Apoptosis (2014) 19:306–315 DOI 10.1007/s10495-013-0936-1
THE UNIVERSE OF DAPK
Post-translational regulation of the cellular levels of DAPK Patricia J. Gallagher • Emily K. Blue
Published online: 2 November 2013 Ó Springer Science+Business Media New York 2013
Abstract Death associated protein kinase (DAPK) is a large, multi-domain ser/thr kinase whose activities converge upon multiple signaling pathways that regulate autophagy, caspase-dependent cell death, cell adhesion and migration. The cellular levels of DAPK are post-translationally regulated by the combined activities of two degradation systems, including the ubiquitin proteasome and an extra-lysosomal proteolysis pathway. At least three distinct E3 ubiquitin ligases target DAPK, including mindbomb1, the chaperone dependent ligase, CHIP (carboxy terminus of Hsp70-interacting protein) and a cullin RING ligase complex, KLHL20-Cul3-RBX1. In addition, it appears that the cellular levels of DAPK are also regulated by an extra-lysosomal protease, cathepsin B. While protein quality control and recycling clearly benefit cells by removal of misfolded or toxic proteins and recycling of their components, the finding that multiple surveillance systems target DAPK suggests that these protein degradation systems also act to fine tune DAPK expression levels in response to specific signaling pathways. Keywords Death associated protein kinase Stability Apoptosis Autophagy Post-translational
P. J. Gallagher (&) Department of Cellular & Integrative Physiology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA e-mail: [email protected] E. K. Blue Department of Pediatrics, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA
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Introduction Death associated protein kinase (DAPK) is a 160 kDa, calcium/calmodulin (Ca2?/CaM)-regulated, multi-domain ser/thr protein kinase that converges upon multiple signaling pathways involved in modulating apoptotic and autophagic cell death as well as cell survival and cancer metastasis [1, 2]. DAPK is the prototypical member of a family of protein kinases that includes 4 additional members: DAPK related protein kinase (DRP-1 or DAPK2), DAPK like kinase/Zipper interacting kinase (DLK, ZIPK), DAPK related apoptosis inducing protein kinases 1 and 2 (DRAK-1/DRAK-2). The kinases in this family share a high degree of homology within their kinase domains, which are all located near the N-terminal region of these proteins, but beyond the kinase domain the protein sequences are dissimilar which likely contributes to the diverse functions of this kinase family [1, 2]. DAPK is comprised of an N-terminal kinase domain, a CaM binding regulatory domain, a series of 8 ankyrin repeats, a cytoskeletal association domain, a Ras of complex proteins (ROC) domain, two nucleotide binding sites (P-loops) involved in GTP binding, and a C-terminal death domain. Located within the CaM binding regulatory domain is an autophosphorylation site (S308) and this must be dephosphorylated by protein phosphatase 2A (PP2A) to allow binding of Ca2?/CaM and activation of the kinase [3– 5]. DAPK has been linked to numerous signaling pathways controlling cell proliferation, apoptosis, autophagy, adhesion, anoikis and its involvement in these signaling pathways provides strong links to pro- and anti-apoptotic functions [4, 6–11]. A number of regulatory mechanisms that govern the activities of DAPK have been identified. These include protein phosphorylation by other kinases as well as autophosphorylation of S308, ubiquitination, and
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Fig. 1 Ubiquitin proteasomal pathway. Shown are the generic steps in generation of a ubiquitinated target protein and subsequent degradation by the 26 s proteasome
protease mediated degradation. These mechanisms ensure that DAPK catalytic activity and stability are carefully regulated in order to appropriately control physiological responses of cells. This review will focus on post-translational mechanisms that control the stability and cellular levels of DAPK in cells. Regulating the levels of cellular proteins is primarily managed through two major intracellular proteolysis systems. These two systems are referred to as the ubiquitin proteasome (UPS) and autophagocytic/lysosomal systems. More recently, an ‘‘extra-lysosomal proteolysis’’ pathway has been described and is characterized by leakage of lysosomal proteases into the cytoplasm to degrade proteins under conditions of stress. Collectively, these three proteolytic degradation systems serve as surveillance mechanisms to identify and remove damaged, misfolded or aggregated proteins, and also modulate the activity of signaling pathways. These quality control pathways are not merely housekeeping mechanisms, but rid cells of toxic proteins and importantly, provide a mechanism for recycling of amino acids within cells. Quality control pathways: be good or be gone In contrast to ‘‘mass’’ degradation of cellular components that occurs via the lysosome, the UPS (Fig. 1) is a more precise pathway to mediate protein degradation, which is directed by the covalent attachment of ubiquitin to individual target proteins. The UPS functions to rapidly remove misfolded, oxidized or otherwise damaged proteins [12–14]. The specificity in the UPS is controlled
through the covalent attachment of a 76-residue ubiquitin chain to the targeted protein, and this tag directs the ubiquitinated protein to the 26S proteasome [14–16]. Ubiquitination of proteins occurs as a result of a trio of enzymatic steps, mediated by E1, E2, and E3 enzymes (Fig. 1). The first step involves the formation of a highenergy thiol-ester bond between the E1 (ubiquitin-activating enzyme) and ubiquitin. Following this, the ubiquitin is transferred to the active site of the E2 enzyme (ubiquitin conjugating enzyme) and then an E3 (ubiquitinligase) mediates the transfer of the activated ubiquitin directly to a lysine reside in the target protein. Once the target protein has been covalently ‘‘tagged’’ by a ubiquitin molecule, the process may repeated several times to generate a polyubiquitin molecule composed of ubiquitin chains usually added to lysine-48 of a previously attached ubiquitin. In addition to K48-linkages, there are several additional types of linkages and configurations that are possible [16, 17]. Polyubiquitin or multi-ubiquitin ‘‘tags’’ direct the protein to the 26S proteasome for degradation, while mono-ubiquitination is generally deemed a signal for endocytosis of membrane proteins such as Notch ligands [16]. The second system responsible for the regulation of proteolysis, autophagy, refers to a catabolic mechanism to degrade non-functional, misfolded, or excess cellular materials [16, 18–23]. Autophagy is a major mechanism for recycling proteins and other macromolecules to provide basic materials and energy for the cell in times of nutrient depletion or stress. There are three types of autophagy: macroautophagy, microautophagy, and chaperone-mediated
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Fig. 2 Autophagy degradation of molecules. Shown is the engulfment of organelles, nucleic acids and aggregated proteins during the formation of the double-membraned autophagosome. Following formation of the autophagosome, fusion with the lysosomal membrane results in destruction of these molecules
autophagy [24–26]. The macroautophagocytic lysosomal pathway is the pathway most relevant to DAPK and refers to a process in which intracellular cytoplasmic constituents are engulfed into a newly formed double membrane vesicle called an autophagosome (Fig. 2) [27]. The autophagosome traffics via microtubules to the lysosome where it fuses with the lysosomal membrane and delivers its contents to the lysosome for degradation. Microautophagy involves direct engulfment of cytoplasmic molecules into the lysosome for degradation and occurs by invagination of the lysosomal membrane, which surrounds the macromolecule(s) [28]. Chaperone-mediated autophagy is a more complex and highly selective pathway, which requires the recognition of the target for degradation by a chaperone, usually HSP70, which localized the target to the lysosomal membrane using a specific receptor called lysosome associated membrane protein 2a (LAMP2a). Following its recognition at the lysosomal membrane, the target protein is unfolded and transferred across the lysosome membrane with the assistance of another chaperone, lysosomal hsc73. Upon translocation to the lysosomal matrix, the target protein is degraded [29–32]. Lysosomes are organelles containing numerous acid hydrolases such as nucleases, phosphatases, proteases, lipases and glycosidases that serve to degrade complex molecules including nucleic acids, proteins, lipids and carbohydrates into their simple components, which are transferred to the cytoplasm for reuse. Of the numerous proteases present in the lysosome, the cathepsin proteases are the most highly characterized and catalyze degradation of proteins into amino acids.
