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• W2034115636 abstract "By recombining subcellular components of 3T3-L1 adipocytes in a test tube, early insulin signaling events dependent on phosphatidylinositol 3-kinase (PI 3-kinase) were successfully reconstituted, up to and including the phosphorylation of glycogen synthase kinase-3 by the serine/threonine kinase, Akt (Murata, H., Hresko, R.C., and Mueckler, M. (2003) J. Biol. Chem. 278, 21607–21614). Utilizing the advantages provided by a cell-free methodology, we characterized phosphoinositide-dependent kinase 2 (PDK2), the putative kinase responsible for phosphorylating Akt on Ser-473. Immunodepleting cytosolic PDK1 from an in vitro reaction containing plasma membrane and cytosol markedly inhibited insulin-stimulated phosphorylation of Akt at the PDK1 site (Thr-308) but had no effect on phosphorylation at the PDK2 site (Ser-473). In contrast, PDK2 activity was found to be highly enriched in a novel cytoskeletal subcellular fraction associated with plasma membranes. Akt isoforms 1–3 and a kinase-dead Akt1 (K179A) mutant were phosphorylated in a phosphatidylinositol 3,4,5-trisphosphate-dependent manner at Ser-473 in an in vitro reaction containing this novel adipocyte subcellular fraction. Our data indicate that this PDK2 activity is the result of a kinase distinct from PDK1 and is not due to autophosphorylation or transphosphorylation of Akt. By recombining subcellular components of 3T3-L1 adipocytes in a test tube, early insulin signaling events dependent on phosphatidylinositol 3-kinase (PI 3-kinase) were successfully reconstituted, up to and including the phosphorylation of glycogen synthase kinase-3 by the serine/threonine kinase, Akt (Murata, H., Hresko, R.C., and Mueckler, M. (2003) J. Biol. Chem. 278, 21607–21614). Utilizing the advantages provided by a cell-free methodology, we characterized phosphoinositide-dependent kinase 2 (PDK2), the putative kinase responsible for phosphorylating Akt on Ser-473. Immunodepleting cytosolic PDK1 from an in vitro reaction containing plasma membrane and cytosol markedly inhibited insulin-stimulated phosphorylation of Akt at the PDK1 site (Thr-308) but had no effect on phosphorylation at the PDK2 site (Ser-473). In contrast, PDK2 activity was found to be highly enriched in a novel cytoskeletal subcellular fraction associated with plasma membranes. Akt isoforms 1–3 and a kinase-dead Akt1 (K179A) mutant were phosphorylated in a phosphatidylinositol 3,4,5-trisphosphate-dependent manner at Ser-473 in an in vitro reaction containing this novel adipocyte subcellular fraction. Our data indicate that this PDK2 activity is the result of a kinase distinct from PDK1 and is not due to autophosphorylation or transphosphorylation of Akt. Insulin initiates a complex array of intracellular signaling events (1Saltiel A.R. Kahn C.R. Nature. 2001; 414: 799-806Crossref PubMed Scopus (3842) Google Scholar). Upon insulin binding, the receptor becomes auto-phosphorylated on several critical intracellular tyrosine residues, thereby activating an intrinsic tyrosine kinase that can then phosphorylate cellular substrates. The major proteins that become tyrosine-phosphorylated by the activated insulin receptor include the insulin receptor substrate proteins (2White M.F. Mol. Cell. Biochem. 1998; 182: 3-11Crossref PubMed Scopus (623) Google Scholar), Shc (3Pelicci G.L. Lanfrancone L. Grignani F. McGlade J. Cauallo F. Forni G. Nicoletti I. Pawson T. Pelicei P.G. Cell. 1992; 70: 93-104Abstract Full Text PDF PubMed Scopus (1131) Google Scholar), and Gab1 (4Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-563Crossref PubMed Scopus (599) Google Scholar). These proteins do not have enzymatic activity. Rather, they serve as adaptor proteins capable of recruiting numerous downstream effectors that contain Src homology 2 domains (1Saltiel A.R. Kahn C.R. Nature. 