InteractiveFly: GeneBrief

multiple ankyrin repeats single KH domain: Biological Overview | References

BIOLOGICAL OVERVIEW

This study shows that Mask, an Ankyrin-repeat and KH-domain containing protein, plays a key role in promoting autophagy flux and mitigating degeneration caused by protein aggregation or impaired ubiquitin-proteasome system (UPS) function. In Drosophila eye models of human tauopathy or amyotrophic lateral sclerosis diseases, loss of Mask function enhanced, while gain of Mask function mitigated, eye degenerations induced by eye-specific expression of human pathogenic MAPT/TAU or FUS proteins. The fly larval muscle, a more accessible tissue, was then used to study the underlying molecular mechanisms in vivo. Mask was found to modulate the global abundance of K48- and K63-ubiquitinated proteins by regulating macroautophagy/autophagy-lysosomal-mediated degradation, but not UPS function. Indeed, upregulation of Mask compensated the partial loss of UPS function. It was further demonstrated that Mask promotes autophagic flux by enhancing lysosomal function, and that Mask is necessary and sufficient for promoting the expression levels of the proton-pumping vacuolar (V)-type ATPases in a TFEB-independent manner. Moreover, the beneficial effects conferred by Mask expression on the UPS dysfunction and neurodegenerative models depend on intact autophagy-lysosomal pathway. These findings highlight the importance of lysosome acidification in cellular surveillance mechanisms and establish a model for exploring strategies to mitigate neurodegeneration by boosting lysosomal function (Zhu, 2017).

Misfolded protein aggregates in and outside of cells in the central nervous system are pathological hallmarks of many neurodegenerative disorders including Alzheimer (AD), Parkinson (PD), Huntington (HD) diseases and amyotrophic lateral sclerosis (ALS). Interestingly, many of the aggregated proteins (such as MAPT (TAU) and APP for Alzheimer disease, SNCA/α-synuclein for Parkinson disease, HTT (Huntingtin) for Huntington disease, FUS, SOD1 and TARDBP/TDP-43 for ALS) can serve as seeds for 'prion-like' spreading of the aggregation within and among cells. It is not entirely clear whether these aggregates are the causes or the results of progressive and cell-type-specific neurodegeneration. However, mounting evidence suggests that clearance and prevention of these toxic protein aggregates are beneficial for meliorating degeneration (Zhu, 2017).

Two major pathways collaborate in regulating intracellular protein degradation: the ubiquitin-proteasome system (UPS) and the autophagy-lysosomal system. Under the normal conditions, UPS serves as the primary route for rapid protein turnover while autophagy mainly degrades long-lived proteins and large cellular organelles under basal conditions and can be robustly induced in face of stresses such as starvation, organelle damage or accumulation of misfolded proteins. However when it comes to degradation of damaged proteins in diseased states, autophagy has been shown to play at least an equally important role as UPS.5. Many of the neurodegenerative disease-related proteins are delivered to autophagic vacuoles and degraded by the autophagy pathway. Meanwhile, impairment of autophagy in the mouse brain causes neurodegeneration associated with ubiquitin-positive protein aggregation. These data suggest that UPS and autophagy are both indispensable in maintaining cellular protein homeostasis. Furthermore, recent studies indicate that UPS and autophagy pathways coordinate with each other to prevent accumulation of toxic protein aggregates, so that enhanced activity of one pathway can compensate if the other is compromised (Zhu, 2017).

Both UPS and autophagy degradation systems are complex processes consisting of chains of sequential events orchestrated by a large group of proteins. To understand their coordinated action, it is necessary to identify novel players that are necessary and sufficient to mediate the compensatory function between the two systems. This study shows Mask, a conserved protein with Ankyrin repeats and a KH domain, as a novel and critical player in such a context. Initially identified as a modulator of receptor tyrosine signaling during Drosophila development (Smith, 2002), Mask has recently been shown to function as a cofactor of the Hippo pathway effector Yorkie and together they regulate target gene transcription with another transcription cofactor (Scalloped) during cell proliferation (Sansores-Garcia, 2013; Sidor, 2013). The human ortholog of Mask, ANKHD1, is highly expressed in several cancer cell lines. Loss of mask function rescues the mitochondrial defects and muscle degeneration observed with pink1 and park mutants (Zhu, 2015). This study shows that in MAPT- and FUS-induced eye degeneration fly models, loss of Mask function enhances degeneration, while gain of Mask function suppresses degeneration. By enhancing V-type ATPase expression, Mask promotes lysosome acidification and autophagic flux; Mask is necessary and sufficient to mediate a compensatory effect for partial loss of UPS function, to increase clearance of ubiquitinated proteins, and to protect against degeneration induced by aggregation-prone mutations (Zhu, 2017).

