Phosphorylation and Activation of Heart 6-Phosphofructo-2-kinase by Protein Kinase B and Other Protein Kinases of the Insulin Signaling Cascades*

(Received for publication, March 27, 1997)

Johan Deprez Dagger §, Didier Vertommen Dagger §, Dario R. Alessi par , Louis Hue Dagger and Mark H. Rider Dagger **

From the Dagger  Hormone and Metabolic Research Unit, Institute of Cellular and Molecular Pathology and the Louvain University Medical School, Avenue Hippocrate, 75, 1200 Brussels, Belgium and the  Medical Research Council Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, Dundee, DD1 4HN, Scotland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


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 (Mr 58,000 and 54,000 on SDS-polyacrylamide gels). The Mr 54,000 protein is an alternatively spliced form, lacking phosphorylation sites for protein kinases. Recombinant enzymes corresponding to the Mr 58,000 (BH1) and Mr 54,000 (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.


INTRODUCTION

Fructose 2,6-bisphosphate (Fru-2,6-P2)1 participates in the regulation of glycolysis in liver, heart, and other mammalian tissues by controlling the activity of 6-phosphofructo-1-kinase (1). For example, in perfused rat hearts, glycolysis and Fru-2,6-P2 concentrations were increased in parallel after stimulation by adrenalin (2) and insulin (3) or by increasing the work load (4). 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 (4). Insulin treatment also activated PFK-2 both in rat heart in vivo (3) and in isolated rat cardiomyocytes (5). To clarify the mechanism of this insulin-induced activation of PFK-2, we have studied the effects of the in 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 (6). As a consequence, the mitogen-activated protein kinase-activated protein kinase-1 (MAPKAP kinase-1) (also called p90 ribosomal S6 kinase, RSK-2, or p90rsk (7)) 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) (8). Finally, a third pathway has recently been described, which also involves phosphatidylinositol 3-kinase activation (9) and which results in the activation of protein kinase B (PKB) (also known as Akt/RAC (11, 12)) (10).

Heart PFK-2/fructose-2,6-bisphosphatase (FBPase-2) differs from the other isozymes of the bifunctional enzyme (13). 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 (14). In bovine heart, two alternatively spliced forms have been described (15, 16), migrating with Mr values of 58,000 and 54,000 on SDS-polyacrylamide gels (15). Peptide sequence analysis revealed that the Mr 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 the Mr 58,000 form (15, 17). The situation in bovine heart is further complicated by the fact that four different mRNAs have been characterized (18). One (B3) lacks exon 15 and is therefore similar to the Mr 54,000 form. The other three (B1, B2, and B4) contain exon 15 and could correspond to the Mr 58,000 form. They differ from each other by minor differences located in domains devoid of any regulatory or catalytic activities (18). Similarly, in rat heart, four mRNA species have been found (19), 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 short Mr 54,000 form as the complete Mr 58,000 form (15). 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 Mr 58,000 form, whereas the latter lacks the sequence encoded by exon 15 and thus corresponds to the short Mr 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.


EXPERIMENTAL PROCEDURES

Materials

Radiochemicals were from Amersham Corp. Activated MAPKAP kinase-1 (350 units/ml), from rabbit skeletal muscle (20), and activated p70s6k (14 units/ml), from the livers of cycloheximide-treated rats (21), were purified as described. Activated PKBalpha (0.64 units/ml) was purified from insulin-like growth factor-1-stimulated 293 cells overexpressing the protein (22). 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 mM EDTA, 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) (23) and protein phosphatase 2A (PP2A; 11,000 units/ml) (24) 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 (15). The inhibitor peptide of PKA (PKI) (25) 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.

Construction of Expression Vectors

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 (18). 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 (26). The cDNA encoding BH1(His)6 was then introduced into the expression vector pET3a as described (18).

Expression and Purification

The BH1 and BH3 isoforms of bovine heart PFK-2/FBPase-2 were expressed and purified as described (26). Following concentration by ultrafiltration, the preparations were dialyzed against 200 volumes of a buffer containing 20 mM HEPES, pH 7.5, 50 mM KCl, 5 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 1 mM potassium phosphate, 15 mM 2-mercaptoethanol, 20% (v/v) glycerol, and 0.5 µg/ml leupeptin and stored at -80 °C.

