From the Department of Cell Biology, Harvard Medical
School, and the § Department of Pediatric Hematology and
Oncology, Dana Farber Cancer Institute,
Boston, Masschusetts 02115
Received for publication, October 31, 2000, and in revised form, December 22, 2000
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ABSTRACT |
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The anti-tumorigenic and
anti-proliferative effects of
N- Cells respond to a vast array of extracellular cues that direct
their intracellular activities and ultimately govern their fates. Such
guided regulation fashions and maintains complex and highly specialized
tissues in multicellular organisms. However, the misregulation or
misinterpretation of such signals can spell disaster for an organism
and is the molecular basis for a number of disease states such as
cancer, autoimmunity, tissue degenerative disorders, and developmental abnormalities.
These extracellular cues include numerous soluble and cell-tethered
ligands for which responsive cells express receptors. For ligands
regulating such diverse processes as cell growth, proliferation,
motility, survival, and differentiation, the "AGC" family of
serine/threonine kinases provides critical links between the
extracellular signals and their intracellular effector molecules (1-4). AGC kinases are so-named as they include protein kinase A
(PKA),1 protein kinase G
(PKG), protein kinase C (PKC), and their nearest kinase relatives.
Although AGC kinases share high structural conservation in their kinase
domains, they exhibit rich disparity in the surrounding regulatory
regions. This permits similar kinase activities to respond to a
spectrum of molecular messengers mobilized by extracellular signals.
Despite such diversity, one critical step in the activation of AGC
kinases is highly conserved: phosphorylation of their autoinhibitory activation loops. The activation loop is a structural feature found
throughout the greater kinase superfamily (5). In their inactive state,
the activation loops of many kinases block the access of substrate to
the catalytic core of the enzyme. However, once phosphorylated, the
activation loop is displaced and substrate is no longer sterically or
electrochemically excluded (6). 3-Phosphoinositide-dependent kinase 1 (PDK1) has recently
been shown to phosphorylate the activation loops of a growing number of
its own AGC kinase family members. These include Akt or protein kinase
B (PKB) (7), the 70-kilodalton ribosomal S6 kinase 1 (S6K1) (8, 9), PKC
isoforms (10-13), serum and glucocorticoid-regulated kinase
(14-16), PKA (17), the 90-kilodalton ribosomal S6 kinase (RSK) (18,
19), the protein kinase C related-kinase (20), and the p21-activated
kinase 1 (21). Additionally it autophosphorylates its own activation
loop (22). Recent work using PDK1 As total PDK1 kinase activity in cells appears largely unaffected by
growth factor stimulation, the rate-limiting steps in PDK1
phosphorylation of AGC kinases involve regulation of their steric and
spatial accessibility to PDK1 (1, 3, 4). Our current understanding of
the molecular mechanics of this accessibility shows rich complexity
when considering the four best-studied PDK1-activated enzymes: Akt,
S6K1, RSK, and PKC isoforms. Akt is activated following the activation
of phosphoinositide 3-kinase (PI 3-K). PI 3-K activity generates
D3-phosphoinositides. Two of these products,
PtdIns-3,4-P2 and PtdIns-3,4,5-P3, bind to the
Akt amino-terminal pleckstrin homology domain and exert a dual
activating effect. First, this binding of PI 3-K lipid products
relieves structural auto-inhibition and exposes the Akt activation loop
to PDK1 (24). Second, as PDK1 harbors a carboxyl-terminal
pleckstrin homology domain, which also binds PI 3-K lipid products, PI
3-K activity co-localizes Akt and PDK1 at the plasma membrane leading
to rapid Akt activation following stimulation (1, 3, 25, 26). S6K1 and
RSK likewise appear to require stimulus-regulated exposition of their
activation loops. Unlike Akt, however, S6K1 and RSK do not contain
pleckstrin homology domains. However, their interaction with PDK1
appears more stable than the interaction between PDK1 and Akt as S6K1 and RSK have been shown to co-immunoprecipitate with PDK1 (11, 18,
27-29). PKC isoforms also interact directly with PDK1 (11, 27), but
activation loops of conventional PKC isoforms appear to be freely
accessible to PDK1 in unstimulated cells. Thus PDK1 is able to
phosphorylate them co-translationally (30). Full activation of
conventional PKCs is obtained after subsequent
agonist-dependent steps (30).
The development of specific inhibitors of AGC kinase members or of
their proximal regulators has been critical in defining their
individual roles in signal transduction. More broadly, inhibitors of
AGC kinases are being pursued as clinical therapeutics. The fungal
macrolide rapamycin is a potent immunosuppressant and inhibits the
activation of S6K1. Rapamycin is now being evaluated in phase III
clinical trials for its ability to synergize with another mechanistically different immunosuppressant, cyclosporin A, in reducing
acute rejection of transplanted tissue (31). As S6K1 mediates important
events for cell growth and proliferation (32-34), the
immunosuppressant activity of rapamycin may be due, at least in part,
to its inhibition of S6K1. To more fully understand the biological role
of S6K1, we previously evaluated the effect of the enzymatic inhibitors
N- Mammalian Cell Culture and Transfection--
E1A-transformed
human embryonic kidney 293 (HEK 293E) cells were maintained in DMEM
supplemented with 10% heat-inactivated fetal bovine serum (FBS).
