Hepatitis C Virus NS5A Inhibits Mixed Lineage Kinase 3 to Block Apoptosis

Hepatitis C virus (HCV) infection results in the activation of numerous stress responses including oxidative stress, with the potential to induce an apoptotic state. Previously we have shown that HCV attenuates the stress-induced, p38MAPK-mediated up-regulation of the K⁺ channel Kv2.1, to maintain the survival of infected cells in the face of cellular stress. We demonstrated that this effect was mediated by HCV non-structural 5A (NS5A) protein, which impaired p38MAPK activity through a polyproline motif-dependent interaction, resulting in reduction of phosphorylation activation of Kv2.1. In this study, we investigated the host cell proteins targeted by NS5A to mediate Kv2.1 inhibition. We screened a phage-display library expressing the entire complement of human SH3 domains for novel NS5A-host cell interactions. This analysis identified mixed lineage kinase 3 (MLK3) as a putative NS5A interacting partner. MLK3 is a serine/threonine protein kinase that is a member of the MAPK kinase kinase (MAP3K) family and activates p38MAPK. An NS5A-MLK3 interaction was confirmed by co-immunoprecipitation and Western blot analysis. We further demonstrate a novel role of MLK3 in the modulation of Kv2.1 activity, whereby MLK3 overexpression leads to the up-regulation of channel activity. Accordingly, coexpression of NS5A suppressed this stimulation. Additionally we demonstrate that overexpression of MLK3 induced apoptosis, which was also counteracted by NS5A. We conclude that NS5A targets MLK3 with multiple downstream consequences for both apoptosis and K⁺ homeostasis.     

Characterization of TrkB Receptor-Mediated Signaling Pathways in Rat Cerebellar Granule Neurons: Involvement of Protein Kinase C in Neuronal Survival

Abstract: TrkB belongs to the Trk family of tyrosine kinase receptors and mediates the response to brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5). Here, we report that both truncated and full-length forms of TrkB receptors are expressed in developing cerebellar granule neurons. BDNF and NT-4/5 increased the survival of cultured cerebellar granule neurons. BDNF and NT-4/5 also induced an autophosphorylation of TrkB receptors and subsequently resulted in a phosphorylation and binding of phospholipase C-γ (PLC-γ) and SH2-containing sequence to the autophosphorylated TrkB receptors. Both contain src homology 2 (SH2) regions. In keeping with a signaling function of PLC-γ, BDNF increased the phosphatidylinositol (PI) turnover and elevated intracellular calcium levels. To investigate the involvement of protein kinase C (PKC) in the survival of granular neurons, we show here activation of PKC after BDNF or TPA treatment and blocking of the observed survival-promoting effects of BDNF and TPA with calphostin C, a specific PKC inhibitor. In addition, BDNF activated c- ras in a concentration-dependent manner. These results suggest that two different pathways, the c- ras and the PLC-γ pathway, are activated by TrkB receptors in primary neurons and that PKC activation is involved in the survival promoting effect of BDNF.     

Mitochondria and Neuronal Death/Survival Signaling Pathways in Cerebral Ischemia

Apoptotic cell death pathways have been implicated in acute brain injuries, including cerebral ischemia, brain trauma, and spinal cord injury, and in chronic neurodegenerative diseases. Experimental ischemia and reperfusion models, such as transient focal/global ischemia in rodents, have been thoroughly studied and suggest the involvement of mitochondria and the cell survival/death signaling pathways in cell death/survival cascades. Recent studies have implicated mitochondria-dependent apoptosis involving pro- and anti-apoptotic protein binding, the release of cytochrome c and second mitochondria-derived activator of caspase, the activation of downstream caspases-9 and –3, and DNA fragmentation. Reactive oxygen species are known to be significantly generated in the mitochondrial electron transport chain in the dysfunctional mitochondria during reperfusion after ischemia, and are also implicated in the survival signaling pathway that involves phosphatidylinositol-3-kinase (PI3-K), Akt, and downstream signaling molecules, like Bad, 14-3-3, and the proline-rich Akt substrate (PRAS), and their bindings. Further studies of these survival pathways may provide novel therapeutic strategies for clinical stroke.Special issue dedicated to Lawrence F. Eng.     

