Activation Of Death-associated Protein Kinase Research Articles

Disassembly of Death-associated Protein Kinase and DANGER Interaction Mediates Hippocampal CA1 Neuron Death in Rat Cerebral Ischemic Reperfusion

Death-associated protein kinase (DAPK) is a Ca2+/CaM-regulated protein kinase that is involved in cell death processes by multiple pathways. It has been reported that DAPK may play a role in brain ischemia-induced neuronal death, but this mechanism is not well understood. DANGER, a membrane-associated protein that binds to DAPK physiologically, inhibits DAPK activation. In the present study, we used a transient global brain ischemia and reperfusion (I/R) rat model to investigate whether the interaction between DAPK and DANGER is involved in neuronal cell death following brain ischemia, and to reveal the mechanism of action. Our results indicate that the DAPK/DANGER interaction in the hippocampal CA1 region was significantly reduced after I/R with a peak reduction at 6 h. We further demonstrate that the NMDA inhibitor MK-801, DAPK inhibitor, or calcineurin inhibitor FK-506 prevented the dissociation of DANGER from DAPK 6 h after I/R. This was accompanied by a significantly decreased I/R-induced dephosphorylation of DAPK(ser-308), inhibiting DAPK catalytic activity. Moreover, the expression of DANGER and the interaction between DANGER and IP3R on the endoplasmic reticulum was significantly increased at I/R 6 h, which may be related to a reduction of DAPK/DANGER binding under I/R condition. Furthermore, MK-801, DAPK inhibitor and FK-506 had neuroprotective effects against hippocampal CA1 neuronal death 5 days after I/R. In conclusion, our data suggest that the dissociation of DANGER from DAPK may mediate DAPK activation, which is involved in DAPK-related neuronal death following I/R injury.

Prolyl oligopeptidase inhibition activates autophagy via protein phosphatase 2A

Prolyl oligopeptidase (PREP) is a serine protease that has been studied particularly in the context of neurodegenerative diseases for decades but its physiological function has remained unclear. We have previously found that PREP negatively regulates beclin1-mediated macroautophagy (autophagy), and that PREP inhibition by a small-molecule inhibitor induces clearance of protein aggregates in Parkinson’s disease models. Since autophagy induction has been suggested as a potential therapy for several diseases, we wanted to further characterize how PREP regulates autophagy. We measured the levels of various kinases and proteins regulating beclin1-autophagy in HEK-293 and SH-SY5Y cell cultures after PREP inhibition, PREP deletion, and PREP overexpression and restoration, and verified the results in vivo by using PREP knock-out and wild-type mouse tissue where PREP was restored or overexpressed, respectively. We found that PREP regulates autophagy by interacting with protein phosphatase 2A (PP2A) and its endogenous inhibitor, protein phosphatase methylesterase 1 (PME1), and activator (protein phosphatase 2 phosphatase activator, PTPA), thus adjusting its activity and the levels of PP2A in the intracellular pool. PREP inhibition and deletion increased PP2A activity, leading to activation of death-associated protein kinase 1 (DAPK1), beclin1 phosphorylation and induced autophagy while PREP overexpression reduced this. Lowered activity of PP2A is connected to several neurodegenerative disorders and cancers, and PP2A activators would have enormous potential as drug therapy but development of such compounds has been a challenge. The concept of PREP inhibition has been proved safe, and therefore, our study supports the further development of PREP inhibitors as PP2A activators.

Calcium/Calmodulin-Dependent Kinase (CaMKII) Inhibition Protects Against Purkinje Cell Damage Following CA/CPR in Mice.

