Phosphoprotein Analysis Using Antibodies Broadly Reactive against Phosphorylated Motifs
2002; Elsevier BV; Volume: 277; Issue: 42 Linguagem: Inglês
10.1074/jbc.m206399200
ISSN1083-351X
AutoresHui Zhang, Xiang‐ming Zha, Yi Tan, Peter Hornbeck, Allison J. Mastrangelo, Dario R. Alessi, Roberto D. Polakiewicz, Michael J. Comb,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoThe substrates of most protein kinases remain unknown because of the difficulty tracing signaling pathways and identifying sites of protein phosphorylation. Here we describe a method useful in detecting subclasses of protein kinase substrates. Although the method is broadly applicable to any protein kinase for which a substrate consensus motif has been identified, we illustrate here the use of antibodies broadly reactive against phosphorylated Ser/Thr-motifs typical of AGC kinase substrates. Phosphopeptide libraries with fixed residues corresponding to consensus motifs RXRXXT*/S* (Akt motif) and S*XR (protein kinase C motif) were used as antigens to generate antibodies that recognize many different phosphoproteins containing the fixed motif. Because most AGC kinase members are phosphorylated and activated by phosphoinositide-dependent protein kinase-1 (PDK1), we used PDK1−/− ES cells to profile potential AGC kinase substrates downstream of PDK1. To identify phosphoproteins detected using the Akt substrate antibody, we characterized the antibody binding specificity to generate a specificity matrix useful in predicting antibody reactivity. Using this approach we predicted and then identified a 30-kDa phosphoprotein detected by both Akt and protein kinase C substrate antibodies as S6 ribosomal protein. Phosphospecific motif antibodies offer a new approach to protein kinase substrate identification that combines immunoreactivity data with protein data base searches based upon antibody specificity. The substrates of most protein kinases remain unknown because of the difficulty tracing signaling pathways and identifying sites of protein phosphorylation. Here we describe a method useful in detecting subclasses of protein kinase substrates. Although the method is broadly applicable to any protein kinase for which a substrate consensus motif has been identified, we illustrate here the use of antibodies broadly reactive against phosphorylated Ser/Thr-motifs typical of AGC kinase substrates. Phosphopeptide libraries with fixed residues corresponding to consensus motifs RXRXXT*/S* (Akt motif) and S*XR (protein kinase C motif) were used as antigens to generate antibodies that recognize many different phosphoproteins containing the fixed motif. Because most AGC kinase members are phosphorylated and activated by phosphoinositide-dependent protein kinase-1 (PDK1), we used PDK1−/− ES cells to profile potential AGC kinase substrates downstream of PDK1. To identify phosphoproteins detected using the Akt substrate antibody, we characterized the antibody binding specificity to generate a specificity matrix useful in predicting antibody reactivity. Using this approach we predicted and then identified a 30-kDa phosphoprotein detected by both Akt and protein kinase C substrate antibodies as S6 ribosomal protein. Phosphospecific motif antibodies offer a new approach to protein kinase substrate identification that combines immunoreactivity data with protein data base searches based upon antibody specificity. phosphoinositide-dependent protein kinase-1 protein kinase C phosphate-buffered saline enzyme-linked immunosorbent assay 12-O-tetradecanoylphorbol-13-acetate 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid wild type matrix-assisted laser desorption ionization time-of-flight insulin-like growth factor 1 glycogen synthase kinase Progress in tracing signaling pathways has been limited by the lack of reagents and methods required to identify substrates of particular protein kinases (1Cohen P. Nat. Cell Biol. 2002; 4: 127-130Crossref PubMed Scopus (792) Google Scholar). The development of broadly reactive phosphotyrosine antibodies allowed identification and characterization of many tyrosine kinase substrates involved in growth factor and cytokine signaling (2Frackelton Jr., A.R. Posner M. Kannan B. Mermelstein F. Methods Enzymol. 1991; 201: 79-92Crossref PubMed Scopus (34) Google Scholar, 3Glenney J.R. Methods Enzymol. 