Swiss3T3 (S3T3) cells were maintained in DMEM supplemented with 5% FBS
and 5% calf serum. NIH3T3 cells harboring Myr-Akt-HA-MER (a gift of R. Roth) were maintained in DMEM supplemented with 10% calf serum and 0.4 µg/ml G418. 32D cells (a gift of M. Meyers) were maintained in RPMI
supplemented with 10% FBS and 5% WEHI-conditioned media as a source
of IL-3. C2C12 myoblasts harboring either empty vector pMV7 or
pMV7-HA-S6K1 were generated by D. Fingar and J. Blenis and were
maintained in 20% FBS and 0.4 mg/ml G418. All cells were maintained in
the presence of penicillin (20 inhibitory units/ml) and streptomycin (20 µg/ml) as antibiotics. Transfection of HEK 293E cells was by
calcium phosphate precipitation with the total amount of DNA per
transfected 60- and 100-mm dish between 3 and 5 µg and 6 and 10 µg,
respectively. After 4-6 h exposure to the precipitate, cells were
washed once with DMEM and then treated as indicated in the figure legends.
Immunoprecipitation, in Vitro Kinase Assays, Immunoblotting, and
Antibodies--
For HEK 293E cells, S3T3 cells, NIH3T3 cells harboring
Myr-Akt-HA-MER, and C2C12 myoblasts harboring HA-S6K1, cells were
washed once with serum-free DMEM and then returned to the same for
24 h prior to drug treatment and stimulation as indicated in the figure legends. For 32D cells, the cells were washed three times in
10-15-ml volumes of RPMI and then incubated in the same for 3-4 h
prior to drug treatment and stimulation as indicated in the figure
legends. Cell lysis, immune complex kinase assays (RSK, Akt, S6K1), and
immunoblotting were performed as described previously (42). PDK1 kinase
assays were performed as for RSK, Akt, and S6K1 except that protein
G-Sepharose was used to immunoprecipitate Myc-tagged PDK1 proteins, a
GST-PKC Metabolic Labeling--
HEK 293E were transfected as described
above. 6 h after transfection, cells were washed once with
serum-free DMEM and returned to DMEM supplemented with 10% FBS for
20 h. The media was then aspirated and cells were placed in
phosphate-free DMEM supplemented with 10% FBS and 4 mCi/10-cm dish of
[32P]orthophosphate (ICN no. 64013). Cells were
radiolabeled for 100 min prior to drug treatments as indicated in
the figure legends.
Apoptosis Assays--
For IL-3 withdrawal-induced apoptosis, 32D
cells were washed three times in 10-25 ml of RPMI and resuspended in
RPMI supplemented with 10% FBS without WEHI-conditioned media for
13-18 h. Cells were then washed once with ice-cold phosphate-buffered
saline and resuspended in phosphate-buffered saline with 40 mg/ml of propidium iodide and analyzed by fluorescence-activated cell sorting. Dead cells stained positive for propidium iodide. For TPCK-induced apoptosis cells were washed as described above and then starved of IL-3
for 2-3 h prior to adding the concentrations of drug indicated in the
figure legends. After incubation with a drug for 20 min, cells were
given recombinant IL-3 (0.5 ng/ml) and then assayed by
fluorescence-activated cell sorting as described above.
Plasmids and Constructs--
pKH3-HA-S6K1 A rapamycin-resistant form of S6K1 was described previously (46)
and was generated by deletion of its amino and carboxyl termini (S6K1
-tosyl-L-phenylalanyl chloromethyl ketone
(TPCK) have been known for more than three decades. Yet little is known
about the discrete cellular targets of TPCK controlling these effects.
Previous work from our laboratory showed TPCK, like the
immunosuppressant rapamycin, to be a potent inhibitor of the
70-kilodalton ribosomal S6 kinase 1 (S6K1), which mediates events
involved in cell growth and proliferation. We show here that rapamycin
and TPCK display distinct inhibitory mechanisms on S6K1 as a
rapamycin-resistant form of S6K1 was TPCK-sensitive. Additionally, we
show that TPCK inhibited the activation of the related kinase and
proto-oncogene Akt. Upstream regulators of S6K1 and Akt include
phosphoinositide 3-kinase (PI 3-K) and 3-phosphoinositide-dependent kinase 1 (PDK1). Whereas TPCK had no effect on either
mitogen-regulated PI 3-K activity or total cellular PDK1 activity, TPCK
prevented phosphorylation of the PDK1 regulatory sites in S6K1 and Akt. Furthermore, whereas both PDK1 and the mitogen-activated protein kinase
(MAPK) are required for full activation of the 90-kilodalton ribosomal
S6 kinase (RSK), TPCK inhibited RSK activation without inhibiting MAPK
activation. Consistent with the capacity of RSK and Akt to mediate a
cell survival signal, in part through phosphorylation of the
pro-apoptotic protein BAD, TPCK reduced BAD phosphorylation and led to
cell death in interleukin-3-dependent 32D cells. Finally, in agreement with results seen in embryonic stem cells lacking PDK1,
protein kinase A activation was not inhibited by TPCK showing TPCK
specificity for mitogen-regulated PDK1 signaling. TPCK inhibition of
PDK1 signaling thus disables central kinase cascades governing diverse
cellular processes including proliferation and survival and provides an
explanation for its striking biological effects.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
embryonic stem cells
demonstrated the necessity for PDK1 in insulin-like growth
factor-1 activation of Akt and S6K1 and in phorbol 12-myristate 13-acetate (PMA) activation of RSK. However, lysates from these cells
showed no loss in PKA activation implying that PDK1 was required for
full activation of Akt, S6K1, and RSK, but PKA could be regulated via a
PDK1-independent mechanism (23).
