From the Department of Pharmacology, University of
California at San Diego, La Jolla, California 92093-0640 and the
¶ Department of Pathology, Beth Israel Deaconess Medical Center,
Harvard Medical School, Boston, Massachusetts 02215
Received for publication, February 13, 2001, and in revised form, March 15, 2001
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ABSTRACT |
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The function of protein kinase C family members
depends on two tightly coupled phosphorylation mechanisms:
phosphorylation of the activation loop by the
phosphoinositide-dependent kinase, PDK-1, followed by
autophosphorylation at two positions in the COOH terminus, the turn
motif, and the hydrophobic motif. Here we address the molecular
mechanisms underlying the regulation of protein kinase C The phosphoinositide-dependent kinase
(PDK-1),1 plays a pivotal
role in cellular signaling by regulating the activation state of
diverse protein kinases (1). Such protein kinases contain a segment
near the entrance to the active site that must be phosphorylated in
order to correctly align residues for catalysis. This segment, the
activation loop, typically blocks the active site in the inactive conformation and moves out following phosphorylation (2). Although some
kinases (e.g. protein kinase A) are able to self-activate by
autophosphorylating at the activation loop (3), a large number of
kinases depend on an upstream activation loop kinase for
phosphorylation. PDK-1 was originally discovered as the activation loop
kinase for Akt/protein kinase B (4). Following on the heels of this
discovery was the finding that PDK-1 is also the upstream kinase for
p70 S6 kinase (5, 6) and both atypical (7, 8) and conventional (9)
isoforms of protein kinase C. The list of PDK-1 substrates continues to
grow, placing PDK-1 in the center of a multitude of signaling pathways,
from protein synthesis to cell growth and survival (10, 11).
Phosphorylation by PDK-1 is the first of three ordered phosphorylations
in the maturation of protein kinase C (9). These phosphorylations are
required to stabilize the catalytically competent conformation of the
enzyme and to localize the mature enzyme to the cytosol (12-15). It is
the phosphorylated species of protein kinase C that transduces the
myriad of signals resulting in generation of diacylglycerol (16, 17).
Mature (i.e. phosphorylated) species of conventional ( In addition to the activation loop, mature protein kinase C is
phosphorylated at two conserved positions in the carboxyl terminus (19): the turn motif (Thr641 in protein kinase C A number of studies suggest that PDK-1 interacts with significant
affinity with its kinase substrates. For example, protein kinase C
isozymes and p70 S6 kinases are present in immune complexes of PDK-1
(7-9). Thus, understanding how PDK-1 recognizes its substrates may
provide a first step in understanding how this master kinase regulates
the function of its substrate kinases.
This study addresses how PDK-1 recognizes and regulates protein kinase
C Materials--
All reagents were obtained from general sources
unless otherwise stated. The large T-antigen transformed human
embryonic kidney cells (tsA201) were the generous gift of Dr. Marlene
Hosey (Northwestern University). The cDNA of rat PKC Plasmid Constructs--
A mammalian expression construct
encoding the NH2-terminal Myc-tagged PDK-1 in pcDNA3
has been described previously (7). The cDNAs encoding the wild-type
and several phosphorylation site mutants of PKC Cell Transfection--
TsA201 cells were maintained in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
containing 10% fetal bovine serum and 1% penicillin/streptomycin at
37 °C in 5% CO2. Transient transfection of tsA201 cells
was carried out using Effectene transfection reagents (Qiagen). The
specific transfection procedures were performed according to the
protocol suggested by Qiagen. Combinations of the different expression
plasmids were used as stated, and 1 µg of each DNA construct was
generally included in the transfection.
GST Fusion Protein Pull-down Assay--
To examine the
interaction between PDK-1 and different domains of protein kinase C
Immunoprecipitation--
TsA201 cells were transiently
transfected with different combinations of protein kinase C We have previously reported that PDK-1 and protein kinase C II by
PDK-1. Co-immunoprecipitation studies reveal that PDK-1 associates
preferentially with its substrate, unphosphorylated protein kinase C,
by a direct mechanism. The exposed COOH terminus of protein kinase C
provides the primary interaction site for PDK-1, with co-expression of
constructs of the carboxyl terminus effectively disrupting the
interaction in vivo. Disruption of this interaction
promotes the autophosphorylation of protein kinase C, suggesting that
the binding of PDK-1 to the carboxyl terminus protects it from
autophosphorylation. Studies with constructs of the COOH terminus
reveal that the intrinsic affinity of PDK-1 for phosphorylated COOH
terminus is over an order of magnitude greater than that for
unphosphorylated COOH terminus, contrasting with the finding that PDK-1
does not bind phosphorylated protein kinase C effectively. However,
effective binding of the phosphorylated species can be induced by the
activated conformation of protein kinase C. This suggests that the
carboxyl terminus becomes masked following autophosphorylation, a
process that can be reversed by the conformational changes accompanying activation. Our data suggest a model in which PDK-1 provides two points
of regulation of protein kinase C: 1) phosphorylation of the activation
loop, which is regulated by the intrinsic activity of PDK-1, and 2)
phosphorylation of the carboxyl terminus, which is regulated by the
release of PDK-1 to allow autophosphorylation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
I,
II, and
) and novel isozymes (
,
,
, and
)
typically localize to the cytosol but translocate to the membrane upon
generation of diacylglycerol. The membrane interaction is mediated by
two membrane-targeting modules, the C1 domain which binds
diacylglycerol and phosphatidylserine, and the C2 domain which, in
conventional isozymes, binds anionic phospholipids in a
Ca2+-dependent manner (18). Engagement of these
domains on the membrane provides the energy to release an
autoinhibitory pseudosubstrate sequence from the substrate-binding
cavity, which results in activation of the enzyme. Extensive
biochemical, biophysical, and cell biological studies over the past two
decades have led to a strong understanding of how mature protein kinase
C is regulated allosterically by lipid second messengers. However, how
the maturation of protein kinase C is regulated remains to be resolved.
