LRRK1-mediated NDEL1 phosphorylation promotes cilia disassembly via dynein-2-driven retrograde intraflagellar transport.

Primary cilia are antenna-like organelles that regulate growth and development via extracellular signals. However, the molecular mechanisms underlying cilia dynamics, particularly those regulating their disassembly, are not well understood. Here, we show that leucine-rich repeat kinase 1 (LRRK1) plays a role in regulating cilia disassembly. The depletion of LRRK1 impairs primary cilia resorption following serum stimulation in cultured cells. Polo-like kinase 1 (PLK1) plays an important role in this process. During ciliary resorption, PLK1 phosphorylates LRRK1 at the primary cilia base, resulting in its activation. We identified nuclear distribution protein nudE-like 1 (NDEL1), which is known to positively regulate cilia disassembly, as a target of LRRK1 phosphorylation. Whereas LRRK1 phosphorylation of NDEL1 on Ser-155 promotes NDEL1 interaction with the intermediate chains of cytoplasmic dynein-2, it is also crucial for triggering ciliary resorption through dynein-2-driven retrograde intraflagellar transport. These findings provide evidence that a novel PLK1-LRRK1-NDEL1 pathway regulates cilia disassembly.

Aurora A and AKT Kinase Signaling Associated with Primary Cilia.

Dysregulation of kinase signaling is associated with various pathological conditions, including cancer, inflammation, and autoimmunity; consequently, the kinases involved have become major therapeutic targets. While kinase signaling pathways play crucial roles in multiple cellular processes, the precise manner in which their dysregulation contributes to disease is dependent on the context; for example, the cell/tissue type or subcellular localization of the kinase or substrate. Thus, context-selective targeting of dysregulated kinases may serve to increase the therapeutic specificity while reducing off-target adverse effects. Primary cilia are antenna-like structures that extend from the plasma membrane and function by detecting extracellular cues and transducing signals into the cell. Cilia formation and signaling are dynamically regulated through context-dependent mechanisms; as such, dysregulation of primary cilia contributes to disease in a variety of ways. Here, we review the involvement of primary cilia-associated signaling through aurora A and AKT kinases with respect to cancer, obesity, and other ciliopathies.

Primary cilia and lipid raft dynamics.

Primary cilia, antenna-like structures of the plasma membrane, detect various extracellular cues and transduce signals into the cell to regulate a wide range of functions. Lipid rafts, plasma membrane microdomains enriched in cholesterol, sphingolipids and specific proteins, are also signalling hubs involved in a myriad of physiological functions. Although impairment of primary cilia and lipid rafts is associated with various diseases, the relationship between primary cilia and lipid rafts is poorly understood. Here, we review a newly discovered interaction between primary cilia and lipid raft dynamics that occurs during Akt signalling in adipogenesis. We also discuss the relationship between primary cilia and lipid raft-mediated Akt signalling in cancer biology. This review provides a novel perspective on primary cilia in the regulation of lipid raft dynamics.

Primary cilia-dependent lipid raft/caveolin dynamics regulate adipogenesis.

Primary cilia play a pivotal role in signal transduction and development and are known to serve as signaling hubs. Recent studies have shown that primary cilium dysfunction influences adipogenesis, but the mechanisms are unclear. Here, we show that mesenchymal progenitors C3H10T1/2 depleted of trichoplein, a key regulator of cilium formation, have significantly longer cilia than control cells and fail to differentiate into adipocytes. Mechanistically, the elongated cilia prevent caveolin-1- and/or GM3-positive lipid rafts from being assembled around the ciliary base where insulin receptor proteins accumulate, thereby inhibiting the insulin-Akt signaling. We further generate trichoplein knockout mice, in which adipogenic progenitors display elongated cilia and impair the lipid raft dynamics. The knockout mice on an extended high-fat diet exhibit reduced body fat and smaller adipocytes than wild-type (WT) mice. Overall, our results suggest a role for primary cilia in regulating adipogenic signal transduction via control of the lipid raft dynamics around cilia.

[Targeting the ubiquitin system for treatment of cilia-related diseases].

