Functional proteomic analysis of a three-tier PKCε-Akt-eNOS signaling module in cardiac protection

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Abstract
Cardiac protective signaling networks have been shown to involve PKCε. However, the molecular mechanisms by which PKCε interacts with other members of these networks to form task-specific modules remain unknown. Among 93 different PKCε-associated proteins that have been identified, Akt and endothelial nitric oxide (NO) synthase (eNOS) are of importance because of their independent abilities to promote cell survival and prevent cell death. The simultaneous association of PKCε, Akt, and eNOS has not been examined, and, in particular, the formation of a module containing these three proteins and the role of such a module in the regulation of NO production and cardiac protection are unknown. The present study was undertaken to determine whether these molecules form a signaling module and, thereby, play a collective role in cardiac signaling. Using recombinant proteins in vitro and PKCε transgenic mouse hearts, we demonstrate the following: 1 ) PKCε, Akt, and eNOS interact and form signaling modules in vitro and in the mouse heart. Activation of either PKCε or Akt enhances the formation of PKCε-Akt-eNOS signaling modules. 2 ) PKCε directly phosphorylates and enhances activation of Akt in vitro, and PKCε activation increases phosphorylation and activation of Akt in PKCε transgenic mouse hearts. 3 ) PKCε directly phosphorylates eNOS in vitro, and this phosphorylation enhances eNOS activity. Activation of PKCε in vivo increased phosphorylation of eNOS at Ser1177, indicating eNOS activation. This study characterizes, for the first time, the physical, as well as functional, coupling of PKCε, Akt, and eNOS in the heart and implicates these PKCε-Akt-eNOS signaling modules as critical signaling elements during PKCε-induced cardiac protection.