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• W2154535504 abstract "General strategies to obtain inactive kinases have utilized mutation of key conserved residues in the kinase core, and the equivalent Lys72 in cAMP-dependent kinase has often been used to generate a “dead” kinase. Here, we have analyzed the consequences of this mutation on kinase structure and function. Mutation of Lys72 to histidine (K72H) generated an inactive enzyme, which was unphosphorylated. Treatment with an exogenous kinase (PDK-1) resulted in a mutant that was phosphorylated only at Thr197 and remained inactive but nevertheless capable of binding ATP. Ser338 in K72H cannot be autophosphorylated, nor can it be phosphorylated in an intermolecular process by active wild type C-subunit. The Lys72 mutant, once phosphorylated on Thr197, can bind with high affinity to the RIα subunits. Thus a dead kinase can still act as a scaffold for binding substrates and inhibitors; it is only phosphoryl transfer that is defective. Using a potent inhibitor of C-subunit activity, H-89, Escherichia coli-expressed C-subunit was also obtained in its unphosphorylated state. This protein is able to mature into its active form in the presence of PDK-1 and is able to undergo secondary autophosphorylation on Ser338. Unlike the H-89-treated wild type protein, the mutant protein (K72H) cannot undergo the subsequent cis autophosphorylation following phosphorylation at Thr197. Using these two substrates and mammalian-expressed PDK-1, we can elucidate a possible two-step process for the activation of the C-subunit: initial phosphorylation on the activation loop at Thr197 by PDK-1, or a PDK-1-like enzyme, followed by second cis autophosphorylation step at Ser338. General strategies to obtain inactive kinases have utilized mutation of key conserved residues in the kinase core, and the equivalent Lys72 in cAMP-dependent kinase has often been used to generate a “dead” kinase. Here, we have analyzed the consequences of this mutation on kinase structure and function. Mutation of Lys72 to histidine (K72H) generated an inactive enzyme, which was unphosphorylated. Treatment with an exogenous kinase (PDK-1) resulted in a mutant that was phosphorylated only at Thr197 and remained inactive but nevertheless capable of binding ATP. Ser338 in K72H cannot be autophosphorylated, nor can it be phosphorylated in an intermolecular process by active wild type C-subunit. The Lys72 mutant, once phosphorylated on Thr197, can bind with high affinity to the RIα subunits. Thus a dead kinase can still act as a scaffold for binding substrates and inhibitors; it is only phosphoryl transfer that is defective. Using a potent inhibitor of C-subunit activity, H-89, Escherichia coli-expressed C-subunit was also obtained in its unphosphorylated state. This protein is able to mature into its active form in the presence of PDK-1 and is able to undergo secondary autophosphorylation on Ser338. Unlike the H-89-treated wild type protein, the mutant protein (K72H) cannot undergo the subsequent cis autophosphorylation following phosphorylation at Thr197. Using these two substrates and mammalian-expressed PDK-1, we can elucidate a possible two-step process for the activation of the C-subunit: initial phosphorylation on the activation loop at Thr197 by PDK-1, or a PDK-1-like enzyme, followed by second cis autophosphorylation step at Ser338. Most protein kinases are themselves phosphoproteins that contain an essential phosphate in the activation loop of the enzyme (1Canagarajah B.J. Khokhlatchev A. Cobb M.H. Goldsmith E.J. Cell. 1997; 90: 859-869Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar, 2Hagopian 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: 275-280Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 3Prowse C.N. Hagopian J.C. Cobb M.H. Ahn N.G. Lew J. Biochemistry. 2000; 39 (Erratum (2000) Biochemistry39, 14002): 6258-6266Crossref PubMed Scopus (51) Google Scholar, 4Johnson D.A. Akamine P. Radzio-Andzelm E. Madhusudan Taylor S. Chemical Reviews. 