II) so
named because it corresponds to a phosphorylation site in protein
kinase A localized at the apex of a turn, and the hydrophobic motif
(Ser660 in protein kinase C
II) which comprises a Ser
flanked by bulky hydrophobic residues. The phosphorylation at the
activation loop that is catalyzed by PDK-1 triggers the intramolecular
autophosphorylation of these two positions (20). Because the first step
mediated by PDK-1 is the rate-limiting step, understanding how it is
regulated is key to understanding the cellular controls of protein
kinase C function.
II in vivo. By examining the ability of PDK-1 to complex with various constructs of protein kinase C and its isolated domains in vivo, we show that PDK-1 interacts primarily with
determinants residing in the COOH-terminal hydrophobic phosphorylation
motif of protein kinase C. The binding of PDK-1 inhibits the
intramolecular autophosphorylation required for the maturation of
protein kinase C, with release of PDK-1 exposing the COOH terminus for
autophosphorylation. Thus, PDK-1 regulates both phosphorylation
switches of protein kinase C: the activation loop by direct
phosphorylation and the COOH-terminal sites by release from protein
kinase C.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
II was a
gift of Dr. Daniel E. Koshland, Jr. (University of California,
Berkeley). A polyclonal antibody against the COOH terminus of PKC
II
was purchased from Santa Cruz Biotechnology. A phospho-specific
antibody (P500) that specifically recognizes the phosphorylated
activation loop of protein kinase C isozymes was generated and
characterized as described previously (9, 21). A phospho-specific
antibody (labeled P660; referred to as Pan-phosphorylated PKC by
vendor) against the phosphorylated Ser660 in the COOH
terminus of PKC
II was obtained from New England Biolabs.
II, including
PKC
II-T447A/T448A/T500A(T500A),2
PKC
II-T634A/T638A/T641A
(T641A),3 PKC
II-S660A,
PKC
II-S660E, PKC
II-T500E/T641E/S660E (E3), and PKC
II-F656A/F659A (FA2), were subcloned into the pcDNA3 vector for expression in mammalian cells (9, 14, 15, 20, 22). The C1 domain
(residues 1-156), the C2 domain (residues 157-296), the catalytic
domain (termed CD, resides 296-673), and the COOH terminus (termed CT,
residues 628-673) of protein kinase C
II were expressed as
glutathione S-transferase (GST) fusion proteins in mammalian
cells following PCR amplification of the relevant sequences using
pcDNA3PKC
II as the template. Specifically, the primers used for
the PCR amplification introduced a BamHI site and a
NotI site at the 5' and 3' ends, respectively. The PCR
products were subcloned into the pEBG vector digested with
BamHI and NotI. For expression of the PKC
II
COOH terminus with mutations S660A, S660E, T641E/S660E (E2), or FA2,
the same PCR amplification procedures were performed except that the
templates containing the corresponding mutations were used. The
resulting expression constructs encoded the following fusion proteins
including GST-CT, GST-CT/S660A, GST-CT/S660E, GST-CT/E2, GST-CT/FA2,
GST-C1, GST-C2, and GST-CD. As a control, the COOH terminus of PRK2
(residues 908-984; previously termed PIF (23)) was PCR amplified from
PRK2 (generous gift of Margaret Chou, University of Pennsylvania) and
subcloned into the pEBG vector for expression as a GST fusion protein
in mammalian cells.
II in vivo, tsA201 cells were transiently transfected
with Myc-tagged PDK-1 and the wild-type or the mutant GST-CTs.
Approximately 40 h post-transfection, the transfected cells were
lysed in buffer A (50 mM Na2HPO4, 1 mM sodium pyrophosphate, 20 mM NaF, 2 mM EDTA, 2 mM EGTA, 1% Triton X-100, 1 mM dithiothreitol, 200 µM benzamidine, 40 µg ml
1 leupeptin, 300 µM
phenylmethylsulfonyl fluoride, and 300 nM okadaic acid).
The lysate was cleared by centrifugation at 13,000 rpm, 5 min, 22 °C
and the resulting supernatant is referred to as the detergent-solubilized cell lysate. Ten percent of the total cell lysate
was kept in SDS sample buffer for further analysis, and the remaining
cell lysate was incubated with glutathione-Sepharose at 4 °C for
overnight. After washing twice in buffer A and twice in buffer B
(buffer A plus 300 mM NaCl), the glutathione-Sepharose bound proteins were analyzed using SDS-PAGE and immunoblotting.