1991; 201: 92-100Crossref PubMed Scopus (12) Google Scholar, 4Kamps M.P. Methods Enzymol. 1991; 201: 101-110Crossref PubMed Scopus (38) Google Scholar, 5White M.F. Backer J.M. Methods Enzymol. 1991; 201: 65-79Crossref PubMed Scopus (25) Google Scholar). However, despite considerable effort, the development of comparable phosphoserine and phosphothreonine specific antibodies has proven much more difficult. Because greater than 90% of protein phosphorylation occurs at serine and threonine residues (6Galski H., De Groot N. Ilan J. Hochberg A.A. Biochim. Biophys. Acta. 1983; 761: 284-290Crossref PubMed Scopus (8) Google Scholar), methods to identify and characterize these sites are needed. Site-specific phosphoserine/threonine antibodies have proven useful in monitoring specific signaling events; however, because these reagents can be prepared only after the precise site of phosphorylation have been mapped (7Czernik A.J. Girault J.A. Nairn A.C. Chen J. Snyder G. Kebabian J. Greengard P. Methods Enzymol. 1991; 201: 264-283Crossref PubMed Scopus (112) Google Scholar), they are not useful in discovery of new sites. Identification of phosphorylation sites by chemical modification (8Oda Y. Nagasu T. Chait B.T. Nat. Biotechnol. 2001; 19: 379-382Crossref PubMed Scopus (760) Google Scholar, 9Zhou H. Ranish J.A. Watts J.D. Aebersold R. Nat. Biotechnol. 2002; 20: 512-515Crossref PubMed Scopus (370) Google Scholar) and/or mass spectrometry (8Oda Y. Nagasu T. Chait B.T. Nat. 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Currently, fewer than 2,000 sites have been identified, emphasizing the need for high throughput and sensitive methods to identify, characterize, and monitor new sites of protein phosphorylation. Growth and survival signals activate phosphatidylinositol 3-kinase, which in turn activates protein kinases that contain phospholipid binding domains such as PDK11(12Toker A. Newton A.C. Cell. 2000; 103: 185-188Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 13Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1400) Google Scholar). In response to phosphatidylinositol 3-kinase and phospholipids, PDK1 phosphorylates and activates AGC protein kinases on sites within their activation loops (13Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1400) Google Scholar), resulting in the activation of many different AGC protein kinases including Akt (14Alessi D.R. Deak M. Casamayor A. Caudwell F.B. Morrice N. 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Here we use peptide libraries representing phosphorylated Ser/Thr protein kinase consensus motifs to two different AGC kinase subfamilies, Akt and PKC, as immunogens to produce phosphospecific antibodies that react selectively with phosphorylated motifs present in a broad array of AGC protein kinase substrates. To demonstrate that the phospho-motif antibodies could identify downstream substrates, we compared immunoreactivity of PDK1−/− ES cell extracts with wild type ES extracts. Combining one- and two-dimensional immunoblotting analysis using Akt and PKC substrate antibodies together with protein data base searching using epitope specificity matrices, we predicted and subsequently identified a 30-kDa protein detected in wild type but not PDK1−/− ES cells as S6 ribosomal protein. Understanding antibody specificity for phosphorylated motifs offers the possibility of using antibody-to-protein recognition features to scan protein databases to predict sites of antibody interaction. Here we show that antibodies recognizing phosphorylated motifs can serve as valuable proteomic tools, interfacing between immunoreactivity data and genome/proteome databases to identify new targets of protein phosphorylation. All the primary and secondary antibodies used in this study were from Cell Signaling Technology (Beverly, MA). The specificity and characterization of primary antibody are described in the Cell Signaling Technology web site (www.cellsignal.com). Materials used in immunoblotting analysis are from Cell Signaling Technology. Protease-inhibitor mixture tablets are from Calbiochem, and tissue culture reagents are from Invitrogen. All other chemicals were from Sigma and Calbiochem. Polyclonal antibodies to specific phosphorylation sites are