S) fails to activate S6K1 (20). This
suggests that membrane targeting of Cdc42 may be required. An
attractive hypothesis is that association with Cdc42 may transiently target S6K1 to a cellular membrane in the course of its activation. This membrane targeting may be important for access to other
membrane-associated S6K1 activators such as PDK1 and PKC. Because
S6K2 is thought to be primarily nuclear, it is notable that the
cytosolic proteins Cdc42 and Rac can regulate this kinase. We
determined that isoprenylation of GST-Cdc42V12 is required for this
effect, because GST-Cdc42V12,C189S fails to activate HA-S6K2 when
cotransfected (Fig. 4). These data suggest that despite localization primarily to the nucleus, S6K2 is
regulated by cytosolic, isoprenylated low molecular weight G proteins.
We hypothesize therefore that S6K2 may shuttle in and out of the
nucleus and target to a membrane during the course of its
activation.
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Atypical PKC Regulates S6K2--
S6K1 associates with and is
regulated by the atypical PKC
(18). PKC
is activated by binding
PI3-K-derived phosphatidylinositol 3,4,5-trisphosphate and by
interaction with and phosphorylation by PDK1 (22, 24). Although PKC
is not sufficient to activate S6K1 under basal conditions, when
coexpressed with PDK1, a strong S6K1 activation is observed (18).
Coimmunoprecipitation of S6K1, PDK1, and PKC
suggests their
participation in a PI3-K-regulated signaling complex (18). To address
whether PKC
can regulate S6K2 in vivo, we cotransfected a
constitutively active FLAG-tagged, myristoylated PKC
(myr-PKC
)
construct with HA-S6K2, which resulted in basal and insulin-stimulated
HA-S6K2 activation (Fig. 5A). There was a notable difference between S6K1 and S6K2, because HA-S6K1
was activated only modestly (up to 2-fold) by cotransfection of
myr-PKC
in quiescent cells (Fig. 5A) and (18), whereas
HA-S6K2 was activated 5-30-fold under basal conditions (Fig.
5A), suggesting that S6K2 may be more sensitive to
regulation by atypical PKCs. Further supporting a role for PKC
in
activation of HA-S6K2 was the observation that insulin-stimulated
activity was inhibited by cotransfection of the dominant negative
FLAG-PKC
K281W (PKC
K/W) (Fig. 5B), as is the case with
S6K1 (18). In addition, HA-S6K2 is cooperatively activated by combined
cotransfection of submaximally activating levels of myr-PKC
and PDK1
(Fig. 6). Under these conditions, the
constitutively active myr-PKC
is not further activated by PDK1 (22),
but the modest overexpression of both activators results in synergistic
activation of S6K2. Although both S6K1 and S6K2 are inhibited by
dominant negative PKC
(K/W) and cooperatively activated by PDK1 and
myr-PKC
, only S6K2 basal activity is substantially activated by
myr-PKC
alone. These data provide the first evidence that S6K1 and
S6K2 can be differentially regulated.
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C-terminal Truncation Potentiates S6K2
Activation--
Structure-function mutational analyses have provided
important insight into regulation and activation of S6K1 (9, 10). We
employed this approach to further study the regulation of S6K2. Because
the C-terminal domain is a region of divergence between S6K1 and S6K2,
we sought to determine the effect of deletion of this domain on
regulation of S6K2. Amino acids 399-482 encoding the pseudosubstrate
and proline-rich regions, as well as the NLS, were deleted from HA-S6K2
(Fig. 1), and the activity of the resulting mutant (HA-S6K2-CT) was
assayed in transfected HEK293 cells. Surprisingly, deletion of the C
terminus resulted in enhanced basal and insulin-stimulated activity
(Figs. 7 and 8). By contrast, the
analogous truncation mutant of S6K1 is mitogen-regulated but is less
active than the full-length kinase and
is not more sensitive to insulin (9, 10) and (Fig. 8). The S6K2
C-terminal truncation did not significantly alter sensitivity of the
kinase to wortmannin (Fig. 7B), suggesting that essential
inputs from the PI3-K pathway are primarily integrated in regions
upstream of amino acid 399.