Autophagy, an evolutionarily conserved cellular mechanism that preserves metabolic homeostasis during nutrient unavailability, is traditionally regarded as a self-eating degradative process with limited selectivity. However, mounting evidence suggests that both micro- and macro-autophagy can play cytoprotective roles to specifically target damaged and toxic organelles and proteins for clearance under pathological conditions. The mechanism of selective autophagy is unclear. There is some evidence that autophagy receptors can recognize ubiquitin-dependent and ubiquitin-independent signals for selective degradation. Autophagy is a multistep process including nucleation, autophagosome formation and fusion with lysosomes and each step can be regulated to enhance degradation of damaged cellular components. Research has emerged showing TFEB is a potent regulator of the autophagy-lysosomal pathway whose activation can promote lysosomal function and mitigate disease in a range of neurodegenerative disorders. This study shows that Mask acts in a TFEB-independent manner to boost the expression of V-ATPase subunits. This study provides novel evidence that lysosome function is not only required for the normal clearance of ubiquitinated and misfolded proteins, but its activity can also be boosted potential through enhanced lysosomal acidification, to mitigate cellular degeneration caused by toxic protein aggregation (Zhu, 2017).

Mask is well positioned to regulate lysosome-mediated clearance of ubiquitinated and misfolded proteins. As a positive regulator of several V-type ATPase V1 subunits expression, Mask function is necessary and sufficient to promote lysosomal acidification and autophagosome degradation in a cell-autonomous manner. When the UPS function is impaired, increased Mask expression is sufficient to increase autophagic flux, which in turn compensates the partial loss of the proteasome-mediated degradation. Interestingly, even when UPS function is intact, levels of Mask activity impact the abundance of UPS-dependent (K48) and -independent (such as K63) ubiquitin-conjugated proteins, suggesting that autophagy and lysosome-mediated degradation plays an important role for basal protein homeostasis. Under pathological conditions such as UPS inactivation or excessive accumulation of disease proteins, upregulation of Mask activity substantially suppressed the cellular degeneration phenotypes in both muscles and photoreceptors, potentially through Mask-mediated increase of autophagy and lysosome activities and subsequent degradation of harmful protein aggregates, as suggested by the current biochemical and genetic analyses. In support of this notion, upregulation of Mask promotes autophagic flux in larval muscles, adult eyes and adult brains (Zhu, 2017).

This work in the Drosophila model organism yielded new insight into Mask-mediated cellular protective mechanisms that regulate lysosomal function in normal and stressed conditions caused by misfolding-prone disease proteins or impaired UPS. Such mechanisms may provide a therapeutic approach for the treatment of a group of neurodegenerative disorders caused by intracellular inclusions (Zhu, 2017).

Binding partners of the kinase domains in Drosophila obscurin and their effect on the structure of the flight muscle

Drosophila obscurin (Unc-89) is a titin-like protein in the M-line of the muscle sarcomere. Obscurin has two kinase domains near the C-terminus, both of which are predicted to be inactive. This study has identified proteins binding to the kinase domains. Kinase domain 1 bound Ballchen (Ball, an active kinase), and both kinase domains 1 and 2 bound MASK (a 400-kDa protein with ankyrin repeats). Ball was present in the Z-disc and M-line of the indirect flight muscle (IFM) and was diffusely distributed in the sarcomere. MASK was present in both the M-line and the Z-disc. Reducing expression of Ball or MASK by siRNA resulted in abnormalities in the IFM, including missing M-lines and multiple Z-discs. Obscurin was still present, suggesting that the kinase domains act as a scaffold binding Ball and MASK. Unlike obscurin in vertebrate skeletal muscle, Drosophila obscurin is necessary for the correct assembly of the IFM sarcomere. Ball and MASK act downstream of obscurin, and both are needed for development of a well defined M-line and Z-disc. The proteins have not previously been identified in Drosophila muscle (Katzemich, 2015).

A stable lattice of thick and thin filaments in striated muscle is needed to maintain the optimum register of the filaments as the fibres contract. Thin filaments from neighbouring sarcomeres are anchored in the Z-disc by α-actinin and other cross-linking proteins, and thick filaments are held in position by cross-links at the M-line in the middle of the sarcomere. The register of thick filaments is also maintained by elastic links between the ends of the filaments and the Z-disc. Large modular proteins of the titin family, associated with thick filaments, contribute to both the stability and the stiffness of the sarcomere. These proteins are made up of tandem immunoglobulin (Ig) and fibronectin-like (Fn3) domains and can have one, or sometimes two, kinase domains near the C-terminus, and there can also be signalling domains (Katzemich, 2015).