2001; 414: 799-806Crossref PubMed Scopus (3842) Google Scholar).The pathway centered on phosphatidylinositol (PI) 1The abbreviations used are: PI, phosphatidylinositol; PIP3, PI 3,4,5-trisphosphate; CYT, cytosol; Ext, extract; HDM, high density microsomes; HiP, high speed pellet; HiS, high speed supernatant; ILK, integrin-linked kinase; LDM, low density microsomes; LoS, low speed supernatant; PDK, phosphoinositide-dependent kinase; PM, plasma membrane; PM(SW), salt-washed PM; GST, glutathione S-transferase.1The abbreviations used are: PI, phosphatidylinositol; PIP3, PI 3,4,5-trisphosphate; CYT, cytosol; Ext, extract; HDM, high density microsomes; HiP, high speed pellet; HiS, high speed supernatant; ILK, integrin-linked kinase; LDM, low density microsomes; LoS, low speed supernatant; PDK, phosphoinositide-dependent kinase; PM, plasma membrane; PM(SW), salt-washed PM; GST, glutathione S-transferase. 3-kinase has emerged as the critical mediator of many of the metabolic responses to insulin, including the stimulation of glucose transport (for review, see Ref. 5Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (835) Google Scholar). The PI 3-kinase mainly relevant to insulin signaling is a heterodimer consisting of a regulatory subunit (p85) and a catalytic subunit (p110). The regulatory subunit contains two Src homology 2 domains that can interact with tyrosine-phosphorylated adaptor proteins, most notably insulin receptor substrate-1 and -2. The major in vivo substrate for PI 3-kinase is phosphatidylinositol 4,5-bisphosphate, found in the cytoplasmic leaflet of the plasma membrane lipid bilayer. Insulin stimulation, thus, leads to the acute increase in the intracellular level of phosphatidylinositol 3,4,5-trisphosphate (PIP3).The activity of the serine/threonine kinase Akt (also known as protein kinase B) is markedly stimulated in a PI 3-kinase-dependent manner (6Lawlor M.A. Alessi D.R. J. Cell Sci. 2001; 114: 2903-2910Crossref PubMed Google Scholar). This phenomenon predominantly relies on the phosphorylation of Akt on two of its amino acid residues, 1) threonine 308 in the activation loop of the kinase catalytic domain and 2) serine 473 in the hydrophobic carboxy-terminal domain (7Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1386) Google Scholar). The phosphorylation of both of these regulatory sites is inhibitable in vivo by the PI 3-kinase inhibitor wort-mannin (8Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2495) Google Scholar). The protein kinase responsible for phosphorylating Akt on Thr-308 is the recently identified phosphoinositide-dependent kinase 1 (PDK1) (9Alessi 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, 10Stephens 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 (910) Google Scholar, 11Alessi D.R. Deak M. Casamayor A. Caudwell F.B. Morrice N. Norman D.G. Gaffney P. et al.Curr. Biol. 1997; 7: 776-789Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar). Despite intense investigative efforts, the kinase responsible for phosphorylating Akt on Ser-473, tentatively termed phosphoinositide-dependent kinase 2 (PDK2), has resisted definitive purification (7Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1386) Google Scholar, 12Brazil D.P. Hemmings B.A. Trends Biochem. Sci. 2001; 26: 657-664Abstract Full Text Full Text PDF PubMed Scopus (1029) Google Scholar, 13Toker A. Newton A.C. Cell. 2000; 103: 185-188Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). At least three models for Ser-473 phosphorylation have been proposed. Alessi and co-workers (14Balendran A. Casamayor A. Deak M. Paterson A. Gaffney P. Currie R. Downes C.P. Alessi D.R. Curr. Biol. 1999; 9: 393-404Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar) demonstrate that PDK1 could be converted in vitro through interaction with a hydrophobic peptide (called PDK1-interacting peptide) into a form capable of phosphorylating Akt on both Thr-308 and Ser-473 (14Balendran A. Casamayor A. Deak M. Paterson A. Gaffney P. Currie R. Downes C.P. Alessi D.R. Curr. Biol. 1999; 9: 393-404Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). Whether this unprecedented mode of regulation occurs in vivo remains unclear. Toker and Newton provide data supporting an Akt autophosphorylation mechanism involving the Ser-473 site (15Toker A. Newton A.C. J. Biol. Chem. 2000; 275: 8271-8274Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar), similar to