The ubiquitin system regulates a wide variety of cellular functions. Not surprisingly, dysregulation of the ubiquitin system is associated with various disorders. Therefore, drugs that can modulate the functions of the ubiquitin system have been actively developed to treat these disorders. Chemical knockdown of pathogenic proteins using the ubiquitin-proteasome system is also a promising approach. The ubiquitin system regulates the assemble and disassemble of primary cilia through balanced control over the ubiquitination and deubiquitination of ciliary proteins. Primary cilia are antenna-like structures present in many vertebrate cells that sense and transduce extracellular cues to control cellular processes such as proliferation and differentiation. Impairment of primary cilia is associated with many diseases, including cancer and ciliopathy, a group of multisystem developmental disorders. In this review, we focus on the role of the ubiquitin system on cilia-related disorders and discuss the possibility of the ubiquitin system as therapeutic targets for these diseases through regulation of primary cilia formation.

Targeting E3 Ubiquitin Ligases and Deubiquitinases in Ciliopathy and Cancer.

Cilia are antenna-like structures present in many vertebrate cells. These organelles detect extracellular cues, transduce signals into the cell, and play an essential role in ensuring correct cell proliferation, migration, and differentiation in a spatiotemporal manner. Not surprisingly, dysregulation of cilia can cause various diseases, including cancer and ciliopathies, which are complex disorders caused by mutations in genes regulating ciliary function. The structure and function of cilia are dynamically regulated through various mechanisms, among which E3 ubiquitin ligases and deubiquitinases play crucial roles. These enzymes regulate the degradation and stabilization of ciliary proteins through the ubiquitin-proteasome system. In this review, we briefly highlight the role of cilia in ciliopathy and cancer; describe the roles of E3 ubiquitin ligases and deubiquitinases in ciliogenesis, ciliopathy, and cancer; and highlight some of the E3 ubiquitin ligases and deubiquitinases that are potential therapeutic targets for these disorders.

Cdc7 kinase stimulates Aurora B kinase in M-phase.

The conserved serine-threonine kinase, Cdc7, plays a crucial role in initiation of DNA replication by facilitating the assembly of an initiation complex. Cdc7 is expressed at a high level and exhibits significant kinase activity not only during S-phase but also during G2/M-phases. A conserved mitotic kinase, Aurora B, is activated during M-phase by association with INCENP, forming the chromosome passenger complex with Borealin and Survivin. We show that Cdc7 phosphorylates and stimulates Aurora B kinase activity in vitro. We identified threonine-236 as a critical phosphorylation site on Aurora B that could be a target of Cdc7 or could be an autophosphorylation site stimulated by Cdc7-mediated phosphorylation elsewhere. We found that threonines at both 232 (that has been identified as an autophosphorylation site) and 236 are essential for the kinase activity of Aurora B. Cdc7 down regulation or inhibition reduced Aurora B activity in vivo and led to retarded M-phase progression. SAC imposed by paclitaxel was dramatically reversed by Cdc7 inhibition, similar to the effect of Aurora B inhibition under the similar situation. Our data show that Cdc7 contributes to M-phase progression and to spindle assembly checkpoint most likely through Aurora B activation.

Primary Cilia as Signaling Hubs in Health and Disease.

Primary cilia detect extracellular cues and transduce these signals into cells to regulate proliferation, migration, and differentiation. Here, the function of primary cilia as signaling hubs of growth factors and morphogens is in focus. First, the molecular mechanisms regulating the assembly and disassembly of primary cilia are described. Then, the role of primary cilia in mediating growth factor and morphogen signaling to maintain human health and the potential mechanisms by which defects in these pathways contribute to human diseases, such as ciliopathy, obesity, and cancer are described. Furthermore, a novel signaling pathway by which certain growth factors stimulate cell proliferation through suppression of ciliogenesis is also described, suggesting novel therapeutic targets in cancer.

Albatross/FBF1 contributes to both centriole duplication and centrosome separation.

