InteractiveFly: GeneBrief

multiple ankyrin repeats single KH domain: Biological Overview | References


Gene name - multiple ankyrin repeats single KH domain

Synonyms - mask

Cytological map position - 95F3-95F5

Function - scaffolding protein

Keywords - Hippo signaling pathway, cofactor of Yorkie, regulation of macroautophagy/autophagy-lysosomal-mediated degradation, genetically interacts with Parkin to modulate mitochondrial morphology, negatively regulates the recruitment of Parkin to mitochondria, promotes autophagic flux by enhancing lysosomal function

Symbol - mask

FlyBase ID: FBgn0043884

Genetic map position - chr3R:24,230,562-24,250,248

Classification - ankyrin repeats, KH domain

Cellular location - nuclear and cytoplasmic



NCBI link: EntrezGene, Nucleotide, Protein
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).

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 mask10.22 was used to generate homozygous mutant eyes with the eyeless.flp FRT Minute system. mask10.22 mutant eyes were much smaller than controls. In the developing larval wing imaginal disc, mask10.22 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 mask10.22 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 mask10.22 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).


REFERENCES

Search PubMed for articles about Drosophila Mask

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


Biological Overview

date revised: 15 January, 2018

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