BH1(His)6 was expressed in Escherichia coli strain BL21(DE3) pLysE. Culture and lysis were as described above. Purification on Ni2+/nitrilotriacetic acid-agarose was carried out as described (26), and the preparations were stored at -80 °C.

Protein Phosphorylation

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 [gamma -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 mM Tris-HCl, pH 6.8 (sample buffer), for SDS-PAGE (27) 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 [gamma -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.

Peptide Phosphorylation

Synthetic peptides were incubated in 0.1 ml of phosphorylation buffer with 0.1 mM [gamma -32P]MgATP (specific radioactivity of 200-1000 cpm/pmol) and each protein kinase as indicated in the legend to Table IV. Aliquots of the reaction mixture (10 µl) were removed between 2 and 10 min for the measurement of 32P incorporation (28).

Table IV. Comparison of the kinetic parameters for the phosphorylation of synthetic peptides by PKB and PKA

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 Km. The results are the means ± S.E. of three separate experiments.
Peptide PKB
PKA
Km Vmax Km Vmax

µM units/mg of protein µM units/mg of protein
PVRMRRNSFT (Ser-466) 3.4  ± 0.7 0.270  ± 0.05 4.2  ± 1.7 2340  ± 480
SNTIRRPRNYSVG (Ser-483) 68  ± 21 0.230  ± 0.02 44  ± 3 1390  ± 460
GRPRTSSFAEG ("crosstide")a 3.1  ± 1.1 0.260  ± 0.04 43  ± 2 900  ± 220

a Ref. 31.

Dephosphorylation by PP2A

Recombinant BH1(His)6 was first phosphorylated by 0.1 mM nonradioactive or [gamma -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.

Enzyme Assays

PFK-2 and FBPase-2 activities were measured (29) 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 by 32P incorporation from [gamma -32P]ATP into peptide or protein substrates (28). These included a peptide related to the C terminus of ribosomal protein S6 for p70s6k and MAPKAP kinase-1 (30), the glycogen synthase kinase peptide GRPRTSSFAEG for PKB (31), 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 (24). One unit of protein kinase or protein phosphatase activity is the amount that catalyzes the (de)phosphorylation of 1 nmol of substrate/min.

Identification of Phosphorylation Sites by MALDI-MS and Edman Microsequencing

BH1(His)6 (50 µg) was phosphorylated with 0.1 mM [gamma -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 (17) and digested in 0.2 ml of 0.1 M Tris-Cl, pH 8.6, and 2 M urea with 1 µg of bovine trypsin overnight at 30 °C. Peptides were separated by narrow bore HPLC (15), collected by hand in Eppendorf tubes, and counted by Cerenkov radiation. Labeled peptides were further purified as described (17). Aliquots of peaks containing radioactivity (0.7 µl) were spotted with 0.7 µl of matrix, consisting of a saturated solution of alpha -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 (32).

Other Methods

Protein was measured by the Bradford method (33) using gamma -globulin as a standard or by the reaction with ninhydrin, after total alkaline hydrolysis (34), 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.


RESULTS

Purification of Recombinant Bovine Heart PFK-2/FBPase-2 Isoforms

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 Mr 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 (35). 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 proline residues located between Arg-477 and Arg-496, identified by MALDI-MS peptide mapping (36) of the intact and truncated forms (data not shown).

To select and isolate the intact protein containing the C-terminal phosphorylation sites, we decided to add a 6-histidine tag at the C terminus of BH1. We previously engineered a polyhistidine tail on the liver PFK-2/FBPase-2 isozyme to facilitate its purification (26). This modification had no effect on PFK-2 or FBPase-2 activity of the liver isozyme (26). The BH1(His)6 recombinant enzyme was expressed in E. coli and purified. Analysis of the recombinant isoform by SDS-PAGE revealed a major band of Mr 61,000 ± 1000 (five preparations) and confirmed that the contamination by truncated forms was greatly reduced, if not abolished.