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Fig. 3 Extra-lysosomal proteolysis. Shown on the left are some of the stimuli that result in lysosomal membrane permeability. The leakage of lysosomal hydrolases into the cytoplasm results in destruction of targets (right). This pathway appears to be committed to promoting cell death
The importance of a third proteolytic, non-ubiquitin degradation pathway (Fig. 3) that primarily functions as a positive regulator of apoptosis has recently become appreciated [33]. This pathway is characterized by the release of hydrolytic enzymes from the lysosome in response to cellular stress and will be referred to as the ‘‘extra-lysosomal pathway’’ [33]. Release of lysosomal enzymes can be provoked by high levels of sphingosine or ceramide resulting from TNF death receptor stimulation, treatment with quinolone antibiotics (e.g. naladixic acid, Cipro, Noroxin), ‘‘lysosomotropic’’ detergents [e.g. O-methyl-serine dodecylamide hydrochloride (MSDH) or siramesine], the production of high levels of free fatty acids and bile salts, some viral proteins, or oxidative stress where reactive oxygen species (ROS) are generated [34–36]. Lysosomal membrane permeability (LMP) is a critical step in this pathway which leads to release of acid hydrolases to the cytoplasm of the cell [33]. As the hydrolytic activities of these enzymes are optimized to function at the acidic pH (pH \ 5)of the lysosome, many are not thought to be active in the more neutral cytoplasmic pH [37]. However, several cathepsins (cathepsin-B, -L, -D) remain catalytically active and can degrade cytoplasmic proteins [37, 38]. Consistent with a function to promote apoptosis [39], some of the known proteolytic targets of the released cathepsins are several pro-apoptotic proteins including Bid which is cleaved to tBid, and DAPK as well as some anti-apoptotic proteins like Bcl2, BclxL, and MCL-1 [34, 39–45]. As LMP proteolytic degradation is believed only to be active under stress conditions, it is likely the major role of this pathway is to promote imminent cell death rather than as a mechanism for recycling cellular constituents.
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Supervising DAPK: balancing activity and cellular levels The first report confirming that the cellular levels of DAPK are regulated by the UPS system came with the discovery of an E3 ubiquitin ligase called DIP-1 (DAPK Interacting Protein1) that interacted with DAPK [46]. DIP-1 was identified using a yeast-2-hybrid screen and the interaction with DAPK was confirmed by co-immunoprecipitation of the endogenous proteins. In addition, biochemical and cellular analysis confirmed that DAPK was a target for poly-ubiquitination and degradation mediated by DIP-1, which sensitized cells to TNF-induced apoptosis [46]. Using deletion and co-immunoprecipitation analysis, DIP1 was found to bind to the ankyrin repeats domain of DAPK [46]. A subsequent study using positional cloning to identify the mutant gene resulting in the mind bomb (mib) zebrafish with severe neural defects revealed that the mutant gene was DIP-1. Based on the large body of information referring to phenotype of mind bomb mutant (mib), DIP-1 is now referred to as Mib1 [47, 48]. Mib1 is a 110-kDa protein with a complex structure that includes a zinc finger, 9 ankyrin repeats, 3 C-terminal RING fingers and a alpha-helical coiled-coil region that separates the 2nd and 3rd RING fingers. The ubiquitination of DAPK by Mib1 can also be modulated by interaction with c-mip (c-maf inducing protein), which binds to Mib1 and inhibits ubiquitination of DAPK to result in upregulation of DAPK cellular levels [49]. The increased levels of DAPK found in response to c-mip expression appear to promote sequestration of ERK in the cytoplasm [49]. Relevant to this, a previous study demonstrated that DAPK could sequester ERK in the cytoplasm, blocking nuclear ERK signaling and promoting anoikis [50]. In this setting, the increased levels of active, cytoplasmic ERK resulted in enhanced ERK phosphorylation of DAPK S735, increased DAPK catalytic activity and advancing cell death via anoikis [50]. Together these findings indicate that the cellular levels of DAPK can be post-translationally downregulated by Mib1 mediated ubiquitination and proteasomal degradation and that signaling pathways can upregulate DAPK expression by preventing binding of Mib1.In addition to ubiquitination of DAPK, Mib1 has a central role in Notch signaling [47, 51–53]. Mib1 monoubiquitination of the Notch ligands Delta (Delta-like, DLL in vertebrates) or Serrate (Jagged in vertebrates) is necessary for endocytosis of these ligands and activation of Notch signaling [47, 51–53]. CHIP (C-terminus of Hsc70-interacting protein) a U-box E3 ubiquitin ligase also ubiquitinates DAPK, but does so in collaboration with the molecular chaperone, heat shock protein 90 (HSP90) (Fig. 4) [54]. CHIP is a 35 kDa protein characterized by the presence of an N-terminal TPR
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(tetratricopeptide) motif that acts as a scaffolding structure for interaction with HSP90 and a U-box ubiquitin ligase domain near its C-terminus [55]. Inhibition of chaperone proteins like HSP90 with agents such as 17-alkyl-amino-17demethoxygeldanamycin (GA) is proposed to block its stabilizing interaction and result in increased proteasomal degradation of client proteins as chaperones often partner with E3 ubiquitin ligases such as CHIP [56]. Treatment of cells with the HSP90 inhibitor 17-alkyl-amino-17-demethoxygeldanamycin (GA) reduced expression of DAPK. This finding suggested that HSP90 had an important role in stabilizing DAPK. A previous study had identified a site within the kinase domain that interacted with HSP90 [57]. This interaction occurs via CHIP’s TPR domain and a TPR acceptor site in HSP90 [58]. A number of studies have confirmed that this interaction results in the degradation of a number of HSP90 client proteins [55, 56, 59–66]. Interestingly, it was found that inhibition of HSP90 with GA resulted in a preferential degradation of the activated form of DAPK in which the inhibitory autophosphorylation site at S308 has been dephosphorylated by PP2A (Fig. 4) [3, 54]. This result directly implied there is a selective mechanism for reducing cellular DAPK activity by targeting degradation