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• W2028268047 abstract "To understand the insulin-induced activation of 6-phosphofructo-2-kinase (PFK-2) of the bifunctional enzyme PFK-2/fructose-2,6-bisphosphatase in heart, the effect of phosphorylation by protein kinases of the insulin signaling pathways on PFK-2 activity was studied. Purified PFK-2/fructose-2,6-bisphosphatase from bovine heart is a mixture of two isoforms (M r 58,000 and 54,000 on SDS-polyacrylamide gels). The M r 54,000 protein is an alternatively spliced form, lacking phosphorylation sites for protein kinases. Recombinant enzymes corresponding to theM r 58,000 (BH1) and M r54,000 (BH3) forms were expressed and used as substrates for phosphorylation. The recombinant BH1 isoform was phosphorylated by p70 ribosomal S6 kinase (p70s6k), mitogen-activated protein kinase-activated protein kinase-1, and protein kinase B (PKB), whereas the recombinant BH3 isoform was a poor substrate for these protein kinases. Treatment with all protein kinases activated PFK-2 in the recombinant BH1 preparation. Phosphorylation of the recombinant BH1 isoform correlated with PFK-2 activation and was reversed by treatment with protein phosphatase 2A. All the protein kinases phosphorylated Ser-466 and Ser-483 in the BH1 isoform, but to different extents: p70s6k preferentially phosphorylated Ser-466, whereas mitogen-activated protein kinase-activated protein kinase-1 and PKB phosphorylated Ser-466 and Ser-483 to a similar extent. We propose that PKB is part of the insulin signaling cascade for PFK-2 activation in heart. To understand the insulin-induced activation of 6-phosphofructo-2-kinase (PFK-2) of the bifunctional enzyme PFK-2/fructose-2,6-bisphosphatase in heart, the effect of phosphorylation by protein kinases of the insulin signaling pathways on PFK-2 activity was studied. Purified PFK-2/fructose-2,6-bisphosphatase from bovine heart is a mixture of two isoforms (M r 58,000 and 54,000 on SDS-polyacrylamide gels). The M r 54,000 protein is an alternatively spliced form, lacking phosphorylation sites for protein kinases. Recombinant enzymes corresponding to theM r 58,000 (BH1) and M r54,000 (BH3) forms were expressed and used as substrates for phosphorylation. The recombinant BH1 isoform was phosphorylated by p70 ribosomal S6 kinase (p70s6k), mitogen-activated protein kinase-activated protein kinase-1, and protein kinase B (PKB), whereas the recombinant BH3 isoform was a poor substrate for these protein kinases. Treatment with all protein kinases activated PFK-2 in the recombinant BH1 preparation. Phosphorylation of the recombinant BH1 isoform correlated with PFK-2 activation and was reversed by treatment with protein phosphatase 2A. All the protein kinases phosphorylated Ser-466 and Ser-483 in the BH1 isoform, but to different extents: p70s6k preferentially phosphorylated Ser-466, whereas mitogen-activated protein kinase-activated protein kinase-1 and PKB phosphorylated Ser-466 and Ser-483 to a similar extent. We propose that PKB is part of the insulin signaling cascade for PFK-2 activation in heart. Fructose 2,6-bisphosphate (Fru-2,6-P2) 1The abbreviations used are: Fru-2,6-P2, fructose 2,6-bisphosphate; PFK-2, 6-phosphofructo-2-kinase; PKA, cyclic AMP-dependent protein kinase; PKB, protein kinase B; MAPK, mitogen-activated protein kinase; MAPKAP kinase-1, mitogen-activated protein kinase-activated protein kinase-1; PKI, PKA inhibitor peptide; FBPase-2, fructose-2,6-bisphosphatase; MOPS, 4-morpholinepropanesulfonic acid; PP2A, protein phosphatase 2A; PAGE, polyacrylamide gel electrophoresis; MALDI-MS, matrix-assisted laser desorption-ionization time-of-flight mass spectrometry; HPLC, high