Death-associated Protein Kinase Is Activated by Dephosphorylation in Response to Cerebral Ischemia

Abstract

Death-associated protein kinase (DAPK) is a calcium calmodulin-regulated serine/threonine protein kinase involved in ischemic neuronal death. In situ hybridization experiments show that DAPK mRNA expression is up-regulated in brain following a global ischemic insult and down-regulated in ischemic tissues after focal ischemia. DAPK is inactive in normal brain tissues, where it is found in its phosphorylated state and becomes rapidly and persistently dephosphorylated and activated in response to ischemia in vivo. A similar dephosphorylation pattern is detected in primary cortical neurons subjected to oxygen glucose deprivation or N-methyl-D-aspartate (NMDA)-induced toxicity. Both a calcineurin inhibitor, FK506, and a selective NMDA receptor antagonist, MK-801, inhibit the dephosphorylation of DAPK after in vitro ischemia. This indicates that DAPK could be activated by NMDA receptor-mediated calcium flux, activation of calcineurin, and subsequent DAPK dephosphorylation. Moreover, concomitantly to dephosphorylation, DAPK is proteolytically processed by cathepsin after ischemia. Furthermore, a selective DAPK inhibitor is neuroprotective in both in vitro and in vivo ischemic models. These results indicate that DAPK plays a key role in mediating ischemic neuronal injury.Ischemic stroke is characterized by apoptotic and necrotic cell death leading to neuronal loss (1). To date, there is no effective neuroprotective drug in clinical use for the treatment of acute ischemic neuronal damage (2). Several biological processes have been proposed to contribute to ischemic cell death, including excitotoxicity, ionic imbalance, oxidative stress, and apoptosis (3). These pathological events in the brain trigger aberrant cell signaling (4) and subsequent gene expression (5), which have been studied to understand the pathophysiology of stroke. We have identified DAPK 2 as a protein regulated by ischemic conditions both in vitro and in vivo.DAPK is a Ca 2ϩ /calmodulin-regulated serine/threonine kinase that acts as a positive mediator of apoptotic pathways, including those involved in neuronal cell death. It is involved in Fas-, INF-␥-, tumor necrosis factor-(6), ceramide- (7), and p53-mediated apoptosis (8), as well as in the disruption of matrix survival signals and suppression of integrin-mediated cell adhesion (9). DAPK appears to function early in the apoptotic pathway prior to the commitment of the cells to apoptosis. DAPK has also been extensively implicated as a tumor suppressor whose inactivation predicts malignancy, as DAPK expression is frequently lost in tumors due to hypermethylation of the DAPK gene (10, 11).DAPK has a unique multidomain structure containing a kinase domain, a calmodulin regulatory segment, eight ankyrin repeats, a cytoskeleton binding region, and a death domain (12). DAPK is negatively regulated by autophosphorylation on serine 308 in the Ca 2ϩ /CaM regulatory domain. This autoregulation of DAPK serves as an inhibitory mechanism in the basal state (13). Dephosphorylation relieves autoinhibition and stim...

Death-Associated Protein Kinase Phosphorylates Mammalian Ribosomal Protein S6 and Reduces Protein Synthesis

“…Although these findings support the notion that calcium/calmodulin-regulated pathways affect the molecular mechanisms that influence synaptic plasticity (47,48), clearly, much remains to be done as a follow up to the novel finding reported here that DAPK is capable of regulating protein synthesis with a possible role in neuronal function. Regardless, the key findings reported here and previously (2,5,8) provide an emerging view of DAPK as a kinase potentially capable of selective modulation of neuronal homeostasis in rapid response to diverse stimuli, with apoptosis as one possible outcome that can be attenuated by targeted therapeutics in disease-relevant events.…”

“…Therapeutic intervention with bioavailable DAPK inhibitors in clinically relevant time windows is neuroprotective in animal models, validating DAPK as a potential drug discovery target for acute and chronic brain injuries (2,4,5). DAPK is highly expressed in the hippocampus, a part of the brain that is essential for memory formation.…”

The tumor suppressor DAP-kinase links cell adhesion and cytoskeleton reorganization to cell death regulation

“…As all of these findings point out a possible role of DAP-kinase in neurodegenerative disorders and ischemic neuronal injury, DAPkinase is now considered as an attractive target for treatment of CNS injury and degenerative diseases. An aminopyridazine-based selective inhibitor of DAP-kinase has been developed recently and this small molecule displays neuroprotective effect in both hypoxia-and ischemia-induced brain injury models [37,38]. In addition to being involved in the pathological death conditions in neurons, recent study also suggests a function of DAP-kinase in developmentally regulated neuronal cell death.…”

“…A number of studies revealed that DAP-kinase not only mediates neuronal death triggered by various death stimuli and pathological conditions, but also is upregulated at the activity or expression level in response to such death stimuli. For instance, DAPkinase has been shown to play a role in neuronal cell death induced by C2-, C6-, and C8-ceramides [34,35], seizure [36], and various ischemia conditions [37,38]. In vivo, retinal ganglion cells for DAP-kinase null mice displays increased survival following the treatment of cytotoxic level of glutamate [33].…”

DAPK1 (death-associated protein kinase 1)

“…DAPK is inactive in normal brain tissues, where it is found in its phosphorylated state and becomes rapidly and persistently dephosphorylated and activated in response to ischemia in vivo (Shamloo et al, 2005). …”