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. 2020 Aug 21;23(9):101491. doi: 10.1016/j.isci.2020.101491

An Amyotrophic Lateral Sclerosis-Associated Mutant of C21ORF2 Is Stabilized by NEK1-Mediated Hyperphosphorylation and the Inability to Bind FBXO3

Yasuaki Watanabe 1,2, Tadashi Nakagawa 2, Tetsuya Akiyama 1, Makiko Nakagawa 2, Naoki Suzuki 1, Hitoshi Warita 1, Masashi Aoki 1, Keiko Nakayama 2,3,∗
PMCID: PMC7481237  PMID: 32891887

Summary

C21ORF2 and NEK1 have been identified as amyotrophic lateral sclerosis (ALS)-associated genes. Both genes are also mutated in certain ciliopathies, suggesting that they might contribute to the same signaling pathways. Here we show that FBXO3, the substrate receptor of an SCF ubiquitin ligase complex, binds and ubiquitylates C21ORF2, thereby targeting it for proteasomal degradation. C21ORF2 stabilizes the kinase NEK1, with the result that loss of FBXO3 stabilizes not only C21ORF2 but also NEK1. Conversely, NEK1-mediated phosphorylation stabilizes C21ORF2 by attenuating its interaction with FBXO3. We found that the ALS-associated V58L mutant of C21ORF2 is more susceptible to phosphorylation by NEK1, with the result that it is not ubiquitylated by FBXO3 and therefore accumulates together with NEK1. Expression of C21ORF2(V58L) in motor neurons induced from mouse embryonic stem cells impaired neurite outgrowth. We suggest that inhibition of NEK1 activity is a potential therapeutic approach to ALS associated with C21ORF2 mutation.

Subject Areas: Biological Sciences, Molecular Biology, Neuroscience

Graphical Abstract

graphic file with name fx1.jpg

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Highlights

  • •

    C21ORF2 is a common substrate of FBXO3 and NEK1

  • •

    NEK1-mediated phosphorylation of C21ORF2 inhibits its ubiquitylation and degradation

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    ALS-associated mutant of C21ORF2 is stable owing to hyperphosphorylation

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    The C21ORF2 mutant protein causes NEK1 accumulation and motor neuron abnormality

Biological Sciences; Molecular Biology; Neuroscience

Introduction

Dysfunction of systems responsible for protein quality control as well as altered RNA metabolism has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS) (Ling et al., 2013; Shahheydari et al., 2017). The ubiquitin-proteasome system is essential for maintenance of protein homeostasis at the level of protein degradation. Indeed, various neurodegenerative diseases including ALS are characterized pathologically by the formation of inclusion bodies consisting of insoluble and unfolded proteins tagged with ubiquitin, suggesting that a defect in or overload of the ubiquitin-proteasome system may play an important role in the formation of these structures (Ardley and Robinson, 2004; Neumann et al., 2006).

The E3 ubiquitin ligases mediate the transfer of ubiquitin to substrate proteins and include CRL1 ligases, also known as S phase kinase associated protein 1 (SKP1)–Cullin 1 (CUL1)-F box protein (SCF) complexes (Wang et al., 2014). In each SCF complex, the COOH terminus of the scaffold protein CUL1 binds the RING-finger protein RBX1 and thereby recruits an E2 enzyme, whereas the NH2 terminus of CUL1 binds SKP1 and an associated F box protein, the latter of which is responsible for substrate recognition (Skaar et al., 2013). The F box domain of F box proteins mediates direct binding to SKP1 (Bai et al., 1996). Approximately 70 F box proteins have been identified in humans, each of which specifically recognizes a distinct group of substrates, with the result that SCF complexes mediate the ubiquitylation and degradation of a large variety of proteins (Nakagawa et al., 2020; Nakayama and Nakayama, 2006). F box proteins are categorized into three classes on the basis of their substrate interaction domains: FBXW proteins (which contain WD40 repeat domains), FBXL proteins (which contain leucine-rich repeat [LRR] domains), and FBXO proteins (whose protein interaction domains are poorly characterized) (Jin et al., 2004).

ALS is characterized by the selective dysfunction and loss of motor neurons (Brown and Al-Chalabi, 2017). Most knowledge of ALS pathogenesis has been obtained by functional analysis of causal or risk genes (Chia et al., 2018; Weishaupt et al., 2016). A recent genome-wide association study and meta-analysis identified the chromosome 21 open reading frame 2 gene (C21ORF2) as being associated with ALS (van Rheenen et al., 2016). The C21ORF2 protein contains thee highly conserved LRR domains (Scott et al., 1998), but the function of these repeats and that of C21ORF2 itself remain largely unexplored. C21ORF2 is known to form a functional complex with NEK1 (never in mitosis gene A related kinase 1) (Wheway et al., 2015). Several homozygous mutations of C21ORF2 and of NEK1 have been identified in individuals with ciliopathies, a group of genetic disorders caused by a defect of primary cilia (Thiel et al., 2011; Wheway et al., 2015). Case-control studies have also revealed that certain missense or loss-of-function mutations of NEK1 are more frequent in individuals with familial or sporadic ALS in European countries and China (Brenner et al., 2016; Gratten et al., 2017; Kenna et al., 2016). NEK1 possesses both serine-threonine and tyrosine kinase activities in vitro (Letwin et al., 1992), and it functions in the repair of DNA damage and in cell cycle regulation (Chen et al., 2008; Fry et al., 2012; Melo-Hanchuk et al., 2017). C21ORF2 and NEK1 may therefore play a cooperative and key role in ALS pathogenesis by contributing to the same signaling pathways.