Ischemic brain damage is triggered by glutamate excitotoxicity resulting in neuronal cell death. Previous research has demonstrated that N-methly-D-aspartate (NMDA) receptor activation triggers downstream calcium-dependent signaling pathways, specifically Ca2+/calmodulin-dependent protein kinase II (CaMKII). Inhibiting CaMKII is protective against hippocampal ischemic injury, but there is little known about its role in the cerebellum. To examine the neuroprotective potential of CaMKII inhibition in Purkinje cells, we subjected C57BL/6 or CaMKIIα KO male mice (8-12weeks old) to cardiac arrest followed by cardiopulmonary resuscitation (CA/CPR). We performed a dose-response study for tat-CN19o and cerebellar injury was analyzed at 7days after CA/CPR. Acute signaling was assessed at 6h after CA/CPR using Western blot analysis. We observed increased phosphorylation of the T286 residue of CaMKII, suggesting increased autonomous activation. Analysis of Purkinje cell density revealed a decrease in cell density at 7days after CA/CPR that was prevented with tat-CN19o at doses of 0.1 and 1mg/kg. However, neuroprotection in the cerebellum required doses that were 10-fold higher than what was needed in the hippocampus. CaMKIIα KO mice subjected to sham surgery or CA/CPR had similar Purkinje cell densities, suggesting CaMKIIα is required for CA/CPR-induced injury in the cerebellum. We also observed a CA/CPR-induced activation of death-associated protein kinase (DAPK1) that tat-CN19o did not block. In summary, our findings indicate that inhibition of autonomous CaMKII activity is a promising therapeutic approach that is effective across multiple brain regions.

Activation of death-associated protein kinase 1 promotes neutrophil apoptosis to accelerate inflammatory resolution in acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is a uniform progression of overwhelming inflammation in lung tissue with extensive infiltration of inflammatory cells. Neutrophil apoptosis is thought to be a significant process in the control of the resolution phase of inflammation. It has been proved that 5-Aza-2′-deoxycytidine (Aza) can inhibit cancer by activating death-associated protein kinase 1 (DAPK1) to promote apoptosis. However, the effect of DAPK1 on neutrophil apoptosis is unclear, and research on the role of Aza in inflammation is lacking. Here, we investigated whether Aza can regulate DAPK1 expression to influence the fate of neutrophils in ARDS. In vitro, we stimulated neutrophil-like HL-60 (dHL-60) cells with different concentrations of Aza for different durations and used RNA interference to up- or downregulate DAPK1 expression. We observed that culturing dHL-60 cells with Aza increased apoptosis by inhibiting NF-κB activation to modulate the expression of Bcl-2 family proteins, which was closely related to the levels of DAPK1. In vivo, ARDS was evoked by intratracheal instillation of lipopolysaccharide (LPS; 3 mg/kg). One hour after LPS administration, mice were treated with Aza (1 mg/kg, i.p.). To inhibit DAPK1 expression, mice were intraperitoneally injected with a DAPK1 inhibitor. Aza treatment accelerated inflammatory resolution in LPS-induced ARDS by suppressing pulmonary edema, alleviating lung injury and decreasing the infiltration of inflammatory cells in bronchoalveolar lavage fluid (BALF). Moreover, Aza reduced the production of proinflammatory cytokines. However, administration of the DAPK1 inhibitor attenuated the protective effects of Aza. Similarly, the proapoptotic function of Aza was prevented when DAPK1 was inhibited either in vivo or in vitro. In summary, Aza promotes neutrophil apoptosis by activating DAPK1 to accelerate inflammatory resolution in LPS-induced ARDS. This study provides the first evidence that Aza prevents LPS-induced neutrophil survival by modulating DAPK1 expression.

Pegylated IFN-α suppresses hepatitis C virus by promoting the DAPK-mTOR pathway

Death-associated protein kinase (DAPK) has been found to be induced by IFN, but its antiviral activity remains elusive. Therefore, we investigated whether DAPK plays a role in the pegylated IFN-α (peg-IFN-α)-induced antiviral activity against hepatitis C virus (HCV) replication. Primary human hepatocytes, Huh-7, and infectious HCV cell culture were used to study the relationship between peg-IFN-α and the DAPK-mammalian target of rapamycin (mTOR) pathways. The activation of DAPK and signaling pathways were determined using immunoblotting. By silencing DAPK and mTOR, we further assessed the role of DAPK and mTOR in the peg-IFN-α-induced suppression of HCV replication. Peg-IFN-α up-regulated the expression of DAPK and mTOR, which was associated with the suppression of HCV replication. Overexpression of DAPK enhanced mTOR expression and then inhibited HCV replication. In addition, knockdown of DAPK reduced the expression of mTOR in peg-IFN-α-treated cells, whereas silencing of mTOR had no effect on DAPK expression, suggesting mTOR may be a downstream effector of DAPK. More importantly, knockdown of DAPK or mTOR significantly mitigated the inhibitory effects of peg-IFN-α on HCV replication. In conclusion, our data suggest that the DAPK-mTOR pathway is critical for anti-HCV effects of peg-IFN-α.

