DAP-kinase: from functional gene cloning to establishment of its role in apoptosis and cancer

DAP-kinase is a pro-apoptotic Ca(2+) calmodulin-regulated serine/threonine kinase that participates in a wide array of apoptotic systems initiated by interferon-gamma, TNF-alpha, activated Fas, and detachment from extracellular matrix. It was isolated by an unbiased functional approach to gene cloning aimed at hitting central mediators of the apoptotic process. This 160 Kd protein kinase is localized to actin microfilaments and carries interesting modules such as ankyrin repeats and the death domain. The death promoting effects of DAP-kinase depend on its intact catalytic activity, the correct intracellular localization, and on the presence of the death domain. A few mechanisms restrain the killing effects of the protein in healthy cells. The enzyme's active site is negatively controlled by an adjacent CaM regulatory domain whose effect is relieved by binding to Ca(2+)-activated calmodulin. A second mode of autoinhibition engages the serine-rich C-terminal tail, spanning the last 17 amino acids of the protein. A link between DAP-kinase and cancer has been established. It was found that the mRNA and protein expression is frequently lost in various human cancer cell lines. Analysis of the methylation status of DAP-kinase's 5' UTR in DNA extracted from fresh tumor samples, showed high incidence of hypermethylation in several human carcinomas and B cell malignancies. The anti-tumorigenic effect of DAP-kinase was also studied experimentally in mouse model systems where the re-introduction of DAP-kinase into highly metastatic mouse lung carcinoma cells who had lost the protein, strongly reduced their metastatic capacity. Thus, it appears that loss of DAP-kinase confers a selective advantage to cancer cells and may play a causative role in tumor progression. A few novel kinases sharing high homology in their catalytic domains with DAP-kinase have been recently identified constituting altogether a novel family of death promoting serine/threonine kinases.     

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

Summary Death-associated protein (DAP)-kinase, an actin-cytoskeleton localized serine/threonine kinase, functions as a novel tumor suppressor and participates in a wide variety of cell death systems. Recent studies indicate that DAP-kinase elicits a potent cytoskeletal reorganization effect and is capable of modulating integrin inside-out signaling. Using this understanding of DAP-kinase protein function as a framework, we discuss the functional mechanisms of this kinase in regulating death-associated morphological and signaling events. Furthermore, a potential role of DAP-kinase to be a drug target is also discussed.     

Hypermethylation-mediated partial transcriptional silencing of DAP-kinase gene in bladder cancer

Death-associated protein kinase (DAP-kinase) is a novel serine/threonine kinase whose expression is required for interferon-γ-induced apoptosis. This study evaluated the methylation pattern and its impact on the expression of the DAP-kinase gene in transitional cell carcinoma of the bladder as hypermethylation is one of the earliest and most frequent alterations leading to cancer. The frequency of hypermethylation of the gene promoter was 37.8%. On correlation with clinicopathological features, methylation was seen mostly in superficial tumours in the group aged?>?60 years (42.9 vs 33.3% of those?≤?60 years) and in smokers (48.1 vs 27.4% of non-smokers). The increased risk of bladder cancer was 6.70-fold (95% confidence interval (CI) 2.09–23.87; p?=?0.000) in those carrying methylated DAP-kinase and it was elevated in patients who smoked (odds ratio 7.87; 95% CI 1.50–54.96; p?=?0.007). This study demonstrated that methylation in the gene promoter on its own could significantly decrease the mRNA expression level of DAP-kinase by 27.68%. Interestingly, patients within the group aged?>?60 years and with a smoking habit showed increased downregulation of mRNA compared with non-smokers of this age group (similar pattern of methylation). Hypermethylation can decrease the expression of DAP-kinase and may be one of the reasons for conversion of normal cells to malignant cells, as the frequency of methylation at the early stage (superficial) of tumours was elevated. Methylation of DAP-kinase can be considered as one of the prognosis indicators for progression and development of bladder cancer.     

DAP-kinase participates in TNF-alpha- and Fas-induced apoptosis and its function requires the death domain.

