Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

DAPK1

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_485

Synonyms

Historical Background

Death-associated protein kinase (DAPK) is the inaugural member of a class of Ser/Thr protein kinases whose members exhibit homologous catalytic domains as well as share cell death-associated functions (Bialik and Kimchi 2006). DAPK was discovered by Kimchi and coworkers as a tumor suppressor gene whose expression is lost in multiple tumor types (Cohen and Kimchi 2001). This attracted the interest of several investigators resulting in an impressive body of literature concerning its function, regulation, and involvement in various diseases and conditions. For example, DAPK participates in apoptotic pathways initiated by interferon-γ, TNFα, activated Fas, and loss of attachment to the extracellular matrix (Bialik and Kimchi 2006). By activating p53 in a p19ARF-dependent fashion DAPK is an intrinsic tumor suppressor that opposes early stage oncogenic transformation (Raveh et al. 2001). However, hypermethylation of the DAPK promoter inactivates this pathway in tumorgenesis such as found in multiple myeloma (Chim et al. 2007) and other cancers (Michie et al. 2010).

Domain Structure of DAPK and Regulation of Enzymatic Activity

DAPK has several functional domains (Fig. 1). These include a series of ankyrin-repeat domains as well as a “death” domain. Between these domains exists a tandem “ROC” and “COR” domain (Carlessi et al. 2011). These domains always occur in tandem. The ROC (Ras of complex proteins) is a GTPase domain resembling the small G-proteins such as Ras, preceding the COR (C-terminal of ROC) domain. The family of ROC-COR proteins has three members that are protein kinases. These include the Parkinson disease-associated kinase LRRK2 (Leucine-rich repeat kinase 2) (Drolet et al. 2011), the closely related LRRK1, and DAPK. Completing the domain structure of DAPK are the catalytic and calmodulin-regulatory domains that are similar to other calmodulin-dependent protein kinases. DAPK activation requires calmodulin (CaM), but it is further modulated by phosphorylation at sites within and outside of the CaM regulatory domain (Michie et al. 2010). Phosphorylation at two sites decrease DAPK activity; Ser-308 is within the CaM regulatory domain, and Tyr-491/492 are within one of the ankyrin repeat domains. The presence of the GTP-binding ROC domain further regulates DAPK activation. In the presence of GTP (which binds at the p-Loop (Fig. 1) DAPK activity is lower as demonstrated with a mutant that lacks the p-Loop and GTP binding (Carlessi et al. 2011). However, upon GTP hydrolysis, the dephosphorylation of Ser-308 by phosphatase PP2a is facilitated (Carlessi et al. 2011), allowing calmodulin activation. Phosphorylation of Ser-735 by activation of the extracellular-regulated kinases ERK1 and 2 results in an increase in DAPK activity (Michie et al. 2010). It is thought that this tight control over DAPK activation is one of the checkpoints for committing a cell to apoptotic/necrotic death.
DAPK1, Fig. 1

Domain structure of DAPK. Shown are the locations of the kinase catalytic (yellow), calmodulin regulatory (light blue), ankyrin repeats (green), ROC COR domain (blue), and death domain (red). The calmodulin regulatory domain is expanded to show the amino acid sequence and the location of the Ser-308 phosphosphorylation site

Regulation of DAPK Expression

The upstream genetic sequences that contain the promoter(s) for the human DAPK gene are diagrammed in Fig. 2a. There are two transcriptional start sites that are controlled by independent promoters. Two alternative translational start sites at -1025 and -622 are followed by termination codons within exon 1 and 1b, respectively, rendering them nonfunctional. These sites precede the active start site in exon 2. There is no TATA box in either promoter, but there are several transcription factor binding sites including AP2, E-box, CAAT box, AP1, and nuclear factor kappa B (NF-kB) (Pulling et al. 2009). A CpG island of 590 bp containing 46 CpG dinucleotides is directly upstream of the translational start site. An additional 100 CpGs are located 1000 bp upstream of this region. Exon 1b contains 17 CpG dinucleotides that may also be methylated in cancer cells (Fig. 2b). Both promoters are active in multiple cell types. Promoter 1 (Exon 1) activity was 40–50% higher than promoter 2- Exon 1b-intron promoter using reporter constructs (Fig. 2c) (Pulling et al. 2009). Methylation analysis in 5 of 15 tumor cell lines revealed that 51–91% of the CpGs in the promoter 1-exon 1 region were methylated and associated with a complete loss of transcription from exon 1. Similarly, there was a good correlation with methylation of promoter 2-exon 1b-intron constructs and loss of expression in 9 of 15 tumor cell lines. The methylation status and location within the DAPK promoters varies greatly in cultured cells but methylation at multiple sites correlates with a decrease or loss of expression. Because of the dual promoters, DAPK expression in one cell type may be more susceptible to methylation than another cell type where the alternative promoter is used. This perhaps explains variability in DAPK promoter hypermethylation in tissue biopsy samples where a mixture of cell types (metastatic versus nonmetastatic) are analyzed. However, the association of DAPK methylation with tumor aggressiveness and disease progression (poor prognosis) is significant (Pulling et al. 2009; Chim et al. 2007). MicroRNAs have also been found to regulate DAPK expression. One, miR-103/107, promotes metastasis in colorectal cancer (Chen et al. 2012). Another, miR-191, inhibits DAPK expression in ovarian endometriosis (Tian et al. 2015). In both cases the miRNA expression is upregulated in the tumor cells resulting in a decrease in DAPK and other tumor suppressors.
DAPK1, Fig. 2

