Oxidant-induced activation of protein kinase A: from biochemistry to the cardiovascular physiology of a novel 'redox-dead' knock-in mouse

Free radicals and oxidising chemicals are produced in cells and tissues. Historically these have been primarily associated with disease or dysfunction. However, over the past decade there has been a growing realisation that these species are not always harmful, and in fact they can play important regulatory roles. We have found in preliminary studies that a protein (known as protein kinase A), which is very important for how the heart is regulated is chemically modified by oxidants and this directly activates it. Here we outline studies that will help fully define the importance of this modification and activation of protein kinase A, both in health and in disease. We will utilise many experimental approaches in this work, including generation of a genetically modified mouse that has been altered so that it cannot be oxidised and directly activated by oxidants. The availability of such mice would be an invaluable resource that will allow us to study fully the importance of oxidant-induced protein kinase A activation in cells, tissues, organs and the animal as a whole.

We have recently found that Type I PKA is subject to reversible cysteine-targeted oxidation, a modification that regulates the activity of this kinase. This oxidative cysteine modification involves a disulfide bond forming between the two regulatory (RIalpha) subunits in the Type I PKA holoenzyme. In this application, which is aimed at further understanding the significance of this oxidative modification of PKA, we outline two primary lines of investigation. (1) A program of experiments that will further characterise the fundamental nature of this mode of kinase activation. (2) A characterisation of a novel ?redox-dead? knock-in mouse line we have generated, in which wild-type RI is replaced with a mutant protein lacking the cysteines required for kinase disulfide bond formation. This Cys17Ser RI PKA mouse is a unique model and will allow us to understand the importance of PKA RI oxidation in health and disease.