Death-associated protein (DAP)-kinase is a calcium/calmodulin regulated serine/threonine kinase that carries ankyrin repeats, a death domain, and is localized to the cytoskeleton. Here, we report that this kinase is involved in tumor necrosis factor (TNF)-alpha and Fas-induced apoptosis. Expression of DAP-kinase antisense RNA protected cells from killing by anti-Fas/APO-1 agonistic antibodies. Deletion of the death domain abrogated the apoptotic functions of the kinase, thus, documenting for the first time the importance of this protein domain. Overexpression of a fragment encompassing the death domain of DAP-kinase acted as a specific dominant negative mutant that protected cells from TNF-alpha, Fas, and FADD/MORT1-induced cell death. DAP-kinase apoptotic function was blocked by bcl-2 as well as by crmA and p35 inhibitors of caspases, but not by the dominant negative mutants of FADD/MORT1 or of caspase 8. Thus, it functions downstream to the receptor complex and upstream to other caspases. The multidomain structure of this serine/threonine kinase, combined with its involvement in cell death induced by several different triggers, place DAP-kinase at one of the central molecular pathways leading to apoptosis.     

DAP-kinase induces apoptosis by suppressing integrin activity and disrupting matrix survival signals

Death-associated protein kinase (DAP-kinase) is a calcium/calmodulin-dependent serine/threonine kinase, and participates in various apoptosis systems. However, its apoptosis-promoting mechanism is poorly understood. Here, we demonstrate that DAP-kinase suppresses integrin-mediated cell adhesion and signal transduction, whereas dominant-negative interference of this kinase promotes adhesion. This effect of DAP-kinase is neither a consequence of apoptosis nor a result of decreased expression of integrins. Rather, DAP-kinase downregulates integrin activity through an inside-out mechanism. We present evidence indicating that this adhesion-inhibitory effect accounts for a major mechanism of the apoptosis induced by DAP-kinase. First, in growth-arrested fibroblasts, DAP-kinase triggers apoptosis in cells plated on fibronectin, but does not affect the death of cells on poly-l-lysine. Second, in epithelial cells, DAP-kinase induces apoptosis in the anoikis-sensitive MCF10A cells, but not in the anoikis-resistant BT474 cells. Most importantly, the apoptosis-promoting effect of DAP-kinase is completely abolished by enforced activation of integrin-mediated signaling pathways from either integrin itself or its downstream effector, FAK. Finally, we show that integrin or FAK activation blocks the ability of DAP-kinase to upregulate p53. Our results indicate that DAP-kinase exerts apoptotic effects by suppressing integrin functions and integrin-mediated survival signals, thereby activating a p53-dependent apoptotic pathway.     

The dependence receptor UNC5H2 mediates apoptosis through DAP-kinase

Netrin-1 receptors UNC5H (UNC5H1-4) were originally proposed to mediate the chemorepulsive activity of netrin-1 during axonal guidance processes. However, UNC5H receptors were more recently described as dependence receptors and, as such, able to trigger apoptosis in the absence of netrin-1. They were also proposed as putative tumor suppressors. Here, we show that UNC5H2 physically interacts with the serine/threonine kinase death-associated protein kinase (DAP-kinase) both in cell culture and in embryonic mouse brains. This interaction occurs in part through the respective death domains of UNC5H2 and DAP-kinase. Moreover, part of UNC5H2 proapoptotic activity occurs through this interaction because UNC5H2-induced cell death is partly impaired in the presence of dominant-negative mutants of DAP-kinase or in DAP-kinase mutant murine embryonic fibroblast cells. In the absence of netrin-1, UNC5H2 reduces DAP-kinase autophosphorylation on Ser308 and increases the catalytic activity of the kinase while netrin-1 blocks UNC5H2-dependent DAP-kinase activation. Thus, the pair netrin-1/UNC5H2 may regulate cell fate by controlling the proapoptotic kinase activity of DAP-kinase.     

DAP-kinase is a Ca2+/calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity.