-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) and
N-
-tosyl-L-lysyl chloromethyl ketone (TLCK) on S6K1
activation given their known anti-proliferative and anti-tumorigenic capacity (35-40). Interestingly, like rapamycin, TPCK (and less potently, TLCK) inhibited the activation of S6K1 by multiple agonists. However, TPCK, but not rapamycin, inhibited S6K1 activation in rapamycin-resistant T-cell lines (41). This suggested that TPCK and
rapamycin inhibited S6K1 via distinct molecular mechanisms. To
elucidate potentially novel regulatory mechanisms acting on S6K1 or its
upstream activators and to define more precisely the molecular
mechanisms whereby TPCK exerts its potent biological effects, we sought
to identify the cellular target(s) of TPCK that control the mitogenic
activation of S6K1.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
fragment was used as substrate, and kinase reactions were
allowed to proceed for 15-20 min. Assays in Fig. 6E
evaluating the direct effect of TPCK on PDK1 in vitro were
performed as follows using baculovirus-purified His6-PDK1 (a gift from A. Toker). Master mixes of His6-PDK1 (25-50
nM) were incubated with either ethanol or 250 µM TPCK at room temperature for 30 min in kinase buffer
without ATP. The final concentration of ethanol did not exceed 3%.
Subsequently, 20 µl of each master mix were aliquoted into tubes
containing 10 µl of a substrate mix consisting of kinase buffer, ATP,
and various concentrations of GST-PKC
as indicated in the figure
legends. Kinase assays were allowed to proceed for 15 min before the
addition of 10 µl of 4× sample buffer. Samples were boiled and
analyzed as indicated in the figure legends. For PKA assays, 5 µl of
cleared lysate were used in a 30-40-µl kinase reaction using
8-Bromo-cAMP (Sigma) and protein kinase inhibitor (Sigma catalog
#8140) as indicated in the figure legends. GST-BAD S112A/S136A and
GST-BAD S136A were used as substrates as indicated. PI 3-K assays were
performed essentially as described previously (43). Briefly, cells were lysed in 25 mM Tris, pH 7.2, 137 mM NaCl, 10%
glycerol, 1% IGEPAL (Nonidet P-40), 25 mM NaF, 10 mM sodium PPi, 1 mM
Na3VO4, 1 mM ZnCl2, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride, 5 µg/ml pepstatin A, and 10 µg/ml leupeptin.
Anti-phosphotyrosine immune complexes were collected using protein
A-Sepharose (Sigma), were washed twice with lysis buffer without NaF,
ZnCl2, phenylmethylsulfonyl fluoride, and pepstatin A, and
were then washed twice with 25 mM Tris, pH 7.2, 137 mM NaCl. Immune complexes were subjected to lipid kinase
reactions for 5 min at room temperature in 10 mM HEPES, pH
7.0, 5 mM MgCl2, 20 µM ATP, 10 µCi of [
-32P]ATP. PtdIns (Avanti) and PtdIns-4-P
(Sigma) were used as substrates with phosphatidyl-serine (Avanti) as a
carrier. The reactions were terminated by adding 1.2 volume of 2 M HCl, and lipid products were then extracted using a 1:1
mixture of CHCl3 and CH3OH. Finally, lipid
products were resolved using thin layer chromatography. Drug treatments
were performed as described in the figure legends with ethanol as the
solvent for each drug except for wortmannin and LY294002 where the
solvent was dimethyl sulfoxide (Me2SO). Vehicle
treatment in all cases was ethanol. Drugs and activators were from the
following sources: TPCK (Sigma and Calbiochem), wortmannin (Sigma),
LY294002 (Eli Lilly), rapamycin (Wyeth-Ayerst Research),
4-hydroxy-tamoxifen (Sigma), EGF (Life Technologies, Inc.), insulin
(Sigma), IL-3 (R & D Systems). Commercial antibodies were from the
following sources: anti-Akt (UBI catalog no. 06-608 for
immunoprecipitation and no. 06-617 for immunoblotting),
anti-phosphotyrosine (UBI no. 05-321), anti-Akt-phosphothreonine-308
and anti-Akt-phosphoserine-473 (NEB). Anti-PDK1 and
anti-PKC
-phosphothreonine-410 antibodies were gifts from A. Toker.
Anti-PKC
-phosphothreonine-500 antibodies cross-reacting with
S6K1-phosphothreonine-229 were a gift from A. Newton. Antibodies raised
against S6K1 (44) and RSK1 (45) were described previously.
Anti-PI 3-K p85 antibodies were a gift of L. Cantley. Results
presented are representative of two or more independent experiments.
