Yohan Oh

Dyrk1A Phosphorylates p53 and Inhibits Proliferation of Embryonic Neuronal Cells

Down syndrome (DS) is associated with many neural defects, including reduced brain size and impaired neuronal proliferation, highly contributing to the mental retardation. Those typical characteristics of DS are closely associated with a specific gene group “Down syndrome critical region” (DSCR) on human chromosome 21. Here we investigated the molecular mechanisms underlying impaired neuronal proliferation in DS and, more specifically, a regulatory role for dual-specificity tyrosine-(Y) phosphorylation-regulated kinase 1A (Dyrk1A), a DSCR gene product, in embryonic neuronal cell proliferation. We found that Dyrk1A phosphorylates p53 at Ser-15 in vitro and in immortalized rat embryonic hippocampal progenitor H19-7 cells. In addition, Dyrk1A-induced p53 phosphorylation at Ser-15 led to a robust induction of p53 target genes (e.g. p21CIP1) and impaired G1/G0-S phase transition, resulting in attenuated proliferation of H19-7 cells and human embryonic stem cell-derived neural precursor cells. Moreover, the point mutation of p53-Ser-15 to alanine rescued the inhibitory effect of Dyrk1A on neuronal proliferation. Accordingly, brains from embryonic DYRK1A transgenic mice exhibited elevated levels of Dyrk1A, Ser-15 (mouse Ser-18)-phosphorylated p53, and p21CIP1 as well as impaired neuronal proliferation. These findings suggest that up-regulation of Dyrk1A contributes to altered neuronal proliferation in DS through specific phosphorylation of p53 at Ser-15 and subsequent p21CIP1 induction.

Proteasome Inhibition Induces α-Synuclein SUMOylation and Aggregate Formation

Parkinsons disease (PD) and Dementia with Lewy Bodies (DLB) are characterized pathologically by intraneuronal inclusions called Lewy bodies (LBs) and Lewy neurites. A major component of these inclusions is the protein α-synuclein, which is natively unfolded but forms oligomers and insoluble fibrillar aggregates under pathological conditions. Although α-synuclein is known to undergo several posttranslational modifications, the contribution of SUMOylation to α-synuclein aggregation and the pathogenesis of α-synucleinopathies have not been elucidated. Here, we provide evidence that aggregates and inclusions formed as a result of impaired proteasome activity contain SUMOylated α-synuclein. Additionally, SUMO1 is present in the halo of LBs colocalizing with α-synuclein in the brains of PD and DLB patients. Interestingly, SUMOylation does not affect the ubiquitination of α-synuclein. These findings suggest that proteasomal dysfunction results in the accumulation of SUMOylated α-synuclein and subsequently its aggregation, pointing to the contribution of this posttranslational modification to the pathogenesis of inclusion formation in α-synucleinopathies.

NF-κB-inducing Kinase Phosphorylates and Blocks the Degradation of Down Syndrome Candidate Region 1

Down syndrome, the most frequent genetic disorder, is characterized by an extra copy of all or part of chromosome 21. Down syndrome candidate region 1 (DSCR1) gene, which is located on chromosome 21, is highly expressed in the brain of Down syndrome patients. Although its cellular function remains unknown, DSCR1 expression is linked to inflammation, angiogenesis, and cardiac development. To explore the functional role of DSCR1 and the regulation of its expression, we searched for novel DSCR1-interacting proteins using a yeast two-hybrid assay. Using a human fetal brain library, we found that DSCR1 interacts with NF-κB-inducing kinase (NIK). Furthermore, we demonstrate that NIK specifically interacts with and phosphorylates the C-terminal region of DSCR1 in immortalized hippocampal cells as well as in primary cortical neurons. This NIK-mediated phosphorylation of DSCR1 increases its protein stability and blocks its proteasomal degradation, the effects of which lead to an increase in soluble and insoluble DSCR1 levels. We show that an increase in insoluble DSCR1 levels results in the formation of cytosolic aggregates. Interestingly, we found that whereas the formation of these inclusions does not significantly alter the viability of neuronal cells, the overexpression of DSCR1 without the formation of aggregates is cytotoxic.