PIM1 Kinase Phosphorylates the Human Transcription Factor FOXP3 at Serine 422 to Negatively Regulate Its Activity under Inflammation

Previous reports have suggested that human CD4⁺ CD25^hiFOXP3⁺ T regulatory cells (Tregs) have functional plasticity and may differentiate into effector T cells under inflammation. The molecular mechanisms underlying these findings remain unclear. Here we identified the residue serine 422 of human FOXP3 as a phosphorylation site that regulates its function, which is not present in murine Foxp3. PIM1 kinase, which is highly expressed in human Tregs, was found to be able to interact with and to phosphorylate human FOXP3 at serine 422. T cell receptor (TCR) signaling inhibits PIM1 induction, whereas IL-6 promotes PIM1 expression in in vitro expanded human Tregs. PIM1 negatively regulates FOXP3 chromatin binding activity by specifically phosphorylating FOXP3 at Ser⁴²². Our data also suggest that phosphorylation of FOXP3 at the Ser⁴¹⁸ site could prevent FOXP3 phosphorylation at Ser⁴²² mediated by PIM1. Knockdown of PIM1 in in vitro expanded human Tregs promoted FOXP3-induced target gene expression, including CD25, CTLA4, and glucocorticoid-induced tumor necrosis factor receptor (GITR), or weakened FOXP3-suppressed IL-2 gene expression and enhanced the immunosuppressive activity of Tregs. Furthermore, PIM1-specific inhibitor boosted FOXP3 DNA binding activity in in vitro expanded primary Tregs and also enhanced their suppressive activity toward the proliferation of T effector cells. Taken together, our findings suggest that PIM1 could be a new potential therapeutic target in the prevention and treatment of human-specific autoimmune diseases because of its ability to modulate the immunosuppressive activity of human Tregs.     

Protein Tyrosine Kinase 6 Directly Phosphorylates AKT and Promotes AKT Activation in Response to Epidermal Growth Factor

Protein tyrosine kinase 6 (PTK6) is a nonmyristoylated Src-related intracellular tyrosine kinase. Although not expressed in the normal mammary gland, PTK6 is expressed in a majority of human breast tumors examined, and it has been linked to ErbB receptor signaling and AKT activation. Here we demonstrate that AKT is a direct substrate of PTK6 and that AKT tyrosine residues 315 and 326 are phosphorylated by PTK6. Association of PTK6 with AKT occurs through the SH3 domain of PTK6 and is enhanced through SH2 domain-mediated interactions following tyrosine phosphorylation of AKT. Using Src, Yes, and Fyn null mouse embryonic fibroblasts (SYF cells), we show that PTK6 phosphorylates AKT in a Src family kinase-independent manner. Introduction of PTK6 into SYF cells sensitized these cells to physiological levels of epidermal growth factor (EGF) and increased AKT activation. Stable introduction of active PTK6 into SYF cells also resulted in increased proliferation. Knockdown of PTK6 in the BPH-1 human prostate epithelial cell line led to decreased AKT activation in response to EGF. Our data indicate that in addition to promoting growth factor receptor-mediated activation of AKT, PTK6 can directly activate AKT to promote oncogenic signaling.Protein tyrosine kinase 6 (PTK6; also known as the breast tumor kinase BRK) is an intracellular Src-related tyrosine kinase (9, 48). Human PTK6 was identified in cultured human melanocytes (32) and breast tumor cells (39), while its mouse orthologue was cloned from normal small intestinal epithelial cell RNA (50). Although PTK6 shares overall structural similarity with Src family tyrosine kinases, it lacks an N-terminal myristoylation consensus sequence for membrane targeting (39, 51). As a consequence, PTK6 is localized to different cellular compartments, including the nucleus (14, 15). PTK6 is expressed in normal differentiated epithelial cells of the gastrointestinal tract (34, 42, 51), prostate (14), and skin (51-53). Expression of PTK6 is upregulated in different types of cancers, including breast carcinomas (6, 39, 54), colon cancer (34), ovarian cancer (47), head and neck cancers (33), and metastatic melanoma cells (16). The significance of apparent opposing signaling roles for PTK6 in normal differentiation and cancer is still poorly understood.In human breast tumor cells, PTK6 enhances signaling from members of the ErbB receptor family (10, 29, 30, 36, 40, 49, 54). In the HB4a immortalized human mammary gland luminal epithelial cell line, PTK6 promoted epidermal growth factor (EGF)-induced ErbB3 tyrosine phosphorylation and AKT activation (29). In response to EGF stimulation, PTK6 promoted phosphorylation of the focal adhesion protein paxillin and Rac1-mediated cell migration (10). PTK6 can be activated by the ErbB3 ligand heregulin and promotes activation of extracellular signal-regulated kinase 5 (ERK5) and p38 mitogen-activated protein kinase (MAPK) in breast cancer cells (40). PTK6 can also phosphorylate p190RhoGAP-A and stimulate its activity, leading to RhoA inactivation and Ras activation and thereby promoting EGF-dependent breast cancer cell migration and proliferation (49). Expression of PTK6 has been correlated with ErbB2 expression in human breast cancers (4, 5, 54).AKT (also called protein kinase B) is a serine-threonine kinase that is activated downstream of growth factor receptors (38). It is a key player in signaling pathways that regulate energy metabolism, proliferation, and cell survival (7, 45). Aberrant activation of AKT through diverse mechanisms has been discovered in different cancers (2). AKT activation requires phosphorylation of AKT on threonine residue 308 and serine residue 473. The significance of phosphorylation of AKT on tyrosine residues is less well understood. Src has been shown to phosphorylate AKT on conserved tyrosine residues 315 and 326 near the activation loop (11). Substitution of these two tyrosine residues with phenylalanine abolished AKT kinase activity stimulated by EGF (11). Use of the Src family inhibitor PP2 impaired AKT activation following IGF-1 stimulation of oligodendrocytes (13). The RET/PTC receptor tyrosine kinase that responds to glial cell-line-derived neurotrophic factor also phosphorylated AKT tyrosine residue 315 promoting activation of AKT (28). AKT tyrosine residue 474 was phosphorylated when cells were treated with the tyrosine phosphatase inhibitor pervanadate, and phosphorylation of tyrosine 474 contributed to full activation of AKT (12). Recently, the nonreceptor tyrosine kinase Ack1 was shown to regulate AKT tyrosine phosphorylation and activation (37).Here we show that AKT is a cytoplasmic substrate of the intracellular tyrosine kinase PTK6. We identify the tyrosine residues on AKT that are targeted by PTK6, and we demonstrate that tyrosine phosphorylation plays a role in regulating association between PTK6 and AKT. In addition, we show that PTK6 promotes AKT activation and cell proliferation in a Src-independent manner.     

