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Nuak family kinase: Biological Overview | References

The inability to remove protein aggregates in post-mitotic cells such as muscles or neurons is a cellular hallmark of aging cells and is a key factor in the initiation and progression of protein misfolding diseases. While protein aggregate disorders share common features, the molecular level events that culminate in abnormal protein accumulation cannot be explained by a single mechanism. This study shows that loss of the serine/threonine kinase NUAK causes cellular degeneration resulting from the incomplete clearance of protein aggregates in Drosophila larval muscles. In NUAK mutant muscles, regions that lack the myofibrillar proteins F-actin and Myosin heavy chain (MHC) instead contain damaged organelles and the accumulation of select proteins, including Filamin (Fil) (Cheerio) and CryAB. NUAK biochemically and genetically interacts with the cochaperone Starvin (Stv), the ortholog of mammalian Bcl-2-associated athanogene 3 (BAG3). Consistent with a known role for the co-chaperone BAG3 and the Heat shock cognate 71 kDa (HSC70)/HSPA8 ATPase in the autophagic clearance of proteins, RNA interference (RNAi) of Drosophila Stv, Hsc70-4, or autophagy-related 8a (Atg8a) all exhibit muscle degeneration and muscle contraction defects that phenocopy NUAK mutants. It was further demonstrated that Fil/Cheerio is a target of NUAK kinase activity and abnormally accumulates upon loss of the BAG3-Hsc70-4 complex. In addition, Ubiquitin (Ub), ref(2)p/p62, and Atg8a are increased in regions of protein aggregation, consistent with a block in autophagy upon loss of NUAK. Collectively, these results establish a novel role for NUAK with the Stv-Hsc70-4 complex in the autophagic clearance of proteins that may eventually lead to treatment options for protein aggregate diseases (Brooks, 2020).

Proteins must fold into an intrinsic three dimensional structure to perform distinct cellular functions. Denatured or misfolded proteins can be refolded by chaperones or are subject to degradation by the ubiquitin-proteasome system (UPS) and/or the autophagosome-lysosome pathway (ALP). The accumulation of misfolded proteins upon genetic mutation or decreased chaperone function causes protein aggregates that are not effectively cleared by the UPS or the ALP. Environmental insults or aging may exacerbate this accumulation of misfolded proteins, resulting in disease and eventual cell death (Brooks, 2020).

A specialized autophagy pathway, termed chaperone-assisted selective autophagy (CASA), has been verified in both Drosophila and mammalian systems. The CASA complex includes BAG3 in concert with the chaperones HSC70/HSPA8 (HSP70 family), HSPB8 (small HSP family), and the ubiquitin (Ub) ligase CHIP/STUB1. CASA regulates the removal and degradation of Fil from the Z-disc in striated muscle or actin stress fibers in non-muscle cells. The N-terminal actin-binding domain (ABD) in Fil is followed by multiple immunoglobulin (Ig)-like repeats which bind numerous proteins to link the internal cytoskeleton to the sarcolemma. Tension exerted by contractile muscle tissue requires continuous folding and refolding of individual Ig-like domains in Fil, eventually damaging the ability of the protein to sense and transmit mechanical strain (Arndt, 2010; Razinia, 2012). The BAG3-HSC70 protein complex binds to the mechanosensor region (MSR) of Fil and upon detection of protein damage, CHIP ensures the addition of polyubiquitin (polyUb) moieties. Rather than promoting delivery to the proteasome, these Ub chains instead recruit the autophagic Ub adapter protein p62/SQSTM1. p62 interacts with Atg8a/LC3 to induce autophagophore formation and the subsequent clearance of Fil through lysosomal degradation. Fil aggregates and a block in autophagosome-lysosome fusion are present in lysosomal associated membrane protein 2 (LAMP2)-deficient muscles, thus linking impaired autophagy to abnormal protein deposits (Brooks, 2020).

Drosophila NUAK encodes for a conserved serine/threonine kinase that is homologous to the mammalian kinases NUAK1/ARK5 and NUAK2/SNARK. These proteins comprise a family of twelve AMP-activated protein kinase (AMPK)-related kinases (NUAK1 and 2, BRSK 1 and 2, QIK, QSK, SIK, MARK 1-4, and MELK) that share a conserved N-terminal kinase domain activated by the upstream liver kinase B1 (LKB1). NUAK1 and NUAK2 proteins are broadly expressed, but enriched in cardiac and skeletal muscle. Muscle contraction and LKB1 phosphorylation can activate both NUAK proteins. NUAK2 activity is additionally stimulated by oxidative stress, AMP, and glucose deprivation in various cell types. Interestingly, NUAK2 expression increases during muscle differentiation and in response to stress or in aging muscle tissue, whereas dominant-negative (DN)-NUAK2 induces atrophy. Homozygous NUAK1 KO mice are embryonic lethal and <10% of NUAK2 homozygotes survive, precluding analysis of post-embryonic contributions. Because of this embryonic lethality, conditional NUAK1 KO mice were generated to examine muscle function . However, no change was observed in muscle mass or fiber size between control or muscle-specific NUAK1 KO mice, likely due to functional redundancy (Brooks, 2020).