II and
Myc-PDK-1. Approximately 40 h post-transfection, the cells were
lysed in buffer A. Ten percent of the total detergent-solubilized cell
lysates was quenched in SDS sample buffer for further analysis, and the
remaining detergent-solubilized cell lysate was incubated with an
anti-Myc monoclonal antibody and protein A/G-agarose (Santa Cruz
Biotechnology) at 4 °C overnight. To examine the direct interaction
between PDK-1 and protein kinase C
II, Sf21 insect cells were
infected with baculovirus encoding 6His-tagged PDK-1 or protein
kinase C
II. The 6His-tagged PDK-1 and protein kinase C
II were purified as described previously (24). The catalytic domain
of protein kinase C was generated by incubation of pure protein kinase
C
II (1.5 µg ml
1) with trypsin (1.2 units
ml
1) for 10 min at 30 °C, in the presence of 1 mM Ca2+, as previously described (25). The
purified proteins were combined in buffer A and incubated with an
anti-PDK-1 antibody (Upstate Biotechnology) in the presence or absence
of protamine sulfate (50 µg ml
1) at 4 °C for
overnight. The immunoprecipitates were washed twice in buffer A and
twice in buffer B. The proteins in the immunoprecipitates were
separated using SDS-PAGE and analyzed using immunoblotting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
II
associate in vivo, as assessed by co-immunoprecipitation
studies in HEK cells co-transfected with the cDNA for each kinase
(9). To further examine the mechanism of this interaction and how it is
regulated, we identified the determinants of protein kinase C that
mediate the interactions and addressed how the interaction is affected
by the phosphorylation state and activation state of protein kinase C. Fig. 1 shows the constructs of PDK-1 and protein kinase C used in this study.
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Fig. 1.
Schematic representation of PDK-1 and protein
kinase C II constructs used in this
study. A, wild-type PDK-1, containing an
NH2-terminal kinase domain and a COOH-terminal PH domain,
was NH2 terminally tagged with a Myc epitope. B,
primary structure of wild-type PKC
II showing the C1 and C2 domains
in the NH2-terminal regulatory moiety and the kinase domain
in the COOH-terminal moiety. Thr500 is in the activation
loop of PKC
II and phosphorylated by PDK-1. Also indicated are the
COOH-terminal constructs (CT) of protein kinase C
II comprising
residues 628 to 673. The autophosphorylation sites, Thr641
and Ser660, in the COOH terminus of PKC
II are
indicated, as are the two hydrophobic residues Phe659 and
Phe661 flanking Ser660. The numbers
at the COOH-terminal end of each construct indicate the last amino acid
residue of the protein.
PDK-1 Preferentially Binds Unphosphorylated Protein Kinase
C--
Myc-tagged PDK-1 and wild-type protein kinase C II were
co-expressed in tsA201 cells and the detergent-soluble supernatant (containing ~90% of the expressed constructs) was subjected to immunoprecipitation using the anti-Myc antibody. Fig.
2, lane 1, shows that a
significant fraction of the protein kinase C co-immunoprecipiated with
PDK-1, consistent with our previous report (9). Two species of protein
kinase C were apparent in the immunoprecipitates (panel A):
a slower migrating band (indicated by double asterisk) which represents fully phosphorylated protein kinase C (see Ref. 19) and was
labeled by the phosphoactivation loop antibody (P500, not shown), and a
faster migrating species (labeled with a dash) which
represents unphosphorylated protein kinase C and was not labeled by the
P500 antibody (not shown). The dephosphorylated species in the immune
complex was significantly enriched relative to the amount in the total
cell lysate: densitometric analysis of six independent experiments
revealed a 2.0 ± 0.7-fold enrichment of the dephosphorylated
species relative to the phosphorylated species in the immune complexes
compared with the ratio of these species in the total lysate. Thus,
PDK-1 selectively bound unphosphorylated protein kinase C.
|
To probe whether the interaction of protein kinase C with PDK-1 was
direct or mediated by a scaffold protein, we examined the interaction
of pure PDK-1 with pure protein kinase C. Both proteins were expressed
in baculovirus-infected insect cells and purified to apparent
homogeneity. Fig. 2, lane 2, shows that protein kinase C
II was present in immune complexes with PDK-1 (panel A).
As observed using cell lysates, the immune complex was enriched in the
unphosphorylated species (dash) of protein kinase C: this species was barely apparent in the starting material (panel
C) but readily apparent in the immune complex (panel
A). The efficiency of immunoprecipitation of protein kinase C by
PDK-1 in cell lysates or in vivo was comparable, suggesting
that direct interaction of the two proteins was the primary mechanism
for the interaction observed in vivo.
Mutations in the Carboxyl-terminal Hydrophobic Motif of Protein
Kinase CII Disrupt the PDK-1/PKC
II Interaction in Vivo--
The
results described above indicate that PDK-1 preferentially associates
with its substrate, unphosphorylated protein kinase C
II, compared
with the mature, fully phosphorylated protein kinase C
II. This
latter species is phosphorylated at three positions: Thr500
on the activation loop, and Thr641 and Ser660
on the carboxyl terminus. To test whether the phosphorylation at these
positions modulates association of protein kinase C with PDK-1, we
examined the ability of constructs mutated at each position for the
ability to form an immune complex with PDK-1. The detergent-soluble cell lysates were immunoprecipitated with the anti-Myc antibody to
precipitate PDK-1 and the immunoprecipitates probed for associated protein kinase C. Fig. 3C
shows that the amount of PDK-1 was similar among all the
immunoprecipitates.
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Fig. 3B shows a Western blot of the wild-type and
phosphorylation site mutants of protein kinase C II expressed in
tsA201 cells. Wild-type protein kinase C (lane 1) migrated
as a doublet, similar to the results in Fig. 2. In contrast, both the
T500A2 and T6413 mutants co-migrated with
unphosphorylated protein kinase C (Fig. 3B, position
indicated by dash). We have previously shown that mutation
of Thr500 to Ala or Thr641 to Ala abolishes
kinase activity and thus results in expression of quantitatively
dephosphorylated protein kinase C (15, 22, 26).4 Mutation of
Ser660 to Ala resulted in the appearance of a major
intermediate migrating band (lane 4, position marked with
asterisk) and a minor slower migrating band (lane
4, position indicated by dash). This is consistent with
previous studies showing that mutation of Ser660 to Ala has
only modest effects on the activity of protein kinase C so that the
majority of the S660A that partitions in the detergent-soluble fraction
is phosphorylated at Thr500 and Thr641: this
phosphorylated species migrates at an intermediate mobility. Replacement of Ser660 with Glu results in unimpaired
phosphorylation of protein kinase C so that the S660E mutant is a good
mimic of wild-type enzyme and co-migrates with wild-type enzyme (Fig.