Thiopental Inhibits Global Protein Synthesis by Repression of Eukaryotic Elongation Factor 2 and Protects from Hypoxic Neuronal Cell Death

Ischemic and traumatic brain injury is associated with increased risk for death and disability. The inhibition of penumbral tissue damage has been recognized as a target for therapeutic intervention, because cellular injury evolves progressively upon ATP-depletion and loss of ion homeostasis. In patients, thiopental is used to treat refractory intracranial hypertension by reducing intracranial pressure and cerebral metabolic demands; however, therapeutic benefits of thiopental-treatment are controversially discussed. In the present study we identified fundamental neuroprotective molecular mechanisms mediated by thiopental. Here we show that thiopental inhibits global protein synthesis, which preserves the intracellular energy metabolite content in oxygen-deprived human neuronal SK-N-SH cells or primary mouse cortical neurons and thus ameliorates hypoxic cell damage. Sensitivity to hypoxic damage was restored by pharmacologic repression of eukaryotic elongation factor 2 kinase. Translational inhibition was mediated by calcium influx, activation of the AMP-activated protein kinase, and inhibitory phosphorylation of eukaryotic elongation factor 2. Our results explain the reduction of cerebral metabolic demands during thiopental treatment. Cycloheximide also protected neurons from hypoxic cell death, indicating that translational inhibitors may generally reduce secondary brain injury. In conclusion our study demonstrates that therapeutic inhibition of global protein synthesis protects neurons from hypoxic damage by preserving energy balance in oxygen-deprived cells. Molecular evidence for thiopental-mediated neuroprotection favours a positive clinical evaluation of barbiturate treatment. The chemical structure of thiopental could represent a pharmacologically relevant scaffold for the development of new organ-protective compounds to ameliorate tissue damage when oxygen availability is limited.     

miR-homoHSV of Singapore Grouper Iridovirus (SGIV) Inhibits Expression of the SGIV Pro-apoptotic Factor LITAF and Attenuates Cell Death

