From the Friedrich Miescher-Institut, Department of Growth Control, P. O. Box 2543, CH-4002, Basel, Switzerland
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ABSTRACT |
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Here we have employed p70s6k truncation and point mutants to elucidate the role played by the carboxyl-terminal autoinhibitory domain S/TP phosphorylation sites in kinase activation. Earlier studies showed that truncation of the p70s6k amino terminus severely impaired kinase activation but that this effect was reversed by deleting the carboxyl terminus, which in parallel led to deregulation of Thr229 phosphorylation in the activation loop (Dennis, P. B., Pullen, N., Kozma, S. C., and Thomas, G. (1996) Mol. Cell. Biol. 16, 6242-6251). In this study, substitution of acidic residues for the four autoinhibitory domain S/TP sites mimics the carboxyl-terminal deletion largely by rescuing kinase activation caused by the amino-terminal truncation. However, these mutations do not deregulate Thr229 phosphorylation, suggesting the involvement of another regulatory element in the intact kinase. This element appears to be Thr389 phosphorylation, because substitution of an acidic residue at this position in the p70s6k variant containing the S/TP mutations leads to a large increase in basal Thr229 phosphorylation and kinase activity. In contrast, an alanine substitution at Thr389 blocks both responses. Consistent with these data, we show that a mutant harboring the acidic S/TP and Thr389 substitutions is an excellent in vitro substrate for the newly identified Thr229 kinase, phosphoinositide-dependent kinase-1 (Pullen, N., Dennis, P. B., Andjelkovic, M., Dufner, A., Kozma, S., Hemmings, B. A., and Thomas, G. (1998) Science 279, 707-710), whereas phosphoinositide-dependent kinase-1 poorly utilizes the two p70s6k variants that have only one set of mutations. These findings indicate that phosphorylation of the S/TP sites, in cooperation with Thr389 phosphorylation, controls Thr229 phosphorylation through an intrasteric mechanism.
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INTRODUCTION |
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The concerted up-regulation of transcription and translation is required for a cell, responding to mitogenic stimuli, to grow and enter the cell cycle (1-3). Recent studies have implicated increased S6 phosphorylation in the selective translation of a subset of essential mRNAs containing an oligopyrimidine tract at their transcriptional start site (4, 5). This event is regulated by p70s6k/p85s6k (6), two mitogen-stimulated protein kinase isoforms that rely on multiple phosphorylation as a principal mechanism for activation (7). The p85s6k isoform is expressed from the same transcript as p70s6k through an alternative translational initiation start site,1 adding a 23-amino acid extension at the amino terminus and constitutively targeting it to the nucleus (8). Little is known about the role of p85s6k in the nucleus; however, S6 has been shown to reside in both the nucleoplasm, in a free form, and in the nucleolus, in preribosomal particles, where it is also phosphorylated in response to mitogens (9). In studies conducted to date, regulation of the nuclear isoform parallels that of p70s6k, so a concomitant role for p85s6k in mitogenesis is predicted (10, 11), perhaps involving transcription or processing of RNA (8).
The identification of intramolecular p70s6k regulatory
elements, in the form of domains and phosphorylation sites, has
increased our understanding of the mechanism by which the kinase
autoregulates (7, 12, 13). To date, eight phosphorylation sites have been identified in the endogenous kinase (14, 15). In initial studies,
Ser411, Ser418, Thr421, and
Ser424, residing within a potential autoinhibitory domain
at the carboxyl terminus of the kinase (16, 17), were found to be
principal sites of mitogen-induced phosphorylation (14). These sites
are characterized by a proline in the +1 position and a hydrophobic residue in the 2 position. More recently, studies have led to the
identification of Ser371 (18) as a phosphorylation site
that shares the same motif, and three additional sites,
Thr229, Thr389, and Ser404, which
are flanked in the +1 and
1 positions by bulky aromatic amino acids
(15). The phosphorylation of these latter sites in response to mitogens
is blocked by treatment of cells with the immunosuppressant rapamycin
or the fungal metabolite wortmannin (15, 19). Based on mutation
studies, Thr229, in the activation loop, as well as
Ser371 and Thr389, in the linker region
coupling the catalytic and autoinhibitory domains, appear to be
critical for kinase activation (15, 18). The activation loop
phosphorylation site, Thr229 in p70s6k, is a
common regulatory element found in many kinases (20, 21). In parallel
studies, we have shown that the phosphorylation of this site is
regulated by the newly described phosphoinositide-dependent protein kinase, PDK12 (22).
