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• W2092022003 abstract "The phosphatidylinositide-3-OH kinase/3-phospho-inositide-dependent protein kinase-1 (PDK1)/Akt and the Raf/mitogen-activated protein kinase (MAPK/ERK) kinase (MEK)/mitogen-activated protein kinase (MAPK) pathways have central roles in the regulation of cell survival and proliferation. Despite their importance, however, the cross-talk between these two pathways has not been fully understood. Here we report that PDK1 promotes MAPK activation in a MEK-dependent manner. In vitro kinase assay revealed that the direct targets of PDK1 in the MAPK pathway were the upstream MAPK kinases MEK1 and MEK2. The identified PDK1 phosphorylation sites in MEK1 and MEK2 are Ser222 and Ser226, respectively, and are known to be essential for full activation. To date, these sites are thought to be phosphorylated by Raf kinases. However, PDK1 gene silencing using small interference RNA demonstrates that PDK1 is associated with maintaining the steady-state phosphorylated MEK level and cell growth. The small interference RNA-mediated down-regulation of PDK1 attenuated maximum MEK and MAPK activities but could not prolong MAPK signaling duration. Stable and transient expression of constitutively active MEK1 overcame these effects. Our results suggest a novel cross-talk between the phosphatidylinositide-3-OH kinase/PDK1/Akt pathway and the Raf/MEK/MAPK pathway. The phosphatidylinositide-3-OH kinase/3-phospho-inositide-dependent protein kinase-1 (PDK1)/Akt and the Raf/mitogen-activated protein kinase (MAPK/ERK) kinase (MEK)/mitogen-activated protein kinase (MAPK) pathways have central roles in the regulation of cell survival and proliferation. Despite their importance, however, the cross-talk between these two pathways has not been fully understood. Here we report that PDK1 promotes MAPK activation in a MEK-dependent manner. In vitro kinase assay revealed that the direct targets of PDK1 in the MAPK pathway were the upstream MAPK kinases MEK1 and MEK2. The identified PDK1 phosphorylation sites in MEK1 and MEK2 are Ser222 and Ser226, respectively, and are known to be essential for full activation. To date, these sites are thought to be phosphorylated by Raf kinases. However, PDK1 gene silencing using small interference RNA demonstrates that PDK1 is associated with maintaining the steady-state phosphorylated MEK level and cell growth. The small interference RNA-mediated down-regulation of PDK1 attenuated maximum MEK and MAPK activities but could not prolong MAPK signaling duration. Stable and transient expression of constitutively active MEK1 overcame these effects. Our results suggest a novel cross-talk between the phosphatidylinositide-3-OH kinase/PDK1/Akt pathway and the Raf/MEK/MAPK pathway. Many growth factors and cytokines have been reported to promote cell survival. Interaction between these factors and their specific receptors trigger the activation of phosphatidyl-inositide-3-OH kinase (PI3K) 1The abbreviations used are: PI3K, phosphatidylinositide-3-OH kinase; AGC, protein kinase A, G, and C; GST, glutathione S-transferase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; PDK1, 3-phosphoinositide-dependent protein kinase-1; PH, pleckstrin homology; PKC, protein kinase C; RSK, p90 ribosomal protein S6 kinase; SGK, serum and glucocorticoid-inducible kinase; siRNA, small interference RNA; WT, wild type; DN, dominant negative; EGF, epidermal growth factor; EGFP, enhanced green fluorescence protein; HA, hemagglutinin; MOPS, 4-morpholinepropanesulfonic acid. 1The abbreviations used are: PI3K, phosphatidylinositide-3-OH kinase; AGC, protein kinase A, G, and C; GST, glutathione S-transferase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; PDK1, 3-phosphoinositide-dependent protein kinase-1; PH, pleckstrin homology; PKC, protein kinase C; RSK, p90 ribosomal protein S6 kinase; SGK, serum and glucocorticoid-inducible kinase; siRNA, small interference RNA; WT, wild type; DN, dominant negative; EGF, epidermal growth factor; EGFP, enhanced green fluorescence protein; HA, hemagglutinin; MOPS, 4-morpholinepropanesulfonic acid. and Ras (1Lee Jr., J.T. McCubrey J.A. Leukemia. 