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• W2073158293 abstract "The identification of phosphoinositide-dependent kinase-1 (PDK-1) as an activating kinase for members of the AGC family of kinases has led to its implication as the activating kinase for cAMP-dependent protein kinase. It has been established in vitro that PDK-1 can phosphorylate the catalytic (C) subunit (19Cheng X. Ma Y. Moore M. Hemmings B.A. Taylor S.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9849-9854Google Scholar), but theEscherichia coli-expressed C-subunit undergoes autophosphorylation. To assess which of these mechanisms occurs in mammalian cells, a set of mutations was engineered flanking the site of PDK-1 phosphorylation, Thr-197, on the activation segment of the C-subunit. Two distinct requirements appeared for autophosphorylation and phosphorylation by PDK-1. Autophosphorylation was disrupted by mutations that compromised activity (Thr-201 and Gly-200) or altered substrate recognition (Arg-194). Conversely, only residues peripheral to Thr-197 altered PDK-1 phosphorylation, including a potential hydrophobic PDK-1 binding site at the C terminus. To address thein vivo requirements for phosphorylation, select mutant proteins were transfected into COS-7 cells, and their phosphorylation state was assessed with phospho-specific antibodies. The phosphorylation pattern of these mutant proteins indicates that autophosphorylation is not the maturation mechanism in the eukaryotic cell; instead, a heterologous kinase with properties resembling the in vitro characteristics of PDK-1 is responsible forin vivo phosphorylation of PKA. The identification of phosphoinositide-dependent kinase-1 (PDK-1) as an activating kinase for members of the AGC family of kinases has led to its implication as the activating kinase for cAMP-dependent protein kinase. It has been established in vitro that PDK-1 can phosphorylate the catalytic (C) subunit (19Cheng X. Ma Y. Moore M. Hemmings B.A. Taylor S.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9849-9854Google Scholar), but theEscherichia coli-expressed C-subunit undergoes autophosphorylation. To assess which of these mechanisms occurs in mammalian cells, a set of mutations was engineered flanking the site of PDK-1 phosphorylation, Thr-197, on the activation segment of the C-subunit. Two distinct requirements appeared for autophosphorylation and phosphorylation by PDK-1. Autophosphorylation was disrupted by mutations that compromised activity (Thr-201 and Gly-200) or altered substrate recognition (Arg-194). Conversely, only residues peripheral to Thr-197 altered PDK-1 phosphorylation, including a potential hydrophobic PDK-1 binding site at the C terminus. To address thein vivo requirements for phosphorylation, select mutant proteins were transfected into COS-7 cells, and their phosphorylation state was assessed with phospho-specific antibodies. The phosphorylation pattern of these mutant proteins indicates that autophosphorylation is not the maturation mechanism in the eukaryotic cell; instead, a heterologous kinase with properties resembling the in vitro characteristics of PDK-1 is responsible forin vivo phosphorylation of PKA. The need for cells to respond to their changing environment requires that they are able to detect change and modify their activity accordingly. The mechanisms used to detect and institute these changes are collectively referred to as signal transduction. Although many different motifs are in place to implement the proper response, protein phosphorylation is one of the most prominent. This event, which involves the transfer of a phosphate from a nucleotide triphosphate to a protein substrate, can dramatically alter the activity of that protein and ultimately the properties of the entire cell. This phosphoryl transfer event is catalyzed enzymatically by the protein kinase family, and these enzymes are themselves often regulated by phosphorylation. The proteins kinases are related through a structurally conserved catalytic core. This core is perhaps best represented by the catalytic subunit (C-subunit) 1The abbreviations used are: C-subunit, catalytic subunit; PKA, cAMP-dependent protein kinase; PDK-1, 3-phosphoinisitide-dependent protein kinase-1; DTT, dithiothreitol; RII-subunit, type II regulatory subunit of cAMP-dependent protein kinase (PKA). PKA is one of the simplest and best understood members in the family (1Beebe S.J. Corbin J.D. The Enzymes: Control by Phosphorylation, Part A. Academic Press, New York1986Google Scholar, 2Taylor S.S. Buechler J.A. Yonemoto W. Annu. Rev. Biochem. 