DAP-kinase was initially identified as a gene whose anti-sense-mediated reduced expression protected HeLa cells from interferon-gamma-induced programmed cell death. It was cloned in our laboratory by a functional gene selection approach. According to its amino acid sequence, this 160 kDa protein was predicted to be a novel type of calmodulin-regulated serine/threonine kinase which carries ankyrin repeats and the death domain. In this work we have shown that the kinase was autophosphorylated and capable of phosphorylating an exogenous substrate in a Ca2+/calmodulin-dependent manner. We proved that calmodulin binds directly to the recombinant kinase, and generated a constitutively active kinase mutant by the deletion of the calmodulin-regulatory domain. By immunostaining and biochemical fractionations we demonstrated that the kinase is localized to the cytoskeleton, in association with the microfilament system, and mapped a region within the protein which is responsible for binding to the cytoskeleton. Several assays attributed a cell death function to the gene. Ectopic expression of wild-type DAP-kinase induced the death of target cells, and the killing property depended strictly on the status of the intrinsic kinase activity. Conversely, a catalytically inactive mutant that carried a lysine to alanine substitution within the kinase domain, displayed dominant-negative features and protected cells from interferon-gamma-induced cell death. DAP-kinase is therefore a novel cytoskeletal-associated cell death serine/threonine kinase whose activation by Ca2+/calmodulin may be linked to the biochemical mechanism underlying the cytoskeletal alterations that occur during cell death.     

The role of calcium in apoptosis

In this chapter various aspects of apoptosis or programmed cell death (PCD) influenced by calcium as a mediator of signal transduction have been reviewed. Attention has been focused on recently described calcium-binding proteins such as ALG-2 or on a new calcium/calmodulin-dependent kinase, the death asso-ciated protein kinase or DAP-kinase. Both play a central role in apoptotic processes. Calcineurin, which normally is involved in the regulation of T-cell proliferation, is reported to interact with the apoptosis protec-tion protein bcl-2. Its possible involvement in the decision process whether T-cell activation leads to prolif-eration or apoptosis is discussed.© Kluwer Academic Publishers     

The regulation of death-associated protein (DAP) kinase in apoptosis

DAP-kinase is a calcium/calmodulin (Ca2+/CaM) serine/threonine kinase which positively mediates programmed cell death in a variety of cell systems. The kinase is localized to the actin microfilament and has a unique, multidomain structure consisting of ankyrin repeats and a death domain. One of the substrates of DAP-kinase was identified as myosin light chain (MLC), the phosphorylation of which mediates membrane blebbing. Another arm in its mode of action leads to the formation of autophagic vesicles. Recent work addressed its mode of regulation and identified a mechanism which restrains its apoptotic function in growing cells and enables its activation during cell death. It involves an inhibitory type of autophosphorylation on serine 308 within the CaM regulatory domain. This negative phosphorylation takes place in growing cells and is strongly reduced upon their exposure to the apoptotic stimulus of C6-ceramide. The substitution of serine 308 to alanine, which mimics the ceramide-induced dephosphorylation at this site, increases Ca2+/CaM-independent substrate phosphorylation, as well as binding and overall sensitivity of the kinase to CaM. At the cellular level, it strongly enhances the death-promoting activity of the kinase. These results are consistent with a molecular model in which phosphorylation on serine 308 stabilizes a locked conformation of the CaM regulatory domain within the catalytic cleft and, simultaneously, also interferes with CaM binding. We propose that this unique mechanism of auto-inhibition evolved to impose a locking device which keeps DAP-kinase silent in healthy cells and ensures its activation only in response to apoptotic signals.     

Death associated protein kinase: From regulation of apoptosis to tumor suppressive functions and B cell malignancies

Death associated protein kinase (DAP-kinase) is a pro-apoptotic calcium/calmodulin-regulated serine/threonine kinase with a multidomain structure that participates in a wide array of apoptotic systems initiated by IFN-, TNF-, activated Fas, and detachment from extracellular matrix. At various stages during tumor development, cells are subjected to apoptosis inducing stimuli and genetic mutations causing inhibition of apoptosis confer a selective advantage to cells. Thus, apoptosis and its regulation play an important role in tumor initiation, progression and metastasis. It has been demonstrated that the tumor-suppressive properties of DAP-kinase operate at two different apoptotic checkpoints in the course of tumor development; first, during the early oncogene-activated apoptotic checkpoint mediated by p19ARF-p53 pathway and second, during the late stages of metastasizing cells entering the circulation after detachment from extracellular matrix. Promoter hypermethylation of DAP-kinase has been observed in a high variety of primary tumors including head and neck tumors, and non-small cell lung cancers, where an association with poor prognosis was also noted. Notably, high frequencies of DAP-kinase methylation have been found in B cell lymphomas and myeloma, where loss of control of c-Myc induced hyperproliferation from inactivated DAP-kinase may possibly play an important role in the pathogenesis of these B cell neoplasms.     