A Novel Recombinant Anti-CD22 Immunokinase Delivers Proapoptotic Activity of Death-Associated Protein Kinase (DAPK) and Mediates Cytotoxicity in Neoplastic B Cells.

The serine/threonine death-associated protein kinases (DAPK) provide pro-death signals in response to (oncogenic) cellular stresses. Lost DAPK expression due to (epi)genetic silencing is found in a broad spectrum of cancers. Within B-cell lymphomas, deficiency of the prototypic family member DAPK1 represents a predisposing or early tumorigenic lesion and high-frequency promoter methylation marks more aggressive diseases. On the basis of protein studies and meta-analyzed gene expression profiling data, we show here that within the low-level context of B-lymphocytic DAPK, particularly CLL cells have lost DAPK1 expression. To target this potential vulnerability, we conceptualized B-cell-specific cytotoxic reconstitution of the DAPK1 tumor suppressor in the format of an immunokinase. After rounds of selections for its most potent cytolytic moiety and optimal ligand part, a DK1KD-SGIII fusion protein containing a constitutive DAPK1 mutant, DK1KD, linked to the scFv SGIII against the B-cell-exclusive endocytic glyco-receptor CD22 was created. Its high purity and large-scale recombinant production provided a stable, selectively binding, and efficiently internalizing construct with preserved robust catalytic activity. DK1KD-SGIII specifically and efficiently killed CD22-positive cells of lymphoma lines and primary CLL samples, sparing healthy donor- or CLL patient-derived non-B cells. The mode of cell death was predominantly PARP-mediated and caspase-dependent conventional apoptosis as well as triggering of an autophagic program. The notoriously high apoptotic threshold of CLL could be overcome by DK1KD-SGIII in vitro also in cases with poor prognostic features, such as therapy resistance. The manufacturing feasibility of the novel CD22-targeting DAPK immunokinase and its selective antileukemic efficiency encourage intensified studies towards specific clinical application. Mol Cancer Ther; 15(5); 971-84. ©2016 AACR.

Death-Associated Protein Kinase Activity Is Regulated by Coupled Calcium/Calmodulin Binding to Two Distinct Sites

SummaryThe regulation of many protein kinases by binding to calcium/calmodulin connects two principal mechanisms in signaling processes: protein phosphorylation and responses to dose- and time-dependent calcium signals. We used the calcium/calmodulin-dependent members of the death-associated protein kinase (DAPK) family to investigate the role of a basic DAPK signature loop near the kinase active site. In DAPK2, this loop comprises a novel dimerization-regulated calcium/calmodulin-binding site, in addition to a well-established calcium/calmodulin site in the C-terminal autoregulatory domain. Unexpectedly, impairment of the basic loop interaction site completely abolishes calcium/calmodulin binding and DAPK2 activity is reduced to a residual level, indicative of coupled binding to the two sites. This contrasts with the generally accepted view that kinase calcium/calmodulin interactions are autonomous of the kinase catalytic domain. Our data establish an intricate model of multi-step kinase activation and expand our understanding of how calcium binding connects with other mechanisms involved in kinase activity regulation.

Death-Associated Protein Kinase Activity Is Regulated by Coupled Calcium/Calmodulin Binding to Two Distinct Sites

Death-Associated Protein Kinase Activity Is Regulated by Coupled Calcium/Calmodulin Binding to Two Distinct Sites

Sphingosine Kinase-1 Protects Multiple Myeloma from Apoptosis Driven by Cancer-Specific Inhibition of RTKs.

Activation of acid sphingomyelinase (ASM) leads to ceramide accumulation and induces apoptotic cell death in cancer cells. In the present study, we demonstrate that the activation of ASM by targeting cancer-overexpressed 67-kDa laminin receptors (67LR) induces lipid raft disruption and inhibits receptor tyrosine kinase (RTK) activation in multiple myeloma cells. Sphingosine kinase 1 (SphK1), a negative regulator of ceramide accumulation with antiapoptotic effects, was markedly elevated in multiple myeloma cells. The silencing of SphK1 potentiated the apoptotic effects of the green tea polyphenol epigallocatechin-3-O-gallate (EGCG), an activator of ASM through 67LR. Furthermore, the SphK1 inhibitor safingol synergistically sensitized EGCG-induced proapoptotic cell death and tumor suppression in multiple myeloma cells by promoting the prevention of RTK phosphorylation and activation of death-associated protein kinase 1 (DAPK1). We propose that targeting 67LR/ASM and SphK1 may represent a novel therapeutic strategy against multiple myeloma.

