Changchuan Xie

PRAK is essential for ras-induced senescence and tumor suppression.

Like apoptosis, oncogene-induced senescence is a barrier to tumor development. However, relatively little is known about the signaling pathways mediating the senescence response. p38-regulated/activated protein kinase (PRAK) is a p38 MAPK substrate whose physiological functions are poorly understood. Here we describe a role for PRAK in tumor suppression by demonstrating that PRAK mediates senescence upon activation by p38 in response to oncogenic ras. PRAK deficiency in mice enhances DMBA-induced skin carcinogenesis, coinciding with compromised senescence induction. In primary cells, inactivation of PRAK prevents senescence and promotes oncogenic transformation. Furthermore, we show that PRAK activates p53 by direct phosphorylation. We propose that phosphorylation of p53 by PRAK following activation of p38 MAPK by ras plays an important role in ras-induced senescence and tumor suppression.

The miR-17-92 Cluster of MicroRNAs Confers Tumorigenicity by Inhibiting Oncogene-Induced Senescence

In mammalian cells, activation of oncogenes usually triggers innate tumor-suppressing defense mechanisms, including apoptosis and senescence, which are compromised by additional mutations before cancers are developed. The miR-17-92 gene cluster, a polycistron encoding six microRNAs (miRNA), is frequently overexpressed in human cancers and has been shown to promote several aspects of oncogenic transformation, including evasion of apoptosis. In the current study, we show a new role of miR-17-92 in inhibiting oncogenic ras-induced senescence. Further dissection of the miRNA components in this cluster reveals that the miR-17/20a seed family accounts for this antisenescence activity. miR-17 and miR-20a are both necessary and sufficient for conferring resistance to ras-induced senescence by directly targeting p21(WAF1), a key effector of senescence. By contrast, these components are not essential for the ability of miR-17-92 to evade Myc-induced apoptosis. Moreover, disruption of senescence by miR-17-92 or its miR-17/20a components leads to enhanced oncogenic transformation by activated ras in primary human cells. Taken together with previous reports that miR-17-92 inhibits apoptosis by suppressing Pten via the miR-19 components, our results indicate that this miRNA cluster promotes tumorigenesis by antagonizing both tumor-suppressing mechanisms, apoptosis, and senescence, through the activities of different miRNA components encoded in this cluster.

Participation of the p38 pathway in Drosophila host defense against pathogenic bacteria and fungi

The signaling network of innate immunity in Drosophila is constructed by multiple evolutionarily conserved pathways, including the Toll- or Imd-regulated NF-κB and JNK pathways. The p38 MAPK pathway is evolutionarily conserved in stress responses, but its role in Drosophila host defense is not fully understood. Here we show that the p38 pathway also participates in Drosophila host defense. In comparison with wild-type flies, the sensitivity to microbial infection was slightly higher in the p38a mutant, significantly higher in the p38b mutant, but unchanged in the p38c mutant. The p38b;p38a double-mutant flies were hypersensitive to septic injury. The immunodeficiency of p38b;p38a mutant flies was also demonstrated by hindgut melanization and larvae stage lethality that were induced by microbes naturally presented in fly food. A canonical MAP3K-MKK cascade was found to mediate p38 activation in response to infection in flies. However, neither Toll nor Imd was required for microbe-induced p38 activation. We found that p38-activated heat-shock factor and suppressed JNK collectively contributed to host defense against infection. Together, our data demonstrate that the p38 pathway-mediated stress response contribute to Drosophila host defense against microbial infection.

Kinases Mst1 and Mst2 positively regulate phagocytic induction of reactive oxygen species and bactericidal activity

