Dominic C. H. Ng

c-Jun N-terminal kinase (JNK) signaling: recent advances and challenges.

c-Jun N-terminal kinases (JNKs), first characterized as stress-activated members of the mitogen-activated protein kinase (MAPK) family, have become a focus of inhibitor screening strategies following studies that have shown their critical roles in the development of a number of diseases, such as diabetes, neurodegeneration and liver disease. We discuss recent advances in the discovery and development of ATP-competitive and ATP-noncompetitive JNK inhibitors. Because understanding the modes of actions of these inhibitors and improving their properties will rely on a better understanding of JNK structure, JNK catalytic mechanisms and substrates, recent advances in these areas of JNK biochemistry are also considered. In addition, the use of JNK gene knockout animals is continuing to reveal in vivo functions for these kinases, with tissue-specific roles now being dissected with tissue-specific knockouts. These latest advances highlight the many challenges now faced, particularly in the directed targeting of the JNK isoforms in specific tissues.

GRIM-19, a cell death regulatory protein, is essential for assembly and function of mitochondrial complex I.

ABSTRACT Mitochondria play essential roles in cellular energy production via the oxidative phosphorylation system (OXPHOS) consisting of five multiprotein complexes and also in the initiation of apoptosis. NADH:ubiquinone oxidoreductase (complex I) is the largest complex that catalyzes the first step of electron transfer in the OXPHOS system. GRIM-19 was originally identified as a nuclear protein with apoptotic nature in interferon (IFN)- and all-trans-retinoic acid (RA)-induced tumor cells. To reveal its biological role, we generated mice deficient in GRIM-19 by gene targeting. Homologous deletion of GRIM-19 causes embryonic lethality at embryonic day 9.5. GRIM-19−/− blastocysts show retarded growth in vitro and, strikingly, display abnormal mitochondrial structure, morphology, and cellular distribution. We reexamined the cellular localization of GRIM-19 in various cell types and found its primary localization in the mitochondria. Furthermore, GRIM-19 is detected in the native form of mitochondrial complex I. Finally, we show that elimination of GRIM-19 destroys the assembly and electron transfer activity of complex I and also influences the other complexes in the mitochondrial respiratory chain. Our result demonstrates that GRIM-19, a gene product with a specific role in IFN-RA-induced cell death, is a functional component of mitochondrial complex I and is essential for early embryonic development.

C-Jun N-terminal kinase controls TDP-43 accumulation in stress granules induced by oxidative stress

BackgroundTDP-43 proteinopathies are characterized by loss of nuclear TDP-43 expression and formation of C-terminal TDP-43 fragmentation and accumulation in the cytoplasm. Recent studies have shown that TDP-43 can accumulate in RNA stress granules (SGs) in response to cell stresses and this could be associated with subsequent formation of TDP-43 ubiquinated protein aggregates. However, the initial mechanisms controlling endogenous TDP-43 accumulation in SGs during chronic disease are not understood. In this study we investigated the mechanism of TDP-43 processing and accumulation in SGs in SH-SY5Y neuronal-like cells exposed to chronic oxidative stress. Cell cultures were treated overnight with the mitochondrial inhibitor paraquat and examined for TDP-43 and SG processing.ResultsWe found that mild stress induced by paraquat led to formation of TDP-43 and HuR-positive SGs, a proportion of which were ubiquitinated. The co-localization of TDP-43 with SGs could be fully prevented by inhibition of c-Jun N-terminal kinase (JNK). JNK inhibition did not prevent formation of HuR-positive SGs and did not prevent diffuse TDP-43 accumulation in the cytosol. In contrast, ERK or p38 inhibition prevented formation of both TDP-43 and HuR-positive SGs. JNK inhibition also inhibited TDP-43 SG localization in cells acutely treated with sodium arsenite and reduced the number of aggregates per cell in cultures transfected with C-terminal TDP-43 162-414 and 219-414 constructs.ConclusionsOur studies are the first to demonstrate a critical role for kinase control of TDP-43 accumulation in SGs and may have important implications for development of treatments for FTD and ALS, targeting cell signal pathway control of TDP-43 aggregation.