2001; 101: 2243-2273Crossref PubMed Scopus (336) Google Scholar, 5Johnson L.N. Noble M.E. Owen D.J. Cell. 1996; 85: 149-158Abstract Full Text Full Text PDF PubMed Scopus (1161) Google Scholar). The activation loop is a conserved motif in the kinase family. The catalytic subunit (C-subunit) of cAMP-dependent kinase (PKA) 1The abbreviations used are: PKA, cAMP-dependent kinase; PKC, protein kinase C; FSBA, p-fluorosulfonylbenzoyl 5′-adenosine; HA, hemagglutinin; MAPK, mitogen-activated protein kinase.1The abbreviations used are: PKA, cAMP-dependent kinase; PKC, protein kinase C; FSBA, p-fluorosulfonylbenzoyl 5′-adenosine; HA, hemagglutinin; MAPK, mitogen-activated protein kinase. has an essential phosphorylation site in the activation loop at Thr197, in addition to Ser338 in the C-terminal tail (6Peters K.A. Demaille J.G. Fischer E.H. Biochemistry. 1977; 16: 5691-5697Crossref PubMed Scopus (134) Google Scholar, 7Shoji S. Titani K. Demaille J.G. Fischer E.H. J. Biol. Chem. 1979; 254: 6211-6214Abstract Full Text PDF PubMed Google Scholar). Fig. 1, A and B, highlight the structure of the activation segment of PKA, including the phosphate on Thr197 and the environment surrounding the Ser338 phosphate. The phosphate on Thr197 in the activation loop of the C-subunit interacts with His87 of the C helix, Arg165 adjacent to the catalytic base, Asp166, Thr195 on the activation loop, and Lys189 in β-strand 9, which positions Arg190 to interact with the A helix. These interactions act in a synergistic fashion with the phosphate, helping to bring these residues to their proper spatial orientation, but they also anchor the phosphate and consequently the activation segment in a conformation required for activity (8Toner-Webb J. van Patten S.M. Walsh D.A. Taylor S.S. J. Biol. Chem. 1992; 267: 25174-25180Abstract Full Text PDF PubMed Google Scholar). Mutagenesis of Thr197 and Ser338 demonstrated their importance for full activity (9Adams J.A. McGlone M.L. Gibson R. Taylor S.S. Biochemistry. 1995; 34: 2447-2454Crossref PubMed Scopus (133) Google Scholar, 10Yonemoto W. McGlone M.L. Grant B. Taylor S.S. Protein Eng. 1997; 10: 915-925Crossref PubMed Scopus (84) Google Scholar). Many protein kinases, the structures of which have been solved in the unphosphorylated inactive form, show that the position of this loop differs from that of the phosphorylated enzyme (Fig. 1C) (8Toner-Webb J. van Patten S.M. Walsh D.A. Taylor S.S. J. Biol. Chem. 1992; 267: 25174-25180Abstract Full Text PDF PubMed Google Scholar, 11Xu W. Doshi A. Lei M. Eck M.J. Harrison S.C. Mol. Cell. 1999; 3: 629-638Abstract Full Text Full Text PDF PubMed Scopus (723) Google Scholar, 12Schindler T. Sicheri F. Pico A. Gazit A. Levitzki A. Kuriyan J. Mol. Cell. 1999; 3: 639-648Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 13Hubbard S.R. Wei L. Ellis L. Hendrickson W.A. Nature. 1994; 372: 746-754Crossref PubMed Scopus (954) Google Scholar, 14Russo A.A. Jeffrey P.D. Pavletich N.P. Nat. Struct. Biol. 1996; 3: 696-700Crossref PubMed Scopus (502) Google Scholar, 15Bishop S.M. Ross J.B. Kohanski R.A. Biochemistry. 1999; 38: 3079-3089Crossref PubMed Scopus (19) Google Scholar). Additionally, studies monitoring the fluorescence of an endogenous Trp on the insulin receptor kinase activation loop showed a change in fluorescence intensity upon phosphorylation of the activation loop and reflect the dynamic properties of the loop (15Bishop S.M. Ross J.B. Kohanski R.A. Biochemistry. 1999; 38: 3079-3089Crossref PubMed Scopus (19) Google Scholar).In many kinases, phosphorylation of the activation loop is a highly dynamic process triggered by a specific signal. In contrast, although PKA is also a phosphoprotein, it is not activated by phosphorylation of its activation loop in response to a signaling event. Instead, the fully phosphorylated enzyme is assembled with inhibitory regulatory subunits. Its activity in cells is thus controlled primarily by regulatory subunits (R-subunits) that bind the C-subunit with high affinity in the absence of cAMP (16Doskeland S.O. Ogreid D. J. Biol. Chem. 