From the Department of Pharmacology and
¶ Biochemistry, The University of Health Science Center, San
Antonio, Texas 78284 and § CLONTECH
Laboratories, Inc., Palo Alto, California 94303
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ABSTRACT |
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Phosphoinositide-dependent protein
kinase-1 (PDK1) is a recently identified serine/threonine kinase that
phosphorylates and activates Akt and p70S6K, two downstream
kinases of phosphatidylinositol 3-kinase. To further study the
potential role of PDK1, we have screened a mouse liver cDNA
library and identified a cDNA encoding the enzyme. The
predicted mouse PDK1 (mPDK1) protein contained 559 amino acids and a
COOH-terminal pleckstrin homology domain. A 7-kilobase mPDK1 mRNA
was broadly expressed in mouse tissues and in embryonic cells. In the
testis, a high level expression of a tissue-specific 2-kilobase transcript was also detected. Anti-mPDK1 antibody recognized multiple proteins in mouse tissues with molecular masses ranging from 60 to 180 kDa. mPDK1 phosphorylated the conserved threonine residue (Thr402) in the activation loop of protein kinase
C- Activation of Akt (also called protein kinase B or PKB) by growth
factors has been shown to be necessary for various cellular processes
including cell growth, differentiation, metabolism, and apoptosis
(1-7). However, the physiological roles of the enzyme and exact
mechanisms by which the enzyme is regulated in the signaling processes
remain elusive. Although some evidence suggests that Akt may be
activated by products of phosphatidylinositol (PI)
3-kinase1 such as
phosphotidylinositol 3, 4-diphosphate (8, 9), other studies have shown
that the activation of the enzyme is primarily caused by
phosphorylation (10-12).
The hypothesis that Akt is activated by phosphorylation is further
supported by the recent identification of a
phosphoinositide-dependent protein kinase-1 or PDK1 (13,
14). The 556-residue enzyme isolated from human tissues (hPDK1)
contains a catalytic domain (residues 84-341) and a COOH-terminal
pleckstrin homology or pleckstrin homology domain (residues 450-550).
hPDK1 phosphorylates Akt at Thr308 and activates the enzyme
in a PI 3-kinase-dependent manner. In addition to Akt,
hPDK1 phosphorylates and activates another PI 3-kinase downstream
target, p70S6K (15).
Although it is generally agreed upon that PI 3-kinase is necessary for
a variety of insulin-mediated metabolic events, some evidence shows
that Akt may not be necessary for insulin-mediated glycogen synthesis
and glucose transport (6, 16). These findings raise a very interesting
question as to how PI 3-kinase is coupled to the downstream targets of
the insulin receptor. Potential candidates that may mediate part of the
phosphatidylinositol 3-kinase (PI 3-kinase) signaling are atypical
isoforms of PKC such as PKC To further understand the mechanism of insulin receptor signal
transduction and regulation, we attempted to clone the pdk1 gene from
mouse tissues. Here we report the cloning and characterization of mouse
PDK1 (mPDK1). We have found that mPDK1 mRNA was broadly expressed
in various mouse tissues and in early development stages. Western blot
analysis suggests there may be isoforms of the protein. We have also
shown that mPDK1 is a kinase that phosphorylates and activates PKC Buffers--
Buffer A consisted of 50 mM Hepes, pH
7.6, 1% Triton X-100, 150 mM NaCl, 1 mM
phenylmethanesulfonyl fluoride, 1 mM sodium orthovanadate,
1 mM sodium fluoride, 1 mM sodium
pyrophosphate, 10 µg/ml aprotinin and 10 µg/ml leupeptin. Buffer B
consisted of 50 mM Tris-HCl, pH 7.5, 5 mM
MgCl2, 1 mM sodium fluoride, 1 mM
sodium orthovanadate, and 1 mM phenylmethanesulfonyl
fluoride. Buffer C consisted of 20 mM Hepes, pH 7.6, 3 mM CaCl2, 5 mM KCl, 7 mM NaHCO3, 107 mM NaCl, 1 mM MgSO4, 10 mM glucose, and 0.1% bovine serum albumin. Buffer D consisted of 20 mM
Na2HPO4, pH 8.6, 0.5% Triton X-100, 0.1% SDS,
0.02% NaN3, and 1 M NaCl.
Cloning of mPDK1--
The EST clone (AA117492) that contains an
insert encoding the COOH terminus of mPDK1 (residues 265-559, see Fig.
