Death-associated protein kinase is activated by dephosphorylation in response to cerebral ischemia

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.     

A protein kinase associated with apoptosis and tumor suppression: structure, activity, and discovery of peptide substrates

Death-associated protein kinase (DAPK) has been implicated in apoptosis and tumor suppression, depending on cellular conditions, and associated with mechanisms of disease. However, DAPK has not been characterized as an enzyme due to the lack of protein or peptide substrates. Therefore, we determined the structure of DAPK catalytic domain, used a homology model of docked peptide substrate, and synthesized positional scanning substrate libraries in order to discover peptide substrates with K(m) values in the desired 10 microm range and to obtain knowledge about the preferences of DAPK for phosphorylation site sequences. Mutagenesis of DAPK catalytic domain at amino acids conserved among protein kinases or unique to DAPK provided a link between structure and activity. An enzyme assay for DAPK was developed and used to measure activity in adult brain and monitor protein purification based on the physical and chemical properties of the open reading frame of the DAPK cDNA. The results allow insight into substrate preferences and regulation of DAPK, provide a foundation for proteomic investigations and inhibitor discovery, and demonstrate the utility of the experimental approach, which can be extended potentially to kinase open reading frames identified by genome sequencing projects or functional genetics screens and lacking a known substrate.     

Death-associated protein kinase phosphorylates mammalian ribosomal protein S6 and reduces protein synthesis

Death-associated protein kinase (DAPK) is a pro-apoptotic, calcium/calmodulin-regulated protein kinase that is a drug discovery target for neurodegenerative disorders. Despite the potential profound physiological role of DAPK in neuronal function and pathophysiology, the endogenous substrate(s) of this kinase and the mechanisms via which DAPK elicits its biological action remain largely unknown. We report here that the mammalian 40S ribosomal protein S6 is a DAPK substrate. Results from immunoprecipitation experiments are consistent with endogenous DAPK being associated with endogenous S6 in rat brain. When S6 is a component of the 40S ribosomal subunit complex, DAPK selectively phosphorylates it at serine 235, one of the five sites in S6 that are phosphorylated by the S6 kinase family of proteins. The amino acid sequence flanking serine 235 matches the established pattern for DAPK peptide and protein substrates. Kinetic analyses using purified 40S subunits revealed a K(m) value of 9 microM, consistent with S6 being a potential physiological substrate of DAPK. This enzyme-substrate relationship has functional significance. DAPK suppresses translation in rabbit reticulocyte lysate, and treatment of neuroblastoma cells with a stimulator of DAPK reduces protein synthesis. In both cases, suppression of translation correlates with increased phosphorylation of S6 at serine 235. These results demonstrate that DAPK is a S6 kinase and provide evidence for a novel role of DAPK in the regulation of translation.     

DAPK activates MARK1/2 to regulate microtubule assembly, neuronal differentiation, and tau toxicity

Death-associated protein kinase (DAPK) is a key player in several modes of neuronal death/injury and has been implicated in the late-onset Alzheimer's disease (AD). DAPK promotes cell death partly through its effect on regulating actin cytoskeletons. In this study, we report that DAPK inhibits microtubule (MT) assembly by activating MARK/PAR-1 family kinases MARK1/2, which destabilize MT by phosphorylating tau and related MAP2/4. DAPK death domain, but not catalytic activity, is responsible for this activation by binding to MARK1/2 spacer region, thereby disrupting an intramolecular interaction that inhibits MARK1/2. Accordingly, DAPK(-/-) mice brain displays a reduction of tau phosphorylation and DAPK enhances the effect of MARK2 on regulating polarized neurite outgrowth. Using a well-characterized Drosophila model of tauopathy, we show that DAPK exerts an effect in part through MARK Drosophila ortholog PAR-1 to induce rough eye and loss of photoreceptor neurons. Furthermore, DAPK enhances tau toxicity through a PAR-1 phosphorylation-dependent mechanism. Together, our study reveals a novel mechanism of MARK activation, uncovers DAPK functions in modulating MT assembly and neuronal differentiation, and provides a molecular link of DAPK to tau phosphorylation, an event associated with AD pathology.     