NT/CT
(27, 46) (rat) and pGEX-4T-1-GST-BAD (murine) wild type and mutant
plasmids (42) were described previously. The pGEX-3X-GST-PKC
fragment was generated by cutting full-length PKC
(rat) using a
5'-internal BglII site and a 3'-vector EcoRI site
and ligated to pGEX-3X digested with BamHI and
EcoRI. pCDNA3-Myc-PDK1 S241E (human) was generated using
the QuikChange approach (Stragene) and introduced an internal
EcoRI site. pCMV6-HA-Akt1 (murine) and pCMV6-Myr-Akt1-HA
were gifts of P. Tsichlis. pCDNA3-HA-Akt1 T308A was a gift of A. Toker. pCDNA3-Myc-PDK1 and pCDNA3-Myc-PDK1 K111I were gifts of
P. Hawkins.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
NT/CT). The basis for the rapamycin resistance of this allele is
proposed to be due to the deletion of a protein phosphatase 2A
(PP2A)-binding region in S6K1 thus preventing PP2A negative regulation
of S6K1. PP2A is itself negatively regulated by the mammalian target of
rapamycin (47). Thus, in the presence of rapamycin the mammalian target
of rapamycin is unable to repress PP2A allowing PP2A to bind to and
inactivate wild type S6K1 but not S6K1
NT/CT. To determine whether
TPCK and rapamycin employed different methods in their inhibition of
S6K1, we asked if the rapamycin-resistant S6K1 was sensitive to TPCK.
This mutant was transiently expressed in E1A-transformed HEK 293E
cells, and its activation was found to be rapamycin-resistant but
TPCK-sensitive (Fig. 1) confirming the
distinct inhibitory mechanisms of rapamycin and TPCK on S6K1.
View larger version (26K):
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Fig. 1.
Rapamycin and TPCK inhibit S6K1 via different
molecular mechanisms. HEK 293E cells were transiently transfected
with a hemagglutinin-tagged (HA), rapamycin-resistant amino- and
carboxyl-terminal deletion mutant of S6K1 (S6K1
NT/CT). Cells were starved of serum for 24 h
and then treated with vehicle (ethanol), 20 nM rapamycin
(RAP) or 50 µM TPCK for 20 min as indicated.
Cells were then stimulated with 100 nM insulin for 20 min
before lysis as indicated. Cell lysates were subjected to either
immunoblotting (upper panel) or immune complex kinase assays
with GST-S6 as substrate using anti-HA antibodies as described under
"Experimental Procedures." The bar graphs represent the
activity of the enzyme, reflecting 32P-labeled phosphate
incorporation into the GST-S6 substrate (not shown) as quantified by
phosphorimaging.
Akt has been shown to positively regulate S6K1 when constitutively
activated by the addition of a membrane-targeting myristoylation sequence (48). This alteration of Akt approximates the Gag-Akt fusion
found in its transforming viral homologue v-Akt (49). A biological
connection between Akt and S6K1 was further suggested as targeted
disruption of the Drosophila alleles of Akt and S6K1 give
strikingly similar small fly phenotypes (50, 51). We therefore asked
whether Akt activation is also prevented by TPCK. Pretreatment of HEK
293E cells with TPCK inhibits insulin activation of Akt to an extent
comparable with the inhibition seen with the PI 3-K inhibitor
wortmannin (Fig. 2A). Similar
results were obtained using Swiss3T3 (S3T3) cells (data not shown). The
concentration of TPCK required to fully inhibit Akt and S6K1 in S3T3
cells and HEK 293E cells was between 25 and 50 µM. The
IC50 for Akt in S3T3 cells was half the IC50
for Akt in HEK 293E cells when compared directly (data not shown).
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The biochemical properties of halomethyl ketones such as TPCK make them effective, irreversible alkylating agents. Alkylation occurs when the structure of the target protein can simultaneously accommodate the drug and bring it into the proximity of the R-group of either a histidine or cysteine residue. TPCK alkylation of histidine occurs at position 1 of the imidazole ring (52), whereas for cysteine TPCK alkylation occurs at the sulfhydryl group (53). To determine whether TPCK is an irreversible inhibitor of Akt activation, S3T3 cells were pretreated with increasing amounts of TPCK or the reversible PI 3-K inhibitor, LY294002 (54). Following treatment, the cells were either washed or maintained in the presence of the inhibitors during a subsequent stimulation with insulin for 30 min. As opposed to the reversible effect seen with LY294002, the effect of TPCK on Akt activation was irreversible (Fig. 2B), implying that TPCK was alkylating either Akt directly or one of its upstream activators.
Inhibition of S6K1 and Akt by similar concentrations of TPCK suggested
that TPCK was inhibiting an upstream regulator common to both S6K1 and
Akt. S6K1 and Akt share at least two upstream activators, PI 3-K and
PDK1 (2). The effect of TPCK on PI 3-K was first analyzed. PI 3-K
(Class IA) signaling is initiated by the association of the
110-kilodalton catalytic subunit (p110)/85-kilodalton regulatory
subunit (p85) heterodimer with phosphotyrosine residues on activated
receptor complexes. This brings the heterodimer into proximity of
substrate at the plasma membrane. This association occurs rapidly
following mitogenic stimuli (3). As a relatively small proportion of
active PI 3-K heterodimers finds access to these receptor complexes, a
good reflection of mitogen-induced PI 3-K activity can be assessed in
lipid kinase reactions following immunoprecipitations using
anti-phosphotyrosine antibodies. As shown in Fig.
