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Originally published In Press as doi:10.1074/jbc.M205141200 on August 12, 2002

J. Biol. Chem., Vol. 277, Issue 42, 39360-39367, October 18, 2002
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Regulation of Kinase Activity of 3-Phosphoinositide-dependent Protein Kinase-1 by Binding to 14-3-3*

Saori SatoDagger , Naoya FujitaDagger , and Takashi TsuruoDagger §

From the Dagger  Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113-0032, Japan and the § Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo 170-8455, Japan

Received for publication, May 27, 2002, and in revised form, July 17, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in activating the protein kinase A, G, and C subfamily. In particular, PDK1 plays an important role in regulating the Akt survival pathway by phosphorylating Akt on Thr-308. PDK1 kinase activity was thought to be constitutively active; however, recent reports suggested that its activity is regulated by binding to other proteins, such as protein kinase C-related kinase-2 (PRK2), p90 ribosomal protein S6 kinase-2 (RSK2), and heat-shock protein 90 (Hsp90). Here we report that PDK1 binds to 14-3-3 proteins in vivo and in vitro through the sequence surrounding Ser-241, a residue that is phosphorylated by itself and is critical for its kinase activity. Mutation of PDK1 to increase its binding to 14-3-3 decreased its kinase activity in vivo. By contrast, mutation of PDK1 to decrease its interaction with 14-3-3 resulted in increased PDK1 kinase activity. Moreover, incubation of wild-type PDK1 with recombinant 14-3-3 in vitro decreased its kinase activity. These data indicate that PDK1 kinase activity is negatively regulated by binding to 14-3-3 through the PDK1 autophosphorylation site Ser-241.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Many growth factors and cytokines have been reported to promote cell survival. Stimulation of cells with these factors activates phosphatidylinositide 3-OH kinase (PI3K),1 and activated PI3K generates the phospholipid second messenger molecules phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate (1-3). These lipids then induce activation of several members of the AGC family of protein kinases including Akt, p70 ribosomal protein S6 kinase (p70S6K), serum, and glucocorticoid-inducible kinases (SGKs), protein kinase A (PKA), and protein kinase C (PKC) isoforms. Activated kinases, then, mediate survival-signal transduction by phosphorylating downstream key regulatory proteins.

3-Phosphoinositide-dependent protein kinase-1 (PDK1) was originally identified as a kinase that could phosphorylate Akt on its activation loop (residue Thr-308) (4-6). Later studies, however, have showed that PDK1 is not just an Akt kinase but also a kinase phosphorylating p70S6K, SGKs, PKC isoforms, and p90 ribosomal protein S6 kinases (RSKs) at the equivalent residues of Thr-308 of Akt (reviewed in Ref. 7). Therefore, PDK1 plays a central role in activating the AGC family of protein kinases. In the case of Akt, the interaction of phosphatidylinositol 3,4,5-trisphosphate with the pleckstrin homology domain recruits Akt to the plasma membrane and promotes conformational change, which results in phosphorylation of Akt at Thr-308 by PDK1 and at Ser-473 by an as yet unidentified kinase (so called PDK2) (7, 8). In the case of p70S6K, SGKs, and PKC isoforms, however, the precise mechanisms for PDK1- dependent phosphorylation and activation are not well understood.

PDK1 itself is also a member of the AGC subfamily of protein kinases and is phosphorylated on the Ser-241 activation loop (equivalent to Thr-308 of Akt) (7). As PDK1 expressed in bacteria is active and is phosphorylated at Ser-241 (9), it is thought to phosphorylate itself at this same site. Further, mutation of Ser-241 to Ala was reported to abolish PDK1 kinase activity, and IGF-I stimulation did not cause further activation of PDK1 (9). According to these results, PDK1 was thought to be constitutively active. Several recent reports, however, suggested that PDK1 kinase activity is controlled by PDK1-associating proteins, such as PKC-related kinase-1 (PRK1)/PRK2 (10), PDK1-interacting fragment of PRK2 (11), RSK2 (12), and Hsp90 (13). The interaction of PDK1 with the PDK1-interacting fragment of PRK2 converts PDK1 from an enzyme that phosphorylates Akt only at Thr-308 into a kinase that phosphorylates both Thr-308 and Ser-473 (11). Further, the association of PDK1 with RSK2 stimulates PDK1 activation and autophosphorylation (12). We recently reported that Hsp90 participates in stability and signaling of PDK1 (13). Therefore, PDK1 is not a constitutively active kinase but a kinase regulated by other interacting proteins.

Members of the 14-3-3 protein family are highly conserved and widely expressed 28-31 kDa proteins that naturally assemble as homodimers or heterodimers. They consists of at least seven isoforms in mammals (beta , gamma , epsilon , eta , sigma , theta , tau , and zeta ). The 14-3-3 proteins have been shown to interact with and to regulate proteins controlling a wide array of signaling pathways, including Raf-1, Bad, FKHRL1, and Cdc25c (reviewed in Ref. 14). Binding of 14-3-3 to its partners depends on phosphorylation of the Ser or Thr residue in the recognition domains. Using peptides derived from Raf-1, Muslin et al. (15) identified that the motif optimal for association with 14-3-3 proteins was RSXpSXP, where pS represents phosphorylated Ser and X represents any amino acid. Moreover, using phosphopeptide libraries, Yaffe et al. (16) and Rittinger et al. (17) revealed that there are two preferred 14-3-3 binding motifs, RSXpSXP and RXXXpSXP, and most of the 14-3-3 partners identified to date contain one of these motifs. It is important to note that several proteins that associate with 14-3-3 proteins do not contain either of these motifs, indicating that 14-3-3 can bind to some variation (e.g. RXXpS in PTPH1 and TH, RX(X)pSXX(X)S in Cbl and PKCµ). Furthermore, 14-3-3 bound to some proteins and peptides in a phosphorylation-independent manner (14, 18).

In the course of searching protein-protein interacting motifs in PDK1, we found four 14-3-3 binding motifs (RXXpS). Thus, we started to examine the interaction between PDK1 and 14-3-3 proteins, and of the five 14-3-3 isoforms we tested, 14-3-3theta and eta  were found to form a complex with PDK1. Using site-directed mutants, we identified the 14-3-3 recognition site in PDK1. Because the association of PDK1 with 14-3-3 reduced PDK1 kinase activity in vitro and in vivo, we concluded that 14-3-3 negatively regulates PDK1 by complex formation.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents-- The recombinant human active PDK1 and inactive SGK proteins were obtained from Upstate Biotechnology (Lake Placid, NY).