Thr229, Ser371, and Thr389, as well
as the domains in which they reside, are strikingly conserved in most
members of the AGC (protein kinases A, G, and C) family of Ser/Thr
kinases (21).
In contrast to the domains containing Thr229, Ser371, and Thr389, the autoinhibitory domain, as well as the carboxyl terminus of p70s6k, are conspicuously absent in the other members of the AGC family of Ser/Thr kinases (21). We have reported that mutation of the S/TP sites within the autoinhibitory domain, as well as Ser404, to alanines or acidic residues modulates kinase activity (15, 19). However, others have claimed little to no effect of similar mutations on the activity of the kinase (23, 24). Indeed, the deletion of the p70s6k carboxyl terminus, containing the S/TP sites, has little effect on either basal or mitogen-induced kinase activity (12, 13, 25), which has also been used to conclude that these sites are not involved in regulating kinase activation at either the G0/G1 or M/G1 transition state of the cell cycle (23, 24). Despite these observations, this domain is completely conserved in all the mammalian forms of p70s6k and is also present in the recently cloned Drosophila homolog, Dp70s6k (26, 27). More notably, a p70s6k amino-terminal truncation, which blocks kinase activation (12, 13, 25) and mitogen-induced Thr229 and Thr389 phosphorylation (13), is rescued by the same carboxyl-terminal deletion that removes the autoinhibitory domain (12, 13, 25). These observations suggest instead that the autoinhibitory domain, and possibly the phosphorylation sites residing within this domain, play a critical role in regulating p70s6k activity in the intact kinase through the modulation of Thr229 and Thr389 phosphorylation.
In this study, we utilized phosphorylation site and truncation mutations to elucidate the role of the autoinhibitory domain S/TP sites in regulating mitogen-induced p70s6k activation. Next, we examined the nature of this event as it relates to the ability of the S/TP sites, along with Thr389, to regulate Thr229 phosphorylation in vivo. Finally, by employing PDK1 in vitro, we have determined that the mechanism by which these carboxyl-terminal phosphorylation sites control Thr229 phosphorylation in vivo is synergistic and intrasteric.
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EXPERIMENTAL PROCEDURES |
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Plasmid Construction and Mutagenesis-- Generation of the amino- and carboxyl-terminal truncation mutants of p70s6k, as well as point mutations at phosphorylation sites, was achieved using the Altered Site II Mutagenesis System (Promega), as described previously (15). All constructs were tagged with a myc epitope and placed immediately following the p70s6k or PDK1 initiator ATG codon (15, 22). Phosphorylation site mutants were placed in the appropriate background by BglII-PstI fragment exchanges. All constructs were subcloned into a cytomegalovirus-driven expression vector after being verified by DNA sequencing.
Cell Culture, Transfection, and Metabolic Labeling--
Human
embryonic kidney 293 cells were maintained in Dulbecco's modified
Eagle's medium supplemented with 10% FCS. Twenty-four hours before
transfection, cells were seeded at a density of 106 cells
per 10-cm-diameter plate. The cells were then transiently transfected,
using a modified calcium phosphate procedure (28, 29), with 1-5 µg
of the appropriate construct. Total DNA transfected was kept at 10 µg
for all experiments using the empty vector. After 12 h, the
transfected cells were placed into serum-free Dulbecco's modified
Eagle's medium for an additional 24 h. Metabolic labeling was
carried out in phosphate-free Dulbecco's modified Eagle's medium with
1-2 mCi 32Pi per 5 ml of medium followed by
extraction either with or without serum stimulation as described
previously (30). Extracts were centrifuged at 12,000 × g for 5 min at 4 °C, and the supernatants were quickly
frozen in liquid N2 and stored at 70 °C.