The presence of single NUAK orthologs in worms (Unc-82) or flies (NUAK/CG43143) allows for the study of NUAK protein function without compensation from additional family members that may mask cellular roles. unc-82 associates with Paramyosin and likely Myosin B to promote proper myofilament assembly in C. elegans. The kinase domain in Drosophila NUAK shares 61% identity and 80% similarity to human NUAK1 and NUAK2. In flies, RNAi knockdown of NUAK phenocopies weak Lkb1 defects in regulating cell polarity during ommatidial formation and actin cone formation in spermatogenesis. NUAK kinase targets or additional functions in other tissues have not been reported (Brooks, 2020).

This study identified Drosophila NUAK as a key regulator of autophagic protein clearance in muscle tissue. NUAK physically interacts with and phosphorylates Fil [encoded by Drosophila cheerio (cher)]. NUAK also genetically and biochemically interacts with the Stv-Hsc-70-4 complex and Stv overexpression is sufficient to rescue NUAK-mediated muscle deterioration. The identification of Fil as a cargo protein that abnormally accumulates in muscle tissue deficient for NUAK, Stv, Hsc70-4, and Atg8a links protein aggregation to defects in autophagic disposal (Brooks, 2020).

Prior to this study, few substrates of NUAK kinase activity had been uncovered. One of these is Myosin phosphatase targeting-1 (MYPT1), a regulatory subunit of myosin light-chain phosphatase. Two Drosophila regulatory subunits, MYPT75D and Myosin binding subunit (Mbs), were tested in Stv NUAK sensitized genetic assay and no protein aggregation and/or muscle degeneration was observed. While negative, this data nevertheless argues that this family of phosphatases likely does not function with NUAK in muscle tissue. Since the mammalian NUAK1-MYPT1 interaction was identified in vitro and further validated in HEK293 cells, NUAK likely has cell and tissue-specific targets that regulate diverse biological outputs (Brooks, 2020).

Based upon the discovery of Fil as a novel NUAK substrate, two scenarios are envisioned that are not mutually exclusive to explain the molecular function of NUAK in preventing protein aggregation. First, the increase in sarcomere number upon muscle-specific NUAK RNAi suggests that at least one role of NUAK may be to negatively regulate the addition of proteins (such as Fil) into sarcomeres. This data is consistent with studies that show C. elegans unc-82 regulates myofilament assembly. Notably, one key feature of the misincorporated proteins in unc-82 mutants is their inclusion into aggregate-like structures, similar to the accumulation of Fil and CryAB in NUAK-/- muscles. An additional, or alternative possibility, is that NUAK phosphorylates unfolded or 'damaged' Fil for removal from the sarcomere, thereby triggering the Stv-Hsc70-4 complex to promote autophagic turnover. Thus, proteins such as Fil that fail to get incorporated into sarcomeres and/or sustain damage due to repeated rounds of tension-induced muscle contraction, may destabilize myofilament architecture and trigger abnormal protein (Brooks, 2020).

In both contractile muscle tissue and in adherent cells subjected to mechanical force, BAG3 acts as a hub to coordinate Fil-induced tension-sensing and autophagosome formation. The MSR of Fil is comprised of Ig repeats whose conformational transitions between open and closed states dictate differential protein-protein interactions and biological outputs. While the chaperones Hsc70/HSPA8 and HSPB8 weakly bind to the MSR of Fil, this biochemical interaction is greatly enhanced in the presence of BAG3. Interestingly, BAG3 interacts with Ig repeats 19-21 in the MSR, while the selected interaction domain of NUAK with Fil comprises Ig repeats 15-18. These data suggest that NUAK and Stv each bind to a separate region of the MSR in Fil (Brooks, 2020).

It remains to be determined if NUAK-mediated phosphorylation is a prerequisite for the removal of damaged Fil protein by BAG3. The rescue results suggest that this phosphorylation event is not required as Stv overexpression alleviates protein aggregation and muscle degeneration upon a loss of NUAK. An alternative possibility is that this excess Stv protein is present in sufficient amounts to interact with Fil and overcome the necessity for phosphorylation by NUAK. The inability of NUAK overexpression to restore muscle defects due to knockdown of Stv, Hsc70-4, or Atg8a suggests that NUAK functions upstream or parallel to this pathway. It seems likely that NUAK has additional target substrates for kinase activity that may regulate autophagic protein clearance in muscle tissue (Brooks, 2020).