DAPK catalytic activity in the hippocampus increases during the recovery phase in an animal model of brain hypoxic-ischemic injury

Death-associated protein kinase (DAPK) is a pro-apoptotic, calmodulin (CaM)-regulated protein kinase whose mRNA levels increase following cerebral ischemia. However, the relationship between DAPK catalytic activity and cerebral ischemia is not known. This knowledge is critical as DAPK function is dependent on the catalytic activity of its kinase domain. Consequently, we examined DAPK catalytic activity in a rat model of neonatal cerebral hypoxia-ischemia (HI). An increase in DAPK specific activity was found in homogenates of the hippocampus from the injured right hemisphere, compared to the uninjured left hemisphere, 7 days after injury. The results raised the possibility that an upregulation of DAPK activity might be associated with the recovery phase of HI, during which neuronal repair and differentiation are initiated. Therefore, we examined the change of DAPK in an experimentally tractable cell culture model of neuronal differentiation. We found that DAPK catalytic activity and protein levels increase after nerve growth factor (NGF)-induced differentiation of rat PC12 cells. These results suggest that DAPK may have a previously unappreciated role in neuronal development or recovery from injury, and that potential future therapies targeting DAPK should consider a restricted time window.     

Structure of the dimeric autoinhibited conformation of DAPK2, a pro-apoptotic protein kinase

The death-associated protein kinase (DAPK) family has been characterized as a group of pro-apoptotic serine/threonine kinases that share specific structural features in their catalytic kinase domain. Two of the DAPK family members, DAPK1 and DAPK2, are calmodulin-dependent protein kinases that are regulated by oligomerization, calmodulin binding, and autophosphorylation. In this study, we have determined the crystal and solution structures of murine DAPK2 in the presence of the autoinhibitory domain, with and without bound nucleotides in the active site. The crystal structure shows dimers of DAPK2 in a conformation that is not permissible for protein substrate binding. Two different conformations were seen in the active site upon the introduction of nucleotide ligands. The monomeric and dimeric forms of DAPK2 were further analyzed for solution structure, and the results indicate that the dimers of DAPK2 are indeed formed through the association of two apposed catalytic domains, as seen in the crystal structure. The structures can be further used to build a model for DAPK2 autophosphorylation and to compare with closely related kinases, of which especially DAPK1 is an actively studied drug target. Our structures also provide a model for both homodimerization and heterodimerization of the catalytic domain between members of the DAPK family. The fingerprint of the DAPK family, the basic loop, plays a central role in the dimerization of the kinase domain.     

DAPK plays an important role in panobinostat-induced autophagy and commits cells to apoptosis under autophagy deficient conditions

The histone deacetylase inhibitor (HDACi) LBH589 has been verified as an effective anticancer agent. The identification and characterization of new targets for LBH589 action would further enhance our understanding of the molecular mechanisms involved in HDACi therapy. The role of the tumor suppressor death-associated protein kinase (DAPK) in LBH589-induced cytotoxicity has not been investigated to date. Stable DAPK knockdown (shRNA) and DAPK overexpressing (DAPK+++) cell lines were generated from HCT116 wildtype colon cancer cells. LBH589 inhibited cell proliferation, reduced the long-term survival, and up-regulated and activated DAPK in colorectal cancer cells. Moreover, LBH589 significantly suppressed the growth of colon tumor xenografts and in accordance with the in vitro studies, increased DAPK levels were detected immunohistochemically. LBH589 induced a DAPK-dependent autophagy as assessed by punctuate accumulation of LC3-II, the formation of acidic vesicular organelles, and degradation of p62 protein. LBH589-induced autophagy seems to be predominantly caused by DAPK protein interactions than by its kinase activity. Caspase inhibitor zVAD increased autophagosome formation, decreased the cleavage of caspase 3 and PARP but didn’t rescue the cells from LBH589-induced cell death in crystal violet staining suggesting both caspase-dependent as well as caspase-independent apoptosis pathways. Pre-treatment with the autophagy inhibitor Bafilomycin A1 caused caspase 3-mediated apoptosis in a DAPK-dependent manner. Altogether our data suggest that DAPK induces autophagy in response to HDACi-treatment. In autophagy deficient cells, DAPK plays an essential role in committing cells to HDACi-induced apoptosis.     