1984; 259: 2291-2301Abstract Full Text PDF PubMed Google Scholar). This mode of activation in which the regulatory and catalytic moieties are separate proteins is unusual in the protein kinase family. Although the C-subunit can be readily phosphorylated in vitro by 3-phosphoinositide-dependent protein kinase-1 (PDK-1), it also undergoes autophosphorylation when it is expressed in E. coli (17Moore M.J. Kanter J.R. Jones K.C. Taylor S.S. J. Biol. Chem. 2002; 277: 47878-47884Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Furthermore, mutant forms of the C-subunit that are defective in autophosphorylation are readily phosphorylated in mammalian cells, whereas mutants that are defective in recognition by PDK-1 are not phosphorylated (18Steinberg R.A. Mol. Cell. Biol. 1991; 11: 705-712Crossref PubMed Scopus (18) Google Scholar). However, nothing is known about the conformation of the C-subunit in its unphosphorylated state, and very little is known about the process by which the inactive dephosphorylated protein is converted into a fully phosphorylated protein prior to its association with regulatory subunits.In addition to these essential phosphorylation sites, there are various key residues in the kinase core that play a significant role in the stability and the functional organization of the kinase. One such residue, Lys72 located in subdomain II in the Hanks classification (19Hanks S.K. Quinn A.M. Hunter T. Science. 1988; 241: 42-52Crossref PubMed Scopus (3782) Google Scholar), represents one of the most conserved residues in the protein kinase core. This was the first residue to be identified in the active site of a protein kinase (Fig. 1D). The absolute conservation of this residue in every protein kinase subsequently reinforced its importance. This residue was first found to be important for kinase function of the C-subunit using an ATP affinity analog FSBA. The alkylating group on FSBA occupies the region of the protein that recognizes the phosphates of ATP (20Jacobson M.A. Colman R.F. J. Biol. Chem. 1984; 259: 1454-1460Abstract Full Text PDF PubMed Google Scholar, 21Bhatnagar D. Hartl F.T. Roskoski Jr., R. Lessor R.A. Leonard N.J. Biochemistry. 1984; 23: 4350-4357Crossref PubMed Scopus (22) Google Scholar), and treatment of the C-subunit with FSBA resulted in inactivation that was protected in the presence of MgATP. Peptide sequencing later identified the modified residue as Lys72 (22Zoller M.J. Nelson N.C. Taylor S.S. J. Biol. Chem. 1981; 256: 10837-10842Abstract Full Text PDF PubMed Google Scholar). Treatment with a hydrophobic carbodiimide, dicyclohexylcarbodiimide, in the absence of MgATP also irreversibly inhibited the C-subunit, due to cross-linking of Lys72 to Asp184, another conserved residue (23Buechler J.A. Taylor S.S. Biochemistry. 1989; 28: 2065-2070Crossref PubMed Scopus (28) Google Scholar).In this work, we have addressed two questions. First, we ask what are the functional consequences of mutation of lysine 72? Second, can this “dead” kinase be used to probe the pathway whereby the C-subunit is activated by phosphorylation? To achieve this, Lys72 was replaced with His, Arg, Ala, and Met. Additionally, to provide a suitable comparison with the inactive C-subunit, the wild type C-subunit was expressed in the presence of a PKA inhibitor, H-89. This C-subunit is not phosphorylated. Using phospho-specific antibodies, we show that both H-89 and the Lys72 mutants are excellent substrates for PDK-1 at Thr197; however, only wild type C-subunit can be phosphorylated at Ser338 and thus converted into an active enzyme. These results define a two-step activation process dependent on an intramolecular autophosphorylation at Ser338. In addition, we show that phosphorylation at Thr197 is sufficient for RIα binding, thus defining a new role for the activation loop independent of its role in coordinating the active site conformation. Thus the dead enzyme is still capable of forming a holoenzyme complex once it is phosphorylated.EXPERIMENTAL PROCEDURESMaterials—Reagents were obtained as