Cell Culture Conditions-- Human embryonic kidney 293T, African green monkey kidney COS-7, human fibrosarcoma HT1080, and mouse fibroblast NIH/3T3 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. In some experiments, cells were precultured for 5 h (for HT1080 cells) or 21 h (for COS-7 cells) in serum-free medium (serum starvation) before serum addition.

Plasmids-- 14-3-3theta , beta , zeta , eta , and epsilon  cDNAs were generated by PCR with human fetal brain cDNA library (Invitrogen, Carlsbad, CA) as the template. The sense (5'-TGGAGAAGACTGAGCTGATCCAG-3') and antisense (5'-TTAGTTTTCAGCCCCTTCTGCCG-3') primers for 14-3-3theta were synthesized according to the sequence from GenBankTM (accession number X56468). The sense (5'-TGACAATGGATAAAAGTGAGCTGG-3') and antisense (5'-TTAGTTCTCTCCCTCCCCAGCGTC-3') primers for 14-3-3beta were synthesized according to the sequence from GenBankTM (accession number X57346). The sense (5'-TGGATAAAAATGAGCTGGTTCAGAAGGCC-3') and antisense (5'-TTAATTTTCCCCTCCTTCTCCTGC-3') primers for 14-3-3zeta were synthesized according to the sequence from GenBankTM (accession number M86400). The sense (5'-ACATCGGGGACCGGGAGCAGCTGCTGCAG-3') and antisense (5'-CCAGGGGATCTGAAGGATCTTCAG-3') primers for 14-3-3eta were synthesized according to the sequence from GenBankTM (accession number L20422). The sense (5'-TGGATGATCGAGAGGATCTGGTGTACC-3') and antisense (5'-TCACTGATTTTCGTCTTCCACGTCCTGC-3') primers for 14-3-3epsilon were synthesized according to the sequence from GenBankTM (accession number U28936). The PCR products were cloned into a pCRII vector (Invitrogen). Substitutions of Arg at 56, 60, or 127 with Ala (R56A, R60A, or R127A, respectively) in 14-3-3theta were accomplished by converting the Arg codon AGG or CGG to the Ala codon GCG by PCR-based mutagenesis using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). These cDNAs were then cut with EcoRI (two sites within the pCRII vector), to release EcoRI-EcoRI fragments and were subcloned into an EcoRI site of a pFLAG-CMV-2 vector (Sigma), a pHM6 vector (Roche Molecular Biochemicals), or a pGEX-6P-3 vector (Amersham Biosciences).

The Myc-tagged, human, full-length wild-type PDK1 cDNA (WT-PDK1) in a pCMV3 vector was kindly provided by Drs. P. Hawkins and K. Anderson (The Babraham Institute, Cambridge, UK) (19). The NH2-terminal deleted PDK1 cDNA that encompassed residues 52-556 (Delta N51-PDK1) was generated by reverse transcription-PCR (RT-PCR) with 293T mRNA as the template and then subcloned into a pFLAG-CMV-2 vector (Sigma) (13). Substitutions of Ser at 241, 410, or 549 with Ala (S241A, S410A, or S549A, respectively), Arg at 238 with Glu (R238E), Ala at 239 with Glu or Gln (A239E or A239Q, respectively), or Val at 243 with Pro (V243P) in PDK1 cDNA was accomplished by PCR-based mutagenesis. The double-point mutant was established by further PCR mutagenesis. The wild type human akt1 cDNAs in a pFLAG-CMV-2 vector or a pHM6 vector were established in our laboratory (20). The NH2-terminal myristoylated (Myr) active mouse akt1 cDNA in a pUSEamp vector was purchased from Upstate Biotechnology. The NH2-terminal-deleted sgk cDNA that encompassed residues 61-431 (Delta N60-SGK) was generated by reverse transcription-PCR (RT-PCR) with Saos-2 mRNA as the template and then subcloned into a pFLAG-CMV-2 vector (Sigma). The wild type human v-raf-1 cDNA in a pCMV vector was purchased from Clontech (Palo Alto, CA). All the plasmid DNAs for transfection were purified using a Qiagen plasmid Maxi kit, according to the manufacturer's protocol (Qiagen, Chatsworth, CA).

Transient Transfection, Immunoprecipitation, and Western Blot Analysis-- Cells were transfected with appropriate plasmids using Superfect reagent (Qiagen) or LipofectAMINE 2000 reagent (Invitrogen), according to the manufacturer's instructions.

Cells were harvested and solubilized in lysis buffer (20 mM Tris-HCl, pH 7.5, 0.2% Nonidet P-40, 10% glycerol, 1 mM EDTA, 1.5 mM magnesium chloride, 137 mM sodium chloride, 50 mM sodium fluoride, 1 mM sodium vanadate, 12 mM beta -glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, and 1 mM aprotinin) (21). For immunoprecipitation, FLAG-tagged proteins were immunoprecipitated with an agarose conjugated with an anti-FLAG M2 antibody (anti-FLAG agarose; Sigma), HA-tagged proteins were immunoprecipitated with an agarose conjugated with an anti-HA antibody (clone F-7) (anti-HA agarose; Santa Cruz Biotechnology, Santa Cruz, CA), and Myc-tagged proteins were immunoprecipitated with an agarose conjugated with an anti-Myc antibody (clone 9E10) (anti-Myc agarose; Santa Cruz Biotechnology) (13, 21). Raf-1 was immunoprecipitated with protein L-agarose (Santa Cruz Biotechnology) that had been conjugated with an anti-Raf-1 antibody (BD Transduction Laboratories, Lexington, KY). In some experiments, cell lysates were incubated with protein A-Sepharose (Zymed Laboratories Inc., South San Francisco, CA) that had been conjugated with a control rabbit IgG or an anti-14-3-3theta antibody (Santa Cruz Biotechnology). Then the immunoprecipitated proteins or the cell lysates were electrophoresed and blotted onto a nitrocellulose or a polyvinylidene difluoride membrane. The membranes were incubated with antibodies to the phospho-14-3-3 binding motif, Akt, or phospho-Akt (Thr-308) (Cell Signaling Technology, Beverly, MA), antibodies to Raf-1 or PDK1 (BD Transduction Laboratories), an antibody to FLAG tag (clone M2; Sigma), an antibody to HA tag (clone 3F10; Roche Molecular Biochemicals), or antibodies to 14-3-3 (clone K-19), 14-3-3theta , glutathione S-transferase (GST), phospho-SGK (Thr-256), or Myc tag (clone 9E10) (Santa Cruz Biotechnology). Subsequently, membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody. After washing several times, the membranes were developed with an enhanced chemiluminescence (ECL) system according to the manufacturer's instructions (Roche Molecular Biochemicals).