DAPK genomic structure, exon 1b sequence, and expression of exon 1 and 1b transcripts. (a) Schematic diagram depicting the location of promoter 1, exon 1 and 1b, promoter 2, and intron 1 in relation to the translational start site within exon 2 is shown. Shaded areas and numbers depict the boundaries and location of each region. The location of the ATGs in exon 1 and 1b that are not translational start sites due to stop codons in these exons are shown as are the location of transcription factor-binding sites that affect reporter activity. (b) Sequence of the 186 bp region designated exon 1b with the 17 CpG dinucleotides bolded is shown. (c) Quantitative expression of DAPK exon 1 and 1b transcripts in H1568, Calu-6, BEC, and keratinocyte (KT) cell lines. Fold expression between exon 1 and 1b transcripts is compared (Reproduced from Pulling et al. (2009) with permission. Transcription factor descriptions were added)

Signal Transduction Pathways Involving DAPK and Its Protein Substrates

Because of its multiple protein interacting domains, DAPK is involved in several cellular processes. Two primary areas of investigative focus are apoptosis and autophagy (Bialik and Kimchi 2006) that will be discussed separately. A summary of well-characterized DAPK substrates is given in Table 1. DAPK phosphorylates regulatory myosin light chains and is involved in membrane blebbing that occurs during programmed cell death (apoptosis) (Bialik and Kimchi 2006). Calmodulin-regulated kinase kinase (CaMKK) is neuronal protein substrate of DAPK. Its phosphorylation site (Ser-511) is near the CaM recognition domain and results in an attenuation of CaM-stimulated activity (Schumacher et al. 2004). Another neuronal substrate is Syntaxin 1A where phosphorylation of Ser-188 is proposed to decrease binding of syntaxin-1A to Munc18-1, a syntaxin-binding protein that regulates a complex (known as the SNARE complex) that is necessary for synaptic vesicle docking and secretion (Tian et al. 2003). The NMDA (N-methyl-d-aspartic acid) receptor NR2B subunit is also phosphorylated by DAPK (Tu et al. 2010). The site is in the regulatory C-terminal end and enhances the Ca2+ conductance of the channel. Thus, if DAPK is activated in presynaptic neurons, two processes may be affected that relate to cellular homeostasis. DAPK phosphorylation of CaMKK is proapoptotic, because this kinase is responsible for activating survival pathways through phosphorylation of the kinase known as AMPK (Kuo et al. 2005), while phosphorylation of syntaxin-1A can lead to inhibition of neurotransmitter secretion (Bialik and Kimchi 2006). DAPK phosphorylation of Zip kinase (DAPK3) activates this kinase leading to changes in cell morphology characteristic of apoptotic cells (Shani et al. 2004). DAPK was found to phosphorylate MCM3, a protein that may be involved in the regulation of DNA replication (Bialik et al. 2008). Pin1 is a proline isomerase that specifically targets phosphor-Ser/Thr-Pro sites in proteins, and phosphorylation of Pin1 at Ser-71 by DAPK fully inactivates Pin1 activity (Lee et al. 2011). The number of proteins/processes that depend upon Pin1 activity includes many tumorogenic and cell cycle-related proteins. Figure 3 summarizes just a few of the proteins within the Pin1 network of interactions. The significance of PIN1 inactivation is that DAPK’s cancer suppressing activity is extended to these processes. Finally, DAPK is one of the several protein kinases that phosphorylates the transcription factor HSF-1 (heat shock factor 1). After stimulation by TNFα, phosphorylation of Ser-230 by DAPK promotes HSF-1 translocation to the nucleus. In turn HSF-1 stimulates expression of DAPK in a positive feedback loop and results in the apoptosis of cancer cells (Benderska et al. 2014).
DAPK1, Table 1

Functional substrates of DAPK

Substrate

Sequence

In vivo?

Function

Reference

MLC

RPQRAT-S-NVF or

RPQRA-T-SNVF

Yes

Activates myosin/ membrane blebbing

(Steinmann et al. 2015)

Zip Kinase

RRRLK-T-RL

YTIK-S-H-S-S-L

PNN-S-YADFERF-S-K

?