pressure liquid chromatography; BH1 and BH3, recombinant bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase long and short isoforms, respectively; BH1(His)6, histidine-tagged BH1. participates in the regulation of glycolysis in liver, heart, and other mammalian tissues by controlling the activity of 6-phosphofructo-1-kinase (1Hue L. Rider M.H. Biochem. J. 1987; 245: 313-324Crossref PubMed Scopus (334) Google Scholar). For example, in perfused rat hearts, glycolysis and Fru-2,6-P2 concentrations were increased in parallel after stimulation by adrenalin (2Hue L. Blackmore P.F. Shikama H. Robinson-Steiner A. Exton J.H. J. Biol. Chem. 1982; 257: 4308-4313Abstract Full Text PDF PubMed Google Scholar) and insulin (3Rider M.H. Hue L. FEBS Lett. 1984; 176: 484-488Crossref PubMed Scopus (28) Google Scholar) or by increasing the work load (4Depre C. Rider M.H. Veitch K. Hue L. J. Biol. Chem. 1993; 268: 13274-13279Abstract Full Text PDF PubMed Google Scholar). Both adrenalin and the work load increased Fru-2,6-P2 by activating 6-phosphofructo-2-kinase (PFK-2), the enzyme responsible for Fru-2,6-P2 synthesis. This activation resulted from phosphorylation by cyclic AMP-dependent protein kinase (PKA) or by the multifunctional Ca2+/calmodulin-dependent kinase II, probably at the same sites (4Depre C. Rider M.H. Veitch K. Hue L. J. Biol. Chem. 1993; 268: 13274-13279Abstract Full Text PDF PubMed Google Scholar). Insulin treatment also activated PFK-2 both in rat heart in vivo (3Rider M.H. Hue L. FEBS Lett. 1984; 176: 484-488Crossref PubMed Scopus (28) Google Scholar) and in isolated rat cardiomyocytes (5Lefebvre V. Méchin M.-C. Louckx M.P. Rider M.H. Hue L. J. Biol. Chem. 1996; 271: 22289-22292Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). To clarify the mechanism of this insulin-induced activation of PFK-2, we have studied the effects of thein vitro phosphorylation of heart PFK-2 by protein kinases of the insulin signaling cascades. On hormone binding, the activation of the insulin receptor causes autophosphorylation at several tyrosine residues. This leads to the docking of the insulin receptor substrate-1 and -2 and their phosphorylation at multiple tyrosine residues by the receptor. Adaptor proteins containing SH2 domains are recruited, which in turn can lead to the activation of at least two signaling pathways. One of these leads to the activation of the mitogen-activated protein kinase (MAPK) cascade, via the adaptor proteins Grb2/Sos and involving the activation of Ras and Raf (6Saltiel A.R. Am. J. Physiol. 1996; 270: E375-E385PubMed Google Scholar). As a consequence, the mitogen-activated protein kinase-activated protein kinase-1 (MAPKAP kinase-1) (also called p90 ribosomal S6 kinase, RSK-2, or p90 rsk (7Lavoinne A. Erickson E. Maller J.L. Price D.L. Avruch J. Cohen P. Eur. J. Biochem. 1991; 199: 723-728Crossref PubMed Scopus (74) Google Scholar)) becomes activated. Another pathway involves activation of the lipid kinase, phosphatidylinositol 3-kinase, through recruitment of its p85 regulatory subunit and the downstream activation of p70 ribosomal S6 kinase (p70s6k) (8Chung J. Grammer T.C. Lemon K.P. Kazlauskas A. Blenis J. Nature. 1994; 370: 71-75Crossref PubMed Scopus (657) Google Scholar). Finally, a third pathway has recently been described, which also involves phosphatidylinositol 3-kinase activation (9Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1880) Google Scholar) and which results in the activation of protein kinase B (PKB) (also known as Akt/RAC (11Belacossa A. Testa J.R. Staal S.P. Tsichlis P.N. Science. 1991; 254: 244-247Google Scholar, 12Jones P.F. Jakubowicz T. Pitossi F.J. Maurer F. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4171-4175Crossref PubMed Scopus (443) Google Scholar)) (10Coffer P.J. Woodgett J.R. Eur. J. Biochem. 1991; 210: 475-481Crossref Scopus (389) Google Scholar). Heart PFK-2/fructose-2,6-bisphosphatase (FBPase-2) differs from the other isozymes of the bifunctional enzyme (13Hue L. Rider M.H. Rousseau G.G. Pilkis S.J. Fructose 2,6-Bisphosphate. CRC Press, Inc., Boca Raton, FL1990: 173-192Google Scholar). The various PFK-2/FBPase-2 isozymes are homodimers and are known as the liver, skeletal muscle, heart, testis, and brain isozymes, and for most, the subunit molecular weight varies between 50,000 and 60,000. The rat gene for the heart isozyme contains 16 exons, one of which (exon 15) codes for a stretch of 64 amino acids containing phosphorylation sites for PKA and protein kinase C (14Darville M.I. Chikri M. Lebeau E. Hue L. Rousseau G.G. FEBS Lett. 1991; 288: 91-94Crossref PubMed Scopus (27) Google Scholar). In bovine heart, two alternatively spliced forms have been described (15Rider M.H. Van Damme J. Lebeau E. Vertommen D. Vidal H. Rousseau G.G. Vandekerckhove J. Hue L. Biochem. J. 1992; 285: 405-411Crossref PubMed Scopus (24) Google Scholar, 16Sakata J. Uyeda K. Biochem. Biophys. Res. Commun. 1991; 180: 470-474Crossref PubMed Scopus (8) Google Scholar), migrating withM r values of 58,000 and 54,000 on SDS-polyacrylamide gels (15Rider M.H. Van Damme J. Lebeau E. Vertommen D. Vidal H. Rousseau G.G. Vandekerckhove J. Hue L. Biochem. J. 1992; 285: 405-411Crossref PubMed Scopus (24) Google Scholar). Peptide sequence analysis revealed that the M r 54,000 form lacks phosphorylation sites for PKA (Ser-466 and Ser-483) and protein kinase C (Ser-466 and Thr-475), which are encoded by exon 15 and thus present in theM r 58,000 form (15Rider M.H. Van Damme J. Lebeau E. Vertommen D. Vidal H. Rousseau G.G. Vandekerckhove J. Hue L. Biochem. J. 1992; 285: 405-411Crossref PubMed Scopus (24) Google Scholar, 17Rider M.H. Van Damme J. Vertommen D. Michel A. Vandekerckhove J. Hue L. FEBS Lett. 1992; 310: 139-142Crossref PubMed Scopus (23) Google Scholar). The situation in bovine heart is further complicated by the fact that four different mRNAs have been characterized (18Vidal H. Crepin K.M. Rider M.H. Hue L. Rousseau G.G. FEBS Lett. 1993; 330: 329-333Crossref PubMed Scopus (16) Google Scholar). One (B3) lacks exon 15 and is therefore similar to the M r 54,000 form. The other three (B1, B2, and B4) contain exon 15 and could correspond to theM r 58,000 form. They differ from each other by minor differences located in domains devoid of any regulatory or catalytic activities (18Vidal H. Crepin K.M. Rider M.H. Hue L. Rousseau G.G. FEBS Lett. 1993; 330: 329-333Crossref PubMed Scopus (16) Google Scholar). Similarly, in rat heart, four mRNA species have been found (19Chikri M. Rousseau G.G. Biochemistry. 1995; 34: 8876-8884Crossref PubMed Scopus (7) Google Scholar), but information on the existence of the corresponding proteins is lacking since the heart PFK-2/FBPase-2 protein has not been purified for sequencing from this species. In this work, we have studied the effects of phosphorylation by several protein kinases on PFK-2 activity of the heart isozyme. With the native bovine heart preparations of PFK-2/FBPase-2 as substrate, the effect of phosphorylation on PFK-2 activity could be partially masked because the preparations contain about twice as much of the shortM r 54,000 form as the completeM r 58,000 form (15Rider M.H. Van Damme J. Lebeau E. Vertommen D. Vidal H. Rousseau G.G. Vandekerckhove J. Hue L. Biochem. J. 1992; 285: 405-411Crossref PubMed Scopus (24) Google Scholar). Therefore, we mainly investigated the effects of