2002; 16: 486-507Crossref PubMed Google Scholar). Activated PI3K generates the phospholipid second messenger molecules phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate (2Rodriguez-Viciana P. Warne P.H. Khwaja A. Marte B.M. Pappin D. Das P. Waterfield M.D. Ridley A. Downward J. Cell. 1997; 89: 457-467Abstract Full Text Full Text PDF PubMed Scopus (957) Google Scholar, 3Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1224) Google Scholar, 4Vanhaesebroeck B. Leevers S.J. Panayotou G. Waterfield M.D. Trends. Biochem. Sci. 1997; 22: 267-272Abstract Full Text PDF PubMed Scopus (831) Google Scholar). These lipids, then, induce the activation of several members of the protein kinase A, G, and C (AGC) family of protein kinases including Akt/protein kinase B, p70 ribosomal protein S6 kinase (p70S6K), protein kinase C isoforms (PKCs), and serum- and glucocorticoid-inducible kinases (SGKs). Activated Ras stimulates Raf translocation from cytosol to the cell membrane by the direct interaction (5Leevers S.J. Paterson H.F. Marshall C.J. Nature. 1994; 369: 411-414Crossref PubMed Scopus (883) Google Scholar, 6Winkler D.G. Cutler R Jr E. Drugan J.K. Campbell S. Morrison D.K. Cooper J.A. J. Biol. Chem. 1998; 273: 21578-21584Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), and the membrane-translocated Raf is phosphorylated and activated by multiple kinases. Activated Raf catalyzes the phosphorylation of its downstream kinases, MEK1 and MEK2 (MEK1/2), through phosphorylation at Ser218 and Ser222 of MEK1 and at Ser222 and Ser226 of MEK2 in their activation loops (7Alessi D.R. Saito Y. Campbell D.G. Cohen P. Sithanandam G. Rapp U. Ashworth A. Marshall C.J. Cowley S. EMBO J. 1994; 13: 1610-1619Crossref PubMed Scopus (466) Google Scholar, 8Zheng C.F. Guan K.L. EMBO J. 1994; 13: 1123-1131Crossref PubMed Scopus (300) Google Scholar). Then activated phospho-MEK1/2 triggers MAPK signaling pathways (9Crews C.M. Alessandrini A. Erikson R.L. Science. 1992; 258: 478-480Crossref PubMed Scopus (739) Google Scholar, 10Crews C.M. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8205-8209Crossref PubMed Scopus (152) Google Scholar). Activated kinases including Akt and MAPK1/2 mediate survival signal transduction and cell cycle progression by phosphorylating downstream key regulatory proteins (11Allan L.A. Morrice N. Brady S. Magee G. Pathak S. Clarke P.R. Nat. Cell Biol. 2003; 5: 647-654Crossref PubMed Scopus (395) Google Scholar, 12Brazil D.P. Park J. Hemmings B.A. Cell. 2002; 111: 293-303Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, 13Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1397) Google Scholar). Because it has been reported that the activity of Akt or MAPK or both are elevated in many cancer cells, these molecules are thought to be suitable as molecular targets of anticancer drugs (1Lee Jr., J.T. McCubrey J.A. Leukemia. 2002; 16: 486-507Crossref PubMed Google Scholar, 14Sebolt-Leopold J.S. Oncogene. 2000; 19: 6594-6599Crossref PubMed Scopus (299) Google Scholar, 15Vivanco I. Sawyers C.L. Nat. Rev. Cancer. 2002; 2: 489-501Crossref PubMed Scopus (5130) Google Scholar, 16West K.A. Castillo S.S. Dennis P.A. Drug Resist. Updat. 2002; 5: 234-248Crossref PubMed Scopus (531) Google Scholar). PDK1 was originally identified as a kinase that could phosphorylate Akt on its activation loop (residue Thr308) (17Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 18Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (972) Google Scholar, 19Stephens 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. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (911) Google Scholar). Later works, however, have shown that PDK1 is not only an Akt kinase but also a kinase responsible for phosphorylating members of the AGC family of protein kinases: p70S6K, SGKs, PKCs, and p90 ribosomal protein S6 kinases (RSKs) at the equivalent residues of Thr308 of Akt (reviewed in Ref. 13Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1397) Google Scholar). PDK1 itself is also a member of the AGC subfamily of protein kinases and is phosphorylated at the Ser241 residue (equivalent to Thr308 of Akt) in the activation loop. Since PDK1 expressed in bacteria was active and was phosphorylated at Ser241, it is thought that PDK1 phosphorylates itself at this site (20Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (291) Google Scholar). During our analysis of the PDK1-mediated signal transduction pathway, we discovered an increase in the phosphorylated MAPK level of PDK1-transfected cells and saw that MAPK activation depended upon MEK activation. Sequence comparison of the residues around the PDK1-mediated phosphorylation sites in AGC kinases and MEK1/2 revealed that MEK1/2 possessed the PDK1-mediated phosphorylation sites. In vitro and in vivo analysis revealed that PDK1 could directly phosphorylate the sites (Ser222 of MEK1 and Ser226 of MEK2). To date, these sites were thought to be phosphorylated by Raf kinases. Because silencing of the PDK1 gene by siRNA decreased the phospho-MEK and phospho-MAPK levels and suppressed cell proliferation, PDK1 regulated the MEK/MAPK pathway by directly phosphorylating MEK1/2. Our results indicate the importance of PDK1 as a MAPK kinase kinase in MEK/MAPK signaling pathways. Reagents, Cell Culture Conditions, and 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide Assay—The recombinant, inactive MAPK2/ERK2, inactive MEK1, active Raf-1, MBP, and purified PP2A proteins were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). The PKC inhibitor rottlerin, myristoylated PKCζ pseudosubstrate inhibitor, and myristoylated PKCθ pseudosubstrate inhibitor were obtained from Calbiochem. Our previously identified PDK1 inhibitor UCN-01 (21Sato S. Fujita N. Tsuruo T. Oncogene. 2002; 21: 1727-1738Crossref PubMed Scopus (209) Google Scholar) was kindly provided by Kyowa Hakko Kogyo (Tokyo, Japan). Recombinant human epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate were purchased from Roche Applied Science and Sigma, respectively. Human embryonic kidney 293T and human fibrosarcoma HT1080 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Human lung cancer A549 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. To assess cell proliferation, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was employed. In brief, cells were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide for 4 h. Formazon products were solubilized with Me2SO, and the optical density was measured at 525 nm using a microplate spectrophotometer (Benchmark Plus; Bio-Rad). Plasmid Construction—The rat wild-type (WT) MEK1 cDNA, rat WT-MEK2 cDNA, and NH2-terminal myristoylated (myr) mouse active form of Akt1 cDNA in pUSEamp vectors were purchased from Upstate Biotechnology. The active form of human v-Raf-1 cDNA containing the membrane-targeting CAAX motif (CAAX-Raf-1) in a pCMV vector was purchased from Clontech (Palo Alto, CA). Substitutions of Lys97 for Ala (K97A), Ser218 for Ala or Asp (S218A or S218D), Ser222 for Ala or Asp (S222A or S222D), or both Ser218 and Ser222 for Asp (S218D/S222D, DD) in MEK1 cDNA were accomplished using the QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla, CA). The Myc-tagged human full-length PDK1 cDNA (WT-PDK1) in a pCMV3 vector was kindly provided by Drs. P. Hawkins and K. Anderson (The Babraham Institute, Cambridge, UK) (22Anderson K.E. Coadwell J. Stephens L.R. Hawkins P.T. Curr. Biol. 1998; 8: 684-691Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). The pleckstrin homology (PH) domain-deleted PDK1 cDNA (ΔPH-PDK1), NH2-terminal-deleted PDK1 cDNA (ΔN51-PDK1), and the kinase-dead form of PDK1 cDNAs (D223A, S241A, and V243P) in a pCMV3 vector or a pFLAG-CMV-2 vector were generated as described previously (21Sato S. Fujita N. Tsuruo T. Oncogene. 