Purification of Recombinant GST and GST·14-3-3theta Proteins-- Cultures of Escherichia coli containing a pGEX 6P-3 plasmid encoding nothing or 14-3-3theta were induced for 2 h with 1 mM isopropyl-beta -D-thiogalactopyranoside at 30 °C with shaking. Cells were harvested, and recombinant proteins were purified using GST Purification Modules according to the manufacturer's instructions (Amersham Biosciences).

Measurement of PDK1 Kinase Activity-- To estimate the autophosphorylation of PDK1, the recombinant human active PDK1 was incubated with GST or GST·14-3-3theta for 2 h at 30 °C, following incubation with 15 µM mouse RSK2 peptides (residues 373-396), non-phosphorylated RSK2 peptide (KKPPSANAHQLFRGFSFVAITSDDE; Ser386 peptide) or phosphorylated RSK2 peptide (KKPPSANAHQLFRGF(pS)FVAITSDDE; pSer386 peptide) for 20 min at 30 °C (12). To estimate the PDK1-dependent SGK phosphorylation in vitro, COS-7 cells were transfected with pCMV3-WT-PDK1 or pCMV3-R238E-PDK1 cDNA. After transfection for 36 h, cells were harvested. The immunoprecipitated Myc-tagged wild type or the mutant PDK1 was incubated with GST or GST·14-3-3theta for 2 h at 4 °C, following incubation with phosphorylated RSK2 peptide for 2 h at 30 °C. Then, 500 ng of inactive SGK was added to the reactions. PDK1 kinase assay was started by adding 20 µM ATP (final concentration) containing 15 µCi [gamma -32P]ATP (13, 21). After incubation for a further 20 min at 30 °C, the reactions were electrophoresed and visualized by autoradiography (12). The relative amounts of incorporated radioactivity were visualized and quantified with a BAS1500 Bio-Imaging analyzer (Fuji Film, Tokyo, Japan).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

14-3-3 Proteins Bind to PDK1 In Vivo and In Vitro-- In order to find novel PDK1-binding proteins that regulate PDK1 kinase activity, we searched protein-protein interaction motifs in PDK1. We found that it has four 14-3-3 binding motifs (RXXpS, pS represents phosphorylated Ser, and X represents any amino acid). Among the four (3RTTS6, 238RANS241, 407RSGS410, 546RYQS549), two Ser residues (Ser-241 and Ser-410) have been reported to be phosphorylated in cells (9). First, we confirmed the presence of 14-3-3 binding motifs in PDK1 by immunoblot analysis using an anti-phospho-14-3-3 binding motif antibody that could preferentially recognize the conserved 14-3-3 recognition motif (RXX(S/T)) in which X may be any amino acid) only when Ser or Thr was phosphorylated (14, 18). As shown in Fig. 1A, Delta N51-PDK1, which lacks the NH2-terminal 51 amino acids, was recognized by the anti-phospho-14-3-3 binding motif antibody (lane 2). The full-length PDK1 was also recognized by the antibody (data not shown). Raf-1 is well known to form a complex with 14-3-3 (14, 18), and we observed that it was also recognized by the antibody (Fig. 1A, lane 4). To date, it has not been reported whether Akt forms a complex with 14-3-3. Fig. 1A shows that Akt was barely recognized in the same analysis (lane 3) although it contains one potential 14-3-3 binding motif (120-RSGpS-124).


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  		Fig. 1.   PDK1 binding to 14-3-3 isoforms in vivo and in vitro. A, 293T cells were transfected with pFLAG-CMV-2 vector encoding nothing (Mock; lane 1), Delta N51-PDK1 (lane 2), or WT-Akt (Akt; lane 3), or with pCMV vector encoding v-Raf-1 (Raf-1; lane 4). The immunoprecipitated FLAG-tagged proteins (lanes 1-3) and Raf-1 protein (lane 4) were immunoblotted with the indicated antibodies. B, 293T cells were transfected with pCMV3 vector containing nothing (Mock) or WT-PDK1 cDNA (WT-PDK1) together with pHM6 vector containing nothing (Mock; lanes 1 and 7), 14-3-3theta (theta ; lanes 2 and 8), 14-3-3beta (beta ; lanes 3 and 9), 14-3-3zeta (zeta ; lanes 4 and 10), 14-3-3eta (eta ; lanes 5 and 11), or 14-3-3epsilon (epsilon ; lanes 6 and 12) cDNAs. Proteins were immunoprecipitated with an anti-Myc agarose and were immunoblotted with the indicated antibodies. Expression level of HA-tagged 14-3-3 isoforms was confirmed by immunoblot analysis (lower panel). C and D, 293T cells were transfected with pFLAG-CMV-2 vector containing nothing (Mock), 14-3-3theta (theta ), 14-3-3beta (beta ), or 14-3-3zeta (zeta ) cDNAs together with pCMV-raf-1 (C) or pHM6-akt (D) plasmid. Proteins were immunoprecipitated with an anti-FLAG agarose and were immunoblotted with the indicated antibodies. E, endogenous 14-3-3theta proteins were immunoprecipitated from 293T and NIH/3T3 cells with an anti-14-3-3theta antibody (alpha -14-3-3theta ). For control experiments, the cell lysates were incubated with protein A-Sepharose conjugated with control rabbit antibody (Cont. IgG). The precipitated proteins (lanes 1, 2, 4, and 5) and each cell lysate (lanes 3 and 6) were immunoblotted with the indicated antibodies. F, GST (lane 1) or GST·14-3-3theta (lane 2) bound to glutathione-Sepharose was incubated with lysates of COS-7 cells expressing WT-PDK1 for 2 h at 30 °C. The precipitate proteins (lanes 1 and 2) and cell lysates (lane 3) were immunoblotted with the indicated antibodies. The positions of molecular mass standards (kDa) are shown on the right.