We have now identified C21ORF2 as a substrate of F box only protein 3 (FBXO3). The SKP1-CUL1-FBXO3 (SCFFBXO3) complex ubiquitylates and thereby triggers the degradation of C21ORF2, but the binding of FBXO3 to C21ORF2 is prevented by NEK1-mediated phosphorylation of C21ORF2. Phosphorylation by NEK1 thus stabilizes the C21ORF2 protein. In a reciprocal manner, we also found that C21ORF2 stabilizes NEK1. Of note, the protein encoded by an ALS-associated mutant form of C21ORF2, C21ORF2(V58L), is not a substrate of SCFFBXO3. Furthermore, mouse embryonic stem cell (ESC)-derived induced motor neurons (iMNs) harboring the corresponding heterozygous missense mutation of mouse C21orf2 manifested a reduced neurite length. Our findings provide biological evidence that C21ORF2 degradation mediated by SCFFBXO3 determines the abundance of NEK1 and contributes to the phenotype of motor neurons.

Results

C21ORF2 Is a Substrate of SCFFBXO3

To investigate the molecular function of C21ORF2 (also known as CFAP410) and its role in ALS pathogenesis, we searched for proteins that associate with C21ORF2. Analysis of published data for C21ORF2-binding proteins identified by affinity purification combined with mass spectrometry revealed that such proteins included SKP1, CUL1, and FBXO3 (Wheway et al., 2015). To validate this finding, we subjected immunoprecipitates of FLAG epitope-tagged C21ORF2 expressed in HEK293T cells to immunoblot analysis. We detected SKP1, CUL1, and FBXO3 in the immunoprecipitates, thereby confirming the interaction between these proteins and C21ORF2 (Figure 1A). Immunoprecipitation analysis with a series of truncated mutants of FBXO3 also confirmed that the F box domain is responsible for binding to the SKP1-CUL1-RBX1 catalytic module of SCFFBXO3 (Figures 1B and 1C), consistent with the general features of F box proteins (Bai et al., 1996). In addition, immunoprecipitation analysis revealed that an FBXO3 mutant comprising amino acids 58 to 278 (ΔNC) bound C21ORF2, whereas FBXO3(ΔNM), consisting of the COOH-terminal 193 amino acids, did not, indicating that residues 58–278 of FBXO3 are responsible for binding to C21ORF2 (Figures 1B and 1D). To test whether C21ORF2 is ubiquitylated by SCFFBXO3, we first examined the ubiquitylation level of C21ORF2 in cells with or without FBXO3 overexpression. Such in vivo ubiquitylation analysis showed that FBXO3 overexpression increased the ubiquitylation level of C21ORF2 (Figure 1E). Moreover, treatment of cells with the proteasome inhibitor MG132 induced the accumulation of C21ORF2 and increased its ubiquitylation level, suggesting that ubiquitylated C21ORF2 is degraded by the proteasome (Figure S1A). We then determined the rate of C21ORF2 degradation in HEK293T cells by blocking protein synthesis with cycloheximide. We found that hemagglutinin epitope (HA)-tagged C21ORF2 was degraded more rapidly in cells overexpressing FBXO3 than in control cells, suggesting that FBXO3 promotes C21ORF2 degradation (Figure 1F). Furthermore, in contrast to cells expressing full-length (FL) FBXO3, cycloheximide treatment did not reveal degradation of C21ORF2 in cells expressing FBXO3(ΔN), which is unable to interact with SKP1 (Figure 1G). We confirmed that proteasome inhibition by MG132 attenuated the degradation of C21ORF2 apparent in cells overexpressing FBXO3(FL) after their exposure to cycloheximide (Figure 1G). Together, these observations indicated that FBXO3 promotes C21ORF2 degradation in a manner dependent on the catalytic activity of both SCFFBXO3 and the proteasome.

Figure 1.

Figure 1

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C21ORF2 Is a Substrate of SCFFBXO3

(A) Lysates of HEK293T cells expressing FLAG-C21ORF2 or transfected with the corresponding empty vector (Vec) were subjected to immunoprecipitation (IP) with antibodies to FLAG, and the resulting precipitates as well as the original cell lysates (Input) were subjected to immunoblot (IB) analysis with antibodies to SKP1, CUL1, FBXO3, FLAG, and HSP90 (loading control).

(B) Schematic representation of truncated variants of human FBXO3. The F box and ApaG domains are indicated. aa, amino acids.

(C) Lysates of HEK293T cells expressing FLAG-tagged FBXO3 variants (FL, ΔC, or ΔN) were subjected to immunoprecipitation with antibodies to FLAG, and the resulting precipitates were subjected to immunoblot analysis with antibodies to SCF components (SKP1, CUL1, and RBX1) and FLAG. The asterisk indicates nonspecific bands.

(D) Lysates of HEK293T cells expressing FLAG-C21ORF2 and Myc epitope-tagged FBXO3 variants (FL, ΔN, ΔC, ΔNC, or ΔNM) were subjected to immunoprecipitation with antibodies to Myc, and the resulting precipitates as well as the original cell lysates were subjected to immunoblot analysis with antibodies to FLAG and Myc.

(E) Lysates of HEK293T cells expressing HA-tagged ubiquitin (ubi), FLAG-C21ORF2, and Myc-FBXO3, as indicated, were subjected to immunoprecipitation with antibodies to FLAG, and the resulting precipitates as well as the original cell lysates were subjected to immunoblot analysis with antibodies to HA, FLAG, and Myc.