3B, lane 5).
Fig. 3A shows that the T500A and T641A constructs were effectively co-immunoprecipitated with PDK-1 (lanes 2 and 3), but the T500A mutant has slightly reduced efficiency compared with wild-type enzyme. Quantitation of several independent experiments revealed that 0.5 ± 0.1 (n = 4) and 0.8 ± 0.1 (n = 3) times as much T500A and T641A, respectively, were present in immune complexes compared with wild-type unphosphorylated protein kinase C (lower band marked by dash). The slightly decreased recognition of protein with Ala at position 500 suggests that the hydroxyl at the phosphoacceptor position may influence the recognition of protein kinase C by PDK-1.
To test whether negative charge at position 660 regulated the
interaction of mature protein kinase C with PDK-1, we examined the
ability of PDK-1 to complex with the S660A and S660E mutants in
vivo. Fig. 3 reveals the presence of intermediate migrating species (marked by one asterisk), the S660A mutant, in the
immune complexes with PDK-1 (Fig. 3A, lane 4). This species
represents protein kinase C phosphorylated at the activation loop and
Thr641. Quantitative analysis of three independent
experiments revealed the fraction of S660A co-immunoprecipitated from
the lysate with PDK-1 was 0.4 ± 0.2-fold lower than the fraction
of wild-type protein kinase C that was co-immunoprecipitated. These
data suggest that replacement of Ser660 with Ala results in
a 2.5-fold reduction in the affinity of mature protein kinase C for
PDK-1. PDK-1 was also able to effectively co-immunoprecipitate the
S660E mutant (Fig. 3A, lane 5). As described previously, the
S660E mutant expressed primarily as the slowest migrating species
co-migrating with the fully phosphorylated wild-type protein kinase C
II (Fig. 3B, lane 5, double asterisk). The fraction of
S660E that co-immunoprecipitated with PDK-1 was similar to the fraction
of fully phosphorylated protein kinase C
II that complexed with
PDK-1 (Fig. 3A, compare lanes 1 and 5;
analysis of three independent experiments revealed that the ratio of
S660E to wild-type protein kinase C in immune complexes was 1.0 ± 0.3). Thus, mature protein kinase C with a Glu or phosphoserine at
position 660 interacted similarly with PDK-1, whereas mature protein
kinase C with a Ala at position 660 interacted less strongly with PDK-1 than protein with phospho-Ser at that position.
Interaction of Different Domains of Protein Kinase C CII with
PDK-1 in Vivo--
In vivo co-precipitation assays reveal
that PDK-1 preferentially recognizes the unphosphorylated conformation
of protein kinase C. To elucidate the contribution of individual
domains in this recognition, we tested the ability of GST fusion
proteins encoding the C1, C2, and catalytic domains of protein kinase C
II to associate with PDK-1 or to disrupt the interaction of PDK-1
with full-length protein kinase C
II in vivo. In
addition, we focused on a series of constructs of the carboxyl-terminal
46 residues to explore the role of the hydrophobic phosphorylation
motif in the PDK-1/protein kinase C interaction. These constructs
included GST-CT, GST-CT/S660A, GST-CT/S660E, GST-CT/E2, and GST-CT/FA2.
We also examined the interaction with PIF, a COOH-terminal construct of
PRK-2 previously shown to bind with high affinity to PDK-1 (23). TsA201
cells were transiently co-transfected with PDK-1 and the GST fusion protein constructs and the interaction with PDK-1 was assessed by GST
pull-down experiments performed using the detergent-solubilized fraction of cells. The total protein expression level of PDK-1 was
comparable in all the transfections (Fig.
4D).
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Fig. 4B reveals that all constructs of the COOH terminus,
including PIF, were expressed in tsA201 cells and effectively
precipitated by glutathione-Sepharose as judged by staining with
anti-GST antibodies. Of these, the GST fusions of the wild-type COOH
terminus and of PIF (Fig. 4A, lanes 2 and 9) were
the most effective at co-precipitating PDK-1. To obtain relatively
balanced signals on the immunoblot, PDK-1 detected in the GST-PIF lane
represented only 30% of the total protein in the glutathione-Sepharose
precipitates. Densitometric analysis of data from three independent
experiments revealed that PDK-1 bound PIF 5-10 times better than the
COOH terminus of protein kinase C II. In contrast to the wild-type
COOH-terminal constructs, neither the S660A, S660E, nor T641E/S660E
constructs of the COOH terminus showed detectable interaction with
PDK-1 (Fig. 4A, lanes 3, 4, and 10). A construct
in which the two Phe flanking Ser660 were mutated to Ala,
GST-F659A/F661A, did not complex with PDK-1 (Fig. 4A, lane
5). A construct of the catalytic domain comprising the kinase core
and the COOH terminus (residues 296-673) was poorly expressed in the
tsA201 cells yet was co-complexed with PDK-1 (Fig. 4B, lanes
8). The GST fusion constructs of the isolated C1 and C2 domains
expressed well in tsA201 cells and GST pull-down experiments revealed
trace binding of PDK-1 to the C1 domain but not to the C2 domain
(lanes 6 and 7). These data suggest that the
kinase domain, and in particular the COOH terminus, provide the primary
determinants in the interaction of protein kinase C with PDK-1, with
some participation of the C1 domain.