Mitochondria need to be juxtaposed to phagosomes for the synergistic production of ample reactive oxygen species (ROS) in phagocytes to kill pathogens. However, how phagosomes transmit signals to recruit mitochondria has remained unclear. Here we found that the kinases Mst1 and Mst2 functioned to control ROS production by regulating mitochondrial trafficking and mitochondrion-phagosome juxtaposition. Mst1 and Mst2 activated the GTPase Rac to promote Toll-like receptor (TLR)-triggered assembly of the TRAF6-ECSIT complex that is required for the recruitment of mitochondria to phagosomes. Inactive forms of Rac, including the human Rac2D57N mutant, disrupted the TRAF6-ECSIT complex by sequestering TRAF6 and substantially diminished ROS production and enhanced susceptibility to bacterial infection. Our findings demonstrate that the TLR-Mst1-Mst2-Rac signaling axis is critical for effective phagosome-mitochondrion function and bactericidal activity.Summary Mitochondria need to be juxtaposted to phagosomes to synergistically produce ample reactive oxygen species (ROS) in phagocytes for pathogens killing. However, how phagosomes transmit signal to recruit mitochondria remains unclear. Here, we report that the kinases Mst1 and Mst2 function to control ROS production by regulating mitochondrial trafficking and mitochondrion-phagosome juxtaposition. Mst1 and Mst2 activate Rac GTPase to promote Toll-like receptor (TLR)-triggered assembly of the TRAF6-ECSIT complex that is required for mitochondrial recruitment to phagosomes. Inactive forms of Rac, including the human Rac2D57N mutant, disrupt the TRAF6-ECSIT complex by sequestering TRAF6, and severely dampen ROS production and greatly increase susceptibility to bacterial infection. These findings demonstrate the TLR-Mst1-Mst2-Rac signalling axis to be critical for effective phagosome-mitochondrion function and bactericidal activity.

beta-actin is required for mitochondria clustering and ROS generation in TNF-induced, caspase-independent cell death

Tumor necrosis factor (TNF)-α induces caspase-independent cell death in the fibrosarcoma cell line L929. This cell death has a necrotic phenotype and is dependent on production of reactive oxygen species (ROS) in the mitochondria. To identify genes involved in this TNF-induced, ROS-dependent cell death pathway, we utilized retrovirus insertion-mediated random mutagenesis to generate TNF-resistant L929 cell lines and we subsequently identified genes whose mutations are responsible for the TNF-resistant phenotype. In one such resistant line, β-actin was disrupted by viral insertion, and subsequent reconstitution of β-actin expression levels in the mutant line Actinmut restored its sensitivity to TNF. Resistance to TNF in Actinmut cells is signal specific since the sensitivity to other death stimuli is either unchanged or even increased. Comparable NF-κB activation and p38 phosphorylation in TNF-treated wild-type and Actinmut cells also indicates that reduced expression of actin only selectively blocked some of the TNF-induced cellular changes. Actin cleavage involved in apoptosis does not occur in TNF-treated L929 cell death, as in HeLa cells. Consistent over-expression of a caspase-cleaved product, a 15 kDa actin fragment, had no effect on TNF-induced necrosis of L929 cell. By contrast, TNF-induced mitochondria clustering and ROS production were dramatically reduced in Actinmut cells, indicating that actin-deficiency-mediated TNF resistance is most likely due to impaired mitochondrial responses to TNF stimulation. Our findings suggest that a full complement of actin is required for transduction of a cell death signal to mitochondria in TNF-treated L929 cells.

Distinct roles of basal steady-state and induced H-ferritin in tumor necrosis factor-induced death in L929 cells

ABSTRACT Tumor necrosis factor (TNF) alpha is a cytokine capable of inducing caspase-dependent (apoptotic) cell death in some cells and caspase-independent (necrosis-like) cell death in others. Here, using a mutagenesis screen for genes critical in TNF-induced death in L929 cells, we have found that H-ferritin deficiency is responsible for TNF resistance in a mutant line and that, upon treatment with TNF, this line fails to elevate levels of labile iron pool (LIP), critical for TNF-induced reactive oxygen species (ROS) production and ROS-dependent cell death. Since we found that TNF-induced LIP in L929 cells is primarily furnished by intracellular storage iron, the lesser induction of LIP in H-ferritin-deficient cells results from a reduction of intracellular iron storage caused by less H-ferritin. Different from some other cell lines, the H-ferritin gene in L929 cells is not TNF inducible; however, when H-ferritin is expressed in L929 cells under a TNF-inducible system, the TNF-induced LIP and subsequent ROS production and cell death were all prevented. Thus, LIP is a common denominator of ferritin both in the enhancement of cell death by basal steady-state H-ferritin and in protection against cell death by induced H-ferritin, thereby acting as a key determinant of TNF-induced cell death.