1990; 59: 971-1005Google Scholar, 3Francis S.H. Corbin J.D. Annu. Rev. Physiol. 1994; 56: 237-272Google Scholar). This is due, in part, to its subunit organization. PKA is composed of two catalytic subunits and a homodimer of two regulatory subunits that can dissociate upon activation by cAMP. This organization allows for a detailed examination of one subunit without any interference from the other subunit. Furthermore, the catalytic subunit is comprised mostly of the conserved core; therefore, it can serve as a blueprint for understanding other protein kinases (4Taylor S.S. Knighton D.R. Zheng J. Ten Eyck L.F. Sowadski J.M. Annu. Rev. Cell Biol. 1992; 8: 429-462Google Scholar, 5Smith C.M. Radzio-Andzelm E. Madhusudan Akamine P. Taylor S.S. Prog. Biophys. Mol. Biol. 1999; 71: 313-341Google Scholar). The catalytic subunit is a 350-amino acid protein (6Shoji S. Parmelee D.C. Wade R.D. Kumar S. Ericsson L.H. Walsh K.A. Neurath H. Long G.L. Demaille J.G. Fischer E.H. Titani K. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 848-851Google Scholar) with the kinase core represented by residues 40–300. Many structurally conserved motifs define the catalytic core. One feature is its bilobal organization (7Knighton D.R. Zheng J.H. ten Eyck L.F. Ashford V.A. Xuong N.H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-414Google Scholar). The smaller N-terminal lobe is comprised mainly of β-sheets and is responsible for nucleotide binding (8Zheng J. Knighton D.R. ten Eyck L.F. Karlsson R. Xuong N. Taylor S.S. Sowadski J.M. Biochemistry. 1993; 32: 2154-2161Google Scholar), whereas the larger C-terminal lobe is composed mostly of α−helices and is responsible for substrate binding and catalysis (7Knighton D.R. Zheng J.H. ten Eyck L.F. Ashford V.A. Xuong N.H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-414Google Scholar). Each lobe contains its own conserved sequence motifs (9Hanks S.K. Hunter T. FASEB J. 1995; 9: 576-596Google Scholar). The small lobe contains the glycine-rich loop, which binds nucleotide, and the C-helix, whose orientation coordinates many parts of the molecule in the proper conformation necessary for optimal catalysis. The large lobe also contains conserved motifs such as the catalytic loop, magnesium positioning loop, activation loop, and the P+1 loop, with the latter three making up the activation segment. Fig.1 A illustrates the amino acid sequence of the activation segment, its functional motifs, and where they fall in Hanks' subdomain classification. The overall architecture of the loop is depicted in Fig. 1 B, and the interactions coordinated by the Thr-197 phosphate are illustrated in Fig.1 C. The activity of many kinases is regulated by the phosphorylation of residues on the activation segment (10Johnson L.N. Noble M.E. Owen D.J. Cell. 1996; 85: 149-158Google Scholar), often by an upstream kinase. A phosphate on the activation loop properly positions the loop for both catalysis and substrate recognition (11Prowse C.N. Lew J. J. Biol. Chem. 2001; 276: 99-103Google Scholar, 12Hagopian J.C. Kirtley M.P. Stevenson L.M. Gergis R.M. Russo A.A. Pavletich N.P. Parsons S.M. Lew J. J. Biol. Chem. 2001; 276: 275-280Google Scholar). Unlike many other kinases, the C-subunit normally is assembled as an active enzyme that is fully phosphorylated on its activation loop. Regulation of the C-subunit occurs through holoenzyme formation with the regulatory subunit and is dependent on the level of cellular cAMP. When purified from mammalian tissue, the C-subunit is always phosphorylated on its activation loop (13Shoji S. Titani K. Demaille J.G. Fischer E.H. J. Biol. Chem. 1979; 254: 6211-6214Google Scholar). What is not well understood is the in vivo mechanism for this phosphorylation. Although C-subunit expressed in Escherichia coli autophosphorylates on both Thr-197 and Ser-338, this most likely does not reflect the mechanism used in the eukaryotic cell (14Cauthron R.D. Carter K.B. Liauw S. Steinberg R.A. Mol. Cell. Biol. 1998; 18: 1416-1423Google Scholar). The identification of 3-phosphoinisitide-dependent protein kinase-1 (PDK-1) (15Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Google Scholar, 16Alessi D.R. Deak M. Casamayor A. Caudwell F.B. Morrice N. Norman D.G. Gaffney P. Reese C.B. MacDougall C.N. Harbison D. Ashworth A. Bownes M. Curr. Biol. 1997; 7: 776-789Google Scholar) was soon followed by the identification of a number of its substrates. PDK-1 belongs to the AGC family of kinases and is a constitutively active kinase regulated in part by a plextrin homology (PH) domain. It was first discovered as the activating enzyme for Akt/PKB. Subsequently, a number of protein kinases have been shown to be substrates of PDK-1. Some of these included PKC (17Dutil E.M. Toker A. Newton A.C. Curr. Biol. 1998; 8: 1366-1375Google Scholar), S6 kinase (18Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Google Scholar), and PKA (19Cheng X. Ma Y. Moore M. Hemmings B.A. Taylor S.