The M-line protein, obscurin, has a similar modular structure in invertebrates and vertebrates, although the number of modules in different isoforms and the position of the signalling domains vary. Both Unc-89 (the obscurin homologue in Caenorhabditis elegans; note that this protein is also known as Unc-89 in Drosophila) and obscurin in Drosophila have SH3 and Rho-GEF signalling domains near the N-terminus and two kinase domains near the C-terminus. In vertebrate obscurin, the signalling domains are near the C-terminus; the isoform obscurin A has an ankyrin-binding domain instead of the two C-terminal kinase domains in obscurin B. Both these isoforms are at the periphery of myofibrils in the M-line region of mature skeletal fibres. Binding of obscurin A to ankyrins creates a link between the sarcoplasmic reticulum (SR) and the myofibril. By contrast, Drosophila obscurin is found throughout the M-line and there is no ankyrin-binding domain, so direct binding to the SR is unlikely. However, in the nematode, loss-of-function mutations in unc89 result in displaced ryanodine receptor and SERCA, as well as abnormal Ca2+ signalling. This suggests that there is a function for Unc-89 in Ca2+ regulation involving the SR. So far, five large isoforms of obscurin have been identified in Drosophila muscles: one expressed in the larva, and four expressed in the pupa and adult. All these isoforms have Ig domains in the tandem Ig region, and at least the first of the kinase domains (denoted Kin1). The indirect flight muscle (IFM) has two isoforms: a major isoform of 475 kDa and a minor isoform that is somewhat smaller. The two remaining isoforms are in other thoracic muscles. Drosophila obscurin is essential for the formation of an M-line, and for the correct assembly of thick and thin filaments in the sarcomere: lack of obscurin in the IFM results in asymmetrical thick filaments and thin filaments of abnormal length and polarity. Paradoxically, vertebrate obscurin is not necessary for normal sarcomere structure, given that obscurin knockout in the mouse had no serious effect on sarcomere assembly or maintenance (Katzemich, 2015).

The kinase domains of titin-like proteins often function as scaffolds binding other proteins, and might or might not be active kinases. In C. elegans, the Unc-89 kinase 1 domain (PK1) is predicted to be inactive because the ATP-binding site lacks essential residues. The Unc-89 kinase 2 domain (PK2) might be active, although a motif contributing to ATP-binding is atypical. Both Unc-89 kinase domains interact with the protein small, C-terminal domain, phosphatase-like 1 (SCPL-1), which is thought to be involved in muscle-specific signalling. Unc-89 PK1 also interacts with the LIM-domain protein, LIM-9; the complex of PK1, SCPL-1 and LIM-9 links Unc-89 to integrin adhesion sites at the cell surface. In Drosophila, both Obscurin kinase domains (denoted Kin1 and Kin2) are predicted to be inactive because the catalytic site lacks the catalytic aspartate and other crucial residues. Both kinase domains in vertebrate obscurin B are predicted to be catalytically active, and can apparently be auto-phosphorylated. The kinase domains are reported to interact with membrane associated proteins: kinase domain 1 (SK1) with the extracellular domain of a Na+/K+-ATPase at adherens junctions, which is not a substrate, and kinase domain 2 (SK2) with the cell-adhesion molecule, N-cadherin, which is an in vitro substrate (Katzemich, 2015).

The kinase domains in titin-like proteins have sequences at the C-terminus that sterically block the active site (the C-terminal regulatory domain). This sequence can inhibit an active kinase, or regulate ligand binding; it can also be part of the structure of the kinase domain, and necessary to maintain the stability of the domain. Titin-like kinases are linked to stretch-activated signalling pathways in muscle. Mechano-sensing by the kinase can result in changes in the C-terminal regulatory domain and transient binding of ligands to the kinase scaffold. The precise mechanism of regulation varies in different species (Katzemich, 2015).

The aim of this study was to identify proteins binding to the two kinase domains in Drosophila obscurin, and to determine the effect of the proteins on the assembly of an ordered sarcomere in IFM. Ball (a protein kinase) was shown to bind to Kin1, and MASK (an ankyrin repeat protein) binds to both Kin1 and Kin2. The kinase ligands are essential for the formation of an intact M-line and Z-disc in the IFM sarcomere (Katzemich, 2015).

The kinase domains of Drosophila Obscurin differ from those of the C. elegans homologue, Unc89, and the vertebrate protein. Both domains are predicted to be inactive as kinases. However, some pseudokinases can become catalytically active by replacing missing residues with residues from neighbouring domains, or from associated ligands. Pseudokinases commonly act as scaffolds for binding proteins involved in signal transduction, and are often tethered to other domains, including Ig and Fn3 domains, which contribute to the binding site. Titin kinase in the M-line region of vertebrate skeletal muscle forms part of a binding site for the autophagy and kinase scaffold proteins, Nbr1 and SQSTM1, and the ubiquitin ligase, MuRF1 (also known as TRIM63). In the case of MuRF1, the site includes the preceding Ig and Fn3 domains, which will also bind MuRF1 without the kinase domain (Katzemich, 2015).

This study has found that Kin1 in Drosophila Obscurin binds Ball, which has the hallmarks of an active serine-threonine kinase. Ball differs from other kinase molecules in having a long extension C-terminal to the kinase sequence. This extension binds to Kin1 of Obscurin, with or without the flanking Ig and regulatory domains. Given that Ball also binds to the Ig domain alone, it is likely the molecule spans a region of Obscurin that includes the Ig domain as well as the kinase. Binding to Kin1 with the regulatory domain was unexpected. However, it is not clear how much of the sequence downstream of the kinase is included in a possible regulatory domain. In pseudokinases that are part of a larger molecule, the association of the regulatory domain with a ligand-binding site can be altered by force applied to the whole molecule. The regulatory domain of Kin1, taken out of its usual context, might not associate with the active site in the same way as it would in vivo. Alternatively, the regulatory domain might be required to stabilise the kinase structure when the muscle is stretched, as suggested for twitchin kinase (Katzemich, 2015).

