Etude de protéines kinases responsables de l'activation de la 6-phosphofructo-2-kinase cardiaque par l'insuline
(2006)
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- Insulin stimulates heart glycolysis via 6-phosphofructo-2-kinase (PFK-2) activation thereby increasing fructose 2,6-bisphosphate concentrations. A PFK-2 kinase was partially purified, called WISK (wortmannin-sensitive and insulin-stimulated protein-kinase) and shown to be distinct from protein-kinase B (PKB). WISK has now been further purified from perfused rat hearts and from HeLa cells. Five consecutive chromatography steps were used. The relative molecular mass of WISK in the purified preparation was estimated at 63,000 by gel filtration. <BR> The WISK preparation phosphorylated and activated recombinant bovine heart PFK-2 and was shown to contain protein kinase C? (PKC?) by immunoblot analysis. Recombinant PKC? phosphorylated and activated PFK-2 in vitro. The phosphorylation sites for WISK were S466 and T475 while the phosphorylation site for PKC? was T475 as identified by mass spectrometry. However, PKC? was not activated by insulin in perfused rat hearts. Moreover, co-transfected kinase-inactive PKC? and wild-type PFK-2 in HEK 293T cells did not prevent insulin-induced heart PFK-2 activation. <BR> In parallel serum and glucocorticoid-stimulated protein kinase-3 (SGK3) was studied as this protein kinase has similar properties to WISK. In rat hearts treated with insulin, SGK3 was activated, as assessed by its activation loop T320 phosphorylation. Moreover, SGK3 activated PFK-2 in vitro by phosphorylating S466 and S483, the same sites phosphorylated by PKB. Co-transfection of HEK 293T cells with SGK3 siRNA indicated that this kinase was not essential for heart PFK-2 activation by insulin. <BR> Lastly, the role of PKB was re-assessed in three different models. Firstly, co-transfection of HEK 293T cells with PKB siRNA indicated that this kinase could participate in heart PFK-2 activation by insulin. Secondly, a model of knock-in mice in which a substrate-docking site of PDK1 was specifically ablated in muscle tissues in a way that prevents activation of its substrates (SGK, p70S6K, p90 RSK) but not PKB was generated in the laboratory of Dr D. Alessi. In these mice, PFK-2 was activated in response to insulin. Thirdly, we studied heart PFK-2 activation in PKB? knock-out mice. Taken together, the results suggest that PKB participates in the activation of heart PFK-2 by insulin
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Mouton, V. (2006). Etude de protéines kinases responsables de l’activation de la 6-phosphofructo-2-kinase cardiaque par l’insuline. https://hdl.handle.net/2078.5/112158