2002; 21: 1727-1738Crossref PubMed Scopus (209) Google Scholar, 23Fujita N. Sato S. Ishida A. Tsuruo T. J. Biol. Chem. 2002; 277: 10346-10353Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 24Sato S. Fujita N. Tsuruo T. J. Biol. Chem. 2002; 277: 39360-39367Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The PDK1-4 siRNA-resistant PDK1 cDNA (PDK1-4Res) in pCMV3 vector was generated by mutating CTCGTG of the PDK1-4 siRNA-targeting sequence (see below) to TTGGTC without mutating the amino acid sequence. The WT and dominant negative (DN) PKCζ cDNA in a pcDNA3 vector were kindly provided by Dr. J. Moscat (Universidad Autónoma, Madrid, Spain) (25Diaz-Meco M.T. Moscat J. Mol. Cell. Biol. 2001; 21: 1218-1227Crossref PubMed Scopus (60) Google Scholar). The human WT-Akt1 cDNA in a pHM6 vector, EGFP cDNA in a pcDNA3 vector, and the active form of NH2-terminal-deleted SGK cDNA that encompassed residues 61–431 (ΔN60-SGK) with an S422D mutation in a pFLAG-CMV-2 vector were established in our laboratory (24Sato S. Fujita N. Tsuruo T. J. Biol. Chem. 2002; 277: 39360-39367Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Transient and Stable Transfection, Immunoprecipitation, and Western Blot Analysis—Cells were transfected with the appropriate plasmids using Superfect transfection reagent (Qiagen, Chatsworth, CA) or LipofectAMINE 2000 reagent (Invitrogen), according to the manufacturers' instructions. Stable mock, WT-MEK1, or DD-MEK1 transfectants were established by transfecting pUSEamp encoding nothing, WT-MEK1, or DD-MEK1 into HT1080 cells. The stable transfectants were selected by cultivating them in medium containing 400 μg/ml Geneticin (Invitrogen). 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 β-glycerophosphate, 1 mm phenylmethylsulfonyl fluoride, and 1 mm aprotinin) (26Sato S. Fujita N. Tsuruo T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10832-10837Crossref PubMed Scopus (836) Google Scholar). Tagged proteins were immunoprecipitated with an anti-FLAG-agarose (Sigma), an anti-HA-agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or an anti-Myc agarose (Santa Cruz Biotechnology) (23Fujita N. Sato S. Ishida A. Tsuruo T. J. Biol. Chem. 2002; 277: 10346-10353Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 26Sato S. Fujita N. Tsuruo T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10832-10837Crossref PubMed Scopus (836) Google Scholar). In some experiments, cell lysates were incubated with normal mouse IgG-conjugated agarose (Santa Cruz Biotechnology), protein A-agarose that had been conjugated with normal rabbit IgG, an anti-Raf-1 antibody-conjugated agarose (C-12; Santa Cruz Biotechnology), or an anti-MEK1 antibody-conjugated agarose (clone H-8; Santa Cruz Biotechnology). Then the immunoprecipitated proteins or the cell lysates were electrophoresed and blotted onto a nitrocellulose membrane. The membranes were incubated with antibodies to Akt, phospho-Akt (Thr308), phospho-MEK1/2 (Ser217/221), phospho-MAPK (Thr202/Tyr204), phospho-c-Myc (Thr58/Ser62), phospho-p90RSK (Thr573) (Cell Signaling Technology, Beverly, MA), c-Raf-1 or PDK1 (BD Transduction Laboratories), MEK1 or phospho-Raf-1 (Ser338) (Upstate Biotechnology), FLAG tag (clone M2) or β-actin (Sigma), HA tag (clone 3F10; Roche Applied Science), Myc tag (clone 9E10), ERK2/MAPK2, phospho-MEK1/2 (Ser218/222), (Santa Cruz Biotechnology), RSK2 (Stressgen, Victoria, Canada), or phospho-MEK1/2 (Ser222) (BIOSOURCE, Camarillo, CA). Subsequently, membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody. After washing, the membranes were developed with an enhanced chemiluminescence system, according to the manufacturer's instructions (Roche Applied Science). Blots were scanned with an EPSON ES-2200 scanner supported by Adobe Photoshop 5.5 and quantified with NIH Image 1.62 software. siRNA Design and Transfection—Five siRNAs were designed from the human PDK1 sequence (PDK1-1 to PDK1-5). The coding strands of the siRNAs were as follows: GAAGCGGCCUGAGGACUUC (PDK1-1; directed to residues 228–246), UGGUGAGGACCCAGACUGA (PDK-1-2; directed to residues 