Ball is found in the Z-disc of IFM, as well as being diffusely distributed in the sarcomere and in some samples, Ball is also detected at the M-line. Ball might migrate to bind to Kin1 in the M-line when the kinase activity of Ball is needed. There are other examples of protein migration in the muscle sarcomere. A transient translocation from the M-line to the Z-disc and cytoplasm has been observed for titin kinase ligands in cardiac muscle. In zebrafish, the myosin chaperone, Unc-45, is associated with myosin during myofibrillogenesis; in the adult, Unc-45 is in the Z-disc in normal fibres and it migrates to the A-band under conditions of stress, where it transiently associates with myosin again. Similarly, Ball might migrate from the Z-disc to the M-line under some conditions. Although this study has shown that Ball is capable of binding to Kin1 in vivo, the conditions necessary for the association are not yet known. Ball is still present in the IFM of obscurin-knockdown flies, though with a less-ordered distribution in the sarcomere, which suggests there are likely to be other binding partners for Ball (Katzemich, 2015).

Kin2 binds MASK, which has two regions with ankyrin repeats, and a relatively long sequence C-terminal to a KH domain. A peptide near the end of the molecule binds to Kin2 with or without the Fn3 domain preceding the kinase. MASK does not bind to the Fn3 domain alone, nor does it bind to Kin2 with sequence C-terminal to the kinase. It is not clear at present whether the C-terminal sequence acts as a regulatory domain (Katzemich, 2015).

The dual position of MASK in both the Z-disc and the M-line of IFM is unlikely to be due to migration of a protein of 400 kDa. The RNA coding for ankyrin-repeat proteins undergoes extensive alternative splicing, which can alter the binding sites of the different ankyrin isoforms. The independence of the binding sites for MASK in the M-line and Z-disc is confirmed by the finding that in obscurin-knockdown flies, MASK is almost eliminated from the M-line but still present in the Z-disc. Thus, Obscurin is needed for MASK to bind in the M-line, but not to the Z-disc. The presence of Obscurin in the M-line of MASK-knockdown flies is consistent with an Obscurin scaffold that binds MASK (Katzemich, 2015).

In addition to MASK, Tropomyosin-1 was identified as a ligand associated with Kin2 expressed in vivo. As tropomyosin is a thin filament protein, the significance of an association with Obscurin, which is at the midline of the thick filament, is not clear. Smaller isoforms of Obscurin have been detected in IFM, and it is possible that Tropomyosin-1 could bind, outside the M-line, to a small isoform containing a Kin2 domain (Katzemich, 2015).

The effect of downregulating Ball or MASK on the structure of the M-line and Z-disc in IFM shows the importance of these proteins in the development of a regular filament lattice. In the IFM of Ball-knockdown flies, the shifted position of the H-zone and M-line is associated with fragmented Z-discs; where the Z-disc is normal within a sarcomere, the H-zone and M-line are at the midline. The aggregates of multiple Z-discs in the IFM of MASK-knockdown flies dominate the sarcomere and there is no regular H-zone. This phenotype differs from the effect of reducing the expression of obscurin in IFM, where the H-zone and M-line are often shifted from the midline, without a corresponding anomaly in the Z-disc. The difference might be due to the presence of Ball and MASK in the Z-disc, whereas Obscurin is solely in the M-line. The high mortality rate at the larval and pupal stages of flies when either Ball or MASK is reduced differs from the survival of flies with reduced obscurin, which is unaffected in RNAi lines. Evidently, the crucial function of Ball and MASK is in binding to the Z-disc. The association of the proteins with Obscurin in the M-line is not necessary for the performance of most muscles, although it is essential for the development of the precise filament lattice that is needed for the performance of the IFM. The function of Obscurin in the assembly of the IFM sarcomere is not seen in vertebrates. Knockouts of obscurin in the mouse have no effect on the assembly of myosin filaments, Z-disc or M-line, but they do impair the assembly of the SR. Whether or not invertebrate and vertebrate obscurin-like proteins are true homologues (with the same functions, rather than just having a similar patterns of domains) is at present uncertain (Katzemich, 2015).

Ball and MASK are reported to be involved in cell proliferation, growth and differentiation in Drosophila. Ball regulates the proliferation and differentiation of germline stem cells and neuroblasts (Herzig, 2014; Yakulov, 2014). However, it is not clear whether this has any relevance to the function of Ball in Drosophila muscle. The VRK family of kinases (of which Ball is a member) phosphorylate the , which is necessary for the correct assembly of chromatin. Comparison of domains in the sequences of Drosophila Ball (also called NHK-1) and VRK in human, mouse, Xenopus and C. elegans, shows that C. elegans and Drosophila are the only homologues with a long C-terminal sequence extension; the other species have a relatively short stretch of sequence following the kinase domain. This suggests that Ball has a different function in the invertebrates; in Drosophila, the C-terminal sequence binds to Kin1 (Katzemich, 2015).