Fig. 4B reveals that the COOH-terminal fusion protein migrated as multiple bands on SDS-PAGE (lane 2), suggesting potential modification by phosphorylation. Expression of the GST-carboxyl-terminal construct resulted in the appearance of 4 bands: a major fastest migrating band and three minor slower migrating bands. These 4 bands are labeled 0, 1, 2, and 3 on Fig. 4B. Both the GST-CT/S660A and S660E constructs migrated as 2 bands, with the S660A construct co-migrating with the two lower wild-type bands (bands 0 and 1) and the S660E construct co-migrating with the two upper wild-type bands (bands 1 and 3). In addition, a construct in which both Thr641 and Ser660 were mutated to Glu (GST-CT/E2) migrated as a single band, whose mobility was the same as that of the slower migrating form of the wild-type construct (band 2). This suggests that a fraction of the COOH-terminal construct becomes phosphorylated at position 660 and/or 641 in vivo. Western blot analysis using an antibody that specifically recognizes phosphorylated Ser660 (P660) revealed that the two slowest upper migrating band (bands 2 and 3) of the wild-type COOH terminus are, indeed, phosphorylated on Ser660 (Fig. 4C, lane 2). This antibody did not label the GST-CT/S660A, GST-CT/S660E, or a double mutant, GST-CT/E2 (Fig. 4C, lanes 3, 4, and 10). These data reveal that a minor fraction (typically 10-20%) of the wild-type COOH terminus is phosphorylated on Ser660, and an even smaller fraction on Ser660 and one other position, possibly Thr641. The wild-type GST-CT is effectively phosphorylated in vitro at Ser660 by protein kinase C suggesting that the weak phosphorylation of this construct observed in vivo may be catalyzed by endogenous protein kinase C (data not shown).
Disruption of PDK-1/PKC Interaction by Co-expression of the
COOH-terminal Fusion Proteins of PKC II--
To further test the
hypothesis that the COOH terminus of protein kinase C is a major
determinant in the interaction of PDK-1 with protein kinase C, we
tested the ability of the various constructs of the COOH terminus,
including PIF, to disrupt the interaction of PDK-1 with protein kinase
C
II in vivo. TsA201 cells were co-transfected with PDK-1
and protein kinase C, and either GST or the GST-COOH-terminal
constructs. PDK-1 was then immunoprecipitated from the
detergent-solubilized fraction and the amount of co-precipitated protein kinase C was determined by Western blot analysis. Fig. 5A shows that protein kinase C
(panel III) was expressed to comparable levels in all
transfections, and the amount of PDK-1 in all the immunoprecipitates
was similar (panel II). Furthermore, the protein kinase C in
the detergent-solubilized fraction migrated as a major slower migrating
species under all transfection conditions (panel III; double
asterisk), revealing fully phosphorylated mature form. Immunoprecipitation of PDK-1 from cells co-transfected with protein kinase C and GST resulted in significant co-immunoprecipitation of
protein kinase C (panel I, lane 1). In contrast,
co-expression of GST-CT reduced the amount of protein kinase C
II
complexed with PDK-1 to barely detectable levels (panel I, lane
2). Similarly, co-expression of PIF caused a marked reduction in
the amount of protein kinase C associated with PDK-1 (panel I,
lane 6). In contrast, the GST-CT/S660A, GST-CT/S660E, and
GST-CT/FA2 constructs had no significant effect on the PDK-1/protein
kinase C interaction (panel I, lanes 3-5).
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Given the unexpected finding that the wild-type COOH-terminal fragment was extremely effective in disrupting the interaction of PDK-1 with protein kinase C in vivo, but that neither the S660A nor S660E constructs interfered with the interaction, we hypothesized that the relevant species involved in disrupting the interaction was the phosphorylated COOH-terminal fragment. To test this, we asked whether the phosphorylated wild-type COOH-terminal construct was selectively co-immunoprecipitated with PDK-1 in the experiments described in Fig. 5A. Fig. 5B shows that only two constructs were present in immune complexes of PDK-1 as detected by the anti-GST antibody: the wild-type COOH terminus and PIF (panel I, lanes 2 and 6). Importantly, the species of COOH-terminal construct in the immune complex was phosphorylated at position Ser660 as judged by labeling with the P660 antibody and its co-migration with the slower migrating species of the COOH-terminal construct (Fig. 5B, panel II). The faster migrating species were not present, revealing that phosphorylation of Ser660 rendered the COOH terminus effective in disrupting the interaction of PDK-1 with protein kinase C. The full-length protein kinase C and the total COOH-terminal fragment expressed to comparable levels in cell lysates (data not shown), with the fraction of phosphorylated COOH-terminal construct representing only about 10% of the total construct (see Figs. 4B and 5B). Thus, the ratio of protein kinase C to phosphorylated COOH-terminal construct was about 10:1. The ability of the Ser660-phosphorylated construct to almost quantitatively disrupt the PDK-1/protein kinase C interaction, despite the 10-fold excess of protein kinase C, suggests that it has a significantly higher affinity for PDK-1 than the full-length protein kinase C.
To further test the hypothesis that Ser660 phosphorylated
GST-CT binds PDK-1 with higher affinity compared with the
unphosphorylated species, we performed a co-immunoprecipitation
experiment using tsA201 cells co-expressing PDK-1 and GST-CT (Fig.