Kinetic Properties of PFK-2 and FBPase-2

The Vmax of PFK-2 in the native bovine heart preparation was ~50 milliunits/mg of protein. However, this value was probably underestimated since the preparation was not pure. The kinetic properties of the recombinant proteins were compared. The Vmax of PFK-2 of the recombinant BH3 isoform was four to five times greater than that of recombinant BH1(His)6, and its Km for Fru-6-P was about half that of the BH1(His)6 isoform (Table I). The Vmax of FBPase-2 of BH1(His)6 was about three times that of BH3, so the PFK-2/FBPase-2 activity ratios were approximately 3 and 40, respectively. The Km of FBPase-2 for Fru-2,6-P2 of the BH1(His)6 preparation was five times higher than that of BH3 (Table I). The FBPase-2 activity of the native bovine heart preparation was too low and did not allow accurate determinations of Km and Vmax to be made.

Table I. Effect 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, p70s6k, and MAPKAP kinase-1 on the kinetic properties of PFK-2 and FBPase-2

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 × Km. FBPase-2 was measured at pH 7.1 in the presence of 3 mM phosphate and concentrations of Fru-2,6-P2 up to 10 times the Km. The results are the means ± S.E. of the number of determinations shown in parentheses.
Enzyme preparation PFK-2 activity
FBPase-2 activity
Km for Fru-6-P Vmax Km for Fru-2,6-P2 Vmax

µM milliunits/mg protein µM milliunits/mg protein
Native
  Control 60  ± 10  (4) 55  ± 3  (4) NDa ND
  +PKA 37  ± 6  (4) 87  ± 11  (4)b ND ND
  +p70s6k 37  ± 8  (4) 116  ± 16  (4)b ND ND
  +MAPKAP kinase-1 25  ± 2  (4)b 56  ± 4  (4) ND ND
BH1(His)6
  Control 86  ± 12  (4) 43  ± 1  (4) 23  ± 5  (3) 16  ± 1  (3)
  +PKA 47  ± 8  (4)b 93  ± 6  (4)b 26  ± 10  (3) 13  ± 3  (3)
  +p70s6k 37  ± 4  (4)b 119  ± 18  (4)b 22  ± 5  (3) 14  ± 2  (3)
  +MAPKAP kinase-1 37  ± 4  (4)b 83  ± 8  (4)b 25  ± 3  (3) 11  ± 2  (3)
BH3
  Control 36  ± 1  (3) 225  ± 22  (3) 5  ± 1  (4) 6  ± 1  (4)
  +PKA 27  ± 6  (3) 199  ± 11  (3) 7  ± 1  (4) 7  ± 1  (4)
  +p70s6k 24  ± 3  (3)b 300  ± 17  (3) 5  ± 1  (4) 7  ± 0  (4)
  +MAPKAP kinase-1 36  ± 4  (3) 204  ± 16  (3) 5  ± 1  (4) 6  ± 1  (4)

a ND, not determined.
b Significant difference (p < 0.05) with respect to the control (unpaired t test).

Phosphorylation by Different Protein Kinases

PKA, p70s6k, MAPKAP kinase-1, PKB, and, to a much lesser extent, MAPK phosphorylated the Mr 58,000 and 54,000 bands of native bovine heart PFK-2/FBPase-2, although the extent of phosphorylation of the Mr 58,000 band greatly exceeded that of the Mr 54,000 band (Fig. 1). In agreement with the principal phosphorylation of the Mr 58,000 band of the native enzyme, the BH1(His)6 preparation was phosphorylated by PKA (0.77 ± 0.05 mol/mol of subunit; n = 3), p70s6k (0.79 ± 0.09 mol/mol of subunit; n = 6), MAPKAP kinase-1 (0.57 ± 0.05 mol/mol of subunit; n = 8), and PKB (0.69 ± 0.04 mol/mol of subunit; n = 12) (Fig. 1). BH1(His)6 was a rather poor substrate of MAPK (0.2 mol/mol of subunit) (Fig. 1), even though exon 15 contains the sequence PLS493P, which is in a suitable consensus for phosphorylation by this protein kinase.