MASK was first identified in the developing eye of Drosophila, where it is required for proliferation and differentiation of photoreceptors. MASK genetically interacts with Corkscrew (CSW), a protein phosphatase that acts downstream of the epidermal growth factor receptor (EGFR) in a signalling pathway involved in myogenesis in Drosophila. Through these interactions, Obscurin can potentially be linked to a receptor tyrosine kinase (RTK) pathway involved in myogenesis. Obscurin and Kettin are present at an early stage in the development of pupal IFM. Both are large titin-like proteins with tandem Ig domains, which have a dual function in myogenesis and in the mature muscle. Obscurin kinase domains appear to be scaffolds for binding MASK. Ankyrin repeats act as adaptor modules, binding cytoskeletal proteins and signalling molecules. The repeats stabilise protein networks, often together with large structural proteins. Therefore, an interaction between obscurin kinases and MASK could provide a platform for the assembly of signalling proteins, and this could be affected by force on the obscurin molecule. Ankyrin-repeat proteins in vertebrate skeletal muscle (MARPs) interact with the N2A elastic region of titin; in the case of Ankrd2 (also known as Arpp), expression is induced by stretch, and MARPs are thought to be involved in stretch-induced signalling pathways. There are two smaller homologues of MASK in human cells: MASK1 (Ankhd1) and MASK2 (Ankrd17), which have the same domain structure as Drosophila MASK. The Drosophila protein (~400 kDA) is larger than the human proteins (~280 kDA), mainly due to a longer stretch of sequence between the ankyrin repeat domains. MASK is a cofactor of Drosophila Yorkie (Yki) and mammalian Yes-associated protein (YAP) in the Hippo signalling pathway, which controls tissue growth. The signalling function in this pathway is similar for Drosophila and human MASK; however, MASK isoforms have not been found it human muscle. There is a sequence in the C. elegans genome that codes for a protein with homology to Drosophila, mouse and human MASK. The protein, ankyrin repeat and KH domain-containing protein is predicted to be 287 kDa, so similar in size to the human protein. The function is unknown (Katzemich, 2015).

The presence of Ball and MASK in mature IFM suggests the proteins have a signalling function in the adult fly. There is turnover of contractile proteins in Drosophila muscles, including the IFM, throughout the life of a fly. The function of obscurin kinase domains as scaffolds for the assembly of signalling proteins is likely to be important in the continual remodelling of the muscle. During contraction, the M-line experiences shearing stress, due to unbalanced forces in the two half sarcomeres, and the M-line is thought to act as a strain sensor. Ball and MASK might be recruited to the M-line in response to mechanical stress sensed by obscurin. Importantly, mutations in human titin kinase lead to a phenotype (Z-disc streaming) similar to that of Ball and MASK knockdowns, possibly by disrupting protein turnover, which supports the finding that Z-disc abnormalities can be an indirect consequence of mutations in proteins associated with the M-line (Katzemich, 2015).

In summary, this study has identified two proteins, Ball and MASK, that are essential for the assembly of an ordered IFM. The pseudokinase domains of obscurin act as scaffolds binding the proteins. This raises the possibility of investigating the regulation of signalling pathways involved in assembly and maintenance of IFM through interaction with obscurin (Katzemich, 2015).

Mask loss-of-function rescues mitochondrial impairment and muscle degeneration of Drosophila pink1 and parkin mutants

PTEN-induced kinase 1 (Pink1) and ubiquitin E3 ligase Parkin function in a linear pathway to maintain healthy mitochondria via regulating mitochondrial clearance and trafficking. Mutations in the two enzymes cause the familial form of Parkinson's disease (PD) in humans, as well as accumulation of defective mitochondria and cellular degeneration in flies. This study shows that loss of function of a scaffolding protein Mask, also known as ANKHD1 (Ankyrin repeats and KH domain containing protein 1) in humans, rescues the behavioral, anatomical and cellular defects caused by pink1 or parkin mutations in a cell-autonomous manner. Moreover, similar rescue can also be achieved if Mask knock-down is induced in parkin adult flies when the mitochondrial dystrophy is already manifested. It was found that Mask genetically interacts with Parkin to modulate mitochondrial morphology and negatively regulates the recruitment of Parkin to mitochondria. Also, loss of Mask activity promotes co-localization of the autophagosome marker with mitochondria in developing larval muscle, and that an intact autophagy pathway is required for the rescue of parkin mutant defects by mask loss of function. Together, these data strongly suggest that Mask/ANKHD1 activity can be inhibited in a tissue- and timely-controlled fashion to restore mitochondrial integrity under PD-linked pathological conditions (Zhu, 2015).