To confirm the interacton between PDK1 and 14-3-3 in cells, we transfected Myc-tagged PDK1 cDNA together with HA-tagged 14-3-3 isoform cDNAs into 293T cells, following immunoprecipitation of PDK1 with an anti-Myc antibody. Immunoblot analysis revealed that 14-3-3theta and eta , but not 14-3-3beta , zeta , or epsilon , were co-immunoprecipitaetd with PDK1 (Fig. 1B). This result indicates that among the five 14-3-3 isoforms we tested, only 14-3-3theta and eta  bind to PDK1 in cells, and 14-3-3theta binds to PDK1 more tightly than 14-3-3eta does (Fig. 1B). We then examined the binding of 14-3-3theta , beta  and zeta  to Raf-1 and found that it bound to all the three isoforms with almost the same affinity (Fig. 1C). On the other hand, Akt bound to none of the three isoforms under the same conditions (Fig. 1D), which is in agreement with the data that Akt was not recognized by the anti-phospho-14-3-3 binding motif antibody (Fig. 1A, lane 3).

To further examine the association between endogenous PDK1 and endogenous 14-3-3theta in cells, we immunoprecipitated the endogenous 14-3-3theta proteins from 293T and NIH/3T3 cells with an anti-14-3-3theta antibody, following immunoblot analysis with an anti-PDK1 antibody (Fig. 1E). We found that endogenous PDK1 was co-immunoprecipitated with 14-3-3theta . In order to reconstitute the interaction between PDK1 and 14-3-3theta in vitro, purified recombinant GST alone or GST-fused 14-3-3theta was incubated for 2 h at 30 °C (Fig. 1F) or at 4 °C (data not shown) with the lysate of COS-7 cells that express Myc-tagged PDK1. Then, GST or GST-fused 14-3-3theta proteins were precipitated by glutathione-Sepharose. PDK1 was co-precipitated with GST·14-3-3theta under both conditions (Fig. 1F, upper panel, lane 2 and data not shown) and, to lesser degree, with GST alone (lane 1). Raf-1 was also co-precipitated with 14-3-3theta , and its association was more specific than PDK1 (Fig. 1F, middle panel, lane 2). These results indicate that PDK1 binds to 14-3-3 in cells and in vitro.

Mutation in 14-3-3theta Impairs the Association between 14-3-3theta and PDK1-- The co-crystal structure of 14-3-3 with synthetic phosphopeptide demonstrated that the phosphopeptide binds along a groove on the inner surface of 14-3-3 (16, 17). Mutational analysis revealed that alteration of the residues exposed on the binding surface, such as Arg-56, Arg-60, and Arg-127, impairs the phosphopeptide binding ability (17, 22). To confirm the interaction of PDK1 and 14-3-3theta mutants in which its binding surface residues were changed to Ala (R56A/R60A and R127A), we transfected these mutant cDNAs into 293T cells together with Delta N51-PDK1 cDNA. Immunoprecipitation of HA-tagged 14-3-3theta or 14-3-3theta mutants followed by immunoblot analysis revealed that PDK1 was co-precipitated with the R56,60A-PDK1 mutant but not the R127A-PDK1 mutant (Fig. 2A). As a control, we also examined Raf-1 binding to 14-3-3theta or its mutants. Consistent with the previous report using a phospho-Ser-containing Raf-1 peptide, Raf-1 did not co-precipitate with either of the 14-3-3theta mutants (Fig. 2B, lanes 3 and 4 and Ref. 23). This result indicates that PDK1 associates with 14-3-3theta in the binding pocket of 14-3-3theta and that Arg-127 is important for binding.


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  		Fig. 2.   Specificity of PDK1 binding to 14-3-3theta mutants. A and B, 293T cells were transfected with pHM6 vector encoding nothing (Mock), 14-3-3theta (WT), R56A/R60A-14-3-3theta (R56,60A), and R127A-14-3-3theta (R127A) together with pFLAG-CMV-2 vector encoding nothing (A, lane 1) or Delta N51-PDK1 (A, lanes 2-5), or pCMV vector encoding v-Raf-1 (B, lanes 1-4). Proteins were immunoprecipitated with an anti-HA agarose and were immunoblotted with the indicated antibodies (upper and middle panels). Expression level of transfected FLAG-tagged Delta N51-PDK1 and Raf-1 proteins were confirmed by immunoblot analysis with an anti-FLAG antibody or an anti-Raf-1 antibody, respectively (lower panels). The positions of molecular mass standards (kDa) are shown on the right.

Identification of the Ser-241 Residue in PDK1 as the 14-3-3 Recognition Site-- As shown in Fig. 3A, 14-3-3 recognizes conserved motifs containing phospho-Ser (e.g. RXRXXpSXP, RSXpSXP, and RXXpS). PDK1 has several reported 14-3-3 binding motifs in its amino acid sequence (3RTTS6, 238RANpS241, 407RSGpS410, 546RYQS549, Ref. 9), so we tried to identify the sites. Substitution at residue Ser-241, Ser-410, or Ser-549 with Ala (S241A, S410A, or S549A, respectively) was introduced to PDK1, and the ability to bind to 14-3-3theta was examined. We did not generate a mutant in which Ser-6 was converted to Ala, since Delta N51-PDK1 that lacks Ser-6 could also interact with 14-3-3 with similar affinity as WT-PDK1 (data not shown). Co-immunoprecipitation analysis indicated that mutation at Ser-241 impairs the PDK1 binding ability for 14-3-3theta and 14-3-3eta (Fig. 3B, upper panel, lane 3, and data not shown). On the other hand, mutations at Ser-410 and Ser-549 did not affect the PDK1-14-3-3theta binding (Fig. 3B, upper panel, lanes 4 and 5, respectively). Consistent with the result of this analysis, S241A was not recognized by the anti-phospho-14-3-3 binding motif antibody (Fig. 3B, second panel, lane 3), but S410A and S549A were recognized (lanes 4 and 5). This result indicates the importance of Ser-241 in PDK1 interaction with 14-3-3theta and 14-3-3eta .