phosphorylation on PFK-2 activity of the recombinant BH1 and BH3 forms. The former is a complete isoform and corresponds to the M r 58,000 form, whereas the latter lacks the sequence encoded by exon 15 and thus corresponds to the short M r 54,000 form. The protein kinases tested were p70s6k, MAPKAP kinase-1, and PKB. The effects of phosphorylation on PFK-2 activity were compared with those induced by PKA. Radiochemicals were from Amersham Corp. Activated MAPKAP kinase-1 (350 units/ml), from rabbit skeletal muscle (20Sutherland C. Campbell D.G. Cohen P. Eur J. Biochem. 1993; 212: 581-588Crossref PubMed Scopus (113) Google Scholar), and activated p70s6k (14 units/ml), from the livers of cycloheximide-treated rats (21Leighton I.A. Dalby K.N. Caudwell F.B. Cohen P.T.W. Cohen P. FEBS Lett. 1995; 375: 289-293Crossref PubMed Scopus (112) Google Scholar), were purified as described. Activated PKBα (0.64 units/ml) was purified from insulin-like growth factor-1-stimulated 293 cells overexpressing the protein (22Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P. Reece C. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar). Glutathione was removed from the PKB preparation by gel filtration on a Superose 12 column (Pharmacia Biotech Inc.) in buffer containing 20 mm MOPS, pH 7, 25 mm KCl, 0.1 mmEDTA, 5% (v/v) glycerol, 0.1% (v/v) 2-mercaptoethanol, and 0.005% (w/v) Brij 35. The catalytic subunits of PKA (800 units/ml) (23Reimann E.M. Beham R.A. Methods Enzymol. 1983; 99: 51-55Crossref PubMed Scopus (173) Google Scholar) and protein phosphatase 2A (PP2A; 11,000 units/ml) (24Cohen P. Alemany S. Hemmings B.A. Resink T.J. Stralfors P. Tung H.Y. Methods Enzymol. 1988; 159: 390-408Crossref PubMed Scopus (387) Google Scholar) were purified from bovine heart as indicated. Activated MAPK (11 units/ml) was kindly donated by Dr. J. Goris (Katholieke Universiteit Leuven, Leuven, Belgium). Native bovine heart PFK-2/FBPase-2 was purified from slaughterhouse tissue (15Rider M.H. Van Damme J. Lebeau E. Vertommen D. Vidal H. Rousseau G.G. Vandekerckhove J. Hue L. Biochem. J. 1992; 285: 405-411Crossref PubMed Scopus (24) Google Scholar). The inhibitor peptide of PKA (PKI) (25Scott J.D. Glaccum M.D. Fischer E.H. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1613-1616Crossref PubMed Scopus (162) Google Scholar) and other peptides were synthesized by Drs. J. Lucchetti and V. Stroobant (Ludwig Institute, Brussels, Belgium). Ni2+/nitrilotriacetic acid-agarose gel was obtained from QIAGEN Inc. All other biochemicals were from Sigma or Boehringer Mannheim. The B1 and B3 PFK-2/FBPase-2 cDNAs were cloned in pBluescript II KS+phagemid (called BH1 and BH3 here) and introduced into the expression vector pET3a (18Vidal H. Crepin K.M. Rider M.H. Hue L. Rousseau G.G. FEBS Lett. 1993; 330: 329-333Crossref PubMed Scopus (16) Google Scholar). The C-terminally polyhistidine-tagged form of BH1, called BH1(His)6, was constructed from the single-stranded form of the phagemid containing the BH1 cDNA following the same procedure as described for the liver isozyme (26Vertommen D. Bertrand L. Sontag B. Di Pietro A. Louckx M. Vidal H. Hue L. Rider M.H. J. Biol. Chem. 1996; 271: 17875-17880Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The cDNA encoding BH1(His)6 was then introduced into the expression vector pET3a as described (18Vidal H. Crepin K.M. Rider M.H. Hue L. Rousseau G.G. FEBS Lett. 1993; 330: 329-333Crossref PubMed Scopus (16) Google Scholar). The BH1 and BH3 isoforms of bovine heart PFK-2/FBPase-2 were expressed and purified as described (26Vertommen D. Bertrand L. Sontag B. Di Pietro A. Louckx M. Vidal H. Hue L. Rider M.H. J. Biol. Chem. 1996; 271: 