InteractiveFly: GeneBrief

Death-associated protein kinase related: Biological Overview | References

Gene name - Death-associated protein kinase related

Synonyms -

Cytological map position - 10C1-10C5

Function - signaling

Keywords - regulation of myosin II dynamics, promotes proper morphogenesis of epithelial tissues during development, maintenance of furrow canal structure and lateral plasma membrane integrity during cellularization, morphogenesis of retina, functions during Myosin II-dependent cell constriction, subsequent multicellular alignment and adherens junction remodelling

Symbol - Drak

FlyBase ID: FBgn0052666

Genetic map position - chrX:11,493,135-11,555,045

Classification - DRAK: Catalytic domain of the Serine/Threonine Kinase, Death-associated protein kinase-Related Apoptosis-inducing protein Kinase

Cellular location - cytoplasmic

NCBI links: Precomputed BLAST | EntrezGene

Morphogenesis of epithelial tissues relies on the interplay between cell division, differentiation and regulated changes in cell shape, intercalation and sorting. These processes are often studied individually in relatively simple epithelia that lack the complexity found during organogenesis when these processes might all coexist simultaneously. To address this issue, this study makes use of the developing fly retinal neuroepithelium. Retinal morphogenesis relies on a coordinated sequence of interdependent morphogenetic events that includes apical cell constriction, localized alignment of groups of cells and ommatidia morphogenesis coupled to neurogenesis. Live imaging was used to document the sequence of adherens junction (AJ) remodelling events required to generate the fly ommatidium. In this context, it was demonstrated that the kinases Rok and Drak function redundantly during Myosin II-dependent cell constriction, subsequent multicellular alignment and AJ remodelling. In addition, it was shown that early multicellular patterning characterized by cell alignment is promoted by the conserved transcription factor Atonal (Ato). Further ommatidium patterning requires the epidermal growth factor receptor (EGFR) signalling pathway, which transcriptionally governs Rho-kinase (rok) and Death-associated protein kinase related (Drak)-dependent AJ remodelling while also promoting neurogenesis. In conclusion, this work reveals an important role for Drak in regulating AJ remodelling during retinal morphogenesis. It also sheds new light on the interplay between Ato, EGFR-dependent transcription and AJ remodelling in a system in which neurogenesis is coupled with cell shape changes and regulated steps of cell intercalation (Robertson, 2012).

In Drosophila, Rok seems to be the main kinase responsible for phosphorylating the Myosin regulatory light chain (Sqh) during epithelial patterning and apical cell constriction. This is the case for the activation of MyoII during intercalation as germband extension proceeds, but also during various instances of compartment boundary formation and cell sorting situations in the embryo and in the wing imaginal disc. The current work reveals that in the constricting cells of the MF, Rok functions redundantly with Drak, a kinase recently shown to phosphorylate Sqh both in vitro and in vivo (Neubueser, 2010). It is noteworthy that previous work has shown that RhoGEF2 is not required for cell constriction in the MF, suggesting that perhaps another guanine exchange factor (GEF) might function redundantly with RhoGEF2 to promote cell constriction. These data on Drak reinforce the idea that redundancies exist in this context. Because the RhoA (Rho1 -- FlyBase) loss of function abolishes this cell response entirely, it would be expected that Drak function is regulated by RhoA. In addition, the current data indicate that Drak acts redundantly with Rok during MyoII-dependent multicellular alignment and AJ remodelling during ommatidia patterning. It will be interesting to test whether Drak functions in other instances of epithelial cell constriction or MyoII-dependent steps of AJ remodelling in other developmental contexts in Drosophila (Robertson, 2012).