1) was obtained from ATCC. A mouse liver cDNA library (19) was
screened using the partial mPDK1 cDNA fragment as a probe. Positive
clones, contained within the pBK-CMV phagemid, were excised in
vivo from the ZAP ExpressTM vector using
ExAssist-XLOLR system (Stratagene, CA), following the protocol
recommended by the manufacturer. DNA was sequenced from both directions
by the Institutional DNA Sequencing Facility.
Northern Blot Analysis--
A mouse multiple Northern blot
containing poly(A) RNA from different tissues of mice aged from 8 to 12 weeks old and an embryonic Northern blot (CLONTECH)
were hybridized under conditions recommended by the manufacturer, using
the radiolabeled EST mPDK1 cDNA fragment as a probe.
cDNA Expression Vectors and Cell Lines--
The full-length
mPDK1 cDNA (except for the last four amino acids) was amplified by
polymerase chain reaction and subcloned into the mammalian expression
vector, pBEX (20), in-frame at its COOH terminus with a sequence
encoding the hemagglutinin (HA)-tag (pBEX/mPDK1). A kinase-inactive
mPDK1 (mPDK1K114G) was generated by replacing the conserved
ATP binding site lysine residue with glycine with the use of a
Quick-change site-directed mutagenesis kit (Stratagene, CA). The
full-length human PKC Anti-mPDK1 Antibody--
The cDNA encoding the COOH terminus
of the enzyme (mPDK1CT, amino acid residues 285-559 of
mPDK1, see Fig. 1) was amplified by polymerase chain reaction and
subcloned into a bacterial expression vector pGEX-4T-1 (Amersham
Pharmacia Biotech). Overexpression and purification of the fusion
protein was carried out according to the manufacturer's protocol.
Polyclonal antibodies to the GST/mPDK1CT fusion protein
were generated by immunizing rabbits. Immobilization of GST and
GST/mPDK1CT to Affi-Gel 10 beads was performed according to
the protocol provided by the manufacturer (Bio-Rad). Anti-mPDK1 antibody was affinity-purified by, first, absorption of the antiserum with GST-immobilized Affi-Gel 10 beads to remove the antibody to GST.
The resulting supernatant was then incubated with
GST/mPDK1CT-immobilized Affi-Gel 10 beads. The beads were
washed extensively with a buffer containing 50 mM Tris-HCl,
pH 7.5, and 0.5 M NaCl. After further washing with 50 mM Tris-HCl, pH 7.5, the anti-mPDK1 antibody bound to the
beads was eluted with a buffer containing 0.1 M glycine, pH
2.8, 0.1% Triton X-100, and 150 mM NaCl, neutralized with
1 M Tris-HCl, pH 8.5, and dialyzed against a buffer
containing 50 mM Hepes, pH 7.6, and 150 mM NaCl.
Western Blot Analysis of mPDK1 in Mouse Tissues--
Tissues
from mice aged from 3 to 6 months old were homogenized in ice-cold
Buffer A. Tissue homogenates were clarified by centrifugation at
18,000 × g for 2 × 30 min. The concentration of
the proteins in the supernatant was determined by the Bradford assay.
Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and detected with the affinity-purified polyclonal antibody to mPDK1.
In Vitro Phosphorylation of Synthetic Peptides by mPDK1--
A
peptide (TFCGTPNYIAPEI) corresponding to the consensus phosphorylation
sequence in the activation loop of PKC Phosphorylation of PKC In Vivo 32P Labeling--
In vivo
labeling was carried out as described previously with some
modifications (21). In brief, cDNAs encoding the wild-type or T402A
mutant PKC Activation of PKC Screening of EST Data Bases and Identification of a Clone Encoding
the COOH Terminus of mPDK1--
To study the functional role of PDK1,
we searched the NCBI dbEST using the hPDK1 sequence (13) as a probe. We
identified a partial cDNA sequence (AA117492) highly homologous
(85% identical) to the sequence encoding the COOH terminus of hPDK1
(amino acid residues 262-556) (13), suggesting that the sequence is
the mouse homologue of hPDK1.
Cloning of the Full-length mPDK1 cDNA--
To clone the
full-length cDNA for mPDK1, we screened a mouse liver cDNA
library using the insert of the mPDK1 EST clone as a probe. Several
positive clones were isolated and characterized. The longest sequence
(clone L3, 1.9 kb) was predicted to encode a protein comprised of 559 amino acids with an estimated molecular mass of 64 kDa (Fig.
