Abstract 17404: Intramolecular Inhibition of Ser473 Phosphorylation by Up-regulated Thr308 Phosphorylation in Akt Contributes to Enlargement of Infarct Size by Chronic Renal Failure

Objective: We previously showed that chronic renal failure (CRF) enlarges infarct size after ischemia/reperfusion (I/R) through impairment of reperfusion-induced Akt activation. Here we examined molecular mechanisms by which CRF impairs Akt activation, focusing on interplay between two phosphorylation sites in Akt, Thr308 and Ser473. Methods and results: Rats received 5/6 nephrectomy (SNx) or a sham operation (Sham). Development of CRF in SNx was confirmed by a 2.2-fold increase in serum creatinine level at 4 weeks after the operation. Infarct size after 20-min coronary occlusion/2-hr reperfusion was larger in SNx than in Sham (53.2±5.8 vs. 41.1±2.4% of risk area, p<0.05), indicating impaired tolerance against I/R injury in CRF. Immunoblot analyses revealed that the level of phospho-Akt-Thr308 was higher by 375% in SNx than in Sham under baseline conditions, while the levels of phospho-Akt-Ser473 were similar in the two groups. Upon reperfusion, phosphorylation of Ser473 and that of Thr308 were similarly enhanced in Sham. However, reperfusion-induced phosphorylation of Ser473, but not that of Thr308, was blunted by 32.8% in SNx compared with that in Sham. Reduction in Akt activity after reperfusion in SNx was indicated by lower levels of phospho-p70S6K and phospho-GSK3β compared with those in Sham. The levels of PTEN, a negative regulator of Akt, and PDK1, a promoter of Thr308 phosphorylation, were similar in SNx and Sham. However, the level of PP2A regulatory subunit B55α, which specifically dephosphorylates Thr308, was lower by 23.6% in SNx than in Sham. To determine whether the constitutive phosphorylation of Thr308 mitigates reperfusion-induced phosphorylation of Ser473, HA-tagged wild-type Akt (HA-WT) and phospho-Thr308-mimetic mutant Akt (HA-T308D) were transiently transfected into HEK293 cells. IGF-1 (10 nmol/L)-induced phosphorylation of Ser473 in HA-T308D was lower by 30.5% than that in HA-WT, whereas that in endogenous Akt at Ser473 was comparable between cells overexpressing HA-T308D and HA-WT. Conclusion: Insufficient activation of Akt by its intramolecular interplay between Thr308 and Ser473 phosphorylation due to down-regulated B55α contributes to increased myocardial susceptibility to I/R injury in CRF.