6). PDK-1 was immunoprecipitated using
anti-Myc antibodies from the detergent-solubilized cell lysates and the
phosphorylation state of the COOH-terminal construct associated with
the immunoprecipitate was examined. The expression of GST-CT and PDK-1
was detected in the cell lysate (Fig. 6, lane 2). Consistent
with the results shown above, GST-CT expressed as multiple bands in the
transfected cells. However, only the species of GST-CT phosphorylated
on Ser660 was co-immunoprecipitated with PDK-1 as assessed
by its labeling with the P660 antibody (Fig. 6B) and
retarded electrophoretic mobility (Fig. 6A, lane 1). These
data further confirm that PDK-1 selectively bound the
Ser660-phosphorylated COOH-terminal fusion protein of PKC
II.
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Accessibility of the COOH terminus of Protein Kinase C Is
Conformationally Sensitive--
The above data reveal that PDK-1
selectively binds unphosphorylated protein kinase C compared with
phosphorylated protein kinase C (Fig. 2). Yet, studies with the
isolated COOH terminus show that the intrinsic affinity of PDK-1 is
much greater for COOH-terminal constructs that are phosphorylated at
Ser660 (Figs. 5 and 6). Because protein kinase C undergoes
global conformational changes following phosphorylation, we addressed
the possibility that the COOH terminus of protein kinase C becomes
masked following the maturation of the enzyme. We have previously shown
that the pseudosubstrate of protein kinase C is exposed in the
unphosphorylated conformation of protein kinase C, masked in the
phosphorylated but inactive conformation, and then exposed again
following activation (24, 26). This led us to explore whether inducing
the "open" conformation of activated protein kinase C might expose
the COOH terminus and promote more efficient binding of PDK-1. Protein kinase C was activated by incubation with protamine sulfate, a cofactor-independent substrate that promotes the activating
conformation of protein kinase C (27). Fig.
7A shows that protamine
sulfate resulted in a significant increase (~3-fold) in the amount of purified protein kinase C II present in immune complexes with purified PDK-1. This effect was specific for full-length protein kinase
C: the proteolytically generated kinase domain of protein kinase C
interacted equally well with PDK-1 in the presence or absence of
protamine sulfate (Fig. 7B). A similar increase in binding
of protein kinase C to PDK-1 was observed following activation by PMA
(data not shown). These data suggest that the open conformation induced by binding protamine increases the binding of PDK-1, presumably by unmasking the COOH-terminal docking site. Since the kinase domain
alone is not masked by the pseudosubstrate or interactions with other
determinants in the regulatory domain, protamine would not be expected
to alter the conformation or accessibility of the COOH terminus of the
kinase domain.
|
Co-expression of the Carboxyl-terminal Fragment of Protein Kinase C
Increased Autophosphorylation of the Full-length Protein Kinase
C--
To examine the effect of co-expression of the COOH terminus of
protein kinase C on the autophosphorylation of the full-length protein
kinase C, tsA201 cells were transfected with PKC II and PDK-1 in the
presence or absence of the COOH-terminal fragment. In the experiment
shown in Fig. 8, protein kinase C
II
expressed as a doublet in cell lysates, with the faster migrating
species (marked by a dash) representing unphosphorylated
protein kinase C and the slower migrating species (marked by
double asterisk) representing fully phosphorylated enzyme
(Fig. 8B). Consistent with the data in Fig. 5, expression of
the COOH-terminal fragment abolished the stable interaction of PDK-1
with protein kinase C (Fig. 8A, lane 3). When the
COOH-terminal fragment was co-expressed, the species of protein kinase
C present in the lysate was shifted exclusively to the slower migrating
form (Fig. 8B, compare lane 3 with lanes
1 and 2). Thus, the COOH-terminal fragment promoted the
maturation of protein kinase C. These data suggest that release of
PDK-1 from protein kinase C upon expression of the COOH terminus promotes the autophosphorylation reactions which cause the mobility shift.
|
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DISCUSSION |
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Co-immunoprecipitation studies reveal that PDK-1 associates preferentially with its substrate, unphosphorylated protein kinase C. Co-expression of specific domains of protein kinase C reveals that the primary determinants that regulate the interaction of protein kinase C with PDK-1 lie in the COOH terminus of protein kinase C, although weak contact with the C1 domain in the regulatory moiety is present. Importantly, the COOH terminus of protein kinase C provides a major determinant for binding PDK-1, and constructs of this domain displace PDK-1 from unphosphorylated protein kinase C, accelerating its maturation. These results show two points of regulation of protein kinase C by PDK-1: by directly phosphorylating the activation loop, and by indirectly controlling the autophosphorylation of the COOH terminus.
PDK-1 Preferentially Binds Unphosphorylated Protein Kinase C-- Analysis of the interaction of PDK-1 with wild-type protein kinase C or phosphorylation site mutants reveals that PDK-1 preferentially binds unphosphorylated protein kinase C compared with phosphorylated, but inactive, enzyme. Studies with pure proteins reveal that the interaction is direct. These results suggest that either the conformation of unphosphorylated protein kinase C is required for optimal interaction with PDK-1, or that phosphate at specific positions sterically or electrostatically disrupts the interaction. The decreased binding of phosphorylated enzyme is unlikely to result from phosphate on the activation loop: the unphosphorylated species of T500E mutant (i.e. negative charge at the activation loop resulting from mutation but no phosphorylation of COOH-terminal sites) interacted to comparable levels with PDK-1 as unphosphorylated wild-type enzyme (data not shown). This led us to explore the role of phosphate at the carboxyl terminus, and of conformation, in regulating the interaction of protein kinase C with PDK-1.