of activated DAPK [54]. Analysis of immune complexes identified two distinct heterocomplexes in cells. One complex contains DAPK with HSP90 and CHIP while a second complex contained only DAPK and Mib1. These results suggested that when DAPK is bound by HSP90, it might be preferentially ubiquitinated by CHIP and in the absence of HSP90 binding DAPK is ubiquitinated by Mib1 [54]. Another binding partner of DAPK that has a role in regulating its protein level is KLHL20, identified in a yeast 2-hybrid screen using the death domain of [67]. KLHL proteins are Kelch-like proteins that contain a BTB (Broad complex, Tamtrack, Bric-a-brac) motif. Cullin-RING-ligases (CRLs) utilize BTB domain proteins such as KLHL20 as substrate adapters to interact with target proteins for ubiquitination [68–75] Consistent with this, in previous studies immune complexes containing DAPK and KLHL20 were also found to contain Cul3 and the Cul3 E3 ubiquitin ligase subunit, RBX1 (Fig. 5) [67]. Cell studies depleting KLHL20 with siRNAs resulted in attenuated ubiquitination of DAPK and in vitro ubiquitination assays confirmed that DAPK is ubiquitinated by the KLHL20-Cul3-RBX1 complex. Interestingly, treatment of cells interferon-alpha or gamma substantially reduced ubiquitination of DAPK and this was associated with KLHL20 being sequestered into peri-nuclear and nuclear locations in PML (promyelocytic leukemia) nuclear bodies [67]. Sequestration of KLHL20 away from DAPK could thus account for the decreased ubiquitination of DAPK and its resultant increased stability provides a satisfying mechanism to explain how or interferon-alpha or –gamma can induce apoptosis in cells.
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Fig. 4 Ubiquitination of DAPK by Mib1 and CHIP. Cellular stimulation by ceramide or tumor necrosis factor (TNF) leads to a rise in the cytoplasmic levels of calcium and results in CaM (calmodulin) binding to the CaM-binding domain in DAPK. Binding of Ca2?/CaM to DAPK requires dephosphorylation of autophosphorylated Ser308 within the CaM-binding domain. Following activation, which may require association with the chaperone HSP90, DAPK phosphorylates substrate proteins. Alternatively, misfolded or altered DAPK can be targeted by the E3 ligases, CHIP or Mib1 for ubiquitination and degradation by the 26 s proteasome. Ubiquitination by Mib1 can be attenuated by Mip1, which sequesters Mib1
Fig. 5 Cullin-RINGubiquitination of DAPK. Shown are the steps in the KLHL20Cullin3-RBX1 ubiquitination of DAPK and subsequent degradation by the 26 s proteasome
Another pathway that has been shown to modulate DAPK degradation is associated with the tuberous sclerosis complex (TSC). The TSC is comprised of two proteins, TSC1 (hamartin) and TSC2 (tuberin), which contain a GTPase activating (GAP) protein domain. When bound together the TSC1/TSC2 complex inhibits the activation of mammalian target of rapamycin complex 1 (mTOR) by
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TSC2. When activated, mTOR acts a sensor of cellular nutrients and energy and modulates protein and lipid synthesis as well as autophagy in response to environmental changes. The principal targets of mTOR are ribosomal protein p70-S6 kinase1 (S6K1) and the eukaryotic initiation factor 4E binding protein-1 (4E-BP1). Both of these targets are central to the formation of the translational
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initiation complex and are key regulators of protein synthesis [76]. Stevens et al. [77] and Lin et al. [78] have shown that the death domain (DD) of DAPK interacts with tuberous sclerosis protein 2 (TSC2) and that this interaction can have one of two outcomes. Either DAPK directly phosphorylates TSC2 leading to dissociation of the TSC1/ TSC2 complex and activation of mTOR to promote protein synthesis or mTOR promotes degradation of DAPK in a lysosome-dependent process. In the Stevens study, a peptide aptamer library was used to screen for peptides binding to the DD of DAPK and TSC2 was identified [77]. The DAPK/TSC2 complex was confirmed by co-immunoprecipitation as was the binding site within the DD of DAPK. Biochemical analysis further demonstrated that TSC2 was phosphorylated at an unidentified site by DAPK. The outcome of TSC2 phosphorylation was a dissociation of TSC1/TSC2 complex and downstream activation of mTOR signaling leading to phosphorylation of S6K1. These studies suggest that DAPK is a positive regulator of mTOR activation and signaling [77]. In the report by Lin et al. [78], the observation that the cellular levels of DAPK are inversely correlated with those of TSC2 was explored. This investigation led to the discovery that TSC2 can regulate the levels of DAPK and does so via a post-translational mechanism. Surprisingly, treatment of cells with the mTOR inhibitor, rapamycin had no effect on the cellular levels of DAPK further suggesting that TSC2 regulation of DAPK was also not an effect of mTOR’s role in modulating protein synthesis. Consistent with this, when a TSC2 mutant lacking GAP activity was expressed in cells, the levels of DAPK were still reduced. To identify the mechanism that resulted in degradation of DAPK, several lysosomal protease inhibitors including leupeptin, E64D, and chloroquine were found to result in increased levels of DAPK while the proteasome inhibitor, MG132 did not [78]. Treatment of cells with the autophagy inhibitor, 3-methyladenine (3-MA) also had no effect on DAPK levels. Collectively these results suggest that regulations of the cellular levels of DAPK are also mediated by lysosomal proteases but independent of autophagy [78]. These results suggested that there is an additional, non-ubiquitin, nonautophagic pathway that has a role in regulation of the cellular levels of DAPK which is consistent with previous study where DAPK was identified as a target of the lysosomal protease, cathepsin B [79], as described below. Lin et al. [79] observed a consistent set of proteolytic fragments of DAPK that was detected by immunoblotting lysates from cells treated with the proteasome inhibitor MG132. As these fragments were observed in the presence of a proteasomal inhibitor, it was predicted that the fragments resulted from a non-proteasomal degradation pathway [79]. Further experiments demonstrated that DAPK has a relatively long half-life (*6 h) compared to other