3, pretreatment of HEK 293 cells with
TPCK did not inhibit the recruitment of p85 to the receptor complex and
did not inhibit phosphotyrosine-associated PI 3-K activity.
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Full activation of Akt requires two PI 3-K-regulated phosphorylation
events: PDK1 phosphorylation of the activation loop at threonine 308 as
well as phosphorylation of serine 473 in the carboxyl terminus (1-4).
The relevant in vivo kinase responsible for phosphorylation
of serine 473 has been referred to as "PDK2" (55). PDK2 activity
has been reported to be a result of autophosphorylation (56), modified
PDK1 activity, (57) as well as heterologous kinase activity (58). To
determine whether one or both of these phosphorylation events was
sensitive to TPCK, we made use of phosphospecific antibodies generated
to specifically recognize either serine 473-phosphorylated Akt or
threonine 308-phosphorylated Akt. As shown in Fig.
4A, pretreatment of HEK 293E
cells with TPCK prevented the phosphorylation of Akt at serine 473. Similar results were seen in 7S3T3 cells (Fig. 7 and data not shown).
The phosphothreonine 308-specific antibody required a higher
concentration of epitope than the one that recognizes phosphoserine
473, so it was necessary to first immunoprecipitate overexpressed
HA-tagged Akt prior to immunoblotting. Fig. 4B shows the
resultant blot indicating that phosphorylation of threonine 308 is also
prevented by treating cells with TPCK prior to stimulation.
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Once stimulated, TPCK treatment of cells diminished Akt activity only
gradually, reducing it to roughly half-maximal after 30 min (Fig.
5A). Thus, activated Akt is
much less sensitive to TPCK. This implies that the gradual decline in
Akt kinase activity observed after TPCK delivery to stimulated cells
represented only the normal kinetics of inactivation via phosphatases
or protein instability. A similar experiment was performed assessing
Akt activity following TPCK treatment of cells expressing either wild type Akt or a myristoylated allele of Akt exhibiting constitutive activity. As shown in Fig. 5B, wild type Akt was fully
inhibited by treating the cells with TPCK prior to stimulation.
However, the activated allele of Akt was resistant to 30 min of TPCK
treatment. Strikingly, however, this same allele, when under the
inducible control of a mutant estrogen receptor (MER) fused to its
carboxyl terminus (48), was fully inhibited by TPCK regardless of
whether TPCK was washed away prior to addition of the inducing agent, 4-hydroxy-tamoxifen (Fig. 5C). Taken together, these data
suggested that TPCK inactivation of Akt was primarily through the
inhibition of an upstream activator of Akt. As a result of these
observations we asked whether TPCK inhibition of Akt activation
represented a unique disruption of PDK1 signaling. To further examine
this possibility, we analyzed the effect of TPCK on the phosphorylation state of the PDK1 site, threonine 229, in S6K1. C2C12 myoblasts harboring either empty vector or HA-tagged S6K1 were starved of serum
for 24 h prior to pretreatment with inhibitors and subsequent stimulation with insulin for 30 min. As shown in Fig. 5D,
pretreatment of cells with TPCK prevented the phosphorylation of
threonine 229 in S6K1. Thus, TPCK is disrupting PDK1 signal
transduction to S6K1 and Akt.
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As TPCK inhibited the phosphorylation of the activation loop sites in
both Akt and S6K1 without inhibiting PI 3-K, it implied TPCK
compromised PDK1 kinase activity. However, as shown in Fig. 6A, TPCK-treated HEK 293E
cells show little to no reduction in total cellular PDK1 activity as
measured by both autophosphorylation and phosphorylation of a
GST-PKC fragment. However, the possibility that TPCK inhibits a
particular pool of PDK1 molecules in the cell cannot be excluded.
Likewise inhibition of PI 3-K had no effect on PDK1 kinase activity
(Fig. 6A), consistent with PI 3-K lipid products having more
bearing on PDK1 localization than on kinase activity per se
(59). Although immunoprecipitated PDK1 kinase activity was not
significantly affected by TPCK, TPCK did strikingly alter its
electrophoretic mobility in SDS-polyacrylamide gels (Fig.
6A). This implied that TPCK was inducing a posttranslational modification that could be responsible for its inability to signal to
substrates. PDK1 overexpressed in HEK 293 cells exhibits constitutive, unregulated phosphorylation at five serine residues (22). When mutated
individually to alanine, only one of these mutations, serine 241 in the
activation loop, affected PDK1 kinase activity toward heterologous
substrates in vitro (22). Mutation of serine 241 to glutamic
acid (S241E) generated a pseudo-phosphorylated, active kinase with
reduced specific activity (22). To determine whether the increased
electrophoretic mobility of PDK1 was due to loss of phosphorylation at
serine 241, we generated the S241E mutant of PDK1 and expressed it in
HEK 293E cells alongside wild type PDK1. We then examined the effect of
TPCK on the respective electrophoretic mobilities and kinase
activities. As seen in Fig. 6B, TPCK increased the
electrophoretic mobility of both of these forms of PDK1. Additionally,
TPCK had no effect on PDK1 autophosphorylation (Fig. 6B). As
expected, the PDK1 S241E mutant has greatly reduced autophosphorylating
activity, but its ability to phosphorylate a GST-PKC
substrate, like
wild type PDK1, was unaffected by TPCK (data not shown). This showed
that loss of phosphorylation at serine 241 was not responsible for the
TPCK-induced mobility profile of PDK1. In a similar experiment a
kinase-inactive lysine 111 to an isoleucine mutant of PDK1 exhibited
the same electrophoretic mobility as both the wild type and S241E (Fig.