Recent studies suggest that PINK1 activates Parkin E3 ubiquitin ligase activity by phosphorylating both Parkin and ubiquitin, and that PINK1 recruits Parkin to the damaged mitochondrial membrane, where Parkin ubiquitinates a pool of outer mitochondrial membrane proteins and promotes mitophagy. These data suggest that mitochondrial dysfunction observed in PD may be the result of compromised mitochondrial quality control mechanisms. Therefore, understanding the pathways of mitochondrial quality control holds the key to unravelling the pathogenesis of PD and other disorders associated with mitochondrial dysfunction (Zhu, 2015).

Flies carrying pink1 or parkin mutations show severe mitochondrial morphological and functional defects in multiple tissues as well as age-dependent  dopaminergic (DA) dysfunction, making it a great genetic model to study mechanisms of mitochondrial homeostasis. Using this model system, previous studies in Drosophila have identified a number of pathways that can be manipulated to rescue the parkin and/or pink1 mutant phenotype. First, increasing mitochondrial fission or decreasing fusion rescues the phenotypes of muscle degeneration and mitochondrial abnormalities in pink1 or parkin mutants. However, manipulation of mitochondrial dynamics causes the opposite effect on loss of parkin or pink1 function in mammalian cells, indicating that Pink1 and Parkin may regulate mitochondrial dynamics in a context-dependent manner. Second, promoting mitochondrial electron transport chain CI activity by overexpressing a yeast NADH dehydrogenase, the CI subunit NDUFA10, the GDNF receptor Ret, Sicily, dNK or Trap1 rescue pink1 mutant mitochondrial defects without affecting parkin mutant phenotypes, suggesting a distinct role of Pink1 in regulating CI activity in addition to its role in Parkin-mediated mitophagy (Zhu, 2015).

This study shows that a highly conserved scaffolding protein Mask, whose normal function is to regulate mitochondrial morphology and selectively inhibit mitophagy, can be targeted in a tissue- and temporal-specific manner to suppress both pink1 and parkin mutant defects in Drosophila. It also shows that such a rescue requires the presence of a functional autophagy pathway. Although tissue- and temporal-specific knock-down of Mask was performed with mainly one mask RNAi line, the mask loss-of-function analysis with mask genetic mutants and another independent RNAi line support the same notion that Mask dynamically regulates mitochondrial morphology. Together, these data suggest that enhancing mitochondrial quality control may serve as a common approach to mitigate mitochondrial dysfunction caused by PD-linked genetic mutations. Consistent with this notion, recent studies show that inhibition of deubiquitinases USP30 and USP15 enhances mitochondrial clearance and quality control, and rescues mitochondrial impairment caused by pink1 or parkin mutations (Zhu, 2015).

It was found that loss of mask function enhances the formation of autophagosome surrounding mitochondria. However, the increase of mCherry-ATG8 did not result in significant increase of free mCherry, suggesting the flux of autophagic degradation is not affected. Further studies are required to elucidate the molecular details by which Mask regulates mitochondrial morphology and function. Recent studies on the connection between Mask and the Hippo pathway demonstrates that Mask physically interacts with the Hippo effector Yorkie, and functions as an essential cofactor of Yorkie in promoting downstream target-gene expression. Interestingly, the Yorkie pathway was also shown to regulate mitochondrial structure and function during fly development. Together, these findings bring up an intriguing possibility that Mask and Yorkie together regulate mitochondrial size during development and disease. It was also shown that reducing Mask activity at the relatively progressed stage of parkin-dependent muscle degeneration mitigates the mitochondrial defects and impairs muscle function, indicating that the human Mask homolog ANKHD1 may serve as a potential therapeutic target for treating PD caused by pink1/parkin mutations (Zhu, 2015).

Mask is required for the activity of the Hippo pathway effector Yki/YAP

The Drosophila Yorkie (Yki) protein and its mammalian homolog Yes-associated protein (YAP) are potent growth promoters, and YAP overexpression is associated with multiple types of cancer. Yki and YAP are transcriptional coactivators and function as downstream effectors of the Hippo tumor suppressor pathway. The regulation of Yki and YAP by the Hippo signaling pathway has been extensively investigated; however, how they regulate gene expression is poorly understood. To identify additional regulators of Yki activity, a genome-wide RNAi screen was performed in Drosophila S2 cells. This screen identified the conserved protein Mask (Multiple ankyrin repeats single KH domain) as a novel promoter of Yki activity in vitro and validated this function in vivo in Drosophila. Mask was shown to be required downstream of the Hippo pathway for Yki to induce target-gene expression and that Mask forms complexes with Yki. The human Mask homolog MASK1 complexes with YAP and is required for the full activity of YAP. Additionally, elevated MASK1 expression is associated with worsened outcomes for breast cancer patients. It is conclude that Mask is a novel cofactor for Yki/YAP required for optimal Yki/YAP activity during development and oncogenesis (Sansores-Garcia, 2013).

