Abstract 17218: ATP Binding to Akt Kinases Confer Dephosphorylation Resistence in Caveolin 3/ Membrane Rafts
Introduction: Akt kinase activation loop phosphorylation (T308 in Akt1) in vivo is regulated by the opposing activities of kinases and phosphatases. To investigate this regulation balance, we showed that the translocation of Akt1 to caveolin-3 (Cav3) containing membrane caveolar/rafts markedly reduced its dephosphorylation by cellular phosphatases. Furthermore, this dephosphorylation resistence was regulated by Akt kinase binding to ATP or ATP-competitive inhibitors.
Methods and Results: In H9C2 cells or in adult cardiac myocytes, we showed that Cav3 overexpression cooperated with insulin to increase T308 phosphorylation by inhibiting Akt dephosphorylation. Cav3 overexpression is known to increase membrane caveolar/raft density. Conversely, treating cells with the cholesterol-depleting lipid raft inhibitor, methyl cyclodextrin (MβCD), enhanced Akt1 dephosphorylation. MβCD also enhanced the dephosphorylation of constitutively phosphorylated myristylated-Akt (Myr-Akt), suggesting that Cav3/lipid rafts protected Akt1 from dephosphorylation. Similarly, an Akt-specific ATP competitive inhibitor (A443654) also cooperated with insulin to increase T308 phosphorylation. Pharmacological and genetic data determined that dephosphorylation resistance depended on the binding of ATP or ATP-competitive inhibitors to Akt kinases. Finally, an in vitro reconstitution assay and mutational analysis revealed that R273 in Akt1 and corresponding R274 in Akt2 were essential for shielding the activation loop against dephosphorylation.
Conclusion: R274 in AKT2, when mutated, is associated with causing autosomal-dominant severe insulin resistance and diabetes mellitus in humans. Our in vitro reconstitution system unambiguously reveals that after Akt binding to membrane Cav3 caveolar or rafts, these ATP and ATP-competitive inhibitors prevent activation loop dephosphorylation via phosphatase access restriction.
- © 2011 by American Heart Association, Inc.