6C) showing that the increase in mobility was independent of
catalytic activity and not due to a loss of cis-autophosphorylation.
Given these results, we analyzed the effect of TPCK on the steady state
levels of PDK1 phosphorylation by [32P]orthophosphate
metabolic labeling of cells expressing either wild type PDK1 or S241E
PDK1. As shown in Fig. 6D, steady state levels of phosphate
incorporation into PDK1 were unaffected by TPCK treatment despite the
noticeable increase in PDK1 electrophoretic mobility. This suggested
the possibility of novel regulatory mechanisms of PDK1 signaling
in vivo. Notwithstanding this dramatic effect in
vivo, Fig. 6E shows that TPCK did not appear to react
significantly with PDK1 in vitro even when subjected to 5×
the concentrations of TPCK used on cells. Additionally, Fig.
6E shows that under these conditions TPCK did not alter PDK1
kinase activity, did not compete directly for substrate, and did not
alter the substrate-induced increase in autophosphorylation previously
observed using a phospho-RSK peptide (29). This peptide contained the
phospho-PDK2 site, the hydrophobic sequence that was shown to
serve as a PDK1 docking site in RSK (29). As PKC
has a glutamic acid
at this residue it is able to likewise induce increased PDK1
autophosphorylation.
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Because TPCK did not significantly affect PDK1 phosphotransferase
activity yet was able to block activation of Akt and S6K1 by preventing
phosphorylation of their activation loops, we asked if TPCK disrupted
PDK1 signaling to RSK. This served as an important control as RSK
activation, unlike the activation of Akt and S6K1, is largely
independent of PI 3-K signaling. Recent work has shown that in addition
to MAPK, PDK1 activity is critical to RSK activation as PDK1
phosphorylates serine 221 (human) in the activation loop of the RSK
amino-terminal kinase domain (18, 19, 23). Additionally, myristoylation of RSK, as with Akt, renders the kinase active even
in the absence of growth factors (42). Such targeting to membrane is
presumed to both locate RSK more proximal to PDK1 as well as remove
autoinhibitory structural constraints on the access of PDK1 to serine
221. Fig. 7 shows TPCK inhibition of RSK
in three different cell types. Interestingly, whereas the concentrations of TPCK used to inhibit Akt, S6K1, and RSK were inconsequential to MAPK activation in 32D and S3T3 cells (data not
shown, (41)), TPCK enhanced MAPK activation in HEK 293E cells (Fig.
7B). Notwithstanding this enhancement, RSK activity was only
marginally increased over baseline in HEK 293E cells treated with TPCK
(Fig. 7B) underscoring the role of PDK1 in the activation of
RSK.
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Recently, in the IL-3-dependent murine hematopoetic cell lines 32D and FL5.12, we showed the ability of RSK to transduce a cell survival signal in part through phosphorylation of the proapoptotic Bcl-2 family member, BAD (42). We thus analyzed the effect of TPCK on BAD phosphorylation as well as its effect on cell survival in 32D cells. 32D cells were starved of IL-3 for 2 h and then treated with either vehicle or TPCK prior to the re-addition of IL-3 for 10 min. Following lysis and immunoprecipitation, immune complexes of endogenous BAD were analyzed by immunoblotting. TPCK prevented the IL-3-induced mobility shift of BAD that corresponded with the loss of RSK phosphotransferase activity (Fig. 7C). In agreement with TPCK inhibition of the IL-3-activated prosurvival kinases RSK and Akt, increasing concentrations of TPCK antagonized IL-3 survival signaling with the amount of cell death after 16 h in the presence of 25 µM TPCK plus IL-3 roughly corresponding to the amount of cell death 16 h after IL-3 withdrawal (Fig. 7D).
PDK1 is able to phosphorylate the activation loop of PKA
in vitro and lead to an increase in PKA activity when
overexpressed in cells (17). PKA, however, can still be activated in
embryonic stem cells lacking PDK1 (23). This provided evidence that
PDK1 is not required for PKA activation despite the similarities
between the primary amino acid sequence surrounding the phosphorylation site in PKA's activation loop and that of other known PDK1 substrates (Fig. 8A). This also proposes
an important control for the specificity of TPCK in PDK1-mediated
signaling. If TPCK were to inhibit PKA it would suggest that TPCK
broadly affects AGC-kinases outside of those that require PDK1 for full
activation. Fig. 8, B and C show that PKA from
both untreated and TPCK-treated cells was similarly activated by
8-Bromo-cAMP as measured by the ability of PKA to phosphorylate
in vitro either GST-BAD S136A or S112A/S136A, which is
consistent with reports showing PKA phosphotransferase activity toward
serine 155 of BAD (60, 61).
