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Casein kinase II phosphorylation regulates SCF(cyclin F) Lys48-specific E3 ligase activity. (a) E3 ligase activity of Lys48-ubiquitylation from immunoprecipitated mCherry–cyclin F in transfected cells treated with CX4945 (4 µM) demonstrated elevated ubiquitylation activity with CK2 inhibition by approximately 1.35-fold (p = 0.0215, n = 3, one-way ANOVA). Immunoblots were carried out following E3 ligase activity assays to demonstrate equivalent levels of immunoprecipitated mCherry–cyclin F used for activity assays, which also revealed cyclin F contained a CK2 phosphorylation motif. (b) Immunoblot analysis of phospho-cyclin F (Ser621) using custom antibodies revealed reduced phosphorylation by approximately 0.68-fold (p = 0.0154, n = 3, one-way ANOVA) with CX4945 treatment. Vertical dashed line indicates cropped lanes. (c) E3 ligase activity of Lys48-ubiquitylation from immunoprecipitated mCherry–cyclin F in cells co-transfected with mCherry–CCNFWT or mCherry–CCNFS621G with scramble or CK2 siRNA demonstrated elevated ubiquitylation activity with CK2 knock down by approximately 1.2-fold (p = 0.0136, n = 3, one-way ANOVA). Immunoblots were carried out following E3 ligase activity assays to demonstrate equivalent levels of immunoprecipitated mCherry–cyclin F used for activity assays. (d) Immunoblot analysis of phospho-cyclin F (Ser621) using custom antibodies revealed reduced phosphorylation by approximately 0.61-fold (p = 0.005, n = 3, one-way ANOVA) with CK2 RNAi knockdown. Data are represented as the mean ± s.e.m. using one-way ANOVA with Tukey's post hoc test.
Source publication
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that is characterized by progressive weakness, paralysis and muscle loss often resulting in patient death within 3–5 years of diagnosis. Recently, we identified disease-linked mutations in the CCNF gene, which encodes the cyclin F protein, in cohorts of patients with familial...
Citations
... Similarly, deubiquitylation of Sec31 antagonizes the formation of large pro-collagen-containing carriers 63 . We previously demonstrated that cyclin F S621G dysregulates ubiquitination at Lys48, disrupting cellular survival and maintenance networks 9,11 . Hence it is possible that aberrant ubiquitination of Sec31 in cyclin F S621G cells impairs the formation of COPII vesicles, forming narrower ERES clusters, as detected here. ...
Amyotrophic lateral sclerosis (ALS) is a severely debilitating neurodegenerative condition that is part of the same disease spectrum as frontotemporal dementia (FTD). Mutations in the CCNF gene, encoding cyclin F, are present in both sporadic and familial ALS and FTD. However, the pathophysiological mechanisms underlying neurodegeneration remain unclear. Proper functioning of the endoplasmic reticulum (ER) and Golgi apparatus compartments is essential for normal physiological activities and to maintain cellular viability. Here, we demonstrate that ALS/FTD-associated variant cyclin FS621G inhibits secretory protein transport from the ER to Golgi apparatus, by a mechanism involving dysregulation of COPII vesicles at ER exit sites. Consistent with this finding, cyclin FS621G also induces fragmentation of the Golgi apparatus and activates ER stress, ER-associated degradation, and apoptosis. Induction of Golgi fragmentation and ER stress were confirmed with a second ALS/FTD variant cyclin FS195R, and in cortical primary neurons. Hence, this study provides novel insights into pathogenic mechanisms associated with ALS/FTD-variant cyclin F, involving perturbations to both secretory protein trafficking and ER-Golgi homeostasis.
... Serine to glycine mutation (p.S621G) is the most studied CCNF mutation to date since this mutation segregates well with disease across generations [2]. Our team previously identified that the cyclin F p.S621G mutation causes an increase in its E3 ubiquitin ligase activity for Lysine(K)48linked polyubiquitylation [19][20][21] which leads to the accumulation of ubiquitylated ALS-associated protein TDP-43 and SCF cyclin F target protein RRM2 in neuronal cells [2]. Notably, we recently reported that mutant p.S621G aberrantly K48-ubiquitylates TDP-43 causing its accumulation in neurons; an aberrant mechanism that likely contributes to the skein-like cytoplasmic TDP-43 aggregates in cyclin F p.S195R patient tissue [22]. ...