follows: pRSETB expression vector (Invitrogen), [γ-32P]ATP (PerkinElmer Life Sciences), Escherichia coli strains BL21(DE3) (Novagen, Madison, WI), H-89 (LC Laboratories, Woburn, MA), Muta-Gene site directed mutagenesis kit and Affi-Gel (Bio-Rad), horseradish peroxidase-conjugated anti-rabbit IgG (Amersham Biosciences), Gammabind G-Sepharose (Amersham Biosciences), pcDNA-3 eukaryotic expression vector (Invitrogen), Super-Signal West Pico chemiluminescent substrate detection kit (Pierce), oligonucleotides (Genosis-Sigma), the PepTag PKA activity assay kit (Promega, Madison, WI), and Effectene transfection kit (Qiagen, Valencia, CA). Mouse monoclonal anti-Myc antibodies (Covance, Princeton, NJ), antibodies that specifically recognize the phosphorylated activation loop of protein kinase C (PKC), were a gift from A. Newton (University of California, San Diego, CA) (24Dutil E.M. Toker A. Newton A.C. Curr. Biol. 1998; 8: 1366-1375Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar). Antibodies against the catalytic subunit of PKA were described previously (10Yonemoto W. McGlone M.L. Grant B. Taylor S.S. Protein Eng. 1997; 10: 915-925Crossref PubMed Scopus (84) Google Scholar). Plasmid pCMV5 containing Myc-tagged PDK1 were the same as described previously (25Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (723) Google Scholar), and DNA sequencing was performed with the ABI Prism 310 Genetic Analyzer from PE Applied Biosystems. The peptide to the Thr197 sequence was synthesized at the Peptide and Oligonucleotide Facility at the University of California, San Diego on a Millagen 9050 PepSys peptide synthesizer using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) methodology activator and purified by high performance liquid chromatography.Mass Spectrometry—Electrospray/mass spectrometry was performed using a Hewlett-Packard 59887A electrospray mass spectrometer. Protein was desalted prior to analysis by narrow bore chromatography.Site-specific Antibodies—Antibodies were generated to distinguish the phosphorylation state of residues at several sites in the C-subunit. The antibody to the unphosphorylated Thr197 (α-Thr197-OH) was generated using a peptide corresponding to the sequence around Thr197: 192KGRTWTLCGTPEYLA206. This peptide was sent to Cocalico Biologicals, Inc. (Reamstown, PA) for antibody generation in rabbit. The peptide antigen and rabbit antibody to the phosphorylated Ser338 (α-Ser338-P) were generated by SynPep Corp. (Dublin, CA) using the peptide sequence IRVpS338INEKAGKE, where pS338 is phosphorylated serine. The α-Ser338-P and α-Thr197-OH antibodies were affinity-purified by conjugating wild type and K72H respectively to Bio-Rad Affi-Gel and following the manufacturer's protocol.Site-directed Mutagenesis of the PKA Catalytic Subunit—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 (26Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4886) Google Scholar, 27Yonemoto W.M. McGlone M.L. Slice L.W. Taylor S.S. Methods Enzymol. 1991; 200: 581-596Crossref PubMed Scopus (49) Google Scholar). cDNA for the C-subunit transfected into COS cells was engineered in the pcDNA-3 expression vector, with a HA epitope tag added at the C terminus of the protein. All mutations were made using the Muta-Gene kit as per the manufacturer's recommendations. DNA sequencing analysis instruments confirmed the presence of the correct mutation.Expression of Murine PKA Catalytic Subunit—Histidine-tagged 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 mm isopropyl-β-d-thiogalactopyranoside for an additional 6 h at 24 °C, collected by centrifugation, and stored frozen. To obtain unphosphorylated C-subunit, 50 μm H-89 was added to the cultures from a 1,000× stock of H-89 in Me2SO at the time of induction. Cells from 500 ml of culture were resuspended in 10-ml lysis buffer (50 mm sodium phosphate, 100 mm NaCl, 5 mm 2-mercaptoethanol, pH 8.0) and lysed by one pass in a French pressure cell at 1,000 p.s.i. Insoluble material was removed by centrifugation at 15,000 rpm in a Beckman JA20 rotor at 4 °C for 