This study demonstrates a two-tiered mechanism regulating the planar polarization of MyoII and Baz. In the constricting cells in the posterior compartment, MyoII and Baz are segregated from one another and this is exacerbated by the wave of cell constriction in the MF. Upon Ato-dependent transcription in the MF cells, this segregated pattern of expression is harnessed and these factors become planar polarized at the posterior margin of the MF. This is independent of the core planar polarity pathway including the Fz receptor and is accompanied by a striking step of multicellular alignment. Previous work has demonstrated that Ato upregulates E-Cad transcription at the posterior boundary of the MF. In addition, apical constriction leads to an increase in E-Cad density at the ZA. The current data are therefore consistent with both hh-dependent constriction and ato-dependent transcriptional upregulation of E-Cad promoting differential adhesion, thus leading to a situation in which the ato+ cells maximize AJ contacts between themselves and minimize contact with the flanking cells that express much less E-Cad at their ZA. This typically leads to a preferential accumulation of cortical MyoII at the corresponding interface. Such actomyosin cables are correlated with increased interfacial tension, and it is proposed that this is in turn responsible for promoting cell alignment. Unfortunately, the very small diameter of these constricted cells precludes direct measurements of the AJ-associated tension using laser ablation experiments (Robertson, 2012).

Supra-cellular cables of MyoII have been previously associated with cell alignment in various epithelia and have also been observed at the boundary of sorted clones, whereby cells align at a MyoII-enriched interface. Interestingly, this study found that the actomyosin cable defining the posterior boundary of the MF is also preferentially enriched for Rok, a component of the T1, MyoII-positive AJ in the ventral epidermis (Simoes Sde, 2010). This indicates an important commonality between actomyosin cable formation during cell sorting and the process of cell intercalation. However, unlike during intercalation, this study found that in the developing retina baz is largely dispensable for directing the pattern of E-Cad and actomyosin planar polarization. Further work will therefore be required to understand better the relationship between Baz and E-Cad at the ZA during ommatidia morphogenesis. It is speculated that the creation of a high E-Cad versus low E-Cad boundary in the wake of the MF might be sufficient to promote Rok and MyoII enrichment at the posterior AJs. This posterior Rok and MyoII enrichment might perhaps prevent E-Cad accumulation by promoting E-Cad endocytosis, as has been recently shown in the fly embryo (Robertson, 2012).

This study has used live imaging to define a conserved step of ommatidia patterning that consists of the coalescence of the ommatidial cells' AJs into a central vertex to form a 6-cell rosette. The corresponding steps of AJ remodelling require Rok, Drak, Baz and MyoII, a situation compatible with mechanisms previously identified during cell intercalation in the developing fly embryo. The steps of AJ remodelling required to transform lines of cells into 5-cell pre-clusters are transcriptionally regulated downstream of EGFR in a ligand-dependent manner. Interestingly, in the eye EGFR signalling is activated in the cells that form lines and type1-arcs in the wake of the MF and, thus, are undergoing AJ remodelling. Previous work examining tracheal morphogenesis in the fly has demonstrated that interfaces between cells with low levels versus high levels of EGFR signalling correlate with MyoII-dependent AJ remodelling in the tracheal placode. This situation resembles that which is described in this study in the wake of the MF. In the eye, however, it was found that EGFR signalling is not required to initiate cell alignment. Nevertheless, taken together with work in the tracheal placode and previous studies related to multicellular patterning in the developing eye, this work indicates a conserved function for the EGFR signalling pathway in promoting MyoII-dependent AJ remodelling. This leaves open several interesting questions; for example, it is not presently clear how EGFR signalling can promote discrete AJ suppression and elongation. It is, however, tempting to speculate that previously described links between EGFR signalling and the expression of E-Cad or Rho1 might play a role during this process (Robertson, 2012).

Drak is required for actomyosin organization during Drosophila cellularization

The generation of force by actomyosin contraction is critical for a variety of cellular and developmental processes. Nonmuscle myosin II is the motor that drives actomyosin contraction, and its activity is largely regulated by phosphorylation of myosin regulatory light chain. During the formation of the Drosophila cellular blastoderm, actomyosin contraction drives constriction of microfilament rings, modified cytokinesis rings. This study found that the kinase Death-associated protein kinase related (Drak) is necessary for most of the phosphorylation of myosin regulatory light chain during cellularization. Drak was shown to be required for organization of myosin II within the microfilament rings. Proper actomyosin contraction of the microfilament rings during cellularization also requires Drak activity. Constitutive activation of myosin regulatory light chain bypasses the requirement for Drak, suggesting that actomyosin organization and contraction are mediated through Drak's regulation of myosin activity. Drak also is involved in the maintenance of furrow canal structure and lateral plasma membrane integrity during cellularization. Together, these observations suggest that Drak is the primary regulator of actomyosin dynamics during cellularization (Chougule, 2016).

