Abstract
Reactive oxygen species (ROS) that accumulate under oxidative pressure cause severe damage to cellular components, and induce various cellular responses, including apoptosis, programmed necrosis and autophagy, depending on the cellular setting. Various studies have described ROS-induced autophagy, but only a few direct factors that regulate autophagy under oxidative stress are known to date. We have identified DAPK and PKD as such regulators by demonstrating their role in the process of autophagy in general, and specifically during oxidative stress. PKD acts as a downstream effector of DAPk in the regulation of autophagy. Furthermore, PKD functions within the autophagic network as an activator of VPS34, by associating with and phosphorylating VPS34, leading to its activation. Significantly, PKD is recruited to the autophagosomal membranes, placing it within proximity of its autophagic target.
Autophagy is a self-digestive process wherein bulk cytosolic components and intra-cellular organelles are sequestered in double-membrane vesicles named autophagosomes. Upon maturation, autophagosomes fuse with lysosomes to create the autolysosome, wherein their contents are degraded by lysosomal proteases. Degradation of cellular components through autophagy is important for maintaining cellular homeostasis and protects the cell from stress conditions, but can also function to eliminate the cell through autophagic cell death when the damage is extensive. Protein kinase D1 (PKD) is a serine/threonine kinase that regulates various cellular processes including transport from the trans-Golgi network (TGN), proliferation, motility and cell death. We have recently demonstrated a novel function of PKD, where it acts as a positive mediator of autophagy. Using a tandem RFP-GFP-LC3 reporter, which allows monitoring both autophagosomes and autolysosomes in the same cell, we found that ectopic expression of PKD induces the formation of both autophagosomes and autolysosomes. This was also observed in electron micrographs, indicating that PKD increases autophagic flux in cells. A direct link between PKD to the autophagic machinery was established through the class III PtdIns3K VPS34. Results from our study show that PKD acts as a VPS34 kinase; by binding and phosphorylating VPS34, PKD induces its activation and consequently, autophagosome formation. Interestingly, in addition to VPS34, PKD phosphorylates another lipid kinase, PtdIns4KIIIβ. Two other substrates of PKD, CERT and OSBP, are also involved in lipid signaling. Notably, immunogold labeling analysis of PKD-expressing cells demonstrates that PKD is recruited to autophagosomes, specifically binding the autophagosomal membranes.
DAPK is a positive mediator of cell death and a bona fide tumor suppressor. In a previous publication from 2007, we have reported that oxidative stress induces DAPK activation, and that under these stress conditions DAPK activates PKD through phosphorylation. This newly discovered path for activation of PKD is an alternate route to a PKC-dependent pathway, formerly described by the Toker group, which has been implicated in cellular survival signaling. Upon activation by DAPK, however, PKD induces caspase-independent cell death. We now show that DAPK and PKD are also mediators of the induction of autophagy under oxidative stress. While treatment of 293T cells with hydrogen peroxide to induce oxidative stress stimulates the appearance of autophagic vesicles in the cells, the percentage of autophagic cells during these oxidizing conditions is markedly decreased when the expression of DAPK or PKD is knocked down, suggesting that both these proteins are required for autophagy during oxidative stress. Furthermore, we have demonstrated that PKD functions downstream to DAPK in the induction of autophagy. In another study from our lab, DAPK was shown to phosphorylate BECN1 and release it from the inhibitory BCL-2 binding. Together, these findings map DAPK as an essential regulator of autophagy that acts through activating the VPS34-BECN1 complex. As cancer cells often generate high levels of reactive oxygen species due to increased metabolism, the identification of DAPK as a redox-sensitive molecule that regulates cell fate through the PKD axis sheds new light on the understanding of the tumor suppressive function of DAPK and could lead to new directions in cancer treatment research.
Our results demonstrate that DAPK and PKD are involved in both necrosis and autophagy under oxidative stress. The relation between autophagy and necrosis is complex. The two processes can either act in parallel or be activated sequentially, and have either common or opposite objectives. It is thus intriguing to consider the crosstalk between these two programs that are initiated by DAPK during oxidative stress. Previous work from our lab has demonstrated that under ER stress, both apoptosis and autophagy are induced in the same cell in a DAPK-dependent manner. In that case, autophagy serves as a second killing mechanism, which acts in concert with apoptosis. Thus, DAPK may function as a node of integration between different programs of cell death, which may mediate coordinated activation of these pathways. Such a multilayered regulation of cell death, in which DAPK and PKD take part, could ensure a robust and fine-tuned response to the stress stimulus. Understanding the crosstalk between different pathways that regulate cell death is highly important in combinatorial drug treatment approaches of human diseases. Identification of a “fingerprint,” which includes the genetic makeup of every cell as well as the environmental cues a cell is exposed to is essential for selecting an effective treatment. Molecules that regulate multiple death pathways, such as DAPK and PKD, are therefore promising targets for such drug design.
| Abbreviations: | ||
| DAPK | = | death-associated protein kinase |
| PtdIns3K | = | phosphatidylinositol 3-kinase |
| PKD | = | protein kinase D |
| TGN | = | trans-Golgi network |
Acknowledgment
This work was supported by a grant from the Flight Attendant Medical Research Institute (FAMRI) Center of Excellence and by the Cooperation Program in Cancer Research of the Deutsches Krebsforschungszentrum (DKFZ) and Israel’s Ministry of Science and Technology (MOST). A.K. is the incumbent of Helena Rubinstein Chair of Cancer Research.