DAPK (death-associated protein kinase) is a newly recognized member of the mammalian family of ROCO proteins, characterized by common ROC (Ras of complex proteins) and COR (C-terminal of ROC) domains. In the present paper, we review our recent work showing that DAPK is functionally a ROCO protein; its ROC domain binds and hydrolyses GTP. Furthermore, GTP binding regulates DAPK catalytic activity in a novel manner by enhancing autophosphorylation on inhibitory Ser308, thereby promoting the kinase ‘off’ state. This is a novel mechanism for in cis regulation of kinase activity by the distal ROC domain. The functional similarities between DAPK and the Parkinson's disease-associated protein LRRK2 (leucine-rich repeat protein kinase 2), another member of the ROCO family, are also discussed.

DAPK (death-associated protein kinase), a new member of the mammalian ROCO family

The ROCO proteins are defined by predicted sequence similarity within two domains that always occur in tandem: the ROC (Ras of complex proteins) domain, a GTPase domain resembling small G-proteins such as Ras, and the COR (C-terminal of ROC) domain [1]. The mammalian subfamily consists of four members, three of which are kinases: the Parkinson's disease-associated kinase LRRK (leucine-rich repeat kinase) 2 and the closely related LRRK1, and DAPK (Figure 1). DAPK is a serine/threonine Ca2+/CaM (calmodulin)-regulated kinase that has been implicated in several important cellular functions, including the programmed cell death pathways of apoptosis and necrosis, autophagy [2], cell adhesion and motility [35], and inflammatory responses [68]. These outcomes can result either from phosphorylation of specific target substrates {e.g. MLC (myosin II regulatory light chain) [9], Beclin 1 [10], PKD (protein kinase D) [11]}, or from direct binding between DAPK and its downstream targets independently of catalytic activity {e.g. MARK1/2 (microtubule-associated protein-regulating kinase 1/2) [12]}. Several of the functional arms activated downstream of DAPK contribute to its capacity as a tumour suppressor [13,14]. In fact, loss of DAPK has been documented in many primary tumours and cancer cell lines, and its expression levels have been shown to inversely correlate with tumour progression, metastasis and survival prognosis [13].

Schematic diagram of protein structures of the mammalian ROCO kinases

Figure 1
Schematic diagram of protein structures of the mammalian ROCO kinases

LRR, leucine-rich region.

Figure 1
Schematic diagram of protein structures of the mammalian ROCO kinases

LRR, leucine-rich region.

We recently investigated the functional and biochemical properties of DAPK's predicted ROC–COR domains, which have established DAPK as a bona fide member of the ROCO family [15]. DAPK's ROC–COR domains lie downstream of its kinase domain and a series of ankyrin repeats, in a region previously identified as responsible for localization to the actin cytoskeleton [16] (Figure 1). In this it differs from other ROCO kinases, in which the kinase domain follows immediately downstream of the ROC–COR domains. It is believed that the close proximity of the kinase and ROC–COR domains in these proteins enables propagation of structural changes that occur during the GTP cycle, resulting in the activation of catalytic activity upon GTP binding, and its shutdown upon GTP hydrolysis [1719]. The structural difference in DAPK suggests that its ROC–COR domains may have a functional role that differs from that of other ROCO family members.

We first confirmed that, similarly to LRRK2, DAPK homodimerizes, an interaction that involves both the ROC and kinase domains [15]. DAPK family members have been shown previously to dimerize via the kinase domain, via a characteristic basic loop within the catalytic domain that is conserved in all members of the DAPK family [20,21]. The ROC domain itself was sufficient to mediate dimerization with either full-length DAPK or itself, but the P-loop motif was not required for this interaction [15]. Using in vitro GTP-binding assays, we demonstrated that DAPK, but not a P-loop mutant or deletant, can bind GTP with a higher affinity than GDP. Moreover, recombinant DAPK ROC–COR domains can hydrolyse GTP in vitro, thus proving that DAPK is a G-protein.