Fig. 1. Phosphorylation of BH1(His)6, BH3, and the native forms of bovine heart PFK-2/FBPase-2 by PKA, p70s6k, MAPKAP kinase-1, and PKB. BH1(His)6 and BH3 (both at 0.08 mg/ml) and native bovine heart PFK-2/FBPase-2 (0.23 mg/ml) were phosphorylated with [gamma -32P]MgATP by PKA (0.32 units/ml), p70s6k (0.56 units/ml), MAPKAP kinase-1 (MAPKAPK-1; 1.4 units/ml), PKB (0.12 units/ml), or MAPK (0.88 units/ml) for 60 min in a final volume of 25 µl. Following SDS-PAGE on 12.5% acrylamide and gel drying, 32P incorporation was calculated by autoradiography as described under "Experimental Procedures."
[View Larger Version of this Image (39K GIF file)]

Only p70s6k and MAPKAP kinase-1 significantly phosphorylated the BH3 preparation (Fig. 1), and as expected, the stoichiometry of phosphorylation was very low (0.02 mol/mol of subunit with p70s6k). Therefore, the major phosphorylation sites reside in the sequence encoded by exon 15.

Effect of Treatment with Protein Kinases on Kinetic Properties of PFK-2 and FBPase-2

Treatment of native bovine heart PFK-2/FBPase-2 with the protein kinases slightly decreased the Km of PFK-2 for Fru-6-P and increased the Vmax, except with MAPKAP kinase-1, which did not affect the latter (Tables I and II).

Table II. Effect of treatment of the native and recombinant BH1(His)6 PFK-2/FBPase-2 preparations with PKB on the kinetic properties of PFK-2

The native and recombinant BH1(His)6 PFK-2/FBPase-2 preparations (0.1 mg/ml) were incubated with 2.5 µM PKI (control) or 0.3 units/ml PKB plus PKI at 30 °C. PFK-2 was measured as described in the legend to Table I. Glutathione was removed from the PKB preparation by gel filtration on a Superose 12 column in buffer containing 20 mM MOPS, pH 7, 25 mM KCl, 0.1 mM EDTA, 5% (v/v) glycerol, 0.1% (v/v) 2-mercaptoethanol, and 0.005% (w/v) Brij 35, and control incubations contained an aliquot of the same buffer. The results are the means ± S.E. of the number of determinations shown in parentheses.
Enzyme preparation PFK-2 activity
Km for Fru-6-P Vmax

µM milliunits/mg protein
    Native
      Control 40  ± 1 (5) 55  ± 3 (5)
      +PKB 28  ± 1(5)a 69  ± 2 (5)a
    BH1(His)6
      Control 120  ± 11 (4) 65  ± 12 (4)
      +PKB 53  ± 4 (4)a 192  ± 23 (4)a

a Significant difference (p < 0.05) with respect to the control (unpaired t test).

Treatment of BH1(His)6 with PKA, p70s6k, MAPKAP kinase-1, or PKB increased the Vmax of PFK-2 and decreased the Km for Fru-6-P (Tables I and II). As expected, the PFK-2 activity of BH3, which was a poor substrate for these protein kinases (Fig. 1), was little affected by treatment with the various protein kinases (Table I and data not shown for PKB). Therefore, this explains why the effects of the protein kinases on the kinetic properties of PFK-2 of the native enzyme were less pronounced than for BH1(His)6. None of the protein kinases tested modified the kinetic properties of FBPase-2 in the BH1(His)6 and BH3 preparations (Table I).

Correlation between Phosphorylation and PFK-2 Activity

We have studied the correlation between the extent of phosphorylation of BH1(His)6 and PFK-2 activity for MAPKAP kinase-1, p70s6k, and PKB. Treatment with MAPKAP kinase-1 led to the time-dependent phosphorylation of BH1(His)6, which paralleled the increase in PFK-2 activity (Fig. 2A). Likewise, phosphorylation by p70s6k or PKB correlated with the increase in PFK-2 activity (Fig. 3).