17875-17880Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Following concentration by ultrafiltration, the preparations were dialyzed against 200 volumes of a buffer containing 20 mmHEPES, pH 7.5, 50 mm KCl, 5 mmMgCl2, 0.5 mm EDTA, 0.1 mm EGTA, 1 mm potassium phosphate, 15 mm2-mercaptoethanol, 20% (v/v) glycerol, and 0.5 μg/ml leupeptin and stored at −80 °C. BH1(His)6 was expressed in Escherichia colistrain BL21(DE3) pLysE. Culture and lysis were as described above. Purification on Ni2+/nitrilotriacetic acid-agarose was carried out as described (26Vertommen D. Bertrand L. Sontag B. Di Pietro A. Louckx M. Vidal H. Hue L. Rider M.H. J. Biol. Chem. 1996; 271: 17875-17880Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), and the preparations were stored at −80 °C. For measurement of the changes in kinetic properties induced by phosphorylation, preparations of bovine heart PFK-2/FBPase-2 were incubated with 1 mm MgATP at 30 °C in phosphorylation buffer containing 10 mm MOPS, pH 7, 0.5 mm EDTA, 10 mm magnesium acetate, 0.1% (v/v) 2-mercaptoethanol, 5 μm PKI (except where PKA was studied), and each protein kinase as indicated in the figure and table legends. After 60 min, the reaction was stopped by 10-fold dilution in 20 mm HEPES, pH 7.5, 50 mm KCl, 0.5 mm EGTA, 5 mm EDTA, 1 mm potassium phosphate, 20% (v/v) glycerol, and 0.1% (v/v) 2-mercaptoethanol (stop buffer) and chilled in ice. Aliquots were taken for PFK-2 or FBPase-2 assay. For each protein kinase, phosphorylation had reached a maximum when the kinetic studies were carried out. For measurements of 32P incorporation, the bovine heart PFK-2/FBPase-2 preparations were incubated as described above with 0.1 mm [γ-32P]MgATP (specific radioactivity of 250 cpm/pmol) and each protein kinase as indicated in the figure legends. The MAPKAP kinase-1 and PKA preparations were diluted prior to use in a buffer containing 20 mm MOPS, pH 7.0, 0.1 mm EDTA, 0.01% (w/v) Brij 35, and 0.1% (v/v) 2-mercaptoethanol. The reactions were terminated by diluting aliquots (5 μl) with 15 μl of stop buffer before boiling for 1 min with 5 μl of 5% (w/v) SDS, 20% (v/v) glycerol, 0.2% (w/v) bromphenol blue, 100 mm dithiothreitol, and 65 mmTris-HCl, pH 6.8 (sample buffer), for SDS-PAGE (27Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207231) Google Scholar) on 12.5% (w/v) acrylamide minigels. To determine 32P incorporation, gels were stained with Coomassie Blue and dried, and bands corresponding to PFK-2/FBPase-2 were counted in a Hewlett-Packard Instant Imager together with spotted dried aliquots of the diluted (1:500) stock solution of [γ-32P]MgATP used in the phosphorylation experiments. Stoichiometries of 32P incorporation (mol/mol of subunit) were calculated from the amount of protein loaded onto the gel as quantified by the ninhydrin method (see below), and the molecular weights of the PFK-2/FBPase-2 subunits which were taken as 61,520 for BH1(His)6 and 53,909 for BH3. Synthetic peptides were incubated in 0.1 ml of phosphorylation buffer with 0.1 mm[γ-32P]MgATP (specific radioactivity of 200–1000 cpm/pmol) and each protein kinase as indicated in the legend to TableIV. Aliquots of the reaction mixture (10 μl) were removed between 2 and 10 min for the measurement of 32P incorporation (28Roskoski Jr., R. Methods Enzymol. 