Fig. 5.

Regulation of PTEN and Akt by ROCK-1. (A) HEK cells were transfected with ROCKΔ1 and its kinase mutant KD (kinase-deficient mutant ROCK-1). Cellular lysates were assessed for phospho-Akt (pAkt) by Western blot analysis. (B) PTEN activities were assessed by Malachite green assay after cells were transfected with full-length ROCK-1 and its mutants. (C) PTEN and pAkt were detected by Western blots after cells were treated by PTEN-specific siRNA. Densitometry analysis for pAkt is shown at Right after normalization by actin expression. (D) In cotransfection with PTEN-specific siRNA and ROCKΔ1, PTEN and pAkt levels were analyzed. The expression levels were normalized to actin and are shown at Right. All experiments were conducted in human HEK cells. Δ1, active Rho kinase ROCK-1; a.u., artificial unit; NS-siRNA, nonspecific siRNA. (E) Levels of pAkt were assessed by Western blot analysis in hearts from WT and ROCK-1^−/− mice subject to either sham surgery or 1 week aortic banding pressure overload. (F) A proposed model outlining the role of caspase-3 cleavage activation of ROCK-1 on cardiac dysfunction. ROCK-1 may be the leading target for activated caspase-3 in failing myocardium. Cleavage-activated ROCK-1 kinase generated a positive feed-forward loop for caspase cascade activation. This proapoptotic effect is associated with the activation of PTEN and the dephosphorylation of Akt.