Primary Structure, Tissue Distribution, and Expression of Mouse Phosphoinositide-dependent Protein Kinase-1, a Protein Kinase That Phosphorylates and Activates Protein Kinase Czeta *

Lily Q. DongDagger , Ruo-bo Zhang§, Paul LanglaisDagger , Huili HeDagger , Matthew ClarkDagger , Li Zhu§, and Feng LiuDagger parallel

From the Dagger  Department of Pharmacology and  Biochemistry, The University of Health Science Center, San Antonio, Texas 78284 and § CLONTECH Laboratories, Inc., Palo Alto, California 94303

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
REFERENCES

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-zeta 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

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 PKCzeta . PKCzeta 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 PKCzeta in rat adipocytes. Furthermore, this insulin-stimulated phosphorylation of PKCzeta was inhibited by PI 3-kinase-specific inhibitors such as LY294002 and wortmannin (18). Although these data suggest that PKCzeta may be activated by PI 3-kinase-dependent phosphorylation, the kinase(s) that phosphorylates PKCzeta has not been identified.

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 PKCzeta . Our findings suggest that PDK1 may be a key enzyme that regulates divergent signaling pathways downstream of PI 3-kinase.

    MATERIALS AND METHODS

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 PKCzeta , except the last valine residue, was subcloned into pcDNA3.1 (Invitrogene, CA) in-frame at its COOH terminus with the Myc-tag (pcDNA/PKCzeta ) and was provided by Dr. R. Lin (UTHSCSA). Mutant forms of PKCzeta 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.

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 PKCzeta (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 PKCzeta , 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 [gamma -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.

Phosphorylation of PKCzeta by mPDK1 in Vitro-- Phosphorylation of PKCzeta 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 PKCzeta 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 PKCzeta 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 [gamma -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 PKCzeta by PDK1 was visualized by autoradiography and quantified by PhosphoImager analysis.

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 PKCzeta 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 PKCzeta 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 PKCzeta was separated by SDS-PAGE, blotted to Immobilon P membrane (Millipore), and visualized by autoradiography.

Activation of PKCzeta by PDK1 in Vitro and in Cells-- The in vitro assay of PKCzeta activity was carried out by a two-step process. First, the immunoprecipitated PKCzeta 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 PKCzeta /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 PKCzeta was then determined as described (18), using the Ser159-PKC-epsilon -(149-164) peptide (Quality Controlled Biochemicals, Inc., Hopkinton, MA) as a substrate. To study the effect of mPDK1 on PKCzeta activation in cells, pcDNA/PKCzeta 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 PKCzeta was immunoprecipitated by the anti-Myc antibody (Santa Cruz, CA) bound to protein G beads. The activity of PKCzeta was determined as described above.

    RESULTS

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.


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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.

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).


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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 beta -actin probe to confirm equal loading of mRNAs from different tissues. SK, skeletal muscle.

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.


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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.

Identification of PKCzeta 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 PKCzeta .


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Fig. 4.   Identification of PKCzeta 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 PKCzeta by mPDK1 in vitro. Extracts from cells expressing the wild-type (WT) or T402A mutant PKCzeta were mixed with those of insulin-treated (+) or nontreated (-) CHO/IR/mPDK1 cells. HA-tagged mPDK1 and Myc-PKCzeta 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 PKCzeta 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 PKCzeta 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 PKCzeta by mPDK1 in cells. The Myc-tagged PKCzeta was immunoprecipitated from insulin-treated (+) or nontreated (-) CHO/IR and CHO/IR/mPDK1 cells transiently expressing the Myc-tagged PKCzeta . After separation by SDS-PAGE and blotting to a membrane, the phosphorylation of PKCzeta was visualized by autoradiography. Data are representative of two independent experiments.

As PKCzeta 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 PKCzeta into CHO/IR cells. We then carried out in vitro phosphorylation studies using the immunoaffinity-purified mPDK1 and PKCzeta . PKCzeta 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 PKCzeta 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 PKCzeta 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.

To identify the phosphorylation site on PKCzeta , we carried out in vitro phosphorylation studies using synthetic peptides corresponding to the activation loop of PKCzeta as substrates. As shown in Fig. 4C, the peptide derived from the wild-type PKCzeta 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 PKCzeta 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 PKCzeta (PKCzeta 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 PKCzeta in vitro (data not shown). Under the this same condition, the insulin-stimulated Akt in vivo phosphorylation was completely blocked (data not shown).

To investigate whether PKCzeta 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, PKCzeta 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 PKCzeta 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 PKCzeta is the site of phosphorylation by PDK1.

To test whether phosphorylation of PKCzeta 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 PKCzeta activity (Fig. 5A, lanes 1 and 2). Overexpression of mPDK1 caused an approximate 2-fold increase in the basal PKCzeta 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 PKCzeta in CHO/IR and CHO/IR/mPDK1 cells (data not shown). These results suggest that although mPDK1 was able to phosphorylate PKCzeta in vitro in an insulin-independent manner, other effector(s) may also be necessary for maximum activation of PKCzeta in cells.


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Fig. 5.   Activation of PKCzeta by PDK1. A, activation of PKCzeta by mPDK1 in cells. The Myc-tagged PKCzeta was immunoprecipitated from insulin-treated (+) or nontreated (-) CHO/IR and CHO/IR/mPDK1 cells transiently expressing the Myc-tagged PKCzeta . PKCzeta 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 PKCzeta by mPDK1 in vitro. The wild-type (lanes 2, 3, and 5) or mutant (T402A, lane 4) PKCzeta 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 PKCzeta was then determined as described under "Materials and Methods." Data are representative of two independent experiments, with bars representing means of duplicate determinations.

To examine whether PDK1 had a direct effect on PKCzeta activity, we examined PKCzeta activity in vitro before and after phosphorylation by PDK1. As shown in Fig. 5B, phosphorylation of PKCzeta by the wild-type PDK1 resulted in a 3-fold increase in the activity of PKCzeta (Fig. 5, lanes 2 and 3). No significant increase in PKCzeta 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 PKCzeta T402A (Fig. 5B, lane 4) had significant effect on the phosphorylation of the peptide substrates.

    DISCUSSION

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 PKCzeta activity in cells (Fig. 5A). One possible explanation for this discrepancy is that although PDK1 was constitutively active and able to phosphorylate and activate PKCzeta 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 PKCzeta . 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 PKCzeta and initiates the activation process. During the reviewing process of our manuscript, two groups reported their findings on the activation of PKCzeta 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 PKCzeta (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.

In summary, available data have shown that PDK1 is an upstream kinase for different effectors including Akt, p70S6K, cAMP-dependent protein kinase, and PKCzeta . 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 PKCzeta 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.

    ACKNOWLEDGEMENTS

We thank Dr. H. Li for the mouse liver cDNA library and Dr. R. Lin for pcDNA/PKCzeta .

    FOOTNOTES

* 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.

parallel 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.

    ABBREVIATIONS

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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