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DAP-kinase—Protector or enemy in apoptotic cell death
The International Journal of Biochemistry & Cell Biology 37 (2005) 1763–1767
Molecules in focus
DAP-kinase—Protector or enemy in apoptotic cell death Regine Schneider-Stock a,∗ , Albert Roessner a , Oliver Ullrich b a
Department of Pathology, Head of Molecular Genetics Division, Otto-von-Guericke University, Leipziger Str. 44, 39120 Magdeburg, Germany b Department of Immunology, Otto-von-Guericke University, Magdeburg, Germany Received 30 December 2004; accepted 23 February 2005
Abstract Death-associated protein (DAP)-kinase, a member of a novel subfamily of pro-apoptotic serine/threonine kinases, was recently identified as a new tumor suppressor gene with multiple functions in programmed cell death. This 160-kDa protein consists of different interaction domains that enable it to participate in seemingly contradictory pathways such as elimination of premalignant cells or cytoprotection in cellular homoeostasis. DAP-kinase is frequently inactivated by aberrant promoter methylation in many cancer types, and its expression was shown to be a useful molecular marker for cancer prognosis. Moreover, DAP-kinase is considered a regulator of neuronal apoptosis. Future investigations should allow for the evaluation of DAP-kinase as a potential target for both pro- and anti-apoptotic therapeutic interventions. © 2005 Elsevier Ltd. All rights reserved. Keywords: DAP-kinase; Apoptosis
1. Introduction A novel subfamily of pro-apoptotic serine/threonine kinases, the death-associated protein (DAP)-kinase family, was described recently (K¨ogel, Prehn, & Scheidtmann, 2001). To date, five members have been identified (Table 1): DAP-kinase (calcium/calmodulinregulated serine/threonine protein kinase), deathassociated protein kinase related protein 1/deathassociated protein kinase 2 (DRP-1/DAPK2), DAPlike kinase/zipper interacting protein kinase (Dlk/ZIP ∗
Corresponding author. Tel.: +49 391 6715060; fax: +49 391 6717839. E-mail address: [email protected] (R. Schneider-Stock). 1357-2725/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2005.02.019
kinase), DAP-kinase related apoptosis-inducing kinases 1 and 2 (DRAK1, DRAK2). All members show a large sequence homology that is mainly restricted to the N-terminal kinase domain (K¨ogel et al., 2001, see Table 1). By contrast, the diverse, adjacent C-terminal regions link individual family members to specific signal transduction pathways. Considering the multidomain structure of DAP-kinase, this molecule might have multiple functions in cells and might participate in the regulation of different types of cell death. 2. DAP-kinase—structure The DAP-kinase is a 160-kDa multidomain protein (Fig. 1) that contains an ankyrin-rich region consist-
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Table 1 The DAP (Death Associated Protein) kinase family Gene name
Gene bank number
Symbol
Gene alias
DAP-kinase 1
NM004938
DAPK1
DAP-kinase 2
NM014326
DAPK2
DRP-1/DAPK2
DAP-kinase 3
NM001348
DAPK3
Dlk/ZIP-kinase
DRAK1
NM004760
DRAK2
NM004226
STK17A serine/threonine kinase 17A STK17B serine/threonine kinase 17B
Death-associated protein kinase Death-associated protein kinase related protein1/death-associated protein kinase 2 DAP-like kinase/zipper interacting protein kinase DAP kinase-related apoptosis inducing protein kinases 1 DAP kinase-related apoptosis inducing protein kinases 2
Sequence homology (%)
Cellular localization
Chromosomal localization 9q34.1
79.8
Actin microfilament Cytoplasmatic
15q22.31
83.3
Nucleus
19p13.3
48.5
Nucleus
7p12-p14
51.9
Nucleus
2q32.3
Fig. 1. Schematic diagram of DAP-kinase protein structure. The 160 kDa Ca2+ /calmodulin (CaM)-regulated Ser/Thr kinase bears a multidomain structure. The catalytic and the calmodulin regulatory domains determine substrate specificity and regulation of kinase catalytic activity, respectively. The non-catalytic association domains, involved in subcellular localization or interactions with other proteins, include the eight ankyrin repeats, two nucleotide-binding P-loops, a cytoskeleton-binding region, and a death domain. The autophosphorylation site was mapped to Ser308 within the CaM-regulatory domain (Shohat et al., 2002).