Immunoblotting, Kinase Assays, and Two-dimensional Phosphopeptide Mapping-- Protein concentrations were measured using the Bio-Rad D/C protein assay. For Western blot analysis, 20 µg of extract protein were resolved by SDS-polyacrylamide gel electrophoresis before transfer onto an Immobilon P membrane (Millipore). Expression of the epitope-tagged proteins was detected by decorating the membrane with the monoclonal 9E10 antibody followed by a secondary rabbit anti-mouse antibody and finally by a fluorescein isothiocyanate-conjugated swine anti-rabbit tertiary antibody. Expression levels were quantified using fluorimetry (Molecular Dynamics) and ImageQuant software (Molecular Dynamics). Activities of the ectopically expressed mutants, immunoprecipitated with the 9E10 antibody, were measured against S6 as a substrate (15). Kinase activities were quantitated using phosphorimagery (Molecular Dynamics) and ImageQuant software. Activities were normalized to the level of expressed kinase and compared with other mutants only when expression levels were similar. Two-dimensional phosphopeptide mapping of 32P-labeled, ectopically expressed p70s6k was performed as described previously (13). The resulting phosphopeptide maps were visualized by phosphorimagery.
In Vitro Phosphorylation--
Myc-tagged p70s6k and
myc-tagged PDK1 were independently transfected in MEK 293 cells as
described above, quiesced, and extracted in Buffer A (50 mM
Tris, pH 7.5, 50 mM NaCl, 10 mM NaF, 10 mM -glycerolphosphate, 10 mM NaPPi, 0.5 mM EGTA, 1 mM DTT, 1 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride, 0.1%
TX-100, and 10 µg/ml leupeptin and aprotinin). Expression was
determined by Western blotting as above, and total extracts containing
PDK1 (2.5 µg) and p70s6k constructs (40 µg) were mixed
in Buffer A before co-immunoprecipitation with 9E10 and protein
G-Sepharose (22). The immunoprecipitates were washed twice with Buffer
A, twice with Buffer A containing 500 mM NaCl, and finally
with Buffer B (50 mM Tris, pH 7.5, 10 mM NaCl,
1 mM DTT, 10% glycerol, 1 mM benzamidine, and
0.2 mM phenylmethylsulfonyl fluoride). The washed
immunoprecipitates were incubated in Buffer B containing 10 mM MgCl2 and 10 µCi
[
-32P]ATP (20 µM) for 30 min at
30 °C. 32P-Labeled proteins were resolved by
SDS-polyacrylamide gel electrophoresis and visualized by
autoradiography on a PhosphorImager.
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RESULTS |
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p70s6kN54 Activation Is Rescued by
Acidic Substitutions in the Carboxyl Terminus--
The inability of
the p70s6k amino-terminal truncation mutant,
p70s6k
N54 (Fig.
1), to respond to mitogens can be largely
rescued by removing the carboxyl terminus (12, 13, 25). To test whether mitogen-induced phosphorylation of the four autoinhibitory
S/TP sites could mimic removal of the carboxyl terminus and
rescue activity, acidic amino acids were substituted for these residues in p70s6k
N54, and the activity of the newly
generated variant,
p70s6k
N54·D3E (Fig. 1), was
measured following transient expression in 293 cells. In contrast to
the double truncation mutant (12, 13, 25),
p70s6k
N54·D3E displayed high
basal activity (Fig. 2), resembling the elevated basal activity previously reported for the same acidic amino
acid substitutions placed in wild-type p70s6k (15, 19).
Serum stimulation increased
p70s6k
N54·D3E activity to
about 50% of the value obtained for the double truncation mutant and
about 10-fold over that detected for the parent,
p70s6k
N54 (Fig. 2). Because the basal
activity of p70s6kD3E can be further augmented
by placing an acidic residue at Thr389 (13, 15), the same
mutation was placed in
p70s6k
N54·D3E
(
N54·D3E·T389E), which further
increased both basal and serum-stimulated activities (Fig. 2).
Together, the results demonstrate that substitution of acidic amino
acids for phosphorylation sites residing in the carboxyl terminus of
p70s6k largely rescues the activity of the amino-terminal
truncation mutant.
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Deregulation of Thr229 Phosphorylation--
Removal of
the amino and carboxyl termini increases basal Thr229
phosphorylation, whereas removal of the amino terminus alone suppresses
Thr229 phosphorylation (13). These findings suggest that
access of the Thr229 kinase is restricted by the
p70s6k carboxyl terminus, possibly through the
phosphorylation state of the S/TP sites in the
autoinhibitory domain. To examine this possibility, Thr229
phosphorylation and kinase activity were first determined for p70s6kC104. In quiescent cells,
p70s6k
C104 had high levels of
Thr229 phosphorylation but undetectable Thr389
phosphorylation (Fig. 3B),
consistent with results obtained for the double truncation mutant (13).