40 min. The proteins were purified via their His tag using Talon metal affinity resin (Clontech). In brief, supernatant was batch-bound to 1.0-ml resin/500-ml culture for 2 h at 4 °C. The resin was batch-washed twice with lysis buffer, a wash with 10 mm imidazole in lysis buffer followed by two 100 mm imidazole elutions and a final 500 mm elution.Catalytic Activity Assays—The PepTag assays were performed according to the manufacturer's instructions. This qualitative 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.Expression of Myc-tagged PDK-1 in 293 Cells—Human 293 cells were propagated at 2 × 106/10-cm dish in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. pCMV5 vector containing Myc-tagged PDK-1 was transfected using Qiagen Effectene transfection kit. 48 h after transfection, the cells were trypsinized and resuspended in buffer A (50 mm Tris-Cl, pH 7.5, 50 mm NaCl, 10 mm NaF, 10 mm β-glycerol phosphate, 10 mm sodium pyrophosphate, 0.5 mm EGTA, 1 mm dithiothreitol, 1 mm benzamidine, 0.5 mm phenylmethylsulfonyl fluoride, 0.1% Triton X-100, 10 μg/ml aprotinin) as described (25Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (723) Google 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. Purified recombinant PDK-1 was used when indicated and was a gift from A. Newton (University of California, San Diego).Immunoprecipitation and PDK-1 Phosphorylation Assays—For the immunoprecipitation experiments, 2 μl of mouse anti-Myc antibody were mixed with the cell extract from 1% of a 10-cm dish of 293 cells transfected with PDK-1 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 dithiothreitol, 10% glycerol, 1 mm benzamidine, 0.2 mm phenylmethylsulfonyl fluoride). For the kinase assay, 0.25 μg of (H-89)-C was mixed with 25 μm ATP, 5 μCi of [γ-32P]ATP, and 10 mm MgCl2 in 25 μl of buffer B and incubated with the immobilized PDK-1 for 45 min at 30 °C with frequent gentle mixing.Autophosphorylation of Ser338—1 μm H6-K72H was used as a substrate for PDK-1 as described above. After a 90-min incubation with PDK-1, 0.2 μm active untagged wild type C-subunit was added for an additional 60 min. Aliquots with and without the addition of wild type enzyme were added to SDS-gel loading buffer and subjected to SDS-PAGE followed by immunoblotting using the α-C-subunit and α-Ser338-P antibodies to determine the phosphorylation state of Ser338. To evaluate the concentration dependence of autophosphorylation, (H-89)-C subunit in varying amounts (1–10 μm) was mixed with 25 μm ATP, 5 μCi of [γ-32P]ATP, and 10 mm MgCl2 in 25 μl of buffer B and incubated for 30 min at 37 °C. The reaction was stopped by the addition of Laemmli loading buffer (4×) and boiled for 5 min. Proteins were separated by SDS-PAGE (10%), and the gel was dried and exposed to x-ray film.Binding to Regulatory Subunit—6 μg of mutant proteins were incubated for 60 min with 5 μg of RIα regulatory subunit (in presence of 5 mm Mg+2 and 5 mm ATP), which act as pseudo substrates in vivo, and run on a non-denaturing Tris/glycine gel. The H6-K72H did not bind to RIα, and a band was observed corresponding to the free R-subunit. The PDK-1-treated H6-K72H catalytic subunit mutant formed a complex with the RIα. The bands ran slightly higher than the wild type catalytic subunit since the mutant had a polyHis tag.Phosphorylation State of Transfected C-subunit Constructs—Wild type, T197A, and S338A in the pcDNA-3 expression vector HA-tagged at the C terminus were transfected into COS cells using the Effectine transfection kit as per the manufacturer's protocol. Cells were resuspended in Buffer A and lysed by three rounds of freeze-thaw followed by centrifugation at 4 °C for 20 min 16,000 rpm. Transfected C-subunit was isolated by immunoprecipitation using a mouse monoclonal anti-HA antibody. These samples were then immunoblotted with the indicated rabbit generated antibodies to determine expression and phosphorylation state.RESULTSPurification of Wild Type C-subunit and Lys72 Mutants— Lys72 is conserved throughout the protein kinase family and is critical