Tight regulation of actomyosin is likely critical for many cellular processes, but how this is accomplished is as yet poorly understood. A key input to the regulation of myosin II is through phosphorylation of the Serine-19, or the Serine-19 and Threonine-18 residues of MRLC (Spaghetti squash). The variety of MRLC kinases might allow different specific aspects of actomyosin dynamics, such as localization, organization and contraction to be regulated independently. Such a system would provide greater flexibility and control than either a single kinase, or multiple kinases acting in concert, regulating all of these functions. drak was found to be required for the organization of myosin II into contractile rings, but is not required for localization of myosin to the cellularization front. Since the majority of Sqh phosphorylation during cellularization is dependent on drak activity, Drak either regulates most aspects of myosin II dynamics during cellularization, or Drak-regulated myosin II organization is required for further function of myosin II, such as contraction (Chougule, 2016).

Myosin II is somewhat less disorganized and Sqh phosphorylation is slightly increased during late cellularization in drak mutants, suggesting that phosphorylation of myosin II by other kinases occurs during late cellularization. Thus other kinases might act synergistically with Drak to regulate actomyosin organization during late cellularization. For example, Drak function has been shown to be partially redundant with Rok function during later development. An alternative possibility is that other kinases that do not normally function in myosin II organization in the microfilament rings might phosphorylate Sqh to some degree and lead to some organization of myosin II in the absence of Drak activity (Chougule, 2016).

Myosin II has been implicated in actin bundling and F-actin organization in some contexts. Since F-actin appears to be organized normally within drak mutant microfilament rings during early cellularization, it is concluded that myosin II does not play a role in initially organizing F-actin within the microfilament rings during cellularization. F-actin is somewhat disorganized during late cellularization in drakdel mutant embryos, but not as severely as myosin II, nor does the pattern of F-actin distribution fit the pattern of myosin II distribution in drakdel mutant embryos. These observations suggest that F-actin disorganization is an indirect consequence of Drak regulation of myosin II activity, and that F-actin disorganization might be due to actomyosin contraction defects or furrow canal structural defects (Chougule, 2016).

Anillin is required for the organization of actomyosin contractile rings during cellularization and cytokinesis. scraps (scra, anillin) mutant embryos have a myosin II organization defect somewhat similar to that of drak mutant embryos: myosin II is found in discrete bars in the actomyosin network. Despite this similarity, myosin II defects differ between scra and drak mutant embryos. Myosin II becomes more disorganized during late cellularization in scra mutant embryos. Myosin II becomes slightly better organized during late cellularization in drak mutant embryos. This organizational difference is likely caused by actomyosin contraction during microfilament ring constriction occurring in a highly disorganized cytoskeleton in scra mutant embryos, and occurring in a disorganized cytoskeleton that has slightly improved during constriction in drak mutant embryos. Anillin only interacts with myosin II when MRLC is phosphorylated. Together with these results, this suggests that Drak phosphorylation of Sqh might be necessary for Anillin-mediated myosin II organization within the contractile ring (Chougule, 2016).

Phosphorylation of MRLC on Serine-19 or Serine-19 and Threonine-18 leads to the unfolding of inactive myosin II hexamers into an open conformation that allows assembly of bipolar myosin II filaments and their association with F-actin to form actomyosin filaments. This is likely how Drak organizes myosin II. Phosphorylation of MRLC on Serine-19 or Serine-19 and Threonine-18 also leads to the activation of the Mg2+-ATPase activity of myosin II that slides actin filaments past each other, causing actomyosin contraction. Three aspects of the drak mutant phenotype support the requirement for Drak in actomyosin contraction: wavy cellularization fronts caused by non-uniform furrow canal depths, abnormal microfilament ring shapes, and failure of microfilament rings to constrict during late cellularization. These are the same defects that suggest an actomyosin contraction defect in src64 mutant embryos. However, src64 mutant embryos do not show myosin II organization defects. Because effective actomyosin contraction likely requires properly organized actomyosin filaments within the contractile ring apparatus, it is unclear whether Drak directly regulates actomyosin contraction or whether Drak only enables actomyosin contraction through proper organization of myosin II within the microfilament rings. One possibility is that phosphorylation of Sqh by Drak both organizes actomyosin filaments into a contractile ring apparatus and directs actomyosin contraction. An alternative possibility is that Drak is directly responsible for organizing actomyosin filaments into a contractile ring by phosphorylating Sqh, but Drak is not directly involved in its contraction and different kinases that phosphorylate Sqh regulate actomyosin contraction. Thus, Drak could be an early regulator of myosin II activity during cellularization, such that further phosphorylation of Sqh and myosin II-driven contraction is dependent on Drak-mediated organization of myosin II. At some level the regulation of actomyosin contraction diverges from the regulation of actomyosin filament organization: Src64 is required for contraction, but has no role in myosin II organization (Chougule, 2016).