ing of eight repeats, a C-terminal death domain, and a region required for the interaction with the actin filaments of the cytoskeleton (Cohen, Feinstein, & Kimchi, 1997). The death domain at the C-terminus has homology to other apoptosis-associated proteins, including TNF receptor and Fas/Apo-1 (Feinstein and Kimchi, 1995b, inTIBS). Furthermore, DAP-kinase has a Ca2+ /calmodulin-binding region adjacent to the kinase domain (Cohen et al., 1997). Only DRP/DAPK2 contains this latter module (Inbal, Shani, Cohen, Kissil, & Kimchi, 2000). By contrast, Dlk/ZIP kinase carries a functional C-terminal leucine zipper motif mediating both homodimerization and interaction with transcription factor ATF4 and novel apoptosis-antagonizing transcription factor (AATF) (Kawai et al., 1998; Page, L¨odige, K¨ogel, & Scheidtmann, 1999). The C-terminal
regions of DRAK1 and DRAK2 do not show any structural motifs similar to other family members. The DAP-kinase family members have a distinct subcellular localization (Table 1). Whereas DAPkinase is associated with the actin filaments of the cytoskeleton, DRP-1/DAPK2 is localized in the cytoplasm, and Dlk/ZIP kinase, DRAK1 and DRAK2 are nuclear proteins (K¨ogel et al., 2001).
3. DAP-kinase—a tumor suppressor All five kinases are ubiquitously expressed in various tissues (Kawai et al., 1999; K¨ogel et al., 1998; Sanjo et al., 1998; Yamamoto et al., 1998). Many tumor cells, including cell lines and biopsies of human
R. Schneider-Stock et al. / The International Journal of Biochemistry & Cell Biology 37 (2005) 1763–1767
tumors, demonstrated loss of DAP-kinase expression, suggesting a role as tumor suppressor (Inbal et al., 1997; Kissil et al., 1997). Loss of DAP-kinase apparently leads to selective advantages for cancer cells, thus playing a causative role in the metastasis of cancer. In this context, mice that failed to express DAP-kinase developed highly metastatic lung carcinoma (Inbal et al., 1997). Re-expression of DAP-kinase in these highly metastatic tumors inhibited their ability to form lung metastases after intravenous injection into syngeneic mice (Inbal et al., 1997). Aberrant methylation of the DAP-kinase promoter region is the underlying mechanism of silenced DAPkinase expression in tumor cells. Hypermethylation of DAP-kinase promoter occurs in many tumor types and preneoplastic lesions (Esteller, 2002). In addition, treatment of cell lines with demethylating agent 5-aza2 deoxycytidine resulted in a significant up-regulation of DAP-kinase protein (Kissil et al., 1997). By contrast, inactivation of DAP-kinase can be associated with homozygous gene deletions as shown in pituitary tumors (Simpson, Clayton, & Farrell, 2002).
4. DAP-kinase—biological function 4.1. Pro-apoptotic role DAP-kinase was recently identified as a positive mediator of apoptosis induced by IFN-␥ in rodents (Raveh, Droguett, Horwitz, Depinho, & Kimchi, 2001), and it counteracts oncogene-induced transformation of mouse primary embryonic fibroblasts by activating p53 in a p19ARF -dependent manner (Deiss, Feinstein, Berissi, Cohen, & Kimchi, 1995; Raveh et al., 2001). In vitro kinase assays showed that DAP-kinase can phosphorylate p19ARF , but not p53 (Raveh et al., 2001). Oncogenes turn on DAP-kinase/p53-dependent apoptotic checkpoint at initial transformation stages (Raveh et al., 2001). Expression of Myc or E2F induced an up-regulation of DAP-kinase, along with p19ARF and p53, in wild-type but not in DAP-kinase deficient mouse primary embryonic fibroblasts (Raveh et al., 2001). Thus, DAP-kinase regulates at an early apoptotic checkpoint designed for eliminating premalignant cells. The human DAP-kinase promoter can be activated by TGF, which links SMADs to mitochondrialbased pro-apoptotic events (Jang et al., 2001). DAP-