Activation of death-associated protein kinase in human peritumoral tissue: A potential therapeutic target

To further understand the molecular mechanisms of N-methyl-D-aspartate receptor 2B (NR2B) phosphorylation and its contribution to glioma-related seizures, we investigated the expression of death-associated protein kinase-1 (DAPK1), which is a kinase known to phosphorylate NR2B at S1303 in glioma and peritumoral tissue. The molecular mechanisms leading to glioma-associated seizures are poorly understood. We recently discovered that NR2B is phosphorylated at S1303 in glioma peritumoral tissue. NR2B is an excitatory glutamate receptor, suggesting that glutamate released from glioma tumor cells may excite the neurons in the peritumoral tissue and contribute to glioma-associated epileptogenesis. DAPK1 levels were assessed in an intracranial mouse model of human glioma and in primary patient peritumoral and glioma tissues using immunohistochemistry. DAPK1 is highly expressed in the peritumoral region, but is poorly expressed in glioma tissues in both a mouse model of human glioma and in the primary patient glioma. In our previous report, we found that NR2B is also highly phosphorylated in the same region. Upregulation of DAPK1 in the peritumoral tissues suggests that DAPK1 can phosphorylate NR2B, increase its excitability, lead to glioma-induced seizures, and could potentially be an important therapeutic target. Furthermore, the xenograft model offers an opportunity to develop and test therapeutic approaches that can block DAPK1 activity in vivo.

Shear stress attenuates apoptosis due to TNFα, oxidative stress, and serum depletion via death-associated protein kinase (DAPK) expression.

BackgroundMisdirected apoptosis in endothelial cells participates in the development of pathological conditions such as atherosclerosis. Tight regulation of apoptosis is necessary to ensure normal cell function. The rate of cell turnover is increased at sites prone to lesion development. Laminar shear stress is protective against atherosclerosis, and helps suppress apoptosis induced by cytokines, oxidative stress, and serum depletion. Current Studies have shown that the pro-apoptotic DAPK expression and function to be regulated in part by shear stress, and that shearing cells already treated with cytokine tumor necrosis factor (TNF) α significantly reduced apoptosis. We investigate further the suppression of endothelial apoptosis by shear stress with other apoptotic triggers, and the involvement of DAPK and caspase 3/7.ResultsWe have shown that exposure to shear stress (12 dynes/cm2 for 6 hrs) suppressed endothelial apoptosis triggered by cytokine (TNFα), oxidative stress (H2O2), and serum depletion, either before or after a long term (18 hr) induction. This is correlated with a parallel decrease of DAPK expression and caspase activity compared to non-sheared cells. We found similar modulation of DAPK and apoptosis by shear stress with other pro-apoptotic signals. Changes in DAPK and caspase 3/7 are directly correlated to changes in apoptosis. Interestingly, shear stress applied to cells prior to induction with apoptosis agents resulted in a higher suppression of apoptosis and DAPK and caspase activity, compared to applying shear stress post induction. This is correlated with a higher expression and activation of DAPK in cells sheared at the end of 24-hr experiment. Also, shear stress alone also induced higher apoptosis and DAPK expression, and the effect is sustained even after 18 hrs incubation in static condition, compared to non-sheared cells.ConclusionsOverall, we show that laminar shear stress inhibits various apoptosis pathways by modulating DAPK activity, as well as caspase activation, in a time-dependent manner. Shear stress could target DAPK as a converging point to exert its effects of suppressing endothelial apoptosis. The temporal shear stress stimulation of DAPK and its role in different apoptosis pathways may help identify key mechanisms of the endothelial mechanotransduction pathway.