The centrosome is a small but important organelle that participates in centriole duplication, spindle formation, and ciliogenesis. Each event is regulated by key enzymatic reactions, but how these processes are integrated remains unknown. Recent studies have reported that ciliogenesis is controlled by distal appendage proteins such as FBF1, also known as Albatross. However, the precise role of Albatross in the centrosome cycle, including centriole duplication and centrosome separation, remains to be determined. Here, we report a novel function for Albatross at the proximal ends of centrioles. Using Albatross monospecific antibodies, full-length constructs, and siRNAs for rescue experiments, we found that Albatross mediates centriole duplication by recruiting HsSAS-6, a cartwheel protein of centrioles. Moreover, Albatross participates in centrosome separation during mitosis by recruiting Plk1 to residue S348 of Albatross after its phosphorylation. Taken together, our results show that Albatross is a novel protein that spatiotemporally integrates different aspects of centrosome function, namely ciliogenesis, centriole duplication, and centrosome separation.

Erbin Suppresses KSR1-Mediated RAS/RAF Signaling and Tumorigenesis in Colorectal Cancer.

Erbin belongs to the LAP (leucine-rich repeat and PDZ domain) family of scaffolding proteins that plays important roles in orchestrating cell signaling. Here, we show that Erbin functions as a tumor suppressor in colorectal cancer. Analysis of Erbin expression in colorectal cancer patient specimens revealed that Erbin was downregulated at both mRNA and protein levels in tumor tissues. Knockdown of Erbin disrupted epithelial cell polarity and increased cell proliferation in 3D culture. In addition, silencing Erbin resulted in increased amplitude and duration of signaling through Akt and RAS/RAF pathways. Erbin loss induced epithelial-mesenchymal transition, which coincided with a significant increase in cell migration and invasion. Erbin interacted with kinase suppressor of Ras 1 (KSR1) and displaced it from the RAF/MEK/ERK complex to prevent signal propagation. Furthermore, genetic deletion of Erbin in Apc knockout mice promoted tumorigenesis and significantly reduced survival. Tumor organoids derived from Erbin/Apc double knockout mice displayed increased tumor initiation potential and activation of Wnt signaling. Results from gene set enrichment analysis revealed that Erbin expression associated positively with the E-cadherin adherens junction pathway and negatively with Wnt signaling in human colorectal cancer. Taken together, our study identifies Erbin as a negative regulator of tumor initiation and progression by suppressing Akt and RAS/RAF signaling in vivoSignificance: These findings establish the scaffold protein Erbin as a negative regulator of EMT and tumorigenesis in colorectal cancer through direct suppression of Akt and RAS/RAF signaling. Cancer Res; 78(17); 4839-52. ©2018 AACR.

Tetraploidy in cancer and its possible link to aging.

Tetraploidy, a condition in which a cell has four homologous sets of chromosomes, is often seen as a natural physiological condition but is also frequently seen in pathophysiological conditions such as cancer. Tetraploidy facilitates chromosomal instability (CIN), which is an elevated level of chromosomal loss and gain that can cause production of a wide variety of aneuploid cells that carry structural and numerical aberrations of chromosomes. The resultant genomic heterogeneity supposedly expedites karyotypic evolution that confers oncogenic potential in spite of the reduced cellular fitness caused by aneuploidy. Recent studies suggest that tetraploidy might also be associated with aging; mice with mutations in an intermediate filament protein have revealed that these tetraploidy-prone mice exhibit tissue disorders associated with aging. Cellular senescence and its accompanying senescence-associated secretory phenotype have now emerged as critical factors that link tetraploidy and tetraploidy-induced CIN with cancer, and possibly with aging. Here, we review recent findings about how tetraploidy is related to cancer and possibly to aging, and discuss underlying mechanisms of the relationship, as well as how we can exploit the properties of cells exhibiting tetraploidy-induced CIN to control these pathological conditions.

EGF receptor kinase suppresses ciliogenesis through activation of USP8 deubiquitinase.