... To evaluate the effect of the ALS and FTD p.S621G mutation on the ubiquitylation of p62, we also conducted ubiquitylation assays with the addition of cyclin F p.S621G in Neuro2A cells. In previous studies, we have shown that the cyclin F p.S621G mutation leads to an increase in the ubiquitylation of substrates [19,22]. Notably, the presence of cyclin F p.S621G led to greater ubiquitylation of p62 compared to WT (Fig. 4B). ...
... In this experiment we also investigated the effect of the cyclin F p.S621G ALS and FTD variant on the ability of cyclin F to ubiquitylate p62. mCherry-cyclin F variants were immunoprecipitated from cell lysates to ensure the SCF complex was intact, which maintains its enzymatic activity with the mCherry-tag [19]. Purified recombinant p62 was incubated with biotinylated-ubiquitin and immunoprecipitated cyclin F in the presence of E1 (UBA1) and E2 (UBE2D3) conjugating enzymes, with and without ATP, and then analyzed by immunoblot. ...
Amyotrophic lateral sclerosis (ALS)- and frontotemporal dementia (FTD)-linked mutations in CCNF have been shown to cause dysregulation to protein homeostasis. CCNF encodes for cyclin F, which is part of the cyclin F-E3 ligase complex SCFcyclinF known to ubiquitylate substrates for proteasomal degradation. In this study, we identified a function of cyclin F to regulate substrate solubility and show how cyclin F mechanistically underlies ALS and FTD disease pathogenesis. We demonstrated that ALS and FTD-associated protein sequestosome-1/p62 (p62) was a canonical substrate of cyclin F which was ubiquitylated by the SCFcyclinF complex. We found that SCFcyclin F ubiquitylated p62 at lysine(K)281, and that K281 regulated the propensity of p62 to aggregate. Further, cyclin F expression promoted the aggregation of p62 into the insoluble fraction, which corresponded to an increased number of p62 foci. Notably, ALS and FTD-linked mutant cyclin F p.S621G aberrantly ubiquitylated p62, dysregulated p62 solubility in neuronal-like cells, patient-derived fibroblasts and induced pluripotent stem cells and dysregulated p62 foci formation. Consistently, motor neurons from patient spinal cord tissue exhibited increased p62 ubiquitylation. We suggest that the p.S621G mutation impairs the functions of cyclin F to promote p62 foci formation and shift p62 into the insoluble fraction, which may be associated to aberrant mutant cyclin F-mediated ubiquitylation of p62. Given that p62 dysregulation is common across the ALS and FTD spectrum, our study provides insights into p62 regulation and demonstrates that ALS and FTD-linked cyclin F mutant p.S621G can drive p62 pathogenesis associated with ALS and FTD.
... The functional role of CCNF in the context of ALS/FTD is not yet fully understood. Initial studies have indicated that ALSassociated variants in CCNF cause UPS dysfunction (20) and increased K48 polyubiquitination, resulting from changes to the E3 ligase activity of the CCNF complex (41)(42)(43). Recently, we have also shown that the expression and aggregation of several ALS-associated proteins lead to UPS dysfunction and perturbed cellular Ub homeostasis (29,30), common features of ALS pathogenesis. ...
... Since the discovery of pathogenic CCNF variants in ALS/FTD, several studies have investigated the disease mechanisms of CCNF S621G , with a focus on the role of CCNF variants in ligase complex activity. Preliminary studies indicated that CCNF S621G may cause ALS through UPS dysfunction (20), with more recent studies drawing attention to the prevention of CCNF S621G phosphorylation, resulting in elevated Lys48-ubiquitination activity (41,42). Overexpression of CCNF has been shown to trigger an upregulation of cell death in zebrafish, with transient overexpression of variant CCNF S621G leading to abnormal axonal outgrowth and impaired motor function (47). ...