Growing evidence demonstrates that various large DNA viruses could encode microRNAs (miRNAs) that regulate host and viral genes to achieve immune evasion. In this study, we report that miR-homoHSV, an miRNA encoded by Singapore grouper iridovirus (SGIV), can attenuate SGIV-induced cell death. Mechanistically, SGIV miR-homoHSV targets SGIV ORF136R, a viral gene that encodes the pro-apoptotic lipopolysaccharide-induced TNF-α (LITAF)-like factor. miR-homoHSV suppressed exogenous and endogenous SGIV LITAF expression, and thus inhibited SGIV LITAF-induced apoptosis. Meanwhile, miR-homoHSV expression was able to attenuate cell death induced by viral infection, presumably facilitating viral replication through the down-regulation of the pro-apoptotic gene SGIV LITAF. Together, our data suggest miR-homoHSV may serve as a feedback regulator of cell death during viral infection. The findings of this study provide a better understanding of SGIV replication and pathogenesis.     

Platelet-derived Growth Factor Differentially Regulates the Expression and Post-translational Modification of Versican by Arterial Smooth Muscle Cells through Distinct Protein Kinase C and Extracellular Signal-regulated Kinase Pathways

The synthesis of proteoglycans involves steps that regulate both protein and glycosaminoglycan (GAG) synthesis, but it is unclear whether these two pathways are regulated by the same or different signaling pathways. We therefore investigated signaling pathways involved in platelet-derived growth factor (PDGF)-mediated increases in versican core protein and GAG chain synthesis in arterial smooth muscle cells (ASMCs). PDGF treatment of ASMCs resulted in increased versican core protein synthesis and elongation of GAG chains attached to the versican core protein. The effects of PDGF on versican mRNA were blocked by inhibiting either protein kinase C (PKC) or the ERK pathways, whereas the GAG elongation effect of PDGF was blocked by PKC inhibition but not by ERK inhibition. Interestingly, blocking protein synthesis in the presence of cycloheximide abolished the PDGF effect, but not in the presence of xyloside, indicating that GAG synthesis that results from PKC activation is independent from de novo protein synthesis. PDGF also stimulated an increase in the chondroitin-6-sulfate to chondroitin-4-sulfate ratio of GAG chains on versican, and this effect was blocked by PKC inhibitors. These data show that PKC activation is sufficient to cause GAG chain elongation, but both PKC and ERK activation are required for versican mRNA core protein expression. These results indicate that different signaling pathways control different aspects of PDGF-stimulated versican biosynthesis by ASMCs. These data will be useful in designing strategies to interfere with the synthesis of this proteoglycan in various disease states.     

The Tyrosine Phosphatase SHP2 Modulates MAP Kinase p38 and Caspase 1 and 3 to Foster Neuronal Survival

1. The Src homology protein tyrosine phosphatase SHP2 is associated with cytoskeletal maintenance, cell division, and cell differentiation, but the role of SHP2 during central nervous system injury requires further definition. We therefore characterized the role of SHP2 during nitric oxide (NO)-induced programmed cell death (PCD).2. Employing primary hippocampal neurons from mice with a dominant negative SHP2 mutant to render the phosphatase site of the SHP2 protein biologically inactive, but functionally capable of binding substrate, neuronal injury was evaluated by trypan blue, DNA fragmentation, membrane phosphatidyl serine (PS) exposure, mitogen-activated protein (MAP) kinase phosphorylation, and cysteine protease activity. NO was administered through the NO generators SIN-1 (300 M) or NOC-9 (300 M).3. Following NO exposure, neuronal survival decreased from 89 ± 3% in untreated controls to 37 ± 2% in wild-type neurons and to 21 ± 4% in SHP2 mutant neurons. In sister cultures following NO exposure, this increased susceptibility to neuronal injury paralleled enhanced genomic DNA degradation and membrane PS exposure with PCD induction increasing in SHP2 mutant neurons by approximately 42% during specified time periods when compared to wild-type neurons. Interestingly, modulation of the MAP kinase p38 appears to represent an initial level of neuronal protection employed by SHP2. In addition, both the rate and degree of caspase 1- and caspase 3-like activities in SHP2 mutant neurons were significantly increased over a 24-h course when compared to wild-type neurons. Inhibition of caspase 1- and caspase 3-like activities reversed the progression of neuronal PCD, suggesting that inhibition of cysteine protease activity is a downstream mechanism for SHP2 to afford neuronal protection.4. Our work supports the premise that the tyrosine phosphatase SHP2 plays a dominant role during NO-induced PCD and may offer a potential molecular checkpoint against neurodegenerative disease.