Recent studies demonstrate that increased autophagic degradation of Fil by BAG3 also induces fil transcription as a compensatory mechanism to ensure steady-state Fil levels. Thus, whether loss of NUAK or Stv alters gene expression upon a block in protein clearance was investigated. While the mRNA levels of cher, CryAB, Hsc70-4, or Atg8a were not altered in NUAK or stv mutants, there was a large increase in p62 transcripts. Thus, this increase in p62 mRNA synthesis may contribute to the elevated p62 protein levels observed upon loss of NUAK or Stv as multiple stress conditions increase p62 transcription, including proteasome inhibition, starvation and atrophic muscle conditions. Data that support a role for an autophagic block include the localization of p62 and Atg8a to regions of protein aggregation (Brooks, 2020).

A model for NUAK is proposed that incorporates these new findings with existing roles for BAG3. Fil and CryAB are physically associated at the Z-disc in Drosophila larval muscle. The phosphorylation of Fil by NUAK may control the incorporation of Fil into the Z-disc during myofibril assembly and/or may be required for the disposal of damaged Fil protein. BAG3 and chaperones such as Hsc70/HSPA8 are thought to monitor the MSR of Fil to detect force-induced damage and to promote the addition of K63-linked polyUb chains. Recruitment of the ubiquitin autophagic adapter p62/SQSTM1 induces autophagosome initiation through the accumulation of Atg8a. Eventual fusion of these autophagosomes with lysosomes promotes protein client complex destruction (Brooks, 2020).

Upon loss of NUAK, excess Fil protein that fails to be incorporated into the Z-disc and/or is damaged due to tension-induced muscle contraction begins to accumulate near the Z-disc. The presence of CryAB in Fil-like aggregates may be due to the normal association of CryAB with Fil at the Z-disc, either to monitor Fil protein damage, or to prevent protein aggregation. It is interesting that while both Fil and CryAB contain actin-binding domains, these associations are lost in NUAK-/- muscle tissue as F-actin is displaced from regions of Fil-CryAB accumulation. At this point it cannot be determined if NUAK preferentially binds to the short (~90kD) and/or long (~240 kD) Fil isoforms since the mapped interaction domains (Ig domains 15-18) are present in both isoforms (Brooks, 2020).

In the initial stages of aggregate formation, nearly all Fil puncta are decorated with Ub. It is hypothesized that the observed decrease in Ub-Fil colocalization in large regions of aggregate formation may be due to intrinsic properties of aggregation-prone proteins whereby protein misfolding triggers aggregation of Fil with itself and other proteins. The accumulation of p62 and circular structures that stain positive for Atg8a in regions of Fil accumulation demonstrate that the autophagosome machinery is recruited to BAG3-client complexes. The absence of lysosomes in these aggregate regions suggest that either fusion and/or transport to sites of degradation are compromised (Brooks, 2020).

CASA-mediated autophagy via the BAG3-client complex includes Hsc70-4/HSPA8, HSPB8, and the E3 ligase CHIP/STUB1, the latter of which ubiquitinates Fil for the subsequent recruitment of p62 to initiate autophagosome formation. However, fibroblasts deficient for CHIP are not defective in autophagy and mice or flies lacking CHIP/STUB1 are viable. A failure to enhance protein aggregation defects upon CHIP RNAi knockdown in the sensitized NUAK+/- or stv+/- backgrounds suggests that additional Ub ligases cooperate with the Stv/BAG3 complex to remove damaged proteins. Future studies will also determine which Drosophila protein is the equivalent of HSPB8 since no genetic interactions were observed with putative CG14207 or Hsp67Bc RNAi lines. This negative data does not rule out the possibility that protein levels are not reduced enough to see phenotypes upon RNAi induction or possible functional redundancy exists between CG14207 and Hsp67Bc (Brooks, 2020).

An interesting hallmark of protein aggregate diseases is the accumulation of specific proteins in affected cells or tissues. Thus, proteins susceptible to aggregation in vivo may possess specific structural characteristics or shared biological functions. This latter feature is evident in a group of protein aggregate diseases termed myofibrillar myopathies (MFM). Laser microdissection of aggregates from normal or affected muscles reveal specificity in the types of proteins that accumulate in patients afflicted with MFMs. Common proteins present in these aggregates include Filamin C (FILC), αB-crystallin (CRYAB), BAG3, and Desmin (DES), among others. The inability of MFM patients to clear these aggregates results in myofibrillar degeneration and a decline in muscle function. Interestingly, mutations in Drosophila NUAK phenocopy both structural and functional deficits observed in MFM patients, including Fil and CryAB accumulation, muscle degeneration, and locomotor defects. The discovery of cellular degeneration and protein aggregation in muscle tissue upon loss of the single fly NUAK ortholog highlights the power of Drosophila as a model. Future studies will focus on identifying kinase targets of NUAK and defining additional proteins that function in NUAK and stv-mediated autophagy for the eventual development of therapeutic targets to treat MFMs and other protein aggregate diseases (Brooks, 2020).