GTP binding regulates DAPK function in a novel manner

Next, we analysed the functional effects of GTP binding on DAPK's enzymatic and cellular activities. This was done using the P-loop deletant of full-length DAPK (ΔPloop), which does not bind GTP. In vitro kinase assays using the canonical DAPK substrate MLC indicated that the GTP-binding-deficient mutant had greater catalytic activity than the wild-type kinase. Consistent with this, expression of the ΔPloop mutant in HEK (human embryonic kidney)-293 cells led to more extensive membrane blebbing and greater cell detachment than wild-type DAPK. These phenotypes represent two separate functional arms of DAPK: membrane blebbing results from increased actomyosin contraction upon phosphorylation of MLC [9], and cell rounding/detachment results from loss of cell adhesion due to inhibition of integrin function [5]. On the basis of these results, in which loss of GTP binding leads to a more active kinase, we can extrapolate that GTP binding to DAPK's ROC domain is inhibitory. This contrasts with the observed effect of GTP binding and hydrolysis on other ROCO proteins.

As a critical regulator of diverse cellular functions, DAPK is subjected to multi-layered regulation. Whereas external regulators, such as ERK (extracellular-signal-regulated kinase), Src, LAR (leucocyte antigen-related) phosphatase and RSK (ribosomal S6 kinase) can influence DAPK activity and function [22], DAPK catalytic activity is primarily determined by two interrelated mechanisms of intrinsic regulation [23]. The first involves the binding of CaM to the CaM-regulatory domain and release of the latter domain from the catalytic cleft, enabling substrate access. In addition, DAPK undergoes autophosphorylation on Ser308 within the CaM domain. This phosphorylation is inhibitory; molecular modelling predicts a tighter interaction between the phosphorylated residue and the catalytic cleft [23]. Dephosphorylation of DAPK is thus critical for its activation. PP2A (protein phosphatase 2A) has been shown to be responsible for dephosphorylating Ser308 and activating DAPK [2426]. In fact, decreased reactivity to a specific phospho-Ser308 antibody on Western blots is often used as a marker for DAPK activation, for example, during ceramide treatment [23], oxidative stress [11], endoplasmic reticulum stress [25], and more. Interestingly, our recent results indicate that phosphorylation of Ser308 occurs in cis; one DAPK monomer phosphorylates itself, but not its partner within the dimer [15].

Binding of GTP to the ROC domain is thus a third level of intrinsic regulation on DAPK catalytic activity. As described above, this effect was curious considering the distance between these two domains and their relative orientation. To see whether the modes of regulation were connected, we examined Ser308 phosphorylation status in GTP-binding mutants [15]. Interestingly, ΔPloop showed extensive dephosphorylation on Ser308, explaining its enhanced catalytic and cellular activity.

We hypothesized several models to potentially explain these results (Figure 2). Decreased phosphorylation of Ser308 can result from attenuated catalytic activity towards the site, or from enhanced dephosphorylation activity of PP2A. Significantly, the PP2A-binding site has been shown to overlap with the ROC domain [26], suggesting that GTP binding may influence PP2A binding or activity. According to the first model (Figure 2A), hydrolysis of GTP leads to the recruitment of PP2A to DAPK, facilitating Ser308 dephosphorylation and activation. This model predicts enhanced association between PP2A and GTP-binding-deficient mutants of DAPK, compared with the GTP-bound wild-type protein. Yet co-immunoprecipitation experiments indicated that PP2A bound somewhat less robustly to these mutants, suggesting that recruitment of PP2A was not regulated by the GTP cycle in this manner [15]. Our second model takes into account these results and predicts constitutive binding to PP2A (Figure 2B). Hydrolysis of GTP would lead to a conformational change in the protein that enables access of PP2A to the CaM domain to facilitate dephosphorylation of Ser308, normally buried within the catalytic cleft. If this model is true, then inhibition of PP2A would abrogate the effect of the P-loop mutations on dephosphorylation. Once again, our experimental results contradicted the predicted model. The P-loop deletant showed reduced phosphorylation on Ser308 in the absence and presence of the PP2A inhibitor okadaic acid, indicating a PP2A-independent mechanism [15]. This left the third model (Figure 2C), in which the catalytic activity towards Ser308 is itself enhanced by GTP binding, and, conversely, is reduced by GTP hydrolysis. This may be due to a conformational change in the CaM-binding domain or the catalytic domain that is induced by GTP hydrolysis at the distal ROC–COR domains. It will be interesting to understand the structural mechanisms by which such a change in conformation can be propagated from such a distant domain. Currently, the crystal structure of DAPK [or its highly related family member DRP-1 (DAPK-related protein 1, also known as DAPK2)] has only been resolved for the kinase domain, with or without part of the CaM-regulatory domain [2730], so this aspect remains to be determined.