Despite the high levels of Thr229 phosphorylation, this
variant has low basal kinase activity (Fig. 3A). Upon serum
stimulation, Thr229 phosphorylation increased approximately
2-fold, whereas Thr389 phosphorylation was greatly enhanced
(Fig. 3C), correlating with increased kinase activity (Fig.
3A). Therefore, truncation of the carboxyl terminus alone is
sufficient to disrupt the regulation of Thr229
phosphorylation in resting cells. To determine whether acidic mutations
of the four S/TP sites in the autoinhibitory domain could
mimic this effect, a variant harboring these mutations, p70s6kD3E, was transiently expressed in 293 cells. In quiescent cells, this mutant displayed elevated kinase
activity, which could be further stimulated with serum (Fig.
4A). However, in contrast to
the p70s6k
C104 truncation mutant, basal
Thr229 phosphorylation in p70s6kD3E
was very low, although it could be stimulated with serum (Fig. 4,
compare B and C). It should be noted that
stronger exposures of the chromatogram depicted in Fig. 4B
showed low levels of Thr389 phosphorylation (data not
shown), reflecting the elevated basal kinase activity and rapamycin
sensitivity of this construct (13). Thus, the acidic mutations alone
are insufficient to increase basal Thr229 phosphorylation,
indicating that another element is necessary for regulating
Thr229 phosphorylation.
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Regulation of Thr229 Phosphorylation by Thr389 Phosphorylation-- An acidic residue substituted for Thr389 potentiates the ability of the S/TP mutations to rescue the p70s6k amino-terminal truncation mutant (Fig. 2), and activation of p70s6kD3E is paralleled by increased Thr389 phosphorylation (Fig. 4C). These findings suggest that Thr389 phosphorylation may be the additional element required to bring about Thr229 phosphorylation. To test this possibility, phosphopeptide analyses of p70s6k were compared with those from p70s6k variants harboring acidic mutations at Thr389 and in the S/TP sites. Phosphopeptide analysis of wild-type p70s6k from quiescent cells revealed low levels of Thr229 phosphorylation, which were dramatically increased by serum stimulation (Fig. 5, compare B and C), as was activity (Fig. 5A). Substitution of a glutamate for Thr389 in the wild-type p70s6k background raised basal kinase activity levels (Fig. 5A) and Thr229 phosphorylation (Fig. 5, compare B and D) approximately 2-fold over that of the wild-type enzyme. The corresponding mutation in the p70s6kD3E variant led to a dramatic increase in basal Thr229 phosphorylation, reaching a value equivalent to the serum-stimulated wild-type kinase (Fig. 5, compare C with E). As previously shown (15), both constructs were further activated by serum (Fig. 5A). In contrast, substitution of an alanine for Thr389 in either p70s6k or p70s6kD3E completely abolished kinase activity (Fig. 5A and Ref. 15) and Thr229 phosphorylation in response to serum (Fig. 6). These results support the hypothesis that in the wild-type kinase, phosphorylation of the autoinhibitory domain sites functions to up-regulate Thr229 phosphorylation by cooperating with Thr389 phosphorylation.
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Intrasteric Regulation of Thr229 Phosphorylation-- The results suggest that phosphorylation sites at the carboxyl terminus synergistically regulate Thr229 phosphorylation. However, the data do not address whether the observed synergy on Thr229 phosphorylation is through a single ordered intrasteric mechanism or is instead regulated in vivo through the interplay of multiple effector molecules. To obtain insight into this issue, advantage was taken of the recently described Thr229 kinase PDK1 (22, 32) and p70s6k mutants harboring the different acidic amino acid substitutions. When tested in vitro against the wild-type p70s6k (Fig. 7A), PDK1 only poorly phosphorylated the kinase (Fig. 7B) and had no effect on activity (data not shown and Ref. 22). Furthermore, even though p70s6kT389E was a slightly better substrate for PDK1 than p70s6kD3E, the response was only marginally enhanced for either variant over that obtained with wild-type p70s6k (Fig. 7B). This finding supports the hypothesis that neither of these individual sets of mutations is sufficient to allow PDK1 to access Thr229. In contrast, substitution of both sets of acidic mutations resulted in synergistic phosphorylation of p70s6kD3E·E389 by PDK1 (Fig. 7B), consistent with the observed effect of the combined mutations on Thr229 phosphorylation in vivo (Fig. 5E). This effect was abolished when an alanine was substituted for Thr389 in the wild-type p70s6k and the p70s6kD3E variant (Fig. 7B and data not shown). The data support the hypothesis that Thr229 phosphorylation is regulated by the collective efforts of the carboxyl-terminal phosphorylation sites, through an intrasteric mechanism, presumably by modulating a domain in p70s6k that blocks access of PDK1 to the activation loop phosphorylation site.