that of certain conventional protein kinase C isoforms (16Behn-Krappa A. Newton A.C. Curr. Biol. 1999; 9: 728-737Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). They suggest that Akt might be partially activated by phosphorylation of Thr-308 due to upstream PDK1, thereby allowing Akt to act upon itself by transferring a phosphate group onto Ser-473 (15Toker A. Newton A.C. J. Biol. Chem. 2000; 275: 8271-8274Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar). Finally, it is possible for PDK2 to be a distinct kinase yet to be characterized. In cells lacking PDK1, growth factor-stimulated phosphorylation of Akt on Thr-308 did not occur, but phosphorylation of Ser-473 still remained intact, suggesting the existence of a PDK2 kinase distinct from PDK1 (17Williams M.R. Arthur J.S.C. Balendran A. van der Kaay J. Poli V. Cohen P. Alessi D.R. Curr. Biol. 2000; 10: 439-448Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar).It is self-evident that these competing models of Akt regulation have mutually exclusive features. However, a clear consensus concerning the nature of PDK2 has not been reached. We chose to address this issue using a novel approach. Using the subcellular components of 3T3-L1 adipocytes, we have recently developed an in vitro assay reconstituting key aspects of PI 3-kinase-dependent insulin signaling (45Murata H. Hresko R.C. Mueckler M. J. Biol. Chem. 2003; 278: 21607-21614Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). We provide evidence obtained through this assay that PDK2 appears to be a cytoskeleton-associated kinase distinct from PDK1.EXPERIMENTAL PROCEDURESCell Culture of 3T3-L1 Adipocytes—3T3-L1 preadipocytes obtained from the American Type Culture Collection were grown to confluence and 48 h later subjected to differentiation as described previously (18Tordjman K.M. Leingang K.A. James D.E. Mueckler M.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7761-7765Crossref PubMed Scopus (115) Google Scholar). 3T3-L1 adipocytes were used 10–14 days after initiating differentiation.Isolation of Subcellular Components—Mature 3T3-L1 adipocytes grown on 10-cm dishes were serum-starved overnight. The cells were then rapidly washed three times with ice-cold serum-free Dulbecco's modified Eagle's medium and maintained further for 15 min at 4 °C in serum-free Dulbecco's modified Eagle's medium in the absence or presence of 1 μm insulin. Cells were then washed 3 times with ice-cold phosphate-buffered saline, scraped in 2 ml of ice-cold HES buffer (50 mm Hepes, pH 7.4, 255 mm sucrose, and 1 mm EDTA)/dish containing protease inhibitors (0.082 trypsin inhibitory units/ml aprotinin (Sigma), 1 μg/ml leupeptin, 1 μg/ml antipain, 1 μg/ml benzamidine, 5 μg/ml trypsin inhibitor, 1 μg/ml chymostatin, 1 μg/ml pepstatin A, and 0.5 mm phenylmethylsulfonyl fluoride) and then homogenized at 4 °C by passing the cells 10 times through a Yamato SC homogenizer at a speed of 1200 rpm. The plasma membrane (PM) fraction was obtained by differential centrifugation and sucrose cushion flotation as described previously (19Piper R.C. Hess L.J. James D.E. Am. J. Physiol. 1991; 260: C570-C580Crossref PubMed Google Scholar) and designated as either PM(–ins) or PM(+ins) according to whether the starting cell source was exposed to insulin. The low density microsomes (LDM) fraction was obtained from basal cells as described previously (19Piper R.C. Hess L.J. James D.E. Am. J. Physiol. 1991; 260: C570-C580Crossref PubMed Google Scholar). PM and LDM, subsequent to their isolation, were resuspended in IC buffer (20 mm Hepes, pH 7.4, 140 mm potassium glutamate, 5 mm NaCl, 1 mm EGTA, and protease inhibitors). A highly concentrated cytosol (CYT) fraction was prepared by washing the 3T3-L1 adipocytes three times with ice-cold IC buffer and then removing the buffer as much as possible by aspiration followed by cell scraping and homogenizing with a ball-bearing homogenizer. The supernatant was recovered after an ultracentrifuge spin for 1 h at 200,000 × g. For the preparation of PM salt-extracted proteins, plasma membranes pelleted after sucrose cushion flotation were resuspended in 200 μlofIC buffer