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DISCUSSION |
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More than 30 years ago (35) and again 20 years ago (38), topically applied TPCK was shown to potently inhibit tumorigenesis initiated in mouse skin by 7,12-dimethylbenz(a)anthracene and promoted by PMA. This anti-tumorigenic effect was also accompanied by a reduction in erythema and leukocyte infiltration at the site of PMA application (35). It was also reported that the survival of mice prone to spontaneous breast cancer was significantly increased when treated with 1 mg of TPCK/week without signs of toxicity after 45 weeks of treatment (35). Additionally, many cell types treated with TPCK have reduced proliferative rates (36, 37, 39). Despite these earlier observations, little was known about the cellular target(s) of TPCK that govern these effects. Given the role of S6K1 in cell growth and proliferation, and as it is a target of the immunosuppressant rapamycin, we evaluated the effect of TPCK on the activity of S6K1 and the signaling pathways regulating S6K1. We found TPCK to potently inhibit not only S6K1 (41) but two additional PDK1-activated enzymes, Akt and RSK, both of which are known mitogenically regulated kinases involved in transducing extracellular signals into diverse cellular responses including proliferation, growth, and survival.
TPCK was originally designed as an in vitro affinity label to establish the identity of critical amino acids in the catalytic core of the serine protease chymotrypsin (62). Subsequently, much of the work published on the in vivo effects of TPCK assumed the involvement of serine protease activity. Although there exists a large body of work describing the chemopreventive effects of protease inhibitors (63), the biochemical properties of TPCK do not directly implicate it as a serine protease inhibitor a priori. Whereas we cannot formally exclude the possibility of chymotrypsin-like serine protease activity regulating PDK1 signaling, we have evaluated the effect of coexpression of Akt with gene products encoding the serine protease inhibitors plasminogen activator inhibitor-2 (64) and the related cowpox virus-encoded cytokine response modifier A (65, 66) and saw no effect on insulin-stimulated Akt activation (data not shown).
However, the inhibitory mechanisms of halomethyl ketones toward serine proteases may permit important inferences about the mechanism of the inhibition of TPCK toward PDK1 signaling. TPCK was shown to irreversibly alkylate chymotrypsin at a one to one molar ratio at histidine 57 (62). The specificity of TPCK for chymotrypsin was accomplished as chymotrypsin proteolyses proteins containing the large hydrophobic and aromatic residues tyrosine or phenylalanine at the P1 position of the target sequence. Additionally, denatured chymotrypsin was not alkylated by TPCK indicating the requirement for chymotrypsin to be in a folded configuration presumably generating a hydrophobic pocket that can accommodate either an aromatic P1 amino acid or the hydrophobic, aromatic rings of TPCK. Along these lines, the lysyl-derivative TLCK has no effect on chymotrypsin but specifically inhibits trypsin. This follows given that the lysyl derivative mimics the trypsin preference for basic amino acids in the P1 position of target proteins (52). If TPCK is acting directly on PDK1 in vivo, it implies that PDK1 contains a hydrophobic pocket that could both accommodate an aromatic compound as well as be structurally important for its activation of or interaction with substrates. Indeed one such pocket was recently identified in PDK1 and was shown to accommodate two phenlylalanine residues from a hydrophobic motif found in PDK1 substrates and thereby facilitated their activation (28). This hydrophobic motif has been shown recently to be essential for PDK1 docking to and subsequent activation of RSK (29). Interestingly, TPCK, TLCK, and the valyl derivitive, TVCK, exert different effects on the activation of S6K1. In cells TPCK fully inhibits S6K1 between 25 and 50 µM, whereas TLCK becomes fully inhibitory at 350 µM (41), and the valyl derivative, TVCK, becomes inhibitory at 500 µM (data not shown). This implies that the structure of the halomethyl ketone is critical to its ability to prevent PDK1 signaling. However, it is formally possible that the primary reason for the observed differences in inhibition of S6K1 by these halomethyl ketones is a function of their solubilities or abilities to penetrate the cell membrane.
A handful of molecules have shown inhibition by TPCK in vitro. However, the concentrations used in vitro far exceeded the concentrations effective to inhibit PDK1 signaling and induce cell death in vivo. For example, TPCK inhibition of PKC in vitro occurs at an IC50 of 8 mM (67). TPCK inhibition of PKA in vitro occurred at concentrations between 340 µM and 1 mM (53) yet we show here that TPCK had no effect on PKA activity from cells treated with concentrations of TPCK that inhibit PDK1 signaling (Fig. 8, B and C). Likewise PKC activation was not inhibited when cells were treated with antiproliferative doses of TPCK (39). We have assayed the in vitro effect of TPCK on PDK1, Akt, and S6K1 purified either from baculoviral expression systems or via immune complexes from mammalian cell lysates. No inhibition of any of these kinases was detected at TPCK concentrations of <500 µM (Fig. 6E, and data not shown), and <50% inhibition was seen after treating the kinases with 1-2 mM TPCK (ref. 41 and data not shown). These data suggest that either the chemical makeup of the cell greatly facilitates TPCK conjugation to PDK1 or that TPCK exerts its effects differentially in vivo and in vitro.