Schematic representation of three hypothetical mechanistic models for GTP-dependent regulation of DAPK

Figure 2
Schematic representation of three hypothetical mechanistic models for GTP-dependent regulation of DAPK

(A) GTP binding blocks the docking of PP2A, consequently inhibiting Ser308 dephosphorylation and DAPK activation. Upon cycling to GDP, the PP2A holoenzyme [composed of catalytic (C), regulatory (B) and structural (A) subunits] can be recruited and DAPK is activated by dephosphorylation. (B) PP2A is constitutively bound to the ROC–COR domain, yet GTP hydrolysis effects a conformational change that enables it to access Ser308 within the CaM domain, leading to dephosphorylation. (C) Conformational change imposed by GTP binding favours DAPK activity towards Ser308 autophosphorylation, bringing about catalytic inactivation. Upon GTP hydrolysis, Ser308 is no longer accessible for autophosphorylation, and the catalytic cleft is now available for substrate phosphorylation.

Figure 2
Schematic representation of three hypothetical mechanistic models for GTP-dependent regulation of DAPK

(A) GTP binding blocks the docking of PP2A, consequently inhibiting Ser308 dephosphorylation and DAPK activation. Upon cycling to GDP, the PP2A holoenzyme [composed of catalytic (C), regulatory (B) and structural (A) subunits] can be recruited and DAPK is activated by dephosphorylation. (B) PP2A is constitutively bound to the ROC–COR domain, yet GTP hydrolysis effects a conformational change that enables it to access Ser308 within the CaM domain, leading to dephosphorylation. (C) Conformational change imposed by GTP binding favours DAPK activity towards Ser308 autophosphorylation, bringing about catalytic inactivation. Upon GTP hydrolysis, Ser308 is no longer accessible for autophosphorylation, and the catalytic cleft is now available for substrate phosphorylation.

Our results thus indicate that GTP binding to the ROC domain provides a novel in cis intramolecular signalling mechanism to restrain the kinase domain. Paradoxically, this occurs through enhanced catalytic activity, but towards a negative autoregulatory site, so that the overall effect is to shut off kinase activity. Thus the GTP-bound kinase represents the ‘off’ state of the kinase. On the basis of this, and the in vitro results that show a greater affinity for GTP than for GDP [15], DAPK should be bound to GTP in the basal unstressed state when it is inactive. GTP hydrolysis would then serve as a regulated event that leads to activation of the kinase. As such, DAPK belongs to a small subclass of G-proteins, including Rheb, which are bound to GTP in the basal state and activated through GTP hydrolysis [31]. It remains to be determined what regulates the GTP-binding/hydrolysis steps within DAPK's ROC domain. For example, is there a specific DAPK GAP (GTPase-activating protein) or GEF (guanine-nucleotide-exchange factor) or are these activities intrinsic to the ROC–COR domains, perhaps via dimerization? What cellular stimuli affect this mode of regulation, and how does it integrate with PP2A-mediated regulation of Ser308 phosphorylation? These exciting questions await resolution in the near future.