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9849-9854Google Scholar). Fig. 2 shows the sequences of the activation loops for those kinases demonstrated to be PDK-1 substrates compared with the two kinases determined not to be substrates. In an effort to identify the PKA kinase, PDK1 was assayed and found to be an excellent in vitro kinase for the C-subunit (19Cheng X. Ma Y. Moore M. Hemmings B.A. Taylor S.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9849-9854Google Scholar). This phosphorylation event proved critical for the activation of the unphosphorylated substrate C-subunit. Whether PDK-1 is the in vivo PKA kinase or not remains to be resolved. However, it is clear from the work of Steinberg and co-workers (14Cauthron R.D. Carter K.B. Liauw S. Steinberg R.A. Mol. Cell. Biol. 1998; 18: 1416-1423Google Scholar) that the C-subunit is not autophosphorylated in mammalian cells; it is phosphorylated by a heterologous kinase. This work attempts to comprehensively examine the role that individual residues in the activation segment contribute to the various functional parameters of the catalytic subunit. Each residue was mutated to alanine, and the resultant mutant proteins were assayed for their ability to affect autophosphorylation in E. coli, phosphorylation of a peptide substrate, binding to a regulatory subunit isoform, and phosphorylation of Thr-197 by PDK-1. The results strengthen the definition of the different motifs of the activation segment and reiterate the functional significance that this conserved loop has in the regulation of this important family of enzymes. In addition, the APE anchor was identified as an important determinant for PDK-1 phosphorylation. The reagents used were pRSETB expression vector (Invitrogen), [γ-32P]ATP (PerkinElmer Life Sciences), E. coli strains BL21(DE3) (Novagen, Madison, WI) and H-89 (LC Laboratories, Woburn, MA), Muta-Gene site-directed mutagenesis kit (Bio-Rad), horseradish peroxidase-conjugated anti-rabbit IgG (Amersham Biosciences), protease inhibitor mixture III (Calbiochem), SuperSignal West Pico chemiluminescent substrate detection kit (Pierce), oligonucleotides (Sigma), the PepTag PKA activity assay kit (Promega, Madison, WI), and the Effectene transfection kit (Qiagen, Valencia, CA). Mouse monoclonal anti-Myc and anti-HA antibodies were from Covance (Princeton, NJ), and antibodies that specifically recognize the phosphorylated activation loop of protein kinase C (PKC), referred to as α-Thr 197-P, were a gift from A. Newton (University of California, San Diego) (20Chou M.M. Hou W. Johnson J. Graham L.K. Lee M.H. Chen C.S. Newton A.C. Schaffhausen B.S. Toker A. Curr. Biol. 1998; 8: 1069-1077Google Scholar). Antibodies against the catalytic subunit of PKA were described previously (21Yonemoto W. McGlone M.L. Grant B. Taylor S.S. Protein Eng. 1997; 10: 915-925Google Scholar). Antibodies directed toward the unphosphorylated Thr-197, α-Thr-197-OH, were described previously. 2M. J. Moore and S. S. Taylor, submitted for publication. Plasmid pCMV5 containing Myc-tagged PDK1 was the same as previously described (18Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Google Scholar). DNA sequencing was performed with the ABI PRISM 310 genetic analyzer from PerkinElmer Life Sciences. cDNA for the murine PKA Cα-subunit in the bacterial expression vector pRESTB was used as a template for Kunkel-based site-directed mutagenesis as described previously (23Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Google Scholar). All mutations were made using the Muta-Gene kit per the manufacturer's recommendations. DNA sequencing analysis instruments confirmed the presence of the correct mutation. Wild-type and mutant C-subunits were expressed in the E. coli strain BL21(DE3). Cells were grown in YT medium containing 100 μg/ml ampicillin at 37 °C to an optical density at 600 nm of 0.5–0.8, induced with 0.5 mmisopropyl-β-d-thiogalactopyranoside (IPTG), incubated for an additional 6 h at 24 °C, collected by centrifugation, and stored frozen. Pellets were resuspended in lysis buffer (50 mm Tris-Cl, pH 7.5, 10 mm NaCl, 1 mm DTT, 0.2 mm phenylmethylsulfonyl fluoride, and 1% Triton X-100 and lysed by 3× freeze thawing and sonication. Insoluble material was removed by centrifugation at 16,000 ×g at 