1). The putative initiation codon at
position 101 was identified based on the presence of a
consensus Kozak sequence (22) and the putative assignment of the human
and rat sequences (13, 14). mPDK1 contains a catalytic domain located between residues 84 and 344 and a COOH-terminal pleckstrin homology domain located between residues 453 and 553. The overall sequence percent identity between mPDK1 and hPDK1 (13) or rat PDK1 (14) is 95 and 98, respectively. However, like both the human and rat PDK1
cDNA sequences, no in-frame upstream stop codons were observed in
mPDK1 cDNA. The delineation of the bona fide open reading frame may
await further investigation.
Tissue Distribution of mPDK1 mRNA--
To assess the
tissue distribution of PDK1 mRNA expression, Northern blot analysis
was performed. A 7-kb mPDK1 transcript was found to be expressed in all
tissues examined, and the highest expression was detected in the heart,
brain, liver, and testis (Fig.
2A). In the testis, a high
level expression of an additional 2-kb transcript was also detected
(Fig. 2A). The high and specific expression of this
transcript in testis is consistent with the hypothesis that there may
be testis-specific isoforms of PDK1 that function in cell survival or
cell death decisions during spermatogenesis (13).
Expression of mPDK1 mRNA in Mouse Embryos--
The expression
of PDK1 mRNA in mouse embryos was also examined. The 7-kb mRNA
transcript could be detected as early as day 7 and maintained constant
to day 17 post-conception (Fig. 2B). The high expression of
mPDK1 mRNA throughout the embryonic stages suggests that the
protein may play important roles in embryonic development.
Expression of mPDK1 Protein in Mouse Tissues--
Using the
affinity-purified polyclonal anti-mPDK1 antibody, we examined
endogenous mPDK1 expression in different mouse tissues (Fig.
3). Protein bands with molecular masses
of approximately of 62 to 64 kDa was detected in most of the tissues
examined, with the highest expression in the testis (Fig. 3, lane
1), brain (Fig. 3, lane 2), heart (Fig. 3, lane
3), spleen (Fig. 3, lane 5), and adipose tissue (Fig.
3, lane 6). Our unpublished data suggest that the
heterogeneity of the protein bands could correspond to mPDK1 isoforms
translated from different mRNAs or generated by alternative use of
initiation sites of translation of the same mRNA. In addition to
these isoforms, the antibody also detected two other protein bands in
most of the tissues examined, with molecular masses of approximately
125 and 180 kDa, respectively (Fig. 3, indicated by arrows).
Preincubation of the antibody with excess amounts of
GST-mPDK1CT antigen eliminated these protein bands,
suggesting that the immunoreactivity was specific.
Identification of PKC
As PKC
To identify the phosphorylation site on PKC
To investigate whether PKC
To test whether phosphorylation of PKC
To examine whether PDK1 had a direct effect on PKC PDK1 is a recently identified PI 3-kinase downstream protein
kinase that phosphorylates and activates Akt, p70S6K and
cAMP-dependent protein kinase (13-15, 23, 24). Although these data suggest that PDK1 may play an important part in cell signaling processes, direct evidence of the role of PDK1 remains elusive.
We report here the cloning of PDK1 in mouse tissue. Our results show
that the mPDK1 mRNA is broadly expressed in various tissues and in
early embryonic stages. These findings suggest that the enzyme may play
a general role in signaling processes and in development. The high
level expression of a testis-specific mPDK1 mRNA also suggests that
the enzyme may be involved in sex differentiation processes. This
hypothesis is consistent with the finding that PDK1 is homologous to
the Drosophila protein kinase DSTPK61, an enzyme that is
implicated in the regulation of sex differentiation, oogenesis, and
spermatogenesis (13).
Western blot analysis using anti-mPDK1 antibody revealed multiple
protein bands in various mouse tissues (Fig. 3), suggesting that there
may be isoforms of mPDK1. This hypothesis is consistent with the
following observations. First, it has been shown that in sheep brain
there were four phosphatidylinositol 3,4,5-trisphosphate-binding proteins with Akt kinase activity (14). Second, PDK1 cDNAs isolated from human (13), rat (14), and mouse (this study) all contain no
5'-upstream stop codon, raising the possibility that they may be
partial DNA sequences of larger cDNAs. In agreement with this observation, protein bands with molecular weights larger than that
encoded by the mPDK1 cDNA were also detected by the antibody (Fig.