Involvement of the Carboxyl Terminus in Regulating the Interaction of PDK-1 with Protein Kinase C-- Studies with GST fusion constructs have revealed that the COOH terminus is a critical determinant in the interaction of protein kinase C with PDK-1. Our data reveal two seemingly contradictory findings: unphosphorylated PKC is much more effectively co-immunoprecipitated with PDK-1 compared with phosphorylated protein kinase C, yet constructs of the isolated carboxyl terminus are much more effective at disrupting the protein kinase C/PDK-1 interaction when phosphorylated. One explanation consistent with both of these findings is that phosphorylation of the carboxyl terminus of protein kinase C causes it to alter its conformation in such a way as to mask it. Consistent with this, inducing the activating open conformation of protein kinase C greatly increases the affinity of phosphorylated protein kinase C for PDK-1. The hypothesis that the COOH terminus is masked in the inactive conformation of mature protein kinase C is also supported by the finding that the phosphatase sensitivity of protein kinase C increases by 2 orders of magnitude upon adopting the active conformation (28). Thus, the interaction of PDK-1 with protein kinase C is regulated in opposing ways by the phosphorylation state of the COOH terminus (with phosphate promoting tighter binding) and the conformation of the full-length protein (with the inactive conformation inhibiting binding). Consistent with this, the mature species of the S660A construct binds less tightly to PDK-1 than mature wild-type protein kinase, which has a phosphate at position 660.
Release of PDK-1 from Protein Kinase C Promotes Carboxyl-terminal Autophosphorylation-- Carboxyl-terminal constructs bearing negative charge (PIF and phosphorylated carboxyl terminus of protein kinase C)5 not only dramatically reduced the amount of protein kinase C present in the PDK-1 immune complexes, but they also increased the fraction of fully phosphorylated protein kinase C in cells. Thus, these constructs did not abolish the interaction of protein kinase C with PDK-1, because phosphorylation on the activation loop still occurred. Rather, by competing for binding to protein kinase C, these constructs promoted the maturation of protein kinase C. This result suggests that displacement of PDK-1 from protein kinase C accelerates the maturation of protein kinase C by promoting autophosphorylation.
A series of recent reports from Alessi and co-workers (23) have also implicated the hydrophobic phosphorylation motif as an important determinant in the regulation of PDK-1 activity. Using a yeast-two hybrid screen, this group identified a region in the COOH terminus of the protein kinase C-related kinase, PRK-2, that bound PDK-1 and named it PIF for PDK-1 interacting fragment (23). This region encompasses the hydrophobic phosphorylation motif except that Asp replaces the Ser at the potential phosphoacceptor position. Co-transfection studies with GST constructs of PIF revealed that Asp at the phosphoacceptor position and the flanking Phe were critical for efficient co-precipitation of PDK-1. The authors showed that inclusion of PIF in phosphorylation assays of Akt and PDK-1 promoted phosphate incorporation into the Ser473 of the hydrophobic phosphorylation motif of Akt. The interpretation of this result was that the interaction of PDK-1 with PIF causes an unprecedented change in substrate specificity causing PDK-1 to gain "PDK-2" activity and phosphorylate Ser473.
Our data unveil an alternate explanation. The binding of PDK-1 to Akt could mask Ser473 which, like protein kinase C, is regulated by autophosphorylation (29). Addition of PIF would effectively compete with binding because PDK-1 has a much higher affinity for PIF, which has a negative charge at the corresponding phosphoacceptor position of the hydrophobic motif, than it does for the COOH terminus of Akt. Release of PDK-1 would then allow autophosphorylation. In support of this, Alessi and co-workers (30) reported that kinase-inactive constructs of Akt are phosphorylated at the activation loop but not Ser473 in the presence of the PDK-1·PIF complex. This is consistent with our data showing that phosphorylation of Ser473 requires the intrinsic catalytic activity of Akt because this site is modified by autophosphorylation. In addition, the basal phosphorylation of Ser473 is increased in ES cells from PDK-1 knock-outs, as would be expected if PDK-1 is no longer blocking the access of the carboxyl terminus to the active site (31). (Note that, like most kinases, Akt has some basal activity even without activation loop phosphorylation; constructs of Akt with Ala instead of Thr at the activation loop have residual activity (32).) Thus, by analogy with protein kinase C, the PDK-2-like activity observed in the presence of PIF likely arises from the ability of PIF to displace PDK-1 from Akt and expose the hydrophobic motif of Akt to autophosphorylation.
More evidence for the importance of the carboxyl terminus in anchoring
PDK-1 to its substrates derives from a second yeast two-hybrid screen
which identified the COOH terminus of PKA as a sequence recognized by
PDK-1 (30). The COOH terminus of PKA contains the turn motif
phosphorylated in protein kinase C and Akt, but ends at the Phe
directly preceding the Ser at the phosphoacceptor position of the
hydrophobic phosphorylation motif (29). This terminal Phe helps anchor
the phosphorylated COOH terminus on the upper lobe of the kinase core
by binding a hydrophobic pocket on the back of the upper lobe (33).
Residues contained in this pocket are conserved in PDK-1, leading
Alessi and co-workers (30) to suggest that PDK-1, which does not have a
hydrophobic phosphorylation motif, has an unoccupied hydrophobic pocket
which binds PIF and other hydrophobic motifs. Related to this finding,
a recent study has shown that the COOH terminus of protein kinase C,
which, like PRK-2, contains an acidic residue at its hydrophobic
phosphorylation motif, is a docking site for PDK-1 (30). Similarly,
phosphorylation of the hydrophobic site on the 90-kDa ribosomal S6
kinase-2 has recently been shown to provide a docking site for PDK-1
(34). The above reports converge on a key role for the hydrophobic
motif in regulating the interaction of PDK-1 with its substrates.