Functional similarities between DAPK and LRRK2

Although DAPK and LRRK1/2 belong to two distinct kinase subfamilies [1], the connection between them may expand beyond the shared ROC–COR homology. Both LRRK1 and LRRK2 have been shown to heterodimerize with DAPK through the corresponding ROC domains [32]. LRRK2 mutations have been associated with both sporadic and familial Parkinson's disease, and, although it is ubiquitously expressed, its primary function seems to be in the brain [33]. High DAPK protein levels are found in the hippocampus and cortex [34,35]. Like LRRK2, DAPK has been linked to several pathologies of the brain and neurodegenerative diseases, as documented, for example, by the association of DAPK variants with late-onset Alzheimer's disease [36]. Interestingly, expression of Drosophila DAPK in the retina results in the loss of photoreceptor neurons, due to activation of PAR-1, the Drosophila MARK1/2 orthologue (see below), and consequent tau toxicity, a typical characteristic of neurodegenerative disease [12]. DAPK is also involved in several additional pathologies associated with neuronal cell death, such as epilepsy [37] and hypoxia/ischaemia acute brain injury [38]. Furthermore, ischaemic injury by excessive activation of NMDA (N-methyl-D-aspartate) glutamate receptors promotes the association of DAPK with these receptors, and disrupting this association reduces damage to the brain [39]. Consistent with this, knockout of DAPK in the mouse protects retinal ganglion cells from glutamate toxicity in vivo [40].

Two proposed functions of LRRK2 in particular have been linked to the aetiology of Parkinson's disease: its effects on cytoskeletal dynamics and its involvement in autophagy [41,42]. Interestingly, DAPK too is a regulator of both processes. Like LRRK2, DAPK has been shown to interact with F-actin (filamentous actin), and can phosphorylate and regulate actin-associated cytoskeletal proteins, such as MLC and tropomyosin, affecting actin filament dynamics and cell morphology [9,4345]. Similarly, DAPK and LRRK2 have both been linked to microtubule assembly. Drosophila LRRK2 interacts with and phosphorylates the Drosophila MAP (microtubule-associated protein) 1 homologue Futsch, a microtubule-binding protein, and may regulate the presynaptic cytoskeleton and axonal trafficking through this interaction [46]. In addition, LRRK2 has been shown to phosphorylate tubulin [41]. DAPK too has been shown to interact with MAP1B [47], and also MARK1/2, the microtubule-binding protein kinases [12]. This interaction results in MARK1/2 activation in neurons, leading to phosphorylation of tau and MAP2/4 and, consequently, microtubule disassembly and inhibition of axon formation in hippocampal neurons.

LRRK2's complex role in autophagy is addressed elsewhere in this issue of Biochemical Society Transactions; in the present paper, we focus on DAPK. DAPK activates autophagy flux, and is necessary for autophagy, particularly in scenarios in which autophagy has been linked to cell death [25,48,49]. We have described previously several mechanisms by which DAPK can activate autophagy (Figure 3). First, DAPK phosphorylates Beclin 1, a component of the Vps34 (vacuolar protein sorting 34) Class III PI3K (phosphoinositide 3-kinase) complex that is essential for formation of the autophagosome membrane [10]. Beclin 1, a BH3 (Bcl-2 homology 3) domain-containing protein, is normally inactivated by interaction with anti-apoptotic Bcl-2 proteins through the latter's BH3-binding domain. Phosphorylation of Beclin 1 by DAPK within the BH3 domain disrupts this interaction, enabling its autophagic activity. In addition, during oxidative stress, DAPK phosphorylates and activates PKD [11], which in turn can phosphorylate Vps34 directly, leading to enhanced generation of PtdIns3P, and, consequently, enhanced autophagosome formation [49]. Thus DAPK activates this important regulatory step in the autophagy signalling pathway by two independent mechanisms. As a modulator of both the actin cytoskeleton [13] and microtubules/microtubule-binding proteins [12,47], DAPK may affect additional steps within the autophagy pathway, such as trafficking of the autophagosome and its fusion to the lysosome. The connection between these functions of DAPK and its interaction with LRRK2 remain an exciting avenue for future research.