3). Because the polyclonal antibody recognizes the COOH terminus of
mPDK1, the antibody-immunoreactive proteins, if they are indeed mPDK1
isoforms, may only differ at their amino termini. In agreement with
this hypothesis, we have cloned a cDNA from mouse
testis.2 This cDNA
encodes a 62-kDa mPDK1 isoform with an amino terminus different from
that of mPDK1 presented in this report (Fig. 1). It is interesting to
notice that multiple isoforms have also been found in many other
protein kinases including the PDK1 downstream effectors such as protein
kinase C (25, 26) and Akt (27). The presence of PDK1 isoforms may thus
have some physiological relevance. It is possible that the amino acid
differences between these isoforms could lead to differences in
specificity between different substrates of the enzyme. In addition,
different isoforms may be differently regulated in cells. Thus, the
existence of multiple and tissue-specific isoforms of PDK1 suggests an
additional level of regulation may be involved. Studies are currently
in progress to test these ideas.
In agreement with the findings of others (13, 15), we have found that
PDK1 was constitutively activated, and insulin treatment did not
further increase its activity in vitro (Fig. 4, B
and C). However, we also observed an additive effect of PDK1
and insulin in PKC In summary, available data have shown that PDK1 is an upstream kinase
for different effectors including Akt, p70S6K,
cAMP-dependent protein kinase, and PKC and activated the enzyme in vitro and in cells. Our
findings suggest that there may be different isoforms of mPDK1 and that
the protein is an upstream kinase that activates divergent pathways
downstream of phosphatidylinositol 3-kinase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
. PKC
is activated in vitro
by phosphatidylserine and polyphosphoinositides, products of PI
3-kinase (17). In addition, insulin provokes a rapid increase in both
the serine phosphorylation and the enzyme activity of PKC
in rat
adipocytes. Furthermore, this insulin-stimulated phosphorylation of
PKC
was inhibited by PI 3-kinase-specific inhibitors such as
LY294002 and wortmannin (18). Although these data suggest that PKC
may be activated by PI 3-kinase-dependent phosphorylation,
the kinase(s) that phosphorylates PKC
has not been identified.
.
Our findings suggest that PDK1 may be a key enzyme that regulates
divergent signaling pathways downstream of PI 3-kinase.
MATERIALS AND METHODS
, except the last valine residue, was subcloned
into pcDNA3.1 (Invitrogene, CA) in-frame at its COOH terminus with
the Myc-tag (pcDNA/PKC
) and was provided by Dr. R. Lin
(UTHSCSA). Mutant forms of PKC
in which the threonine residue at
position 402 was changed either to alanine (PKCT402A) or
glutamate (PKCT402E) were also generated by site-directed
mutagenesis. To establish cell lines expressing both the IR and mPDK1,
the recombinant pBEX/mPDK1 plasmid was transfected into CHO/IR cells
with LipofectAMINE (Life Technologies, Inc.). Stable cell lines
expressing high levels of mPDK1 (CHO/IR/mPDK1) were selected with 10 µg/ml puromycin. Positives were identified by Western blot using the
antibody to the HA-tag (Babco, CA) and cloned by limiting dilution.
(residues 402 to 414) was
chemically synthesized and used as a substrate for mPDK1. To facilitate
the in vitro assays, three arginine residues were added to
the ends of the peptide (one at the NH2 terminus and two at
the COOH terminus). To identify the PDK1 phosphorylation site in
PKC
, we synthesized a peptide with the potential phosphorylation site threonine (Thr402) changed to alanine (T402A). Two
positive control peptides with the sequences derived from the
activation loop of Akt (KTFCGTPEYLAPEVRR) and the
cAMP-dependent protein kinase (PKA, RTLCGTPEYLAPEIRR) were
also synthesized. For in vitro kinase assays, the HA-tagged mPDK1 was immunoprecipitated from CHO/IR/mPDK1 cells by the anti-HA antibody (BABCO, CA) or control normal mouse immunoglobulin bound to
protein-G-agarose beads. Kinase reactions were initiated by the
addition of 30 µl of Buffer B containing 4 µCi of
[
-32P]ATP and 10 µl of 0.4 mM substrate
or control peptides. After incubation for 30 min at 30 °C, the
incorporation of 32P into peptides was determined by
absorption of the positively charged peptides to P81 phosphocellulose
membrane and checked by Cerenkov counting.
by mPDK1 in Vitro--
Phosphorylation
of PKC
by mPDK1 was performed using a protocol similar to that used
in the study of PDK1-catalyzed p70S6K phosphorylation (14). In
brief, the Myc-tagged PKC
was transiently expressed in CHO/IR cells.
