Necdin Promotes Ubiquitin-Dependent Degradation of PIAS1 SUMO E3 Ligase

Necdin, a pleiotropic protein that promotes differentiation and survival of mammalian neurons, is a member of MAGE (melanoma antigen) family proteins that share a highly conserved MAGE homology domain. Several MAGE proteins interact with ubiquitin E3 ligases and modulate their activities. However, it remains unknown whether MAGE family proteins interact with SUMO (small ubiquitin-like modifier) E3 ligases such as PIAS (protein inhibitor of activated STAT) family, Nsmce2/Mms21 and Cbx4/Pc2. In the present study, we examined whether necdin interacts with these SUMO E3 ligases. Co-immunoprecipitation analysis revealed that necdin, MAGED1, MAGEF1 and MAGEL2 bound to PIAS1 but not to Nsmce2 or Cbx4. These SUMO E3 ligases bound to MAGEA1 but failed to interact with necdin-like 2/MAGEG1. Necdin bound to PIAS1 central domains that are highly conserved among PIAS family proteins and suppressed PIAS1-dependent sumoylation of the substrates STAT1 and PML (promyelocytic leukemia protein). Remarkably, necdin promoted degradation of PIAS1 via the ubiquitin-proteasome pathway. In transfected HEK293A cells, amino- and carboxyl-terminally truncated mutants of PIAS1 bound to necdin but failed to undergo necdin-dependent ubiquitination. Both PIAS1 and necdin were associated with the nuclear matrix, where the PIAS1 terminal deletion mutants failed to localize, implying that the nuclear matrix is indispensable for necdin-dependent ubiquitination of PIAS1. Our data suggest that necdin suppresses PIAS1 both by inhibiting SUMO E3 ligase activity and by promoting ubiquitin-dependent degradation.

Necdin is a member of the MAGE protein family bearing a large homologous region known as the MAGE homology domain (MHD) [14,15]. Placental mammals possess .30 MAGE genes per genome [14,15], whereas only a single MAGE gene has been identified in invertebrates such as the fruit fly (Drosophila melanogaster) [16] and non-mammalian vertebrates such as the zebrafish (Danio rerio) [17] and chicken (Gallus gallus) [18]. In yeasts, the MAGE protein has been identified as Nse3, a subunit of the SMC (structural maintenance of chromosomes) 5/6 complex involved in the homologous recombination-based repair of DNA damage [19][20][21]. Thus, it is speculated that mammalian MAGE genes evolved from a single ancestral MAGE gene [22,23]. Nonmammalian MAGE genes in the fruit fly [24,25], zebrafish [17] and chicken [18] are expressed during neurogenesis, suggesting that MAGE family genes are involved in neuronal development.
Mammalian MAGE family proteins are divided into two classes based on the sequence similarities of the MHDs and gene expression patterns [15]: Type I MAGEs such as MAGEA, MAGEB, and MAGEC subfamilies are expressed in cancer and male germ cells but not in normal cells, and their genes are located on chromosome X [26]. In contrast, Type II MAGEs such as necdin, necdin-like 2, MAGED, MAGEE, MAGEF, MAGEH, and MAGEL are expressed in normal cells including neural cells. Among Type II MAGE proteins, necdin, MAGEL2 [27] and necdin-like 2 [28] are encoded by the genes that are closely located on human chromosome 15. Human necdin (NDN) and MAGEL2 genes are located at chromosome 15q11-12, a region responsible for the pathogenesis of the classic genomic imprinting-associated neurodevelopmental disorder Prader-Willi syndrome [27,[29][30][31][32]. Both necdin and MAGEL2 are expressed only from the paternal alleles and implicated in the neurodevelopmental significance based on the phenotypes of gene knockout mice [12,27,33,34]. In contrast to necdin and its homologous MAGE proteins, there is limited information about biochemical functions of most MAGE family proteins.