... Here, we show that expression of a pathogenic CCNF variant also perturbs Ub homeostasis, with NSC-34 cells expressing CCNF S621G exhibiting large cytosolic aggregates and decreased levels of mobile Ub (specifically, free monomeric Ub) in comparison to controls. This depletion is consistent with previous findings that the pathogenic CCNF S621G variant causes an accumulation of ubiquitinated proteins through increased activity of the CCNF complex (41,42). Although disrupting the ability of CCNF to form an active E3 ligase complex did not seem to significantly alter the distribution of the entire mobile Ub pool, disrupting the ligase activity of CCNF did significantly improve the availability of free monomeric Ub to cells. ...
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that share pathological features, including the aberrant accumulation of ubiquitinated protein inclusions within motor neurons. Previously, we have shown that the sequestration of ubiquitin (Ub) into inclusions disrupts Ub homeostasis in cells expressing ALS-associated variants SOD1, FUS and TDP-43. Here, we investigated whether an ALS/FTD-linked pathogenic variant in the CCNF gene, encoding the E3 Ub ligase Cyclin F (CCNF), also perturbs Ub homeostasis. The presence of a pathogenic CCNF variant was shown to cause ubiquitin-proteasome system (UPS) dysfunction in induced pluripotent stem cell (iPSC)-derived motor neurons harbouring the CCNFS621G mutation. The expression of the CCNFS621G variant was associated with an increased abundance of ubiquitinated proteins and significant changes in the ubiquitination of key UPS components. To further investigate the mechanisms responsible for this UPS dysfunction, we overexpressed CCNF in NSC-34 cells and found that the overexpression of both wild-type (WT) and the pathogenic variant of CCNF (CCNFS621G) altered free Ub levels. Furthermore, double mutants designed to decrease the ability of CCNF to form an active E3 Ub ligase complex significantly improved UPS function in cells expressing both CCNFWT and the CCNFS621G variant, and were associated with increased levels of free monomeric Ub. Collectively, these results suggest that alterations to the ligase activity of the CCNF complex and the subsequent disruption to Ub homeostasis play an important role in the pathogenesis of CCNF-associated ALS/FTD.
... However, the p.S621G variant located in the PEST sequence has been reported repeatedly in Caucasian FALS/FTD patients [5,22]. The p.S621G variant prevents the phosphorylation of casein kinase II (CK2) and increases the lys48-ubiquitin activity, resulting in dysfunction of the autophagy degradation pathway [5,29,30]. It was also found that zebrafish with the p.S621G variant had disrupted axonal growth, suggesting a toxic gain-of-function mechanism in the pathogenesis of ALS [31]. ...
... It was also found that zebrafish with the p.S621G variant had disrupted axonal growth, suggesting a toxic gain-of-function mechanism in the pathogenesis of ALS [31]. The p.S621G variant was not found in our cohort, and all variants identified in our cohort were not located at the identified phosphorylation sites [29]. Therefore, based on the results predicted by software and the current literature review, we considered these seven variants located in the PEST sequence in our cohort to be likely benign, but more basic experimental studies are needed to explore the pathogenicity and pathogenesis of CCNF variants located in the PEST sequence. ...
Cyclin F (CCNF) variants have been found to be associated with amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). However, the genetic and clinical characteristics of ALS patients who carry CCNF variants are largely unknown. Genetic analysis was performed for 1587 Chinese ALS patients, and missense variants were predicted by software analyses. Additionally, we searched PubMed, Embase, and Web of Science for relevant literature and conducted a meta-analysis of the frequency of variants. In our ALS cohort, we identified 29 nonsynonymous variants in 41 ALS patients. Among these ALS patients, 18 (1.1%) were carriers of 15 rare missense variants that were considered probably pathogenic variants, and 11 of 15 variants were novel. Seven relevant studies were identified, and a total of 43 CCNF variants in 59 ALS patients with a frequency of 0.8% were reported. The ratio of males to females in our cohort (10/8) was similar to that in Caucasian populations (4/7) and significantly higher than that in Asian populations (10/1). The proportion of bulbar onset in Caucasian CCNF carriers was similar to our cohort (25.0 vs. 27.8%); however, bulbar onset had never been reported in previous Asian studies (0/11). FTD was not found in CCNF carriers in previous Asian studies and our cohort, but it has been reported in a FALS cohort (1/75) of Caucasian individuals. There were some differences in the clinical characteristics among different ethnic ALS populations. More basic scientific studies are needed to elucidate the pathogenic mechanisms and genotype-phenotype associations of CCNF variants.