(F) HEK293T cells expressing HA-C21ORF2 with or without FLAG-FBXO3 were treated with cycloheximide (CHX, 25 μg/mL) for the indicated times, lysed, and subjected to immunoblot analysis with antibodies to HA, FLAG, and HSP90. Representative immunoblots (upper) as well as quantitative analysis of the band intensity for HA-C21ORF2 normalized by that for HSP90 (lower) are shown. Quantitative data are mean ± SEM values from three independent experiments. The p value was determined by two-way ANOVA.

(G) HEK293T cells coexpressing HA-C21ORF2 and FLAG-FBXO3 (FL or ΔN) were incubated in the absence or presence of cycloheximide (25 μg/mL) or 10 μM MG132 (or dimethyl sulfoxide [DMSO] vehicle) for 4 h, lysed, and subjected to immunoblot analysis with antibodies to HA, FLAG, and β-actin (loading control). Representative immunoblots (upper) as well as quantitative analysis of the percentage of HA-C21ORF2 remaining (normalized by β-actin) in cycloheximide-treated cells compared with the corresponding cells without cycloheximide treatment (lower) are shown. Quantitative data are mean ± SEM values from four independent experiments. The p value was determined by one-way ANOVA followed by Tukey's post hoc test.

See also Figure S1.

To identify the sites of C21ORF2 ubiquitylation, we expressed a mutant protein in which all eight lysine residues were replaced with arginine (8KR) in HEK293T cells (Figure S1B). However, we found that the 8KR mutant was ubiquitylated at a level similar to that apparent for the wild-type (WT) protein (Figure S1C), suggesting that C21ORF2 is ubiquitylated at sites other than lysine residues, possibly including the NH2-terminal amine group (McDowell and Philpott, 2013).

The ALS-Associated V58L Mutant of C21ORF2 Is Not a Substrate of SCFFBXO3

An ALS-associated missense mutation of C21ORF2 results in a V58L substitution in the second of the three LRRs (van Rheenen et al., 2016) (Figure S2A). We tested whether this mutant protein is also a substrate of SCFFBXO3. FBXO3 bound to the NH2-terminal region of C21ORF2(WT) containing the LRRs as well as to the corresponding COOH-terminal region containing the LRR COOH-terminal (LRRCT) domain (Figure S2), whereas FBXO3 did not bind to C21ORF2(V58L) and the mutant protein was not ubiquitylated in HEK293T cells (Figures 2A and 2B), suggesting that FBXO3 is the only ubiquitin ligase for C21ORF2 and that the ALS-related V58L mutation prevents the ubiquitylation of C21ORF2. Given that SCFFBXO3 targets C21ORF2 for proteasomal degradation, C21ORF2(V58L) might be expected to be more stable than the WT protein. To compare the stability of the WT and V58L proteins in neuronal cells, we generated SH-SY5Y cell lines in which the expression of HA-tagged forms of these proteins was inducible by doxycycline (DOX) with the use of the Tet-on system (rtTA-TetP-HA-C21ORF2-WT and -V58L, respectively). The cells were incubated with DOX for 2 days to induce the expression of C21ORF2(WT) or C21ORF2(V58L), after which further transcription was blocked by removal of DOX and the cells were subjected to immunoblot analysis 2, 4, or 6 h later. The percentage amount of C21ORF2(V58L) remaining during the time course was significantly greater than that of C21ORF2(WT) (Figure 2C), indicating that C21ORF2(V58L) is indeed more stable than is C21ORF2(WT) as a result of not being a substrate of SCFFBXO3.

Figure 2.

Figure 2

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The ALS-Associated V58L Mutant of C21ORF2 Is not a Substrate of SCFFBXO3

(A) Immunoprecipitation and immunoblot analysis of the interaction between FLAG-tagged C21ORF2 (WT or V58L) and Myc-tagged FBXO3 in HEK293T cells.

(B) In vivo ubiquitylation analysis of FLAG-tagged C21ORF2 (WT or V58L) in HEK293T cells.

(C) SH-SY5Y cells engineered to express HA-tagged C21ORF2 (WT or V58L) on exposure to DOX were incubated with or without (−) DOX (1 μg/mL) for 2 days. The cells cultured with DOX were then incubated without DOX for the indicated times, after which they were lysed and subjected to immunoblot analysis with antibodies to HA and HSP90. Representative blots (upper) and quantitative analysis of the band intensity for HA-C21ORF2 normalized by that for HSP90 (lower) are shown. Quantitative data are means ± SEM from three independent experiments. The p value was determined by two-way ANOVA.

(D) In vitro binding assay for glutathione S-transferase (GST)-tagged FBXO3 and His6-tagged C21ORF2 (WT or V58L). The recombinant proteins were incubated together overnight at 4°C, after which GST or GST-FBXO3 removed by precipitation with glutathione beads as well as a portion of the His6-C21ORF2 input (10%) were subjected to immunoblot analysis with antibodies to His6 and to GST.

See also Figure S2.

To confirm that C21ORF2 binds FBXO3 directly and that the V58L mutation of C21ORF2 disrupts this binding, we performed a pull-down assay with recombinant proteins expressed in and purified from Escherichia coli. Unexpectedly, we found that not only C21ORF2(WT) but also the V58L mutant bound to FBXO3 (Figure 2D). Given that SCF complexes often recognize substrates in a modification-dependent manner (Skaar et al., 2013), this finding suggested the possibility that either posttranslational modification such as phosphorylation of C21ORF2 or some other molecule regulates the interaction between C21ORF2 and FBXO3.