Induction of autophagy in hepatocellular carcinoma cells by SB203580 requires activation of AMPK and DAPK but not p38 MAPK

SB203580 is a well-known inhibitor of p38 mitogen-activated protein kinase (MAPK). However, it can suppress cell proliferation in a p38 MAPK independent manner. The inhibitory mechanism remains unknown. Here, we showed that SB203580 induced autophagy in human hepatocellular carcinoma (HCC) cells. SB203580 increased GFP-LC3-positive cells with GFP-LC3 dots, induced accumulation of autophagosomes, and elevated the levels of microtubule-associated protein light chain 3 and Beclin 1. It stimulated the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and p53, but inhibited the phosphorylation of death-associated protein kinase (DAPK). Inhibition of AMPK, p53, or DAPK attenuated SB203580-induced autophagy. AMPK activation appeared to predate the DAPK signal. The activation of both AMPK and DAPK prompted the phosphorylation of p53 and enhanced Beclin 1 expression. Neither the downregulation of p38 MAPK by its siRNA or chemical inhibitor nor the upregulation of p38 MAPK by p38 MAPK DNA transfection affected B203580-induced autophagy. Collectively, the findings demonstrate a novel function of SB203580 to induce autophagy via activating AMPK and DAPK but independent of p38 MAPK. The induction of autophagy can thus account for the antiproliferative effect of SB203580 in HCC cells.     

The tumor suppressor DAP kinase is a target of RSK-mediated survival signaling

The viability of vertebrate cells depends on a complex signaling interplay between survival factors and cell-death effectors. Subtle changes in the equilibrium between these regulators can result in abnormal cell proliferation or cell death, leading to various pathological manifestations. Death-associated protein kinase (DAPK) is a multidomain calcium/calmodulin (CaM)-dependent Ser/Thr protein kinase with an important role in apoptosis regulation and tumor suppression. The molecular signaling mechanisms regulating this kinase, however, remain unclear. Here, we show that DAPK is phosphorylated upon activation of the Ras-extracellular signal-regulated kinase (ERK) pathway. This correlates with the suppression of the apoptotic activity of DAPK. We demonstrate that DAPK is a novel target of p90 ribosomal S6 kinases (RSK) 1 and 2, downstream effectors of ERK1/2. Using mass spectrometry, we identified Ser-289 as a novel phosphorylation site in DAPK, which is regulated by RSK. Mutation of Ser-289 to alanine results in a DAPK mutant with enhanced apoptotic activity, whereas the phosphomimetic mutation (Ser289Glu) attenuates its apoptotic activity. Our results suggest that RSK-mediated phosphorylation of DAPK is a unique mechanism for suppressing the proapoptotic function of this death kinase in healthy cells as well as Ras/Raf-transformed cells.     

The tumor suppressor death-associated protein kinase targets to TCR-stimulated NF-kappa B activation

Death-associated protein kinase (DAPK) is a unique multidomain kinase acting both as a tumor suppressor and an apoptosis inducer. The molecular mechanism underlying the effector function of DAPK is not fully understood, while the role of DAPK in T lymphocyte activation is mostly unknown. DAPK was activated after TCR stimulation. Through the expression of a dominant-negative and a constitutively active form of DAPK in T cells, we found that DAPK negatively regulated T cell activation. DAPK markedly affected T cell proliferation and IL-2 production. We identified TCR-induced NF-kappaB activation as a target of DAPK. In contrast, IL-1beta- and TNF-alpha-triggered NF-kappaB activation was not affected by DAPK. We further found that DAPK selectively modulated the TCR-induced translocation of protein kinase Ctheta, Bcl-10, and IkappaB kinase into membrane rafts. Notably, the effect of DAPK on the raft entry was specific for the NF-kappaB pathway, as other raft-associated molecules, such as linker for activation of T cells, were not affected. Our results clearly demonstrate that DAPK is a novel regulator targeted to TCR-activated NF-kappaB and T cell activation.     

Role of DAPK in neuronal cell death

Neuronal cell death happens as a result of the normal physiological process that occurs during development, or as part of the pathological process that occurs during disease. Death-associated protein kinase (DAPK) is an intracellular protein that mediates cell death by its serine/threonine kinase activity, and transmits apoptotic cell death signals in various cells, including neurons. DAPK is elevated in injured neurons in acute models of injury such as ischemia and seizure. The absence of DAPK has been shown to protect neurons from a wide variety of acute toxic insults. Moreover, DAPK also regulates neuronal cell death during central nervous system development. Neurons are initially overproduced in the developing nervous system, following which approximately one-half of the original cell population dies. This “naturally-occurring” or “programmed” cell death is essential for the construction of the developing nervous system. In this review, we focus on the role of DAPK in neuronal cell death after neuronal injury. The participation of DAPK in developmental neuronal death is also explained.     