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signaling proteins like p53 (*20 min) or interferon regulatory factor (IRF-1; *40 min). Further, the addition of both a proteasome inhibitor (MG132) and a cathepsin B inhibitor (benzyloxycarbonyl-FA-fluoromethyl ketone) extended the lifetime of DAPK in cultured cells. These results suggested the presence of another degradation pathway in addition to the proteasomal pathway that likely involved proteolysis by the lysosomal protease cathepsin B. However, the finding that overexpression of cathepsin B had no effect on the endogenous levels of DAPK, but attenuated the expression of exogenous DAPK was surprising. This result was explained by proposing that steady state, endogenous levels of DAPK may exist in multiprotein complexes that shield the kinase from cathepsin B proteolysis and when overexpressed, some DAPK is not sequestered in these protective complexes. In support of this it was found that exogenous expression of TNFR-1, which can induce lysosomal permeabilization [80], resulted in the formation of a complex between DAPK and cathepsin B and relocalization from the insoluble cytoskeletal fraction to the soluble cytosolic fraction [79]. The consequence of this complex formation was an increase in cytosolic cathepsin B and decrease in DAPK expression. Using deletion mutants of DAPK, the binding site for cathepsin B was identified in a region residing at the C-terminus between the cytoskeletal binding and death domains, and expression of this ‘‘miniprotein’’ blocked formation of the complex. Although cathepsin B is predominantly found in the lysosome, there is data supporting the occurrence of lysosomal membrane ‘‘leakage’’ allowing these lysosomal hydrolases access to the cytoplasm particularly when cells are undergoing apoptosis or in stress conditions [44, 81–83]. The are also at least some data suggesting that acidic proteases such as cathepsin B can function in the more neutral pH environment of the cytosol [84, 85]. Together these data suggest that DAPK can be degraded both by lysosomal hydrolases within the lysosome and under some conditions by these hydrolases after they are released into the cytoplasm of a stressed or apoptotic cell.
Conclusions and perspectives There are several systems monitoring the structure and functionality of proteins in cells. These systems include the ubiquitin-proteasome, autophagic/lysosomal and extralysosomal protein degradation. In recent years, these protein degradation systems have become appreciated to have not only a housekeeping role to rid the cell of toxic, misfolded or aggregated proteins, but to also play a key role in control signaling pathways by fine-tuning the cellular levels of critical signaling molecules. The ‘‘logic’’ behind
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Fig. 6 Summary of the posttranslational mechanisms that regulate the cellular levels of DAPK. Shown are the three ubiquitin ligases (Mib1, CHIP, KLHL20-Cul3-RBX1) that target DAPK for ubiquitination and destruction by the 26 s proteasome. Also shown is the extra-lysosomal proteolysis pathway in which DAPK is proteolyzed by cathepsin B leakage from the lysosome
these processes is not yet completely appreciated, but this is a good example of the cellular ‘economy’ of utilizing the same mechanisms to rid the cell of noxious proteins and to limit the activity of signaling pathways. Key to understanding how signaling pathways are both controlled by, and control, the protein degradation machinery will be an understanding the signals that link the two processes. Already there are indications that the conformational change that occurs in DAPK upon activation may itself be a signal that is recognized by one of these chaperone and/or degradation systems. Other obvious examples can be seen with the Cullin-RING-ligase system whereby some ubiquitin targets require post-translational modifications such as phosphorylation to be recognized by the Cullin-RINGligases [86–88]. Currently, we understand some of the degradation and quality control pathways that assess the suitability of DAPK and regulate its posttranslational stability (Fig. 6). Finding that several distinct monitoring pathways regulate DAPK levels indicate that the cellular levels of DAPK like p53 must be carefully regulated. The work summarized in this review indicates that several E3 RING ubiquitin ligases (Mib1, CHIP, KLHL20-Cul3-RBX1) as well as the extralysosomal protease cathepsin B all monitor the cellular levels of DAPK. In addition, the surveillance of DAPK also involves at least one chaperone (HSP90), which likely functions to assist DAPK in attaining and sustaining a mature, functional 3D configuration. The fact that several degradative pathways focus on DAPK suggest there must be
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additional complexities involved in determining which pathway is active where and when. This likely involves regulation of cellular localization or involvement in higher order complex structures or use of scaffolding molecules to perhaps direct the activity of specific degradation complexes to different intracellular pools of DAPK. Do these multiple surveillance systems indicate the need to limit certain pools of DAPK activity in order to perhaps promote cell migration or proliferation without inducing autophagic or caspase-dependent cell death? Exactly how do these multiple surveillance systems serve to regulate the activities of DAPK in response to different environmental stimuli? Are these degradation systems merely a way to terminate DAPK signaling? At least one study has supported this possibility by demonstrating that activated but not inactive DAPK is targeted by CHIP for ubiquitination and recycling [54]. This could serve to limit DAPK activity even when cellular levels of calcium remain elevated. Clearly there are still many issues to be resolved regarding the role of, and the mechanisms underlying, the post-translational regulation of the cellular levels of DAPK. Acknowledgments This work was supported by Grants from the National Institutes of Health HL54118 and DK062810 to P. J. G., the Indiana University Diabetes and Obesity Research Training Grant DK064466 and American Heart Association Pre-doctoral fellowship grant 0815608G to E. K. B. Conflict of Interest of interest.