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kinase is also involved in TNF␣- and Fas-induced cell death (Cohen et al., 1999). The death-promoting effects of DAP-kinase are regulated by at least two distinct autoinhibitory mechanisms controlled by the CaM domain and the serine-rich C-terminal tail. Inhibition of the CaM domain is relieved upon binding of Ca/Calmodulin, and deletion of the C-terminal tail creates a super-killing mutant (Cohen et al., 2001; Raveh et al., 2001). 4.2. Anti-apoptotic role Only few authors regard DAP-kinase as an antiapoptotic factor (Jin & Gallagher, 2003). DAP-kinase is highly expressed in non-apoptotic tissues such as brain cortex and hippocampus (Schumacher, Velentza, Watterson, & Wainwright, 2002; Yamamoto et al., 1999). There is a significant portion of peripheral blood cells in patients without any malignancy that has low but detectable levels of DAP-kinase promoter hypermethylation (Reddy et al., 2003). Furthermore, it has been reported that the anti-sense DAP-kinase fragment, protecting cells from IFN-␥-induced apoptosis, did not protect these cells from TNF-induced apoptosis (Deiss et al., 1995; Jang et al., 2002). The overexpression of DAP-kinase does not induce apoptosis under normal growth conditions (Inbal et al., 1997; Jin, Blue, Dixon, Shao, & Gallagher, 2002). In summary, these studies strongly suggest a cytoprotective role for DAP-kinase in cellular homeostasis (Jin & Gallagher, 2003). It is a major challenge to elucidate and to distinguish these seemingly contradictory roles of DAP-kinase to understand its function in programmed cell death and tumorigenesis. The identification of the substrates of DAP-kinase may help to delineate the regulatory pathways and the down-stream targets.
5. Clicinal aspects 5.1. DAPK-kinase and tumor prognosis Methylation-dependent DAP-kinase expression might be a useful molecular marker for cancer prognosis (Satoh et al., 2002) and plays an important role in determining the biological aggressiveness of early-stage non-small-cell lung cancer (Tang et al., 2000) and primary biliary tract carcinoma (Tozawa
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et al., 2004). DAP-kinase protein expression strongly indicates high recurrence rates in bladder cancer (Tada et al., 2002) and is an independent predictor of breast cancer prognosis and survival (Levy et al., 2004). Thus, DAP-kinase expression may be useful in identifying aggressive tumors that are more likely to spread and that alter the patients’ long-term survival. The clinical usefulnesss of DAP-kinase inactivation for cancer detection is based on highly sensitive methods measuring aberrant promoter methylation in accessible body fluids such as urine sediments of bladder cancer patients (Friedrich et al., 2004) or sera of breast cancer patients (Dulaimi, Hillinck, Ibanez de Caceres, Saleem, & Cairns, 2004).
5.2. DAP-kinase and therapeutic implications Since DAP-kinase mediates signals in the early pathways of apoptosis (Raveh et al., 2001), it may be a useful therapeutic target for the prevention of diseases, including cell death. In this context, DAP-kinase is considered a strong positive regulator of neuronal apoptosis in vitro and in vivo (Pelled et al., 2002; Schori et al., 2002; Yamamoto, Hioki, Ishii, Nakajima-Iijima, & Uchino, 2002). Moreover, DAP-kinase is particularly abundant in adult brain (Bialek et al., 2004), with a protein expression pattern limited to cortical areas and areas of the hippocampus and olfactory bulb (Tian, Das, & Sheng, 2003). This suggests that DAP-kinase expression in the CNS is rigidly regulated, strictly controlled and suppressed, thus preventing neuronal cell death in healthy CNS of adults. However, during pathological conditions associated with neuronal cell death such as cerebral ischemia (Schumacher et al., 2002) or epilepsy (Henshall et al., 2004), DAP-kinase expression is increased, thus defining this enzyme as a potential neuroprotective drug target for the treatment of CNS diseases. The crystal structure of DAP-kinase was identified recently. Therefore, therapeutic approaches aim at designing specific inhibitors directed to the catalytic ATP-site of DAP-kinase, which is a validated drug target (Cohen, 2002) and attractive for drug development (Schumacher et al., 2002). An alkylated 3amino-6-phenylpyridazine with an IC50 of 13 M was developed recently, causing neuroprotective effects in animal hypoxia–ischemia models, even when administered 6 h following injury (Velentza et al., 2003).
In contrast to neuroprotective interventions, DAPkinase-based strategies in tumor therapy will aim at restoring DAP-kinase activity by demethylating agents, thus driving cancer cells into apoptosis. Another strategy might be specific activation of residual DAP-kinase in tumors by interfering with DAP-kinase auto-inhibitory mechanism (against the self inhibitory activity of the C-terminal tail or by blocking the binding of Ser308 of the CaM regulatory domain). However to date, such concepts have been purely hypothetical (Bialik & Kimchi, 2004).
