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Death associated protein kinase (DAPK; also called DAPK1) was originally isolated in an unbiased antisense based genetic screen for genes whose protein products were necessary for interferon (IFN)-γ-induced death in HeLa cells. Following the discovery of DAPK, four other kinases that share different degrees of homology to DAPK’s catalytic domain were identified. These five kinases compose the DAPK family (Figure 1). The two closest family members are DRP-1 (DAPK related protein 1, also called DAPK2) and ZIPK (zipper-interacting protein kinase, also called Dlk or DAPK3). They share approximately 80 % identity in their kinase domains with DAPK. Two other members, DRAK1 and DRAK2 (DAPK related apoptosis inducing kinase 1 and 2, also called STK17A and STK17B), are more distantly related. They share only approximately 50 % identity with DAPK, and have been less studied. Examination of the human kinome reveals that the DAPK family falls into the branch of calmodulin (CaM)-regulated kinases, positioned close to myosin light chain kinase (MLCK), which shares 44 % identity with the DAPK catalytic domain. Consistent with their high homology to MLCK, all members of the DAPK family can phosphorylate the myosin II regulatory light chain (MLC), and their overexpression in cells results in extensive membrane blebbing.
Figure 1. The DAPK Family of Proteins.
As its name implies, DAPK has been implicated as a regulator of cell death, both caspase-dependent and independent. DAPK is necessary for apoptosis induced by various death signals, and has also been linked to the activation of autophagy. It functions as a tumor suppressor whose expression is lost in many cancers because of promoter methylation. This can be attributed to its ability to suppress cellular transformation at early stages of tumor development, and to inhibit metastasis. Part of its latter capability stems from its cytoskeletal functions. In this respect, it has been elegantly shown that DAPK inhibits cell motility and cell adhesion through interfering with integrin function. Moreover, through activation of myosin II, DAPK promotes membrane blebbing and stress fiber formation. DAPK also plays a role in neuronal pathologies; it has been implicated in ischemia-induced neuronal cell death, as well as in the development of Alzheimer’s disease. At the mechanistic level, it has been shown that DAPK binds and phosphorylates the NMDA receptors in response to cerebral injury leading to neuronal cell death. Recently, DAPK has been shown to be involved in the regulation of inflammation and immune function.
DAPK and autophagy
Expression of DAPK in various cell types results in the enhanced formation of autophagosomes. This was observed by electron microscopy as an increase in the appearance of double membrane vesicles enclosing cytoplasmic contents, indicative of autophagosomes in varying states of maturation, including autolysosomes. Furthermore, increased accumulation of the autophagy marker GFP-LC3 in puncta representing the autophagosome membrane was observed upon DAPK expression.
Remarkably, DAPK is activated by various stimuli that induce autophagy. Activation of DAPK has been shown to involve several inter-related mechanisms that include binding of Ca2+-activated CaM to the CaM regulatory domain, dephosphorylation of Ser308 within the CaM regulatory domain through the PP2A phosphatase, and potentially, hydrolysis of GTP to GDP through the Ras of complex proteins (ROC)–C-terminal of ROC (COR) domains. Reductions in Ser308 phosphorylation were also observed during autophagy induced by several anti-neoplastic drugs, such as the histone deacetylase inhibitor LBH589 (Panobinostat) in colon cancer and the phase II clinical drug PM02734 (Elisidepsin) in non-small cell lung cancer (NSCLC) cells. Similar effects on DAPK’s phosphorylation/activation state were observed in hepatocellular carcinoma cells treated with the p38 MAPK inhibitor SB203580. Interestingly, the anti-proliferative and autophagy effects of this drug are independent of p38, and may instead involve activation of AMPK and inhibition of Akt/PKB.
Figure 2. Pathways linking DAPK1 to the nucleation step of autophagy
DAPK in cancer
Like many other tumor suppressors, the function of DAPK is compromised in various cancers. But different from genes like p53, the dysfunction of DAPK in cancer is usually because of loss of expression rather than mutation. DAPK expression loss is mainly caused by hypermethylation at the 5’UTR of DAPK gene, although less frequently it can also be a result of the homozygous deletion. DAPK gene methylation has been found in over 30 types of cancers, albeit the methylation rates vary dramatically. Nevertheless, it was found that in primary tissues and cell lines of NSCLC, DAPK protein can still be expressed in the presence of hypermethylation. This miscorrelation of hypermethylation status and protein expression have also been described in other cancers such as renal cell carcinoma (RCC) and chronic lymphoid leukemia (CLL), suggesting a post-translational regulation of DAPK may also be crucial in certain cancer types for DAPK protein expression control.
Despite the wide literature support of DAPK as a tumor suppressor, there are studies showing the opposite. Gallagher et al. have discovered a splice variant of mouse DAPK (DAPK-β) that can promote cell survival. But considering the active roles the isoforms of other tumor suppressors like p53 and p73 in cancer, it is possible that there are more splice variants of DAPK in cancer that can antagonize the wild-type DAPK function and thereby protect cells from death. More controversially, Yukawa et al. showed DAPK is essential for the survival of several types of uterine cancer cells, and the knockdown of DAPK enhanced 5-fluorouracil (5-FU) and Fas induced apoptosis in human endometrial adenocarcinoma cells. Lately, Zhao et al. reported that DAPK is important for the survival of estrogen receptor (ER) negative breast cancer cells carrying p53 mutation. All these studies suggest that in specific tissue or genetic environment, DAPK may process pro-survival function and promote tumorigenicity.
DAPK as a therapeutic target
In summary, DAPK is actively involved in multiple diseases with pleiotropic cellular functions, which make it an interesting therapeutic target. For complex diseases such as cancer, where DAPK has diverse or even opposing functions, tissue specificity may be a direction to find tumors that DAPK is more functionally important and thus a better target. For instance, although methylation of DAPK gene is reported in many different types of cancer, there is a big difference among tissues for the frequency of methylation. It is logical to assume the dysfunction of DAPK may be more important for the tumorigenesis of cancers like follicular lymphoma and targeting DAPK in these tumors may be more likely to bring clinical benefits.