Sangjune Kim

Macro Histone H2A1.2 (MacroH2A1) Protein Suppresses Mitotic Kinase VRK1 during Interphase

Background: VRK1 phosphorylates mitotic histone H3 at Thr-3 and Ser-10, but its negative regulator was not elucidated during interphase. Results: The macrodomain of macroH2A1 interacts with VRK1, and this suppresses enzymatic activity of VRK1 during interphase. Conclusion: Specific binding between VRK1 and macroH2A1 is required to regulate the cell cycle-dependent histone H3 phosphorylation. Significance: Understanding epigenetic regulation of histone H3 during the cell cycle is important in cancer development. VRK1-mediated phosphorylation of histone H3 should be restricted in mitosis for consistent cell cycling, and defects in this process trigger cellular catastrophe. However, an interphasic regulator against VRK1 has not been actually investigated so far. Here, we show that the histone variant macrodomain-containing histone H2A1.2 functions as a suppressor against VRK1 during interphase. The level of macroH2A1.2 was markedly reduced in the mitotic phase, and the macroH2A1.2-mediated inhibition of histone H3 phosphorylation occurred mainly during interphase. We also found direct interaction and binding features between VRK1 and macroH2A1.2 by NMR spectroscopy. Hence, our findings might provide valuable insight into the underlying molecular mechanism regarding an epigenetic regulation of histone H3 during the cell cycle.

Therapeutic Approaches for Inhibition of Protein Aggregation in Huntington's Disease

Huntingtons disease (HD) is a late-onset and progressive neurodegenerative disorder that is caused by aggregation of mutant huntingtin protein which contains expanded-polyglutamine. The molecular chaperones modulate the aggregation in early stage and known for the most potent protector of neurodegeneration in animal models of HD. Over the past decades, a number of studies have demonstrated molecular chaperones alleviate the pathogenic symptoms by polyQ-mediated toxicity. Moreover, chaperone-inducible drugs and anti-aggregation drugs have beneficial effects on symptoms of disease. Here, we focus on the function of molecular chaperone in animal models of HD, and review the recent therapeutic approaches to modulate expression and turn-over of molecular chaperone and to develop anti-aggregation drugs.

Modulation of exosome-mediated mRNA turnover by interaction of GTP-binding protein 1 (GTPBP1) with its target mRNAs

Eukaryotic mRNA turnover is among most critical mechanisms that affect mRNA abundance and are regulated by mRNA‐binding proteins and the cytoplasmic exosome. A functional protein, guanosinetriphosphate‐binding protein 1 (GTPBP1), which associates with both the exosome and target mRNAs, was identified. The overexpression of GTPBP1 accelerated the target mRNA decay, whereas the reduction of the GTPBP1 expression with RNA interference stabilized the target mRNA. GTPBP1 has a putative guanosinetriphosphate (GTP)‐binding domain, which is found in members of the G‐protein family and Ski7p, a well‐known core factor of the exosome‐mediated mRNA turnover pathway in yeast. Analyses of protein interactions and mRNA decay demonstrated that GTPBP1 modulates mRNA degradation via GTP‐binding‐dependent target loading. Moreover, GTPBP1‐knockout models displayed multiple mRNA decay defects, including elevated nocturnal levels of Aanat mRNA in pineal glands, and retarded degradation of TNF‐α mRNA in lipopolysaccharide‐treated splenocytes. The results of this study suggest that GTPBP1 is a regulator and adaptor of the exosome‐mediated mRNA turnover pathway.—Woo, K.‐C., Kim, T.‐D., Lee, K.‐H., Kim, D.‐Y. Kim, S., Lee, H.‐R., Kang, H.‐J., Chung, S. J., Senju, S., Nishimura, Yasuharu, Kim, K.‐T. Modulation of exosome‐mediated mRNA turnover by interaction of GTP‐binding protein 1 (GTPBP1) with its target mRNAs. FASEB J. 25, 2757‐2769 (2011). www.fasebj.org

Protein kinase Cδ regulates vaccinia-related kinase 1 in DNA damage-induced apoptosis.

A pro-apoptotic function of activated PKCδ may be mediated by several downstream nuclear regulators involved in apoptotic cell death. Vaccinia-related kinase 1 (VRK1) is a new nuclear target of PKCδ in the regulation of apoptotic cell death induced by DNA damage.

Vaccinia-related kinase 2 mediates accumulation of polyglutamine aggregates via negative regulation of the chaperonin TRiC

ABSTRACT Misfolding of proteins containing abnormal expansions of polyglutamine (polyQ) repeats is associated with cytotoxicity in several neurodegenerative disorders, including Huntingtons disease. Recently, the eukaryotic chaperonin TRiC hetero-oligomeric complex has been shown to play an important role in protecting cells against the accumulation of misfolded polyQ protein aggregates. It is essential to elucidate how TRiC function is regulated to better understand the pathological mechanism of polyQ aggregation. Here, we propose that vaccinia-related kinase 2 (VRK2) is a critical enzyme that negatively regulates TRiC. In mammalian cells, overexpression of wild-type VRK2 decreased endogenous TRiC protein levels by promoting TRiC ubiquitination, but a VRK2 kinase-dead mutant did not. Interestingly, VRK2-mediated downregulation of TRiC increased aggregate formation of a polyQ-expanded huntingtin fragment. This effect was ameliorated by rescue of TRiC protein levels. Notably, small interference RNA-mediated knockdown of VRK2 enhanced TRiC protein stability and decreased polyQ aggregation. The VRK2-mediated reduction of TRiC protein levels was subsequent to the recruitment of COP1 E3 ligase. Among the members of the COP1 E3 ligase complex, VRK2 interacted with RBX1 and increased E3 ligase activity on TRiC in vitro. Taken together, these results demonstrate that VRK2 is crucial to regulate the ubiquitination-proteosomal degradation of TRiC, which controls folding of polyglutamine proteins involved in Huntingtons disease.