References Bialik, S., & Kimchi, A. (2004). DAP-kinase as a target for drug design in cancer and diseases associated with accelerated cell death. Seminars in Cancer Biology, 14, 283–294. Cohen, O., Feinstein, E., & Kimchi, A. (1997). DAP-kinase is a Ca2+ /calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity. EMBO Journal, 16, 998–1008. Cohen, O., Inbal, B., Kissil, J. L., Raveh, T., Berissi, H., SpivakKroizaman, T., et al. (1999). DAP-kinase participates in TNF-␣and Fas-induced apoptosis and its function requires the death domain. The Journal of Cell Biology, 146, 141–148. Cohen, P. (2002). Protein kinases—The major drug targets of the 21st century. Nature Reviews, 1, 309–315. Deiss, L. P., Feinstein, E., Berissi, H., Cohen, O., & Kimchi, A. (1995). Identification of a novel serine/threonine kinase and a novel 15-kDa protein as potential mediators of the gamma interferon-induced cell death. Genes Development and Cancer, 9, 15–30. Dulaimi, E., Hillinck, J., Ibanez de Caceres, I., Al-Saleem, T., & Cairns, P. (2004). Tumor suppressor gene promoter hypermethylation in serum of breast cancer patients. Clinical Cancer Research, 10, 6189–6193. Esteller, M. (2002). CpG island hypermethylation and tumor suppressor genes: A booming present, a brighter future. Oncogene, 21, 5427–5440. Friedrich, M. G., Weisenberger, D. J., Cheng, J. C., Chandrasoma, S., Siegmund, K. D., Gonzalgo, M. L., et al. (2004). Detection of methylated apoptosis-associated genes in urine sediments of bladder cancer patients. Clinical Cancer Research, 10, 7457–7465. Henshall, D. C., Schindler, C. K., So, N. K., Lan, J. Q., Meller, R., & Simon, R. P. (2004). Death-associated protein kinase expression in human temporal lobe epilepsy. Annals of Neurology, 55, 485–494. Inbal, B., Cohen, O., Polak-Charcon, S., Kopolovic, J., Vadai, E., Eisenbach, L., et al. (1997). DAP kinase links the control of apoptosis to metastasis. Nature, 390, 180–184. Inbal, B., Shani, G., Cohen, O., Kissil, J. L., & Kimchi, A. (2000). Death-associated protein kinase-related protein 1, a novel ser-
R. Schneider-Stock et al. / The International Journal of Biochemistry & Cell Biology 37 (2005) 1763–1767 ine/threonine kinase involved in apoptosis. Molecular Cell Biology, 20, 1044–1054. Jang, C. W., Chen, C. H., Chen, C. C., Chen, J., Su, Y. H., & Chen, R. H. (2001). TGF-beta induces apoptosis through Smad-mediated expression of DAP-kinase. Nature Cell Biology, 4, 51–58. Jin, Y., Blue, E. K., Dixon, S., Shao, Z., & Gallagher, P. J. (2002). A death-associated protein kinase (DAPK)-interacting protein, DIP-1, is an E3 ubiquitin ligase that promotes tumor necrosis factor-induced apoptosis and regulates the cellular levels of DAPK. Journal of Biological Chemistry, 277, 46980–46986. Jin, Y., & Gallagher, P. J. (2003). Antisense depletion of deathassociated protein kinase promotes apoptosis. Journal of Biological Chemistry, 278, 51587–51593. Kissil, J. L., Feinstein, E., Cohen, O., Jones, P. A., Tsai, Y. C., Knowles, M. A., et al. (1997). DAP-kinase loss of expression in various carcinoma and B-cell lymphoma cell lines: Possible implications for role as tumor suppressor gene. Oncogene, 15, 403–407. K¨ogel, D., Prehn, J. H. M., & Scheidtmann, K. H. (2001). The DAP kinase family of pro-apoptotic proteins: Novel players in the apoptotic game. BioEssays, 23, 352–358. Levy, D., Plu-Bureau, G., Decroix, Y., Hugol, D., Rostene, W., Kimchi, A., et al. (2004). Death-associated protein kinase loss of expression is a new marker for breast cancer prognosis. Clinical Cancer Research, 10, 