Multiple cell death programs: Charon's lifts to Hades

Cells use different pathways for active self-destruction as reflected by different morphology: while in apoptosis (or "type I") nuclear fragmentation associated with cytoplasmic condensation but preservation of organelles is predominant, autophagic degradation of cytoplasmic structures preceding nuclear collapse is a characteristic of a second type of programmed cell death (PCD). The diverse morphologies can be attributed--at least to some extent--to distinct biochemical and molecular events (e.g. caspase-dependent and -independent death programs; DAP-kinase activity, Ras-expression). However, apoptosis and autophagic PCD are not mutually exclusive phenomena. Rather, diverse PCD programs emerged during evolution, the conservation of which apparently allows cells a flexible response to environmental changes, either physiological or pathological.     

Autophosphorylation restrains the apoptotic activity of DRP-1 kinase by controlling dimerization and calmodulin binding

DRP-1 is a pro-apoptotic Ca2+/calmodulin (CaM)-regulated serine/threonine kinase, recently isolated as a novel member of the DAP-kinase family of proteins. It contains a short extra-catalytic tail required for homodimerization. Here we identify a novel regulatory mechanism that controls its pro-apoptotic functions. It comprises a single autophosphorylation event mapped to Ser308 within the CaM regulatory domain. A negative charge at this site reduces both the binding to CaM and the formation of DRP-1 homodimers. Conversely, the dephosphorylation of Ser308, which takes place in response to activated Fas or tumour necrosis factor-alpha death receptors, increases the formation of DRP-1 dimers, facilitates the binding to CaM and activates the pro-apoptotic effects of the protein. Thus, the process of enzyme activation is controlled by two unlocking steps that must work in concert, i.e. dephosphorylation, which probably weakens the electrostatic interactions between the CaM regulatory domain and the catalytic cleft, and homodimerization. This mechanism of negative autophosphorylation provides a safety barrier that restrains the killing effects of DRP-1, and a target for efficient activation of the kinase by various apoptotic stimuli.     

The DAP-kinase interactome

DAP-kinase (DAPK) is a Ca²⁺/calmodulin regulated Ser/Thr kinase that activates a diverse range of cellular activities. It is subject to multiple layers of regulation involving both intramolecular signaling, and interactions with additional proteins, including other kinases and phosphatases. Its protein stability is modulated by at least three distinct ubiquitin-dependent systems. Like many kinases, DAPK participates in several signaling cascades, by phosphorylating additional kinases such as ZIP-kinase and protein kinase D (PKD), or Pin1, a phospho-directed peptidyl-prolyl isomerase that regulates the function of many phosphorylated proteins. Other substrate targets have more direct cellular effects; for example, phosphorylation of the myosin II regulatory chain and tropomyosin mediate some of DAPK’s cytoskeletal functions, including membrane blebbing during cell death and cell motility. DAPK induces distinct death pathways of apoptosis, autophagy and programmed necrosis. Among the substrates implicated in these processes, phosphorylation of PKD, Beclin 1, and the NMDA receptor has been reported. Interestingly, not all cellular effects are mediated by DAPK’s catalytic activity. For example, by virtue of protein–protein interactions alone, DAPK activates pyruvate kinase isoform M2, the microtubule affinity regulating kinases and inflammasome protein NLRP3, to promote glycolysis, influence microtubule dynamics, and enhance interleukin-1β production, respectively. In addition, a number of other substrates and interacting proteins have been identified, the physiological significance of which has not yet been established. All of these substrates, effectors and regulators together comprise the DAPK interactome. By presenting the components of the interactome network, this review will clarify both the mechanisms by which DAPK function is regulated, and by which it mediates its various cellular effects.     