1983; 99: 3-6Crossref PubMed Scopus (692) Google Scholar).Table IEffect of treatment of native bovine heart PFK-2/FBPase-2 and the recombinant BH1(His) 6 and BH3 PFK-2/FBPase-2 preparations with PKA, p70 s6k, and MAPKAP kinase-1 on the kinetic properties of PFK-2 and FBPase-2Enzyme preparationPFK-2 activityFBPase-2 activityK m for Fru-6-PV maxK m for Fru-2,6-P2V maxμmmilliunits/mg proteinμmmilliunits/mg proteinNative Control60 ± 10 (4)55 ± 3 (4)ND1-aND, not determined.ND +PKA37 ± 6 (4)87 ± 11 (4)1-bSignificant difference (p < 0.05) with respect to the control (unpaired t test).NDND +p70s6k37 ± 8 (4)116 ± 16 (4)1-bSignificant difference (p < 0.05) with respect to the control (unpaired t test).NDND +MAPKAP kinase-125 ± 2 (4)1-bSignificant difference (p < 0.05) with respect to the control (unpaired t test).56 ± 4 (4)NDNDBH1(His)6 Control86 ± 12 (4)43 ± 1 (4)23 ± 5 (3)16 ± 1 (3) +PKA47 ± 8 (4)1-bSignificant difference (p < 0.05) with respect to the control (unpaired t test).93 ± 6 (4)1-bSignificant difference (p < 0.05) with respect to the control (unpaired t test).26 ± 10 (3)13 ± 3 (3) +p70s6k37 ± 4 (4)1-bSignificant difference (p < 0.05) with respect to the control (unpaired t test).119 ± 18 (4)1-bSignificant difference (p < 0.05) with respect to the control (unpaired t test).22 ± 5 (3)14 ± 2 (3) +MAPKAP kinase-137 ± 4 (4)1-bSignificant difference (p < 0.05) with respect to the control (unpaired t test).83 ± 8 (4)1-bSignificant difference (p < 0.05) with respect to the control (unpaired t test).25 ± 3 (3)11 ± 2 (3)BH3 Control36 ± 1 (3)225 ± 22 (3)5 ± 1 (4)6 ± 1 (4) +PKA27 ± 6 (3)199 ± 11 (3)7 ± 1 (4)7 ± 1 (4) +p70s6k24 ± 3 (3)1-bSignificant difference (p < 0.05) with respect to the control (unpaired t test).300 ± 17 (3)5 ± 1 (4)7 ± 0 (4) +MAPKAP kinase-136 ± 4 (3)204 ± 16 (3)5 ± 1 (4)6 ± 1 (4)Native bovine heart PFK-2/FBPase-2 and the recombinant BH1(His)6 and BH3 PFK-2/FBPase-2 preparations (0.1 mg/ml) were incubated with 5 μm PKI (control), 0.64 units/ml PKA, 0.84 units/ml p70s6k plus PKI, or 2.1 units/ml MAPKAP kinase-1 plus PKI at 30 °C. After 60 min, the reactions were stopped by dilution in stop buffer, and aliquots were taken for the measurement of PFK-2 and FBPase-2 activities. PFK-2 was measured at pH 7.1 in the presence of 5 mm phosphate, 5 mm MgATP, and concentrations of Fru-6-P up to 10 × K m . FBPase-2 was measured at pH 7.1 in the presence of 3 mmphosphate and concentrations of Fru-2,6-P2 up to 10 times theK m . The results are the means ± S.E. of the number of determinations shown in parentheses.1-a ND, not determined.1-b Significant difference (p < 0.05) with respect to the control (unpaired t test). Open table in a new tab Figure 2Time course of changes in phosphorylation and PFK-2 activity of BH1(His)6 on incubation with MAPKAP kinase-1 (A) and dephosphorylation by treatment with PP2A (B). A, BH1(His)6 (0.16 mg/ml) was incubated with 0.1 mm [γ-32P]MgATP and MAPKAP kinase-1 (3.5 units/ml) as described under “Experimental Procedures.” Aliquots (5 μl) were removed at the indicated times for SDS-PAGE, gel drying, and phosphorimaging for the measurement of32P incorporation (▪). In parallel incubations under control conditions (○) or with MAPKAP kinase-1 (•) and with 1 mm nonradioactive MgATP, aliquots were removed at the indicated times and diluted 10-fold in stop buffer for the assay of PFK-2 at pH 7.1 under suboptimal conditions with 40 μmFru-6-P and 5 mm MgATP. The results are the means of two separate experiments. B, BH1(His)6 was incubated as described above, except that 5 units/ml MAPKAP kinase-1 was used. After 60 min of incubation, the reaction was stopped with 10 mm EDTA, and 70 units/ml PP2A was added. Aliquots were removed at the indicated times for the measurement of32P-protein (▪) and PFK-2 activity (•) as described above. Control incubations for PFK-2 activity (○) were conducted in the absence of MAPKAP kinase-1. The results are the means of two separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IVComparison of the kinetic parameters for the phosphorylation of synthetic peptides by PKB and