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Another marked difference was that basal and insulin-stimulated
HA-S6K2-CT activity was dramatically potentiated by cotransfection with Myc-PDK1 (30-fold basal activation; Fig. 7A). In
contrast, HA-S6K1-
CT was more modestly activated by PDK1 (4-fold
basal activation; data not shown) (18). Insulin-stimulated
HA-S6K2-
CT activity is also more sensitive to activation by Cdc42V12
than HA-S6K2 wild type (Fig. 8). As with PDK1 activation, Cdc42V12 stimulation of the S6K2 C-terminal mutant is stronger than of the
analogous S6K1 mutant under both starved and stimulated conditions (Fig. 8). These data demonstrate that the presence of the intact C-terminal domain exerts a more potent inhibitory influence on S6K2
than on S6K1, because deletion of the domain greatly facilitates S6K2
activation by insulin or PI3-K effectors.
There is a striking difference (10-25-fold) in the specific activity
of S6K1 and S6K2. In our immune complex kinase assays, with equivalent
protein expression levels, S6K2 is a significantly less active kinase
toward GST-S6 or histone H2B substrates (Figs. 2B, 3,
5A, and 8 and Ref. 6). However, the insulin-stimulated specific activity of HA-S6K2-CT upon cotransfection with Myc-PDK1 or
Cdc42V12 was similar to the specific activity of wild type HA-S6K1
(Figs. 7A and 8). These data show that the intrinsic
specific activity of the S6K2 kinase domain is not less than that of
S6K1 but that S6K2 kinase activity is subject to repression in
vivo, mediated by the C-terminal domain. Thus, a second major
difference between these closely related S6 kinases is the role of the
C terminus in the activation process.
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DISCUSSION |
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In vivo studies suggest that S6K1 and S6K2 may serve
both distinct and overlapping physiological functions, because it
appears that S6K2 can only partly compensate in cells lacking S6K1 (4). The primarily nuclear localization of S6K2 (7) also suggests that S6K2
may serve unique functions. Given the likely functional differences, as
well as regions of divergence in primary sequence, it is important to
understand the upstream signaling pathways and intermediates that
regulate each S6 kinase to coordinate these physiological responses.
Our studies of the recently identified S6K1 homolog, S6K2, reveal
similarities as well as differences in the regulation of these related
kinases. Both are activated by common effectors of the PI3-K pathway,
including Akt (7), Cdc42, Rac, PKC, and PDK1 (this report). In
addition, we demonstrate that regions of sequence divergence,
particularly in the C terminus, dramatically influence the specific
activity and growth factor activation of S6K2.
The PI3-K pathway is critical to activation of S6 kinases, because
PI3-K inhibition with wortmannin or LY294002 potently inhibits activation of both S6K1 and S6K2 (4-7). Here, we show that PI3-K effectors known to participate in S6K1 activation also activate S6K2.
In addition, Akt overexpression activates both S6K1 and S6K2 (7).
Because the various PI3-K effectors examined here exhibit distinct
patterns of cytosolic, membrane, and nuclear localization, it is
surprising that all were implicated in S6K2 activation. Because there
is evidence that Akt and PKC can translocate to the nucleus in
stimulated cells (25, 26), it is reasonable to hypothesize that these
kinases may regulate S6K2, which is primarily nuclear. However, Cdc42,
Rac, and PDK1, thought to be cytosolic and associated with membranes
(21, 27), are potent activators of S6K2. It is likely that this
regulation is not merely due to overexpression of these proteins,
because inhibition of endogenous Cdc42 or PDK1 by expression of
dominant negative mutants inhibits S6K2 (this report and Ref. 6). We
therefore hypothesize that S6K2 may shuttle between the nucleus and the
cytosol during the course of its activation. We and others do not
detect a stable change in subcellular localization in HA-S6K2 by
immunofluorescence in quiescent versus growth
factor-stimulated cells (data not shown and Ref. 7), suggesting that
any cytosolic translocation may be rapid and transient and/or involve
levels of protein below the range of detection of this method. Such
nuclear/cytosolic shuttling models have been suggested for other
signaling proteins and kinases including Ste5, MEK, and extracellular
signal-regulated kinase (28, 29). Furthermore, our data suggest that
S6K2 may transiently associate with a cytosolic membrane during the
activation process, as a prenylation-deficient Cdc42 mutant that does
not associate with membranes fails to activate S6K2. Additionally, all
of the PI3-K effectors implicated in S6K2 regulation can associate with
cellular membranes in growth factor-stimulated cells (23).