Ciliogenesis is generally inhibited in dividing cells, however, it has been unclear which signaling cascades regulate the phenomenon. Here, we report that epidermal growth factor receptor (EGFR) kinase suppresses ciliogenesis by directly phosphorylating the deubiquitinase USP8 on Tyr-717 and Tyr-810 in RPE1 cells. These phosphorylations elevate the deubiquitinase activity, which then stabilizes the trichoplein-Aurora A pathway, an inhibitory mechanism of ciliogenesis. EGFR knockdown and serum starvation result in ciliogenesis through downregulation of the USP8-trichoplein-Aurora A signal. Moreover, primary cilia abrogation, which is induced upon IFT20 or Cep164 depletion, ameliorates the cell cycle arrest of EGFR knockdown cells. The present data reveal that the EGFR-USP8-trichoplein-Aurora A axis is a critical signaling cascade that restricts ciliogenesis in dividing cells, and functions to facilitate cell proliferation. We further show that usp8 knockout zebrafish develops ciliopathy-related phenotypes including cystic kidney, suggesting that USP8 is a regulator of ciliogenesis in vertebrates.

Sphingosine 1-phosphate signaling through its receptor S1P5 promotes chromosome segregation and mitotic progression.

Sphingosine kinase 1 (SphK1) promotes cell proliferation and survival, and its abundance is often increased in tumors. SphK1 produces the signaling lipid sphingosine 1-phosphate (S1P), which activates signaling cascades downstream five G protein-coupled receptors (S1P1-5) to modulate vascular and immune system function and promote proliferation. We identified a new function of the SphK1-S1P pathway specifically in the control of mitosis. SphK1 depletion in HeLa cells caused prometaphase arrest, whereas its overexpression or activation accelerated mitosis. Increasing the abundance of S1P promoted mitotic progression, overrode the spindle assembly checkpoint (SAC), and led to chromosome segregation defects. S1P was secreted through the transporter SPNS2 and stimulated mitosis by binding to and activating S1P5 on the extracellular side, which then activated the intracellular phosphatidylinositol 3-kinase (PI3K)-AKT pathway. Knockdown of S1P5 prevented the S1P-induced spindle defect phenotype. RNA interference assays revealed that the mitotic kinase Polo-like kinase 1 (PLK1) was an important effector of S1P-S1P5 signaling-induced mitosis in HeLa cells. Our findings identify an extracellular signal and the downstream pathway that promotes mitotic progression and may indicate potential therapeutic targets to inhibit the proliferation of cancer cells.

Mechanisms of ciliogenesis suppression in dividing cells.

The primary cilium is a non-motile and microtubule-enriched protrusion ensheathed by plasma membrane. Primary cilia function as mechano/chemosensors and signaling hubs and their disorders predispose to a wide spectrum of human diseases. Most types of cells assemble their primary cilia in response to cellular quiescence, whereas they start to retract the primary cilia upon cell-cycle reentry. The retardation of ciliary resorption process has been shown to delay cell-cycle progression to the S or M phase after cell-cycle reentry. Apart from this conventional concept of ciliary disassembly linked to cell-cycle reentry, recent studies have led to a novel concept, suggesting that cells can suppress primary cilia assembly during cell proliferation. Accumulating evidence has also demonstrated the importance of Aurora-A (a protein originally identified as one of mitotic kinases) not only in ciliary resorption after cell-cycle reentry but also in the suppression of ciliogenesis in proliferating cells, whereas Aurora-A activators are clearly distinct in both phenomena. Here, we summarize the current knowledge of how cycling cells suppress ciliogenesis and compare it with mechanisms underlying ciliary resorption after cell-cycle reentry. We also discuss a reciprocal relationship between primary cilia and cell proliferation.

Desmin phosphorylation by Cdk1 is required for efficient separation of desmin intermediate filaments in mitosis and detected in murine embryonic/newborn muscle and human rhabdomyosarcoma tissues.