... Among the cyclin F substrates identified so far are CP110, NuSAP1, RRM2, CDC6, SLBP, RBPJ, activator E2Fs, and E2F7 [6][7][8][9][10][11][12][13]. Additionally, other interaction partners that modulate the function of, or are themselves modulated by cyclin F have also been identified; which include b-Myb, p27 Kip1 , Akt, Casein kinase II, β-TrCP, and VCP [14][15][16][17][18]. Cyclin F functionally interacts with these substrates and interaction partners to chiefly regulate genomic and chromosomal stability. ...
Cyclin F, unlike canonical and transcriptional cyclins, does not bind or activate any cyclin-dependent kinases. Instead, it harbors an F-box motif and primarily functions as the substrate recognition subunit of the Skp1-Cul1-F-box E3 ubiquitin ligase complex, SCFCyclin F. By targeting specific proteins for ubiquitin-mediated proteasomal degradation, cyclin F plays a critical role in the regulation of centrosomal duplication, DNA replication and repair, and maintenance of genomic stability. Cyclin F abundance and activity are tightly regulated throughout the cell cycle. However, the molecular mechanisms regulating cyclin F are scantily understood. Here, we identify the deubiquitylase USP7 as a novel cyclin F-interacting protein. We observe that USP7 stabilizes cyclin F protein and that this function is independent of the deubiquitylase activity of USP7. Additionally, our data suggest that USP7 is also involved in the regulation of cyclin F mRNA. Pharmacological inhibition of the deubiquitylase activity of USP7 resulted in downregulation of cyclin F mRNA.
... For example, five different diGly sites in MAP1B, an α-tubulin binding protein, were found to be down-regulated with a log 2 FC < −1.5 in N2a cells, whereas several actin-binding proteins such as MSN, FSCN1, and WDR1 showed decreased ubiquitination in CCNF D628V LCL cells ( Fig 1F). Notably, Ub diGly sites representing K63and K48-linked chains were decreased in both LCLs (RPS27A_K63 in V335M; RPS27A_K48 in D628V) which is different from reports describing elevated Ub K48 levels in cells expressing the ALS-linked Cyclin-F mutation S621G (34). Taking into account that the four cell four respective eight biological replicates in N2a, SH-SY5Y, and LCLs described in Fig 1D. ...
The founding member of the F-box protein family, Cyclin-F, serves as a substrate adaptor for the E3 ligase Skp1-Cul1-F-box (SCF) Cyclin-F which is responsible for ubiquitination of proteins involved in cell cycle progression, DNA damage and mitotic fidelity. Missense mutations in CCNF encoding for Cyclin-F are associated with amyotrophic lateral sclerosis (ALS). However, it remains elusive whether CCNF mutations affect the substrate adaptor function of Cyclin-F and whether altered SCF Cyclin-F –mediated ubiquitination contributes to pathogenesis in CCNF mutation carriers. To address these questions, we set out to identify new SCF Cyclin-F targets in neuronal and ALS patient–derived cells. Mass spectrometry–based ubiquitinome profiling of CCNF knockout and mutant cell lines as well as Cyclin-F proximity and interaction proteomics converged on the HSP90 chaperone machinery as new substrate candidate. Biochemical analyses provided evidence for a Cyclin-F–dependent association and ubiquitination of HSP90AB1 and implied a regulatory role that could affect the binding of a number of HSP90 clients and co-factors. Together, our results point to a possible Cyclin-F loss-of-function–mediated chaperone dysregulation that might be relevant for ALS.
