Phosphorylation and activation of heart 6-phosphofructo-2-kinase by protein kinase B and other protein kinases of the insulin signaling cascades.
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 (p70(s6k)), 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: p70(s6k) 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.
Deprez
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Hormone and Metabolic Research Unit, Institute of Cellular and Molecular Pathology and the Louvain University Medical School, Avenue Hippocrate, 75, 1200 Brussels, Belgium.
Vertommen
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Alessi
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Hue
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Rider
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation; D007328:Insulin / Q000502:physiology*; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D025481:Phosphofructokinase-2; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D019893:Ribosomal Protein S6 Kinases; D038744:Ribosomal Protein S6 Kinases, 90-kDa; D015398:Signal Transduction
D007328:Insulin; D007527:Isoenzymes; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D025481:Phosphofructokinase-2; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019893:Ribosomal Protein S6 Kinases; D038744:Ribosomal Protein S6 Kinases, 90-kDa; D017868:Cyclic AMP-Dependent Protein Kinases