Rescue of myosin II organization, actomyosin contraction and F-actin distribution defects in drak mutant embryos by the mono-phosphorylated SqhE21 phosphomimetic suggests that Drak-mediated mono-phosphorylation of Sqh at Serine-21 is sufficient for regulation of actomyosin dynamics during cellularization. Although the diphosphorylated SqhE20E21 phosphomimetic also rescues myosin II organization and actomyosin contraction defects, it does not rescue F-actin distribution defects in drak mutant embryos. These results are consistent with Drak primarily phosphorylating Sqh at Serine-21, and are consistent with reports that DAPK family members phosphorylate MRLC mainly at Serine-19 (Chougule, 2016).

The normal teardrop shape of the furrow canals in early cellularization is likely caused by actomyosin contraction in the microfilament rings. In drak mutant embryos, unexpanded early cellularization furrow canals and failure of many late cellularization furrow canals to expand further suggest that Drak is required for proper furrow canal structure. Some of the furrow canal structural defects in drak mutant embryos are similar to those of nullo mutant embryos: collapsed furrow canals and blebbing. However, nullo mutant embryos, as well as RhoGEF2 or dia mutant embryos, have other, more severe furrow canal defects: missing or regressing furrow canals and compromised lateral membrane-furrow canal compartment boundaries. Furthermore, cytochalasin treatment causes similar defects, suggesting that reduced F-actin levels in the furrow canals are responsible for these defects. Thus Nullo, RhoGEF2 and Dia regulate F-actin and its levels in furrow canals. These observations suggest that Drak regulates myosin II and thereby regulates actomyosin organization and contraction, and that these are necessary for structural integrity and expansion of the furrow canals, but not for their continued existence (Chougule, 2016).

The furrow canals of drak mutant embryos during late cellularization show extensive blebbing into the lumens. This is consistent with a defect in furrow canal membrane or cortex integrity. Blebs can be formed by local rupture of the cortical cytoskeleton or detachment of the plasma membrane from the cortical actomyosin cytoskeleton. Actomyosin contraction has been implicated in bleb formation. Therefore, it is proposed that blebbing in furrow canals is caused by aberrant localized actomyosin contraction during late cellularization in the disorganized actomyosin cytoskeleton of drak mutant embryos. Contraction is presumably driven by phosphorylation of Sqh by kinases other than Drak. Since actomyosin contraction occurs in a disorganized actomyosin cytoskeleton, it does not lead to uniform constriction of the microfilament rings, but instead leads to localized contraction that produces cytoplasmic blebs. However, other causes for furrow canal defects are possible. Plasma membrane attachment sites might not form or function properly in the disorganized furrow canal cytoskeleton in drak mutant embryos. The disorganized cytoskeleton might inhibit vesicle trafficking. Vesicle trafficking itself might be defective: mammalian DAPKs have been shown to be involved in membrane trafficking and in phosphorylation of syntaxin A1. Vesiculated lateral plasma membrane in drak mutant embryos during late cellularization suggests that the plasma membrane breaks down. Intriguingly, scra mutant embryos have lines of vesicles where the closely apposed lateral plasma membranes would have been. However in scra mutant embryos, vesiculation is observed during early cellularization, but to a lesser extent than during late cellularization. drak mutant embryos do not show lateral plasma membrane vesiculation defects until late cellularization. drak mutant defects in both the furrow canal membrane and the lateral plasma membrane might reflect a general defect in membrane integrity. It will be interesting to investigate the potential role of myosin II organization in furrow canal structure and plasma membrane integrity (Chougule, 2016).