... In accordance with the idea that loss of ATR activity results in cyclin F phosphorylation, we observed increased phosphorylation in the ATR inhibitor-treated MuSCs (Fig. 5D). Phosphorylation of cyclin F may alter its function or target it for degradation (49)(50)(51). We found that cyclin F levels were markedly decreased in ATR cKO MuSCs relative to ATR WT MuSCs (Fig. 5E), suggesting that cyclin F is phosphorylated and subsequently degraded upon ATR loss. ...
... family of transcription factors and RRM2 ("ribonucleotide reductase M2") (50,(52)(53)(54). To first test whether cyclin F targets E2F1 or RRM2 for degradation, we knocked down cyclin F in MuSCs using a lentiviral construct expressing shRNA targeting cyclin F (shCCNF) or a scrambled nontargeting control (shCTRL). ...
... Casein kinase II (CK2) dependent phosphorylation as well as anaphase promoting complex (APC) dependent ubiquitination and degradation can control cyclin F protein levels (51,56). CK2 phosphorylation of cyclin F can also mediate the ubiquitin ligase activity of the SCF complex (50). To first investigate whether either CK2 or APC lie downstream of ATR in promoting MuSC quiescence, we measured EdU incorporation of MuSCs treated ex vivo with the CK2specific inhibitor, CX-4945, or the APC-specific inhibitor, TAME, in addition to ATR inhibition. ...
Significance
The replication stress response protein, ATR, is active in quiescent muscle stem cells in response to DNA:RNA hybrids, and this activity maintains quiescence by ensuring degradation of key cell-cycle transition factors by the E3 ubiquitin ligase cyclin F–SCF complex. This is critical for understanding how stem cells regulate a state of prolonged and reversible cell-cycle arrest and how genome integrity is maintained over time. Together, these studies offer a unique picture of a molecular mechanism controlling stem cell quiescence.
... Its phosphorylation by CK2 at S621 negatively controls the E3 ligase activity of the complex (Fig. 3e); consistently S621G mutation leads to the stimulation of the activity and the consequent aberrant increase of proteins ubiquitination, a hallmark of ALS and FTD. 183 In general, the hypothesis of CK2 targeting in neurodegeneration is still premature. It is based on some observations, as in the case of ataxin-3 phosphorylation in SCA3 models, 175 or SET phosphorylation 162 and axonal transport inhibition 170 in AD. ...
CK2 is a constitutively active Ser/Thr protein kinase, which phosphorylates hundreds of substrates, controls several signaling pathways, and is implicated in a plethora of human diseases. Its best documented role is in cancer, where it regulates practically all malignant hallmarks. Other well-known functions of CK2 are in human infections; in particular, several viruses exploit host cell CK2 for their life cycle. Very recently, also SARS-CoV-2, the virus responsible for the COVID-19 pandemic, has been found to enhance CK2 activity and to induce the phosphorylation of several CK2 substrates (either viral and host proteins). CK2 is also considered an emerging target for neurological diseases, inflammation and autoimmune disorders, diverse ophthalmic pathologies, diabetes, and obesity. In addition, CK2 activity has been associated with cardiovascular diseases, as cardiac ischemia–reperfusion injury, atherosclerosis, and cardiac hypertrophy. The hypothesis of considering CK2 inhibition for cystic fibrosis therapies has been also entertained for many years. Moreover, psychiatric disorders and syndromes due to CK2 mutations have been recently identified. On these bases, CK2 is emerging as an increasingly attractive target in various fields of human medicine, with the advantage that several very specific and effective inhibitors are already available. Here, we review the literature on CK2 implication in different human pathologies and evaluate its potential as a pharmacological target in the light of the most recent findings.
... The exact site of Sec31 ubiquitination may not be crucial for regulation of COPII vesicle tra cking, but it is a signal to recruit other effectors to assemble the coat or to regulate its catalytic activity. We previously demonstrated that mutant cyclin F S621G dysregulates ubiquitination at Lys48, resulting in disruption of biological networks responsive for cellular survival and maintenance [11,13]. Here, we demonstrate that in cells expressing mutant cyclin F, ubiquitination of Sec31 was reduced and bulk secretion of large proteins (> 100kDa) was inhibited, although the secretion of proteins smaller than this was unchanged. ...