supplemented with NaCl to a final concentration of 1 m. Samples were incubated on ice for 30 min and then subjected to centrifugation in a TLA-100.3 fixed angle rotor at 37,000 × g for 20 min. The pellet formed from this spin (PM(SW)) was resuspended in IC buffer. The 1 m NaCl was removed from the supernatant (Ext-LoS) using a 1-ml Sephadex G-25 spin column that was pre-equilibrated with 2 ml of IC buffer. Equilibrated columns were centrifuged for 1 min at 300 × g before applying the sample to the top of the resin. Centrifugation of the sample through the spin column for 1 min at 300 × g removed the 1 m NaCl. For certain experiments, Ext-LoS was further centrifuged in a TLA-100.3 fixed angle rotor at 200,000 × g for 1 h to produce a supernatant (Ext-HiS) and a pellet (Ext-HiP). ExtHiP was resuspended in IC buffer, and Ext-HiS was desalted with a 1-ml Sephadex G-25 spin column.In Vitro Assay—Reactions were prepared in a manner described previously (45Murata H. Hresko R.C. Mueckler M. J. Biol. Chem. 2003; 278: 21607-21614Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Samples were prepared on ice by mixing in various combinations of LDM (∼2.5 mg/ml final concentration), CYT (∼ 3 mg/ml final concentration), and PM (∼0.5 mg/ml final concentration). Reactions were initiated with the addition of an ATP-regenerating system (final reaction concentrations, 1 mm ATP, 8 mm creatine phosphate, 30 units/ml creatine phosphokinase, and 5 mm MgCl2) and then incubated with rotation at 37 °C for 15 min. The reactions were quenched by the addition of an equal volume of buffer A (50 mm Hepes, pH 7.4, 150 mm NaCl, 2 mm sodium vanadate, 100 mm NaF, and 10 mm sodium pyrophosphate) containing 2% SDS and 1 mm EDTA. Certain in vitro reactions as indicated included 10 μm PIP3 (Calbiochem) in a sonication mixture of 100 μm phosphatidylcholine (Avanti Polar Lipids) and 100 μm phosphatidylserine (Avanti Polar Lipids). All phosphorylated phosphatidylinositol lipids were obtained from Calbiochem. Bovine liver PI was obtained from Avanti Polar Lipids. For the preparation of PDK1-immunodepleted CYT, pre-cleared CYT was incubated for 1.5 h at 4 °C with protein G-agarose (Upstate Biotechnology) bound with anti-PDK1 polyclonal sheep IgG (Upstate Biotechnology catalog #06–637; 5 μg of IgG/mg of CYT). For experiments addressing the salt requirement for Akt phosphorylation, the salt content of the IC buffer used in the in vitro reactions (normally 140 mm potassium glutamate and 5 mm NaCl) was altered as indicated.Immunoblot Analysis and Immunoprecipitation—Protein samples from the in vitro assay were subjected to SDS-PAGE and transferred to nitrocellulose. Phospho-specific antibodies recognizing the phosphorylated forms of Akt were obtained from New England Biolabs. Antibodies directed against integrin-linked kinase (ILK), paxillin, and integrin β1 receptor were purchased from Pharmingen. PDK1 antibody used for immunoblot analysis (catalog #06-906) as well as antibodies specific for vinculin, insulin receptor, and caveolin were purchased from Upstate Biotechnology. Actin antibody was obtained from Chemicon. The Arp3 antibody was a kind gift from Dr. John Cooper (Washington University School of Medicine).Expression of Glutathione S-Transferase (GST)-Akt1 and GST-Akt1(K179A) in 293 Cells—pEBG2t-Akt1 and pEBG2t-Akt1(K179A) DNA constructs that have been used to overexpress GST-Akt fusion proteins in human embryonic kidney 293 cells were obtained from Dr. Dario Alessi (University of Dundee) (9Alessi 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). 