Localization and dimerization

(Steinmann et al. 2015)

Syntaxin 1A

GHMDSSI-S-KQA

Yes

Synaptic vesicle membrane fusion

(Steinmann et al. 2015)

CaMKK

RREERSL-S-APG

Yes

Inhibits activation

(Steinmann et al. 2015)

Beclin 1A

SRRLKV-T-GDLF

Yes

Dissociates Beclin from Bcl-xL proteins

(Steinmann et al. 2015)

MCM3

TIERRY-S-DLT

Yes

Unknown

(Bialik et al. 2008)

PIN1

RRP-S-SWRQ

Yes

Inhibits activity

(Lee et al. 2011)

NR2B (NMDA receptor

KLRRQH-S-YDTF

Yes

Damaging Ca2+ influx in stroke

(Tu et al. 2010)

S6

AKRRRL-S-SLRAS

Yes

Suppression of translation

(Schumacher et al. 2006)

P53

LSQE-T-F-S-DLWK

Yes

Stimulates transcriptional activity

(Steinmann et al. 2015)

HSF-1

YSRQF-S-LE

Yes

Stimulates transcriptional activity

(Benderska et al. 2014)

DAPK1, Fig. 3

Pin1 interaction network. Illustrated is a portion of the Pin1 interactions created using String V10 and its associated database (string-db.org)

DAPK and Autophagy

Figure 4a summarizes the initial phase of autophagy and the role that DAPK and its substrate Beclin 1 play in this process. Autophagy is a highly conserved process that is characterized by the formation of membrane enclosed “autophagosomes” that function to engulf intracellular organelles and other constituents and deliver them to the lysosomes for degradation. Thus, autophagy is the cell’s intrinsic “recycling” machinery that serves to provide material for cell metabolism during periods when extracellular sources of nutrients are low. Beclin 1 is an essential autophagic protein that binds BcL-2 family proteins through its BH3 domain. The phosphorylation site for DAPK (Thr-119) is located within the BH3 domain, and phosphorylation promotes dissociation of Beclin 1 from Bcl-2/XL (Fig. 4b). Dissociated Beclin 1 promotes activation of autophagic machinery by interacting with a complex centered upon phosphatidylinositol-3-kinase (PI-3 Kinase) (Fig. 4a). This multiprotein complex participates in autophagosome nucleation (Funderburk et al. 2010).
DAPK1, Fig. 4

(a) Diagram of the early stages of autophagy that lead to membrane nucleation. From pathways created by +Cell Signaling Technology reproduced with permission. (b) A model of phosphorylation events which regulate the interaction between Beclin 1 and BCL-2/XL leading to the induction of autophagy. DAPK-mediated phosphorylation on Beclin 1’s T119 residue and JNK1-mediated phosphorylation on residues T69, S70, and S87 on Bcl-2, each reduces Beclin 1’s interaction with its inhibitors leading to autophagy (Reproduced from Hu et al. (2010) by permission from the Authors)

DAPK Protein Interactions and Apoptosis

DAPK is linked through direct binding to a number of proteins that participate in apoptosis by one or more pathways (Bialik and Kimchi 2006; Bialik and Kimchi 2014). Not all of the pathways are functional in a given cell type. The first pathway is through the binding of Fas to the death-initiator, FADD (Fig. 5). DAPK interacts with FADD by way of its death domain (Fig. 1). This interaction results in the downstream activation of caspases (CASP3) that lead to cell death (Fig. 5). The involvement of DAPK was established in multiple ways: (1) Expression of a fragment of DAPK containing the death domain protects cells from Fas-mediated cell death, (2) Expression of a DAPK mutant lacking the death domain does not promote cell death, (3) DAPK mutants that lack CaM regulation (deletion of CaM segment) resulted in massive apoptosis (Bialik and Kimchi 2006; Bialik and Kimchi 2014). Thus, both the death domain and DAPK catalytic activity are necessary for Fas-mediated cell death. A second pathway mediated by DAPK is also initiated from other signals such as UNC5H2 (Fig. 5). UNC52B (netrin 1 receptor) when bound to ligand (NTN1) blocks DAPK activation (Bialik and Kimchi 2014). However, when netrin is absent, UNC5H2 reduces the phosphorylation of DAPK at Ser-308 which induces its activation by Ca2+ CaM (Bialik and Kimchi 2006; Bialik and Kimchi 2014). Thus, netrin1/UNC5H2 functions as a DAPK switch. (Bialik and Kimchi 2014). In some cell types, however, depletion of DAPK by mRNA interference can promote apoptosis (Jin and Gallagher 2003). This may be mediated by a DAPK isoform that has a truncation near the C-terminus of the protein that inhibits the activity of the death domain (Bialik and Kimchi 2006). It is not clear what mediates the expression of this isoform called DAPKβ. Two examples of upregulated DAPK expression that might involve this isoform are p53 mutant cancers (Zhao et al. 2015) and human peritumoral tissues that results in phosphorylation of NR2B (Table 1) and increased exitability that is thought to lead to seizures (Gao et al. 2015).
DAPK1, Fig. 5

DAPK network and apoptosis. Illustrated is a portion of the DAPK interaction network focused on apoptosis. DAPK engages several proteins in a complex that receives inputs from potential death signals (FADD) as well as survival signals (UNC5H2 –Netrin receptor). This network was created using String v10 and its associated database (string-db.org)

Summary

DAPK is a fascinating kinase with respect to its involvement in multiple cellular processes that have not been fully studied. For example, although some substrates have been identified the timing and duration of DAPK activation may be critically important with respect to cellular commitment to apoptosis.

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Department of PharmacologyNorthwestern UniversityChicagoUSA