Background
Mutations in the CCNF gene encoding cyclin F are associated with sporadic and familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, but the underlying pathophysiological mechanisms are unknown. Proper functioning of the endoplasmic reticulum (ER) is essential for physiological cellular function.
Methods
We used human neuroblastoma SH-SY5Y and human embryonic kidney HEK293T cell lines and mouse primary neurons-overexpressing two familial ALS cyclin F mutants to examine whether mutant ALS/FTD-associated cyclin F perturbs key functions of the ER and Golgi compartments. Specific cellular assays were used to examine ER-Golgi transport (VSVGts045), the budding of vesicles from ER membranes and ER-associated degradation (ERAD). Immunocytochemistry was used to examine the morphology of the Golgi and ER-exit sites, and to detect ER stress and apoptosis. Western blotting was used to examine the content of vesicles budding from ER membranes and the interaction between Sec 31 and cyclin F. Flow cytometry was used to examine cell death.
Results
We demonstrated that mutant cyclin F inhibited protein transport from the ER to Golgi apparatus by a mechanism involving aberrant vesicle sorting from the ER. It also impeded ER-associated degradation, whereby misfolded ER proteins are ubiquitinated and degraded by the proteasome. This was associated with induction of ER stress and Golgi fragmentation, leading to apoptosis.
Conclusion
Together, these results demonstrate that ER dysfunction is a pathogenic pathway associated with ALS/FTD-variant cyclin F.
... Currently, there are relatively few known interaction partners of cyclin F. These proteins are generally associated with cell cycle function, including substrates such as ribonucleosidediphosphate reductase subunit M2 (RRM2) (15), nucleolar and spindle-associated protein 1 (NuSAP1) (16), centriolar coiled-coil protein of 110 kDa (CP110) (17), cell division control protein 6 homolog (18), stem-loop binding protein (19), exonuclease 1 (Exo1) (20), fizzy-related protein homolog (21) and transcription factors E2F1, E2F2 and E2F3A (22). In addition, known interaction partners of cyclin F include Skp1 (forming part of the ubiquitin ligase complex), Myb-related protein B (B-Myb) (23) and Casein kinase II (CKII) (24). In recent years, cyclin F has also been demonstrated to interact with ALS/FTD-associated proteins TDP-43, Sequestosome-1 (SQSTM1) and Transitional endoplasmic reticulum ATPase (VCP) (25). ...
Previously, we identified missense mutations in CCNF that are causative of familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Hallmark features of these diseases include the build-up of insoluble protein aggregates as well as the mislocalisation of proteins such as TDP-43. In recent years, the dysregulation of SFPQ has also emerged as a pathological hallmark of ALS/FTD. CCNF encodes for the protein cyclin F, a substrate recognition component of an E3-ubiquitin ligase. We have previously shown that ALS/FTD-linked mutations in CCNF cause disruptions to overall protein homeostasis that leads to a build-up of K48-linked ubiquitylated proteins as well as defects in autophagic machinery. To investigate further processes that may be affected by cyclin F, we used a protein-proximity ligation method, known as BioID, standard immunoprecipitations and mass spectrometry (MS) to identify novel interaction partners of cyclin F and infer further process that may be affected by the ALS/FTD-causing mutation. Results demonstrate that cyclin F closely associates with proteins involved with RNA metabolism as well as a number of RNA binding proteins previously linked to ALS/FTD, including splicing factor proline and glutamine rich (SFPQ). Notably, the overexpression of cyclin F(S621G) led to the aggregation and altered subcellular distribution of SFPQ in HEK293 cells, whilst leading to altered degradation in primary neurons. Overall, our data links ALS/FTD-causing mutations in CCNF to converging pathological features of ALS/FTD and provides a link between defective protein degradation systems and the pathological accumulation of a protein involved in RNA processing and metabolism.
