Abstract 21013: Caveolin 3-Containing Lipid Rafts Synergized With Insulin to Activate Akt Kinase by Blocking Akt Dephosphorylation
Introduction: Cardiac overexpression of muscle specific caveolin-3 (Cav3) protects against ischemic/reperfusion injury through Akt kinase activation. However, the mechanism by which Cav3 regulates Akt activation remains unclear. We tested the hypothesis that Cav3 regulates Akt activity by directly modulating Akt activation loop phosphorylation (T308 in Akt1).
Methods and Results: Adult mouse cardiac myocytes were infected with an adeno-virus that expressed Cav3 and GFP (Ad-GFP/Cav3). In the absence of serum, Cav3 overexpression did not significantly promote T308 phosphorylation. However, when myocytes were stimulated with insulin for 15 minutes, Cav3 synergized with insulin to induce T308 phosphorylation (Ad-GFP-In: 6.9±1.3, Ad-GFP/Cav3-In: 13.7±0.8, p<0.01). The cholesterol-depleting lipid raft inhibitor, cyclodextrin (CyDex), prevented Cav3-mediated Akt activation, suggesting that Cav3 regulates Akt activation through lipid rafts. To test whether lipid rafts directly modulate Akt-T308 phosphorylation, we expressed membrane-targeted Myr-Akt1 which were constitutively phosphorylated at T308. After treating the cells with CyDex to disrupt lipid rafts, we isolated Cav3-containing lipid rafts and non-raft membranes and found that CyDex treatment preferentially reduced Myr-Akt1 phosphorylation in Cav3-containing lipid rafts (non-raft mem: 21±5% inhibition, Cav3/lipid raft: 52±6% inhibition, p<0.05), suggesting that Cav3/lipid rafts protected Myr-Akt1 from de-phosphorylation. To confirm, we pharmacologically inhibited PDK1, the kinase responsible for Akt activation loop phosphorylation, and found that after PDK1 inhibition, Myr-Akt1 in non-raft membranes were dephosphorylated at a faster rate when compared to Myr-Akt1 localized in Cav3/lipid rafts (non-raft mem: 63±3% inhibition, Cav3/ lipid rafts: 18±10% inhibition, p<0.05).
Conclusions: We showed that insulin and Cav3 cooperated to induce Akt activation through lipid rafts. The basis for this synergy is that insulin stimulation promotes PDK1 to phosphorylate Akt activation loop. On the other hand, Cav3/lipid rafts complement PDK1 action by blocking Akt dephosphorylation.
- © 2010 by American Heart Association, Inc.