83–101), UCCUUGGGGAAGGCUCUUU (PD-K1-3; directed to residues 260–278), GAGACCUCGUGGAGAAACU (P-DK1-4; directed to residues 929–947), and UGGAAGGAUACGGACCUCU (PDK1-5; directed to residues 989–1007). In our experiments to suppress human PDK1 expression in 293T, HT1080, and A549 cells, we used the most effective siRNA (PDK1-4) for further analysis. Nonsilencing control siRNA was purchased from Qiagen. The oligonucleotides had 3′ dTdT overhangs. The sequences of siRNAs targeted to both Akt1 and Akt2 (Aktc) or Raf-1 genes were reported previously (27Cioca D.P. Aoki Y. Kiyosawa K. Cancer Gene Ther. 2003; 10: 125-133Crossref PubMed Scopus (120) Google Scholar, 28Katome T. Obata T. Matsushima R. Masuyama N. Cantley L.C. Gotoh Y. Kishi K. Shiota H. Ebina Y. J. Biol. Chem. 2003; 278: 28312-28323Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Cells were transfected with siRNAs using the LipofectAMINE 2000 reagent, according to the manufacturer's instruction. Immunostaining—Cells were transfected with pUSEamp-WT-MEK2 and pcDNA3-EGFP together with a pFLAG-CMV-2 vector encoding nothing (Mock) or ΔN51-PDK1. 24 h after transfection, cells were plated onto collagen-coated culture dishes. After incubation for a further 24 h, cells were washed with phosphate-buffered saline and fixed in 3.7% formaldehyde for 15 min. Permeabilization was carried out in 0.2% Triton X-100 for 5 min. After incubation for 1 h in phosphate-buffered saline supplemented with 1% bovine serum albumin, the labeling was carried out by incubation overnight with a rabbit polyclonal anti-phospho-MAPK (Thr202/Tyr204) antibody (Cell Signaling Technology) followed by a 90-min incubation with AlexaFluor 568-conjugated goat anti-rabbit IgG (Molecular Probes, Inc., Eugene, OR). After washing the cells, we visualized them using a fluorescence microscope (Olympus IX-70; Olympus, Tokyo, Japan) equipped with a CCD camera. Purification of Recombinant GST-ΔN51-PDK1 and GST-ΔN51-PDK1 (K111A/D223A) Proteins—Cultures of BL21 Star Escherichia coli (Invitrogen) containing a pGEX 6P-3 plasmid encoding ΔN51-PDK1 or kinase-dead form of ΔN51-PDK1 (K111A/D223A) were induced for 2 h with 1 mm isopropyl-β-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), as described previously (24Sato S. Fujita N. Tsuruo T. J. Biol. Chem. 2002; 277: 39360-39367Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The proteins were further treated with PreScission protease (Amersham Biosciences) to remove the GST tag. In Vitro Kinase Assay—To perform the in vitro kinase assay, recombinant, active Raf-1 or immunopurified, FLAG-tagged ΔN51-PDK1 was incubated with recombinant inactive MEK1 (0.3 μg/assay), recombinant inactive MAPK2 (1 μg/assay), or both in 50 μl (final volume) of kinase reaction buffer for PDK1 (40 mm Tris-HCl (pH 7.5), 0.1 mm EGTA, 0.1 mm EDTA, 0.1% 2-mercaptoethanol, 1 μm microcystin-LR, 8 mm magnesium acetate, 15 mm magnesium chloride, and 180 μm ATP) or for Raf-1 (16 mm MOPS (pH 7.2), 20 mm β-glycerol phosphate, 4 mm EGTA, 0.8 mm sodium orthovanadate, 0.8 mm dithiothreitol, 15 mm magnesium chloride, and 100 μm ATP) for 1 h at 30 °C. 5 μl of the mixture was then reacted with 20 μg of MBP in the presence of 100 μm ATP (final concentration) containing 15 μCi of [γ-32P]ATP for 10 min at 30 °C. Reactions were spotted onto P81 phosphocellulose paper, washed three times with 0.75% phosphoric acid, air-dried, and subjected to Cerenkov counting. PDK1-mediated MEK phosphorylation was also estimated by incubating immunoprecipitated, FLAG-tagged ΔN51-PDK1, or the kinase-dead form of ΔN51-PDK1 (S241A) with recombinant inactive MEK1 (0.8 μg/assay) in kinase reaction buffer for PDK1 for 1 h at 30 °C. Appropriate amounts of recombinant ΔN51-PDK1 and the kinase-dead form of ΔN51-PDK1 (K111A/D223A) were also incubated with recombinant inactive MEK1 (1 μg/assay) in kinase reaction buffer for PDK1 for 2 h at 37 °C. Reactions were electrophoresed and immunoblotted with antibodies to phospho-MEK, MEK1, phospho-PDK1 (Ser241) (Cell Signaling Technology), and