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  		Fig. 3.   Identification of phospho-Ser-241 in PDK1 as a 14-3-3theta recognition site. A, schematic representation of the 14-3-3 binding motifs of the known 14-3-3 binding partners, the potential 14-3-3 binding sequences in PDK1, and the sequences of the generated PDK1 mutants. *S represents phosphorylated Ser, and X represents any amino acids. B, 293T cells were transfected with pFLAG-CMV-2 vector encoding nothing (Mock, lane 1), Delta N51-PDK1 (WT, lane 2), S241A-PDK1 (S241A, lane 3), S410A-PDK1 (S410A, lane 4), and S549A-PDK1 (S549A, lane 5) together with pHM6 vector containing 14-3-3theta cDNA (+; lanes 1-5). Proteins were immunoprecipitated with an anti-FLAG agarose and were immunoblotted with the indicated antibodies. Expression level of transfected HA-tagged 14-3-3theta protein was confirmed by immunoblot analysis with an anti-HA antibody (lower panel). C and D, 293T cells were transfected with pCMV3 vector encoding nothing (Mock), WT-PDK1 (WT), or the indicated PDK1 mutants together with pHM6 vector containing 14-3-3theta cDNA. Double stands for a double PDK1 point mutant (S241A/V243P-PDK1). Proteins were immunoprecipitated with an anti-Myc agarose and were immunoblotted with the indicated antibodies. Expression level of transfected HA-tagged 14-3-3theta protein was confirmed by immunoblot analysis with an anti-HA antibody (lower panels). The positions of molecular mass standards (kDa) are shown on the right.

To further confirm the role of residues surrounding Ser-241 of PDK1, we generated other PDK1 point mutants in which these residues were changed (Fig. 3A). Substitution of Arg-238, equivalent to conserved Arg in the 14-3-3 binding motif, with Glu (R238E) decreased 14-3-3theta binding to PDK1 (Fig. 3C, upper panel, lane 4). In contrast, substitution of Val-243 with Pro (V243P), a residue conserved in many 14-3-3 targets, such as Raf-1 and Bad (Fig. 3A), dramatically increased the amount of 14-3-3theta bound to PDK1 (Fig. 3C, upper panel, lane 7). Changes in the binding ability of R238E and V243P were similarly observed when these mutants were incubated in vitro with GST·14-3-3theta (data not shown). Additional mutation of Ser-241 to Ala in V243P (V243P/S241A; Double) reduced the binding affinity of PDK1 to 14-3-3theta (Fig. 3D, upper panel, lane 4), indicating the importance of phospho-Ser-241 for PDK1-14-3-3theta binding. The mutation of Ala-239 to Glu (A239E) or to Gln (A239Q) did not affect the PDK1-14-3-3 complex formation (Fig. 3C, upper panel, lane 5 or 6, respectively). Therefore, 14-3-3theta and 14-3-3eta recognized and bound to the 238-RANpS-241 residue in PDK1. Moreover, Arg-238 and phospho-Ser-241 residues are important for 14-3-3 binding.

Regulation of PDK1 Kinase Activity by Binding to 14-3-3-- Ser-241 of PDK1 was reported to be phosphorylated by itself (9). Phosphorylation of PDK1 at Ser-241 is suggested to be essential for its kinase activity because the conversion of Ser-241 to Ala dramatically reduces PDK1 kinase activity (9). Therefore, it is possible that 14-3-3theta regulates PDK1 kinase activity by associating with PDK1 at Ser-241 residue. We then investigated the role of PDK1-14-3-3theta complex formation on PDK1 kinase activity. PDK1 mutant cDNAs were co-transfected with WT-akt cDNA into COS-7 cells, and the kinase activity of PDK1 and its mutants were estimated by the amount of the phosphorylated form of Akt at Thr-308 after serum stimulation. Consistent with a previous report (9), S241A exhibited weak phosphorylation activity when compared with WT-PDK1 (Fig. 4A, upper panel, compare lanes 4 and 3). It is important to note that mutating Arg-238 to Glu (R238E) to decrease the interaction between PDK1 and 14-3-3 resulted in a slight increase in PDK1 kinase activity (Fig. 4A, upper panel, lane 5). By contrast, converting Val-243 to Pro (V243P) to increase the association with 14-3-3theta reduced that activity (Fig. 4A, upper panel, lane 6). Using HT1080 cells, we obtained almost the same result (data not shown). To exclude the possibility that these changes in kinase activity were due to the changes in the affinity of the mutants to Akt, we investigated the binding of the PDK1 mutants to Akt. We did this by immunoprecipitating PDK1 following immunoblot analysis. Fig. 4B shows that no change in binding ability of R238E and V243P, or in S241A, was observed. Moreover, we saw no change in Akt binding to mutant PDK1 proteins when Akt was immunoprecipitated from the cell lysate (data not shown). Because SGK was also known to be phosphorylated by PDK1 at the Thr-256 residue (7), we estimated the kinase activities of PDK1 point mutants by transfecting Delta N60-sgk cDNA together with PDK1 mutant cDNAs into COS-7 cells. Immunoblot analysis with an anti-phospho-SGK (Thr-256) antibody revealed that mutation of Ser-241 to Ala in PDK1 abrogated PDK1 activity in phosphorylating Delta N60-SGK (Fig. 4C, upper panel, lane 4). We also observed that converting Val-243 to Pro (V243P) to increase the association with 14-3-3theta decreased the PDK1-dependent phosphorylation of SGK at Thr-256 (Fig. 4C, upper panel, lane 6), compared with WT-PDK1 (lane 3). These results suggest that in cells, 14-3-3 proteins negatively regulate PDK1 kinase activity by binding to PDK1 at the residues surrounding Ser-241, but phosphorylation of PDK1 at Ser-241 positively regulates its activity.


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  		Fig. 4.   Negative regulation of PDK1 kinase activity by binding to 14-3-3 in vivo. A and B, COS-7 cells were transfected with pCMV3 vector encoding nothing (Mock), WT-PDK1 (WT), or the indicated PDK1 point mutants together with pHM6 vector containing nothing (-) or WT-akt (+) cDNA. After transfection for 24 h, cells underwent serum stimulation for 10 min after serum-starvation for 21 h. Each cell lysate was immunoblotted with the indicated antibodies (A). Proteins were immunoprecipitated with an anti-Myc-agarose and were immunoblotted with the indicated antibodies (B). Expression level of transfected HA-tagged Akt protein was confirmed by immunoblot analysis with an anti-HA antibody (B, lower panel). C, COS-7 cells were transfected with pCMV3 vector encoding nothing (Mock; lanes 1 and 2), WT-PDK1 (WT; lane 3), or the indicated PDK1 point mutants (lanes 4-6) together with pFLAG-CMV-2 vector containing nothing (-; lane 1) or Delta N60-sgk (+; lanes 2-6) cDNA. Cells were treated as described in A, and each cell lysate was immunoblotted with the indicated antibodies. D, COS-7 cells were transfected with pUSEamp vector encoding Myr-Akt (+; lanes 1-3) together with pHM6 vector containing nothing (-; lane 1), 14-3-3theta (theta ; lane 2), or 14-3-3eta (eta ; lane 3) cDNA. After transfection for 24 h, cells were harvested, and each cell lysate was immunoblotted with the indicated antibodies. The positions of molecular mass standards (kDa) are shown on the right.