Scheme of the stages of the autophagy signalling pathway and placement of DAPK as a regulator of Vps34 complex activity

Figure 3
Scheme of the stages of the autophagy signalling pathway and placement of DAPK as a regulator of Vps34 complex activity

In brief, following an autophagic trigger, the autophagosome forms de novo from the phagophore, which expands to engulf cytoplasmic contents. The closed autophagosome undergoes further maturation, involving trafficking to and fusion with endosomal compartments to form the amphisome, which fuses with the lysosome, forming the autolysome, wherein its contents are released for degradation by lysosomal enzymes. The nucleation step is controlled by a lipid kinase complex consisting of Vps34, Beclin 1 and other associated proteins. DAPK can phosphorylate Beclin 1, resulting in release of inhibitory Bcl-2. In addition, DAPK activates a kinase cascade involving phosphorylation of PKD, which phosphorylates Vps34, to activate its lipid kinase activity. DAP-kinase, death-associated protein kinase; PI, phosphatidylinositol; PI(3)P, phosphatidylinositol 3-phosphate; PI(3)K, phosphoinositide 3-kinase.

Figure 3
Scheme of the stages of the autophagy signalling pathway and placement of DAPK as a regulator of Vps34 complex activity

In brief, following an autophagic trigger, the autophagosome forms de novo from the phagophore, which expands to engulf cytoplasmic contents. The closed autophagosome undergoes further maturation, involving trafficking to and fusion with endosomal compartments to form the amphisome, which fuses with the lysosome, forming the autolysome, wherein its contents are released for degradation by lysosomal enzymes. The nucleation step is controlled by a lipid kinase complex consisting of Vps34, Beclin 1 and other associated proteins. DAPK can phosphorylate Beclin 1, resulting in release of inhibitory Bcl-2. In addition, DAPK activates a kinase cascade involving phosphorylation of PKD, which phosphorylates Vps34, to activate its lipid kinase activity. DAP-kinase, death-associated protein kinase; PI, phosphatidylinositol; PI(3)P, phosphatidylinositol 3-phosphate; PI(3)K, phosphoinositide 3-kinase.

In conclusion, we have shown that DAPK is a bona fide G-protein, a functional member of the ROCO family. These studies highlight a novel mechanism by which GTP binding affects catalytic activity. The main issues to resolve now are to identify the physiological triggers that serve to regulate the GTP-binding activity of the ROC–COR domains, and to determine the precise functional connection to LRRK2.

LRRK2: Function and Dysfunction: A Biochemical Society Focused Meeting held at Royal Holloway, University of London, Egham, UK, 28–30 March 2012. Organized and Edited by Patrick Lewis (University College London, U.K.) and Dario Alessi (Dundee, U.K.).

Abbreviations

     
  • BH3

    Bcl-2 homology 3

  •  
  • CaM

    calmodulin

  •  
  • COR

    C-terminal of Ras of complex proteins

  •  
  • DAPK

    death-associated protein kinase

  •  
  • LRRK

    leucine-rich repeat kinase

  •  
  • MAP

    microtubule-associated protein

  •  
  • MARK1/2

    microtubule-associated protein-regulating kinase 1/2

  •  
  • MLC

    myosin II regulatory light chain

  •  
  • PKD

    protein kinase D

  •  
  • PP2A

    protein phosphatase 2A

  •  
  • ROC

    Ras of complex proteins

  •  
  • Vps34

    vacuolar protein sorting 34

Funding

Our work is supported by a grant from Merck-Serono and by a Center of Excellence grant from the Flight Attendant Medical Research Institute (FAMRI). A.K. is the incumbent of the Helena Rubinstein Chair of Cancer Research.

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