A novel DRAK inhibitor, SC82510, promotes axon branching of adult sensory neurons in vitro

Recently, a new potent protein kinase inhibitor, SC82510, was identified acting on DRAK2 and stimulating axon outgrowth at low concentrations. DRAK is the Drosophila homologue of death-associated protein kinase that phosphorylates myosin-II regulatory light chain in a similar fashion as ROCK, the downstream target of RhoA mediating axon outgrowth inhibition. While higher concentrations of this novel compound exhibited toxic effects, significant promotion of process outgrowth of PC12 cells and of adult primary neurons was observed at 1 nM which could be further enhanced by addition of a neuronal growth factor (FGF-2). Unlike the effects of ROCK inhibitors on axon outgrowth that stimulate both, elongation and branching, SC82510 primarily promoted axon branching, whereas axon elongation was not increased in this cell culture model of peripheral axon regeneration (Marvaldi, 2014).

Overlapping roles of Drosophila Drak and Rok kinases in epithelial tissue morphogenesis

Dynamic regulation of cytoskeletal contractility through phosphorylation of the nonmuscle Myosin-II regulatory light chain (MRLC) provides an essential source of tension for shaping epithelial tissues. Rho GTPase and its effector kinase ROCK have been implicated in regulating MRLC phosphorylation in vivo, but evidence suggests that other mechanisms must be involved. This study reports the identification of a single Drosophila homologue of the Death-associated protein kinase (DAPK) family, called Drak, as a regulator of MRLC phosphorylation. Based on analysis of null mutants, this study found that Drak broadly promotes proper morphogenesis of epithelial tissues during development. Drak activity is largely redundant with that of the Drosophila ROCK orthologue, Rok, such that it is essential only when Rok levels are reduced. These two kinases synergistically promote phosphorylation of Spaghetti squash (Sqh), the Drosophila MRLC orthologue, in vivo. The lethality of drak/rok mutants can be rescued by restoring Sqh activity, indicating that Sqh is the critical common effector of these two kinases. These results provide the first evidence that DAPK family kinases regulate actin dynamics in vivo and identify Drak as a novel component of the signaling networks that shape epithelial tissues (Neubueser, 2010).

Drak and Rok have partially redundant or overlapping functions. Although rok heterozygotes are phenotypically normal, removing just one functional copy of rok in a drak mutant background leads to fully-penetrant lethality. Both drak-/-;rok+/- and drak-/-;rok-/- animals show morphological defects that are not present in either single mutant alone. Both kinases play a role in shaping imaginal disc epithelia, as drak-/-;rok+/- discs show morphological defects and massive apoptosis. Interestingly, mutations in the genes encoding Moesin, a protein that links the actin cytoskeleton to membrane proteins, and its activating kinase Slik both have remarkably similar consequences on disc morphology and apoptosis, reflecting a failure in epithelial integrity (Speck, 2003; Hipfner, 2004). No reductions were observed in Moesin phosphorylation in drak or drak/rok mutants, ruling this out as a possible cause of the defect. It may be that reduced anchoring of the actin cytoskeleton and reduction of actomyosin contractility both have the same consequence on disc morphology, in that both impair the ability of cells to generate tension. drak,rok double mutants also fail to complete head involution and have problems with formation of the tracheal tree. The processes that are impaired in drak,rok mutants (head involution, tracheal morphogenesis, disc eversion) all involve dramatic changes in cell shape. This spectrum of phenotypes suggests that Drak, like Rok, is broadly involved in the dynamic changes in epithelial cell organization that accompany tissue morphogenesis (Neubueser, 2010).

Although Rok appears to be the predominant kinase in many morphogenetic processes, genetic analyses indicate that Drak also compensates for the absence of Rok, though to a lesser extent. For example, the fully-penetrant head involution defect in drak-/-;rok-/- embryos is not observed in rok mutants alone, indicating that Drak can partially substitute for Rok in this process. Similarly, the relatively mild phenotype associated with rok mutant wing imaginal disc clones compared with the dramatic loss of epithelial integrity observed in drak-/-;rok+/- wing discs suggests that drak compensates to a large extent for the loss of Rok in this tissue (Neubueser, 2010).