Severe Heart Failure and Early Mortality in a Double-Mutation Mouse Model of Familial Hypertrophic Cardiomyopathy

Background— Familial hypertrophic cardiomyopathy (FHC) is characterized by genetic and clinical heterogeneity. Five percent of FHC families have 2 FHC-causing mutations, which results in earlier disease onset, increased cardiac dysfunction, and a higher incidence of sudden death events. These observations suggest a relationship between the number of gene mutations and phenotype severity in FHC. Methods and Results— We sought to develop, characterize, and investigate the pathogenic mechanisms in a double-mutant murine model of FHC. This model (designated TnI-203/MHC-403) was generated by crossbreeding mice with the Gly203Ser cardiac troponin I (TnI-203) and Arg403Gln α-myosin heavy chain (MHC-403) FHC-causing mutations. The mortality rate in TnI-203/MHC-403 mice was 100% by age 21 days. At age 14 days, TnI-203/MHC-403 mice developed a significantly increased ratio of heart weight to body weight, marked interstitial myocardial fibrosis, and increased expression of atrial natriuretic factor and brain natriuretic peptide compared with nontransgenic, TnI-203, and MHC-403 littermates. By age 16 to 18 days, TnI-203/MHC-403 mice rapidly developed a severe dilated cardiomyopathy and heart failure, with inducibility of ventricular arrhythmias, which led to death by 21 days. Downregulation of mRNA levels of key regulators of Ca2+ homeostasis in TnI-203/MHC-403 mice was observed. Increased levels of phosphorylated STAT3 were observed in TnI-203/MHC-403 mice and corresponded with the onset of disease, which suggests a possible cardioprotective response. Conclusions— TnI-203/MHC-403 double-mutant mice develop a severe cardiac phenotype characterized by heart failure and early death. The presence of 2 disease-causing mutations may predispose individuals to a greater risk of developing severe heart failure than human FHC caused by a single gene mutation.

Stat3 promotes directional cell migration by regulating Rac1 activity via its activator βPIX

Stat3 is a member of the signal transducer and activator of transcription family, which is important for cytokine signaling as well as for a number of cellular processes including cell proliferation, anti-apoptosis and immune responses. In recent years, evidence has emerged suggesting that Stat3 also participates in cell invasion and motility. However, how Stat3 regulates these processes remains poorly understood. Here, we find that loss of Stat3 expression in mouse embryonic fibroblasts leads to an elevation of Rac1 activity, which promotes a random mode of migration by reducing directional persistence and formation of actin stress fibers. Through rescue experiments, we demonstrate that Stat3 can regulate the activation of Rac1 to mediate persistent directional migration and that this function is not dependent on Stat3 transcriptional activity. We find that Stat3 binds to βPIX, a Rac1 activator, and that this interaction could represent a mechanism by which cytoplasmic Stat3 regulates Rac1 activity to modulate the organization of actin cytoskeleton and directional migration.

Tracking protein aggregation and mislocalization in cells with flow cytometry

We applied pulse-shape analysis (PulSA) to monitor protein localization changes in mammalian cells by flow cytometry. PulSA enabled high-throughput tracking of protein aggregation, translocation from the cytoplasm to the nucleus and trafficking from the plasma membrane to the Golgi as well as stress-granule formation. Combining PulSA with tetracysteine-based oligomer sensors in a cell model of Huntingtons disease enabled further separation of cells enriched with monomers, oligomers and inclusion bodies.