PDK1. In some experiments, WT or point-mutated MEK1 proteins were immunoprecipitated and incubated with recombinant, inactive MAPK2 (0.5 μg/assay) in kinase reaction buffer for Raf-1 for 30 min at 30 °C, following immunoblot analysis with antibodies to phospho-MAPK and MAPK. In other experiments, MAPK activity was measured using a MAPK immunoprecipitation kinase assay kit (Upstate Biotechnology), according to the manufacturer's instructions. Activation of MEK/MAPK Signal Transduction Pathway by PDK1—To analyze the MEK/MAPK signaling pathway, 293T cells were transfected with pUSEamp-MEK2 plasmid together with EGFP-expressing plasmid to detect the transfected cells. Overexpression of MEK2 alone could not induce MAPK phosphorylation (Fig. 1A, yellow arrowheads). Interestingly, co-expression of ΔN51-PDK1, which possesses almost the same activity as full-length WT-PDK1 (data not shown), resulted in the increased phospho-MAPK amount in the transfected cells (Fig. 1A, blue arrowheads). We then examined whether or not PDK1 directly activates MAPK. In vitro incubation of PDK1 with recombinant, inactive MAPK2 did not induce MAPK2 activation (Fig. 1B). We observed PDK1-induced MAPK activation in vitro only in the presence of both inactive MEK1 and inactive MAPK2, as we found with Raf-1 (Fig. 1B). Therefore, PDK1 cannot directly activate MAPK; it needs MEK for in vitro activation. The fact that the immunoprecipitated, kinase-dead form of PDK1 (S241A) failed to phosphorylate and activate MEK1 indicates that PDK1-dependent MEK1 activation is not mediated by other co-precipitated kinases (Fig. 1C) (data not shown). Moreover, in vitro incubation of recombinant inactive MEK1 with purified recombinant ΔN51-PDK1, but not the kinase-dead form of ΔN51-PDK1, increased the phospho-MEK1 levels in a dose-dependent manner (Fig. 1D). The results suggest that the target of PDK1 in the MAPK signaling pathway is not MAPK itself but rather its upstream kinase MEK. PDK1 was originally isolated as a kinase responsible for the phosphorylation of Akt on its activation loop (Thr308 residue) (17Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 18Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (972) Google Scholar, 19Stephens 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. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (911) Google Scholar). Later studies have shown that PDK1 is not only an Akt kinase, but it also regulates multiple kinases, which belong to the AGC family of protein kinases through phosphorylating Ser or Thr residues equivalent to Thr308 of Akt (13Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1397) Google Scholar, 29Belham C. Wu S. Avruch J. Curr. Biol. 1999; 9: R93-R96Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Sequence comparison of the residues around Ser222 in human MEK1 and Ser226 in human MEK2 in their activation loops showed that these residues had homology to the previously reported PDK1 phosphorylation sites in human Akt1, SGK1, and PAK1 (Fig. 1E) (13Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1397) Google Scholar, 29Belham C. Wu S. Avruch J. Curr. Biol. 1999; 9: R93-R96Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). To prove that MEK phosphorylation was PDK1-dependent, we examined the change in phospho-MEK1/2 levels with an anti-phospho-MEK antibody. The Cell Signaling antibody used recognized MEK1/2 only when MEK1/2 were phosphorylated at Ser218 and/or Ser222 in MEK1 and at Ser222 and/or Ser226 in MEK2. Immunoblot analysis showed that co-transfection of PDK1 increased the phospho-MEK1/2 levels in cells (Fig. 1F). Similar results were obtained using a Santa Cruz Biotechnology antibody (sc-7995-R) that could recognize MEK1/2 when MEK1/2 were phosphorylated at Ser218 and/or Ser222 in MEK1 and at Ser222 and/or Ser226 in MEK2 (data not shown). We also found increased phospho-MEK levels when we used a BIOSOURCE antibody that specifically recognized phosphorylated MEK1 (Ser222) or MEK2 (Ser226) (data not shown). To