Evaluation of the role of protein kinase Cζ in insulin-induced heart 6-phosphofructo-2-kinase activation

Abstract

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 Cζ (PKCζ). Both WISK and recombinant PKCζ were inhibited by a pseudo-substrate peptide inhibitor of PKCζ. WISK and PKCζ phosphorylated and activated recombinant heart PFK-2 by increasing its V max. The phosphorylation sites in heart PFK-2 for WISK were Ser466 and Thr475, whereas PKCζ 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 PKCζ 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 PKCζ Thr410 phosphorylation was detected. Lastly in HEK 293T cells transfected with heart PFK-2, co-transfection with a kinase-inactive PKCζ construct failed to prevent insulin-induced PFK-2 activation. Therefore, it is unlikely that PKCζ is required for PFK-2 activation by insulin in heart.