Posttranslational modifications of proteins with ubiquitin and SUMO (small ubiquitin-related modifier) modulate their stability, intracellular localization, and biological function [35]. Furthermore, a crosstalk between ubiquitination and sumoylation plays key roles in the regulation of various cellular functions [36,37]. Several human MAGE proteins bind to RING (really interesting new gene)-type ubiquitin E3 ligases and promote ubiquitination of their substrates [38,39]. Thus, MAGE family proteins may serve as adaptor proteins that regulate protein degradation and turnover via the ubiquitin-proteasome pathway. Both ubiquitination and sumoylation utilize E1, E2, and E3 enzymes for their covalent modifications of target proteins [40]. Although numerous ubiquitin E3 ligases are involved in the specific substrate recognition, sumoylation relies on only a small number of E3 ligases such as PIAS family [41], Cbx4 (Chromobox homolog 4)/Pc2 (Polycomb 2) [42], Nsmce2 (non-SMC element 2)/Mms21 [43,44], and RanBP2 (Ran-binding protein 2) [45]. However, there is little information about physical and functional interactions between MAGE proteins and SUMO E3 ligases in mammalian cells. These findings prompted us to investigate whether necdin interacts with these SUMO E3 ligases.
We report here that necdin interacts with PIAS1, a typical RING-type SUMO E3 ligase involved in various biological events [46,47]. We demonstrate that necdin suppresses PIAS1 via two distinct mechanisms whereby necdin suppresses sumoylation of PIAS1 substrate proteins and promotes PIAS1 degradation in the ubiquitin-proteasome pathway. We also show that the amino (N)and carboxyl (C)-terminal regions of PIAS1 affect necdindependent ubiquitination. The present results provide novel insights into the regulatory mechanism of PIAS SUMO E3 ligase family by necdin and other MAGE family proteins.
We then analyzed the PIAS1-binding region of necdin using HEK293A cells transfected with truncated necdin mutants (Fig. 2C). PIAS1 interacted with full-length necdin and the Nterminally truncated mutant (CT) but not with the N-terminal region (NT). We also found that full-length necdin and CT reduced expression levels of Myc-PIAS1, suggesting that coexpressed necdin destabilizes the PIAS1 protein (Fig. 2C, third panel). To confirm whether the necdin MHD is required for PIAS1 binding, we used the p53 binding-defective mutant necdinDH4/5 (Fig. 2D). This mutant failed to bind to PIAS1 or p53 (control), suggesting that the H4/5 region of the MHD is indispensable for the interaction between necdin and PIAS1.

Necdin inhibits sumoylation of PIAS1 substrates
Because necdin interacted with the PINIT and SP-RING domains that constitute the catalytic region of PIAS1 [50], we investigated whether necdin affects the SUMO E3 ligase activity of PIAS1. We examined the effects of necdin on PIAS1-dependent sumoylation using HEK293A cells transfected with SUMO1 and the PIAS1 substrates STAT1 [41] and PML [51]. Because the PIAS1 protein level was decreased by co-expressed necdin, we used twice the amount of the PIAS1-expressing vector to avoid reducing the PIAS1 enzyme level. In this assay, faint bands of sumoylated STAT1 were detected at ,95 kDa in the absence of PIAS1, and PIAS1 promoted STAT1 sumoylation to 5.2 times the control level (Fig. 3A, B). Co-expression of necdin suppressed the PIAS1-promoted STAT1 sumoylation level by 57%. Similarly, PIAS1 markedly increased the level of sumoylated PML (6.6 times the control level) detected as smeared bands at .150 kDa, and cotransfection of necdin decreased the PML sumoylation level by 54% (Fig. 3C, D). These data suggest that necdin suppresses the SUMO E3 ligase activity of PIAS1.

Necdin promotes PIAS1 degradation via the ubiquitinproteasome pathway
In the preceding experiments, PIAS1 protein levels were reduced when necdin was co-expressed. Thus, we examined whether necdin affects the stability of PIAS1 using transfected HEK293A cells (Fig. 4A). Necdin reduced the PIAS1 level in a dose-dependent manner. To examine whether necdin degrades the PIAS1 protein via the ubiquitin-proteasomal pathway, we used the proteasome inhibitor MG132. Treatment with MG132 protected PIAS1 from necdin-promoted degradation (Fig. 4B), suggesting that necdin promotes PIAS1 degradation in the proteasome.