A calmodulin-regulated protein kinase linked to neuron survival is a substrate for the calmodulin-regulated death-associated protein kinase

Death-associated protein kinase (DAPK) is a calmodulin (CaM)-regulated protein kinase and a drug-discovery target for neurodegenerative diseases. However, a protein substrate relevant to neuronal death had not been described. We identified human brain CaM-regulated protein kinase kinase (CaMKK), an enzyme key to neuronal survival, as the first relevant substrate protein by using a focused proteomics- and informatics-based approach that can be generalized to protein kinase open reading frames identified in genome projects without prior knowledge of biochemical context. First, DAPK-interacting proteins were detected in yeast two-hybrid screens and in immunoprecipitates of brain extracts. Second, potential phosphorylation site sequences in yeast two-hybrid hits were identified on the basis of our previous results from positional-scanning synthetic-peptide substrate libraries and molecular modeling. Third, reconstitution assays using purified components demonstrated that DAPK phosphorylates CaMKK with a stoichiometry of nearly 1 mol of phosphate per mole of CaMKK and a K(m) value of 3 microM. Fourth, S511 was identified as the phosphorylation site by peptide mapping using mass spectrometry, site-directed mutagenesis, and Western blot analysis with a site-directed antisera targeting the phosphorylated sequence. Fifth, a potential mechanism of action was identified on the basis of the location of S511 near the CaM recognition domain of CaMKK and demonstrated by attenuation of CaM-stimulated CaMKK autophosphorylation after DAPK phosphorylation. The results raise the possibility of a CaM-regulated protein kinase cascade as a key mechanism in acute neurodegeneration amenable to therapeutic targeting.     

Regulation of inflammation by DAPK

Death-associated protein kinase (DAPK) is a tumor suppressor and negatively regulates several activation signals. Consistent with its potential anti-inflammatory activity, DAPK promotes the formation of IFN-γ-activated inhibitor of translation (GAIT) complex that suppresses the translation of selected inflammatory genes. DAPK has been found to inhibit tumor necrosis factor-α (TNF-α)- or lipopolysaccharides (LPS)-induced NF-κB activation and pro-inflammatory cytokine expression. Inflammation is always associated with T cell activation, while DAPK attenuates T cell activation by a selective suppression in T cell receptor-triggered NF-κB activation. Recent studies, however, also reveal a contribution of DAPK to pro-inflammatory processes. DAPK is shown to mediate pro-inflammatory signaling downstream of TNF-α, LPS, IL-17, or IL-32. In addition, DAPK is required for the full formation of NLRP3 inflammasome, essential for the generation of IL-1β and IL-18. These results suggest the complicated role of DAPK in the regulation of inflammation that is likely dependent on cell types and environmental cues.     

Structure–activity relationship of novel DAPK inhibitors identified by structure-based virtual screening

Death-associated protein kinase (DAPK) is a serine/threonine protein kinase implicated in diverse programmed cell death pathways. DAPK is a promising target protein for the treatment of ischemic diseases. We identified novel potent and selective DAPK inhibitors efficiently by structure-based virtual screening, then further developed the hit compounds. In this paper, we describe the development of the hit compounds and the structure–activity relationship studies of the DAPK inhibitors in detail, including calculation of the solvated interaction energy (SIE), and verification of selectivity using a kinase panel.     

Regulation of death-associated protein kinase. Stabilization by HSP90 heterocomplexes

Death-associated protein kinase (DAPK) has been found associated with HSP90, and inhibition of HSP90 with 17-alkylamino-17-demethoxygeldanamycin reduced expression of DAPK. These results were extended to determine whether the degradation of DAPK in the absence of HSP90 activity is dependent on the ubiquitin-proteasome pathway. Our results show that treatment of cells with geldanamycin (GA) leads to degradation of DAPK, and this degradation is attenuated by the proteasome inhibitor, lactacystin. GA-induced DAPK degradation is also dependent on phosphorylation of DAPK at Ser(308), and the cellular levels of phospho(Ser(308))-DAPK dramatically increase in response to GA treatment. Expression of two distinct ubiquitin E3 ligases, carboxyl terminus of HSC70-interacting protein (CHIP) or DIP1/Mib1, enhanced DAPK degradation, and conversely, short interfering RNA depletion of either CHIP or DIP1/Mib1 attenuated DAPK degradation. In vitro ubiquitination assays confirmed that DAPK is targeted for ubiquitination by both CHIP and DIP. Consistent with these results, DAPK is found in two distinct immune complexes, one containing HSP90 and CHIP and a second complex containing only DIP1/Mib. Collectively, these results indicate that strict modulation of DAPK activities is critical for regulation of apoptosis and cellular homeostasis.     