To further confirm the role of 14-3-3 in PDK1 function, we examined the amount of the phosphorylated form of Akt at Thr-308 in COS-7 cells by transfecting the myristoylated form of akt cDNA (Myr-Akt) together with 14-3-3theta or 14-3-3eta cDNA. Tagging Akt with c-Src-derived residues required for myristoylation could induce the constitutive targeting of Myr-Akt to the plasma membrane in a PI3K-independent manner. As shown in Fig. 4D, transfection of 14-3-3theta (lane 2) or 14-3-3eta (lane 3) decreased the phospho-Akt (Thr-308) level of transfected Myr-Akt protein. A decrease in the amount of the phosphorylated form of Akt was also observed when WT-Akt was co-transfected with 14-3-3theta or 14-3-3eta cDNA (data not shown). These results suggest that 14-3-3 proteins suppress PDK1 kinase activity in cells, and the 14-3-3 binding to PDK1 is important for cellular signal transduction.

PDK1 Activity Was Down-regulated by Binding to 14-3-3theta In Vitro-- To confirm the negative regulation of PDK1 kinase activity by 14-3-3, we incubated recombinant PDK1 with recombinant GST·14-3-3theta in vitro and estimated the change of PDK1 autophosphorylation activity. After the incubation with GST or GST-fused 14-3-3theta at 30 °C for 2 h, a condition sufficient to reconstitute specific interaction of PDK1 with 14-3-3theta in vitro (Fig. 1F), PDK1 was incubated with phosphorylated or non-phosphorylated RSK2 peptide. Then, the autophosphorylation activity was examined by autoradiography. As shown in Fig. 5A, preincubation with GST·14-3-3theta down-regulated PDK1 autophosphorylation activity in a dose-dependent manner. As reported previously (12), PDK1 kinase activity was elevated by adding phosphorylated RSK2 peptide (pS386 peptide). Under this condition, GST·14-3-3theta also decreased PDK1 autophosphorylation activity (Fig. 5A). Similarly, incubation of the immunoprecipitated Myc-tagged WT-PDK1 with GST·14-3-3theta caused a reduction of PDK1 kinase activity to phosphorylate a PDK1 substrate, SGK (Fig. 5B). In contrast, the kinase activity of R238E-PDK1, which associated less with 14-3-3theta (Fig. 3C), was not affected by preincubation with 14-3-3theta (Fig. 5B). These results indicate that 14-3-3 negatively regulates PDK1 kinase activity by association with the residues surrounding the PDK1 Ser-241 residue.


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  		Fig. 5.   Incubation of PDK1 with 14-3-3theta in vitro decreases PDK1 kinase activity. A, the recombinant PDK1 was incubated with the indicated amount of GST or GST·14-3-3theta for 2 h at 30 °C, following incubation with 15 µM non-phosphorylated RSK2 peptide (non-phospho) or phosphorylated RSK2 peptide (phospho) for 20 min at 30 °C. PDK1 autophosphorylation assay was performed, as described under "Experimental Procedures." The relative amounts of incorporated radioactivity were quantified with a BAS1500 Bio-Imaging analyzer. B, COS-7 cells were transfected with pCMV3 vector encoding nothing (-), WT-PDK1 (WT) or R238E-PDK1 (R238E). The immunoprecipitated Myc-tagged proteins were incubated with the indicated amounts of GST or GST·14-3-3theta for 2 h at 4 °C, following incubation with 15 µM phosphorylated RSK2 peptide (phospho-RSK2 peptide). Then, 500 ng of recombinant inactive SGK was added to the reactions. PDK1 kinase assay was performed, as described under "Experimental Procedures." The relative amounts of 32P-SGK were quantified with a BAS1500 Bio-Imaging analyzer. The amount of the immunoprecipitated Myc-tagged PDK1 proteins was confirmed by immunoblot analysis with an anti-Myc antibody (lower panel). The positions of molecular mass standards (kDa) are shown on the right.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It is clear that PDK1 plays a central role in activating the AGC subfamily of protein kinases (7, 24). These kinases then mediate intracellular signaling such as cell survival, cell growth, protein synthesis, and gene expression. PDK1 phosphorylates AGC kinase members at the residues equivalent to Thr-308 of Akt (known as activation loop or T-loop) (7). PDK1 is, itself, a member of the AGC subfamily of protein kinases. Thus, PDK1 phosphorylates itself at its activation loop (Ser-241), thereby activating itself (9). Although PDK1 kinase activity has been thought to be constitutively active and not further activated by growth factor stimulation, recent findings suggest that its activity and its character are controlled by interaction with other proteins. For example, when PDK1 interacts with the PDK1-interacting fragment of PRK2, it is converted to exhibit PDK2-like activity (11). The association of PDK1 with RSK2 up-regulates PDK1 kinase activity and autophosphorylation (12). We recently reported that PDK1 binds to Hsp90, and its binding prevents PDK1 from proteasome-dependent degradation and keeps it in a soluble and active conformational state (13). Moreover, PDK1 kinase activity is promoted by phosphorylation at tyrosine residues, presumably by a member of the Src kinase family (25, 26).

Here we provide evidence that PDK1 binds to 14-3-3 in vivo and in vitro through the residues surrounding the autophosphorylation site Ser-241 and that the association is achieved only when Ser-241 has been phosphorylated (Fig. 3). Although PDK1 contains other 14-3-3 binding motifs in its amino acid sequence (3RTTS6, 407RSGS410, 546RYQS549), site-directed mutagenesis of the target Ser revealed that these sites were not involved in the PDK1-14-3-3 complex formation (Fig. 3B). It should be noted that 14-3-3 also bound to the S410A-PDK1 mutant in which Ser-410 was converted to Ala although the Ser-410 residue has been reported to be phosphorylated in vivo (9). There may be some conformational state essential for the 14-3-3 binding in addition to Ser phosphorylation in 14-3-3 binding motifs.

