Doublecortin X (DCX) serine 28 phosphorylation is a regulatory switch, modulating association of DCX with microtubules and actin filaments

Doublecortin X (DCX) plays essential roles in neuronal development via its regulation of cytoskeleton dynamics. This is mediated through direct interactions between its doublecortin (DC) domains (DC1 and DC2) with microtubules (MTs) and indirect association with actin filaments (F-ACT). While the regulatory role of the DCX C-terminus following DC2 (i.e. DCX residues 275–366) has been established, less is known of the possible contributions made by the DCX N-terminus preceding DC1 (i.e. DCX residues 1–44). Here, we assessed the influence of DCX Ser28 within the DCX N-terminus, on the association of DCX with MTs and F-ACT. We compared the cytoskeletal interactions of the DCX S28E phosphomimetic and DCX S28A phospho-resistant mutants and wild-type DCX. Immunoprecipitation and colocalisation analyses indicated increased association of DCX S28E with F-ACT but decreased interaction with MTs, and conversely enhanced DCX S28A association with MTs but decreased association with F-ACT. To evaluate the impact of DCX mutants on cytoskeletal filaments we performed fluorescence recovery after photobleaching (FRAP) studies on SiR-tubulin and β-actin-mCherry and observed comparable tubulin and actin exchange rates in the presence of DCX WT and DCX S28A. However, we observed faster tubulin exchange rates but slower actin exchange rates in the presence of DCX S28E. Moreover, DCX S28E enhanced the association with the actin-binding protein spinophilin (Spn) suggesting the shift to favour association with both F-ACT and Spn in the presence of DCX S28E. Taken together, our results highlight a new role for DCX S28 as a regulatory switch for cytoskeletal organisation

Endothelin-1, phorbol esters and phenylephrine stimulate MAP kinase activities in ventricular cardiomyocytes

AbstractET-1 stimulated MBP kinase activity in cultured cardiomyocytes. Maximal activation (3.5-fold) was at 5 min. EC50 was 0.2 nM. PMA or PE also increased MBP kinase (4- or 2.5-fold, respectively). Pre-treatment with PMA down-regulated the subsequent response to ET-1 or PMA. ET-1- or PMA-stimulated MBP kinase was resolved into 2 major (peaks II and IV) and 2 minor peaks by FPLC on Mono Q. Peaks II and IV were inactivated by either LAR or PP2A. Renatured MBP kinase activities following SDS-PAGE in MBP-containing gels and immunoblot analysis showed that peak II was a p42 MAP kinase and peak IV was a p44 MAP kinase

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

<p>Abstract</p> <p>Background</p> <p>TDP-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.</p> <p>Results</p> <p>We 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.</p> <p>Conclusions</p> <p>Our 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.</p

Uses for JNK: the Many and Varied Substrates of the c-Jun N-Terminal Kinases

The c-Jun N-terminal kinases (JNKs) are members of a larger group of serine/threonine (Ser/Thr) protein kinases from the mitogen-activated protein kinase family. JNKs were originally identified as stress-activated protein kinases in the livers of cycloheximide-challenged rats. Their subsequent purification, cloning, and naming as JNKs have emphasized their ability to phosphorylate and activate the transcription factor c-Jun. Studies of c-Jun and related transcription factor substrates have provided clues about both the preferred substrate phosphorylation sequences and additional docking domains recognized by JNK. There are now more than 50 proteins shown to be substrates for JNK. These include a range of nuclear substrates, including transcription factors and nuclear hormone receptors, heterogeneous nuclear ribonucleoprotein K, and the Pol I-specific transcription factor TIF-IA, which regulates ribosome synthesis. Many nonnuclear substrates have also been characterized, and these are involved in protein degradation (e.g., the E3 ligase Itch), signal transduction (e.g., adaptor and scaffold proteins and protein kinases), apoptotic cell death (e.g., mitochondrial Bcl2 family members), and cell movement (e.g., paxillin, DCX, microtubule-associated proteins, the stathmin family member SCG10, and the intermediate filament protein keratin 8). The range of JNK actions in the cell is therefore likely to be complex. Further characterization of the substrates of JNK should provide clearer explanations of the intracellular actions of the JNKs and may allow new avenues for targeting the JNK pathways with therapeutic agents downstream of JNK itself

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 Ras 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

Respiratory Syncytial Virus Matrix Protein Is Sufficient and Necessary to Remodel Host Mitochondria in Infection

Although respiratory syncytial virus (RSV) is the most common cause of respiratory infection in infants, immunosuppressed adults and the elderly worldwide, there is no licensed RSV vaccine or widely applicable antiviral therapeutics We previously reported a staged redistribution of mitochondria with compromised respiratory activities and increased reactive oxygen species (ROS) generation during RSV infection. Here, we show for the first time that the RSV matrix protein (M) is sufficient and necessary to induce these effects. Ectopically expressed M, but not other RSV proteins, was able to induce mitochondrial perinuclear clustering, inhibition of mitochondrial respiration, loss of mitochondrial membrane potential (Δψm), and enhanced generation of mitochondrial ROS (mtROS) in infection. Truncation and mutagenic analysis revealed that the central nucleic acid-binding domain of M is essential for the effects on host mitochondria, with arginine/lysine residues 170/172 being critically important. Recombinant RSV carrying the arginine/lysine mutations in M was unable to elicit effects on host mitochondria. Further, wild-type but not mutant RSV was found to inhibit the mRNA expression of genes encoding mitochondrial proteins, including Complex I subunits. Importantly, the RSV mutant was impaired in virus production, underlining the importance of M-dependent effects on mitochondria to RSV infection. Together, our results highlight M’s unique ability to remodel host cell mitochondria and its critical role in RSV infection, representing a novel, potential target for future anti-RSV strategies

Subversion of Host Cell Mitochondria by RSV to Favor Virus Production is Dependent on Inhibition of Mitochondrial Complex I and ROS Generation

Respiratory syncytial virus (RSV) is a key cause of severe respiratory infection in infants, immunosuppressed adults, and the elderly worldwide, but there is no licensed vaccine or effective, widely-available antiviral therapeutic. We recently reported staged redistribution of host cell mitochondria in RSV infected cells, which results in compromised respiratory activities and increased reactive oxygen species (ROS) generation. Here, bioenergetic measurements, mitochondrial redox-sensitive dye, and high-resolution quantitative imaging were performed, revealing for the first time that mitochondrial complex I is key to this effect on the host cell, whereby mitochondrial complex I subunit knock-out (KO) cells, with markedly decreased mitochondrial respiration, show elevated levels of RSV infectious virus production compared to wild-type cells or KO cells with re-expressed complex I subunits. This effect correlates strongly with elevated ROS generation in the KO cells compared to wild-type cells or retrovirus-rescued KO cells re-expressing complex I subunits. Strikingly, blocking mitochondrial ROS levels using the mitochondrial ROS scavenger, mitoquinone mesylate (MitoQ), inhibits RSV virus production, even in the KO cells. The results highlight RSV&rsquo;s unique ability to usurp host cell mitochondrial ROS to facilitate viral infection and reinforce the idea of MitoQ as a potential therapeutic for RSV