4 °C for 20 min. The PepTag assays were performed according to the manufacturer's instructions. This assay uses the Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) peptide substrate tagged with a fluorescent dye. Upon phosphorylation, the net charge of this peptide changes from +1 to −1, which then alters the migration of the peptide when run on an agarose gel. Briefly, lysed bacterial supernatant expressing the wild type or mutant proteins was incubated with the tagged Kemptide substrate and activator buffers at 30 °C, and the reaction was run on a 1% agarose gel at 100 V. Active protein was detected by its substrate migrating toward the anode. Human 293 cells were propagated at 2 × 106 per 10-cm dish in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. A pCMV5 vector containing Myc-tagged PDK1 was transfected using the Qiagen Effectene transfection kit. 48 h post transfection, the cells were trypsinized and resuspended in Buffer A (50 mmTris-Cl, 7.5, 50 mm NaCl, 10 mm NaF, 10 mm β-glycerol phosphate, 10 mm sodium pyrophosphate, 0.5 mm EGTA, 1 mm DTT, 1 mm benzamidine, 0.5 mm phenylmethylsulfonyl fluoride, 0.1% Triton X-100, and 10 μg/ml aprotinin) as described (18Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Google Scholar). The cells were then subjected to three rounds of freeze-thaw cycle followed by centrifugation at 50,000 rpm in a Beckman TLA 100 rotor for 30 min at 4 °C. For the immunoprecipitation experiments, 2 μl of mouse anti-Myc antibody were mixed with the cell extract of 1% of a 10-cm dish in 25 μl of Buffer A for 2 h on ice. The immunocomplex was then transferred to 10 μl (bed volume) of protein G-Sepharose resin and mixed for 1 h on a rotating wheel at 4 °C. The immunoprecipitates then were washed at room temperature five times, twice with buffer A, twice with buffer A plus 0.5 m NaCl, and once with Buffer B (50 mm Tris-Cl, pH 7.5, 10 mm NaCl, 1 mm DTT, 10% glycerol, 1 mm benzamidine, and 0.2 mm phenylmethylsulfonyl fluoride). For the kinase assay, 0.25 μg of H-89-C was mixed with 25 μm ATP, 5μCi of [γ-32P]ATP, and 10 mmMgCl2 in 25 μl of Buffer B and incubated with the immobilized PDK1 for 45 min at 30 °C with frequent gentle mixing. Aliquots of each reaction were subject to SDS-PAGE, and 32P incorporation was observed by autoradiography. COS cells were plated into 6-well plates the day prior to transfections and transfected with Effectene reagent. 30 h later, the cells were rinsed with phosphate-buffered saline and scraped on ice in lysis buffer containing 50 mm Tris-Cl, pH 7.5, 50 mmNaCl, 10 mm MgCl, 10 mm NaF, 10 mmsodium pyrophosphate, 0.5 mm EGTA, 1 mm DTT, 1% Triton X-100, and protease inhibitor mixture III (final concentrations were 100 μm4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), 80 nm aprotinin, 5 μm bestatin,1.5 μm E-64, 2 μm leupeptin, and 1 μm pepstatin A). Scraped cells sat with lysis buffer for 15 min and were then centrifuged at 14,000 rpm in a microfuge for 20 min to pellet any insoluble material. The supernatants were removed and incubated overnight in the cold with a mouse monoclonal antibody to the HA tag. The following morning protein G-Sepharose was added for 2 h, and immunoprecipitates were collected by centrifugation, washed 2 times in lysis buffer with 0.1% Triton, and finally resuspended in SDS sample buffer for electrophoresis. Samples were run on Nu-Page minigels from Invitrogen, transferred to nitrocellulose membranes, and probed for either the total C-subunit of PKA or the Thr-197 phosphorylation state using a rabbit α-C-subunit and a rabbit α-Thr-197-P, respectively. Two classes of mutant proteins were engineered in the C-subunit. Our primary focus was the region extending from the activation loop through the anchor motif to the large lobe, residues 194–208. This corresponds to subdomain VIII in Hanks' designation (24Hanks S.K. Quinn A.M. Hunter T. Science. 1988; 241: 42-52Google Scholar). Each residue in this segment was replaced with alanine. Also, two additional mutants outside the activation segment, F347A and F350A, were used to characterize the PDK-1 specificity. In order for these mutants to be tested as substrates for PDK-1, they needed to be isolated in their unphosphorylated state. Each mutant thus was expressed in E. coli and, upon induction with isopropyl-1-thio-β-d-galactopyranoside (IPTG), was treated with the ATP analog H-89. H-89 is an inhibitor of C-subunit activity and prevents autophosphorylation (14Cauthron R.D. Carter K.B. Liauw S. Steinberg R.A. Mol. Cell. Biol. 1998; 18: 1416-1423Google Scholar, 25Chijiwa T. Mishima A. Hagiwara M. Sano M. Hayashi K. Inoue