produced by immunizing rabbits with a synthetic phosphorylated peptide (KLH (keyhole limpet hemocyanin) coupled) corresponding to residues surrounding phosphorylation sites. Antibodies are then purified by protein A column chromatography on anAmersham Biosciences ÄKTA fast protein liquid chromatograph to isolate the IgG antibody fraction. Affinity chromatography is then performed using peptides coupled to SulfoLink resin from Pierce according to manufacturer. Both phospho-peptide-containing resin and the corresponding non-phospho-peptide resin were prepared. Two rounds of subtractive purification were performed using the non-phospho-peptide resin; protein A eluate was incubated with non-phospho-peptide resin by rotation in a sealed column at room temperature for 1 h to remove antibodies reactive with the non-phospho version of the protein antigen. The column was drained, and the flow-through (containing the desired antibody) was incubated with fresh non-phospho-peptide resin. The flow-through from this second subtractive step was next purified by incubation with phospho-peptide resin. After the phospho-peptide column was washed twice with PBS, phospho-specific antibody (bound to the resin) was eluted with 0.1m glycine, pH 2.7, and pooled fractions were neutralized with 1 m Tris-HCl, pH 9.5 (∼1–2% of the fraction volume). The eluted phospho-specific antibody was then dialyzed overnight in PBS at 4 °C. The antibody against the consensus Akt substrate was raised against the following synthetic peptide antigen CXXXRXRXXT*XXXX, whereX represents a position in the peptide synthesis where a mixture of all 20 amino acids (excluding cysteine) were used, and T* represents phosphothreonine. The synthetic phospho-peptide was conjugated to keyhole limpet hemocyanin and injected into rabbits. Once rabbits showed high phospho-specific titers, serum was then purified by protein A chromatography. Phospho-Akt substrate antibody was found to be highly phospho-specific as crude serum, so that a subtraction step on a column containing the non-phospho-peptide was not necessary, and the elution from the protein A column was used directly for affinity chromatography on a phospho-peptide-containing column. To develop antibodies against the consensus conventional PKC substrate motif, the following synthetic degenerate peptide library was constructed CXXX(K/R)(K/R)S*(F/L/V)(K/R)(K/R)XXX, where K/R means lysine or arginine are present at that position in equal moles, F/L/V means phenylalanine, leucine, or valine are present at equal mole amounts, X represents any amino acid except Trp and Cys, and S* is phosphoserine. The synthetic phospho-peptide library was conjugated to keyhole limpet hemocyanin and injected into rabbits. Phosphospecific antibodies were purified as described as above. The sequence specificity for polyclonal antibodies made against the consensus Akt substrate motif was determined using the orientated-peptide library approach (29Yaffe M.B. Cantley L.C. Nature. 1999; 402: 30-31Crossref PubMed Scopus (71) Google Scholar, 30Songyang Z. Cantley L.C. Methods Mol. Biol. 1998; 87: 87-98PubMed Google Scholar, 31Songyang Z. Methods Enzymol. 2001; 332: 171-183Crossref PubMed Scopus (12) Google Scholar). The first peptide library was synthesized as AXXXXXXXT*XXXXXXXAKKK, whereX stands for a mixture of 19 amino acids (Cys was omitted). Sequence analysis of this peptide library indicates that it contains all 19 amino acids with less than a 3-fold variation for each amino acid at each degenerate position (data not shown). Phospho-substrate antibodies were bound to protein A beads by incubating 1 mg of affinity-purified antibody with 200 μl of 50% slurry of pre-swelled protein A beads at 4 °C overnight with gentle agitation. The beads were then washed 3 times with 1 ml of PBS with 0.5% of Nonidet P-40 followed by 2 washes with 1 ml of PBS. The beads were transferred to a microspin column (Bio-Rad) and washed 3 times with 1 ml of PBS. 1 mg of peptide library (30 mg/ml) was loaded to the antibody column and incubated at room temperature for 10 min followed by 1.5 h at 4 °C. The antibody column was rapidly washed twice with 1 ml of ice-cold PBS + 0.5% Nonidet P-40 and twice with 1 ml of ice-cold PBS. The bound peptides were eluted with 30% acetic acid at room