The Interaction between FBXO3 and C21ORF2 Is Prevented by NEK1-Mediated Phosphorylation of C21ORF2

Given that NEK1 was previously shown to bind C21ORF2 and is implicated in the pathogenesis of ALS, we examined whether NEK1 might phosphorylate C21ORF2 and thereby regulate its interaction with FBXO3. Immunoprecipitation analysis showed that both C21ORF2(WT) and C21ORF2(V58L) bound NEK1 in HEK293T cells, although the extent of the interaction with NEK1 was less pronounced for the V58L mutant than for C21ORF2(WT) (Figure 3A). To investigate whether NEK1 directly phosphorylates C21ORF2, we performed an in vitro kinase assay with recombinant C21ORF2 purified from bacteria and an immunoprecipitated fragment of NEK1 that consists of the NH2-terminal 811 amino acids and includes the kinase domain. Phos-tag SDS-poylacrylamide gel electrophoresis (PAGE), which separates phosphorylated forms of proteins as higher molecular mass bands (Kinoshita et al., 2009), revealed multiple upward band shifts of C21ORF2(WT) after its incubation with NEK1-N811 and ATP (Figure 3B). Such band shifts were not detected after treatment of the reaction mixture with lambda (λ) phosphatase or in the presence of the kinase-dead K33R mutant of NEK1-N811 instead of the WT fragment, suggesting that C21ORF2(WT) was phosphorylated at multiple sites by NEK1. Of note, overexpression of NEK1(WT) inhibited the binding of C21ORF2 to FBXO3 as well as attenuated the ubiquitylation of C21ORF2 in HEK293T cells, whereas forced expression of NEK1(K33R) increased both C21ORF2-FBXO3 binding and C21ORF2 ubiquitylation (Figures 3C and 3D). These results suggested that the FBXO3-mediated ubiquitylation of C21ORF2 is negatively regulated by NEK1-dependent phosphorylation of C21ORF2. In addition, NEK1 knockdown resulted in a reduction in the abundance of C21ORF2 in HEK293T cells, and this effect was prevented by proteasome inhibition induced by MG132 treatment (Figure 3E), suggesting that phosphorylation by NEK1 stabilizes C21ORF2 by protecting it from proteasome-dependent degradation.

Figure 3.

Figure 3

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FBXO3-Mediated C21ORF2 Ubiquitylation Is Negatively Regulated by NEK1

(A) Immunoprecipitation and immunoblot analysis of the interaction between FLAG-tagged C21ORF2 (WT or V58L) and Myc-tagged NEK1 in HEK293T cells.

(B) In vitro kinase assay of recombinant His6-C21ORF2 phosphorylation by Myc-tagged NEK1-N811 (WT or K33R) immunoprecipitates prepared from HEK293T cells. The reaction was performed for 15 min at 30°C, after which the reaction mixtures were treated (or not) with λ phosphatase and then subjected to Phos-tag or Laemmli SDS-PAGE followed by immunoblot analysis with antibodies to His6 and Myc.

(C) Immunoprecipitation and immunoblot analysis of the effect of Myc-tagged NEK1 (WT or K33R) expression on the interaction of FLAG-C21ORF2 with Myc-FBXO3 in HEK293T cells. Myc-FBXO3 and Myc-NEK1 were distinguished on the basis of molecular size.

(D) In vivo assay of the effect of Myc-tagged NEK1 (WT or K33R) expression on the ubiquitylation of FLAG-C21ORF2 in HEK293T cells.

(E) HEK293T cells were transfected first with two different small interfering RNAs (siRNAs) for NEK1 or with a control siRNA (day 1) and then with an expression vector for HA-C21ORF2 (day 2), incubated with 10 μM MG132 or DMSO vehicle for 3 h (day 3), lysed, and subjected to immunoblot analysis with antibodies to HA and NEK1. The band intensity for HA-C21ORF2 (normalized by that of HSP90) was quantified by densitometry.

(F) HEK293T cells expressing FLAG-tagged C21ORF2 (WT or V58L) and Myc-tagged NEK1 (WT or K33R) were lysed, treated (or not) with λ phosphatase, and subjected to Phos-tag or Laemmli SDS-PAGE followed by immunoblot analysis with antibodies to FLAG and Myc.

(G) In vivo assay of the effect of Myc-NEK1(K33R) expression on the ubiquitylation of FLAG-C21ORF2(V58L) in HEK293T cells.

Given that C21ORF2(V58L) did not bind FBXO3 in vivo but did so in vitro (Figures 2A and 2D) and that the interaction of NEK1 with C21ORF2(V58L) was less pronounced than was that with C21ORF2(WT) (Figure 3A), we predicted that C21ORF2(V58L) might be phosphorylated to a greater extent by NEK1 compared with C21ORF2(WT), resulting in its inability to bind to and to undergo ubiquitylation by FBXO3 in vivo. To assess this possibility, we compared the intracellular phosphorylation levels of C21ORF2(WT) and its V58L mutant. The extent of C21ORF2(V58L) phosphorylation was indeed greater than that of C21ORF2(WT) in HEK293T cells overexpressing NEK1 (Figure 3F). Of note, dominant negative inhibition of endogenous NEK1 function by forced expression of NEK1(K33R) promoted the ubiquitylation of C21ORF2(V58L) (Figure 3G). Together, these data suggested that ubiquitylation of C21ORF2 by SCFFBXO3 is negatively regulated by NEK1-mediated phosphorylation of C21ORF2. Moreover, the binding of C21ORF2(V58L) to and its ubiquitylation by FBXO3 in vivo are prevented as a result of its increased susceptibility to phosphorylation by NEK1 compared with C21ORF2(WT).