T. Naito K. Toshioka T. Hidaka H. J. Biol. Chem. 1990; 265: 5267-5272Google Scholar). The single alanine mutants engineered at positions 194–208 were tested first for their ability to autophosphorylate. It has been established that when the C-subunit is expressed in E. coli, processing into the active form occurs through an autophosphorylation mechanism (21Yonemoto W. McGlone M.L. Grant B. Taylor S.S. Protein Eng. 1997; 10: 915-925Google Scholar). To assess the effect that each mutation might have on autophosphorylation, each mutant protein was expressed in E. coli without the addition of H-89. The soluble fraction of lysed cells expressing each mutant protein was used for two immunoblots (Fig.3 A). The first was probed using the antibody that recognizes the phosphorylated activation loop, α-Thr-197-P, and the other with an antibody raised against the C-subunit, α-C-subunit. The α-C-subunit immunoblot shows that there are similar amounts of protein for each mutant, but the α-Thr-197-P blot indicated that some of the mutant proteins are not capable of undergoing autophosphorylation. Only three of these do not phosphorylate, i.e. R194A, G200A, and T201A. The mutation of Arg-194 would abolish the consensus sequence for C-subunit phosphorylation and likely account for the inability of this mutant protein to be phosphorylated. The latter two mutant proteins are not only a part of the peptide-positioning loop, but they contact residues in the core that are required for catalysis. The same mutant proteins used to evaluate autophosphorylation were tested for their effect on activity using the PepTag PKA activity assay (Fig.3 B). This assay uses the PKA peptide substrate Kemptide (26Kemp B.E. J. Biol. Chem. 1979; 254: 2638-2642Google Scholar) tagged with a fluorescent dye. Upon phosphorylation, the net charge of this peptide changes from +1 to −1. When these samples are loaded onto an agarose gel, phosphorylated peptide migrates in the opposite direction of the unphosphorylated peptide. Just as was done in the autophosphorylation assays, the soluble fractions containing mutant proteins expressed in the absence of H-89 were assayed for activity. Several of these mutants are compromised in their activity. Because phosphorylation is required for activity, those mutants that could not autophosphorylate, were also inactive, i.e. R194A, G200A, and T201A. Of the mutant proteins that could autophosphorylate, there were two that had compromised activity, namely Y204A and E208A. These mutants are unique because they autophosphorylated and yet are not capable of phosphorylating a peptide substrate. These mutant proteins were also assessed for their ability to bind to the R-subunit. The soluble fractions of theE. coli expression extracts were incubated with a purified His-tagged RII-subunit mutant, R213K. This RIIα subunit has a mutation in the cAMP-binding site of cAMP binding domain A and thus forms a holoenzyme complex that is more resistant to dissociation by cAMP. It can be used for the purification of the C-subunit. The R-subunits were then pulled down via their His-tag using Ni2+ affinity resin. The complexes were then subject to SDS-PAGE and immunoblot analysis using an α-C-subunit antibody (Fig. 3 C). Mutants that were incapable of binding the R-subunit were, again, R194A, G200A, and T201A. These were the same mutants that were unable to autophosphorylate. These results are consistent with data indicating that phosphorylation of Thr-197 on the activation loop is required for R-subunit binding (27Levin L.R. Zoller M.J. Mol. Cell. Biol. 1990; 10: 1066-1075Google Scholar). Y204A and E208A were also capable of binding to RIIα, even though they did not display catalytic activity. Nearly all H-89-treated mutant proteins were expressed at levels comparable with wild type C-subunit and were soluble. The PDK-1 used in these assays was immunoprecipitated from 293 cells transfected with a Myc-tagged PDK-1 using an anti-Myc antibody and GammaBind G-Sepharose (19Cheng X. Ma Y. Moore M. Hemmings B.A. Taylor S.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9849-9854Google Scholar). Assays were conducted with PDK-1 on the resin in a reaction buffer containing[γ-32P]ATP, and the soluble fraction of the bacterial lysate containing the individual C-subunit mutants as substrates. Residues contributing to substrate recognition were identified by reduced 32P incorporation following incubation with PDK-1. The most striking observation initially, shown in Fig.4, was that most of the mutant proteins were substrates for PDK-1 based on this qualitative assay. This is particularly true for residues 194–205, which includes the entire peptide-positioning loop. This is a region that is highly conserved in all of the known substrates of PDK-1 (28Chan T.O. Rittenhouse S.E. Tsichlis P.N. Annu. Rev. Biochem. 