Determinants That Control the Specific Interactions between TAB1 and p38α

ABSTRACT Previous studies have revealed that transforming growth factor-β-activated protein kinase 1 (TAB1) interacts with p38α and induces p38α autophosphorylation. Here, we examine the sequence requirements in TAB1 and p38α that drive their interaction. Deletion and point mutations in TAB1 reveal that a proline residue in the C terminus of TAB1 (Pro412) is necessary for its interaction with p38α. Furthermore, a cryptic D-domain-like docking site was identified adjacent to the N terminus of Pro412, putting Pro412 in the φB+3 position of the docking site. Through mutational analysis, we found that the previously identified hydrophobic docking groove in p38α is involved in this interaction, whereas the CD domain and ED domain are not. Furthermore, chimeric analysis with p38β (which does not bind to TAB1) revealed a previously unidentified locus of p38α comprising Thr218 and Ile275 that is essential for specific binding of p38α to TAB1. Converting either of these residues to the corresponding amino acid of p38β abolishes p38α interaction with TAB1. These p38α mutants still can be fully activated by p38α upstream activating kinase mitogen-activated protein kinase kinase 6, but their basal activity and activation in response to some extracellular stimuli are reduced. Adjacent to Thr218 and Ile275 is a site where large conformational changes occur in the presence of docking-site peptides derived from p38α substrates and activators. This suggests that TAB1-induced autophosphorylation of p38α results from conformational changes that are similar but unique to those seen in p38α interactions with its substrates and activating kinases.

The transcriptional coactivator TAZ regulates reciprocal differentiation of TH17 cells and Treg cells

An imbalance in the lineages of immunosuppressive regulatory T cells (Treg cells) and the inflammatory TH17 subset of helper T cells leads to the development of autoimmune and/or inflammatory disease. Here we found that TAZ, a coactivator of TEAD transcription factors of Hippo signaling, was expressed under TH17 cell–inducing conditions and was required for TH17 differentiation and TH17 cell–mediated inflammatory diseases. TAZ was a critical co-activator of the TH17-defining transcription factor RORγt. In addition, TAZ attenuated Treg cell development by decreasing acetylation of the Treg cell master regulator Foxp3 mediated by the histone acetyltransferase Tip60, which targeted Foxp3 for proteasomal degradation. In contrast, under Treg cell–skewing conditions, TEAD1 expression and sequestration of TAZ from the transcription factors RORγt and Foxp3 promoted Treg cell differentiation. Furthermore, deficiency in TAZ or overexpression of TEAD1 induced Treg cell differentiation, whereas expression of a transgene encoding TAZ or activation of TAZ directed TH17 cell differentiation. Our results demonstrate a pivotal role for TAZ in regulating the differentiation of Treg cells and TH17 cells.

The p38 pathway regulates oxidative stress tolerance by phosphorylation of mitochondrial protein IscU.

Background: The p38 pathway is an evolutionarily conserved signaling pathway that responds to a variety of stresses. Results: dIscU can be phosphorylated by dMK2, thereby impacting mitochondrial respiratory complex I activity. Conclusion: Iron-sulfur cluster protein IscU phosphorylation by MK2 downstream of p38 signaling may regulate oxidative stress tolerance. Significance: IscU is a novel substrate of MK2, mechanistically connecting the p38 pathway and mitochondria iron-sulfur clusters for the first time. The p38 pathway is an evolutionarily conserved signaling pathway that responds to a variety of stresses. However, the underlying mechanisms are largely unknown. In the present study, we demonstrate that p38b is a major p38 MAPK involved in the regulation of oxidative stress tolerance in addition to p38a and p38c in Drosophila. We further show the importance of MK2 as a p38-activated downstream kinase in resistance to oxidative stresses. Furthermore, we identified the iron-sulfur cluster scaffold protein IscU as a new substrate of MK2 both in Drosophila cells and in mammalian cells. These results imply a new mechanistic connection between the p38 pathway and mitochondria iron-sulfur clusters.

c-Src phosphorylation and activation of hexokinase promotes tumorigenesis and metastasis

It is well known that c-Src has important roles in tumorigenesis. However, it remains unclear whether c-Src contributes to metabolic reprogramming. Here we find that c-Src can interact with and phosphorylate hexokinases HK1 and HK2, the rate-limiting enzymes in glycolysis. Tyrosine phosphorylation dramatically increases their catalytic activity and thus enhances glycolysis. Mechanistically, c-Src phosphorylation of HK1 at Tyr732 robustly decreases its Km and increases its Vmax by disrupting its dimer formation. Mutation in c-Src phosphorylation site of either HK1 or HK2 remarkably abrogates the stimulating effects of c-Src on glycolysis, cell proliferation, migration, invasion, tumorigenesis and metastasis. Due to its lower Km for glucose, HK1 rather than HK2 is required for tumour cell survival when glucose is scarce. Importantly, HK1-Y732 phosphorylation level remarkably correlates with the incidence and metastasis of various clinical cancers and may serve as a marker to predict metastasis risk of primary cancers.