3124–3130. Page, G., L¨odige, I., K¨ogel, D., & Scheidtmann, K. H. (1999). AATF, a novel transcription factor that interacts with Dlk/ZIP kinase and interferes with apoptosis. FEBS Letters, 462, 187–191. Pelled, D., Raveh, T., Riebeling, C., Fridkin, M., Berissi, H., Futerman, H., et al. (2002). Death-associated protein (DAP) kinase plays a central role in ceramide-induced apoptosis in cultured hippocampal neurons. Journal of Biological Chemistry, 277, 1957–1961. Raveh, T., Droguett, G., Horwitz, M. S., Depinho, R. A., & Kimchi, A. (2001). DAP kinase activates a p19ARF /p53-mediated apoptotic checkpoint to suppress oncogenic transformation. Nature Cell Biology, 3, 1–7. Reddy, A. N., Jiang, W. W., Kim, M., Benoit, N., Taylor, R., Clinger, J., et al. (2003). Death-associated protein kinase promoter hypermethylation in normal human lymphocytes. Cancer Research, 63, 7694–7698. Satoh, A., Toyota, M., Itoh, F., Kikuchi, T., Obata, T., Sasaki, Y., et al. (2002). DNA methylation and histone deacetylation associated with silencing DAP kinase gene expression in colorectal and gastric cancers. British Journal of Cancer, 86, 1817–1823.
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Schori, H., Yoles, E., Wheeler, L. A., Raveh, T., Kimchi, A., & Schwartz, M. (2002). Immune-related mechanims participating in resistance and susceptibility to glutamate toxicity. European Journal of Neuroscience, 16, 557–564. Schumacher, A. M., Velentza, A. V., Watterson, D. M., & Wainwright, M. S. (2002). DAPK catalytic activity in the hippocampus increases during the recovery phase in an animal model of brain hypoxic-ischemic injury. Biochemica Biophysica Acta, 1600, 128–137. Shohat, G., Shani, G., Eisenstein, M., & Kimchi, A. (2002). The DAP-kinase family of proteins: Study of a novel group of calcium-regulated, death-promoting kinases. Biochemica Biophysica Acta, 1600, 45–50. Simpson, D. J., Clayton, R. N., & Farrell, W. E. (2002). Preferential loss of death-associated protein kinase expression in invasive pituitary tumours is associated with either CpG island methylation or homozygous deletion. Oncogene, 21, 1217– 1224. Tada, Y., Wada, M., Taguchi, K., Mochida, Y., Kinugawa, N., Tsuneyoshi, M., et al. (2002). The association of deathassociated protein kinase hypermethylation with early recurrence in superficial bladder cancers. Cancer Research, 62, 4048– 4053. Tang, X., Khuri, F. R., Lee, J. J., Kemp, B. L., Liu, D., Hong, W. K., et al. (2000). Hypermethylation of the death-associated protein (DAP) kinase promoter and aggressiveness in stage I non-smallcell lung cancer. Journal of the National Cancer Institute, 92, 1511–1516. Tian, J. H., Das, S., & Sheng, Z. H. (2003). Ca2+ —Dependent phosphorylation of syntaxin-1a by the death-associated protein (DAP) kinase regulates its interaction with munc18. Journal of Biological Chemistry, 278, 26265–26274. Tozawa, T., Tamura, G., Honda, T., Nawata, S., Kimura, W., Makino, N., et al. (2004). Promoter hypermethylation of DAP-kinase is associated with poor survival in primary biliary tract carcinoma patients. Cancer Science, 95, 736–740. Velentza, A. V., Wainwright, M. S., Zasadzki, M., Miroeva, S., Schumacher, A. M., & Haiech, J. (2003). An aminopyridazinebased inhibitor of a pro-apoptotic protein kinase attenuates hypoxia–ischemia-induced acute brain injury. Bioorganisms Medical Chemistry Letters, 13, 3465–3470. Yamamoto, M., Hioki, T., Ishii, T., Nakajima-Iijima, S., & Uchino, S. (2002). DAP kinase activity is critical for C(2)-ceramide-induced apoptosis in PC12 cells. European Journal of Biochemistry, 269, 139–147.