1999; 68: 965-1014Google Scholar). Surprisingly, however, the mutant proteins that represent the highly conserved residues immediately following the phosphorylation site did not disrupt PDK-1 phosphorylation. In contrast, mutations at Pro-207 and Glu-208, located at the very end of the activation segment, did interfere with PDK-1 phosphorylation. Immunoblotting was used to demonstrate that there were equivalent amounts of protein in each assay using the anti-C-subunit antibody. In addition to the high degree of similarity between the residues following the site of phosphorylation in PDK-1 substrates, all the known substrates of PDK-1 appear to have a FXXF motif at the C terminus. This sequence was shown to be important for interaction with PDK-1 (29Biondi R.M. Cheung P.C. Casamayor A. Deak M. Currie R.A. Alessi D.R. EMBO J. 2000; 19: 979-988Google Scholar). The hydrophobic motif is thought to dock to the small domain of PDK-1 and position the enzyme for phosphorylation by PDK-1 on its activation loop. This motif corresponds to the last four residues of the C-subunit. Phe-347 and Phe-350 residues were mutated to Ala to assess their effects on autophosphorylation and phosphorylation by PDK-1 (Fig.5). When expressed in the absence of H-89 and probed with the α-Thr-197-P antibody, both of these mutant proteins are phosphorylated at Thr-197. Alternatively, when expressed in the presence of H-89 and incubated with PDK-1 and [γ-32P]ATP, there is no incorporation of32P, indicating no recognition by PDK-1. Although the in vitro results nicely map out distinct requirements for autophosphorylation and phosphorylation by PDK-1, they do not address phosphorylation that occurs in a eukaryotic cell. To observe which residues are required for phosphorylationin vivo and to make a comparison with the in vitro results, select mutant proteins were transfected into COS cells, and their phosphorylation state was determined using phospho-specific antibodies. To avoid cross-reactivity with the endogenous C-subunit, an HA tag was placed at the C terminus of the C-subunit, and transfected proteins were immunoprecipitated before being subject to Western blot analysis. The Thr-197 phosphorylation state and expression levels were determined using the α-Thr-197-P and α-C-subunit antibodies, respectively. Wild type and the following four mutant proteins were selected for transfection: R194A for disrupting autophosphorylation, E208A for disrupting in vitro PDK-1 phosphorylation, and F347A and F350A for altering the PDK-1 docking site as well as for compromising the in vitro PDK-1 phosphorylation. As indicated in Fig.6, the phosphorylation pattern observed here closely matches that for the in vitro PDK-1 phosphorylation assays, providing further evidence that PDK-1 is indeed responsible for C-subunit phosphorylation in vivo. The activation segment of protein kinases is a conserved and essential motif in the protein kinase family (24Hanks S.K. Quinn A.M. Hunter T. Science. 1988; 241: 42-52Google Scholar). One of the central features of this segment is phosphorylation of one or more residues in the activation loop (10Johnson L.N. Noble M.E. Owen D.J. Cell. 1996; 85: 149-158Google Scholar). This event is a crucial on/off step for the activation of many kinases. Although this on/off event is not a part of the in vivo cAMP-mediated activation mechanism for PKA, the phosphate is critical for C-subunit activity (30Adams J.A. McGlone M.L. Gibson R. Taylor S.S. Biochemistry. 1995; 34: 2447-2454Google Scholar). How the C-subunit is initially assembled as a fully phosphorylated protein in the holoenzyme has been poorly understood. Those kinases whose structure have been solved in their unphosphorylated and inactive state highlight the changes in the loop's position and demonstrate the central role that a single phosphate plays in orchestrating a fully functional active site (31Zhang F. Strand A. Robbins D. Cobb M.H. Goldsmith E.J. Nature. 1994; 367: 704-711Google Scholar, 32De Bondt H.L. Rosenblatt J. Jancarik J. Jones H.D. Morgan D.O. Kim S.H. Nature. 