We then examined whether necdin promotes PIAS1 ubiquitination by immunoprecipitation assay using HEK293A cells transfected with ubiquitin cDNA (Fig. 4C), Ubiquitinated PIAS1 was detected as multiple bands at 100-250 kDa. Although the ubiquitinated PIAS1 level was reduced, presumably owing to the degradation of the PIAS1 protein, in the absence of MG132, necdin significantly promoted PIAS1 ubiquitination in the presence of MG132 (Fig. S1A). We also found that the necdin DH4/5 mutant failed to promote PIAS1 ubiquitination (Fig. S1B). These data indicate that necdin promotes PIAS1 degradation in the ubiquitin-proteasome system. To analyze the specificity of necdin in PIAS1 degradation, we examined the effects of MAGEA1 and necdin-like 2 on PIAS1 ubiquitination in the presence of MG132. MAGEA1 promoted ubiquitination of PIAS1 to a significant but lesser extent than necdin, whereas necdin-like 2 exerted little or no ubiquitination-promoting effect (Fig. 4D, Fig.  S1C).
We next examined the subcellular localization of PIAS1 and necdin by immunocytochemistry using transfected HEK293A cells. PIAS1 was localized exclusively in the nucleus, whereas necdin was in both the nucleus and the cytoplasm (Fig. 5A). Although co-expression of necdin and PIAS1 did not alter their subcellular localization patterns, co-expressed necdin markedly reduced nuclear PIAS1 signals. We then examined the effects of MG132 on the distribution and expression levels of PIAS1 (Fig. 5B). MG132 per se had little or no effect on the localization of PIAS1 or necdin, but suppressed the necdin-induced reduction of nuclear PIAS1 levels. We classified PIAS1(Myc)-expressing cells into three groups by relative fluorescence intensities; undetectable or barely detectable, low, and high ( Fig. 5C, D). The PIAS1 immunoreactivity in 55% of necdin-positive cells was undetectable or barely detectable in the absence of MG132, whereas 52% of necdin-immunopositive cells had high fluorescence intensities in the presence of MG132, suggesting that necdin promotes degradation of nuclear PIAS1 in the proteasome.
To examine whether ectopic expression of necdin promotes degradation of endogenous PIAS1, we infected H1299 cells, a human non-small cell lung carcinoma cell line that endogenously expresses PIAS1 at a relatively high level, with necdin-expressing lentivirus vector and analyzed the PIAS1 level by Western blotting (Fig. 6). Under the conditions where most of the cells were infected

PIAS1 N-and C-terminal regions are required for necdindependent ubiquitination
To determine the PIAS1 regions responsible for necdindependent degradation, we transfected HEK293A cells with PIAS1 mutants lacking the N-terminus (DNT) and C-terminus (DCT) (Fig. 7A). As expected, these truncated mutants, which contain the central conserved domains, bound to necdin (Fig. 7B). Although necdin significantly reduced the full-length PIAS1 level, PIAS1DNT and DCT mutants were resistant to necdin-promoted degradation (Fig. 7C, Fig. S1D). We also examined whether these mutants undergo necdin-dependent ubiquitination (Fig. 7D). PIAS1DNT was resistant to necdin-promoted ubiquitination, whereas PIAS1DCT underwent robust ubiquitination in a necdin-independent manner. These results suggest that these PIAS1 terminal regions mediate the susceptibility of PIAS1 to necdin-dependent ubiquitination. To investigate the mechanism whereby the terminal truncation of PIAS1 affects necdin-dependent ubiquitination, we analyzed the subcellular localization of ectopically expressed PIAS1 mutants in HEK293A cells. The full-length PIAS1 and PIAS1DNT were distributed in the nucleus, whereas PIAS1DCT localized predominantly in the cytoplasm (Fig. 8A). All of the full-length and mutant PIAS1 (Myc)-transfected cells showed similar distribution patterns.