10 μg of Akt-DNA and 10 μg of pEGFP-C1 (Clontech) were co-transfected into a 6-cm diameter dish of 293 cells using the MBS Mammalian Transfection Kit (Stratagene). Pooled transfectants were selected by growth in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 300 μg/ml G418. GST-Akt1 and GST-Akt1(K179A) fusion proteins were purified from the transfected 293 cells as previously described (9Alessi 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).RESULTS AND DISCUSSIONExamination of Akt Phosphorylation Using an in Vitro Assay—Early insulin-stimulated events downstream of PI 3-kinase, including Akt activation, occur with reasonable efficiency in our novel cell-free system (45Murata H. Hresko R.C. Mueckler M. J. Biol. Chem. 2003; 278: 21607-21614Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). This assay involves recombining in various combinations three subcellular fractions of 3T3-L1 adipocytes prepared by differential centrifugation; that is, the plasma membrane PM(–ins) or PM(+ins), depending on whether the intact cell source had been exposed to insulin at 4 °C before fractionation, LDM, and CYT. After mixing of the components, the in vitro reaction is initiated by the addition of an exogenous ATP source and then incubated at 37 °C for up to 15 min. By using this methodology, insulin-stimulated Akt phosphorylation on Thr-308 and Ser-473 is reconstituted in a manner dependent on PI 3-kinase and ATP.We utilized this system to investigate the molecular regulation of Akt, taking advantage of experimental manipulations made possible by unhindered access to all reaction components. To clarify the role of PDK1 in the phosphorylation of Akt on Ser-473, we performed our in vitro reaction using a CYT fraction from which PDK1 had been immunodepleted. Among the reaction components used, PDK1 was found predominantly in the CYT fraction (Fig. 1A), consistent with localization observed by others (7Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1386) Google Scholar, 20Currie R.A. Walker K.S. Gray A. Deak M. Casamayor A. Downes C.P. Cohen P. Alessi D.R. Lucocq J. Biochem. J. 1999; 337: 575-583Crossref PubMed Scopus (272) Google Scholar). The faint band with a slightly retarded mobility observed in the PM fraction might be either a cross-reacting protein or a post-translationally modified form of PDK1. The LDM was essentially devoid of PDK1. Immunodepletion of CYT with an anti-PDK1 antibody successfully removed PDK1 (CYT-PDK1). In contrast, mock immunodepletion of CYT with an irrelevant antibody had no effect on PDK1 content (CYT-CON).In an in vitro reaction combining immunodepleted CYT with PM, the lack of PDK1 resulted in greatly diminished insulin-stimulated phosphorylation of Akt on Thr-308. However, insulin-stimulated Ser-473 phosphorylation occurred normally (Fig. 1B, left panels). There is some evidence to suggest that the phosphorylation of Akt in vivo takes place after its recruitment to cellular membranes (7Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1386) Google Scholar). To address the possibility that PDK2 might be membrane-associated, a reaction was performed by combining CYT with PM that had been salt-washed with 1 m NaCl (PM(SW)). Salt washing of PM abrogated insulin-stimulated Ser-473 phosphorylation. However, insulin-stimulated Thr-308 still occurred (Fig. 1B, middle panels). The inclusion of LDM to the reaction rescued insulin-stimulated Ser-473 phosphorylation from being inhibited by salt extraction of PM (Fig. 1B, right panels).Collectively, the data strongly suggest that PDK2 might be a kinase distinguishable from PDK1. This was supported by the fact that independent manipulations (i.e. immunodepletion of PDK1 and salt extraction of PM) segregated these two kinase activities in complementary fashion (i.e. inhibiting PDK1 while leaving PDK2 intact and vice versa). In contrast to the predominantly cytosolic localization of PDK1, PDK2 appeared to be associated with PM and LDM and was largely absent from the cytosol.ILK has been recently identified as a candidate for PDK2. The activity of ILK is apparently increased by insulin stimulation in a PI 3-kinase-dependent manner (21Delcommenne M. Tan C. Gray V. Ruel L. Woodgett J. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11211-11216Crossref PubMed Scopus (941) Google Scholar). Using transfected cells, S. Dedhar and co-workers provide evidence suggesting that ILK can phosphorylate Akt on Ser-473 (22Persad S. Attwell S. Gray V. Mawji N. Deng J.T. Leung D. Yan J. Sanghera J. Walsh M.P. Dedhar S. J. Biol. Chem. 2001; 276: 27462-27469Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar). However, the data regarding ILK are not conclusive. There is even uncertainty that ILK is a functional kinase, as several critical residues normally found in the catalytic domain of protein kinases are not conserved in ILK (23Lynch D.K. Ellis C.A. Edwards P.A.W. Hiles I.D. Oncogene. 