The authors declare that they have no conflict
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THE UNIVERSE OF DAPK
Post-translational regulation of the cellular levels of DAPK Patricia J. Gallagher • Emily K. Blue
Published online: 2 November 2013 Ó Springer Science+Business Media New York 2013
Abstract Death associated protein kinase (DAPK) is a large, multi-domain ser/thr kinase whose activities converge upon multiple signaling pathways that regulate autophagy, caspase-dependent cell death, cell adhesion and migration. The cellular levels of DAPK are post-translationally regulated by the combined activities of two degradation systems, including the ubiquitin proteasome and an extra-lysosomal proteolysis pathway. At least three distinct E3 ubiquitin ligases target DAPK, including mindbomb1, the chaperone dependent ligase, CHIP (carboxy terminus of Hsp70-interacting protein) and a cullin RING ligase complex, KLHL20-Cul3-RBX1. In addition, it appears that the cellular levels of DAPK are also regulated by an extra-lysosomal protease, cathepsin B. While protein quality control and recycling clearly benefit cells by removal of misfolded or toxic proteins and recycling of their components, the finding that multiple surveillance systems target DAPK suggests that these protein degradation systems also act to fine tune DAPK expression levels in response to specific signaling pathways. Keywords Death associated protein kinase Stability Apoptosis Autophagy Post-translational
P. J. Gallagher (&) Department of Cellular & Integrative Physiology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA e-mail: [email protected] E. K. Blue Department of Pediatrics, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA
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Introduction Death associated protein kinase (DAPK) is a 160 kDa, calcium/calmodulin (Ca2?/CaM)-regulated, multi-domain ser/thr protein kinase that converges upon multiple signaling pathways involved in modulating apoptotic and autophagic cell death as well as cell survival and cancer metastasis [1, 2]. DAPK is the prototypical member of a family of protein kinases that includes 4 additional members: DAPK related protein kinase (DRP-1 or DAPK2), DAPK like kinase/Zipper interacting kinase (DLK, ZIPK), DAPK related apoptosis inducing protein kinases 1 and 2 (DRAK-1/DRAK-2). The kinases in this family share a high degree of homology within their kinase domains, which are all located near the N-terminal region of these proteins, but beyond the kinase domain the protein sequences are dissimilar which likely contributes to the diverse functions of this kinase family [1, 2]. DAPK is comprised of an N-terminal kinase domain, a CaM binding regulatory domain, a series of 8 ankyrin repeats, a cytoskeletal association domain, a Ras of complex proteins (ROC) domain, two nucleotide binding sites (P-loops) involved in GTP binding, and a C-terminal death domain. Located within the CaM binding regulatory domain is an autophosphorylation site (S308) and this must be dephosphorylated by protein phosphatase 2A (PP2A) to allow binding of Ca2?/CaM and activation of the kinase [3– 5]. DAPK has been linked to numerous signaling pathways controlling cell proliferation, apoptosis, autophagy, adhesion, anoikis and its involvement in these signaling pathways provides strong links to pro- and anti-apoptotic functions [4, 6–11]. A number of regulatory mechanisms that govern the activities of DAPK have been identified. These include protein phosphorylation by other kinases as well as autophosphorylation of S308, ubiquitination, and
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Fig. 1 Ubiquitin proteasomal pathway. Shown are the generic steps in generation of a ubiquitinated target protein and subsequent degradation by the 26 s proteasome
protease mediated degradation. These mechanisms ensure that DAPK catalytic activity and stability are carefully regulated in order to appropriately control physiological responses of cells. This review will focus on post-translational mechanisms that control the stability and cellular levels of DAPK in cells. Regulating the levels of cellular proteins is primarily managed through two major intracellular proteolysis systems. These two systems are referred to as the ubiquitin proteasome (UPS) and autophagocytic/lysosomal systems. More recently, an ‘‘extra-lysosomal proteolysis’’ pathway has been described and is characterized by leakage of lysosomal proteases into the cytoplasm to degrade proteins under conditions of stress. Collectively, these three proteolytic degradation systems serve as surveillance mechanisms to identify and remove damaged, misfolded or aggregated proteins, and also modulate the activity of signaling pathways. These quality control pathways are not merely housekeeping mechanisms, but rid cells of toxic proteins and importantly, provide a mechanism for recycling of amino acids within cells. Quality control pathways: be good or be gone In contrast to ‘‘mass’’ degradation of cellular components that occurs via the lysosome, the UPS (Fig. 1) is a more precise pathway to mediate protein degradation, which is directed by the covalent attachment of ubiquitin to individual target proteins. The UPS functions to rapidly remove misfolded, oxidized or otherwise damaged proteins [12–14]. The specificity in the UPS is controlled
through the covalent attachment of a 76-residue ubiquitin chain to the targeted protein, and this tag directs the ubiquitinated protein to the 26S proteasome [14–16]. Ubiquitination of proteins occurs as a result of a trio of enzymatic steps, mediated by E1, E2, and E3 enzymes (Fig. 1). The first step involves the formation of a highenergy thiol-ester bond between the E1 (ubiquitin-activating enzyme) and ubiquitin. Following this, the ubiquitin is transferred to the active site of the E2 enzyme (ubiquitin conjugating enzyme) and then an E3 (ubiquitinligase) mediates the transfer of the activated ubiquitin directly to a lysine reside in the target protein. Once the target protein has been covalently ‘‘tagged’’ by a ubiquitin molecule, the process may repeated several times to generate a polyubiquitin molecule composed of ubiquitin chains usually added to lysine-48 of a previously attached ubiquitin. In addition to K48-linkages, there are several additional types of linkages and configurations that are possible [16, 17]. Polyubiquitin or multi-ubiquitin ‘‘tags’’ direct the protein to the 26S proteasome for degradation, while mono-ubiquitination is generally deemed a signal for endocytosis of membrane proteins such as Notch ligands [16]. The second system responsible for the regulation of proteolysis, autophagy, refers to a catabolic mechanism to degrade non-functional, misfolded, or excess cellular materials [16, 18–23]. Autophagy is a major mechanism for recycling proteins and other macromolecules to provide basic materials and energy for the cell in times of nutrient depletion or stress. There are three types of autophagy: macroautophagy, microautophagy, and chaperone-mediated
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Fig. 2 Autophagy degradation of molecules. Shown is the engulfment of organelles, nucleic acids and aggregated proteins during the formation of the double-membraned autophagosome. Following formation of the autophagosome, fusion with the lysosomal membrane results in destruction of these molecules
autophagy [24–26]. The macroautophagocytic lysosomal pathway is the pathway most relevant to DAPK and refers to a process in which intracellular cytoplasmic constituents are engulfed into a newly formed double membrane vesicle called an autophagosome (Fig. 2) [27]. The autophagosome traffics via microtubules to the lysosome where it fuses with the lysosomal membrane and delivers its contents to the lysosome for degradation. Microautophagy involves direct engulfment of cytoplasmic molecules into the lysosome for degradation and occurs by invagination of the lysosomal membrane, which surrounds the macromolecule(s) [28]. Chaperone-mediated autophagy is a more complex and highly selective pathway, which requires the recognition of the target for degradation by a chaperone, usually HSP70, which localized the target to the lysosomal membrane using a specific receptor called lysosome associated membrane protein 2a (LAMP2a). Following its recognition at the lysosomal membrane, the target protein is unfolded and transferred across the lysosome membrane with the assistance of another chaperone, lysosomal hsc73. Upon translocation to the lysosomal matrix, the target protein is degraded [29–32]. Lysosomes are organelles containing numerous acid hydrolases such as nucleases, phosphatases, proteases, lipases and glycosidases that serve to degrade complex molecules including nucleic acids, proteins, lipids and carbohydrates into their simple components, which are transferred to the cytoplasm for reuse. Of the numerous proteases present in the lysosome, the cathepsin proteases are the most highly characterized and catalyze degradation of proteins into amino acids.