PIAS1DNT partially accumulated as nuclear speckles that were immunopositive for PML (Fig. S2), consistent with the previous observation that a PIAS1 mutant lacking the SAP domain localizes to the PML bodies [52]. Because PIAS4 localizes in the nuclear matrix via its N-terminal SAP domain [53], we immunocytochemically examined the localization of these mutants in the nuclear matrix (Fig. 8B). Necdin localized in the nuclear matrix and accumulated as distinct speckles, consistent with the previous findings [54]. Full-length PIAS1 localized in the nuclear matrix of all of the transfected immunopositive cells, whereas PIAS1 deletion mutants were totally undetectable in the nuclear matrix, suggesting that the N-and C-terminal regions of PIAS1 are indispensable for its nuclear matrix localization.

Discussion
The present study has demonstrated that necdin interacts with PIAS1 SUMO E3 ligase and suppresses its catalytic activity. Other MAGE proteins, except necdin-like 2, also bound to PIAS1, whereas Nsmce2 and Cbx4 SUMO E3 ligases bound only to MAGEA1 (Fig.1D). We have found that necdin interacts with RanBP2, another well-studied SUMO E3 ligase associated with the nuclear pore complex (KF and KY, unpublished observation). Several MAGE proteins bind to RING-type ubiquitin E3 ligases such as TRIM27 and TRIM28 to promote ubiquitination of their substrates, but necdin is unable to interact with these ubiquitin E3 ligases [38,39]. We confirmed the previous findings [38,55] that Nsmce1, the ubiquitin E3 ligase in the SMC5/6 complex, interacts with necdin-like 2 and MAGEF1 but not with necdin (Fig. 1A). Necdin-like 2 shows biochemical and functional characteristics similar to those of necdin [56]. Additionally, chicken and Drosophila MAGE proteins, which resemble mammalian necdin-like 2, exhibit functional similarities to necdin [18,25]. In contrast to these similarities among MAGE family proteins, it is likely that mammalian MAGE proteins are diversified to interact with their specific ubiquitin and SUMO E3 ligases (Fig. S3).
The present study has also shown that necdin promotes degradation of PIAS1 via the ubiquitin-proteasome pathway. Necdin is likely to enhance PIAS1 ubiquitination by interacting with endogenous ubiquitin E3 ligases. PIAS1 is ubiquitinated by the ubiquitin E3 ligase hSiah2 (human homologues of seven in absentia) [57]. Necdin binds to the ubiquitin E3 ligase Mdm2 and promotes degradation of the proapoptotic protein CCAR1/ CARP1 (cell cycle apoptosis regulatory protein) [58]. Thus, we examined whether hSiah2 and Mdm2 mediate necdin-promoted PIAS1 ubiquitination. However, we found that these ubiquitin E3 ligases were unable to ubiquitinate PIAS1 in a necdin-dependent manner (I.G. and K.Y., unpublished observations). Although detailed molecular mechanisms underlying necdin-promoted PIAS1 ubiquitination remain to be elucidated, we assume that necdin, like other MAGE proteins [38], serves as an adaptor to form a necdin-ubiquitin E3 ligase complex for PIAS1 ubiquitination. We found that PIAS1 promoted PML sumoylation (Fig. 3C), consistent with the previous report [51]. PIAS1-dependent PML sumoylation may promote PML degradation by RNF4, a SUMOdirected E3 ubiquitin ligase [51,59,60]. Thus, it is possible that necdin prevents PML degradation by suppressing PIAS1-dependent PML sumoylation. Because PML regulates cell fate during neocortical development [61], we speculate that necdin stabilizes PML by suppressing endogenous PIAS1 during neuronal differentiation.