The extracts of these cells were mixed with those of insulin-treated or
-nontreated CHO/IR cells expressing also the wild-type or mutant
(K114G) mPDK1. The Myc-tagged PKC
and the HA-tagged mPDK1 were
co-immunoprecipitated by appropriate antibodies to the tags. In
vitro phosphorylation was initiated with the addition of 30 µl
of Buffer B containing 2 µCi of [
-32P]ATP and
incubated for 30 min at 30 °C. After washing with an ice-cold buffer
containing 50 mM Hepes, pH 7.4, 150 mM NaCl,
and 0.1% Triton X-100, the immunoprecipitated proteins were heated at
95 °C for 3 min, separated by SDS-PAGE, and blotted to a
nitrocellulose membrane. Phosphorylation of PKC
by PDK1 was
visualized by autoradiography and quantified by PhosphoImager analysis.
were transiently transfected into either CHO/IR or
CHO/IR/mPDK1 cells. After transfection (36 h), cells in 10-cm diameter
plates were incubated in 4 ml of phosphate-free Buffer C for 30 min at
37 °C and then radiolabeled with 0.3 mCi of carrier-free
[32P]orthophosphate/plate for 4 h at 37 °C. After
treatment with or without insulin (10 nM) for an additional
5 min, cells were washed twice with ice-cold Buffer C and lysed with
0.4 ml/plate lysis Buffer A. After centrifugation at 12,000 × g for 10 min at 4 °C, the 32P-labeled PKC
was immunoprecipitated with anti-Myc antibody bound to protein
G-agarose beads (Amersham Pharmacia Biotech). The immunoprecipitates were washed twice with Buffer D and then twice with the same buffer, except that the NaCl concentration in the buffer was changed to 0.15 M. The radiolabeled PKC
was separated by SDS-PAGE,
blotted to Immobilon P membrane (Millipore), and visualized by autoradiography.
by PDK1 in Vitro and in Cells--
The
in vitro assay of PKC
activity was carried out by a
two-step process. First, the immunoprecipitated PKC
was activated by
purified mPDK1 in the presence of Buffer B containing 25 µM ATP. After incubation for 30 min at 30 °C, the
phosphorylated PKC
/protein G beads were washed twice with an
ice-cold buffer containing 50 mM Hepes, pH 7.4, 150 mM NaCl, and 0.1% Triton X-100 and once with Buffer B. The
activity of PKC
was then determined as described (18), using the
Ser159-PKC-
-(149-164) peptide (Quality Controlled
Biochemicals, Inc., Hopkinton, MA) as a substrate. To study the effect
of mPDK1 on PKC
activation in cells, pcDNA/PKC
plasmid was
transfected into CHO/IR or CHO/IR/mPDK1 cells by electroporation. After
transfection (48 h), the cells were serum-starved for 1 h, treated
with or without 10
8 M insulin for 5 min, and
then lysed in Buffer A. The transiently expressed PKC
was
immunoprecipitated by the anti-Myc antibody (Santa Cruz, CA) bound to
protein G beads. The activity of PKC
was determined as described above.
RESULTS
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Fig. 1.
Deduced amino acid sequence of
mPDK1. The catalytic domain and the pleckstrin homology domain are
underlined and italicized, respectively.
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Fig. 2.
Tissue expression of mPDK1 mRNA. A
mouse multiple-tissue Northern blot (CLONTECH)
(A) and a mouse embryonic-tissue blot
(CLONTECH) (B) were hybridized to the
radiolabeled mPDK1 insert derived from EST clone AA117492. The
membranes were stripped from the probe and reblotted with a 2-kb mouse
-actin probe to confirm equal loading of mRNAs from different
tissues. SK, skeletal muscle.
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Fig. 3.
Expression of mPDK1 isoforms in mouse
tissues. Cell lysates (200 µg of proteins/lane) from
mouse testis (lane 1), brain (lane 2), heart
(lane 3), kidney (lane 4), spleen (lane
5), adipose tissue (lane 6), liver (lane 7),
and skeletal muscle (lane 8) were resolved by SDS-PAGE,
transferred to a nitrocellulose membrane, and blotted with an
affinity-purified polyclonal anti-mPDK1 antibody. The expression of
mPDK1 isoforms were visualized by alkaline phosphatase-catalyzed
chromatogenous substrate reactions. M.M., molecular
mass.
as a Potential Substrate for
PDK1--
hPDK1 has been shown to phosphorylate Akt and p70S6K
at Thr308 (14) and Thr229 (15), respectively.