PKAPeptidePKBPKAK mV maxK mV maxμmunits/mg of proteinμmunits/mg of proteinPVRMRRN S FT (Ser-466)3.4 ± 0.70.270 ± 0.054.2 ± 1.72340 ± 480SNTIRRPRNY S VG (Ser-483)68 ± 210.230 ± 0.0244 ± 31390 ± 460GRPRTS S FAEG (“crosstide”)4-aRef. 31.3.1 ± 1.10.260 ± 0.0443 ± 2900 ± 220Synthetic peptides were phosphorylated by PKB (3 microunits with peptide 3 as substrate) in the presence of 2.5 μm PKI or by PKA (4 microunits with histone IIA as substrate) as described under “Experimental Procedures.” The concentration range for each peptide was 0.5–10 times the K m . The results are the means ± S.E. of three separate experiments.4-a Ref. 31Cross D.A.E. Alessi D.R. Cohen P. Andjelkovic M. Hemmings B.A. Nature. 1995; 378: 229-233Crossref PubMed Scopus (4376) Google Scholar. Open table in a new tab Native bovine heart PFK-2/FBPase-2 and the recombinant BH1(His)6 and BH3 PFK-2/FBPase-2 preparations (0.1 mg/ml) were incubated with 5 μm PKI (control), 0.64 units/ml PKA, 0.84 units/ml p70s6k plus PKI, or 2.1 units/ml MAPKAP kinase-1 plus PKI at 30 °C. After 60 min, the reactions were stopped by dilution in stop buffer, and aliquots were taken for the measurement of PFK-2 and FBPase-2 activities. PFK-2 was measured at pH 7.1 in the presence of 5 mm phosphate, 5 mm MgATP, and concentrations of Fru-6-P up to 10 × K m . FBPase-2 was measured at pH 7.1 in the presence of 3 mmphosphate and concentrations of Fru-2,6-P2 up to 10 times theK m . The results are the means ± S.E. of the number of determinations shown in parentheses. Synthetic peptides were phosphorylated by PKB (3 microunits with peptide 3 as substrate) in the presence of 2.5 μm PKI or by PKA (4 microunits with histone IIA as substrate) as described under “Experimental Procedures.” The concentration range for each peptide was 0.5–10 times the K m . The results are the means ± S.E. of three separate experiments. Recombinant BH1(His)6was first phosphorylated by 0.1 mm nonradioactive or [γ-32P]MgATP in phosphorylation buffer, in which the magnesium acetate and EDTA concentrations were reduced to 1 and 0.1 mm, respectively, and protein kinase as indicated in the figure legends. After 60 min, the reaction was stopped by adding an excess of EDTA (10 mm), followed by the indicated amount of PP2A. Aliquots were removed at the indicated times and diluted 3- or 10-fold in stop buffer, in which the KCl was replaced by 50 mm KF, for the measurement of 32P incorporation by autoradiography (see above) or PFK-2 activity, respectively. PFK-2 and FBPase-2 activities were measured (29Crepin K.M. Vertommen D. Dom G. Hue L. Rider M.H. J. Biol. Chem. 1993; 268: 15277-15284Abstract Full Text PDF PubMed Google Scholar) under the conditions described in the figure and table legends. One unit of PFK-2 or FBPase-2 activity corresponds to the formation of 1 μmol of product/min. The protein kinases were assayed by32P incorporation from [γ-32P]ATP into peptide or protein substrates (28Roskoski Jr., R. Methods Enzymol. 1983; 99: 3-6Crossref PubMed Scopus (692) Google Scholar). These included a peptide related to the C terminus of ribosomal protein S6 for p70s6k and MAPKAP kinase-1 (30Price D.J. Nemenoff R.A. Avruch J. J. Biol. Chem. 1989; 264: 13825-13833Abstract Full Text PDF PubMed Google Scholar), the glycogen synthase kinase peptide GRPRTSSFAEG for PKB (31Cross D.A.E. Alessi D.R. Cohen P. Andjelkovic M. Hemmings B.A. Nature. 1995; 378: 229-233Crossref PubMed Scopus (4376) Google Scholar), and histone IIA (1.25 mg/ml) or myelin basic protein (0.5 mg/ml) for the catalytic subunit of PKA and MAPK, respectively. PP2A was assayed with 4-nitrophenyl phosphate as substrate (24Cohen P. Alemany S. Hemmings B.A. Resink T.J. Stralfors P. Tung H.Y. Methods Enzymol. 