Mask proteins are cofactors of Yorkie/YAP in the Hippo pathway

The Hippo signaling pathway acts via the Yorkie (Yki)/Yes-associated protein (YAP) transcriptional coactivator family to control tissue growth in both Drosophila and mammals. Yki/YAP drives tissue growth by activating target gene transcription, but how it does so remains unclear. This study identified Mask as a novel cofactor for Yki/YAP. Drosophila Mask forms a complex with Yki and its binding partner, Scalloped (Sd), on target-gene promoters and is essential for Yki to drive transcription of target genes and tissue growth. Furthermore, the stability and subcellular localization of both Mask and Yki is coregulated in response to various stimuli. Finally, Mask proteins are functionally conserved between Drosophila and humans and are coexpressed with YAP in a wide variety of human stem/progenitor cells and tumors (Sidor, 2013).

The Hippo signaling pathway is an emerging tumor suppressor pathway in Drosophila and mice. The key effector of the Hippo pathway is the Drosophila Yorkie (Yki)/mammalian Yes-associated protein (YAP). Yki/YAP is a transcriptional coactivator whose nuclear localization and activity is inhibited upon phosphorylation by the Wts kinase. Yki/YAP is an oncogenic component of the pathway that is sufficient to drive tissue overgrowth when overexpressed in either Drosophila tissues or mouse tissues such as liver and intestine. In addition, YAP is nuclear in human cancer cell lines but translocates to the cytoplasm upon contact inhibition of cell proliferation at confluence. Drosophila Yki acts by binding to transcription factors, such as Scalloped (Sd), Homothorax, and Teashirt, to activate the expression of genes promoting cell proliferation and survival, such as cyclin E, myc, DIAP1, and bantam, as well as genes encoding upstream components of the Hippo pathway, such as expanded, merlin, kibra, and four-jointed . Thus, Yki promotes tissue growth by regulating the transcription of target genes, but how it does so is poorly understood (Sidor, 2013).

In an in vivo RNAi screen based on the Vienna Drosophila RNAi Center library, the mask (CG33106) gene was shown to have a strong undergrowth phenotype in the wing and eye when silenced by expression of an inverted-repeat (IR) hairpin RNAi transgene (mask-IR), as opposed to the effect of wts-IR, which causes tissue overgrowth. To confirm these results, a null mutation in the mask gene mask^10.22 was used to generate homozygous mutant eyes with the eyeless.flp FRT Minute system. mask^10.22 mutant eyes were much smaller than controls. In the developing larval wing imaginal disc, mask^10.22 mutant clones proliferated poorly, growing to only 10% of the size of their wild-type 'twin-spot' clones. These data show that mask is essential for cell proliferation and tissue growth. The mask loss-of-function phenotype is similar to that caused by mutants in the Hippo pathway component yki but distinct from that of other signaling pathways such as the Ras pathway (Sidor, 2013).

Whether mask is required for the expression of Yki target genes was examined. Silencing of mask in the posterior compartment of the wing disc by expression of mask-IR with hh.gal4 led to a significant reduction in the expression of four-jointed.lacZ and DIAP1.lacZ reporters. In mask^10.22 mutant clones, the levels of expression of four-jointed.lacZ, DIAP1.lacZ, and expanded.lacZ were also reduced compared to their levels in wild-type surrounding cells. These results show that mask is required for the expression of Yki target genes (Sidor, 2013).

The mask gene encodes a very large protein of 4,001 amino acids containing two highly conserved ankyrin-repeat domains and a KH domain. The KH domain was first identified in the hnRNP K protein, which was originally found in association with RNA in ribonucleoprotein particles but later found to bind to DNA via its KH domain and to act as a transcriptional cofactor for p53 by facilitating assembly of the transcription factor complex on DNA. Similarly, the NF-κB transcription factor must form a complex with the KH-domain protein RPS3 on certain target genes to drive transcription. Whether Mask could interact with Yki on target-gene promoters was tested. In immunoprecipitation experiments, it was found that the ankyrin-repeat domains of Mask are able to bind to an EGFP-tagged form of Yki. In addition, a DNA pull-down experiment was performed with a biotin-tagged 583 bp fragment of the DIAP1 promoter that contains multiple Sd binding sites. When the DNA is pulled down with streptavidin-coated beads from S2 cells transfected with Yki and Sd, it was found that endogenous Mask binds with Yki and Sd to the DIAP1 promoter, but not to a negative control actin sequence. RNAi knockdown of Sd reduced the binding of Mask to the DIAP1 promoter. These results indicate that Mask is able to complex with Yki/Sd on target-gene promoters (Sidor, 2013).

Henetic epistasis experiments were performed. Overexpression of Yki in clones of cells is sufficient to cause a strong overproliferation phenotype. In contrast, when Yki is overexpressed in mask mutant clones, it is no longer able to induce overproliferation. Overexpressed Yki accumulates in the cytoplasm and nucleus of both wild-type and mask^10.22 mutant cells, indicating that Mask is not essential for Yki to enter the nucleus. The overgrowth of clones mutant for wts or expressing nonphosphorylatable or nuclear-localized Yki is also partially suppressed by mutation of mask. Furthermore, the activation of Yki target-gene transcription in wts mutant clones is suppressed by mutation of mask. These results show that Mask is not required for activation or nuclear localization of Yki but is instead required for Yki to normally activate transcription of its target genes. However, Yki still retains some activity in the absence of Mask, as indicated by the fact that wts, mask double mutants grow larger than mask single mutant clones (Sidor, 2013).