Molecules in focus
DAP-kinase—Protector or enemy in apoptotic cell death Regine Schneider-Stock a,∗ , Albert Roessner a , Oliver Ullrich b a
Department of Pathology, Head of Molecular Genetics Division, Otto-von-Guericke University, Leipziger Str. 44, 39120 Magdeburg, Germany b Department of Immunology, Otto-von-Guericke University, Magdeburg, Germany Received 30 December 2004; accepted 23 February 2005
Abstract Death-associated protein (DAP)-kinase, a member of a novel subfamily of pro-apoptotic serine/threonine kinases, was recently identified as a new tumor suppressor gene with multiple functions in programmed cell death. This 160-kDa protein consists of different interaction domains that enable it to participate in seemingly contradictory pathways such as elimination of premalignant cells or cytoprotection in cellular homoeostasis. DAP-kinase is frequently inactivated by aberrant promoter methylation in many cancer types, and its expression was shown to be a useful molecular marker for cancer prognosis. Moreover, DAP-kinase is considered a regulator of neuronal apoptosis. Future investigations should allow for the evaluation of DAP-kinase as a potential target for both pro- and anti-apoptotic therapeutic interventions. © 2005 Elsevier Ltd. All rights reserved. Keywords: DAP-kinase; Apoptosis
1. Introduction A novel subfamily of pro-apoptotic serine/threonine kinases, the death-associated protein (DAP)-kinase family, was described recently (K¨ogel, Prehn, & Scheidtmann, 2001). To date, five members have been identified (Table 1): DAP-kinase (calcium/calmodulinregulated serine/threonine protein kinase), deathassociated protein kinase related protein 1/deathassociated protein kinase 2 (DRP-1/DAPK2), DAPlike kinase/zipper interacting protein kinase (Dlk/ZIP ∗
Corresponding author. Tel.: +49 391 6715060; fax: +49 391 6717839. E-mail address: [email protected] (R. Schneider-Stock). 1357-2725/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2005.02.019
kinase), DAP-kinase related apoptosis-inducing kinases 1 and 2 (DRAK1, DRAK2). All members show a large sequence homology that is mainly restricted to the N-terminal kinase domain (K¨ogel et al., 2001, see Table 1). By contrast, the diverse, adjacent C-terminal regions link individual family members to specific signal transduction pathways. Considering the multidomain structure of DAP-kinase, this molecule might have multiple functions in cells and might participate in the regulation of different types of cell death. 2. DAP-kinase—structure The DAP-kinase is a 160-kDa multidomain protein (Fig. 1) that contains an ankyrin-rich region consist-
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Table 1 The DAP (Death Associated Protein) kinase family Gene name
Gene bank number
Symbol
Gene alias
DAP-kinase 1
NM004938
DAPK1
DAP-kinase 2
NM014326
DAPK2
DRP-1/DAPK2
DAP-kinase 3
NM001348
DAPK3
Dlk/ZIP-kinase
DRAK1
NM004760
DRAK2
NM004226
STK17A serine/threonine kinase 17A STK17B serine/threonine kinase 17B
Death-associated protein kinase Death-associated protein kinase related protein1/death-associated protein kinase 2 DAP-like kinase/zipper interacting protein kinase DAP kinase-related apoptosis inducing protein kinases 1 DAP kinase-related apoptosis inducing protein kinases 2
Sequence homology (%)
Cellular localization
Chromosomal localization 9q34.1
79.8
Actin microfilament Cytoplasmatic
15q22.31
83.3
Nucleus
19p13.3
48.5
Nucleus
7p12-p14
51.9
Nucleus
2q32.3
Fig. 1. Schematic diagram of DAP-kinase protein structure. The 160 kDa Ca2+ /calmodulin (CaM)-regulated Ser/Thr kinase bears a multidomain structure. The catalytic and the calmodulin regulatory domains determine substrate specificity and regulation of kinase catalytic activity, respectively. The non-catalytic association domains, involved in subcellular localization or interactions with other proteins, include the eight ankyrin repeats, two nucleotide-binding P-loops, a cytoskeleton-binding region, and a death domain. The autophosphorylation site was mapped to Ser308 within the CaM-regulatory domain (Shohat et al., 2002).