exclude the possibility that MEK phosphorylation was accomplished by autophosphorylation, we generated a kinase-dead form of MEK1 by mutating Lys97 to Ala. As shown in Fig. 1G, PDK1 also increased the phospho-MEK1 (Ser218/Ser222) levels in KD-MEK1. Of note, the anti-phospho-MEK1/2 antibody used did not recognize DD-MEK in which both Ser218 and Ser222 residues had been substituted for the phosphomimic Asp residues. These results indicate that PDK1 activates the MEK/MAPK signaling pathway by directly phosphorylating MEK1/2. Kinase Activity of PDK1 Is Essential for MEK Phosphorylation—Since PDK1 increased the phospho-MEK levels in MEK-transfected 293T cells (Fig. 1), we investigated whether PDK1 promoted the phosphorylation of endogenous MEK. When 293T and HT1080 cells were transfected with PDK1 alone, endogenous MEK1/2 were phosphorylated in a PDK1 dose-dependent manner, like Raf-1 did (Fig. 2A). Membrane targeting of PDK1 is important for phosphorylation and activation of Akt (22Anderson K.E. Coadwell J. Stephens L.R. Hawkins P.T. Curr. Biol. 1998; 8: 684-691Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). However, membrane targeting was not necessary for PDK1-mediated endogenous MEK phosphorylation, because PH domain-deleted PDK1 (ΔPH-PDK1) also increased the phospho-MEK level (Fig. 2B). Transfection of kinase-dead forms of PDK1 (S241A-, V243P-, or D223A-PDK1) (17Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 20Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (291) Google Scholar, 24Sato S. Fujita N. Tsuruo T. J. Biol. Chem. 2002; 277: 39360-39367Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) did not increase the endogenous phospho-MEK levels (Fig. 2B). Consistent with the results, phosphorylation of MEK was dependent on the amounts of co-transfected WT-PDK1 but not the kinase-dead (S241A) form of ΔN51-PDK1 in cells (Fig. 2C). As reported previously (20Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (291) Google Scholar), S241A-PDK1 had a weak kinase activity. That might be the reason why a slight increase in phospho-MEK level was observed when MEK1 was co-transfected with S241A-PDK1 (Fig. 2C). Because c-Raf-1 phosphorylation was not affected by WT- or ΔPH-PDK1 transfection (Fig. 2B), PDK1 would phosphorylate MEK without affecting c-Raf-1 activity. Consistent with the results, our previously identified PDK1 inhibitor UCN-01 (21Sato S. Fujita N. Tsuruo T. Oncogene. 2002; 21: 1727-1738Crossref PubMed Scopus (209) Google Scholar) dose-dependently suppressed the PDK1-mediated MEK phosphorylation, in addition to Akt, in cells (Fig. 2D). Thus, kinase activity of PDK1 was essential for MEK phosphorylation. Because PDK1 is known to phosphorylate and activate multiple downstream kinases (13Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1397) Google Scholar, 29Belham C. Wu S. Avruch J. Curr. Biol. 1999; 9: R93-R96Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar), we investigated the role of PDK1 downstream kinases on MEK phosphorylation. Akt and SGK were known to act as main mediators of PI3K/PDK1-regulated biological responses (12Brazil D.P. Park J. Hemmings B.A. Cell. 2002; 111: 293-303Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, 13Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1397) Google Scholar). The increase in phospho-MEK levels was not observed in 293T cells that had been transfected with the active form of Akt1 or SGK1 cDNA although transfection of PDK1 or the active form of Raf-1 cDNA induced MEKl phosphorylation (Fig. 3A). Thus, Akt and SGK might not be involved in MEK phosphorylation and MAPK activation. PDK1 is also known to phosphorylate and activate PKCs (13Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1397) Google Scholar, 18Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (972) Google Scholar, 29Belham C. Wu S. Avruch J. Curr. Biol. 1999; 9: R93-R96Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar), especially PKCζ," @default.
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