Activation of phosphatidylinositol 3-kinase is required for transcriptional activity of F-type 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: assessment of the role of protein kinase B and p70 S6 kinase.
Previous studies have demonstrated that the F isoform of<hsp sp=0.5>6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase(6PF2K/Fru-2,6-BPase) is transcriptionally regulated by growth factors. The aim of this study was to investigate the importance of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway in the regulation of 6PF2K/Fru-2,6-BPase gene expression. We have completed studies using chemical inhibitors and expression vectors for the proteins involved in this signalling cascade. Treatment of cells with LY 294002, an inhibitor of PI 3-kinase, blocked the epidermal growth factor (EGF)-dependent stimulation of 6PF2K/Fru-2,6-BPase gene transcription. Transient transfection of a constitutively active PI 3-kinase was sufficient to activate transcription from the F-type 6PF2K/Fru-2,6-BPase promoter. In contrast, co-transfection with a dominant-negative form of PI 3-kinase completely abrogated the stimulation by EGF, and down-regulated the basal promoter activity. In an attempt to determine downstream proteins that lie between PI 3-kinase and 6PF2K/Fru-2,6-BPase gene expression, the overexpression of a constitutively active form of protein kinase B (PKB) was sufficient to activate 6PF2K/Fru-2,6-BPase gene expression, even in the presence of either a dominant-negative form of PI 3-kinase or LY 294002. The over-expression of p70/p85 ribosomal S6 kinase or the treatment with its inhibitor rapamycin did not affect 6PF2K/Fru-2,6-BPase transcription. We conclude that PI 3-kinase is necessary for the transcriptional activity of F-type 6PF2K/Fru-2,6-BPase, and that PKB is a downstream effector of PI 3-kinase directly involved in the regulation of 6PF2K/Fru-2,6-BPase gene expression.
Fernández de Mattos
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S
Departament de Bioquímica i Biologia Molecular, Div. IV, Facultat de Farmàcia, Universitat de Barcelona, Av. Diagonal 643, E08028 Barcelona, Catalunya, Spain.
de los Pinos E
E
E
Joaquin
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M
Tauler
A
A
D000818:Animals; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D002867:Chromones / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D005799:Genes, Dominant; D015151:Immunoblotting; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D025481:Phosphofructokinase-2; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D019893:Ribosomal Protein S6 Kinases / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection
D015500:Chloramphenicol O-Acetyltransferase; D002867:Chromones; D004791:Enzyme Inhibitors; D004815:Epidermal Growth Factor; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D025481:Phosphofructokinase-2; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010744:Phosphoric Monoester Hydrolases; D017853:Phosphotransferases (Alcohol Group Acceptor); D020033:Protein Isoforms; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D019893:Ribosomal Protein S6 Kinases; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one