1999; 18: 8024-8032Crossref PubMed Scopus (183) Google Scholar). ILK may, thus, regulate the phosphorylation of Akt on Ser-473 through an indirect mechanism (23Lynch D.K. Ellis C.A. Edwards P.A.W. Hiles I.D. Oncogene. 1999; 18: 8024-8032Crossref PubMed Scopus (183) Google Scholar).To address the role of ILK in our system, immunoblot analysis using an ILK antibody was performed on 50 μg of each of the subcellular fractions (Fig. 1C). ILK was found enriched in the PM fraction relative to the CYT and LDM. However, the amount of ILK in the cytosolic fraction was significant considering the fact that our in vitro reactions typically contained five times the amount of cytosolic proteins relative to PM proteins. This observation is inconsistent with our expected profile for PDK2, which should be absent from the cytosol. In addition, although the amount of ILK in the PM was reduced with salt washing, there was still a significant amount remaining. This behavior did not correlate with the PDK2 activity that was observed. These data indicated that the presence of ILK might not be sufficient for Ser-473 phosphorylation, but they did not rule out the possibility that ILK might be a necessary cofactor.The localization of PDK2 was also independently confirmed by another approach. We investigated whether the addition of exogenous PIP3 to our in vitro reaction could bypass the requirement for PI 3-kinase altogether, thus allowing the phosphorylation of Akt to occur in a non-insulin-dependent manner (Fig. 2). The addition of PIP3 to CYT alone resulted in the efficient phosphorylation of Thr-308, consistent with the localization of PDK1 predominantly in the cytosol. However, the phosphorylation of Ser-473 occurred only marginally, consistent with PDK2 being largely absent from the cytosol. When PIP3 was added to a reaction containing CYT and LDM, the Thr-308 signal was not further enhanced relative to that produced by a reaction containing CYT alone. In contrast, PIP3 addition to CYT and LDM produced a robust Ser-473 signal, confirming the presence of PDK2 activity associated with the LDM. Heating the LDM at 65 °C for 10 min completely inhibited the PIP3-stimulated Ser-473 activity, suggesting that a catalytic protein was responsible for the PDK2 activity in the LDM rather than an ancillary thermostable cofactor (data not shown). Similar effects were observed when PIP3 was added to a reaction containing CYT and PM. The presence of PM significantly enhanced the phosphorylation of Ser-473 but not Thr-308. Salt extraction of the LDM did not affect Thr-308 phosphorylation and only marginally reduced Ser-473 phosphorylation. Approximately 50% of the total protein in the LDM could be extracted with 1 m NaCl. In contrast, salt extraction of the PM almost completely suppressed PIP3-induced Ser-473 phosphorylation but had no effect on Thr-308 phosphorylation. In this case, ∼30% of the PM proteins were removed with the salt wash. The data again support the idea that PDK2, in contrast to PDK1, was mainly associated with the membrane fractions and absent in soluble form.Fig. 2The addition of PIP3 to the in vitro reaction can bypass the requirement for PI 3-kinase. 10 μm PIP3 in a sonication mixture of 100 μm phosphatidylcholine and 100 μm phosphatidylserine was added or not to various 3T3-L1 adipocyte components isolated from basal cells and incubated for 15 min at 37 °C in the presence of ATP. PM(SW) and LDM(SW) are PM and LDM, respectively, that were salt-washed with 1 m NaCl during the isolation procedure. The reaction samples were subjected to immunoblot analysis using phospho-Akt antibodies.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In addition to PIP3, we tested other phosphatidylinositol lipids for their ability to stimulate Akt phosphorylation. Only PIP3 and PI 3,4-bisphosphate, but not PI 4,5-bisphosphate, PI 3-phosphate, or PI, could stimulate Thr-308 and Ser-473 phosphorylation (Fig. 3), confirming the class Ia PI 3-kinase-dependent nature of both PDK1 and PDK2 (5Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (835) Google Scholar). Notably, the PDK2 activity associated with the PM did not significantly differ in lipid specificity compared with the PDK2 activity associated with the LDM.Fig. 3Phosphoinositide specificity of PDK1 and PDK2. 