ASK1 Negatively Regulates the 26 S Proteasome

The 26 S proteasome, composed of the 20 S core and 19 S regulatory particle, plays a central role in ubiquitin-dependent proteolysis. Disruption of this process contributes to the pathogenesis of the various diseases; however, the mechanisms underlying the regulation of 26 S proteasome activity remain elusive. Here, cell culture experiments and in vitro assays demonstrated that apoptosis signal-regulating kinase 1 (ASK1), a member of the MAPK kinase kinase family, negatively regulated 26 S proteasome activity. Immunoprecipitation/Western blot analyses revealed that ASK1 did not interact with 20 S catalytic core but did interact with ATPases making up the 19 S particle, which is responsible for recognizing polyubiquitinated proteins, unfolding them, and translocating them into the 20 S catalytic core in an ATP-dependent process. Importantly, ASK1 phosphorylated Rpt5, an AAA ATPase of the 19 S proteasome, and inhibited its ATPase activity, an effect that may underlie the ability of ASK1 to inhibit 26 S proteasome activity. The current findings point to a novel role for ASK1 in the regulation of 26 S proteasome and offer new strategies for treating human diseases caused by proteasome malfunction.

Human Polycomb protein 2 promotes α-synuclein aggregate formation through covalent SUMOylation

Parkinsons disease (PD) manifests from the impairment of motor systems due to the specific loss of dopaminergic neurons and the appearance of intracellular filamentous inclusions called Lewy bodies (LBs). α-Synuclein, a major component of LBs, is known to contribute to the pathogenesis of PD. Although α-synuclein is known to be a target of diverse posttranslational modifications, the contribution of α-synuclein SUMOylation and its functional consequences have not yet been fully characterized. Here, we demonstrate that human Polycomb protein 2 (hPc2) binds to α-synuclein and may function as a SUMO E3 ligase to promote the SUMOylation of α-synuclein. In addition, hPc2 promotes the SUMOylation of α-synuclein in the presence of MG-132-induced proteasome inhibition, which consequently promotes α-synuclein aggregate formation. Furthermore, the increased formation of intracellular α-synuclein aggregates, which predominantly contain SUMOylated α-synuclein, significantly reduces the death of fibroblast cells in response to staurosporine. In summary, the results from this study demonstrate that the hPc2-induced SUMOylation of α-synuclein could function as a cytoprotector by increasing α-synuclein aggregate formation within fibroblast cells.

Neddylation positively regulates the ubiquitin E3 ligase activity of parkin.

Mutations in the parkin gene underlie a familial form of Parkinsons disease known as autosomal recessive juvenile Parkinsonism (AR‐JP). Dysfunction of parkin, a ubiquitin E3 ligase, has been implicated in the accumulation of ubiquitin proteasome system‐destined substrates and eventually leads to cell death. However, regulation of parkin enzymatic activity is incompletely understood. Here we investigated whether the ubiquitin E3 ligase activity of parkin could be regulated by neddylation. We found that parkin could be a target of covalent modification with NEDD8, a ubiquitin‐like posttranslational modifier. In addition, NEDD8 attachment caused an increase of parkin activity through the increased binding affinity for ubiquitin‐conjugating E2 enzyme as well as the enhanced formation of the complex containing parkin and substrates. These findings point to the functional importance of NEDD8 and suggest that neddylation is one to the diverse modes of parkin regulation, potentially linking it to the pathogenesis of AR‐JP.

α-Synuclein overexpression reduces gap junctional intercellular communication in dopaminergic neuroblastoma cells

Alpha-synuclein has been implicated in the pathology of certain neurodegenerative diseases, including Parkinson disease (PD) and dementia with Lewy bodies (LBs). Overexpression of human alpha-synuclein in neuronal cells reduces cell viability, but the precise cellular and molecular mechanisms remain poorly understood. Gap junctional intercellular communication (GJIC) is thought to be essential for maintaining cellular homeostasis and growth control. In the present study, the effect of alpha-synuclein overexpression on GJIC in human dopaminergic neuroblastoma SH-SY5Y cells was investigated. Cells overexpressing wild-type alpha-synuclein were more vulnerable to hydrogen peroxide and 6-hydroxydopamine. GJIC was decreased in cells overexpressing alpha-synuclein. In addition, alpha-synuclein binds directly to connexin-32 (Cx32). As such, the post-translational modification of Cx32 was enhanced in cells overexpressing alpha-synuclein. These findings suggest that alpha-synuclein can modulate GJIC in a dopaminergic neuronal cell line through specific binding to Cx32.