ing of eight repeats, a C-terminal death domain, and a region required for the interaction with the actin filaments of the cytoskeleton (Cohen, Feinstein, & Kimchi, 1997). The death domain at the C-terminus has homology to other apoptosis-associated proteins, including TNF receptor and Fas/Apo-1 (Feinstein and Kimchi, 1995b, inTIBS). Furthermore, DAP-kinase has a Ca2+ /calmodulin-binding region adjacent to the kinase domain (Cohen et al., 1997). Only DRP/DAPK2 contains this latter module (Inbal, Shani, Cohen, Kissil, & Kimchi, 2000). By contrast, Dlk/ZIP kinase carries a functional C-terminal leucine zipper motif mediating both homodimerization and interaction with transcription factor ATF4 and novel apoptosis-antagonizing transcription factor (AATF) (Kawai et al., 1998; Page, L¨odige, K¨ogel, & Scheidtmann, 1999). The C-terminal
regions of DRAK1 and DRAK2 do not show any structural motifs similar to other family members. The DAP-kinase family members have a distinct subcellular localization (Table 1). Whereas DAPkinase is associated with the actin filaments of the cytoskeleton, DRP-1/DAPK2 is localized in the cytoplasm, and Dlk/ZIP kinase, DRAK1 and DRAK2 are nuclear proteins (K¨ogel et al., 2001).
3. DAP-kinase—a tumor suppressor All five kinases are ubiquitously expressed in various tissues (Kawai et al., 1999; K¨ogel et al., 1998; Sanjo et al., 1998; Yamamoto et al., 1998). Many tumor cells, including cell lines and biopsies of human
R. Schneider-Stock et al. / The International Journal of Biochemistry & Cell Biology 37 (2005) 1763–1767
tumors, demonstrated loss of DAP-kinase expression, suggesting a role as tumor suppressor (Inbal et al., 1997; Kissil et al., 1997). Loss of DAP-kinase apparently leads to selective advantages for cancer cells, thus playing a causative role in the metastasis of cancer. In this context, mice that failed to express DAP-kinase developed highly metastatic lung carcinoma (Inbal et al., 1997). Re-expression of DAP-kinase in these highly metastatic tumors inhibited their ability to form lung metastases after intravenous injection into syngeneic mice (Inbal et al., 1997). Aberrant methylation of the DAP-kinase promoter region is the underlying mechanism of silenced DAPkinase expression in tumor cells. Hypermethylation of DAP-kinase promoter occurs in many tumor types and preneoplastic lesions (Esteller, 2002). In addition, treatment of cell lines with demethylating agent 5-aza2 deoxycytidine resulted in a significant up-regulation of DAP-kinase protein (Kissil et al., 1997). By contrast, inactivation of DAP-kinase can be associated with homozygous gene deletions as shown in pituitary tumors (Simpson, Clayton, & Farrell, 2002).
4. DAP-kinase—biological function 4.1. Pro-apoptotic role DAP-kinase was recently identified as a positive mediator of apoptosis induced by IFN-␥ in rodents (Raveh, Droguett, Horwitz, Depinho, & Kimchi, 2001), and it counteracts oncogene-induced transformation of mouse primary embryonic fibroblasts by activating p53 in a p19ARF -dependent manner (Deiss, Feinstein, Berissi, Cohen, & Kimchi, 1995; Raveh et al., 2001). In vitro kinase assays showed that DAP-kinase can phosphorylate p19ARF , but not p53 (Raveh et al., 2001). Oncogenes turn on DAP-kinase/p53-dependent apoptotic checkpoint at initial transformation stages (Raveh et al., 2001). Expression of Myc or E2F induced an up-regulation of DAP-kinase, along with p19ARF and p53, in wild-type but not in DAP-kinase deficient mouse primary embryonic fibroblasts (Raveh et al., 2001). Thus, DAP-kinase regulates at an early apoptotic checkpoint designed for eliminating premalignant cells. The human DAP-kinase promoter can be activated by TGF, which links SMADs to mitochondrialbased pro-apoptotic events (Jang et al., 2001). DAP-
1765
kinase is also involved in TNF␣- and Fas-induced cell death (Cohen et al., 1999). The death-promoting effects of DAP-kinase are regulated by at least two distinct autoinhibitory mechanisms controlled by the CaM domain and the serine-rich C-terminal tail. Inhibition of the CaM domain is relieved upon binding of Ca/Calmodulin, and deletion of the C-terminal tail creates a super-killing mutant (Cohen et al., 2001; Raveh et al., 2001). 4.2. Anti-apoptotic role Only few authors regard DAP-kinase as an antiapoptotic factor (Jin & Gallagher, 2003). DAP-kinase is highly expressed in non-apoptotic tissues such as brain cortex and hippocampus (Schumacher, Velentza, Watterson, & Wainwright, 2002; Yamamoto et al., 1999). There is a significant portion of peripheral blood cells in patients without any malignancy that has low but detectable levels of DAP-kinase promoter hypermethylation (Reddy et al., 2003). Furthermore, it has been reported that the anti-sense DAP-kinase fragment, protecting cells from IFN-␥-induced apoptosis, did not protect these cells from TNF-induced apoptosis (Deiss et al., 1995; Jang et al., 2002). The overexpression of DAP-kinase does not induce apoptosis under normal growth conditions (Inbal et al., 1997; Jin, Blue, Dixon, Shao, & Gallagher, 2002). In summary, these studies strongly suggest a cytoprotective role for DAP-kinase in cellular homeostasis (Jin & Gallagher, 2003). It is a major challenge to elucidate and to distinguish these seemingly contradictory roles of DAP-kinase to understand its function in programmed cell death and tumorigenesis. The identification of the substrates of DAP-kinase may help to delineate the regulatory pathways and the down-stream targets.