The necdin-interacting region of PIAS1 consists of two known domains: the PINIT domain that contains the Pro-Ile-Asn-Ile-Thr motif in the middle of the PIAS-conserved sequence stretch [62] and the SP-RING domain similar to the RING-finger domain found in many ubiquitin E3 ligases [46,63]. These two domains are highly conserved among PIAS family members [47]. Although PIAS1 interacts with SUMO-modified substrates via its distinct regions throughout the whole molecule [46], the SUMO ligase activity relies solely on these central domains [50]. As expected, necdin inhibited PIAS1-dependent sumoylation of STAT1 [41] and PML [51] (Fig. 3). Thus, it is likely that necdin-mediated suppression of the PIAS1 catalytic activity is attributed to its specific binding region in PIAS1.
The necdin deletion mutant that lacks aa 191-222 (DH4/5) in the MHD (aa116-280) failed to bind to PIAS1 (Fig. 2D), indicating that necdin interacts with PIAS1 via its MHD. Homology modeling based on the X-ray crystallographic data of necdin-like 2 [38] revealed that the mouse necdin aa 191-222 region corresponds to helices 4 and 5 in the winged-helix motif B (WH-B) (Fig. S4A, B). The H4/5 region in necdin-like 2 is predicted to form a hydrophobic pocket that interacts with NSMCE4A [49]. We have previously found that this deletion mutant neither induces cell growth suppression nor associates with the nuclear matrix [48]. Because MAGE proteins such as MAGEA1, MAGED1, MAGEF1 and MAGEL2 interact with PIAS1 (Fig. 1D), it is possible that the PIAS1-binding domains of these MAGE proteins take configurations similar to that of necdin. In contrast, necdin-like 2, which shows structural and functional similarities to necdin [56], failed to interact with PIAS1. Necdin-  like 2 bears five amino acid substitutions in the H4/5 region compared with PIAS1-interacting MAGE proteins (Fig. S4C). It is noteworthy that three Thr residues are present in and near the H5 region of necdin-like 2, whereas other MAGE proteins contain only one or no Thr residue in their H4/5 regions. We speculate that necdin-like 2 lacks PIAS1 binding owing to its unique configuration of the H4/5 region.
The N-terminal deletion of PIAS1 lost both ubiquitination and responsiveness to necdin (Fig.7). The N-terminal domain contains a highly conserved SAP domain that mediates association with the nuclear matrix. Thus, PIAS1 is likely to interact with the nuclear matrix via the SAP domain. We found that the PIAS1 N-terminal deletion mutant was dissociated from the nuclear matrix and translocated to the PML bodies (Fig. 8B, Fig. S2), consistent with the previous report [52]. We have previously shown that necdin localizes in the nuclear matrix and interacts with the nuclear matrix-associated protein SAF-A/hnRNP U [54]. These findings suggest that necdin and PIAS1 are colocalized in the nuclear matrix, where necdin modulates PIAS1 ubiquitination. Intriguingly, PIAS1 C-terminal deletion enhanced ubiquitination in a necdin-independent manner (Fig. 7), implying that the PIAS1 Cterminus negatively controls necdin-promoted ubiquitination. Because C-terminally truncated PIAS1 was translocated to the cytoplasm (Fig. 8), the C-terminus of PIAS1 is likely to contribute to the nuclear retention. We speculate that these terminal regions are required for the translocation of PIAS1 to the nuclear matrix that contributes to the necdin-dependent ubiquitination.
Necdin plays anti-apoptotic and pro-survival roles in various cell types such as neurons and neural stem/progenitor cells through interactions with various proteins [5,6,[8][9][10][11][12]64]. Previous studies have shown that PIAS1 promotes apoptosis in response to various stimuli [52,65,66]. These observations suggest that necdin targets PIAS1 to suppress its proapoptotic activities. Because the necdinbinding region in PIAS1 is highly conserved among PIAS family members, it is possible that necdin also interacts with other PIAS family proteins to regulate their functions. Although information on the regulation of PIAS family proteins during neuronal development is currently limited, we speculate that necdin interacts with PIAS family proteins to modulate sumoylation of their target proteins involved in mammalian neuron development.