to kinase activity. When this residue is mutated, activity is dramatically decreased. Mutation of the equivalent Lys in other kinase family members often serves as the traditional “kinase-dead” mutant. To probe the function of Lys72 and to explore its phosphorylation state, four different mutations (K72A, K72H, K72M, and K72R) were made at the Lys72 position. The mutant proteins were purified by the addition of a polyHis tag at the N terminus followed by affinity chromatography. A qualitative PepTag activity assay was used to determine whether these proteins were active. Although all proteins were purified in equivalent amounts, only the wild type subunit was active (Fig. 2B). Based on densitometry and the coupled kinase assay, the activity of these mutants was less than 1% of the wild type C-subunit.Fig. 2Characteristics of Lys72 mutant proteins. The purified Lys72 mutant proteins were assayed for their ability to act as substrates of PDK-1 and for activity. In A, wild type C-subunit and Lys72 mutant proteins were incubated with immunoprecipitated Myc-PDK-1 in the presence of MgATP and [γ-32P]ATP. 32P incorporation was visualized by autoradiography. B, the activity of these proteins was determined using the PepTag activity assay. Fluorescent tagged Kemptide substrate peptide was run on an agarose gel, where phosphorylation is determined by a shift in the direction of mobility. Experiments were carried out multiple times with similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mass spectroscopy of the mutant proteins indicated that their mass was consistent for each mutant protein in an unphosphorylated state (Table I). To determine whether these dead kinases could be phosphorylated by a heterologous protein kinase, the Lys72 mutant proteins were all incubated with PDK-1 and [32P]ATP. As indicated in Fig. 2A, all four mutant proteins were phosphorylated by PDK-1, in contrast to the wild type C-subunit, which is already fully phosphorylated prior to incubation with [32P]ATP. However, although these mutant proteins were good substrates for PDK-1, activity was not restored upon phosphorylation (data not shown).Table IMass spectroscopy of Lys72 mutant proteinsMutantTheoretical mass (two phosphates)Theoretical mass (unphosphorylated)Actual massH6K72A42,704.742,544.742,543.6H6K72H42,770.842,610.842,611.1H6K72M42,764.842,604.842,604.8H6K72R42,789.842,629.842,626.4 Open table in a new tab Phosphospecific Antibodies—To specifically characterize the phosphorylation state of the C-subunit, antibodies were generated that could distinguish the phosphorylation at Thr197 and Ser338. It was established previously that an antibody generated against a phosphorylated peptide corresponding to Thr500 in the activation loop of protein kinase C was also able to discriminate the phosphorylation state of Thr197 in the activation loop of the C-subunit (28Cheng X. Shaltiel S. Taylor S.S. Biochemistry. 1998; 37: 14005-14013Crossref PubMed Scopus (41) Google Scholar). This segment is highly conserved in PKA and PKC. This antibody will be referred to here as α-Thr197-P. Additional peptides were synthesized to generate antibodies specific to other phosphorylation sites. Peptides were synthesized corresponding to the unphosphorylated Thr197 of the activation loop as well as the phosphorylated site in the C-terminal tail at Ser338 and used as the antigen for generation of antibodies in rabbits. These antibodies are designated as α-Thr197-OH and α-Ser338-P.Autophosphorylation of Wild Type and Mutant C-subunit— The ability of the proteins to undergo autophosphorylation following phosphorylation with PDK-1 was tested using the antibodies specific to the two phosphorylation sites, Thr197 and Ser338. To compare wild type with the Lys72 mutants, we needed a wild type control protein that is not phosphorylated. To obtain dephosphorylated C-subunit, the wild type protein was expressed in the presence of an inhibitor of the C-subunit. The addition of H-89, an ATP analog, at the time of induction inhibits the C-subunit and