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DISCUSSION |
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Of the eight known p70s6k phosphorylation sites, the
first to be identified were the S/TP sites in the
carboxyl-terminal autoinhibitory domain of the kinase (14). Although we
reported that neutral and acidic mutations at these sites lower and
raise basal kinase activity, respectively (15, 19), others found little
to no effect of similar mutations (23, 24). This study emphasizes the
importance of the autoinhibitory domain phosphorylation sites in
p70s6k activation. First, acidic amino acid substitutions
at these sites largely rescue the activity of an amino-terminal
truncation mutant, and second, these sites cooperate with an acidic
mutation at Thr389 to synergistically regulate
phosphorylation of the activation loop site, Thr229.
Although the p70s6kC104 mutant is still
regulated by mitogens, indicating that other elements are involved in
p70s6k activation, it does not exclude a role for the
autoinhibitory domain S/TP in the activation of the intact
kinase. Indeed, the synergistic effect conferred by the
S/TP and Thr389 acidic mutations on
Thr229 phosphorylation suggests a possible mechanism for
the sensitive control of Thr229 phosphorylation, which is
dependent on the stoichiometry of S/TP site phosphorylation
in the autoinhibitory domain (see below). This is further supported by
the observation that truncation of the carboxyl terminus largely
deregulates Thr229 phosphorylation (Fig. 3).
The ability of the acidic mutations of the S/TP sites to
rescue p70s6kN54 activity was unexpected,
because deletion of this domain did not significantly affect the
serum-induced phosphorylation of these sites (13). However, this may be
due to the degree of phosphorylation at the autoinhibitory domain
S/TP sites in response to mitogen stimulation. Mitogen
stimulation is hypothesized to first lead to an increase in
S/TP site phosphorylation in the autoinhibitory domain,
which functions together with the amino terminus to facilitate
Thr389 phosphorylation (Fig.
8). In the absence of the amino terminus, the level of mitogen-induced S/TP site phosphorylation may
not be sufficient to promote a net increase in Thr389
phosphorylation and subsequent Thr229 phosphorylation,
attenuating kinase activation. However, substitution of an acidic amino
acid at each of the S/TP sites would raise the overall of
negative charge of this domain and overcome the effect of the
amino-terminal truncation, triggering Thr389
phosphorylation. In support of this model, phosphopeptide maps show
that Thr389 phosphorylation is rescued when acidic
S/TP site mutations are placed in the
p70s6k
N54
background.3 Thus,
Thr389 phosphorylation would act as an intermediary step
between autoinhibitory S/TP and activation loop site
phosphorylation, which would be the final step in mitogen-induced
p70s6k activation (Fig. 8).
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Although a number of candidate autoinhibitory domain S/TP
kinases have been suggested, including cyclin-dependent
kinase-1 and the mitogen-activated kinases
p42mapk/p44mapk (33), their requirement has not
been substantiated to date. Indeed, utilization of interfering mutants
of p21ras and p74raf, as well as deletion
mutants of the platelet-derived growth factor receptor, demonstrated
that p42mapk/p44mapk were not effectors of the
p70s6k signaling pathway (11). It has been shown that
over-expression of a kinase dead p70s6k blocks
Thr229, Thr389, and Ser404
phosphorylation as well as strongly suppressing Ser411 and
Thr421 phosphorylation in the autoinhibitory domain (5,
34), a pattern of inhibition resembling that induced by rapamycin (15). Furthermore, it was found that over-expression of kinase dead or
wild-type p70s6k blocks the same sites of phosphorylation
in the suppressor of protein synthesis initiation factor 4E,
eIF4E-binding protein-1, as does rapamycin (34). These results suggest
that overexpression of p70s6k might sequester a common
upstream kinase that is also responsible for phosphorylating
eIF4E-binding protein-1 (34). Consistent with this hypothesis, the
phosphorylation sites in eIF4E-binding protein-1 also display
S/TP motifs (35), and recently, it was shown that these
sites are phosphorylated in vitro by the mammalian target of
rapamycin (36). However, it is unlikely that mammalian target of
rapamycin is the S/TP kinase for p70s6k,
because rapamycin has no effect on serum-induced S/TP
phosphorylation in the amino-terminal truncation mutant,
p70s6kN54.4
Interest in the identity of the S/TP kinase has been
further increased by the recent observation that Pin1, the conserved
mitotic peptidyl-prolyl isomerase, binds to p70s6k,
apparently through phosphorylated Ser411 (37).