Desmin is a type III intermediate filament (IF) component protein expressed specifically in muscular cells. Desmin is phosphorylated by Aurora-B and Rho-kinase specifically at the cleavage furrow from anaphase to telophase. The disturbance of this phosphorylation results in the formation of unusual long bridge-like IF structures (IF-bridge) between two post-mitotic (daughter) cells. Here, we report that desmin also serves as an excellent substrate for the other type of mitotic kinase, Cdk1. Desmin phosphorylation by Cdk1 loses its ability to form IFs in vitro. We have identified Ser6, Ser27, and Ser31 on murine desmin as phosphorylation sites for Cdk1. Using a site- and phosphorylation-state-specific antibody for Ser31 on desmin, we have demonstrated that Cdk1 phosphorylates desmin in entire cytoplasm from prometaphase to metaphase. Desmin mutations at Cdk1 sites exhibit IF-bridge phenotype, the frequency of which is significantly increased by the addition of Aurora-B and Rho-kinase site mutations to Cdk1 site mutations. In addition, Cdk1-induced desmin phosphorylation is detected in mitotic muscular cells of murine embryonic/newborn muscles and human rhabdomyosarcoma specimens. Therefore, Cdk1-induced desmin phosphorylation is required for efficient separation of desmin-IFs and generally detected in muscular mitotic cells in vivo.

Ndel1 suppresses ciliogenesis in proliferating cells by regulating the trichoplein-Aurora A pathway.

Primary cilia protrude from the surface of quiescent cells and disassemble at cell cycle reentry. We previously showed that ciliary reassembly is suppressed by trichoplein-mediated Aurora A activation pathway in growing cells. Here, we report that Ndel1, a well-known modulator of dynein activity, localizes at the subdistal appendage of the mother centriole, which nucleates a primary cilium. In the presence of serum, Ndel1 depletion reduces trichoplein at the mother centriole and induces unscheduled primary cilia formation, which is reverted by forced trichoplein expression or coknockdown of KCTD17 (an E3 ligase component protein for trichoplein). Serum starvation induced transient Ndel1 degradation, subsequent to the disappearance of trichoplein at the mother centriole. Forced expression of Ndel1 suppressed trichoplein degradation and axonemal microtubule extension during ciliogenesis, similar to trichoplein induction or KCTD17 knockdown. Most importantly, the proportion of ciliated and quiescent cells was increased in the kidney tubular epithelia of newborn Ndel1-hypomorphic mice. Thus, Ndel1 acts as a novel upstream regulator of the trichoplein-Aurora A pathway to inhibit primary cilia assembly.

Phospho-Specific Antibody Probes of Intermediate Filament Proteins.

Intermediate filaments (IFs) form one of the major cytoskeletal systems in the cytoplasm or beneath the nuclear membrane. Accumulating data have suggested that IF protein phosphorylation dramatically changes IF structure/dynamics in cells. For the production of an antibody recognizing site-specific protein phosphorylation (a site- and phosphorylation state-specific antibody), we first employed a strategy to immunize animals with an in vitro-phosphorylated polypeptide or a phosphopeptide (corresponding to a phosphorylated residue and its surrounding sequence of amino acids), instead of a phosphorylated protein. Our established methodology not only improves the chance of obtaining a phospho-specific antibody but also has the advantage that one can predesign a targeted phosphorylation site. It is now applied to the production of an antibody recognizing other types of site-specific posttranslational modification, such as acetylation or methylation. The use of such an antibody in immunocytochemistry enables us to analyze spatiotemporal distribution of site-specific IF protein phosphorylation. The antibody is of great use to identify a protein kinase responsible for in vivo IF protein phosphorylation and to monitor intracellular kinase activities through IF protein phosphorylation. Here, we present an overview of our methodology and describe stepwise approaches for the antibody characterization. We also provide some examples of analyses for IF protein phosphorylation involved in mitosis and signal transduction.

Vimentin Phosphorylation Underlies Myofibroblast Sensitivity to Withaferin A In Vitro and during Corneal Fibrosis.