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Fig. 3 Extra-lysosomal proteolysis. Shown on the left are some of the stimuli that result in lysosomal membrane permeability. The leakage of lysosomal hydrolases into the cytoplasm results in destruction of targets (right). This pathway appears to be committed to promoting cell death
The importance of a third proteolytic, non-ubiquitin degradation pathway (Fig. 3) that primarily functions as a positive regulator of apoptosis has recently become appreciated [33]. This pathway is characterized by the release of hydrolytic enzymes from the lysosome in response to cellular stress and will be referred to as the ‘‘extra-lysosomal pathway’’ [33]. Release of lysosomal enzymes can be provoked by high levels of sphingosine or ceramide resulting from TNF death receptor stimulation, treatment with quinolone antibiotics (e.g. naladixic acid, Cipro, Noroxin), ‘‘lysosomotropic’’ detergents [e.g. O-methyl-serine dodecylamide hydrochloride (MSDH) or siramesine], the production of high levels of free fatty acids and bile salts, some viral proteins, or oxidative stress where reactive oxygen species (ROS) are generated [34–36]. Lysosomal membrane permeability (LMP) is a critical step in this pathway which leads to release of acid hydrolases to the cytoplasm of the cell [33]. As the hydrolytic activities of these enzymes are optimized to function at the acidic pH (pH \ 5)of the lysosome, many are not thought to be active in the more neutral cytoplasmic pH [37]. However, several cathepsins (cathepsin-B, -L, -D) remain catalytically active and can degrade cytoplasmic proteins [37, 38]. Consistent with a function to promote apoptosis [39], some of the known proteolytic targets of the released cathepsins are several pro-apoptotic proteins including Bid which is cleaved to tBid, and DAPK as well as some anti-apoptotic proteins like Bcl2, BclxL, and MCL-1 [34, 39–45]. As LMP proteolytic degradation is believed only to be active under stress conditions, it is likely the major role of this pathway is to promote imminent cell death rather than as a mechanism for recycling cellular constituents.
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Supervising DAPK: balancing activity and cellular levels The first report confirming that the cellular levels of DAPK are regulated by the UPS system came with the discovery of an E3 ubiquitin ligase called DIP-1 (DAPK Interacting Protein1) that interacted with DAPK [46]. DIP-1 was identified using a yeast-2-hybrid screen and the interaction with DAPK was confirmed by co-immunoprecipitation of the endogenous proteins. In addition, biochemical and cellular analysis confirmed that DAPK was a target for poly-ubiquitination and degradation mediated by DIP-1, which sensitized cells to TNF-induced apoptosis [46]. Using deletion and co-immunoprecipitation analysis, DIP1 was found to bind to the ankyrin repeats domain of DAPK [46]. A subsequent study using positional cloning to identify the mutant gene resulting in the mind bomb (mib) zebrafish with severe neural defects revealed that the mutant gene was DIP-1. Based on the large body of information referring to phenotype of mind bomb mutant (mib), DIP-1 is now referred to as Mib1 [47, 48]. Mib1 is a 110-kDa protein with a complex structure that includes a zinc finger, 9 ankyrin repeats, 3 C-terminal RING fingers and a alpha-helical coiled-coil region that separates the 2nd and 3rd RING fingers. The ubiquitination of DAPK by Mib1 can also be modulated by interaction with c-mip (c-maf inducing protein), which binds to Mib1 and inhibits ubiquitination of DAPK to result in upregulation of DAPK cellular levels [49]. The increased levels of DAPK found in response to c-mip expression appear to promote sequestration of ERK in the cytoplasm [49]. Relevant to this, a previous study demonstrated that DAPK could sequester ERK in the cytoplasm, blocking nuclear ERK signaling and promoting anoikis [50]. In this setting, the increased levels of active, cytoplasmic ERK resulted in enhanced ERK phosphorylation of DAPK S735, increased DAPK catalytic activity and advancing cell death via anoikis [50]. Together these findings indicate that the cellular levels of DAPK can be post-translationally downregulated by Mib1 mediated ubiquitination and proteasomal degradation and that signaling pathways can upregulate DAPK expression by preventing binding of Mib1.In addition to ubiquitination of DAPK, Mib1 has a central role in Notch signaling [47, 51–53]. Mib1 monoubiquitination of the Notch ligands Delta (Delta-like, DLL in vertebrates) or Serrate (Jagged in vertebrates) is necessary for endocytosis of these ligands and activation of Notch signaling [47, 51–53]. CHIP (C-terminus of Hsc70-interacting protein) a U-box E3 ubiquitin ligase also ubiquitinates DAPK, but does so in collaboration with the molecular chaperone, heat shock protein 90 (HSP90) (Fig. 4) [54]. CHIP is a 35 kDa protein characterized by the presence of an N-terminal TPR
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(tetratricopeptide) motif that acts as a scaffolding structure for interaction with HSP90 and a U-box ubiquitin ligase domain near its C-terminus [55]. Inhibition of chaperone proteins like HSP90 with agents such as 17-alkyl-amino-17demethoxygeldanamycin (GA) is proposed to block its stabilizing interaction and result in increased proteasomal degradation of client proteins as chaperones often partner with E3 ubiquitin ligases such as CHIP [56]. Treatment of cells with the HSP90 inhibitor 17-alkyl-amino-17-demethoxygeldanamycin (GA) reduced expression of DAPK. This finding suggested that HSP90 had an important role in stabilizing DAPK. A previous study had identified a site within the kinase domain that interacted with HSP90 [57]. This interaction occurs via CHIP’s TPR domain and a TPR acceptor site in HSP90 [58]. A number of studies have confirmed that this interaction results in the degradation of a number of HSP90 client proteins [55, 56, 59–66]. Interestingly, it was found that inhibition of HSP90 with GA resulted in a preferential degradation of the activated form of DAPK in which the inhibitory autophosphorylation site at S308 has been dephosphorylated by PP2A (Fig. 4) [3, 54]. This result directly implied there is a selective mechanism for reducing cellular DAPK activity by targeting degradation of activated DAPK [54]. Analysis