DAPK and cytoskeleton-associated functions

Death-associated protein kinase (DAPK) undergoes activation in response to various death stimuli, and they have been associated with an increase in DAPK catalytic activity. One of the most prominent features of DAPK-induced cell death is the effect on the cytoskeleton, including loss of matrix attachment, and membrane blebbing. One known cytoskeletal-associated substrate of DAPK is the myosin-II light chain, phosphorylated by DAPK on Ser¹⁹, thus stabilizing actin stress fibres. Moreover, paxillin, a component of focal adhesions, was found to be localized in close proximity to the tips of the DAPK-positive filaments, indicating that stress fibres containing DAPK extend to focal contacts. Forced expression of DAPK in multiple cell types results in morphological changes such as cell rounding, membrane blebbing, shrinking and detachment. During directed migration, DAPK functions as a potent inhibitor of cell polarization, as evidenced by its perturbation of the formation of static protrusion at the leading edge. Furthermore, DAPK inhibits random migration by suppressing directional persistence. One of the studies considered DAPK as an anoikis inducer. Others showed that DAP-kinase inhibits the activities of cell surface integrins by converting them into an inactive conformation. Biochemical experiments have established the DAPK binding to Syntaxin1 and its subsequent phosphorylation at Ser¹⁸⁸ in a Ca²⁺ dependent manner. This phosphorylation event has been shown to decrease the binding of Syntaxin to MUNC18-1, a protein critically involved in synaptic vesicle docking. Here, we have investigated the structural interactions that modulate DAPK phosphorylation with Syntaxin and its functional role in binding to the MUNC18-1 to regulate vesicle docking. This review will summarize our current knowledge of the role of DAPK on cytoskeleton reorganization and report the mechanisms that regulate these changes.     

DAP-kinase and autophagy

DAP-kinase (DAPK) is a Ca²⁺-calmodulin regulated kinase with various, diverse cellular activities, including regulation of apoptosis and caspase-independent death programs, cytoskeletal dynamics, and immune functions. Recently, DAPK has also been shown to be a critical regulator of autophagy, a catabolic process whereby the cell consumes cytoplasmic contents and organelles within specialized vesicles, called autophagosomes. Here we present the latest findings demonstrating how DAPK modulates autophagy. DAPK positively contributes to the induction stage of autophagosome nucleation by modulating the Vps34 class III phosphatidyl inositol 3-kinase complex by two independent mechanisms. The first involves a kinase cascade in which DAPK phosphorylates protein kinase D, which then phosphorylates and activates Vps34. In the second mechanism, DAPK directly phosphorylates Beclin 1, a necessary component of the Vps34 complex, thereby releasing it from its inhibitor, Bcl-2. In addition to these established pathways, we will discuss additional connections between DAPK and autophagy and potential mechanisms that still remain to be fully validated. These include myosin-dependent trafficking of Atg9-containing vesicles to the sites of autophagosome formation, membrane fusion events that contribute to expansion of the autophagosome membrane and maturation through the endocytic pathway, and trafficking to the lysosome on microtubules. Finally, we discuss how DAPK's participation in the autophagic process may be related to its function as a tumor suppressor protein, and its role in neurodegenerative diseases.     

The DAPK family: a structure–function analysis

DAP-kinase (DAPK) is the founding member of a family of highly related, death associated Ser/Thr kinases that belongs to the calmodulin (CaM)-regulated kinase superfamily. The family includes DRP-1 and ZIP-kinase (ZIPK), both of which share significant homology within the common N-terminal kinase domain, but differ in their extra-catalytic domains. Both DAPK and DRP-1 possess a conserved CaM autoregulatory domain, and are regulated by calcium-activated CaM and by an inhibitory auto-phosphorylation within the domain. ZIPK’s activity is independent of CaM but can be activated by DAPK. The three kinases share some common functions and substrates, such as induction of autophagy and phosphorylation of myosin regulatory light chain leading to membrane blebbing. Furthermore, all can function as tumor suppressors. However, they also each possess unique functions and intracellular localizations, which may arise from the divergence in structure in their respective C-termini. In this review we will introduce the DAPK family, and present a structure/function analysis for each individual member, and for the family as a whole. Emphasis will be placed on the various domains, and how they mediate interactions with additional proteins and/or regulation of kinase function.     