Death‐associated protein kinase mediates myofibril degeneration and myocyte apoptosis induced by beta‐adrenergic receptors (404.3)

Background: In cardiac muscle, persistent stimulation of β‐adrenergic receptors (β‐ARs) and calcium (Ca)2+ signaling plays a central role in regulating contractility, hypertrophy, and apoptosis. The Ca2+/calmodulin‐regulated Serine/Threonine kinase, death‐associated protein kinase (DAPK), regulates cytoskeletal dynamics and cell death. However, the role and activation mechanisms of DAPK in cardiac myocytes and heart failure (HF) are unknown. This study examines the role of DAPK in myocyte death induced by β‐AR stimulation.Methods and Results: We found increased DAPK expression and activation in human failing hearts and in hearts of cardiac‐specific β1‐ARs transgenic mice. This activation of DAPK correlated with myofibril degeneration, myocyte death and cardiac contractile dysfunction, pointing to a possible role of DAPK in the development of HF. We therefore assessed whether DAPK activation was involved in myocyte apoptosis secondary to excess exposure of catecholamine. Chronic treatment with β1‐AR agonist or adenoviral expression of β1‐ARs induced DAPK activation, which was dependent on intracellular Ca(2+) elevation and calcineurin activation. Adenoviral expression of wild‐type DAPK or a constitutively active DAPK mutant significantly enhanced myofibril degeneration and myocyte apoptosis induced in response to β1‐AR stimulation. In contrast, expression of a dominant negative DAPK mutant blocked β1‐AR‐induced myocyte apoptosis and promoted myocyte hypertrophy and myofibril organization.Conclusions: These findings reveal an unprecedented role of DAPK as a regulator of myofibril organization and myocyte apoptosis induced by persistent stimulation of β1‐AR. Thus, modulating DAPK activation to attenuate myofibril degeneration and myocyte death could be considered as a therapy target to treat patients with HF.Grant Funding Source: NIH R01 360998

Suppression of VEGF expression through interruption of the HIF-1α and Akt signaling cascade modulates the anti-angiogenic activity of DAPK in ovarian carcinoma cells

Death-associated protein kinase (DAPK) plays an important role in apoptosis regulation and has been shown to maintain antitumor and metastasis suppressor properties. In the present study, we investigated whether DAPK overexpression may mediate vascular endothelial growth factor (VEGF)/hypoxia-inducible factor-1α (HIF-1α) expression and angiogenic activity in the human carcinoma cell model system. VEGF plays a pivotal role in tumor angiogenesis and tumorigenesis. We found that DAPK significantly downregulated VEGF-induced endothelial cell proliferation, migration and tube formation as well as VEGF receptor-2 (VEGFR-2) phosphorylation in vitro. In addition, DAPK exhibited potent anti-angiogenic activity and clearly decreased the levels of VEGF and HIF-1α expression, a key regulator for angiogenesis. Notably, our results strongly indicated that DAPK can disturb VEGFR-2 transcriptional activity by inhibiting VEGFR-2 phosphorylation through the PI3K/Akt signaling cascade. Collectively, our study identified a novel function of DAPK in regulating cellular VEGF/HIF-1α activity during tumorigenesis, which may act together with its anti-angiogenic function to inhibit tumor progression.

The role of DAPK–BimEL pathway in neuronal death induced by oxygen–glucose deprivation

Death-associated protein kinase (DAPK) has been found promoting cell death under stress conditions, including cell death during brain ischemia. However, little is known about the mechanisms how DAPK is involved in the neuronal death-promoting process during ischemia. The present study was to examine the DAPK signal transduction pathways using an ischemia mimicking model, oxygen glucose deprivation (OGD). OGD was induced by incubating SH-SY5Y neuroblastoma cells in glucose-free culture medium flushed with a mixture of N2 and CO2. DAPK expression was inhibited by transfection of SH-SY5Y cells with DAPK short hairpin RNA (shRNA). Cell death induced by OGD exposure was assessed by Annexin V-FITC and propidium iodide (PI) assay. Protein expressions were examined by Western blot and protein interactions were detected with immunoprecipitation followed by Western blot. OGD treatment resulted in neuronal death and led to DAPK activation as demonstrated by increase of DAPK (active form) and decrease of phospho-DAPK (inactive form). The activation of DAPK in turn led to BimEL up-regulation and endoplasmic reticulum (ER) stress activation. Further analyses showed that DAPK mediated BimEL expression through extracellular signal-regulated protein kinase1/2 (ERK1/2) inactivation and c-Jun-N-terminal kinase1/2 (JNK1/2) activation. These findings revealed novel signal transduction pathways leading to neuronal death in response to OGD.