Comparison of the sequences surrounding these phosphorylation sites
revealed a high homology between these two enzymes (Fig.
4A). This finding suggests
that PDK1 recognizes a consensus sequence that may also exist in other
protein kinases. To test this hypothesis, we searched the NCBI
Brookhaven protein data bank using the Akt phosphorylation consensus
sequence TFCGTPEYLAPE (Fig. 4A) as a probe. In addition to
the two known PDK1 substrates Akt and P70S6K, we identified
a number of kinases including the catalytic domain of
cAMP-dependent protein kinase (PKA, protein data bank
accession number P05206), PKC isoforms, cell cycle protein kinase CDC5 (G416768), cGMP-dependent protein kinase (P00516), the
polo-like Ser/Thr protein kinase PLK (P53350), and
spermatozoon-associated protein kinase (P21901) (Fig. 4A).
This finding was very interesting as it suggests that PDK1 may be a
potential upstream kinase for these kinases, including PKC
.
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Fig. 4.
Identification of PKC
as a potential substrate for PDK1. A, comparison
of conserved sequences in the activation loop region of a number of
protein kinases. The potential threonine residue phosphorylation site
is in bold. CGK, cGMP-dependent
protein kinase; PLK1, polo-like Ser/Thr protein kinase;
SAK, spermatozoon-associated protein kinase. B,
phosphorylation of PKC
by mPDK1 in vitro. Extracts from
cells expressing the wild-type (WT) or T402A mutant PKC
were mixed with those of insulin-treated (+) or nontreated (
)
CHO/IR/mPDK1 cells. HA-tagged mPDK1 and Myc-PKC
were
co-immunoprecipitated with appropriate antibodies to each epitope tag.
In vitro phosphorylation was initiated by the addition of kinase buffer
containing [32P]ATP. The phosphorylated proteins were
separated by SDS-PAGE and blotted to a nitrocellulose membrane, and the
phosphorylation of PKC
was visualized by autoradiography
(upper panel). The same membrane was reblotted with anti-Myc
(middle panel) or anti-mPDK1 (bottom panel)
antibodies, and the expression of mPDK1 and PKC
was visualized by
alkaline phosphatase-catalyzed chromatogenous substrate reaction. Data
are representative of two independent experiments. Nlg,
normal immunoglobulin C, phosphorylation of the wild-type
(WT) or T402A peptides by mPDK1 in vitro.
HA-tagged mPDK1 was immunoprecipitated from CHO/IR/mPDK1 cells with
anti-HA antibody (+ mPDK1) or the control normal mouse immunoglobulin
(
mPDK1). In vitro phosphorylation was performed as
described under "Materials and Methods." The incorporation of
32P was quantified by Cerenkov counting. Data are
representative of three independent experiments, with bars
representing means of duplicate determinations. D,
phosphorylation of PKC
by mPDK1 in cells. The Myc-tagged PKC
was
immunoprecipitated from insulin-treated (+) or nontreated (
) CHO/IR
and CHO/IR/mPDK1 cells transiently expressing the Myc-tagged PKC
.
After separation by SDS-PAGE and blotting to a membrane, the
phosphorylation of PKC
was visualized by autoradiography. Data are
representative of two independent experiments.
has been shown to undergo PI 3-kinase-dependent
phosphorylation and activation, we attempted to examine whether this enzyme was a substrate of mPDK1. We established a stable Chinese hamster ovary cell line expressing both the IR and an HA-tagged mPDK1
(CHO/IR/mPDK1). We also transiently expressed mutant mPDK1 (mPDK1K114G) and Myc-tagged PKC
into CHO/IR cells. We
then carried out in vitro phosphorylation studies using the
immunoaffinity-purified mPDK1 and PKC
. PKC
was significantly
phosphorylated in the presence of mPDK1 (Fig. 4B,
lanes 3 and 4) but not in the absence of the enzyme (Fig. 4B, lanes 1 and 2) or in the
presence of a kinase-inactive mutant mPDK1 (mPDK1K114G,
Fig. 4B, lanes 7 and 8). The in
vitro phosphorylation of PKC
by mPDK1 was insulin-independent
as a pretreatment of CHO/IR/mPDK1 cells with insulin had no significant
effect on mPDK1 activity (Fig. 4B), although under the same
conditions the insulin-stimulated insulin receptor and IRS-1 tyrosine
phosphorylation as well as Akt activation were all significantly
increased (data not shown). PhosphoImager analysis showed that the
amount of phosphate incorporated into PKC
was 20-fold in the
presence of mPDK1 over that in the absence of the enzyme. The wild-type
mPDK1 also underwent significant autophosphorylation in
vitro and in cells (data not shown). However, whether the
phosphorylation plays a role in the regulation of the activity of the
enzyme remain to be established.