1993; 363: 595-602Google Scholar). This change displaces many residues critical for activity. Alanine-scanning mutagenesis in this region was used to help define how individual amino acids contribute to the maturation process. The ability to obtain an E. coli-expressed C-subunit that is soluble, active, and purified in high yield has allowed for extensive characterization of this enzyme (33Slice L.W. Taylor S.S. J. Biol. Chem. 1989; 264: 20940-20946Google Scholar). The recombinant enzyme, autophosphorylated on Thr-197 and Ser-338, is fully active, has a stable conformation, and is very soluble. When expressing activation segment mutant proteins, there appear to be two requirements necessary for autophosphorylation to occur, namely activity and substrate recognition. The inability of G200A and T201A to autophosphorylate is undoubtedly related to their disruption of interactions critical for activity. The crystallographic molecular model shows that the hydroxyl moiety of Thr-201 is hydrogen-bonding distance from Lys-168 and, in some cases, actually bridges Lys-168 to Asp-166 (8Zheng J. Knighton D.R. ten Eyck L.F. Karlsson R. Xuong N. Taylor S.S. Sowadski J.M. Biochemistry. 1993; 32: 2154-2161Google Scholar). Lys-168 is the single link from the large lobe to the γ-phosphate of ATP and is an essential part of the phosphoryl transfer process; Asp-166 orients the substrate hydroxyl to accept the phosphate and is a required component of the activation site. A mutation at Thr-201 disrupts this interaction and most likely the organization of the active site, resulting in an inactive enzyme. Thr-201 is conserved in all Ser/Thr protein kinases (24Hanks S.K. Quinn A.M. Hunter T. Science. 1988; 241: 42-52Google Scholar). Replacing the Gly-200 with Ala may increase the rigidity of the loop and prevent the adjacent residue, Thr-201, from making its essential interaction; however, it is more likely that this mutation abolishes activity because of steric factors. Furthermore the backbone amide of Gly-200 interacts with the P + 1 residue of the substrate. Thus, these two residues coordinate the phosphoryl transfer site by binding to the peptide (Gly-200) and may organize, through Thr-201, the two residues in the catalytic loop that bind to the two substrates,i.e. ATP (via Lys-168) and the peptide hydroxyl (via Asp-166). Fig. 7 A depicts the backbone interactions that involve Thr-201. The second requirement, substrate recognition, is observed with the mutation of Arg-194, situated at the P-3 position. General PKA substrate recognition involves two Arg residues, either at the P-2 and P-3 or P-3 and P-6 positions. Removal of the P-3 Arg-194 abolishes the only basic residue in the consensus sequence and would certainly alter recognition of Thr-197 as a site of phosphorylation. Complementary characterization of the mutant proteins reiterates the importance of the Thr-197 phosphate as well as that of particular residues. The ability to bind the RII-subunit appeared to depend entirely on the presence of a phosphate in coprecipitation experiments. This correlates with previous observations (27Levin L.R. Zoller M.J. Mol. Cell. Biol. 1990; 10: 1066-1075Google Scholar), but stresses that in these qualitative assays it contributes to RII-subunit binding more than any particular residue. Not surprising is the correlation between phosphorylation state and activity. There are two additional mutant proteins that have diminished activity, Y204A and E208A. Tyr-204 appears to be crucial for substrate recognition as well as stabilizing the P+1 loop. 3M. J. Moore, J. A. Adams, and S. S. Taylor, submitted for publication. The highly conserved Glu-208 serves as an anchor to the large lobe through its interaction with Arg-280. Disruption of this anchor could alter the P+1 loop and substrate recognition and perhaps even the interactions made by the Thr-197 phosphate. The identification of PDK-1 and its substrates has provided new understanding about the activation of kinases in the AGC superfamily (35Toker A. Newton A.C. Cell. 2000; 103: 185-188Google Scholar). Its relationship as an in vitro kinase for the C-subunit has allowed for new thinking as to the processing of the C-subunit in eukaryotic cells. The most compelling evidence still supports the prediction that the C-subunit is not autophosphorylated in mammalian cells but instead is phosphorylated by a heterologous protein kinase (14Cauthron R.D. Carter K.B. Liauw S. Steinberg R.A. Mol. Cell. Biol. 1998; 18: 1416-1423Google Scholar). PDK-1 or a homolog of PDK-1 is still a candidate for anin vivo PKA kinase, even though embryonic stem cells with the PDK-1 gene knocked out did not completely abolish phosphorylation of the C-subunit (36Williams M.R. Arthur J.S. Balendran A. van der Kaay J. Poli V. Cohen P. Alessi D.R. Curr. Biol. 2000; 10: 439-448Google Scholar). We thus compared sequence requirements for autophosphorylation versus requirements for phosphorylation