10 μm PIP3, PI 3,4-bisphosphate (P2), PI 4,5-bisphosphate, PI 3-phosphate (P), or PI in a sonication mixture of 100 μm phosphatidylcholine and 100 μm phosphatidylserine were added or not to an in vitro reaction containing PM and CYT or LDM and CYT isolated from basal cells. Reactions were incubated for 15 min at 37 °C in the presence of ATP. Quenched reaction samples were then subjected to immunoblot analysis using phospho-Akt antibodies.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effect of Chloride on PDK1 and PDK2 Activities—The definitive identification of PDK2 has remained elusive despite intensive efforts by many investigators over the past several years (7Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1386) Google Scholar, 12Brazil D.P. Hemmings B.A. Trends Biochem. Sci. 2001; 26: 657-664Abstract Full Text Full Text PDF PubMed Scopus (1029) Google Scholar, 13Toker A. Newton A.C. Cell. 2000; 103: 185-188Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). The enrichment of PDK2 in membrane fractions may contribute to technical difficulties experienced in attempts at purifying this activity. Our initial efforts at reconstituting PDK2 activity from the salt extract of membranes were unsuccessful until we discovered that high concentrations of chloride (>100 mm) could completely inhibit PDK2 activity. This observation is illustrated in Fig. 4. In vitro reactions containing PM and CYT were carried out in the presence of 140 mm potassium glutamate, 5 mm NaCl (KGlu), 140 mm potassium acetate, 5 mm NaCl (KAc), 140 mm KCl, 5 mm NaCl (KCl), or 145 mm NaCl. Insulin-stimulated phosphorylation of both Thr-308 and Ser-473 were almost completely suppressed in the presence of 145 mm chloride (Fig. 4, top panels). To address whether PDK1 and PDK2 activities were directly inhibited by chloride as opposed to an indirect effect involving an earlier signaling step, in vitro reactions were performed using exogenous PIP3 (Fig. 4, lower panels). The robust PIP3-induced Ser-473 phosphorylation observed in the presence of potassium glutamate or potassium acetate was completely inhibited at high chloride concentrations. The PIP3-stimulated Thr-308 phosphorylation was also severely inhibited at high chloride concentrations but was readily apparent with longer film exposures. The effect of chloride is probably not observed in vivo because the intracellular concentration of this ion is low (<5 mm). Nevertheless, buffers containing chloride in excess of 100 mm are routinely used in kinase assays and protein purification, which may in part explain some of the past difficulty in identifying PDK2.Fig. 4Chloride inhibits PDK1 and PDK2 enzymatic activity. PM and CYT were incubated for 15 min at 37 °C. INSULIN + and – refers to whether the PM used in the reactions was derived from cells pretreated or not on ice with insulin as described under “Experimental Procedures.” PIP3 (+ and –) denotes whether 10 μm PIP3 was added or not to PM(–ins) and CYT. KGlu, KAc, KCl, NaCl refers to reactions carried out in the presence of 140 mm potassium glutamate, 5 mm NaCl; 140 mm potassium acetate, 5 mm NaCl; 140 mm potassium chloride, 5 mm NaCl; or 145 mm sodium chloride, respectively. The reaction samples were subjected to immunoblot analysis using phospho-Akt antibodies.View Large Image" @default.
• W2034115636 created "2016-06-24" @default.
• W2034115636 creator A5054724495 @default.
• W2034115636 creator A5075900186 @default.
• W2034115636 creator A5079562644 @default.
• W2034115636 date "2003-06-01" @default.
• W2034115636 modified "2023-09-28" @default.
• W2034115636 title "Phosphoinositide-dependent Kinase-2 Is a Distinct Protein Kinase Enriched in a Novel Cytoskeletal Fraction Associated with Adipocyte Plasma Membranes" @default.
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