Vimentin is a newly recognized target for corneal fibrosis. Using primary rabbit corneal fibroblasts and myofibroblasts we show that myofibroblasts, unlike fibroblasts, display impaired cell spreading and cell polarization, which is associated with increased levels of soluble serine-38 phosphorylated vimentin (pSer38Vim). This pSer38Vim isoform is inefficiently incorporated into growing vimentin intermediate filaments (IFs) of myofibroblasts during cell spreading, and as a result, myofibroblasts maintain higher soluble pSer38Vim levels compared to fibroblasts. Moreover, the soluble vimentin-targeting small molecule and fibrotic inhibitor withaferin A (WFA) causes a potent blockade of cell spreading selectively in myofibroblasts by targeting soluble pSer38Vim for hyperphosphorylation. WFA treatment does not induce vimentin hyperphosphorylation in fibroblasts. This hyperphosphorylated pSer38Vim species in WFA-treated myofibroblasts becomes complexed with adaptor protein filamin A (FlnA), and these complexes appear as short squiggles when displaced from focal adhesions. The extracellular-signal regulated kinase (ERK) is also phosphorylated (pERK) in response to WFA, but surprisingly, pERK does not enter the nucleus but remains bound to pSer38Vim in cytoplasmic complexes. Using a model of corneal alkali injury, we show that fibrotic corneas of wild type mice possess high levels of pERK, whereas injured corneas of vimentin-deficient (Vim KO) mice that heal with reduced fibrosis have highly reduced pERK expression. Finally, WFA treatment causes a decrease in pERK and pSer38Vim expression in healing corneas of wild type mice. Taken together, these findings identify a hereto-unappreciated role for pSer38Vim as an important determinant of myofibroblast sensitivity to WFA.

Cytokinetic Failure-induced Tetraploidy Develops into Aneuploidy, Triggering Skin Aging in Phosphovimentin-deficient Mice.

Tetraploidy, a state in which cells have doubled chromosomal sets, is observed in ∼20% of solid tumors and is considered to frequently precede aneuploidy in carcinogenesis. Tetraploidy is also detected during terminal differentiation and represents a hallmark of aging. Most tetraploid cultured cells are arrested by p53 stabilization. However, the fate of tetraploid cells in vivo remains largely unknown. Here, we analyze the ability to repair wounds in the skin of phosphovimentin-deficient (VIM(SA/SA)) mice. Early into wound healing, subcutaneous fibroblasts failed to undergo cytokinesis, resulting in binucleate tetraploidy. Accordingly, the mRNA level of p21 (a p53-responsive gene) was elevated in a VIM(SA/SA)-specific manner. Disappearance of tetraploidy coincided with an increase in aneuploidy. Thereafter, senescence-related markers were significantly elevated in VIM(SA/SA) mice. Because our tetraploidy-prone mouse model also exhibited subcutaneous fat loss at the age of 14 months, another premature aging phenotype, our data suggest that following cytokinetic failure, a subset of tetraploid cells enters a new cell cycle and develops into aneuploid cells in vivo, which promote premature aging.

Sphingolipids inhibit vimentin-dependent cell migration.

The sphingolipids, sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), can induce or inhibit cellular migration. The intermediate filament protein vimentin is an inducer of migration and a marker for epithelial-mesenchymal transition. Given that keratin intermediate filaments are regulated by SPC, with consequences for cell motility, we wanted to determine whether vimentin is also regulated by sphingolipid signalling and whether it is a determinant for sphingolipid-mediated functions. In cancer cells where S1P and SPC inhibited migration, we observed that S1P and SPC induced phosphorylation of vimentin on S71, leading to a corresponding reorganization of vimentin filaments. These effects were sphingolipid-signalling-dependent, because inhibition of either the S1P2 receptor (also known as S1PR2) or its downstream effector Rho-associated kinase (ROCK, for which there are two isoforms ROCK1 and ROCK2) nullified the sphingolipid-induced effects on vimentin organization and S71 phosphorylation. Furthermore, the anti-migratory effect of S1P and SPC could be prevented by expressing S71-phosphorylation-deficient vimentin. In addition, we demonstrated, by using wild-type and vimentin-knockout mouse embryonic fibroblasts, that the sphingolipid-mediated inhibition of migration is dependent on vimentin. These results imply that this newly discovered sphingolipid-vimentin signalling axis exerts brake-and-throttle functions in the regulation of cell migration.