of immune complexes identified two distinct heterocomplexes in cells. One complex contains DAPK with HSP90 and CHIP while a second complex contained only DAPK and Mib1. These results suggested that when DAPK is bound by HSP90, it might be preferentially ubiquitinated by CHIP and in the absence of HSP90 binding DAPK is ubiquitinated by Mib1 [54]. Another binding partner of DAPK that has a role in regulating its protein level is KLHL20, identified in a yeast 2-hybrid screen using the death domain of [67]. KLHL proteins are Kelch-like proteins that contain a BTB (Broad complex, Tamtrack, Bric-a-brac) motif. Cullin-RING-ligases (CRLs) utilize BTB domain proteins such as KLHL20 as substrate adapters to interact with target proteins for ubiquitination [68–75] Consistent with this, in previous studies immune complexes containing DAPK and KLHL20 were also found to contain Cul3 and the Cul3 E3 ubiquitin ligase subunit, RBX1 (Fig. 5) [67]. Cell studies depleting KLHL20 with siRNAs resulted in attenuated ubiquitination of DAPK and in vitro ubiquitination assays confirmed that DAPK is ubiquitinated by the KLHL20-Cul3-RBX1 complex. Interestingly, treatment of cells interferon-alpha or gamma substantially reduced ubiquitination of DAPK and this was associated with KLHL20 being sequestered into peri-nuclear and nuclear locations in PML (promyelocytic leukemia) nuclear bodies [67]. Sequestration of KLHL20 away from DAPK could thus account for the decreased ubiquitination of DAPK and its resultant increased stability provides a satisfying mechanism to explain how or interferon-alpha or –gamma can induce apoptosis in cells.
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Fig. 4 Ubiquitination of DAPK by Mib1 and CHIP. Cellular stimulation by ceramide or tumor necrosis factor (TNF) leads to a rise in the cytoplasmic levels of calcium and results in CaM (calmodulin) binding to the CaM-binding domain in DAPK. Binding of Ca2?/CaM to DAPK requires dephosphorylation of autophosphorylated Ser308 within the CaM-binding domain. Following activation, which may require association with the chaperone HSP90, DAPK phosphorylates substrate proteins. Alternatively, misfolded or altered DAPK can be targeted by the E3 ligases, CHIP or Mib1 for ubiquitination and degradation by the 26 s proteasome. Ubiquitination by Mib1 can be attenuated by Mip1, which sequesters Mib1
Fig. 5 Cullin-RINGubiquitination of DAPK. Shown are the steps in the KLHL20Cullin3-RBX1 ubiquitination of DAPK and subsequent degradation by the 26 s proteasome
Another pathway that has been shown to modulate DAPK degradation is associated with the tuberous sclerosis complex (TSC). The TSC is comprised of two proteins, TSC1 (hamartin) and TSC2 (tuberin), which contain a GTPase activating (GAP) protein domain. When bound together the TSC1/TSC2 complex inhibits the activation of mammalian target of rapamycin complex 1 (mTOR) by
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TSC2. When activated, mTOR acts a sensor of cellular nutrients and energy and modulates protein and lipid synthesis as well as autophagy in response to environmental changes. The principal targets of mTOR are ribosomal protein p70-S6 kinase1 (S6K1) and the eukaryotic initiation factor 4E binding protein-1 (4E-BP1). Both of these targets are central to the formation of the translational
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initiation complex and are key regulators of protein synthesis [76]. Stevens et al. [77] and Lin et al. [78] have shown that the death domain (DD) of DAPK interacts with tuberous sclerosis protein 2 (TSC2) and that this interaction can have one of two outcomes. Either DAPK directly phosphorylates TSC2 leading to dissociation of the TSC1/ TSC2 complex and activation of mTOR to promote protein synthesis or mTOR promotes degradation of DAPK in a lysosome-dependent process. In the Stevens study, a peptide aptamer library was used to screen for peptides binding to the DD of DAPK and TSC2 was identified [77]. The DAPK/TSC2 complex was confirmed by co-immunoprecipitation as was the binding site within the DD of DAPK. Biochemical analysis further demonstrated that TSC2 was phosphorylated at an unidentified site by DAPK. The outcome of TSC2 phosphorylation was a dissociation of TSC1/TSC2 complex and downstream activation of mTOR signaling leading to phosphorylation of S6K1. These studies suggest that DAPK is a positive regulator of mTOR activation and signaling [77]. In the report by Lin et al. [78], the observation that the cellular levels of DAPK are inversely correlated with those of TSC2 was explored. This investigation led to the discovery that TSC2 can regulate the levels of DAPK and does so via a post-translational mechanism. Surprisingly, treatment of cells with the mTOR inhibitor, rapamycin had no effect on the cellular levels of DAPK further suggesting that TSC2 regulation of DAPK was also not an effect of mTOR’s role in modulating protein synthesis. Consistent with this, when a TSC2 mutant lacking GAP activity was expressed in cells, the levels of DAPK were still reduced. To identify the mechanism that resulted in degradation of DAPK, several lysosomal protease inhibitors including leupeptin, E64D, and chloroquine were found to result in increased levels of DAPK while the proteasome inhibitor, MG132 did not [78]. Treatment of cells with the autophagy inhibitor, 3-methyladenine (3-MA) also had no effect on DAPK levels. Collectively these results suggest that regulations of the cellular levels of DAPK are also mediated by lysosomal proteases but independent of autophagy [78]. These results suggested that there is an additional, non-ubiquitin, nonautophagic pathway that has a role in regulation of the cellular levels of DAPK which is consistent with previous study where DAPK was identified as a target of the lysosomal protease, cathepsin B [79], as described below. Lin et al. [79] observed a consistent set of proteolytic fragments of DAPK that was detected by immunoblotting lysates from cells treated with the proteasome inhibitor MG132. As these fragments were observed in the presence of a proteasomal inhibitor, it was predicted that the fragments resulted from a non-proteasomal degradation pathway [79]. Further experiments demonstrated that DAPK has a relatively long half-life (*6 h) compared to other