The synergistic nature of the interaction between drak and rok is reflected at molecular as well as phenotypic levels. In both embryos and wing imaginal discs, simultaneous reduction of drak and rok leads to a synergistic decrease in Sqh phosphorylation. It is this reduction in Sqh phosphorylation rather than misregulation of other potential targets that causes the morphogenetic defects, as expression of the phosphomimetic form of Sqh rescued the viability and morphology of drak-/-;rok+/- animals. It remains to be seen how the activity of Drak and Rok are temporally and spatially integrated to control Sqh activity within cells and more broadly within tissues. Mammalian ROCK1 can phosphorylate ZIPK at two sites in vitro, promoting its catalytic activity. However, neither site is conserved in Drak. The fact that reducing rok gene dosage has such a strong phenotypic effect in the absence of Drak suggests that the primary effect of Rok on Sqh phosphorylation in vivo is unlikely to involve direct regulation of Drak. The synergistic nature of the genetic interactions is more consistent with a model in which Drak and Rok act through parallel but convergent signaling pathways. This convergence could be at the level of several different targets identified as potential substrates of both ROCK and DAPK family kinases in mammals, including Sqh/MRLC, MYPT1, and CPI-17, although the ultimate common effector of both kinases is Sqh/MRLC. Studies of MLCK, ROCK, and DAPK1 in cells suggest that these kinases regulate distinct pools of MRLC. Interestingly, whereas MLCK regulates the formation of cortical actin bundles, both ROCK and DAPK1 control assembly of more centrally-located stress fibers, and overexpression of DAPK1 can overcome cell morphology defects caused by treatment of cells with a ROCK inhibitor. Overlapping subcellular distribution of Rok and Drak could also explain, at least in part, the partial functional redundancy between these kinases (Neubueser, 2010).

The current results represent the first evidence for the involvement of DAPK family kinases in actin cytoskeleton regulation in vivo. Given the ability of mammalian DAPKs to phosphorylate MRLC, it is likely to be a physiologically important target of at least some of the mammalian kinases during tissue morphogenesis as well. However, the results indicate that the analysis of the functions of mammalian DAPKs in vivo may be complicated not only by redundancy between DAPK family members but also with ROCKs (Neubueser, 2010).


Search PubMed for articles about Drosophila Drak

Chougule, A.B., Hastert, M.C. and Thomas, J.H. (2016). Drak is required for actomyosin organization during Drosophila cellularization. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 26818071.

Hipfner, D. R., Keller, N. and Cohen, S. M. (2004). Slik Sterile-20 kinase regulates Moesin activity to promote epithelial integrity during tissue growth. Genes Dev 18: 2243-2248. PubMed ID: 15371338

Marvaldi, L., Hausott, B., Auer, M., Leban, J. and Klimaschewski, L. (2014). A novel DRAK inhibitor, SC82510, promotes axon branching of adult sensory neurons in vitro. Neurochem Res 39: 403-407. PubMed ID: 24407843

Neubueser, D. and Hipfner, D. R. (2010). Overlapping roles of Drosophila Drak and Rok kinases in epithelial tissue morphogenesis. Mol Biol Cell 21: 2869-2879. PubMed ID: 20573980

Robertson, F., Pinal, N., Fichelson, P. and Pichaud, F. (2012). Atonal and EGFR signalling orchestrate rok- and Drak-dependent adherens junction remodelling during ommatidia morphogenesis. Development 139: 3432-3441. PubMed ID: 22874916

Simoes Sde, M., Blankenship, J. T., Weitz, O., Farrell, D. L., Tamada, M., Fernandez-Gonzalez, R. and Zallen, J. A. (2010). Rho-kinase directs Bazooka/Par-3 planar polarity during Drosophila axis elongation. Dev Cell 19: 377-388. PubMed ID: 20833361

Speck, O., Hughes, S. C., Noren, N. K., Kulikauskas, R. M. and Fehon, R. G. (2003). Moesin functions antagonistically to the Rho pathway to maintain epithelial integrity. Nature 421: 83-87. PubMed ID: 12511959

Biological Overview

date revised: 5 April 2016

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