Death-associated protein kinase 2: Regulator of apoptosis,autophagy and inflammation

Death-associated protein kinase 2 (DAPK2/DRP-1) belongs to a family of five related serine/threonine kinases that mediate a range of cellular processes, including membrane blebbing, apoptosis, and autophagy, and possess tumour suppressive functions. The three most conserved family members DAPK1/DAPK, DAPK2 and DAPK3/ZIPK share a high degree of homology in their catalytic domain, but differ significantly in their extra-catalytic structures and tissue-expression profiles. Hence, each orthologue binds to various unique interaction partners, localizes to different subcellular regions and controls some dissimilar cellular functions. In recent years, mechanistic studies have broadened our knowledge of the molecular mechanisms that activate DAPK2 and that execute DAPK2-mediated apoptosis, autophagy and inflammation. In this “molecules in focus” review on DAPK2, the structure, modes of regulation and various cellular functions of DAPK2 will be summarized and discussed.     

DAPK3 inhibits gastric cancer progression via activation of ULK1-dependent autophagy

Dysregulation of the balance between cell proliferation and cell death is a central feature of malignances. Death-associated protein kinase 3 (DAPK3) regulates programmed cell death including apoptosis and autophagy. Our previous study showed that DAPK3 downregulation was detected in more than half of gastric cancers (GCs), which was related to tumor invasion, metastasis, and poor prognosis. However, the precise molecular mechanism underlying DAPK3-mediated tumor suppression remains unclear. Here, we showed that the tumor suppressive function of DAPK3 was dependent on autophagy process. Mass spectrometry, in vitro kinase assay, and immunoprecipitation revealed that DAPK3 increased ULK1 activity by direct ULK1 phosphorylation at Ser556. ULK1 phosphorylation by DAPK3 facilitates the ULK1 complex formation, the VPS34 complex activation, and autophagy induction upon starvation. The kinase activity of DAPK3 and ULK1 Ser556 phosphorylation were required for DAPK3-modulated tumor suppression. The coordinate expression of DAPK3 with ULK1 Ser556 phosphorylation was confirmed in clinical GC samples, and this co-expression was correlated with favorable survival outcomes in patients. Collectively, these findings indicate that the tumor-suppressor roles of DAPK3 in GC are associated with autophagy and that DAPK3 is a novel autophagy regulator, which can directly phosphorylate ULK1 and activate ULK1. Thus, DAPK3 might be a promising prognostic autophagy-associated marker.Subject terms: Tumour-suppressor proteins, Macroautophagy     

Bidirectional signals transduced by DAPK-ERK interaction promote the apoptotic effect of DAPK

Death-associated protein kinase (DAPK) is a death domain-containing serine/threonine kinase, and participates in various apoptotic paradigms. Here, we identify the extracellular signal-regulated kinase (ERK) as a DAPK-interacting protein. DAPK interacts with ERK through a docking sequence within its death domain and is a substrate of ERK. Phosphorylation of DAPK at Ser 735 by ERK increases the catalytic activity of DAPK both in vitro and in vivo. Conversely, DAPK promotes the cytoplasmic retention of ERK, thereby inhibiting ERK signaling in the nucleus. This reciprocal regulation between DAPK and ERK constitutes a positive feedback loop that ultimately promotes the apoptotic activity of DAPK. In a physiological apoptosis system where ERK-DAPK interplay is reinforced, downregulation of either ERK or DAPK suppresses such apoptosis. These results indicate that bidirectional signalings between DAPK and ERK may contribute to the apoptosis-promoting function of the death domain of DAPK.     

Site-directed mutagenesis of the glycine-rich loop of death associated protein kinase (DAPK) identifies it as a key structure for catalytic activity

Death associated protein kinase (DAPK) is a calmodulin (CaM)-regulated protein kinase that is a therapeutic target for central nervous system (CNS) disorders. We report here the results of studies that test the hypothesis of McNamara et al. (2009) that conformational selection in DAPK's glycine-rich region is key for catalytic activity. The hypothesis was tested by site-directed mutagenesis of glutamine-23 (Q23) in the middle of this loop. The glycine-rich loop exhibits localized differences in structure among DAPK conformations that correlate with different stages of the catalytic cycle. Changing the Q23 to a Valine (V23), found at the corresponding position in another CaM regulated protein kinase, results in a reduced catalytic efficiency. High resolution X-ray crystal structures of various conformations of the Q23V mutant DAPK and their superimposition with the corresponding conformations from wild type catalytic domain reveal localized changes in the glycine-rich region. The effect of the mutation on DAPK catalytic activity and the finding of only localized changes in the DAPK structure provide experimental evidence implicating conformational selection in this domain with activity. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.