by PDK-1. The residues that compromised PDK-1 phosphorylation were distal to Thr-197, namely P207A and E208A, which are located in the activation loop anchor. The identification of Pro-207 and Glu-208 as recognition determinants for PDK-1 has not been previously described, and these residues lie outside the limits of most peptide screens. Pro-207 certainly conveys rigidity to the anchor, and replacing it with Ala would make it more flexible. The E208A mutation would abolish the interaction with Arg-280. Neither mutant protein altered autophosphorylation, despite the compromised activity of E208A. We predict that the Pro-Glu motif may be a previously unrecognized determinant for PDK-1 in addition to its requirements for a peripheral hydrophobic docking site. Other residues that immediately follow the phosphorylation site are highly conserved in PDK-1 substrates (24Hanks S.K. Quinn A.M. Hunter T. Science. 1988; 241: 42-52Google Scholar) but did not disrupt PDK-1 phosphorylation. The high degree of conservation in this region thus appears to be more important for function than PDK-1 recognition. Previous work established that PDK-1 could interact with some of its substrates through a C-terminal hydrophobic docking site (29Biondi R.M. Cheung P.C. Casamayor A. Deak M. Currie R.A. Alessi D.R. EMBO J. 2000; 19: 979-988Google Scholar, 34Ziegler W.H. Parekh D.B. Le Good J.A. Whelan R.D. Kelly J.J. Frech M. Hemmings B.A. Parker P.J. Curr. Biol. 1999; 9: 522-529Google Scholar, 37Balendran A. Casamayor A. Deak M. Paterson A. Gaffney P. Currie R. Downes C.P. Alessi D.R. Curr. Biol. 1999; 9: 393-404Google Scholar). The FXXF motif occurs in PDK-1 substrates (Fig. 2) and corresponds to the last four residues of the C-subunit (Fig.7 B). A yeast two-hybrid screen using full-length PDK-1 as bait was used to identify the C terminus of the C-subunit as a PDK-1 binding partner (29Biondi R.M. Cheung P.C. Casamayor A. Deak M. Currie R.A. Alessi D.R. EMBO J. 2000; 19: 979-988Google Scholar). This interaction was disrupted when either Phe-347 or Phe-350 were mutated to Ala. Mutations were engineered at Phe-347 and Phe-350 to address their contributions to phosphorylation at Thr-197 in the substrate form of the C-subunit. Previous work demonstrated that these two mutant proteins exhibit compromised activity (22Batkin M. Schvartz I. Shaltiel S. Biochemistry. 2000; 39: 5366-5373Google Scholar). Nevertheless, both F347A and F350A mutant proteins autophosphorylated when expressed in bacteria; in contrast, these mutant proteins were not phosphorylated by PDK-1 in vitro. This supports the prediction that PDK-1 or a homolog of PDK-1 may indeed be the in vivo kinase. Additionally, this motif is absent in calcium/calmodulin-dependent protein kinase IV (CaMKIV) and 5′-AMP-activated kinase (AMPK) (Fig. 2), and that is probably why they do not serve as substrates of PDK-1 despite the similarity in the activation segment residues. Clearly the requirements for autophosphorylation and phosphorylation by PDK-1 are quite distinct. Because in vitro experiments allow for the characterization of a particular type of phosphorylation mechanism but do not address phosphorylation in the eukaryotic environment, select mutant proteins that correspond to residues important for autophosphorylation or PDK-1 phosphorylation were transfected into a mammalian cell line, and their phosphorylation states were determined. Those mutant proteins that disrupted PDK-1 phosphorylation in vitro also disrupted phosphorylation in vivo, whereas the autophosphorylation-disrupting mutant protein was properly phosphorylated in vivo. Not only does this discount autophosphorylation as the in vivo mechanism for PKA phosphorylation, but the PKA kinase activity observed in COS cells also has similar substrate requirements as PDK-1 does in vitro. Clearly PDK-1 is an excellent in vitro kinase for the unphosphorylated C-subunit. When the contributions individual amino acids make to C-subunit phosphorylation were examined, separate requirements were observed for autophosphorylation and for phosphorylation by PDK-1. Moreover, those residues important for PDK-1 phosphorylation were also required for phosphorylation in vivo. As our understanding of the cellular regulation of PDK-1 continues to expand, so might our understanding of how the maturation process of the unphosphorylated C-subunit contributes to its own regulation." @default.
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• W2073158293 title "Phosphorylation of the Catalytic Subunit of Protein Kinase A" @default.
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