prevents autophosphorylation (29Cauthron R.D. Carter K.B. Liauw S. Steinberg R.A. Mol. Cell. Biol. 1998; 18: 1416-1423Crossref PubMed Scopus (63) Google Scholar).Both K72H- and H-89-treated wild type were incubated with PDK-1, and samples of each, before and after PDK-1 treatment, were subjected to Western blot analysis. All three antibodies indicated that neither protein was phosphorylated before PDK-1 treatment (Fig. 3). Following incubation with PDK-1, each substrate protein was phosphorylated, but phosphorylation was not equivalent. The α-Thr197-P antibody indicated that both proteins were phosphorylated at this site. The antibody to the unphosphorylated Thr197 demonstrated, furthermore, that phosphorylation was essentially complete. Although phosphorylation of wild type (H-89) protein was complete, over 80% of K72H was phosphorylated. A significant difference between the two proteins was phosphorylation at the Ser338 site. Indeed, after PDK-1 treatment, only the wild type (H-89) protein underwent this second phosphorylation event. Only 2% of Lys72 mutant was phosphorylated, whereas over 74% of wild type (H-89) protein was phosphorylated at Ser338. A blot using an antibody to the C-subunit showed that all these proteins were of equal intensity before and after phosphorylation.Fig. 3Specificity of antibodies directed to phosphorylation sites in the C-subunit of PKA. Phosphorylated wild type (Wt), unphosphorylated wild type (H-89), and the unphosphorylated K72H mutant protein were used to demonstrate the specificity of phospho-specific antibodies by Western blot analysis. The first panel illustrates a probe using the α-Thr197-P antibody, the second panel illustrates a probe using the α-Thr197-OH antibody, and the third panel illustrates a probe using α-Ser338-P antibody. A blot using the α-C-subunit demonstrates equivalent protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Stoichiometry of Phosphorylation—The rate of phosphorylation of the Lys72 mutant proteins by PDK-1 was then compared with phosphorylation of the wild type (H-89) protein. Both proteins are rapidly phosphorylated, but more 32P was incorporated into the wild type (H-89) substrate as compared with the K72H mutant protein, despite an equivalent amount of protein (Fig. 4A). It was established previously that in vitro, the only site of phosphorylation by PDK-1 is Thr197 (30Cheng X. Ma Y. Moore M. Hemmings B.A. Taylor S.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9849-9854Crossref PubMed Scopus (191) Google Scholar), yet there is another phosphorylation event necessary to produce the fully phosphorylated and active C-subunit. This other phosphorylation event is likely brought about by autophosphorylation, which the mutant protein is incapable of undergoing.Fig. 4Phosphorylation and activity of C-subunit substrates by PDK-1 as a function of time.A, equivalent amounts of wild type (Wt) (H-89) and K72H (0.25 μg) were incubated with immunoprecipitated Myc-PDK-1 (5 nm) in the presence of MgATP and [γ-32P]ATP. Aliquots were removed at the indicated times and, after SDS-PAGE, 32P incorporation was visualized by autoradiography. B, PDK-1 was incubated with both wild type (H-89) and K72H (1 μm). Aliquots were removed at the indicated times, subjected to SDS-PAGE, and then immunoblotted with the α-Thr197-P, α-Thr197-OH, and α-Ser338-P antibodies. C, a time course was also performed using the PepTag assay on aliquots taken from reactions at the indicated times. Experiments were performed in triplicate with similar results.View Large Image Figure ViewerDownload Hi-res" @default.
• W2154535504 created "2016-06-24" @default.
• W2154535504 creator A5045962448 @default.
• W2154535504 creator A5061396000 @default.
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• W2154535504 date "2005-03-01" @default.
• W2154535504 modified "2023-09-30" @default.
• W2154535504 title "Consequences of Lysine 72 Mutation on the Phosphorylation and Activation State of cAMP-dependent Kinase" @default.
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