Among the 14-3-3-binding partners, some proteins, like Raf-1, bind to almost all 14-3-3 isoforms with nearly equivalent affinity (18). However, others, like A20 and Cdc25B, bind to different 14-3-3 isoforms with significantly different affinities (27, 28). We recently reported that p27Kip1 binds to 14-3-3 after Akt-mediated phosphorylation at the COOH-terminal Thr-198 (29). The phosphorylated p27Kip1 could form complexes with 14-3-3theta , eta , and epsilon  but not with 14-3-3beta and zeta  (29), suggesting that 14-3-3 isoforms have different affinities to their partners. Among the five isoforms (beta , epsilon , eta , theta , and zeta ), only 14-3-3theta and eta  interacted with PDK1, and 14-3-3theta bound more tightly than 14-3-3eta (Fig. 1B). The difference among 14-3-3theta , eta  and other isoforms that accounts for the different affinities for PDK1 is still unclear, since many of the residues that form the ligand-binding groove are conserved in the seven isoforms. However, comparing the residues in 14-3-3theta necessary for the interaction with Raf-1 and PDK1 did reveal some differences. To form a complex with Raf-1, Arg-56, Arg-60, and Arg-127 were necessary (Fig. 2B, lanes 3 and 4), a finding consistent with a previous report (23). In the case of PDK1, Arg-56 and Arg-60 were not essential but Arg-127 was (Fig. 2A). By contrast, both Arg-56 and Arg-60 are necessary but Arg-127 is not for p27Kip1 binding to 14-3-3 (Ref. 29).2 These results indicate that the residues necessary for ligand-14-3-3 binding vary with the ligand. Moreover, there may be some residues essential for the association with ligands that are not conserved in the 14-3-3 isoforms. However, we could not exclude the possibility that the differences we observed above were a result of the differences between the ligand that possessed at least two 14-3-3 binding motifs (Raf-1) and the one that had only one (PDK1). The distinct mechanism of isoform-specific interaction is a problem to be solved in future studies.

The general mechanisms by which 14-3-3 regulates partner protein functions are 4-fold: 1) regulation of subcellular localization of the target protein; 2) direct regulation of catalytic activity of the binding partner; 3) protection of the ligands from proteolysis or dephosphorylation; and 4) regulation of interaction between bound protein and other molecules. The identified 14-3-3 binding residue of PDK1 (Ser-241) is thought to be phosphorylated by itself and it be essential for its kinase activity (9). Consistent with previous reporting (9), we confirmed that S241A-PDK1 exhibited weak Akt and SGK phosphorylation activity when compared with WT-PDK1 (Fig. 4). We thus generated PDK1 point mutants in which residues other than Ser-241 are mutated (Fig. 3C) and examined their activity to phosphorylate Akt (Fig. 4A) or SGK (Fig. 4C). Mutation of PDK1 to promote 14-3-3 (V243P-PDK1) association exhibited reduced PDK1 kinase activity to phosphorylate Akt and SGK in cells, while mutation of PDK1 to inhibit the interaction (R238E-PDK1) slightly increased its activity (Fig. 4). Moreover, incubation with recombinant 14-3-3theta decreased PDK1 autophosphorylation activity (Fig. 5A) and kinase activity to phosphorylate SGK (Fig. 5B) in a dose-dependent manner in vitro. These results indicate that 14-3-3 is not only a PDK1-associating protein but also a protein that negatively regulates PDK1 kinase activity.

Several reports indicate that PDK1 binds to phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate through its pleckstrin homology domain (19, 30). Because mutants of PDK1 deleted in its pleckstrin homology domain have been reported to prevent translocation of Akt, the relocalization of PDK1 from cytosol to the plasma membrane is suggested to play an important role in the recruitment of Akt to the plasma membrane and the subsequent Akt activation in stimulated cells (30). PDK1 catalytic activity is not required for the PDK1 translocation to the plasma membrane (30). Although mutating PDK1 to increase its binding to 14-3-3 (V243P) reduced kinase activity to phosphorylate Akt at Thr-308, the mutation did not affect the binding affinity of PDK1 to Akt (Fig. 4). Therefore, the decrease in kinase activity of V243P-PDK1 to phosphorylate Akt in cells may be caused by down-regulation of its kinase activity but not by suppressing translocation. This notion was supported by the fact that the amount of the phosphorylated form of Myr-Akt was reduced by 14-3-3 protein expression even though Myr-Akt constitutively targeted the plasma membrane (Fig. 4D). In the case of SGK, translocation to the plasma membrane is not required for PDK1-dependent phosphorylation of SGK at Thr-256 (31). SGK phosphorylation at Thr-256 was also suppressed by PDK1 mutation to increase its binding to 14-3-3 (V243P; Fig. 4C). These results strongly suggest that inhibition of PDK1 activity but not PDK1 translocation is the main mechanism of 14-3-3-mediated inactivation of PDK1-dependent signal transduction.

    ACKNOWLEDGEMENTS

We thank Drs. Philip Hawkins and Karen Anderson for providing the pCMV3-PDK1.

    FOOTNOTES

* This study was supported in part by a special grant for Advanced Research on Cancer from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to T. T.), by Foundation for Promotion of Cancer Research in Japan (to N. F.), and by a grant for Research Fellowships of the Japanese Society for the Promotion of Science for Young Scientists (to S. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-3-5841-7861; Fax: 81-3-5841-8487; E-mail: ttsuruo@iam.u-tokyo.ac.jp.