Retraction: PM02734 (Elisidepsin) Induces Caspase-Independent Cell Death Associated with Features of Autophagy, Inhibition of the Akt/mTOR Signaling Pathway, and Activation of Death-Associated Protein Kinase

The authors wish to retract the article titled “PM02734 (Elisidepsin) Induces Caspase-Independent Cell Death Associated with Features of Autophagy, Inhibition of the Akt/mTOR Signaling Pathway, and Activation of Death-Associated Protein Kinase,” which was published in the August 15, 2011, issue of Clinical Cancer Research (1).After an unidentified concerned reader made allegations of image mishandling in several figures, the authors requested that their medical school conduct an investigation as per its established protocol. The investigators concluded that there was enhancement of bands in some of the questioned figures and that the changes were subtle and seen only on special analysis. One of the coauthors, who since has returned to his home country, accepted full responsibility for the changes. He never mentioned the enhancements to any of the authors before manuscript submission. In no case would the conclusions from the experiments have been different if no alterations had been made. Independent repetition of key experiments by another senior investigator showed reproducibility of the major findings. In view of the reproducibility of the original data by a third party, the authors continue to stand by the conclusions of the article. However, because the submission did not meet the standards for manuscript submission to Clinical Cancer Research as described in the Instructions to Authors, the authors voluntarily retract this article. The authors apologize to the readers for the inconvenience that this oversight may have caused.Yi-He LingMiguel AracilYiyu ZouZiqiang YuanBo LuJosé JimenoAna Maria CuervoRoman Perez-Soler

Structural and Functional Characterization of the Recombinant Death Domain from Death-Associated Protein Kinase

Death-associated protein kinase (DAPk) is a calcium/calmodulin-regulated Ser/Thr-protein kinase that functions at an important point of integration for cell death signaling pathways. DAPk has a structurally unique multi-domain architecture, including a C-terminally positioned death domain (DD) that is a positive regulator of DAPk activity. In this study, recombinant DAPk-DD was observed to aggregate readily and could not be prepared in sufficient yield for structural analysis. However, DAPk-DD could be obtained as a soluble protein in the form of a translational fusion protein with the B1 domain of streptococcal protein G. In contrast to other DDs that adopt the canonical six amphipathic α-helices arranged in a compact fold, the DAPk-DD was found to possess surprisingly low regular secondary structure content and an absence of a stable globular fold, as determined by circular dichroism (CD), NMR spectroscopy and a temperature-dependent fluorescence assay. Furthermore, we measured the in vitro interaction between extracellular-regulated kinase-2 (ERK2) and various recombinant DAPk-DD constructs. Despite the low level of structural order, the recombinant DAPk-DD retained the ability to interact with ERK2 in a 1∶1 ratio with a K d in the low micromolar range. Only the full-length DAPk-DD could bind ERK2, indicating that the apparent ‘D-motif’ located in the putative sixth helix of DAPk-DD is not sufficient for ERK2 recognition. CD analysis revealed that binding of DAPk-DD to ERK2 is not accompanied by a significant change in secondary structure. Taken together our data argue that the DAPk-DD, when expressed in isolation, does not adopt a classical DD fold, yet in this state retains the capacity to interact with at least one of its binding partners. The lack of a stable globular structure for the DAPk-DD may reflect either that its folding would be supported by interactions absent in our experimental set-up, or a limitation in the structural bioinformatics assignment of the three-dimensional structure.

Identification of DAPK as a scaffold protein for the LIMK/cofilin complex in TNF-induced apoptosis

The role of cytoskeleton-associated proteins during TNF-induced apoptosis is not fully understood. A potential candidate kinase that might connect TNF signaling to actin reorganization is the death-associated protein kinase (DAPK). To identify new DAPK interaction partners in TNF-induced apoptosis, we performed a peptide array screen. We show that TNF-treatment enhanced the phosphorylation of LIMK at threonine508 and its downstream target cofilin at serine3 (p-cofilinSer3). Modulation of DAPK activity and expression by DAPK inhibitor treatment, siRNA knockdown, and overexpression affected the phosphorylation of both proteins. We propose a 3D structural model where DAPK functions as a scaffold for the LIMK/cofilin complex and triggers a closer interaction of both proteins under TNF stimulation. Upon TNF a striking redistribution of LIMK, DAPK, and cofilin to the perinuclear compartment was observed. The pro-apoptotic DAPK/LIMK/cofilin multiprotein complex was abrogated in detached cells, indicating that its signaling was no longer needed if cells committed to apoptosis. P-cofilinSer3 was strongly accumulated in cells with condensed chromatin, pronounced membrane blebs and Annexin V up-regulation. From studying different cofilinSer3 mutants we suggest that p-cofilinSer3 is an indicator of TNF-induced apoptosis. Collectively, our findings identify a novel molecular cytoskeleton-associated mechanism in TNF-induced DAPK-dependent apoptosis.