Fig. 2. Time 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 [gamma -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 of 32P incorporation (black-square). In parallel incubations under control conditions (open circle ) or with MAPKAP kinase-1 (bullet ) 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 µM Fru-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 of 32P-protein (black-square) and PFK-2 activity (bullet ) as described above. Control incubations for PFK-2 activity (open circle ) were conducted in the absence of MAPKAP kinase-1. The results are the means of two separate experiments.
[View Larger Version of this Image (15K GIF file)]


Fig. 3. Correlation between the phosphorylation of BH1(His)6 by p70s6k and PKB and the increase in PFK-2 activity. BH1(His)6 was incubated as described in the legends to Fig. 2A and Table II with 1.4 units/ml p70s6k or 0.4 units/ml PKB. Aliquots were taken at 2, 5, 10, 20, 30, 40, and 60 min for the determination of 32P incorporation (see legend to Fig. 2A) or PFK-2 activity, which was measured under optimal conditions at pH 7.1 with 1 mM (PKB) or 2 mM (p70s6k) Fru-6-P and 5 mM MgATP. Two different BH1(His)6 preparations with slightly different PFK-2 activities were used in the experiment.
[View Larger Version of this Image (13K GIF file)]

After phosphorylation of BH1(His)6 by MAPKAP kinase-1, the protein kinase reaction was stopped with EDTA prior to incubation with PP2A. The dephosphorylation of BH1(His)6 by PP2A correlated with the loss of PFK-2 activity (Fig. 2B). However, the activation of PFK-2 by MAPKAP kinase-1 was not totally reversed because BH1(His)6 was not completely dephosphorylated in this experiment. The small changes in kinetic properties of PFK-2 in the BH3 preparation seen after treatment with p70s6k (Table I) were not related to the phosphorylation state of the protein (data not shown).

Phosphorylation Site Identification

BH1(His)6 was phosphorylated with [gamma -32P]MgATP and the protein kinases under conditions that gave maximum phosphorylation and digested with trypsin. Following peptide purification by narrow bore HPLC, three labeled peaks were identified (Fig. 4). The labeled peptides were further purified and analyzed by Edman microsequencing and MALDI-MS (Table III). Solid-phase sequencing indicated that in peaks I and II, the burst of radioactivity occurred at Ser-466 (Fig. 4). The phosphorylation of Ser-466 by PKA has previously been demonstrated by Edman sequencing (17, 37). In peak III, the burst of radioactivity occurred at Ser-483 (Fig. 4), and we previously demonstrated the phosphorylation of Ser-483 by PKA (17).


Fig. 4. Narrow bore HPLC profile of tryptic fragments of BH1(His)6 phosphorylated by p70s6k. The narrow bore HPLC profile of BH1(His)6 phosphorylated by p70s6k and [gamma -32P]MgATP and digested with trypsin is shown. The aqueous phase was 0.1% (v/v) trifluoroacetic acid, and peptides were eluted in an acetonitrile gradient. The elution profiles of BH1(His)6 phosphorylated by PKA, MAPKAP kinase-1, and PKB were the same. The sequences of radioactive peptides purified from peaks I-III for the four protein kinases are given in Table III. Aliquots of the purified 32P-labeled peptides in peaks I (p70s6k), II (MAPKAP kinase-1), and III (MAPKAP kinase-1) were analyzed by solid-phase sequencing. AU, absorbance units.
[View Larger Version of this Image (31K GIF file)]

Table III. Edman microsequencing and MALDI-MS of tryptic peptides from BH1(His)6 phosphorylated by PKA, p70s6k, MAPKAP kinase-1, and PKB

BH1(His)6 was phosphorylated with [gamma -32P]MgATP and either PKA, p70s6k, MAPKAP kinase-1, or PKB and digested with trypsin as described under "Experimental Procedures". The positions of the labeled peaks in the HPLC elution profile are shown in Fig. 4. The labeled peaks were counted by Cerenkov radiation and further purified for Edman microsequencing and MALDI-MS. S* indicates a low yield of phenylthiohydantoin-Ser and the presence of phenylthiohydantoin-dehydroalanine, indicative of a phosphorylated Ser residue. X represents a gap in the sequence. Solid-phase sequencing confirmed the position of the phosphorylated amino acids (Fig. 4).
Protein kinase Peak cpm × 10-3 Sequence Measured mass Calculated mass