One difference we have identified in PI3-K-mediated regulation of
full-length S6Ks is that S6K2 is more sensitive to activation by PKC
than is S6K1. Because the mechanism by which PKC
activates S6K1 is
not yet understood, why the effect may be stronger on S6K2 is
uncertain. The ability of PKC
to translocate to the nucleus suggests
a potential mechanism (26). We have previously shown that PKC
activates S6K1 lacking the C-terminal MEK-dependent sites
(18). Similarly, HA-S6K2-D3, in which C-terminal
MEK-dependent sites are replaced with aspartic acid
residues, is activated by PKC
(data not shown). These data suggest
that PKC
-mediated extracellular signal-regulated kinase activation
(30-32) is not a likely mechanism for preferential S6K2
versus S6K1 regulation. In addition, we do not detect
activation of extracellular signal-regulated kinase 1/2 by transfection
of myr-PKC
in our experiments (data not shown and Ref. 18).
We report here a significant difference in the role of the C-terminal
domain in regulation of S6K1 and S6K2. Both contain a pseudosubstrate
region with high homology to S6. Autoinhibition of S6K1 by this
pseudosubstrate domain is thought to be relieved by phosphorylation of
four proline-directed sites (Ser411, Ser418,
Ser421, and Ser423) within this region.
Deletion of the C-terminal domain of S6K1 results in a kinase with
activity slightly lower than that of full-length S6K1, which is still
sensitive to activation by mitogens and inhibition by wortmannin (9,
10). By contrast, the analogous C-terminal truncation of S6K2 is also
sensitive to wortmannin but confers elevated basal activity and
hypersensitivity to mitogens or PI3-K effectors. Thus, the greatly
reduced specific activity of S6K2 relative to S6K1 appears to be
mediated in part by the S6K2 C terminus, because HA-S6K2-CT can be
activated by PDK1 and growth factors to levels similar to S6K1 activity.
The C termini of S6K1 and S6K2 are highly homologous (73% identity) in
the pseudosubstrate region. However, there is only 25% sequence
identity between the kinases from the end of this region to the extreme
C terminus (S6K2 II amino acids 429-482) (5). It is likely that
sequences within this divergent region account for the particularly
strong inhibition of S6K2. Of note, this region of S6K2 encodes a
polyproline-rich domain not found in S6K1. This domain could
potentially interact with SH3 domain containing proteins that may
confer unique regulation to S6K2. However, Gout et al. (5)
cite unpublished data indicating that selective deletion of this
proline-rich domain does not alter activity of S6K2. It is not known
whether the effects of coexpressed PDK1 or other PI3-K effectors might
be greater on this mutant, as is the case for S6K2-
CT.
One hypothesis for the enhanced activity of the S6K2-CT mutant is
that deletion of the C-terminal NLS and consequent cytosolic localization facilitates S6K2 activation. Immunofluorescence studies indicate that this mutant is in fact localized to the cytosol (data not
shown). We have recently examined the roles of the unique S6K2
C-terminal features in an accompanying study (33). We report that
disruption of the unique S6K2 C-terminal NLS by point mutation potentiates S6K2 activation but is not responsible for the dramatic effects observed upon truncation of the entire C-terminal domain. Instead, we find that MEK-dependent regulation of three
C-terminal proline-directed phosphorylation sites is the critical
regulatory influence on this domain.
PI3-K-derived lipid messengers mediate signals affecting the critical
processes of cell growth and size, proliferation, and survival. We
demonstrate here that despite distinct patterns of subcellular
localization and divergences in primary sequence, S6K1 and S6K2 are
regulated similarly by effectors of the PI3-K pathway. However, we also
identify differences in activity and regulation of these related
kinases, such as reduced specific activity of S6K2 compared with S6K1,
greater S6K2 sensitivity to atypical PKC, and a differential
C-terminal domain regulatory mechanism. The phenotype of the mice
lacking S6K1 through homologous recombination suggests that S6K1 and
S6K2 share common as well as nonredundant functions. Our regulatory
data further support the possibility that these related kinases may
regulate both common and distinct cellular substrates and functions.
Differential response to downstream effectors of common pathways, along
with discrete subcellular localization, may confer specificity toward
potentially unique substrates of these S6 kinases.
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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.
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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.
Glaxo Wellcome Gertrude B. Elion Fellow of the Leukemia and
Lymphoma Society of America.
** 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.M006969200
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ABBREVIATIONS |
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The abbreviations used are: S6K, ribosomal S6 kinase; PI3-K, phosphoinositide 3-kinase; mTOR, mammalian target of rapamycin; PDK1, phosphoinositide-dependent kinase 1; MEK, mitogen-activated protein-extracellular signal-regulated kinase kinase; NLS, nuclear localization signal; PKC, protein kinase C; myr-PKC, myristoylated PKC; GST, glutathione S-transferase; HA, hemagglutinin.
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