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signaling proteins like p53 (*20 min) or interferon regulatory factor (IRF-1; *40 min). Further, the addition of both a proteasome inhibitor (MG132) and a cathepsin B inhibitor (benzyloxycarbonyl-FA-fluoromethyl ketone) extended the lifetime of DAPK in cultured cells. These results suggested the presence of another degradation pathway in addition to the proteasomal pathway that likely involved proteolysis by the lysosomal protease cathepsin B. However, the finding that overexpression of cathepsin B had no effect on the endogenous levels of DAPK, but attenuated the expression of exogenous DAPK was surprising. This result was explained by proposing that steady state, endogenous levels of DAPK may exist in multiprotein complexes that shield the kinase from cathepsin B proteolysis and when overexpressed, some DAPK is not sequestered in these protective complexes. In support of this it was found that exogenous expression of TNFR-1, which can induce lysosomal permeabilization [80], resulted in the formation of a complex between DAPK and cathepsin B and relocalization from the insoluble cytoskeletal fraction to the soluble cytosolic fraction [79]. The consequence of this complex formation was an increase in cytosolic cathepsin B and decrease in DAPK expression. Using deletion mutants of DAPK, the binding site for cathepsin B was identified in a region residing at the C-terminus between the cytoskeletal binding and death domains, and expression of this ‘‘miniprotein’’ blocked formation of the complex. Although cathepsin B is predominantly found in the lysosome, there is data supporting the occurrence of lysosomal membrane ‘‘leakage’’ allowing these lysosomal hydrolases access to the cytoplasm particularly when cells are undergoing apoptosis or in stress conditions [44, 81–83]. The are also at least some data suggesting that acidic proteases such as cathepsin B can function in the more neutral pH environment of the cytosol [84, 85]. Together these data suggest that DAPK can be degraded both by lysosomal hydrolases within the lysosome and under some conditions by these hydrolases after they are released into the cytoplasm of a stressed or apoptotic cell.
Conclusions and perspectives There are several systems monitoring the structure and functionality of proteins in cells. These systems include the ubiquitin-proteasome, autophagic/lysosomal and extralysosomal protein degradation. In recent years, these protein degradation systems have become appreciated to have not only a housekeeping role to rid the cell of toxic, misfolded or aggregated proteins, but to also play a key role in control signaling pathways by fine-tuning the cellular levels of critical signaling molecules. The ‘‘logic’’ behind
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Fig. 6 Summary of the posttranslational mechanisms that regulate the cellular levels of DAPK. Shown are the three ubiquitin ligases (Mib1, CHIP, KLHL20-Cul3-RBX1) that target DAPK for ubiquitination and destruction by the 26 s proteasome. Also shown is the extra-lysosomal proteolysis pathway in which DAPK is proteolyzed by cathepsin B leakage from the lysosome
these processes is not yet completely appreciated, but this is a good example of the cellular ‘economy’ of utilizing the same mechanisms to rid the cell of noxious proteins and to limit the activity of signaling pathways. Key to understanding how signaling pathways are both controlled by, and control, the protein degradation machinery will be an understanding the signals that link the two processes. Already there are indications that the conformational change that occurs in DAPK upon activation may itself be a signal that is recognized by one of these chaperone and/or degradation systems. Other obvious examples can be seen with the Cullin-RING-ligase system whereby some ubiquitin targets require post-translational modifications such as phosphorylation to be recognized by the Cullin-RINGligases [86–88]. Currently, we understand some of the degradation and quality control pathways that assess the suitability of DAPK and regulate its posttranslational stability (Fig. 6). Finding that several distinct monitoring pathways regulate DAPK levels indicate that the cellular levels of DAPK like p53 must be carefully regulated. The work summarized in this review indicates that several E3 RING ubiquitin ligases (Mib1, CHIP, KLHL20-Cul3-RBX1) as well as the extralysosomal protease cathepsin B all monitor the cellular levels of DAPK. In addition, the surveillance of DAPK also involves at least one chaperone (HSP90), which likely functions to assist DAPK in attaining and sustaining a mature, functional 3D configuration. The fact that several degradative pathways focus on DAPK suggest there must be
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additional complexities involved in determining which pathway is active where and when. This likely involves regulation of cellular localization or involvement in higher order complex structures or use of scaffolding molecules to perhaps direct the activity of specific degradation complexes to different intracellular pools of DAPK. Do these multiple surveillance systems indicate the need to limit certain pools of DAPK activity in order to perhaps promote cell migration or proliferation without inducing autophagic or caspase-dependent cell death? Exactly how do these multiple surveillance systems serve to regulate the activities of DAPK in response to different environmental stimuli? Are these degradation systems merely a way to terminate DAPK signaling? At least one study has supported this possibility by demonstrating that activated but not inactive DAPK is targeted by CHIP for ubiquitination and recycling [54]. This could serve to limit DAPK activity even when cellular levels of calcium remain elevated. Clearly there are still many issues to be resolved regarding the role of, and the mechanisms underlying, the post-translational regulation of the cellular levels of DAPK. Acknowledgments This work was supported by Grants from the National Institutes of Health HL54118 and DK062810 to P. J. G., the Indiana University Diabetes and Obesity Research Training Grant DK064466 and American Heart Association Pre-doctoral fellowship grant 0815608G to E. K. B. Conflict of Interest of interest.
The authors declare that they have no conflict
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