5. Clicinal aspects 5.1. DAPK-kinase and tumor prognosis Methylation-dependent DAP-kinase expression might be a useful molecular marker for cancer prognosis (Satoh et al., 2002) and plays an important role in determining the biological aggressiveness of early-stage non-small-cell lung cancer (Tang et al., 2000) and primary biliary tract carcinoma (Tozawa
1766
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et al., 2004). DAP-kinase protein expression strongly indicates high recurrence rates in bladder cancer (Tada et al., 2002) and is an independent predictor of breast cancer prognosis and survival (Levy et al., 2004). Thus, DAP-kinase expression may be useful in identifying aggressive tumors that are more likely to spread and that alter the patients’ long-term survival. The clinical usefulnesss of DAP-kinase inactivation for cancer detection is based on highly sensitive methods measuring aberrant promoter methylation in accessible body fluids such as urine sediments of bladder cancer patients (Friedrich et al., 2004) or sera of breast cancer patients (Dulaimi, Hillinck, Ibanez de Caceres, Saleem, & Cairns, 2004).
5.2. DAP-kinase and therapeutic implications Since DAP-kinase mediates signals in the early pathways of apoptosis (Raveh et al., 2001), it may be a useful therapeutic target for the prevention of diseases, including cell death. In this context, DAP-kinase is considered a strong positive regulator of neuronal apoptosis in vitro and in vivo (Pelled et al., 2002; Schori et al., 2002; Yamamoto, Hioki, Ishii, Nakajima-Iijima, & Uchino, 2002). Moreover, DAP-kinase is particularly abundant in adult brain (Bialek et al., 2004), with a protein expression pattern limited to cortical areas and areas of the hippocampus and olfactory bulb (Tian, Das, & Sheng, 2003). This suggests that DAP-kinase expression in the CNS is rigidly regulated, strictly controlled and suppressed, thus preventing neuronal cell death in healthy CNS of adults. However, during pathological conditions associated with neuronal cell death such as cerebral ischemia (Schumacher et al., 2002) or epilepsy (Henshall et al., 2004), DAP-kinase expression is increased, thus defining this enzyme as a potential neuroprotective drug target for the treatment of CNS diseases. The crystal structure of DAP-kinase was identified recently. Therefore, therapeutic approaches aim at designing specific inhibitors directed to the catalytic ATP-site of DAP-kinase, which is a validated drug target (Cohen, 2002) and attractive for drug development (Schumacher et al., 2002). An alkylated 3amino-6-phenylpyridazine with an IC50 of 13 M was developed recently, causing neuroprotective effects in animal hypoxia–ischemia models, even when administered 6 h following injury (Velentza et al., 2003).
In contrast to neuroprotective interventions, DAPkinase-based strategies in tumor therapy will aim at restoring DAP-kinase activity by demethylating agents, thus driving cancer cells into apoptosis. Another strategy might be specific activation of residual DAP-kinase in tumors by interfering with DAP-kinase auto-inhibitory mechanism (against the self inhibitory activity of the C-terminal tail or by blocking the binding of Ser308 of the CaM regulatory domain). However to date, such concepts have been purely hypothetical (Bialik & Kimchi, 2004).
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