Activation of signal transducer and activator of transcription (STAT) pathways in failing human hearts

OBJECTIVES The signal transduction pathways mediating the progression to failure have been intensively studied in a variety of in vitro and in vivo animal models. Recently, acute activation of the Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) has been observed in the heart, but whether this is sustained in ischemic heart disease (IHD) or dilated cardiomyopathy (DCM) has not been previously addressed. METHODS We assessed the tyrosine phosphorylation of STAT1, 3, 5 and 6 in ventricular samples of explanted human hearts with IHD (n=11) and DCM (n=9) as an indication of STAT activation. Samples from normal donor hearts (n=9) acted as controls. In parallel, we also assessed protein expression and phosphorylation of three major families of mitogen-activated protein kinases (MAPKs); ERK, p38 MAPK and c-Jun NH(2)-terminal kinase (JNK). RESULTS All STAT isoforms were significantly phosphorylated in DCM. In contrast, only the phosphorylation of STATs 1 and 5 were significantly enhanced in IHD. Expression of total STAT protein remained unchanged. For the MAPKs, significant phosphorylation of p38(MAPK) was only observed in IHD. In contrast, there was no change in ERK or JNK activation despite abundant protein expression. CONCLUSIONS We have shown that different members of the STAT transcription factor family are chronically phosphorylated in the failing heart as a result of IHD (STAT1 and 5) or DCM (STAT1, 3, 5 and 6). In contrast, IHD but not DCM showed significant p38(MAPK) phosphorylation. Whilst the differences noted between IHD and DCM may reflect different initiating events, the common activation of STATs 1 and 5 suggests that these transcription factors may play a common role regulating the progression of heart failure.

Taking the Cell by Stealth or Storm Protein Transduction Domains (PTDs) as Versatile Vectors for Delivery

A cell delivery system is increasing in use in many areas of cell and molecular biology and bio-medicine. This system is based on a number of naturally occurring protein motifs and/or sequences which show the remarkable ability to rapidly cross the mammalian cell membrane without compromising its structure or function. These so-called Protein Transduction Domains (PTDs) offer unprecedented advantages for intracellular delivery. These advantages include, but are not limited to, their applicability to all cell types (no cell type has yet been described which is not transduced by these PTDs), and the range of cargoes that can be transduced (including peptides, small proteins, full-length enzymes, DNA oligomers, peptide-nucleic acid oligomers, liposomes, and magnetic nanoparticles). Furthermore, the PTDs have been demonstrated to be suitable for in vivo delivery including delivery across the blood brain barrier, and have been shown to cross the plasma membrane rapidly and enter the cytoplasm and nuclear regions of the cell. In this review, the general properties of the most commonly used PTDs are described. The strategies currently being undertaken also highlight that improvements in membrane transduction are possible despite our lack of understanding of the exact biochemical and/or physical mechanisms of transduction. Recent examples of the range of potential applications are also discussed.

WD40-repeat protein 62 is a JNK-phosphorylated spindle pole protein required for spindle maintenance and timely mitotic progression

Summary 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.

The Mechanism of Heat Shock Activation of ERK Mitogen-activated Protein Kinases in the Interleukin 3-dependent ProB Cell Line BaF3

We have investigated heat shock stimulation of MAPK cascades in an interleukin 3-dependent cell line, BaF3. Following exposure to 42 °C, the stress-activated JNK MAPKs were phosphorylated and activated, but p38 MAPKs remained unaffected. Surprisingly, heat shock also activated ERK MAPKs in a potent (>60-fold), delayed (>30 min), and sustained (≥120 min) manner. These characteristics suggested a novel mechanism of ERK MAPK activation and became the focus of this study. A MEK-specific inhibitor, PD98059, inhibited heat shock ERK MAPK activation by >75%. Surprisingly, a role for Ras in the heat shock response was eliminated by the failure of a dominant-negative RasAsn-17 mutant to inhibit ERK MAPK activation and the failure to observe increases in Ras·GTP. Heat shock also failed to stimulate activation of A-, B-, and c-Raf. Instead, a serine/threonine phosphatase inhibitor, okadaic acid, activated ERK MAPK in a similar manner to heat shock. Furthermore, pretreatment with suramin, generally recognized as a broad range inhibitor of growth factor receptors, inhibited both okadaic acid-stimulated and heat shock-stimulated ERK MAPK activity by >40%. Inhibiting ERK MAPK activation during heat shock with PD98059 enhanced losses in cell viability. These results demonstrate Ras- and Raf-independent ERK MAPK activation maintains cell viability following heat shock.