temperature for 10 min. Peptides were separated from antibody by passing the eluent through a Centricon (10-kDa cut off) twice with 0.4 ml of 30% acetic acid. The solution was lyophilized or evaporated to dryness on a SpeedVac apparatus. The pellet was resuspended in 80 μl of water, 0.5 μl of aliquots were checked by MALDI-TOF to ensure the presence of eluted peptides, and 40-μl aliquots were used for sequencing. We always performed a control experiment simultaneously by performing library screening on protein A beads alone and protein A beads immobilized with another irrelevant antibody, phosphotyrosine antibody, which does not bind the peptide in library. Peptides eluted from the antibody columns were analyzed by automated amino acid sequencing (Applied Biosystems). The abundance of each amino acid at a given cycle in the sequence of the bound peptide mixture was divided by the abundance of the same amino acid in the same cycle of the starting peptide library. These raw preference values were then summed and normalized to the total number of amino acids in the degenerate position. A second peptide library was synthesized as sequence MAXXXRXXT*XGGGAKK, whereX is all 18 amino acids except Cys and Trp, after the first library screening resulted in a strong selection for arginine at −3 position. The same procedure was repeated with the secondary library. ELISA was performed after the standard ELISA protocol (48Perlmann H. Perlmann P. Cell Biology: A Laboratory Handbook. Academic Press, Inc., San Diego, CA1994Google Scholar). Briefly, 50 μl of 1 μmsynthetic phospho- and non-phospho-peptides were used to coat each well in 96-well plates. Coating was carried out overnight at 4 °C. Phospho-Akt substrate and phospho-PKC substrate antibodies were used at 1:1000 dilution. The plates were incubated at 37 °C for 2 h after the addition of primary antibody. An alkaline phosphatase-conjugated goat anti-rabbit antibody (Cell Signaling Technology) was used as secondary antibody, andp-nitrophenyl phosphate tablets (Sigma) were used for color development. Absorbance at 405 nm was read on an ELISA plate reader (PerkinElmer Model 1420-018). Generation of PDK1−/− cells was described previously (15Williams M.R. Arthur J.S. 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 (396) Google Scholar). PDK1+/+ and PDK1−/− ES cells were cultured to confluence on 15-cm diameter dishes in KnockOutTMDulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% KnockOutTM SR (Invitrogen), 0.1 mmnonessential amino acids, antibiotics (100 units of penicillin G, 100 mg/ml streptomycin), 2 mml-glutamine, 0.1 mm 2-mercaptoethanol, and 1000 units/ml ESGROTM. The cells were then serum-starved for 3 h by incubating in medium minus KnockOutTM SR and ESGROTM. The cells were then pretreated for 1 h with or without 100 μm LY294002 (LY) or 5 μmRo318220. The cells were then left unstimulated or stimulated with either IGF1 (100 ng/ml) or 12-O-tetradecanoylphorbol-13-acetate (TPA, 0.2 μg/ml) for 15 min. The cells were then lysed in 1.0 ml of ice-cold Buffer A and centrifuged at 4 °C for 10 min at 13,000 × g. The supernatants were frozen in liquid nitrogen and stored at −80 °C until use. Buffer A was 50 mm Tris-HCl, pH 7.5, 1 mm EGTA, 1 mm EDTA, 1% (by mass) Triton X-100, 1 mm sodium orthovanadate, 50 mm sodium fluoride, 5 mm sodium pyrophosphate, 0.27 msucrose, 1 μm microcystinLR, 0.1% (by volume) β-mercaptoethanol, and Complete proteinase inhibitor mixture (1 tablet/50 ml, Roche Molecular Biochemicals). A constitutively active form of Akt was generated using the PCR containing the Akt-coding sequence modified by the addition of the Src myristoylation signal and the hemagglutinin epitope. This form of Akt was subsequently subcloned into a pBABE-Puro retroviral vector, and viral particles were generated by co-transfection of ecotropic packaging into HEK293 cells. The MyrAkt virus was then used to infect exponentially growing NIH 3T3 cells, which then selected with puromycin (1.5 μg/ml). Pools of mock-infected and MyrAkt-infected NIH3T3 cells were starved in serum-free containing medium for 24 h before cell extracts were harvested for immunoblotting. 