C21ORF2 Stabilizes NEK1

To investigate the cellular function of C21ORF2, we inactivated C21ORF2 in HEK293T cells with the CRISPR/Cas9 system. We confirmed that C21ORF2 protein was depleted (Figures 4A and S3) and that the amount of C21ORF2 mRNA was reduced (Figure 4B), probably as a result of nonsense-mediated decay, in two knockout (KO) clones. We noticed that the abundance of endogenous NEK1 protein, but not that of NEK1 mRNA, was also significantly reduced in these C21ORF2 KO clones (Figures 4A and 4B), suggesting that C21ORF2 stabilizes NEK1 at the protein level. Examination of the dynamics of Myc-NEK1 degradation in cycloheximide-treated HEK293T cells with or without C21ORF2 overexpression revealed that C21ORF2 suppressed the degradation of NEK1 (Figure 4C). Given that C21ORF2(V58L) was more stable than C21ORF2(WT) in SH-SY5Y cells (Figure 2C), we hypothesized that the increased abundance of C21ORF2 that results from such ALS-associated C21ORF2 mutation might result in an increase in the amount of endogenous NEK1. To test further whether the abundance of endogenous NEK1 is affected by the amount of C21ORF2, we induced the expression of HA-C21ORF2 in engineered SH-SY5Y cells by DOX treatment. We found that the abundance of endogenous NEK1 was indeed increased by such DOX treatment (Figure 4D). Conversely, the abundance of endogenous NEK1 decreased in parallel with the decline in the expression of HA-C21ORF2 in these cells after DOX washout (Figure 4E). Collectively, these results indicated that C21ORF2 stabilizes NEK1 by suppressing its degradation.

Figure 4.

Figure 4

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C21ORF2 Stabilizes NEK1

(A) Immunoblot analysis of NEK1 in two C21ORF2 KO clones of HEK293T cells as well as in the parental cells. Representative immunoblots (left, with the arrowhead indicating the endogenous C21ORF2 band and the asterisk indicating a nonspecific band) and quantification of the band intensity for NEK1 normalized by that of α-tubulin (right) are shown. Quantitative data are means ± SEM for five independent experiments. The p values were determined by one-way ANOVA followed by Tukey's post hoc test.

(B) Reverse transcription (RT) and quantitative polymerase chain reaction (qPCR) analysis of the abundance of C21ORF2 and NEK1 mRNAs (normalized by that of GAPDH mRNA) in cells as in (A). Data are means ± SEM for three independent experiments. The p values were determined by one-way ANOVA followed by Tukey's post hoc test. N.S., not significant.

(C) Cycloheximide chase analysis of Myc-NEK1 in HEK293T cells expressing (or not) FLAG- C21ORF2.

(D) SH-SY5Y cells engineered to express HA-C21ORF2 on exposure to DOX were incubated with DOX (0, 0.5, or 1 μg/mL) for 2 days, lysed, and subjected to immunoblot analysis with antibodies to NEK1 and HA.

(E) Cells incubated with DOX (1 μg/mL) as in (D) were subsequently incubated in the absence of DOX for 0, 12, 24, or 48 h before immunoblot analysis.

See also Figure S3.

C21ORF2 and NEK1 Are Up-Regulated in FBXO3 KO Cells

To examine whether FBXO3 regulates NEK1 abundance indirectly by promoting the proteasomal degradation of C21ORF2, we inactivated FBXO3 in SH-SY5Y cells with the use of the CRISPR-Cas9 system. We found that the abundance of C21ORF2 protein was increased in two FBXO3 KO cell clones (Figures 5A and S4), whereas that of C21ORF2 mRNA was not significantly altered (Figure S5A), consistent with the notion that the loss of FBXO3 results in the stabilization of C21ORF2 protein. The abundance of NEK1 protein was also increased in the FBXO3 KO cell clones (Figures 5A and S4). Given that the amount of NEK1 mRNA was also increased in these cells (Figure S5A), both this effect and the accumulation of C21ORF2 protein likely contributed to the up-regulation of NEK1 protein induced by FBXO3 ablation. The expression of FLAG-FBXO3 in the FBXO3 KO cells reduced the abundance of both C21ORF2 and NEK1 (Figure 5B) without affecting the amounts of the corresponding mRNAs (Figure S5B). Together, these results provide a link between our observations that FBXO3 promotes the proteasomal degradation of C21ORF2 and that C21ORF2 stabilizes NEK1. In other words, our findings suggest that FBXO3 negatively regulates the abundance of both C21ORF2 and NEK1.

Figure 5.

Figure 5

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C21ORF2 and NEK1 Are Up-Regulated in FBXO3 KO Cells

(A) Immunoblot analysis of C21ORF2, NEK1, and FBXO3 in two FBXO3 KO clones of SH-SY5Y cells as well as in the parental cells. Representative blots (left) and quantitative analysis of the band intensity for C21ORF2 and NEK1 normalized by that for tubulin (right) are shown. The arrowhead and asterisk indicate the band for endogenous C21ORF2 and a nonspecific band, respectively. The arrows indicate the bands for isoforms of endogenous FBXO3. Quantitative data are means ± SEM from five independent experiments. The p values were determined by one-way ANOVA followed by Tukey's post hoc test.