1988; 159: 390-408Crossref PubMed Scopus (387) Google Scholar). One unit of protein kinase or protein phosphatase activity is the amount that catalyzes the (de)phosphorylation of 1 nmol of substrate/min. BH1(His)6 (50 μg) was phosphorylated with 0.1 mm [γ-32P]MgATP (specific radioactivity of 500 cpm/pmol) and 1.6 units/ml PKA, 2.8 units/ml p70s6k, 7 units/ml MAPKAP kinase-1, or 0.7 units/ml PKB as described above. The final incubation volumes were 0.15 ml for PKA, p70s6k, and MAPKAP kinase-1 and 0.3 ml for PKB. After 2 h at 30 °C, protein was precipitated (17Rider M.H. Van Damme J. Vertommen D. Michel A. Vandekerckhove J. Hue L. FEBS Lett. 1992; 310: 139-142Crossref PubMed Scopus (23) Google Scholar) and digested in 0.2 ml of 0.1 m Tris-Cl, pH 8.6, and 2 murea with 1 μg of bovine trypsin overnight at 30 °C. Peptides were separated by narrow bore HPLC (15Rider M.H. Van Damme J. Lebeau E. Vertommen D. Vidal H. Rousseau G.G. Vandekerckhove J. Hue L. Biochem. J. 1992; 285: 405-411Crossref PubMed Scopus (24) Google Scholar), collected by hand in Eppendorf tubes, and counted by Cerenkov radiation. Labeled peptides were further purified as described (17Rider M.H. Van Damme J. Vertommen D. Michel A. Vandekerckhove J. Hue L. FEBS Lett. 1992; 310: 139-142Crossref PubMed Scopus (23) Google Scholar). Aliquots of peaks containing radioactivity (0.7 μl) were spotted with 0.7 μl of matrix, consisting of a saturated solution of α-cyano-4-hydroxycinnamic acid in CH3CN and 0.1% (v/v) trifluoroacetic acid (2:1) plus substance P as an internal standard (1 pmol; M + H+ = 1348.7), and allowed to dry on the target. The mass spectrometer was a LASERMAT 2000 (Finnigan MAT Ltd., San Jose, CA), and masses were calculated from 20–30 cumulated spectra. Edman microsequencing was performed with an Applied Biosystems 477A gas-phase sequencer equipped with a phenylthiohydantoin detector. Solid-phase sequencing of peptides was carried out as described (32Stokoe D. Campbell D.G. Nakielny S. Hidaka H. Leevers S.J. Marshall C. Cohen P. EMBO J. 1992; 11: 3985-3994Crossref PubMed Scopus (392) Google Scholar). Protein was measured by the Bradford method (33Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar) using γ-globulin as a standard or by the reaction with ninhydrin, after total alkaline hydrolysis (34Allen G. Work T.S. Burdon R.H. Sequencing of Proteins and Peptides. Elsevier Science Publishers B. V., Amsterdam1989: 140-141Google Scholar), using bovine serum albumin as a standard. Kinetic constants were calculated by computer fitting of the data to a hyperbola describing the Michaelis-Menten equation by nonlinear least-squares regression. The two recombinant isoforms of bovine heart PFK-2/FBPase-2, BH1 and BH3, were compared with the native enzyme purified from bovine heart myocardium. Analysis of the purified recombinant BH1 and BH3 preparations by SDS-PAGE revealed that they contained major bands of M r 58,000 and 54,000, respectively, each one corresponding to the two forms of native bovine heart PFK-2/FBPase-2 (data not shown). However, truncated forms of BH1 were also observed, which have been noted previously (35Abe Y. Minami Y. Li Y. Nguyen C. Uyeda K. Biochemistry. 1995; 34: 2553-2559Crossref PubMed Scopus (9) Google Scholar). Electroblotting and N-terminal microsequencing of the intact and truncated proteins from SDS-polyacrylamide gels gave the same amino acid sequence, SGNPASSSEQ, suggesting that truncation resulted from the loss of a C-terminal fragment. This C-terminal truncation could result from a premature arrest of translation due to the presence of consecutive pro" @default.
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