Alternatively, TPCK may not be acting via direct inhibition of the kinase activity of PDK1 or its kinase substrates but may be preventing a productive PDK1-substrate interaction inside the cell. This is consistent with the data presented in Fig. 5 that suggest TPCK is operating at the level of upstream activators of Akt in vivo. This is also consistent with the observation that the activation of conventional PKC isoforms is insensitive to TPCK (39) as they are co-translationally phosphorylated by PDK1 (30), and thus TPCK treatment for 20-30 min prior to stimulation would have little effect on conventional PKC activity. We are aware of only one protein that has been directly conjugated to TPCK in vivo, the transforming protein E7 from the human papillomavirus-18 (68). Concentrations of ~200 µM TPCK or 250 µM TLCK were required to fully modify cysteine 27 of the E7 protein stabley expressed in a human foreskin keratinocyte cell line (68). Interestingly, such a modification increased the E7 electrophoretic mobility. This increase in mobility was reversed by mutating cysteine 27 to glycine (68).
TPCK has been shown to induce cell death in a number of cell types at concentrations that should inhibit PDK1 signaling (40, 69, 70). It is interesting that protection from TPCK-induced death has been observed in cells overexpressing Bcl-XL (69) and c-Rel (69) or when flooded with a high concentration of n-acetyl-cysteine (40), perhaps generating competition for TPCK binding. This argues against TPCK-induced cytotoxicity and for its inhibition of signaling pathways that mediate cell survival including kinase cascades that modulate mitochondrial integrity via phosphorylation of Bcl-2 family members.
In recent years TPCK has been shown to be a potent inhibitor of nuclear
factor B (NF
B) signaling (69, 71). Although the exact mechanism
behind this inhibition is unknown, TPCK has been shown to inhibit the
phosphorylation of the inhibitor of NF
B (I
B), implying TPCK is
acting at either the level of the kinases that phosphorylate I
B
(IKK) or their upstream activators. If the effect of TPCK on NF
B
signaling were via its inhibition of PDK1 signaling then the simplest
explanation is explained by the inhibition of the PDK1-activated
kinases Akt and RSK, both of which have been shown to positively
regulate NF
B signaling (72, 73). However, we find it an intriguing
possibility that PDK1 plays a more direct role in NF
B signaling
perhaps by directly phosphorylating the activation loops of IKK-1 and
IKK-2. The activation loops of IKK-1 and -2 contain two regulated
serine residues, serines 176 and 180 for IKK-1 and serines 177 and 181 for IKK-2 (both human). The phosphorylation of these residues is
required for full kinase activity (71). Comparison of the amino acid
context surrounding the second phosphorylated serine in the activation loops of IKK-1 and -2 with the sequence surrounding known PDK1 phosphorylation sites shows remarkable similarity (Fig. 8A).
This raises the possibility that TPCK exerts such a potent inhibitory effect on NF
B signaling by disabling multiple
PDK1-dependent inputs.
Considerable research has been conducted using the halomethyl
ketone TPCK, and the observed effects of TPCK have extended into
multiple aspects of normal and aberrant cellular processes with little
knowledge of its specific cellular target molecules. The results
presented here show that TPCK disrupts PDK1 signaling to Akt, S6K1, and
RSK and thereby disrupts central pathways involved in transducing
extracellular stimuli into meaningful cellular events such as growth,
proliferation, and survival. This understanding serves not only as a
starting point for further elucidation of its precise inhibitory
mechanism on PDK1 signaling but invites re-examination of the potential
use of TPCK as a therapeutic agent as a means of controlling
aberrations resulting from inappropriate PDK1 signal propagation.
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ACKNOWLEDGEMENTS |
---|
We thank D. Fruman and L. Cantley for reagents and assistance with the PI 3-K assays, A. Toker for reagents and insights, A. Newton for reagents, D. Fingar for the C2C12 cells, T. Grammer and J. Powers for important communications, and members of the Blenis laboratory for advice and critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants CA45695 and 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.
¶ To whom correspondence should be addressed. Tel.: 617-432-4848; Fax: 617-432-1144; E-mail: jblenis@hms.harvard.edu.
Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M009939200
2 Contact the corresponding for website address.
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ABBREVIATIONS |
---|
The abbreviations used are:
PKA, protein kinase
A;
PKB, protein kinase B;
PKC, protein kinase C;
PKG, protein kinase G;
PDK1, 3-phosphoinositide-dependent kinase 1;
PDK2, 3-phosphoinositide-dependent kinase 2;
S6K1, ribosomal S6 kinase 1;
RSK, ribosomal S6 kinase;
PMA, phorbol
12-myristate 13-acetate;
PtdIns, phosphoinositide;
PI 3-K, phosphoinositide 3-kinase;
TPCK, N--tosyl-L-phenylalanyl chloromethyl
ketone;
TLCK, N-
-tosyl-L-lysyl chloromethyl ketone;
HEK, human embryonic kidney;
DMEM, Dulbecco's modified Eagle's medium;
FBS, fetal bovine serum;
IL, interleukin;
PP2A, protein phosphatase 2A;
S3T3, Swiss3T3 cells;
NF
B, nuclear factor
B;
I
B, NF
B
inhibitor;
IKK, I
B phosphorylating kinases;
PAGE, polyacrylamide gel
electrophoresis;
HA, hemagglutinin.
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