(B) An FBXO3 KO clone of SH-SY5Y cells was infected with a lentivirus encoding FLAG-FBXO3 or with the corresponding empty virus (Vector), lysed, and subjected to immunoblot analysis with antibodies to NEK1, C21ORF2, FLAG, and FBXO3. The arrowhead and asterisk indicate the band for endogenous C21ORF2 and a nonspecific band, respectively.

See also Figures S4 and S5.

Mutation of C21orf2 Induces NEK1 Stabilization in ESCs and an Aberrant Phenotype in iMNs

Given that the ALS-associated C21ORF2(V58L) mutant is stabilized as a result of its not being targeted by SCFFBXO3 for degradation, we hypothesized that the accumulation of the mutant protein might have adverse effects on motor neurons in patients with ALS. To assess this hypothesis, we analyzed the phenotype of motor neurons (iMNs) differentiated from mouse ESCs expressing green fluorescent protein (GFP) under the control of the Hb9 promoter (Hb9::GFP), which is selectively active in motor neurons (Wichterle et al., 2002). The valine-58 residue of human C21ORF2 that is replaced with leucine in association with ALS is conserved in the mouse protein (Figure 6A). To achieve robust and homogeneous induction of motor neuron differentiation in ESCs, we generated DOX-NIL ESCs, in which polycistronic expression of the transcription factors NGN2, ISL1, and LHX3 (NIL factors) is induced in response to DOX treatment, with these factors having been shown to be sufficient for such differentiation (Mazzoni et al., 2013; Velasco et al., 2017). We then subjected these DOX-NIL ESCs to heterozygous knockin of the V58L mutation of C21orf2, thereby generating VLKI ESCs (Figure 6B). The extent of alkaline phosphatase staining (Figure S6A) as well as the amounts of Oct4 and Fgf4 mRNAs (Figure S6B) did not differ between VLKI ESCs and the parental DOX-NIL ESCs, indicating that knockin of the C21orf2 mutation had no effect on ESC pluripotency. Consistent with our finding with HEK293T cells that C21ORF2(V58L) is stabilized as a result of its impaired interaction with FBXO3 (Figure 2), we found that the abundance of endogenous C21ORF2 was increased in VLKI ESCs compared with parental cells (Figure 6C), whereas the amount of C21orf2 mRNA was reduced in VLKI ESCs (Figure 6D), possibly as a result of negative feedback by the encoded protein. Cycloheximide chase analysis confirmed that total C21ORF2 protein was relatively stable in VLKI ESCs (Figure S6C). We also found that the amount of NEK1 protein was increased in VLKI ESCs (Figure 6C), without an increase in the abundance of the corresponding mRNA (Figure 6D), consistent with our earlier finding that C21ORF2 stabilizes NEK1 (Figure 4). To determine the possible effect of the C21orf2 mutation on the phenotype of motor neurons, we generated iMNs from VLKI ESCs by exposing the cells to DOX in order to induce expression of NIL factors. Immunoblot analysis of the VLKI cells at 2 days after DOX addition confirmed the accumulation of C21ORF2 and NEK1 compared with the levels of these proteins apparent in parental cells (Figure S6D). We measured the length of neurites formed by the GFP-positive cells and found that the average neurite length for VLKI iMNs was significantly shorter than that for iMNs differentiated from the parental ESCs (Figures 6E and 6F). Although the detailed mechanism underlying this phenotype of VLKI iMNs remains to be determined, the accumulation of C21ORF2 or NEK1 induced by C21orf2 mutation likely triggers aberrant differentiation or dysfunction of iMNs.

Figure 6.

Figure 6

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C21orf2 Mutation Induces NEK1 Stabilization in ESCs and Impairs Neurite Outgrowth in iMNs

(A) Amino acid sequence alignment of vertebrate C21ORF2 orthologs. Residue 58 (arrowhead) is enclosed in the red box.

(B) Sanger sequencing of DNA extracted from parental (DOX-NIL) and VLKI ESCs. The codon for valine-58 of C21orf2 is shown within the dashed box.

(C) Immunoblot analysis of endogenous C21ORF2 and NEK1 in parental and VLKI ESCs. Representative blots (left) and quantitative analysis of the band intensity for C21ORF2 and NEK1 normalized by that for HSP90 (right) are shown. Quantitative data are means ± SEM from three independent experiments. The p values were determined with Student's t test.

(D) RT-qPCR analysis of C21orf2 and Nek1 mRNA abundance (normalized by that of Gapdh mRNA) in parental and VLKI ESCs. Data are means ± SEM for three independent experiments. The p values were determined by Student's t test.

(E) Representative immunofluorescence analysis of parental and VLKI iMNs generated after exposure of the corresponding ESCs to DOX (3 μg/mL) for 2 days. The cells were stained with antibodies to βIII-tubulin (neuronal marker), and the fluorescence of GFP was monitored directly. Scale bar, 50 μm.

(F) Quantification of neurite length for parental and VLKI iMNs in images similar to those in (E). The length of neurites formed by iMNs differentiated from parental and VLKI ESCs was measured in eight wells (n = 8: four wells of a six-well plate in each of two independent experiments). Total neurite length for at least 15 iMNs per well was measured, with the average values being calculated and plotted for each genotype. The internal line and box boundaries indicate the median and quartile values. The p value was determined with Student's t test.

