Biochemistry and Pharmacology - Research Publications

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    c-Jun N-terminal Kinase Phosphorylation of Stathmin Confers Protection against Cellular Stress
    Ng, DCH ; Zhao, TT ; Yeap, YYC ; Ngoei, KR ; Bogoyevitch, MA (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2010-09-10)
    The cell stress response encompasses the range of intracellular events required for adaptation to stimuli detrimental to cell survival. Although the c-Jun N-terminal kinase (JNK) is a stress-activated kinase that can promote either cell survival or death in response to detrimental stimuli, the JNK-regulated mechanisms involved in survival are not fully characterized. Here we show that in response to hyperosmotic stress, JNK phosphorylates a key cytoplasmic microtubule regulatory protein, stathmin (STMN), on conserved Ser-25 and Ser-38 residues. In in vitro biochemical studies, we identified STMN Ser-38 as the critical residue required for efficient phosphorylation by JNK and identified a novel kinase interaction domain in STMN required for recognition by JNK. We revealed that JNK was required for microtubule stabilization in response to hyperosmotic stress. Importantly, we also demonstrated a novel cytoprotective function for STMN, as the knockdown of STMN levels by siRNA was sufficient to augment viability in response to hyperosmotic stress. Our findings show that JNK targeting of STMN represents a novel stress-activated cytoprotective mechanism involving microtubule network changes.
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    Identification and characterization of bi-thiazole-2,2′-diamines as kinase inhibitory scaffolds
    Ngoei, KRW ; Ng, DCH ; Gooley, PR ; Fairlie, DP ; Stoermer, M ; Bogoyevitch, MA (ELSEVIER SCIENCE BV, 2013-06)
    Based on bioinformatics interrogation of the genome, >500 mammalian protein kinases can be clustered within seven different groups. Of these kinases, the mitogen-activated protein kinase (MAPK) family forms part of the CMGC group of serine/threonine kinases that includes extracellular signal regulated kinases (ERKs), cJun N-terminal kinases (JNKs), and p38 MAPKs. With the JNKs considered attractive targets in the treatment of pathologies including diabetes and stroke, efforts have been directed to the discovery of new JNK inhibitory molecules that can be further developed as new therapeutics. Capitalizing on our biochemical understanding of JNK, we performed in silico screens of commercially available chemical databases to identify JNK1-interacting compounds and tested their in vitro JNK inhibitory activity. With in vitro and cell culture studies, we showed that the compound, 4'-methyl-N(2)-3-pyridinyl-4,5'-bi-1,3-thiazole-2,2'-diamine (JNK Docking (JD) compound 123, but not the related compound (4'-methyl-N~2~-(6-methyl-2-pyridinyl)-4,5'-bi-1,3-thiazole-2,2'-diamine (JD124), inhibited JNK1 activity towards a range of substrates. Molecular docking, saturation transfer difference NMR experiments and enzyme kinetic analyses revealed both ATP- and substrate-competitive inhibition of JNK by JD123. In characterizing JD123 further, we noted its ATP-competitive inhibition of the related p38-γ MAPK, but not ERK1, ERK2, or p38-α, p38-β or p38-δ. Further screening of a broad panel of kinases using 10μM JD123, identified inhibition of kinases including protein kinase Bβ (PKBβ/Aktβ). Appropriately modified thiazole diamines, as typified by JD123, thus provide a new chemical scaffold for development of inhibitors for the JNK and p38-γ MAPKs as well as other kinases that are also potential therapeutic targets such as PKBβ/Aktβ.
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    Intracellular mobility and nuclear trafficking of the stress-activated kinase JNK1 are impeded by hyperosmotic stress
    Misheva, M ; Kaur, G ; Ngoei, KRW ; Yeap, YY ; Ng, IHW ; Wagstaff, KM ; Ng, DCH ; Jans, DA ; Bogoyevitch, MA (ELSEVIER SCIENCE BV, 2014-02)
    The c-Jun N-terminal kinases (JNKs) are a group of stress-activated protein kinases that regulate gene expression changes through specific phosphorylation of nuclear transcription factor substrates. To address the mechanisms underlying JNK nuclear entry, we employed a semi-intact cell system to demonstrate for the first time that JNK1 nuclear entry is dependent on the importin α2/β1 heterodimer and independent of importins α3, α4, β2, β3, 7 and 13. However, quantitative image analysis of JNK1 localization following exposure of cells to either arsenite or hyperosmotic stress did not indicate its nuclear accumulation. Extending our analyses to define the dynamics of nuclear trafficking of JNK1, we combined live cell imaging analyses with fluorescence recovery after photobleaching (FRAP) protocols. Subnuclear and subcytoplasmic bleaching protocols revealed the slowed movement of JNK1 in both regions in response to hyperosmotic stress. Strikingly, while movement into the nucleus of green fluorescent protein (GFP) or transport of a GFP-T-antigen fusion protein as estimated by initial rates and time to reach half-maximal recovery (t1/2) measures remained unaltered, hyperosmotic stress slowed the nuclear entry of GFP-JNK1. In contrast, arsenite exposure which did not alter the initial rates of nuclear accumulation of GFP, GFP-T-antigen or GFP-JNK1, decreased the t1/2 for nuclear accumulation of both GFP and GFP-JNK1. Thus, our results challenge the paradigm of increased nuclear localization of JNK broadly in response to all forms of stress-activation and are consistent with enhanced interactions of stress-activated JNK1 with scaffold and substrate proteins throughout the nucleus and the cytosol under conditions of hyperosmotic stress.
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    WD40-repeat protein 62 is a JNK-phosphorylated spindle pole protein required for spindle maintenance and timely mitotic progression
    Bogoyevitch, MA ; Yeap, YYC ; Qu, Z ; Ngoei, KR ; Yip, YY ; Zhao, TT ; Heng, JI ; Ng, DCH (COMPANY BIOLOGISTS LTD, 2012-11-01)
    The impact of aberrant centrosomes and/or spindles on asymmetric cell division in embryonic development indicates the tight regulation of bipolar spindle formation and positioning that is required for mitotic progression and cell fate determination. WD40-repeat protein 62 (WDR62) was recently identified as a spindle pole protein linked to the neurodevelopmental defect of microcephaly but its roles in mitosis have not been defined. We report here that the in utero electroporation of neuroprogenitor cells with WDR62 siRNAs induced their cell cycle exit and reduced their proliferative capacity. In cultured cells, we demonstrated cell-cycle-dependent accumulation of WDR62 at the spindle pole during mitotic entry that persisted until metaphase-anaphase transition. Utilizing siRNA depletion, we revealed WDR62 function in stabilizing the mitotic spindle specifically during metaphase. WDR62 loss resulted in spindle orientation defects, decreased the integrity of centrosomes displaced from the spindle pole and delayed mitotic progression. Additionally, we revealed JNK phosphorylation of WDR62 is required for maintaining metaphase spindle organization during mitosis. Our study provides the first functional characterization of WDR62 and has revealed requirements for JNK/WDR62 signaling in mitotic spindle regulation that may be involved in coordinating neurogenesis.