Sumoylation assay
HEK293A cells (1610 6 cells per 60 mm dish) were transfected with combinations of expression vectors for HA-SUMO1, Myc-PIAS1, necdin, FLAG-STAT1 and FLAG-PML and harvested 24 hrs after transfection. For analysis of PML sumoylation, cells were treated with 10 mM MG132 (Peptide Institute) 4 hrs prior to harvest as described [51]. Cells were lysed in the lysis buffer supplemented with 20 mM N-ethylmaleimide, and the lysates (300 mg) were incubated at 4uC for 2 hrs with anti-FLAG antibody (M2; Sigma-Aldrich). The protein-antibody complexes were pelleted with protein A-Sepharose, separated by 10% SDS-PAGE, and detected by Western blotting as above. Signal intensities of sumoylated proteins were quantitated by densitometry with ImageJ 1.48 software. The protein concentration was determined by the Bradford method (Bio-Rad).

Ubiquitination assay
HEK293A cells (1610 6 cells per 60 mm dish) were transfected with combinations of expression vectors for FLAG-tagged ubiquitin, HA-tagged ubiquitin, Myc-tagged full-length PIAS1, Myc-tagged PIAS1 deletion mutants, necdin, FLAG-tagged necdin, FLAG-tagged MAGEA1, and FLAG-tagged necdin-like 2. Transfected cells were incubated for 20 hrs, treated with 30 mM MG132 or DMSO (vehicle control) for 4 hrs, and harvested. Cells lysates (250 mg) were incubated at 4uC for 2 hrs with the anti-Myc antibody, and the protein-antibody complexes were pelleted with protein A-Sepharose, separated by 10% SDS-PAGE, and detected by Western blotting as above. Signal intensities of ubiquitinated proteins were quantitated by densitometry.

Lentivirus infection
Recombinant lentiviruses were produced in HEK293FT cells by transfecting SIN vector plasmids and two helper plasmids as described previously [70,71]. Necdin cDNAs were subcloned into pENTR1A entry vector (Life Technologies) to construct CSII-EF1a-necdin-IRES-EmGFP, in which Emerald Green Fluorescent Protein (EmGFP) (Life Technologies) was used for an expression indicator [6]. The viral titer was measured by serial dilution on HEK293FT cells (Life Technologies) and determined as GFP-positive cell population by immunocytochemistry. Lentivirus vectors for EmGFP (empty vector) or necdin (+EmGFP) were infected into H1299 cells at a multiplicity of infection (moi) of 12, and the infected cells were incubated for 72 hrs prior to analyses. Expressed proteins were analyzed by Western blotting using antibodies against PIAS1 (EPR2518Y, Abcam; 1:5000), necdin, and c-tubulin. Signal intensities of proteins were quantitated by densitometry.

Statistics
Statistical significance was tested using Tukey-Kramer multiple comparison method unless otherwise stated. A significance of p, 0.05 was required for rejection of the null hypothesis. Figure S1 Quantification of necdin-promoted ubiquitination and degradation of PIAS1. (A) Effects of necdin on PIAS1 ubiquitination. Ubiquitinated PIAS1 signals shown in Fig. 4C were quantified by densitometry, and the signal intensities of ubiquitinated proteins were normalized with those of Myc-PIAS1. Images with two different exposure times for each sample were analyzed. (B) Effect of necdin DH4/5 mutant on PIAS1 ubiquitination. PIAS1 was ubiquitinated by wild-type necdin (WT) and necdin DH4/5 mutant (DH4/5) in the presence of MG132, and ubiquitinated PIAS1 signals were quantified as above. (C) Effects of MAGEA1 and necdin-like 2 on PIAS1 ubiquitination. Ubiquitinated PIAS1 signals shown in Fig. 4D was ubiquitinated as above. (D) Effects of necdin on protein levels of PIAS1 and its mutants. Signals of ubiquitinated PIAS1 and its mutants shown in Fig. 7C