, we carried out in
vitro phosphorylation studies using synthetic peptides
corresponding to the activation loop of PKC
as substrates. As shown
in Fig. 4C, the peptide derived from the wild-type PKC
activation loop sequence was readily phosphorylated by mPDK1 in
vitro (lanes 5 and 6). Similar levels of
phosphorylation were observed for the peptide substrates derived from
Akt and PKA (Fig. 4C, lanes 7 and 8). Replacement
of Thr402 with alanine in PKC
peptide reduced the
phosphorylation to the basal level (Fig. 4C, lanes
1-4), suggesting that Thr402 was the site of
phosphorylation by PDK1. Consistent with this observation, the
phosphorylation of the mutant PKC
(PKC
T402A) by mPDK1
was significantly decreased (Fig. 4B, lanes 5 and
6). Similar results were also obtained for the
PKCT402E mutant (data not shown). Treatment of the cells
with either wortmannin (50 nM, 1 h) or LY294002 (50 µM, 1 h) had no significant effect on the in
vitro mPDK1 activity toward its peptide substrates nor did it
affect PDK1-mediated phosphorylation of PKC
in vitro
(data not shown). Under the this same condition, the insulin-stimulated Akt in vivo phosphorylation was completely blocked (data not shown).
is an in vivo substrate for
PDK1 and whether Thr402 is the site of phosphorylation by
PDK1 in cells, we performed in vivo labeling experiments. As
shown in Fig. 4D, PKC
was phosphorylated in CHO/IR cells,
and insulin-treatment stimulated the phosphorylation of the enzyme
(Fig. 4D, lanes 1 and 2).
Overexpressing mPDK1 increased the basal phosphorylation of the enzyme
(Fig. 4D, lanes 3 and 4). In agreement
with the in vitro phosphorylation results, the phosphorylation of PKC
T402A mutant was significantly
decreased by mPDK1 in the CHO/IR/mPDK1 cells (Fig. 4D,
lanes 5 and 6). These data provide further
evidence that Thr402 in the activation loop of PKC
is
the site of phosphorylation by PDK1.
affected its enzymatic
activity, we transiently expressed the Myc-tagged enzyme in CHO/IR or
CHO/IR/mPDK1 cells. In the parental CHO/IR cells, insulin stimulation
resulted in an approximate 1.6-fold increase in PKC
activity (Fig.
5A, lanes 1 and
2). Overexpression of mPDK1 caused an approximate 2-fold
increase in the basal PKC
activity (Fig. 5A, lanes
1 and 3), and insulin treatment further activated the enzyme (Fig. 4D, lane 4). A Western blot showed
similar levels of PKC
in CHO/IR and CHO/IR/mPDK1 cells (data not
shown). These results suggest that although mPDK1 was able to
phosphorylate PKC
in vitro in an insulin-independent
manner, other effector(s) may also be necessary for maximum activation
of PKC
in cells.
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Fig. 5.
Activation of PKC by
PDK1. A, activation of PKC
by mPDK1 in cells. The
Myc-tagged PKC
was immunoprecipitated from insulin-treated (+) or
nontreated (
) CHO/IR and CHO/IR/mPDK1 cells transiently expressing
the Myc-tagged PKC
. PKC
activity was assayed as described under
"Materials and Methods." Data are representative of two independent
experiments, with bars representing means of duplicate
determinations. B, activation of PKC
by mPDK1 in
vitro. The wild-type (lanes 2, 3, and
5) or mutant (T402A, lane 4) PKC
were purified
by affinity purification, incubated with kinase buffer containing 25 µM ATP (lane 2), or incubated with kinase
buffer, ATP, and the wild-type (lane 3) or mutant (K114G,
lane 4) mPDK1. After phosphorylation, the activity of PKC
was then determined as described under "Materials and Methods."