Protein kinase A phosphorylates Down syndrome critical region 1 (RCAN1)

The Down syndrome critical region 1 (DSCR1) gene encodes a regulator of the calcineurin 1 (RCAN1) protein, and the elevated levels of RCAN1 are associated with Alzheimers disease (AD) and Down syndrome (DS). In this report, we found that protein kinase A (PKA) was able to phosphorylate RCAN1 in vitro and in vivo. In addition, we found that the phosphorylation of RCAN1 by PKA caused an increase of RCAN1 expression by increasing of the half-life of the protein. Consistently, the pharmacological inhibition of intracellular PKA using H-89 and the knockdown of the endogenous PKA catalytic subunit with siRNA decreased the expression of RCAN1. Furthermore, the phosphorylation of RCAN1 by PKA enhanced the inhibitory function of RCAN1 on calcineurin-mediated gene transcription. Our data provide the first evidence that PKA acts as an important regulatory component in the control of RCAN1 function through phosphorylation.

Small Ubiquitin-like Modifier (SUMO) Modification of Zinc Finger Protein 131 Potentiates Its Negative Effect on Estrogen Signaling

Background: A poorly characterized zinc finger protein, ZNF131, is implicated as a negative regulator of estrogen receptor α target gene expression. Results: ZNF131 is a target of covalent SUMOylation via hPc2, which potentiates its inhibitory effect on estrogen signaling. Conclusion: Estrogen signaling is down-regulated through the SUMOylation of ZNF131. Significance: Covalent SUMO1 modification of ZNF131 negatively regulates estrogen receptor-mediated signaling and estrogen-induced cell proliferation in breast cancer cells. Like ubiquitin, small ubiquitin-like modifier (SUMO) covalently attaches to specific target proteins and modulates their functional properties, including subcellular localization, protein dimerization, DNA binding, and transactivation of transcription factors. Diverse transcriptional co-regulator complexes regulate the ability of estrogen receptors to respond to positive and negative acting hormones. Zinc finger protein 131 (ZNF131) is poorly characterized but may act as a repressor of estrogen receptor α (ERα)-mediated trans-activation. Here, we identify ZNF131 as a target for SUMO modification and as a substrate for the SUMO E3 ligase human polycomb protein 2 (hPc2). We report that the SUMO-interacting motif 1 (SIM1) and the C-box of hPc2 are critical regions required for ZNF131 SUMOylation and define the ZNF131 SUMOylation site as lysine 567. We further show that SUMO modification potentiates the negative effect of ZNF131 on estrogen signaling and consequently attenuates estrogen-induced cell growth in a breast cancer cell line. Our findings suggest that SUMOylation is a novel regulator of ZNF131 action in estrogen signaling and breast cancer cell proliferation.

UHRF2, a Ubiquitin E3 Ligase, Acts as a Small Ubiquitin-like Modifier E3 Ligase for Zinc Finger Protein 131

Background: The nuclear protein UHRF2 is a member of the RING-type ubiquitin E3 ligase family. Results: UHRF2 promotes covalent SUMOylation of ZNF131 regardless of its ubiquitin E3 ligase activity. Conclusion: UHRF2 acts as a novel SUMO E3 ligase for ZNF131. Significance: UHRF2 is a dual-functional E3 ligase for covalent ubiquitin and SUMO conjugation. Small ubiquitin-like modifier (SUMO), a member of the ubiquitin-related protein family, is covalently conjugated to lysine residues of its substrates in a process referred to as SUMOylation. SUMOylation occurs through a series of enzymatic reactions analogous to that of the ubiquitination pathway, resulting in modification of the biochemical and functional properties of substrates. To date, four mammalian SUMO isoforms, a single heterodimeric SUMO-activating E1 enzyme SAE1/SAE2, a single SUMO-conjugating E2 enzyme ubiquitin-conjugating enzyme E2I (UBC9), and a few subgroups of SUMO E3 ligases have been identified. Several SUMO E3 ligases such as topoisomerase I binding, arginine/serine-rich (TOPORS), TNF receptor-associated factor 7 (TRAF7), and tripartite motif containing 27 (TRIM27) have dual functions as ubiquitin E3 ligases. Here, we demonstrate that the ubiquitin E3 ligase UHRF2 also acts as a SUMO E3 ligase. UHRF2 effectively enhances zinc finger protein 131 (ZNF131) SUMOylation but does not enhance ZNF131 ubiquitination. In addition, the SUMO E3 activity of UHRF2 on ZNF131 depends on the presence of SET and RING finger-associated and nuclear localization signal-containing region domains, whereas the critical ubiquitin E3 activity RING domain is dispensable. Our findings suggest that UHRF2 has independent functional domains and regulatory mechanisms for these two distinct enzymatic activities.