10 μg of protein lysate in SDS sample buffer (2% SDS, 10% glycerol, 80 mm Tris, pH 6.8,0.15m β-mercaptoethanol, 0.02% bromphenol blue) were subjected to 12% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Polyvinylidene difluoride membranes were used for two-dimensional immunoblotting. Blots were blocked with 5% nonfat dry milk in TBST (10 mm Tris, pH 7.5,150 mm NaCl, 0.05% Tween 20) for 1 h at room temperature, incubated with primary antibody overnight at 4 °C, incubated with goat anti-rabbit secondary antibody conjugated to horseradish peroxidase for 1 h at room temperature, and visualized using the chemiluminescent Western detection kit. Membrane stripping was carried out by incubating membranes in stripping buffer (37.5 mm Tris, pH 6.8, 2% SDS, 1% β-mercaptoethanol at 56 °C for 20 min. Stripped membranes were washed 3× with PBS with Tween followed by immunoblotting. 100 μg of wild type or PDK1−/− cell lysate were incubated with Phospho-(Ser/Thr) Akt substrate antibody overnight at 4 °C and protein A-agarose beads for 2 h. Beads were washed twice with cell lysis buffer (20 mm Tris, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1 mmEGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm Na3VO4, 1 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride) and twice with phosphate-buffered saline, and proteins were eluted with SDS sample buffer for Western blot analysis. 70 μg of wild type or PDK1−/− ES cells lysate (2 mg/ml) was precipitated by methanol and resuspended in 125 μl of urea lysis buffer (9m urea, 0.25% Triton X-100, 5 mm CHAPS, 2% ampholytes 4–7 or 6–11, 2% β-mercaptoethanol, protease inhibitor mixture, and 0.1 μm calyculin A). The lysate was then used to rehydrate 7-cm IPG strips after the manufacturer's recommendations (Amersham Biosciences). The rehydration process was carried out overnight at room temperature. For the first dimension (isoelectric focus), strips were run at 200 V for 0.01 h followed by 3500 V for 1.30 h and 3500 V for 3.30 h. Strips were then incubated with SDS buffer and subjected to the second dimension on 10% SDS gel at 130 V for 1.5 h. The gel was then transferred to polyvinylidene difluoride membranes and analyzed by Western bloting as described above. To identify components of the Akt- and PKC-signaling pathways, we prepared antibodies reactive with short linear motifs phosphorylated by the Akt and PKC subfamilies of AGC protein kinases. Fig.1 shows evolutionary relationships between the Akt subfamily, including Akt, p70 S6 kinase, ribosomal S6 kinase, and serum and glucocorticoid-inducible kinase (SGK). These enzymes phosphorylate consensus motifs of the form RXRXX(T*/S*), where X represents any amino acid, R represents Arg (sometimes Lys), and S*/T* represents phosphorylated serine/threonine (24Park J. Leong M.L. Buse P. Maiyar A.C. Firestone G.L. Hemmings B.A. EMBO J. 1999; 18: 3024-3033Crossref PubMed Scopus (482) Google Scholar, 33Alessi D.R. Caudwell F.B. Andjelkovic M. Hemmings B.A. Cohen P. FEBS Lett. 1996; 399: 333-338Crossref PubMed Scopus (552) Google Scholar, 34Kobayashi T. Deak M. Morrice N. Cohen P. Biochem. J. 1999; 344: 189-197Crossref PubMed Scopus (335) Google Scholar, 35Leighton I.A. Dalby K.N. Caudwell F.B. Cohen P.T. Cohen P. FEBS Lett. 1995; 375: 289-293Crossref PubMed Scopus (112) Google Scholar, 36Obata T. Yaffe M.B. Leparc G.G. Piro E.T. Maegawa H. Kashiwagi A. Kikkawa R. Cantley L.C. J. Biol. Chem. 2000; 275: 36108-36115Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). Conventional PKC family members phosphorylate substrates at serine or threonine in the context of arginine or lysine at the +2 and/or −2 positions (37Nishikawa K. Toker A. Johannes F.J. Songyang Z. Cantley L.C. J. Biol. Chem. 1997; 272: 952-960Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar). The primary sequence around the site of phosphorylation is an important determinant of downstream kinase specificity in vivo (38Songyang Z. Blechner S. Hoagland N. Hoekstra M.F. Piwnica-Worms H. Cantley L.C. Curr. Biol. 1994; 4: 973-982Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar). Phosphopeptide libraries with fixed residues corresponding to consensus Akt motifXXXRXRXXT*XXXand the conventional PKC motifXXXRXS*XRXXXX were used as antigens to generate polyclonal antisera in rabbits. Phosphospecific antibodies were purified from rabbit sera as described under "Experimental Procedures," and their specificity was