The finding that in the carboxyl-terminal deletion mutant,
p70s6k C104, kinase activity and
Thr389 phosphorylation are tightly regulated suggests the
existence of an additional regulatory element in p70s6k
that is controlled by the phosphorylation of the S/TP and
Thr389 sites. This element would function to modulate
Thr229 phosphorylation and activity. An obvious candidate
for such an element is the linker region, which couples the carboxyl
and catalytic domains of p70s6k (Fig. 1). Previously, it
was noted that many members of the AGC family of protein kinases (21)
contain a site homologous to Thr389, as well as the
conserved motif surrounding this site (15). In a more recent study, it
was also pointed out that this conservation extends through the entire
linker region (18). Within this region we identified a novel site,
Ser371, the phosphorylation of which appears to be critical
for kinase activation (18). The site equivalent to Ser371
has been identified as a major autophosphorylation site in protein kinase C
II (38, 39) and protein kinase C
(40),
Thr641 and Thr638, respectively. In both cases,
there is a proline in the +1 position and a hydrophobic residue in the
2 position, as with Ser371 (18). Furthermore, modeling
studies with protein kinase C have suggested that the conserved linker
region may interact with the amino terminus and that Thr641
is juxtaposed to the active site allowing autophosphorylation by an
intramolecular reaction (41-43). Although Ser371 is not an
autophosphorylation site in p70s6k (18), the modeling
studies suggest that it could be strategically placed to modulate
potential interactions between the amino terminus, the catalytic
domain, and the autoinhibitory region. Indeed, mutation of this site to
either an alanine or an aspartic acid blocked both Thr389
phosphorylation and kinase activation, but surprisingly, it did not
affect Thr229 phosphorylation (18). This may indicate that
mutations at Ser371 disrupt the normal function of the
linker region in regulating Thr389 and Thr229
phosphorylation.
PDK1 has been identified as the common activation loop kinase for Thr229 phosphorylation in p70s6k (22) and Thr308 in protein kinase B (32). In a manner similar to the p70s6k S/TP and Thr389 phosphorylation sites, it has been shown that the access of PDK1 to the activation loop of protein kinase B is controlled by the binding of specific phosphatidylinositides to its PH domain (31, 32). Therefore, the two kinases are linked by a common, constitutively active upstream kinase that appears to catalyze the final event in activation. However, distinct internal regulatory elements control the differential activity of each kinase by regulating the access of PDK1 to the activation loop. This mechanism may provide an economical way for the two kinases to share a common upstream activator without sacrificing their ability to be independently regulated.
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ACKNOWLEDGEMENTS |
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We thank Drs. Heidi Lane and Timothy Myles as well as Almut Dufner for their critical reading of the manuscript. We are also grateful to Mike Rothnie for preparing the figures.
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FOOTNOTES |
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* This work was supported in part by a grant from the Human Frontier Science Program Organization (to G. T.).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.
Recipients of long term postdoctoral fellowships from the Human
Frontier Science Program Organization.
§ Present address: Peter MacCallum Cancer Institute, Locked Bag No. 1, A' Beckett St., Melbourne, Victoria 3000, Australia.
¶ To whom correspondence should be addressed. Tel.: 41-61-697-3012; Fax: 41-61-697-6681.
1 Y. Chen, C. D. Hoemann, G. Thomas, and S. C. Kozma, submitted for publication.
2 The abbreviations used are: PDK1, phosphoinositide-dependent kinase-1; FCS, fetal calf serum.
3 P. B. Dennis and G. Thomas, unpublished results.
4 P. B. Dennis, N. Pullen, R. B. Pearson, S. C. Kozma, and G. Thomas, unpublished data.
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REFERENCES |
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