A Novel Multi-Target Drug Screening Strategy Directed Against Key Proteins of DAPk Family

Screening and identifying multi-target ligands becomes a daunting task when there are very few matching pharmacophoric features among the proteins. Herein, we describe a novel screening strategy to identify multi-target ligands for proteins having varying pharmacophoric features with their ligands. This strategy was adopted to identify multi-target ligands for death-associated protein kinase (DAPk) family. The role of the kinase activity of DAPk in eukaryotic cell apoptosis and the ability of bioavailable DAPk inhibitors to rescue neuronal death after brain injury have made it a drug-discovery target for neurodegenerative disorders. In this work, we employed a novel strategy using the existing computational approaches to design multi-target inhibitors, which can potentially inhibit one or any combination of the three DAPk family members. The strategy employs a combination of merged pharmacophore matching, database screening and molecular docking to reliably identify potential multi-target inhibitors targeted against DAPk protein family.

Effect of shear stress and substrate on endothelial DAPK expression, caspase activity, and apoptosis

BackgroundIn the vasculature, misdirected apoptosis in endothelial cells leads to pathological conditions such as inflammation. Along with biochemical and molecular signals, the hemodynamic forces that the cells experience are also important regulators of endothelial functions such as proliferation and apoptosis. Laminar shear stress inhibits apoptosis induced by serum depletion, oxidative stress, and tumor necrosis factor α (TNFα). Death associated protein kinase (DAPK) is a positive regulator of TNFα induced apoptotic pathway. Here we investigate the effect of shear stress on DAPK in endothelial cells on glass or silicone membrane substrate. We have already shown a link between shear stress and DAPK expression and apoptosis in cells on glass. Here we transition our study to endothelial cells on non-glass substrates, such as flexible silicone membrane used for cyclic strain studies.ResultsWe modified the classic parallel plate flow chamber to accommodate silicone membrane as substrate for cells, and validated the chamber for cell viability in shear stress experiments. We found that adding shear stress significantly suppressed TNFα induced apoptosis in cells; while shearing cells alone also increased apoptosis on either substrate. We also found that shearing cells at 12 dynes/cm2 for 6 hours resulted in increased apoptosis on both substrates. This shear-induced apoptosis correlated with increased caspase 3/7 activities and DAPK expression and activation via dephosphorylation of serine 308.ConclusionThese data suggest that shear stress induced apoptosis in endothelial cells via increased DAPK expression and activation as well as caspase-3/7 activity. Most in vitro shear stress studies utilize the conventional parallel plate flow chamber where cells are cultured on glass, which is much stiffer than what cells encounter in vivo. Other mechanotransduction studies have utilized the flexible silicone membrane as substrate, for example, in cyclic stretch studies. Thus, this study bridges the gap between shear stress studies on cells plated on glass to studies on different stiffness of substrates or mechanical stimulation such as cyclic strain. We continue to explore the mechanotransduction role of DAPK in endothelial apoptosis, by using substrates of physiological stiffness for shear stress studies, and by using silicone substrate in cyclic stretch devices.

Sodium selenite-induced activation of DAPK promotes autophagy in human leukemia HL60 cells

Autophagy has been suggested as a possible mechanism for non-apoptotic death despite evidence from many species that autophagy represents a survival strategy of cells under stress. From our previous findings that supranutritional doses of sodium selenite induced apoptosis in human leukemia cells, now we show autophagic cell death occurred after selenite exposure in HL60, suggested an alternative mechanism for the potential therapeutic properties of selenite. Additionally, Death-associated Protein Kinase (DAPK) performed a significantly increased expression during this process, concomitantly with gradually decreased phosphorylation at Ser(308). We further reveal that the up-regulation of DAPK which depends on selenite-activated ERK had no effect on autophagy. However, activation of DAPK via PP2A-mediated dephosphorylation at Ser(308) serves as a new strategy for autophagy induction. In conclusion, these results indicate that PP2A-mediated activated DAPK sensitizes HL60 cells to selenite, ultimately triggers autophagic cell death pathway to commit cell demise.