characterized as described below. Purified phosphospecific polyclonal antisera prepared against the Akt consensus substrate motif was immobilized on protein A beads and used to select peptides from a synthetic oriented-peptide library, AXXXXXXXT*XXXXXXXAKKK, where X stands for a mixture of 19 amino acids (cystine omitted). Eluted peptides were sequenced, and the relative enrichment of each amino acid at different positions over the applied peptide library is shown in Fig. 2 A. The Akt substrate antibody was strongly selective for arginine at position −3 (see Fig. 2 A). To further determine antibody specificity, a second peptide library was synthesized where arginine at position −3 was fixed MAXXXRXXT*XGGGAKK, and peptides bound to the antibody were eluted and sequenced. The relative preference of each amino acid at different positions relative to the fixed phosphothreonine over the applied peptide library is shown in Fig. 2 B. These data indicate some selection for arginine at positions −6, −5, and −4. The same peptide library with fixed phosphoserine at position 0 was also synthesized, and the relative preference of each amino acid position over the applied peptide library (data not shown) is similar to the phosphothreonine library results shown in Fig. 2. In contrast, when phosphotyrosine antibody was used to select peptides from the same peptide libraries, no peptides were present in eluent from antibody as determined by MALDI-TOF mass spectrometry (data not shown). Epitope mapping of the Akt consensus substrate motif antibody was further determined by ELISA analysis together with arrays of synthetic phospho- and non-phospho-peptides. ELISA reactivities relative to phospho-GSK3 peptide are shown in TableI. Non-phospho-control peptides normally score in the range of 3–5% of phospho-GSK3. Akt consensus substrate motif antibody bound only to phospho-peptides. For phosphothreonine-containing peptides, arginine at position −3 is required, although lysine can substitute for arginine with weaker binding. For phosphoserine-containing peptides, arginine appears to be required at position −3 and at position −5 or −2. Good antibody binding was associated with hydrophobic amino acids at position +1, small non-charged residues at position −1, and either small residues or arginine/lysine at position −2.Table ISpecificity of Akt substrate antibody as analyzed using various phospho- and nonphospho-peptides containing different versions of the consensus Akt substrate motif as determined by ELISA Open table in a new tab ELISA analysis of synthetic peptides was also used to determine the binding specificity of the PKC substrate motif antibody. ELISA readings for each peptide are presented in TableII as a percentage relative to phospho-AFX peptide. The data indicate that this antibody binds only phosphoserine-containing peptides, where phosphoserine is followed by arginine or lysine at position +2. In addition, the antibody appears selective for hydrophobic amino acids at position +1.Table IISpecificity of PKC substrate antibody to consensus PKC substrate motif containing peptides determined by ELISA Open table in a new tab Because the phospho-Akt and -PKC substrate antibodies appeared to be generally reactive with Akt and PKC substrates, we next tested their reactivity with cell extracts. NIH3T3 cell extracts prepared 15 min after platelet-derived growth factor treatment showed an increase in immunoreactive proteins when analyzed by Western blotting using the Akt substrate antibody (Fig.3 A). Antibody reactivity to a subset of proteins was diminished or lost upon treatment with the phosphatidylinositol 3-kinase inhibitor, LY 294002 (Fig.3 A). Analysis with phospho-specific antibodies to Akt Ser-473 and Thr-308 indicated elevated Akt phosphorylation in the same extracts (data not shown). Similar Western analysis was carried out using the PKC substrate antibody and WEHI231 and Jurkat cells treated with TPA, an activator of PKC. TPA treatment stimulated antibody reactivity to a number of different proteins (Fig. 3 B). Antibody detection was compared with 32P incorporation using cell extracts phosphorylated in vitro with purified active Akt kinase (Fig. 3 C). Western blotting sensitivity using the
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