See also Figure S6.

Discussion

Multiple SCF-type ubiquitin ligases play pivotal roles in regulation of the cell cycle and suppression of tumorigenesis, with the functions of these enzymes having been studied mostly in relation to cancer development (Nakayama and Nakayama, 2006; Wang et al., 2014). On the other hand, emerging evidence implicates SCF complexes in the development of several neurodegenerative diseases. Mutations in the F box protein FBXO7 (encoded by PARK15), for example, have been shown to give rise to early-onset autosomal recessive Parkinson's disease (Shojaee et al., 2008), and SCFFBXL5 has been found to catalyze ubiquitylation of α-synuclein, which contributes to the Lewy body-like pathology of Parkinson's disease (Gerez et al., 2019). In addition, down-regulation of CUL1 and SKP1 has been observed in animal models of Huntington's disease and spinocerebellar ataxia type 3 (SCA3) (Bhutani et al., 2012), and SCFFBXO33 modulates the ubiquitylation and solubility of the polyglutamine protein SCA3 (Chen et al., 2019). However, no biological data have been available on the potential role of SCF-type ubiquitin ligases in the pathogenesis of ALS. Although CCNF, which encodes cyclin F (also known as FBXO1), is a causative gene of ALS (Williams et al., 2016), we previously found that SCFFBXO1 does not mediate the ubiquitylation of other major ALS-related proteins (Yu et al., 2019). We now demonstrate that an SCF-type ubiquitin ligase is associated with ALS pathogenesis by showing that C21ORF2 is a substrate of SCFFBXO3 in vivo, whereas its ALS-associated mutant V58L is not.

The substrates and biological roles of FBXO3 remain to be fully characterized. FBXO3 regulates gene expression associated with cytokine-driven inflammation as well as bone morphogenetic protein (BMP) signaling by mediating the proteasomal degradation of FBXL2 and SMAD-specific E3 ubiquitin ligase 1 (SMURF1), respectively (Chen et al., 2013; Li et al., 2015; Mallampalli et al., 2013). FBXO3 also increases the transcriptional activity of autoimmune regulator (AIRE) at genes for tissue-specific antigens by mediating its ubiquitylation (Shao et al., 2016). Although the ApaG domain in the COOH terminal region of FBXO3 is indispensable for recognition of FBXL2 (Mallampalli et al., 2013), our results now show that FBXO3 binds to its substrate C21ORF2 not via the ApaG domain but through the region upstream of this domain (Figure 1B). At least 21 conserved homology domains including the ApaG domain have been identified as sites of substrate binding in FBXO-type F box proteins (Jin et al., 2004; Skaar et al., 2013). Our data now indicate that FBXO3 possesses at least one other substrate recognition domain in addition to its ApaG domain. In general, substrate recognition by F box proteins is regulated by posttranslational modification (Skaar et al., 2013). In most cases, phosphorylation of a degron in the substrate facilitates its interaction with the F box protein. Phosphorylation of threonine-404 in FBXL2 and that of threonine-68 and serine-156 in AIRE are thus indispensable for the interaction with FBXO3 (Chen et al., 2013; Shao et al., 2016). However, we have now found that the binding of FBXO3 to C21ORF2 is negatively regulated by NEK1-catalyzed phosphorylation of C21ORF2, again suggesting that FBXO3 possesses at least two distinct domains for substrate recognition and that these domains differ in their phosphorylation requirement. Binding between an F box protein and its substrate, such as that between FBXO11 and CDT2 or between FBXL2 and p85β (Kuchay et al., 2013; Rossi et al., 2013), has previously been found to be inhibited by substrate phosphorylation.

Functional interaction between C21ORF2 and NEK1 has been previously suggested. The impaired repair of DNA damage apparent in C21ORF2-depleted HeLa cells was thus normalized by overexpression of NEK1 (Fang et al., 2015). In addition, a ciliopathy phenotype of zebrafish induced by injection of a NEK1 antisense morpholino-oligonucleotide was rescued by co-injection of human C21ORF2 mRNA (Wheway et al., 2015). These studies suggest that C21ORF2 and NEK1 play complementary roles in the same biological pathways. Given that we have now shown that C21ORF2 and NEK1 stabilize each other in a manner dependent on SCFFBXO3, our study may provide a possible explanation for the functional interaction between C21ORF2 and NEK1 observed in HeLa cells and zebrafish. Of note, we found that the V58L mutant of C21ORF2 undergoes excessive phosphorylation by NEK1, which attenuates its interaction with FBXO3 and thereby increases its stability, resulting in turn in the stabilization of NEK1 and in an aberrant motor neuron phenotype. NEK1 is thus a potential target for a kinase inhibitor that might be expected to limit the accumulation of C21ORF2 and thereby rescue neuronal function.

C21ORF2 is widely expressed among tissues, but its function has not been well elucidated (Wang et al., 2016). A reduced level of C21ORF2 expression has been observed in the brain of individuals with Down syndrome (Shim et al., 2003), suggesting that it may play a key role in neurons. C21ORF2 mutations have been found to cause early-onset retinal dystrophy, Jeune syndrome, and axial spondylometaphyseal dysplasia, the latter of which is also caused by NEK1 mutations (Khan et al., 2015; Wang et al., 2016, 2017; Wheway et al., 2015). These conditions are all categorized as ciliopathies, given that they result from dysfunction of the primary cilium, a solitary organelle located at the cell surface that regulates various cellular functions (Fliegauf et al., 2007). The proportion of lumbar motor neurons with a primary cilium was found to be reduced in a transgenic mouse model of ALS (Ma et al., 2011). Although the function of the primary cilium of motor neurons remains unknown, the accumulation of C21ORF2 in motor neurons of individuals with ALS-associated C21ORF2 mutations might induce dysfunction of the primary cilium and thereby contribute to ALS pathogenesis.