Da Da
p70s6k I 160 RNS*FTPLSSSNXIRRP ... NDa
RNS*FTPLSSSNXIR 1742.0 1739.7
p70s6k II 79 RNS*FTPLSSSNTIR 1660.1 1659.7
p70s6k III 61 NYS*VGSRPLQPLSPLR 1863.7 1864.0
PKA II 133 ND 1658.7 1659.7
PKA III 143 ND 1862.4 1864.0
MAPKAP kinase-1 II 131 ND 1659.1 1659.7
MAPKAP kinase-1 III 124 ND 1860.9 1864.0
PKB II 169 RNS*FTP ... 1658.2 1659.7
PKB III 139 NYS*VG ... 1861.4 1864.0

a ND, not determined.

Analysis of the extent of phosphorylation of the three peaks, after treatment with the various protein kinases, indicated that PKA, MAPKAP kinase-1, and PKB labeled the same peaks (II and III), i.e. Ser-466 and Ser-483, to the same extent, whereas p70s6k mainly labeled peak I (Ser-466) and peak II (also Ser-466). Moreover, the phosphorylation of Ser-483 by p70s6k was about four times less than that of Ser-466 (Table III). Interestingly, microsequencing of the peptides in peak I, which were only phosphorylated by p70s6k at Ser-466, suggested that Thr-475 was also phosphorylated since a gap in the sequence was found at this residue and, during solid-phase sequencing, a burst of radioactivity occurred at this position (Fig. 4). However, phosphoamino acid analysis of the peptides in peak I, following total acid hydrolysis and thin-layer chromatography, indicated that the phosphorylation of Thr-475 was only ~30% of that of Ser-466 (data not shown). Thr-475 has been shown to be phosphorylated by protein kinase C in native PFK-2/FBPase-2 (8, 17, 37), but treatment with protein kinase C was without effect on PFK-2 or FBPase-2 activity (15, 38).


DISCUSSION

In this paper, we show that the native and recombinant BH1(His)6 forms of bovine heart PFK-2/FBPase-2 are new substrates of p70s6k, MAPKAP kinase-1, and PKB and that phosphorylation activates PFK-2 (Tables I and II). With recombinant BH1(His)6, all the protein kinases tested decreased the Km for Fru-6-P and increased the Vmax.

A comparison of the extent of phosphorylation of the recombinant BH1(His)6 and BH3 preparations suggested that the major phosphorylation sites reside in the sequence encoded by exon 15. Following phosphorylation of BH1(His)6 and trypsin digestion, sequence analysis and MALDI-MS suggested that PKA, MAPKAP kinase-1, and PKB phosphorylated Ser-466 and Ser-483 to a similar extent, whereas p70s6k treatment preferentially labeled Ser-466. The stoichiometry of phosphorylation by the protein kinases tended toward 1 mol of phosphate incorporated per mol of subunit, but never reached the expected value of 2 mol of incorporation on the basis of complete phosphorylation of Ser-466 and Ser-483 in each subunit. Moreover, we were not able to increase the stoichiometry of phosphorylation, even after prolonged incubation with the protein kinases. This might indicate "half-of-the-sites" phosphorylation. Alternatively, only 50% of the recombinant protein might have been in the correct conformation for phosphorylation.

The amino acid sequences surrounding Ser-466 and Ser-483 are similar to those of Ser-9 of rabbit muscle glycogen synthase kinase-3beta and Ser-21 of rabbit glycogen synthase kinase-3alpha : bovine heart PFK-2, RMRRNS466FT; bovine heart PFK-2, RPRNYS483VG; muscle glycogen synthase kinase-3beta , RPRTTS9FA; and muscle glycogen synthase kinase-3alpha , RARTSS21FA.