Model--
Taken together with previous findings, the data in this
contribution lead us to propose the following model for the interaction of PDK-1 with protein kinase C. PDK-1 associates with newly
synthesized, unphosphorylated protein kinase C. This interaction is
mediated primarily through the COOH terminus of protein kinase C, with the hydrophobic phosphorylation motif playing a key role in the tethering of PDK-1. PDK-1 then phosphorylates protein kinase C II on
Thr500 of the activation loop, the first and rate-limiting
step in the processing of protein kinase C by phosphorylation (9) (Fig. 9, left panel). PDK-1 then
dissociates from protein kinase C (Fig. 9, middle panel),
unmasking the carboxyl terminus and making it available for the
intramolecular autophosphorylation of Thr641 and
Ser660 (20). The phosphorylated COOH terminus shifts its
position in such a way as to become masked to PDK-1 (Fig. 9,
bottom panel). Thus, although PDK-1 has intrinsically a much
higher affinity for the isolated COOH terminus when it is
phosphorylated compared with unphosphorylated, its apparent affinity
for the phosphorylated COOH terminus is reduced in the context of the
mature, inactive, protein kinase C. Activation of the mature enzyme
results in conformational changes that once again expose the COOH
terminus for more efficient binding to PDK-1. However, more studies are
required to elucidate the role of protein kinase C activation in
binding to PDK-1 in a cellular context.
|
The release of PDK-1 from protein kinase C is greatly accelerated by
the presence of isolated COOH-terminal fragments that contain negative
charge at the hydrophobic motif such as PIF or the phosphorylated
carboxyl terminus of protein kinase C II. Whether there are
accessible phosphorylated or Glu-containing hydrophobic sequences
in vivo that regulate the interaction of PDK-1 with protein
kinase C remains to be explored. If so, this could provide a regulatory
mechanism for fine-tuning phosphorylation at the COOH terminus of
protein kinase C. One interesting possibility is that the activated
conformation of protein kinase C serves as a sink for PDK-1, since this
conformation has an exposed, phosphorylated COOH terminus that is
highly effective at binding PDK-1. If this is the case, activation of
protein kinase C would be predicted to modulate the processing of newly
synthesized protein kinase C. Curiously, once phosphorylated at the
COOH terminus, phosphorylation of the activation loop has little effect
on either the basal or cofactor stimulated activity of the mature
enzyme in vitro (9). This suggests that the interaction of
activated protein kinase C with PDK-1 is not likely to directly
regulate the catalytic function of protein kinase C. Further study is
required to elucidate whether activation of protein kinase C in
vivo does, indeed, promote PDK-1 binding and, if so, what the
functional consequences of this interaction are.
In summary, our study has identified the COOH terminus as a key
determinant in regulating the interaction of protein kinase C and PDK-1
in vivo. Our data suggest that the carboxyl terminus of
unphosphorylated protein kinase C docks PDK-1, with release of PDK-1
unmasking the carboxyl terminus to allow autophosphorylation. This
release is promoted by phosphorylated versions of the carboxyl terminus, which effectively compete for binding to PDK-1 given the
higher intrinsic affinity of PDK-1 for negative charge at the
hydrophobic motif. This model could account not only for why PIF
promotes phosphorylation of the hydrophobic site of Akt, but also why
in attempts to purify a putative upstream kinase for conventional
protein kinase Cs, protein kinase C (which contains an acidic
residue at its hydrophobic site and so might be expected to displace
PDK-1 from conventional protein kinase Cs) was identified in a protein
complex (35).
The above results suggest two key steps in the regulation of protein
kinase C by PDK-1: 1) phosphorylation of the activation loop which is
regulated by the activity of PDK-1, and 2) autophosphorylation of the
COOH-terminal sites, including the hydrophobic site, which is regulated
by release of PDK-1 from protein kinase C. The tight coupling of
COOH-terminal autophosphorylation to the activation loop
phosphorylation catalyzed by PDK-1 likely accounts for the confusion in
the literature regarding whether PDK-1 or PDK-1-like (PDK-2) kinases
phosphorylates this site (4, 23, 36).
![]() |
FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants P01 DK54441 (to A. C. N.) and CA75134 (to A. 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.
§ Supported in part by a Leukemia Research Foundation postdoctoral fellowship.
To whom correspondence should be addressed. Tel.:
858-534-4527; Fax: 858-534-6020; E-mail: anewton@ucsd.edu.
Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M101357200
2 All three potential phosphorylation sites around the PDK-1 site were mutated to prevent compensating phosphorylations resulting from mutation of Thr500 to Ala.
3 Surrounding phosphorylation sites were mutated to prevent compensating phosphorylations that occur when Thr641 is mutated to Ala (15).
4 The T641A mutant is a substrate for PDK-1, however, it is highly phosphatase sensitive and not phosphorylated in vivo under our cell culture conditions (15).
5 Although PIF contains an acidic residue (Asp) at the phosphoacceptor position in the hydrophobic motif, COOH-terminal constructs of protein kinase C with Ser660 mutated to Glu were much less effective than PIF in binding PDK-1. Effective binding required phosphate at position 660. Note that PIF contains an additional acidic residue at the P-2 position, so the double negative charge of phosphate may be required to mimic the PIF sequence.
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ABBREVIATIONS |
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The abbreviations used are: PDK-1, phosphoinositide-dependent kinase 1; PAGE, polyacrylamide gel electrophoresis; PKC, protein kinase C; PCR, polymerase chain reaction; GST, glutathione S-transferase.
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