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 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.     

Phosphorylation of Beclin 1 by DAP-kinase promotes autophagy by weakening its interactions with Bcl-2 and Bcl-XL

Beclin 1, an essential autophagic protein, is a BH3-only protein that binds Bcl-2 anti-apoptotic family members. The dissociation of Beclin 1 from the Bcl-2

inhibitors is essential for its autophagic activity, and therefore is tightly controlled. We recently revealed a novel phosphorylation-based mechanism by which Death

Associated Protein Kinase (DAPk) regulates this process. We found that DAPk phosphorylates Beclin 1 on T119, a critical residue within its BH3 domain, and thus

promotes Beclin 1 dissociation from Bcl-X_L and autophagy induction.¹ Here we report that T119 phosphorylation also reduces the interaction between Beclin 1 and Bcl-2, in

line with the high degree of structural homology between the BH3 binding pockets of Bcl-2 and Bcl-XL proteins. Our results reveal a new phosphorylation-based

mechanism that reduces the interaction of Beclin 1 with its inhibitors to activate the autophagic machinery.     

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: 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 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.     

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.     

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.     

Boning up on autophagy: The role of autophagy in skeletal biology

From an evolutionary perspective, the major function of bone is to provide stable sites for muscle attachment and affording protection of vital organs, especially the heart and lungs (ribs) and spinal cord (vertebrae and intervertebral discs). However, bone has a considerable number of other functions: serving as a store for mineral ions, providing a site for blood cell synthesis and participating in a complex system-wide endocrine system. Not surprisingly, bone and cartilage cell homeostasis is tightly controlled, as is the maintenance of tissue structure and mass. While a great deal of new information is accruing concerning skeletal cell homeostasis, one relatively new observation is that the cells of bone (osteoclasts osteoblasts and osteocytes) and cartilage (chondrocytes) exhibit autophagy. The focus of this review is to examine the significance of this process in terms of the functional demands of the skeleton in health and during growth and to provide evidence that dysregulation of the autophagic response is involved in the pathogenesis of diseases of bone (Paget disease of bone) and cartilage (osteoarthritis and the mucopolysaccharidoses). Delineation of molecular changes in the autophagic process is uncovering new approaches for the treatment of diseases that affect the axial and appendicular skeleton.     

DAP-kinase is a mediator of endoplasmic reticulum stress-induced caspase activation and autophagic cell death

Damage to endoplasmic reticulum (ER) homeostasis that cannot be corrected by the unfolded protein response activates cell death. Here, we identified death-associated protein kinase (DAPk) as an important component in the ER stress-induced cell death pathway. DAPk-/- mice are protected from kidney damage caused by injection of the ER stress-inducer tunicamycin. Likewise, the cell death response to ER stress-inducers is reduced in DAPk-/- primary fibroblasts. Both caspase activation and autophagy induction, events that are activated by ER stress and precede cell death, are significantly attenuated in the DAPk null cells. Notably, in this cellular setting, autophagy serves as a second cell killing mechanism that acts in concert with apoptosis, as the depletion of Atg5 or Beclin1 from fibroblasts significantly protected from ER stress-induced death when combined with caspase-3 depletion. We further show that ER stress promotes the catalytic activity of DAPk by causing dephosphorylation of an inhibitory autophosphorylation on Ser(308) by a PP2A-like phosphatase. Thus, DAPk constitutes a critical integration point in ER stress signaling, transmitting these signals into two distinct directions, caspase activation and autophagy, leading to cell death.     

Radiation-induced autophagy: mechanisms and consequences

Autophagy is an evolutionary conserved, indispensable, lysosome-mediated degradation process, which helps in maintaining homeostasis during various cellular traumas. During stress, a context-dependent role of autophagy has been observed which drives the cell towards survival or death depending upon the type, time, and extent of the damage. The process of autophagy is stimulated during various cellular insults, e.g. oxidative stress, endoplasmic reticulum stress, imbalances in calcium homeostasis, and altered mitochondrial potential. Ionizing radiation causes ROS-dependent as well as ROS-independent damage in cells that involve macromolecular (mainly DNA) damage, as well as ER stress induction, both capable of inducing autophagy. This review summarizes the current understanding on the roles of oxidative stress, ER stress, DNA damage, altered mitochondrial potential, and calcium imbalance in radiation-induced autophagy as well as the merits and limitations of targeting autophagy as an approach for radioprotection and radiosensitization.     

Liver autophagy: physiology and pathology

Autophagy has long been thought of as a bulk degradation system in which cytoplasmic components are sequestered by double-membrane structures called autophagosomes, and the contents are then degraded after autophagosomes fuse with lysosomes. Genetic experiments in yeast identified a set of Autophagy-related (ATG) genes that are essential for autophagy. We have since elucidated many of the molecular underpinnings of autophagy and the physiologic roles of these processes in various systems. This review summarizes the physiologic roles of autophagy with a particular focus on liver autophagy based on analyses of knockout mice lacking Atg genes.     

GTP binding to the ROC domain of DAP-kinase regulates its function through intramolecular signalling

Death-associated protein kinase (DAPk) was recently suggested by sequence homology to be a member of the ROCO family of proteins. Here, we show that DAPk has a functional ROC (Ras of complex proteins) domain that mediates homo-oligomerization and GTP binding through a defined P-loop motif. Upon binding to GTP, the ROC domain negatively regulates the catalytic activity of DAPk and its cellular effects. Mechanistically, GTP binding enhances an inhibitory autophosphorylation at a distal site that suppresses kinase activity. This study presents a new mechanism of intramolecular signal transduction, by which GTP binding operates in cis to affect the catalytic activity of a distal domain in the protein.     

Avoiding death by autophagy: Interactions of Listeria monocytogenes with the macrophage autophagy system

Autophagy restricts the growth of a variety of intracellular pathogens. However, cytosol-adapted pathogens have evolved ways to evade restriction by this innate immune mechanism. Listeria monocytogenes is a Gram-positive bacterial pathogen that utilizes a cholesterol-dependent pore-forming toxin, listeriolysin O (LLO), to escape from the phagosome. Autophagy targets L. monocytogenes in LLO-damaged phagosomes and also in the cytosol under some experimental conditions. However, this bacterium has evolved multiple mechanisms to evade restriction by autophagy, including actin-based motility in the cytosol and an as yet undefined mechanism mediated by bacterial phospholipases C’s (PLCs). A population of L. monocytogenes with inefficient LLO activity forms Spacious Listeria-containing Phagosomes (SLAPs), which are autophagosome-like compartments that do not mature, allowing slow bacterial growth within enlarged vesicles. SLAPs may represent a stalemate between bacterial LLO action and the host autophagy system, resulting in persistent infection.

Addendum to: Birmingham CL, Canadien V, Gouin E, Troy EB, Yoshimori T, Cossart P, Higgins DE, Brumell JH. Listeria monocytogenes evades killing by autophagy during colonization of host cells. Autophagy 2007; 3:442-51.andBirmingham CL, Canadien V, Kaniuk NA, Steinberg BE, Higgins DE, Brumell JH. Listeriolysin O allows Listeria monocytogenes replication in macrophage vacuoles. Nature 2008; 451:350-4.