Evaluation of the role of protein kinase Czeta in insulin-induced heart 6-phosphofructo-2-kinase activation.
A wortmannin-sensitive and insulin-stimulated protein kinase (WISK) that phosphorylates and activates heart 6-phosphofructo-2-kinase (PFK-2) was purified from serum-fed HeLa cells and found to contain protein kinase Czeta (PKCzeta). Both WISK and recombinant PKCzeta were inhibited by a pseudo-substrate peptide inhibitor of PKCzeta. WISK and PKCzeta phosphorylated and activated recombinant heart PFK-2 by increasing its Vmax. The phosphorylation sites in heart PFK-2 for WISK were Ser466 and Thr475, whereas PKCzeta phosphorylated only Thr475. In perfused rat hearts, insulin activated protein kinase B (PKB) 16-fold compared with the untreated controls. However in the same experiments, no change in phosphorylation state of the activation loop Thr410 residue of PKCzeta was observed. By contrast, in incubations of isolated rat epididymal adipocytes, where insulin activated PKB 30-fold compared with the untreated controls, a 50% increase in PKCzeta Thr410 phosphorylation was detected. Lastly in HEK 293T cells transfected with heart PFK-2, co-transfection with a kinase-inactive PKCzeta construct failed to prevent insulin-induced PFK-2 activation. Therefore, it is unlikely that PKCzeta is required for PFK-2 activation by insulin in heart.
Mouton
Véronique
V
Hormone and Metabolic Research Unit, Christian de Duve Institute of Cellular Pathology and Université catholique de Louvain, Avenue Hippocrate, 75, B-1200 Brussels, Belgium.
Vertommen
Didier
D
Bertrand
Luc
L
Hue
Louis
L
Rider
Mark H
MH
D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology* / Q000502:physiology; D008297:Male; D009206:Myocardium / Q000201:enzymology*; D025481:Phosphofructokinase-2 / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000502:physiology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000378:metabolism; D000099221:Protein Kinase C zeta
D007328:Insulin; D025481:Phosphofructokinase-2; D011493:Protein Kinase C; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins; D000099221:Protein Kinase C zeta; C408700:wortmannin-sensitive and insulin-sensitive protein kinase, rat