Drosophila LKB1 is required for the assembly of the polarized actin structure that allows spermatid individualization

In mammals, a testis-specific isoform of the protein kinase LKB1 is required for spermiogenesis, but its exact function and specificity are not known. Human LKB1 rescues the functions of Drosophila Lkb1 essential for viability, but these males are sterile, revealing a new function for this genes in fly. A testis-specific transcript was identified, generated by an alternative promoter; it only differs by a longer 5'UTR. dLKB1 is required in the germline for the formation of the actin cone, the polarized structure that allows spermatid individualization and cytoplasm excess extrusion during spermiogenesis. Three of the nine LKB1 classical targets in the Drosophila genome (AMPK, NUAK and KP78b) are required for proper spermiogenesis, but later than dLKB1. dLkb1 mutant phenotype is reminiscent of that of myosin V mutants, and both proteins show a dynamic localization profile before actin cone formation. Together, these data highlight a new dLKB1 function and suggest that dLKB1 posttranscriptional regulation in testis and involvement in spermatid morphogenesis are evolutionarily conserved features (Couderc, 2017).

Search PubMed for articles about Drosophila Nuak

Arndt, V., Dick, N., Tawo, R., Dreiseidler, M., Wenzel, D., Hesse, M., Furst, D. O., Saftig, P., Saint, R., Fleischmann, B. K., Hoch, M. and Hohfeld, J. (2010). Chaperone-assisted selective autophagy is essential for muscle maintenance. Curr Biol 20(2): 143-148. PubMed ID: 20060297

Banerjee, S., Zagorska, A., Deak, M., Campbell, D. G., Prescott, A. R. and Alessi, D. R. (2014). Interplay between Polo kinase, LKB1-activated NUAK1 kinase, PP1betaMYPT1 phosphatase complex and the SCFbetaTrCP E3 ubiquitin ligase. Biochem J 461(2): 233-245. PubMed ID: 24785407

Brooks, D., Naeem, F., Stetsiv, M., Goetting, S. C., Bawa, S., Green, N., Clark, C., Bashirullah, A. and Geisbrecht, E. R. (2020). Drosophila NUAK functions with Starvin/BAG3 in autophagic protein turnover. PLoS Genet 16(4): e1008700. PubMed ID: 32320396

Chen, Y., Xie, X., Wang, C., Hu, Y., Zhang, H., Zhang, L., Tu, S., He, Y. and Li, Y. (2020). Dual targeting of NUAK1 and ULK1 using the multitargeted inhibitor MRT68921 exerts potent antitumor activities. Cell Death Dis 11(8): 712. PubMed ID: 32873786

Couderc, J. L., Richard, G., Vachias, C. and Mirouse, V. (2017). Drosophila LKB1 is required for the assembly of the polarized actin structure that allows spermatid individualization. PLoS One 12(8): e0182279. PubMed ID: 28767695

Hoppe, P. E., Chau, J., Flanagan, K. A., Reedy, A. R. and Schriefer, L. A. (2010). Caenorhabditis elegans unc-82 encodes a serine/threonine kinase important for myosin filament organization in muscle during growth. Genetics 184(1): 79-90. PubMed ID: 19901071

Inazuka, F., Sugiyama, N., Tomita, M., Abe, T., Shioi, G. and Esumi, H. (2012). Muscle-specific knock-out of NUAK family SNF1-like kinase 1 (NUAK1) prevents high fat diet-induced glucose intolerance. J Biol Chem 287(20): 16379-16389. PubMed ID: 22418434

Kolliopoulos, C., Raja, E., Razmara, M., Heldin, P., Heldin, C. H., Moustakas, A. and van der Heide, L. P. (2019). Transforming growth factor beta (TGFbeta) induces NUAK kinase expression to fine-tune its signaling output. J Biol Chem 294(11): 4119-4136. PubMed ID: 30622137

Razinia, Z., Makela, T., Ylanne, J. and Calderwood, D. A. (2012). Filamins in mechanosensing and signaling. Annu Rev Biophys 41: 227-246. PubMed ID: 22404683

Schiller, N. R., Duchesneau, C. D., Lane, L. S., Reedy, A. R., Manzon, E. R. and Hoppe, P. E. (2017). The Role of the unc-82 Protein Kinase in Organizing Myosin Filaments in Striated Muscle of Caenorhabditis elegans. Genetics 205(3): 1195-1213. PubMed ID: 28040740

date revised: 20 October 2020

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