Data are representative of two independent experiments, with
bars representing means of duplicate determinations.
activity, we
examined PKC
activity in vitro before and after
phosphorylation by PDK1. As shown in Fig. 5B,
phosphorylation of PKC
by the wild-type PDK1 resulted in a 3-fold
increase in the activity of PKC
(Fig. 5, lanes 2 and
3). No significant increase in PKC
activity was observed
when the enzyme was preincubated with the kinase-inactive mutant
mPDK1K114G (Fig. 5B, lane 5). Under
this assay condition, neither mPDK1 itself (Fig. 5B,
lane 1) nor the mutant PKC
T402A (Fig.
5B, lane 4) had significant effect on the
phosphorylation of the peptide substrates.
DISCUSSION
activity in cells (Fig. 5A). One
possible explanation for this discrepancy is that although PDK1 was
constitutively active and able to phosphorylate and activate PKC
in vitro and in cells, other insulin-dependent
mechanism(s), such as phosphorylation by other kinase(s) or
translocation to a specific cellular compartment, may also be required
for the full activation of PKC
. Consistent with this idea, our
unpublished data showed that overexpression of mPDK1 in cells resulted
in an increase in Akt activity but did not stimulate its
phosphorylation at Ser473 in cells, whose phosphorylation
(by an unknown kinase) has been shown to be necessary for the full
activation of the enzyme (24, 27). Our results are also consistent with
those of Alessi et al. (13) who found that overexpression of
PDK1 activated Akt and potentiated the IGF-1-induced increase of Akt
activity in cells. In addition, previous studies have shown that
phosphorylation of the conserved threonine residue in the activation
loop of PKC isoforms resulted in a conformational change of the enzymes
and rendered the inactive PKCs to become the cofactor-activable, mature form (28, 29). Our studies provide evidence that PDK1 is the kinase
that phosphorylates the conserved threonine residue in PKC
and
initiates the activation process. During the reviewing process of our
manuscript, two groups reported their findings on the activation of
PKC
by PDK1 (30, 31). By using antibodies specific to the
phosphothreonine residue in the activation loop of PKC, both groups
showed that Thr410 in rat PKC
(equivalent to
Thr402 in the human version enzyme) was indeed the in
vivo phosphorylation site of PDK1. Our results are in full
agreement with these findings.
. These findings
suggest that PDK1 may be a site for divergence of different signaling pathways downstream of PI 3-kinase. There is evidence suggesting that
PKC
is downstream of PI 3-kinase and may contribute to
insulin-stimulated glucose transport in 3T3-L1 adipocytes (17, 18, 32).
Activation of PKC isoforms by PDK1 may thus provide an alternative
pathway to mediate some PI 3-kinase-dependent downstream
events such as glycogen synthesis and GLUT4 translocation in cells. The
finding that PDK1 recognizes the consensus activation loop sequence in various protein kinases suggests that other PKC isoforms may also be
substrates of PDK1. Further studies will help us to define the
importance of the PDK1/PKC pathway in insulin and other growth factor-mediated signaling processes.
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ACKNOWLEDGEMENTS |
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We thank Dr. H. Li for the mouse liver
cDNA library and Dr. R. Lin for pcDNA/PKC.
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FOOTNOTES |
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* This research was supported in part by Grant DK52933 from the National Institutes of Health (to F. L. and L. Q. D.) and by an Award to the University of Texas Health Science Center at San Antonio from the Research Resource Program for Medical Schools of the Howard Hughes Medical Institute (to F. L.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF086625.
To whom correspondence should be addressed: Dept. of
Pharmacology, The University of Texas Health Science Center at San
Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78284-7764. Tel.:
210-567-3097; Fax: 210-567-4226; E-mail: liuf{at}uthscsa.edu.
2 L. Q. Dong and F. Liu, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: PI 3-kinase, phosphatidylinositol 3-kinase; CHO, Chinese hamster ovary; EST, expressed sequence tag; GST, glutathione S-transferase; IR, insulin receptor; PAGE, polyacrylamide gel electrophoresis; PKC, protein kinase C; PKA, protein kinase A; PDK1, phosphoinositide-dependent protein kinase-1; hPDK1, human PDK1; mPDK1, mouse PDK1; HA, hemagglutinin; kb, kilobase(s).
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REFERENCES |
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