Published, JBC Papers in Press, August 12, 2002, DOI 10.1074/jbc.M205141200

2 N. Fujita, S. Sato, and T. Tsuruo, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: PI3K, phosphatidylinositide 3-OH kinase; AGC, protein kinase A, G, and C; GST, glutathione S-transferase; p70S6K, p70 ribosomal protein S6 kinase; PDK1, 3-phosphoinositide-dependent protein kinase-1; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PRK, PKC-related kinase; RSK, p90 ribosomal protein S6 kinase; SGK, serum and glucocorticoid-inducible kinase; WT, wild type; HA, hemagglutinin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Vanhaesebroeck, B., Leevers, S. J., Panayotou, G., and Waterfield, M. D. (1997) Trends Biochem. Sci. 22, 267-272[CrossRef][Medline] [Order article via Infotrieve]
2. Toker, A., and Cantley, L. C. (1997) Nature 387, 673-676[CrossRef][Medline] [Order article via Infotrieve]
3. Rodriguez-Viciana, P., Warne, P. H., Khwaja, A., Marte, B. M., Pappin, D., Das, P., Waterfield, M. D., Ridley, A., and Downward, J. (1997) Cell 89, 457-467[CrossRef][Medline] [Order article via Infotrieve]
4. Alessi, D. R., James, S. R., Downes, C. P., Holmes, A. B., Gaffney, R. P. J., Reeese, C. B., and Cohen, P. (1997) Curr. Biol. 7, 261-269[CrossRef][Medline] [Order article via Infotrieve]
5. Stephens, L., Anderson, K., Stokoe, D., Erdjument-Bromage, H., Painter, G. F., Holmes, A. B., Gaffney, P. R., Reese, C. B., McCormick, F., Tempst, P., Coadwell, J., and Hawkins, P. T. (1998) Science 279, 710-714[Abstract/Free Full Text]
6. Le Good, J. A., Ziegler, W. H., Parekh, D. B., Alessi, D. R., Cohen, P., and Parker, P. J. (1998) Science 281, 2042-2045[Abstract/Free Full Text]
7. Vanhaesebroeck, B., and Alessi, D. R. (2000) Biochem. J. 346, 561-576[CrossRef][Medline] [Order article via Infotrieve]
8. Franke, T. F., Kaplan, D. R., and Cantley, L. C. (1997) Cell 88, 435-437[CrossRef][Medline] [Order article via Infotrieve]
9. Casamayor, A., Morrice, N. A., and Alessi, D. R. (1999) Biochem. J. 342, 287-292[Medline] [Order article via Infotrieve]
10. Wick, M. J., Dong, L. Q., Riojas, R. A., Ramos, F. J., and Liu, F. (2000) J. Biol. Chem. 275, 40400-40406[Abstract/Free Full Text]
11. Balendran, A., Casamayor, A., Deak, M., Paterson, A., Gaffney, P., Currie, R., Downes, C. P., and Alessi, D. R. (1999) Curr. Biol. 9, 393-404[CrossRef][Medline] [Order article via Infotrieve]
12. Frodin, M., Jensen, C. J., Merienne, K., and Gammeltoft, S. (2000) EMBO J. 19, 2924-2934[CrossRef][Medline] [Order article via Infotrieve]
13. Fujita, N., Sato, S., Ishida, A., and Tsuruo, T. (2002) J. Biol. Chem. 277, 10346-10353[Abstract/Free Full Text]
14. Tzivion, G., and Avruch, J. (2002) J. Biol. Chem. 277, 3061-3064[Free Full Text]
15. Muslin, A. J., Tanner, J. W., Allen, P. M., and Shaw, A. S. (1996) Cell 84, 889-897[CrossRef][Medline] [Order article via Infotrieve]
16. Yaffe, M. B., Rittinger, K., Volinia, S., Caron, P. R., Aitken, A., Leffers, H., Gamblin, S. J., Smerdon, S. J., and Cantley, L. C. (1997) Cell 91, 961-971[CrossRef][Medline] [Order article via Infotrieve]
17. Rittinger, K., Budman, J., Xu, J., Volinia, S., Cantley, L. C., Smerdon, S. J., Gamblin, S. J., and Yaffe, M. B. (1999) Mol. Cell 4, 153-166[CrossRef][Medline] [Order article via Infotrieve]
18. Yaffe, M. B. (2002) FEBS Lett. 513, 53-57[CrossRef][Medline] [Order article via Infotrieve]
19. Anderson, K. E., Coadwell, J., Stephens, L. R., and Hawkins, P. T. (1998) Curr. Biol. 8, 684-691[CrossRef][Medline] [Order article via Infotrieve]
20. Sato, S., Fujita, N., and Tsuruo, T. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 10832-10837[Abstract/Free Full Text]
21. Sato, S., Fujita, N., and Tsuruo, T. (2002) Oncogene 21, 1727-1738[CrossRef][Medline] [Order article via Infotrieve]
22. Zhang, L., Wang, H., Liu, D., Liddington, R., and Fu, H. (1997) J. Biol. Chem. 272, 13717-13724[Abstract/Free Full Text]
23. Thorson, J. A., Yu, L. W., Hsu, A. L., Shih, N. Y., Graves, P. R., Tanner, J. W., Allen, P. M., Piwnica-Worms, H., and Shaw, A. S. (1998) Mol. Cell. Biol. 18, 5229-5238[Abstract/Free Full Text]
24. Williams, M. R., Arthur, J. S., Balendran, A., van der Kaay, J., Poli, V., Cohen, P., and Alessi, D. R. (2000) Curr. Biol. 10, 439-448[CrossRef][Medline] [Order article via Infotrieve]
25. Grillo, S., Gremeaux, T., Casamayor, A., Alessi, D. R., Le, Marchand-Brustel, Y., and Tanti, J. F. (2000) Eur. J. Biochem. 267, 6642-6649[Medline] [Order article via Infotrieve]
26. Park, J., Hill, M. M., Hess, D., Brazil, D. P., Hofsteenge, J., and Hemmings, B. A. (2001) J. Biol. Chem. 276, 37459-37471[Abstract/Free Full Text]
27. Vincenz, C., and Dixit, V. M. (1996) J. Biol. Chem. 271, 20029-20034[Abstract/Free Full Text]
28. Mils, V., Baldin, V., Goubin, F., Pinta, I., Papin, C., Waye, M., Eychene, A., and Ducommun, B. (2000) Oncogene 19, 1257-1265[CrossRef][Medline] [Order article via Infotrieve]
29. Fujita, N., Sato, S., Katayama, K., and Tsuruo, T. (2002) J. Biol. Chem. 277, 28706-28713[Abstract/Free Full Text]
30. Filippa, N., Sable, C. L., Hemmings, B. A., and Van Obberghen, E. (2000) Mol. Cell. Biol. 20, 5712-5721[Abstract/Free Full Text]
31. Kobayashi, T., and Cohen, P. (1999) Biochem. J. 339, 319-328[CrossRef][Medline] [Order article via Infotrieve]

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