Preparation and evaluation of BBB-permeable trehalose derivatives as potential therapeutic agents for Huntington's disease†

The BBB-permeable trehalose derivative (TD-G6) was found to efficiently prevent the aggregation of polyQ in the transfected HEK293 cells. Furthermore, it was also found to significantly prolong lifespan, improve motor functions, and reduce the inclusion bodies in a transgenic mouse model (Tg R6/2), when given ad libitum.

Stress-induced nuclear translocation of CDK5 suppresses neuronal death by downregulating ERK activation via VRK3 phosphorylation.

Although extracellular signal-related kinase 1/2 (ERK 1/2) activity is generally associated with cell survival, prolonged ERK activation induced by oxidative stress also mediates neuronal cell death. Here we report that oxidative stress-induced cyclin-dependent kinase 5 (CDK5) activation stimulates neuroprotective signaling via phosphorylation of vaccinia-related kinase 3 (VRK3) at Ser 108. The binding of vaccinia H1-related (VHR) phosphatase to phosphorylated VRK3 increased its affinity for phospho-ERK and subsequently downregulated ERK activation. Overexpression of VRK3 protected human neuroblastoma SH-SY5Y cells against hydrogen peroxide (H2O2)-induced apoptosis. However the CDK5 was unable to phosphorylate mutant VRK3, and thus the mutant forms of VRK3 could not attenuate apoptotic process. Suppression of CDK5 activity results in increase of ERK activation and elevation of proapoptotic protein Bak expression in mouse cortical neurons. Results from VRK3-deficient neurons were further confirmed the role of VRK3 phosphorylation in H2O2-evoked ERK regulation. Importantly, we showed an association between phospho-VRK3 levels and the progression of human Alzheimer’s disease (AD) and Parkinson’s disease (PD). Together our work reveals endogenous protective mechanism against oxidative stress-induced neuronal cell death and suggest VRK3 as a potential therapeutic target in neurodegenerative diseases.

Vaccinia-Related Kinase 2 Controls the Stability of the Eukaryotic Chaperonin TRiC/CCT by Inhibiting the Deubiquitinating Enzyme USP25

ABSTRACT Molecular chaperones monitor the proper folding of misfolded proteins and function as the first line of defense against mutant protein aggregation in neurodegenerative diseases. The eukaryotic chaperonin TRiC is a potent suppressor of mutant protein aggregation and toxicity in early stages of disease progression. Elucidation of TRiC functional regulation will enable us to better understand the pathological mechanisms of neurodegeneration. We have previously shown that vaccinia-related kinase 2 (VRK2) downregulates TRiC protein levels through the ubiquitin-proteasome system by recruiting the E3 ligase COP1. However, although VRK2 activity was necessary in TRiC downregulation, the phosphorylated substrate was not determined. Here, we report that USP25 is a novel TRiC interacting protein that is also phosphorylated by VRK2. USP25 catalyzed deubiquitination of the TRiC protein and stabilized the chaperonin, thereby reducing accumulation of misfolded polyglutamine protein aggregates. Notably, USP25 deubiquitinating activity was suppressed when VRK2 phosphorylated the Thr680, Thr727, and Ser745 residues. Impaired USP25 deubiquitinating activity after VRK2-mediated phosphorylation may be a critical pathway in TRiC protein destabilization.

Brazilin Isolated from Caesalpinia sappan Suppresses Nuclear Envelope Reassembly by Inhibiting Barrier-to-Autointegration Factor Phosphorylation

To date, many anticancer drugs have been developed by directly or indirectly targeting microtubules, which are involved in cell division. Although this approach has yielded many anticancer drugs, these drugs produce undesirable side effects. An alternative strategy is needed, and targeting mitotic exit may be one alternative approach. Localization of phosphorylated barrier-to-autointegration factor (BAF) to the chromosomal core region is essential for nuclear envelope compartment relocalization. In this study, we isolated brazilin from Caesalpinia sappan Leguminosae and demonstrated that it inhibited BAF phosphorylation in vitro and in vivo. Moreover, we demonstrated direct binding between brazilin and BAF. The inhibition of BAF phosphorylation induced abnormal nuclear envelope reassembly and cell death, indicating that perturbation of nuclear envelope reassembly could be a novel approach to anticancer therapy. We propose that brazilin isolated from C. sappan may be a new anticancer drug candidate that induces cell death by inhibiting vaccinia-related kinase 1–mediated BAF phosphorylation.

Glycogen synthase kinase 3β suppresses polyglutamine aggregation by inhibiting Vaccinia-related kinase 2 activity.

Huntington’s disease (HD) is a neurodegenerative disorder caused by an abnormal expansion of polyglutamine repeats in the N-terminal of huntingtin. The amount of aggregate-prone protein is controlled by various mechanisms, including molecular chaperones. Vaccinia-related kinase 2 (VRK2) is known to negatively regulate chaperonin TRiC, and VRK2-facilitated degradation of TRiC increases polyQ protein aggregation, which is involved in HD. We found that VRK2 activity was negatively controlled by glycogen synthase kinase 3β (GSK3β). GSK3β directly bound to VRK2 and inhibited the catalytic activity of VRK2 in a kinase activity-independent manner. Furthermore, GSK3β increased the stability of TRiC and decreased the formation of HttQ103-GFP aggregates by inhibiting VRK2. These results indicate that GSK3β signaling may be a regulatory mechanism of HD progression and suggest targets for further therapeutic trials for HD.