Drosophila Mask has two human homologs, which have been named Mask1 (also called ANKHD1) and Mask2 (also called ANKRD17). Both Mask1 and Mask2 can coimmunoprecipitate with FLAG-tagged YAP. YAP5SA strongly induces CTGF expression in human cells, but not when the cells are cotransfected with siRNAs targeting either YAP or both Mask1 and Mask 2. Mask1 and Mask2 colocalize with YAP in the nucleus of sparsely plated HEK293 cells and cytoplasm of densely confluent HEK293 cells. Translocation of Mask1, Mask2, and YAP was also observed in Caco2 cells. These results show that human Mask proteins bind to and colocalize with YAP to promote target-gene expression. A similar coregulation of Mask and Yki localization and stability was observed in Drosophila. Finally, the expression of YAP, Mask1, and Mask2 was examined in human epithelial tissue sections. The three proteins are strongly expressed in the basal cell layer of epithelial tissues, where stem/progenitor cells are located. A systematic survey of YAP and Mask1 expression confirms that they are coexpressed in a wide variety of human tissues and tumors (Sidor, 2013).

In conclusion, Mask proteins are novel cofactors for Yki/YAP whose function is conserved between Drosophila and humans. Mask acts in a similar fashion to other KH-domain cofactors for NF-κB and p53, being coregulated with their cognate transcription factor in response to signals and assembling with their cognate transcription factor on promoters to drive transcription. Thus, KH-domain cofactors represent a novel class of control mechanism for signal-regulated transcription factors. Given the essential role of Mask in Yki/YAP function and the increasing evidence implicating human YAP in stem cell proliferation and tumorigenesis, human Mask proteins are candidate targets for new cancer therapies (Sidor, 2013).

MASK, a large ankyrin repeat and KH domain-containing protein involved in Drosophila receptor tyrosine kinase signaling

The receptor tyrosine kinases Sevenless (SEV) and the Epidermal growth factor receptor (EGFR) are required for the proper development of the Drosophila eye. The protein tyrosine phosphatase Corkscrew (CSW) is a common component of many RTK signaling pathways, and is required for signaling downstream of SEV and EGFR. In order to identify additional components of these signaling pathways, mutations that enhanced the phenotype of a dominant negative form of Corkscrew were isolated. This genetic screen identified the novel signaling molecule MASK, a large protein that contains two blocks of ankyrin repeats as well as a KH domain. MASK genetically interacts with known components of these RTK signaling pathways. In the developing eye imaginal disc, loss of MASK function generates phenotypes similar to those generated by loss of other components of the SEV and EGFR pathways. These phenotypes include compromised photoreceptor differentiation, cell survival and proliferation. Although MASK is localized predominantly in the cellular cytoplasm, it is not absolutely required for MAPK activation or nuclear translocation. Based on these results, it is proposed that MASK is a novel mediator of RTK signaling, and may act either downstream of MAPK or transduce signaling through a parallel branch of the RTK pathway (Smith, 2002).

Search PubMed for articles about Drosophila Mask

Katzemich, A., West, R. J., Fukuzawa, A., Sweeney, S. T., Gautel, M., Sparrow, J. and Bullard, B. (2015). Binding partners of the kinase domains in Drosophila obscurin and their effect on the structure of the flight muscle. J Cell Sci 128(18): 3386-3397. PubMed ID: 26251439

Sansores-Garcia, L., Atkins, M., Moya, I. M., Shahmoradgoli, M., Tao, C., Mills, G. B. and Halder, G. (2013). Mask is required for the activity of the Hippo pathway effector Yki/YAP. Curr Biol 23(3): 229-235. PubMed ID: 23333314

Sidor, C. M., Brain, R. and Thompson, B. J. (2013). Mask proteins are cofactors of Yorkie/YAP in the Hippo pathway. Curr Biol 23(3): 223-228. PubMed ID: 23333315

Smith, R. K., Carroll, P. M., Allard, J. D. and Simon, M. A. (2002). MASK, a large ankyrin repeat and KH domain-containing protein involved in Drosophila receptor tyrosine kinase signaling. Development 129(1): 71-82. PubMed ID: 11782402

Zhu, M., Li, X., Tian, X. and Wu, C. (2015). Mask loss-of-function rescues mitochondrial impairment and muscle degeneration of Drosophila pink1 and parkin mutants. Hum Mol Genet 24(11): 3272-3285. PubMed ID: 25743185

Zhu, M., Zhang, S., Tian, X. and Wu, C. (2017). Mask mitigates MAPT- and FUS-induced degeneration by enhancing autophagy through lysosomal acidification. Autophagy 14:1-15. PubMed ID: 28806139

date revised: 22 February 2022

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