We found that stabilization of C21ORF2 protein resulted in an increased abundance of NEK1 and impaired the growth of neurites in iMNs. However, there is no genetic or biological evidence indicating that overexpression of NEK1 leads to dysfunction of motor neurons. Indeed, heterozygous frameshift mutations of NEK1 have been identified as causal mutations of ALS, suggesting that NEK1 haploinsufficiency gives rise to the development of ALS (Brenner et al., 2016). Consistent with this notion, motor neurons derived from human induced pluripotent stem cells harboring a heterozygous 2434A>T mutation of NEK1 were found to contain half the normal amount of NEK1 protein and to manifest impairment of DNA damage repair (Higelin et al., 2018). However, the effects of several other ALS-associated missense mutations, such as p.Asn181Ser located in the kinase domain, on NEK1 function remain to be determined. Demonstration that some such missense mutations increase the kinase activity of NEK1 would provide genetic support for our suggestion that overexpression of NEK1 may contribute to the development of ALS.

With regard to potential substrates of NEK1 that might play a role in the pathogenesis of ALS, NEK1 binds ALS2 and vesicle-associated membrane protein B (VAPB), both of which function in the endoplasmic reticulum transport system and are implicated as ALS-related proteins (Cirulli et al., 2015). In addition, kinesin family member 3A (KIF3A) was identified as an interacting protein for NEK1 and harbors putative phosphorylation sites (Surpili et al., 2003). KIF3A is a subunit of the heterotrimeric KIF3 motor complex that is essential for the elongation of neurites and which is down-regulated in the motor cortex of individuals with sporadic ALS (Hirokawa et al., 2009; Pantelidou et al., 2007). It is possible that pathogenic effects of NEK1 haploinsufficiency or hyperactivity are due to altered phosphorylation of these or other proteins.

Together, our data thus show that SCFFBXO3 targets C21ORF2 for proteasomal degradation and that the ubiquitylation of C21ORF2 is negatively regulated by NEK1-mediated phosphorylation of C21ORF2. Cross talk between ubiquitylation and phosphorylation thus regulates the abundance of C21ORF2. On the other hand, C21ORF2 suppresses the degradation of NEK1, which means that SCFFBXO3 limits the amounts of both C21ORF2 and NEK1. The ALS-associated V58L mutant of C21ORF2 is more stable than is the WT protein because it is phosphorylated to a greater extent by NEK1 and therefore resistant to ubiquitylation by FBXO3. It therefore mediates more robust inhibition of NEK1 degradation and gives rise to an aberrant phenotype of motor neurons. The accumulation of C21ORF2 and NEK1 thus likely contributes to the pathogenesis of ALS caused by mutation of C21ORF2 (Figure 7).

Figure 7.

Figure 7

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Proposed Model for the Mechanism by Which C21ORF2 Mutation Might Give Rise to ALS

Cross talk between FBXO3-mediated ubiquitylation and NEK1-catalyzed phosphorylation determines the intracellular abundance of C21ORF2, which in turn stabilizes NEK1. The V58L mutation of C21ORF2 disrupts the operation of this regulatory system, resulting in the accumulation of both C21ORF2 and NEK1 and giving rise to an aberrant motor neuron phenotype.

Limitations of the Study

The results of this study were obtained with cultured cells. Validation of our findings will thus be required with animal models.

Resource Availability

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Keiko Nakayama (nakayak2@med.tohoku.ac.jp).

Materials Availability

All unique and stable reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement.

Data and Code Availability

Original data from replicated experiments have been deposited to Mendeley Data: https://doi.org/10.17632/gg2d67hm43.1.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.

Acknowledgments

This work was supported by KAKENHI grants (15K18365, 17K14955, and 19K07837 to T.N.; 15H05667 and 18K07519 to N.S.; 16H05318 to M.A.; and 18H05215 and 17H04035 to K.N.) from Japan Society for the Promotion of Science (JSPS). We thank Y. Nagasawa and M. Kikuchi for general technical assistance, other laboratory members for discussion, and K. Eggan (Harvard University) for providing mouse ESCs (Hb9::GFP).

Author Contributions

Y.W. designed and performed most of the experiments as well as co-wrote the manuscript. T.N. and K.N. directed and coordinated the study, oversaw collection and analysis of the results, and co-wrote the manuscript. M.N. assisted with experiments. T.A., N.S., H.W., and M.A. provided advice. All authors discussed the results and commented on the manuscript.

Declaration of Interests

The authors declare no competing interests.

Published: September 25, 2020

Footnotes

Supplemental Information can be found online at https://doi.org/10.1016/j.isci.2020.101491.

Supplemental Information

Document S1. Transparent Methods, Figures S1–S6, and Table S1
mmc1.pdf (1.2MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Transparent Methods, Figures S1–S6, and Table S1
mmc1.pdf (1.2MB, pdf)

Data Availability Statement

Original data from replicated experiments have been deposited to Mendeley Data: https://doi.org/10.17632/gg2d67hm43.1.

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