Störz, Peter

Increases in reactive oxygen species (ROS) have been implicated in age-related diseases, including cancer. The serine/threonine kinase protein kinase D1 (PKD1) is a stress-responsive kinase and sensor for reactive oxygen species, which can initiate cell survival through NF-κB signaling. We have previously shown that in response to ROS, PKD1 is activated at the mitochondria. However, the initial signaling events leading to localization of PKD1 to the mitochondria are unknown. Here, we show that formation of mitochondrial diacylglycerol (DAG) and its binding to PKD1 is the means by which PKD1 is localized to the mitochondria in response to ROS. Interestingly, DAG to which PKD1 is recruited in this pathway is formed downstream of phospholipase D1 (PLD1) and a lipase-inactive PLD1 or inhibition of PLD1 by pharmacological inhibitors blocked PKD1 activation under oxidative stress. To date it has been viewed that monosaturated and saturated DAG formed via PLD1 have no signaling function. However, our data describe a role for PLD1-induced DAG as a competent second messenger at the mitochondria that relays ROS to PKD1-mediated mitochondria-to-nucleus signaling.

The Par-1 protein kinases are conserved from yeast to humans, where they function as key polarity determinants. The mammalian Par-1 family is comprised of 4 members (Par-1a, -b, -c, and -d). Previously, we demonstrated that atypical protein kinase C (aPKC) phosphorylates the Par-1 kinases on a conserved threonine residue (T595) to regulate localization and kinase activity. Here, we demonstrate that Par-1b is also regulated by another arm of the PKC pathway, one that involves novel PKCs (nPKC) and protein kinase D. Treatment of cells with the PKC activator phorbol-12-myristate-13-acetate (PMA) potently stimulated phosphorylation of Par-1b on serine 400 (S400), a residue that is conserved in all 4 mammalian Par-1 kinases as well as the fly ortholog. We demonstrate that PMA stimulates nPKC to activate PKD, which in turn directly phosphorylates Par-1b on S400 to positively regulate 14-3-3 binding and to negatively regulate membrane association. Thus, 2 arms of the PKC pathway regulate interactions between Par-1b and 14-3-3 proteins: one involving aPKC and the other nPKC/PKD.

Mitochondria-generated reactive oxygen species have been implicated as a common feature that connects aging of organisms and age-related diseases. Efficient elimination of these radicals by antioxidants correlates with increased life span. Understanding how the mitochondrion signals to the nucleus to regulate antioxidant proteins might be a key to aging processes and treatment of human diseases.