DAP-kinase-mediated morphological changes are localization dependent and involve myosin-II phosphorylation

DAP-kinase (DAPk) is a Ser/Thr kinase that regulates cytoplasmic changes associated with programmed cell death. It is shown here that a GFP-DAPk fusion, which partially localized to actin stress fibers, induced extensive membrane protrusions. This phenotype correlated with changes in myosin-II distribution and with increased phosphorylation of the myosin-II regulatory light chain (RLC). A mutant lacking the cytoskeletal-interacting region (GFP-DAPkDeltaCyto) displayed diffuse cytoplasmic localization, and induced peripheral membrane blebbing, instead of the extensive protrusions. In contrast, deletion of the ankyrin repeats led to mislocalization of the kinase to focal contacts, where it failed to elicit any changes in cell morphology. While both wild-type DAPk and DAPkDeltaCyto induced RLC phosphorylation independently of the Rho-activated kinase ROCK, only the wild type led to increases in stress-fiber associated phospho-RLC. Thus, the precise intracellular localization of DAPk is critical for exposure to its substrates, including the RLC, which mediate varying morphologic changes.     

Gtdap-1 and the role of autophagy during planarian regeneration and starvation

Planarians have been established as an ideal model organism for stem cell research and regeneration. Planarian regeneration and homeostasis require an exquisite balancing act between cell death and cell proliferation as new tissues are made (epimorphosis) and existing tissues remodeled (morphallaxis). Some of the genes and mechanisms that control cell proliferation and pattern formation are known. However, studies about cell death during remodeling are few and far between. We have studied the gene Gtdap-1, the planarian ortholog of human death-associated protein-1 or DAP-1. DAP-1 together with DAP-kinase has been identified as a positive mediator of programmed cell death induced by gamma-interferon in HeLa cells. We have found that the gene functions at the interface between autophagy and cell death in the remodeling of the organism that occurs during regeneration and starvation in sexual and asexual races of planarians. Our data suggest that autophagy of existing cells may be essential to fuel the continued proliferation and differentiation of stem cells by providing the necessary energy and building blocks to neoblasts.     

The DAP-kinase family of proteins: study of a novel group of calcium-regulated death-promoting kinases

DAP-kinase (DAPk) is a Ca(2+)/calmodulin (CaM)-regulated Ser/Thr kinase that functions as a positive mediator of programmed cell death. It associates with actin microfilament and has a unique multidomain structure. One of the substrates of DAPk was identified as myosin light chain (MLC), the phosphorylation of which mediates membrane blebbing. Four additional kinases have been identified based on the high homology of their catalytic domain to that of DAPk. Yet, they differ in the structure of their extracatalytic domains and in their intracellular localization. One member of this family, DRP-1, also shares with DAPk both the property of activation by Ca(2+)/CaM and a specific phosphorylation-based regulatory mechanism. The latter involves an inhibitory type of autophosphorylation on a conserved serine at position 308, in the CaM regulatory domains of these two kinases. This phosphorylation, which occurs in growing cells, restrains the death-promoting effects of these kinases, and is specifically removed upon exposure of cells to various apoptotic stimuli. The dephosphorylation at this site increases the binding and sensitivity of each of these two kinases to their common activator-CaM. In DAPk, the dephosphorylation of serine 308 also increases the Ca(2+)/CaM-independent substrate phosphorylation. In DPR-1, it also promotes the formation of homodimers necessary for its full activity. These results are consistent with a molecular model in which phosphorylation on serine 308 stabilizes a locked conformation of the CaM regulatory domain within the catalytic cleft and simultaneously also interferes with CaM binding. In DRP-1, it introduces an additional locking device by preventing homodimerization. We propose that this unique mechanism of autoinhibition, evolved to keep these death-promoting kinases silent in healthy cells and ensures their activation only in response to apoptotic signals.