Serine 9 in glycogen synthase kinase-3beta is phosphorylated by p70s6k and MAPKAP kinase-1 (39) and PKB (31). Indeed, sequences of the type RXRXXSXX, with arginine residues at positions n-3 and n-5 of the phosphorylated serine, are recognized by p70s6k and MAPKAP kinase-1 (21, 40). Serine 466 is in a favorable consensus for phosphorylation by PKB (Arg-Xaa-Arg-Yaa-Zaa-(Ser/Thr)-Hyd, where Xaa is any amino acid, Yaa and Zaa are small residues other than glycine, and Hyd is a bulky hydrophobic residue (41)). Indeed, a synthetic peptide containing Ser-466 was a good substrate of PKB (Table IV). By contrast, Ser-483 is in a rather unfavorable consensus for phosphorylation by PKB, especially due to the presence of the bulky tyrosine residue at position n-1 of Ser-483 and the lack of a large hydrophobic residue at position n+1 (41), despite the fact that its phosphorylation was clearly demonstrated in the intact protein (Table III). Moreover, a synthetic peptide containing Ser-483 was a poorer substrate for PKB, with a Km 20-fold higher than that of the peptide containing Ser-466 (Table IV). Therefore, the primary sequence around Ser-483 may not be the sole factor determining its phosphorylation by PKB. An interesting possibility is that phosphorylation of Ser-483 would require prior phosphorylation of Ser-466.

While this work was in progress, we were investigating the insulin signaling pathway leading to PFK-2 activation in isolated rat cardiomyocytes (5). The activation of PFK-2 by insulin was sensitive to wortmannin, a phosphatidylinositol 3-kinase inhibitor, but was not blocked by rapamycin, which prevents the activation of p70s6k, or by PD 98059, a compound that blocks the MAPK cascade and hence MAPKAP kinase-1 activation. Therefore, the insulin signaling pathway for PFK-2 activation probably involves phosphatidylinositol 3-kinase activation, and indeed, our data show that insulin activates this enzyme in rat heart (5). The insensitivity of the insulin-induced activation of PFK-2 to rapamycin and PD 98059 in cardiomyocytes suggests that the effect is unlikely to be mediated by p70s6k or MAPKAP kinase-1.

A good candidate for mediating the insulin-induced activation of heart PFK-2 is therefore PKB, as already described for the insulin-induced inactivation of glycogen synthase kinase-3 in a skeletal muscle cell line (31). Indeed, our preliminary results indicate that PKB is activated in rat hearts in response to insulin. Furthermore, the changes in PFK-2 kinetic properties induced by phosphorylation of BH1(His)6 were similar to those observed in vivo (4), but are partially masked in the native bovine heart enzyme, which contains more of the BH3 isoform. However, rat heart may contain proportionately more of the long isoform corresponding to BH1. In conclusion, we propose that PKB is part of the insulin signaling cascade leading to PFK-2 activation in heart.


FOOTNOTES

*   This work was supported in part by the Belgian State Program on Interuniversity Poles of Attraction, Prime Minister's Office, Federal Office for Scientific, Technical, and Cultural Affairs; by the D. G. Higher Education and Scientific Research, French Community of Belgium; by the Fund for Medical Scientific Research (Belgium); and by the Medical Research Council (United Kingdom).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   Supported by the Fund for Scientific Development of the University of Louvain and by the Fund for Scientific Research in Industry and Agriculture.
par    Supported by the Medical Research Council (United Kingdom).
**   Research Associate of the National Fund for Scientific Research (Belgium). To whom correspondence should be addressed: HORM Unit, ICP-UCL 75.29, Avenue Hippocrate, 75, 1200 Brussels, Belgium. Tel.: 32-2-764-74-86; Fax: 32-2-762-74-55; E-mail: rider{at}horm.ucl.ac.be.
1   The 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.

ACKNOWLEDGEMENTS

We thank Professor P. Cohen for interest and Dr. B. Caudwell (University of Dundee) for help with the solid-phase sequencing. For the gas-phase sequencing, the help of H. Degand and Dr. M. Boutry (University of Louvain) is gratefully acknowledged. Finally, we thank Dr. M.-C. Méchin for initial phosphorylation trials and M. P. Louckx for technical help.


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